·THE PHYSIOLOGY AND BIOCHEVISTRY OF LACTATION

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·THE PHYSIOLOGY
AND BIOCHEMISTRY OF
LACTATION
BY
S J FOLLEY
DSc PhD FRS
Head of the Physiology Department
National Institute for Research in Dairying
Shunfield Reading Berks
OLIVER AND BOYD
EDINBURGH TWEEDDALE COURT
LONDON 39A WELBECK STREET WI

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·PREFACE TO THE ENGLISH EDITION In prepanng an English version of my book Recherches Recentes sur la Physiologue et la Biochimie de la Secretion Lactee which was published in December 1954 as No 18 of the series Actualites Biochimiques under the imprint of Editions Desoer of Liege and Masson et Cic of Paris, I have brought the text up to date by inserting bref references to papers published since that time which appear to be important Apart from this and the correction of such crrors as have been brought to my attention the text remains substantially unchanged In addı tion to the acknowledgements made previously I should lhe to express my thanks to Mrs Gwen Stephen of the Classics Department University of Reading for information about the mythology of the origin of the galaxy After completion of the manuscript unfortunate circum stances beyond my control intervened to prevent me from seeing this book through the press This arduous and exact ing task was at once willingly undertaken by my colleague, Dr A T Cowie to whom my grateful thanks are due Without his help, unstintingly given, this book could never have been published I am also grateful to Miss Anne Pendree and Mrs M C Lloyd for help in the preparation of the index The subject matter of these lectures embraces a field which has occupied the Physiology Department of the National Institute for Research in Dairying for many years and in offering them in the form of a book I hope that it will be found useful by medical and veterinary students particularly those taking the degree of B Sc in Physiology or Biochemistry Shinfield June 1955 S J FOLLEY

·FIRST ENGLISH EDITION 1956
PRINTED IN HOLLAXD
SY KONINKLIKE VERENIGDE DRUKKERIJEN ROITSEMA NY
TOR OLIVER AND BOYD LYD EDINBLAGH
GRONINGEN

·FORET ORD VII Lister Institute, London as well as to Dr Raymond Michel of the Collège de France who also helped me considerably in vanous ways with the preparation of my lectures The expenses entailed in connection with the translation and preparation of these lectures for publication were met by grants from the Bureau Scientifique de l Ambassade de France in London, through the courtesy of Vonsieur Rene Vann, Conseiller Culturel and M&S A N Vidal Hall and by the Ciba Foundation through the courtesy of the Director Dr GEW Wolstenholme O BE To both these bodies I express my sincere thanks I would also like to acknowledge the great debt I owe to my collaborators whose work forms the basis of the greater part of these lectures and who lindly gave me permission to quote their unpublished expenments Their names will be found in appropriate places in the text It is a pleasure also to thank Mr G Machin for drawing the figures and Mr N Gruber for skilled photographic work and my secretary Mrs Jean King My thanks are also due to all who have given me permission to reproduce material from published papers SJ FOLLEY ·FOREWORD The lectures which comprise this book were delivered at the College de France, where I was invited as a visiting Professor in December, 1953 In preparing them for publication I have changed them as little as possible from their original form I should like first to express to Monsieur 1 Administrateur, M Edmond Faral, and the Professors of the Collège de France my deep appreciation of the honour accorded to me in issuing their invitation My gratitude is especially due to Professor Jean Roche who gave me the hospitality of his Department during my stay it is to him that I owe the suggestion that these lectures might be published I am greatly in his debt for his invaluable assistance in preparing this edition for publi cation in the series Actualites Brochimiques I should also like to record my indebtedness to Professor Robert Courrier, Membre de l'Institut who not only introduced me to my audience but in various ways did much to make my stay in Paris pleasant There would have been no question of my being in a position to accept the invitation to lecture in the College de France had I not been able to enlist the moral support and encouragement of Professor A G Lehmann of the Department of French Studies, University of Reading who kindly put at my disposal the valuable help of two of the staff of his Department Monsieur Jean Gautier who willingly undertook the difficult task of translating my lectures into French and Mrs Antoinette Hunt, who toob endless trouble to improve my French pronunciation I would be to express my sincere gratitude to them For help with the translation of technical terms not found in dictionaries my best thanks are due to Professor Marc Klein of the University of Strasbourg, and Dr L Sazbo then of the ·CHAPTER I RECENT STUDIES ON THE DEVELOPMENT OF THE MAMMARY GLAND NORMAL POST NATAL DEVELOPMENT Earlier comparative studies on the normal development of the mammary gland in different reproductive phases, whuch laid the foundations of our knowledge of the endocrine control of mammary development, suggested that growth of the mammary duct system depended upon the presence of oestro gen and that for extensive lobule alveolar development luteal influence was necessary in addition Later, when expenmental analysis of the hormonal control of mammary growth by ex periments involving the administration of pure hormones to spayed females or males became possible, these generalities resulting from observational studies though substantiated in broad outline were found to be over simplifications in the case of some species The reader is referred to the review by Folley (1952) for an account of these observational and ex perimental studies Recently there has been a quickening of interest in the development of quantitative and objective methods for studying mammary growth One reason for this is the prospect of practical application of fundamental knowledge of the hor monal control of the mammary gland to the artificial stimula tion of udder growth and lactation in dairy cattle Such tech niques should also prove valuable to workers in the cancer field who are interested in the influences determining the architecture of the mammary gland in various strains of mouse The subject has reached a stage of development at which the subjective and qualitative methods that have served in the past can no longer be expected to lead to further progress the extension of knowledge demands the application of quantita tive methods ·CONTENTS PAGB CHAPTER I RECENT STUDIES IN THE DEVELOPMENT OF THE MAM MARY GLAND Normal post natal development Experimental analysis of hormonal influences 1 II THE INITIATION OF MILK SECRETION (LACTOGENESIS) The biochemistry of lactogenesis Thc hormonal control of lactation induction at parturition 23 34 III The MAINTENANCE OF MILK SECRETION - GALACTO POIESIS Prolactin and somatotrophın The pituitary adrenal mechanism The pituitary thyroid mechanism 47 53 62 73 IN THE PHYSIOLOGY OF SUCKLING AND MILKING The milk ejection reflex Evidence for the neuroendocrinc regulation of milk ejection Nature of the milk ejection hormone The effector contractile tissue of the mammary gland 77 86 88 93 v The BIOSYNTHESIS OF MILK FAT AND ITS HORMONAL CONTROL Earlier theories of the origin of milk fat Synthesis of fatty acids from small molecules in the mammary gland The origin of the glycerol of the milk glycerides Hormonal control of lipogenesis in the mammary gland 96 IIO v RECENT STUDIES OF THE BIOSYNTHESIS OF LACTOSE AND MILK PROTEINS Thc ongin of the lactose of mulk Thc biosynthesis of milk proteins 119 130 ·DEVELOPMENT OF MAMMARY GLAND 3 and mouse (Flux, 19540) The whole mount technique is of course not applicable to three dimensional glands like the udder of the ruminant the human breast or thc mamma of the guinea pig, and other methods must be used for these Mr KC Richardson of the Department of Anatomy, University College London with whom we have had the advantage of working in collaboration, has evolved a tech nique for the estimation of the total alveolar surface area in the goat udder based on a method onginally developed by another worker for application to lung tissue Figure I 1 shows the excellent linear relationship between total alveolar area and mill yield in a series of hormonally developed goat half udders (Richardson 1953), thus allustrating the usefulness of Richardson s technique This technique, since it also affords an estimate of the area of the secretory epithelium per unit volume of tissue ('porosity index ) also gives a measure of the mean diameter of the alveoli This type of measurement as applied to the goat udder, has been further developed by Benson Cowie, Cox Flux and Folley (1955) Though it has long been known that in many species the mammary duct system undergoes a slow extension during the period from birth to puberty it has generally been believed that mammary growth remains substantially in abeyance until puberty when the beginning of ovarian cycles was thought to initiate a phase of a rapid mammary development (see review by Folley, 1952) Quantitative studies of the rate of increase in the mammary gland area in relation to the rate of enlargement of the body surface show that this is not the case for the rat and mouse Nor as was shown some years ago, is it true for the monkey It probably does not hold for any mammal The first use of relative growth analysis which was de veloped in France by G Teissier and in England by J S Huxley, for the quantitative study of mammary growth in the normal female was made many years ago in the monkey by Folley, Guthkelch and Zucherman (1939) Table I 1 ex plains the principles of relative growth analysis and gives the terminology proposed by Huxley and Teissier (1936) The value of the equilibrium constant , which can be evaluated by a simple graphical method indicates whether the organ ·2 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION In species, such as the mouse, rat and rhesus monkey, in which the mammae, save in late pregnancy and lactation, may for practical purposes be regarded as more or less Alat sheets of tissue so that they can be prepared for histological study as whole mounts, changes in the mammary gland area, that is the area bounded by a line joining the periphery of the ducts give a measure of the rate of general extension of the duct tree These measurements of course provide no informa tion about morphological changes within that area such as increased branching of the duct system or the formation or multiplication of alveoli In 1947 Dr A T Cowie and I attempted to devise a scoring system, semi quantitative and susceptible of statistical analysis, which we hoped would af ford some measure of qualitative changes of this nature (Cowie & Folley, 1947) More recently, improved and more ob jective techniques for estimating the degree of branching have been developed in our laboratory for the rat (Silver, 1953) 45 33 30 O Surface area of secretory epithelium (mº) o Control uhol. udders Half udd rs deweloped arhficially * 14 16 6 8 10 1. Milk yleld (litres/week) Fig I 1 Relation between milk production and total alveolar arca in a series of half udders from goats (from Richardson 1953) ·DEVELOPMENT OF MAMMARY GLAND 5 showed that over a wide range of body weights the breast in the non pregnant female macaque grows faster than the body according to the simple allometric law presumably under the specific influence of ovanan hormones More detailed studies of the dynamics of mammary growth using relative growth analysis were later made in our laboratory in the rat (Cowe, 1949) As Figure I 2 shows Dr Cowie found that in the female rat the total area of all the mammary glands in creased at approximately the same rate as the body surface, that is isometrically, until the 22nd-23rd day when a phase of rapid allometric growth abruptly set in In this phase « had a value of 3 03 which means that the mammae were increasing in area about three times faster than the body surface Ovarı ectomy at the 22nd day prevented the onset of the allometric phase and isometric growth (a not significantly different from unity) continued Since in our strain of rat the vagina does not open nor oestrous cycles begin unul the 35th-42nd day It is clear that the mammary gland begins to grow faster than the body well before puberty a result which probably would not have been revealed merely by the conventional qualita tive examnation of whole mounts or thin sections In the immature male rat, the increase of mammary area on body surface was very slightly but significantly allometric since TABLE I 2 Equilibrium corzstants for mammary growth relative to body growth in the Norway rat (from Silver, 19534) Equihbrum constant Significance of the Group (a) Silver (b) Cowie difference (1953) (1949) (a) (b) Females (intact) 3 40+017 303+013 Males (intact) 1 1740 05 II+0 03 P>01 Females (ovariectomized) I 45+0 09 1 05 10 10 Males (castrated) I 34+008 I 06H012 P=09 P=0005 P=007 ·4 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION TABLE II Relatue growth analysis Law of simple allometric growth (Huxley, Teissier) brº or log y o log + log b y total mammary gland area * arta of reference standard (body) equilibrium constant 6 constant If the law is obeyed, a plut of log y against log & gives a straight line with the slope o a>r positive allometry a<1 negative allometry a *1 isometry under study is growing faster than the body as a whole, if it is then « is greater than unity In our study of the monkey, we i. Bats Age 2-100 days o Controls . Ovaricctomized 303 O! 1 OS : O 10 108,0 total mammary gland area (mm) 110 06 22-6 23* daj & 20 $2 16 OS L0316 [body weight (2)] Fig 12 Relation between total mammary gland area and (body weight) in the female rat (fross Cours 1949) ·DEVELOPMENT OF MAMVARY GLAND 7 Evidence beanng on this question has recently been ob tained in our laboratory (Silver, 19536) Mrs Silver found that the mammary gland of the suchling rat shows no growth response to physiological doses of oestrogen administered between the 17th and 19th days of age a finding which is in conflict with the earlier results of Astwood, Geschichter and Rausch (1937) who reported that oestrogen would begin to evole growth of the rat mammary gland as early as the 16th day Howcver, it should be noted that the doses used by Astwood and his colleagues were relatively high and probably unphysiological It was further found that low doses of oestro gen administered together with an anterior pituitary extract, ineffective by itself caused appreciable growth of the mammae in the young suchling rat Similarly Sykes and Wrenn (1950) have found that the mammary growth promoting effect of oestrogen and progesterone in young calves was potentiated by anterior pituitary extract If, therefore as has been suggested the role of the anterior pituitary in mammary growth is to sensitize the mammae to the mammogenic effect of oestrogen it would appear that in the suckling rat the pituitary is not yet fully functional in this respect These experiments how ever do not exclude the possibility that in the rat at least some factor ingested in the mill perhaps androgen, is respon sible for the failure of the pituitary to play its part in mammary growth Relative growth analysis has also added to our knowledge of the factors governing the growth of the mamma in the normal male rat The fact that the gland of the male rat grows isometri cally or nearly so, and that its specific growth rate is not affected by castration demonstrates more decisively than had previously been possible that the testis has very little effect on the growth of the mammary duct system in the intact male rat However castration at 21 days presents for a time the development of the lobules of als eoli which constitute a remarkable and char acteristic feature of the mammary gland in normal male rats first described long ago by Turner and Schultze (1931) AI veolar developinent in the mamma of the male rat would thus seem to depend on the presence of the testis Nevertheless, as we showed some years ago, (Cowie and Folley, 1947) some alveoli eventually develop in the mammae of immaturely ha ·6 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION had a value of it and the relative growth rate was not signs ficantly altered by castration at 22 days These findings have since been confirmed and extended in our laboratory with the same strain of rat by Mfrs Marian Silver She found, however, as shown in Table 1 2 which gives her equilibrium constants together with those of Dr Cowie, that ovariectomy at ten days of age, that is cleven days earlier than in the experiments of Cowie, was eventually followed by a phase of slightly allo metric growth of the mammae (Silver 1953a) This might be due to the action of mammogenic steroids secreted by the adrenal cortices under unremitting stimulation by a pituitary released from ovarian control Similar studies carried out in our laboratory by Dr DS Flux on female mice of Strong s CHI strain showed that here too the mammae grew isometrically over the 7th-21st days of life As in the rat, a phase of marked allometry, the onset of which could be prevented by ovariectomy, set in at about 24 days The value obtained by Dr Flux for the equilibrium constant during the allometric phase (« = 522) indicates that after weaning the mouse mammary gland grows about five times faster than the body surface by contrast with that of the rat which grows only three times faster than the body (Flux 1954a) Whether this species difference in specific growth rate is in any way connected with the relative basal metabolic rates of the two forms is a matter for speculation In this strain of mouse the phase of rapid mammary duct growth does not precede the onset of cyclic ovarian activity, which occurs at about 28 days, by so long an interval as in our strain of rat While these studies establish the necessity of the ovary for the abrupt change from isometric to allometric mammary growth the nature of the mechanism involved and the reason why allomeric growth begins just when it does are not known with certainty It is possible to discuss this quesuon in terms of the ume of maturation of pituitary gonadotrophic function and of ovarian responsiveness to pituitary influence Neverthe less the fact that the change from isometry to allometry closely follows weaning may not be coincidental and raises the ques tion whether the female rat secretes in her milk a substance which antagonises the mammogenic acuon of oestrogen

·I LATE 13
2
3
4
10 mm
The figures represent whole mounts of approximately half a mammary
sland from a male rabbit (from Scharf and Lyons 1941)
30 Desirone per day
2 240
30 14 Destrone -
1 mg progesterone per day
5 240
6960
3 960

·8 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION gonadectomized rats and just as in the case of the female spayed at 10 days already referred to, it seems reasonable to seek the cause in enhanced production by the adrenal cortex of mammogenic steroids, such as androgens or perhaps pro gesterone, due to the disturbance in the endocrne balance brought about by gonadectomy EXPERIMENTAL ANALYSIS OF HORMONAL INFLUENCES Olarran hormones We now come to the experimental analysis of hormonal influences in mammary growth and will consider first the ovarian hormones Early work involving hormone administration pointed to the general conclusion that of the ovarian hormones oestrogen is more particularly responsible for the growth of the mammary duct system while pro gesterone, acting in concert with oestrogen, is necessary for full alveolar growth Later work has shown that in forms such as the mouse, rat and monkey progesterone alone, if enough is given, will evoke development of the alveolt and perhaps of the ducts in spayed animals unprimed with oestrogen For a review of the above mentioned studies the reader should con sult Folley (1952) The general conclusion emerging from the early studies a conclusion which incidentally remains unshaken by the latest work, was that both oestrogen and progesterone are needed for the experimental growth of a gland comparable morphologic ally with that of mid pregnancy However it was apparent from the outset that various species show considerable differ ences in the nature of the growth response of the mammary rudiment to oestrogen and on this basis three broad categories among laboratory and domestic animals can be disunguished though we must not forget that interpretation may be complı cated by the possibility that spayed animals may still possess a source of progesterone in the adrenal cortex The first category includes the mouse rat, rabbit and cat in all of which the male gland is equipotendal sath that of the female In the mammae of spayed females or of intact or cas trated males of this group oestrogen in physiological doses evokes primanly and mainly duct growth, alveolt only appear ing when hugher doses are given and when administration is prolonged This last point is illustrated by the rabbit in which, ·DEVELOP\ENT OF MAMARY GLAND 9 as Lewis and Tumer (1941) and others have shown, mammary alveoli can be descloped by high doses of oestrogen if applied for long enough It might therefore be questioned whether the rabbit should be included in the present category However, the striking synergum between oestrogen and progesterone shown in the beautifully clear-cut results of Lyons and his collaborators (Lyons and VicGinty 1941, Scharf and Lyons, 1941), some of which are shown in Plate 13 effectively disposes of such doubts This Plate shows whole mounts of mammary glands of male rabbits some of which had received oestrone and others oestrone and progesterone The synergistic action of progesterone in causing lobule alveolar growth is quite evident. 40 38 O-Rats rowing Hic strigarh Regression line for the intact centr Is - Regression in for the narut maatris - 3 . 3-0 Log10 total mammary gland area (mm) Os 8 2 OR 15 12 i ti Log. [body weight (g)] Fig 14 Relation between the total mammary gland area and (body weight)' in the ovanectomized rat receiving a dose of oestradiol dipropionate which was increased stepwise with the body weight (from Super 19534)

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·DEVELOPMENT OF MAMMARY CLAND normal mammary growth process as judged by the equili brium constant and by the increase in duct buds and branches by starung with a dose of o i ug oestradiol dipropionate every other day when the rats were 21 days old and increasing the dose stepwise with the body weight Figure 1 4 shows how closely she was able to imitate the relative mammary growth rate of the intact female rat Mrs Silver was also able to obtain evidence that as R Hertz first showed for the oviduct of the fowl and for the rat uterus folic acid is concerned in some way in the growth response of the mammary ducts to oestrogen This is illustrated in Figure I 5 which shows that the relative rate of mammary growth in response to the administration of oestradiol dipropionate is diminished by the administration of aminopterin, a powerful folic acid antagonist, even when the food intake of the control rats was restricted to that of those receiving the antagonist (Silver, 1954) TABLE 13 Effect of oestrone on the area of the mammary glands of CHI mice (from Flux 1954a) 6 ovariectomized mice per group Total mammary gland area Treatment daily injections area af 42 days of age from 21-42 days of age 0 ml arachis oul oor ug oestrone in o 1 ml oil 0 055 ug oestrone in o 1 ml oil Oro ug oestrone in o 1 ml oil Intact controls 64 129 669 675 565 In the mouse Dr Flux has found in our laboratory as shown in Table I 3 that our ug oestrone daily from 21 to 42 days of age about doubled the total mammary area over that of ovariectomized controls at 43 days so that the threshold dose for the mouse must be somewhat lower than this (Flux, 1954a) However, somewhat higher doses o 055 kg/day, were ·10 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION Information about the body levels of oestrogen which prob ably obtain dunng the allometric phase of mammary growth in the immature normal female rat has been obtained by Mrs Silver in our laboratory by means of relative growth analysis She found (Silver, 19534) the threshold dose of oestrogen for the induction of allometric mammary growth in spayed immature female rats to be in the neighbourhood of 0 05ug oestradiol dipropionate injected subcutaneously every two days In the young spayed rat she could best mimic the 31 30H 29 28 O 27 O O 26 Logic total mammary gland area (mm) 25 24 оо 23 22 21 20 09 10 13 12 L0810 [body weight (8)] Fug 15 Effect of aminopterin on the gronth of the mammary gland in the rat (from Silver 1954) Group Aminopterin +e stradiol dipropionate O stradiol dipropionate alone (pair fed controls) Equilibrium constanta 2 29 5 0 08 poor 3 45 $ O OG

·PLATE 6
OES
PRC
OES O IMC
PRC
4
معا
DES
PAG 2 4 MG
OES I MG
PAC 24 MG
Sections at the same magnification of mammary glands from ovant
ectomized virgin guinea pigs which had secerved daily doses as indicated
for 68 days of oestrone (OES ) progesterone (PRG) or oestronet
progesterone (photograph provided by Dr å 7 Comie)

·12 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION necessary to attain mammary arcas not significantly different from those of comparable intact mice The second of the aforementioned categories comprises forms in which oestrogen in more or less physiological doses evokes extensive growth of the mammary lobule alveolar sys tem as well as of the duct trec The earliest known example is the guinea pig in which, as is well known, ſuncuonal mammae can readily be developed in either sex simply by oestrogen administration Though carlier workers believed that com bined treatment with oestrogen and progesterone had no greater effect on the guinea pig mamma than treatment with oestrogen alone recent work by Dr Cowie, who gave the two hormones in various ratios to spayed females and sub mitted serial sections of the mammae to a semi quantitative system of gland assessment indicates that both progesterone and oestrogen in the correct rauo are necessary for optimal lobule alveolar development in the guinea pig Some of his results (Cowie 1951) are illustrated in Plate I 6 whuch shows typical sections from the mammae of guinea pigs re ceiving the hormone treatments indicated The effect of progesteronc on lobule alveolar growth can be seen by comparing the upper and lower right hand figures Also included in this category are the domestic ruminants, in particular the cow and goat in which however the male mammary rudiment is not equipotential with that of the female The extensive udder der elopment obtainable with oestrogen alone was first demonstrated in the intact goat in our labora tory fifteen years ago (Folley Scott Watson and Bottomley, 1940, 1941) and similar results werc later obtained by numerous workers in extensive experiments on cattle In most of the earlier investigations no detailed morpho logical studies were made, the occurrence of extensive alveolar growth in response to oestrogen being inferred from the con siderable mulk yields often given by the treated animals and sometimes from macroscopic inspection of sliced udders (Plate I 7) Moreover, since intact animals were mostly used the possibility that progesterone of ovanan origin participated in the responses could not be discounted Nevertheless the magnitude of the mill yields, the appearance of macro udder slices from the treated animals, (cs Plate I 7) and such ·DEVELOPMENT OF MAMMARE GLAND 13 histological observations as those of Lewis and Turner (1942) and Alixner and Turner (1943) on oestrogen treated goats, left no doubt that treatment with oestrogen alone caused extensive lobule alveolar development in the ruminant That this conclusion was justified as shown by our later experiments on gonadectomized goats, one of which is illustrated in Plate I 8 which shows a section of a half udder prepared by Mr K G Richardson from an ovaniectomized virgin goat which was treated with oestrogen for 20 weeks extensive alveolar development occurred in this gland However the great variability of the lactational responses to a given treatment observed by most workers, and the fact that even the best milk yields were below those to be expected from similar animals after parturition, suggested that udders artificially developed by oestrogen treatment tend to exhibit some abnormality or deficiency in glandular structure Indeed, over ten years ago Afiner and Turner (1943) reported histo logical abnormalities in the glands of virgin goats treated for long periods with stilboestrol These mainly comprised grossly dılated or cystic alveoli with papillomatous epithelial out growths Combinations of oestrogen and progesterone, on the other hand gave glands in which cystic alveoli were not so prominent Recently our group (Cowie, Folley, Aſalpress and Richardson 1952) have made extensive morphological stu dies, by the aforementioned special methods developed for the purpose by Mr Richardson, of udders grown in imma turely ovariectomized virgin goats by treatment with com binations of hexoestrol and progesterone in various proportions and at different dose levels in comparison with the udders resulting from treatment with hexoestrol alone The showed various histological abnormalities of which the most striking and probably most significant functionally was a marhed deficiency of total epithelial surface probably because most of the alveol were too large as compared with those found in the udders of normally lactating goats A section showing such dilated alveoli is shown at the bottom of Plate Ig The middle section shows that they are largely absent from udders grown with suitable combinations of oestrogen and Progesterone, both these may be compared with the top section which is from a normal goat udder All are photograph

·PLATE 17
HLOR]
Suction of the udder developed by treatment with heroestrol from a hcifer
Malpress 1944)
mr Hey u 1

·DEVELOPMENT OF MAMVARY GLAND 15 indication which was upheld by morphological studies of these udders Syles and Wrenn (1951) have descubed somewhat similar histological abnormalties in the aruficially grown udders of calves and cows receiving stelboestrol and found that pro gesterone in sufficient doses largely abolishes these abnormal manifestations Coreover, Reincle Alertes Cairy and Huffman (1952) have succeeded in developing in heifers and cows by treatment with oestrogen and progesteronc udders which, because they have yielded much more milk than has been ob taned in previous studies involving treatment wsch ocstrogen tlone must presumably be more comparable structurally with the udder resulting from normal pregnancy The last of our three categories of animals comprises those in which ocstrogen alone in physiological doses causes little or no mammary de clopment of cren of ducts The only instance which has been adequately reported is the bitch włuch was studied by Trentin de Vsta and Gardner (1952), but since Hammond and Marshall (1930) found very little mammary duct growth in the oestrous ferret es en aſter prolonged oestrus It seems licly that the ferret belongs to this class also Anterior pituitary hormones Let us now turns to the consider ation of the role of anterior pituitary hormones in mammary growth The relation of the anterior pituitary to mammary development became a live issue soon after the discovery by Stricker & Grieter (1928) in Strasbourg of the lactogenic action of anterior pituitary extracts The early report of Corner (1930) that anterior pituitary extracts developed the mammae of spayed rabbits was the forerunner of 3 number of similar claims For instance WR Lyons has always maintained that lactogenic pituitary preparations would cause mammary development and his ingenious demonstration (Lyons 1942) that punfied prolacun will cause localized al veolar hyperplasia in those and only those sectors of the rabbit mammary gland into which minute quantities of the hormone solution have been injected would seem to afford good evidence (provided the prolactin used was of high purity) that at least one anterior pituitary hormone can exert direct mammogenic effects at any rate on a gland which is under the influence of a normal picuitary ·14 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION ed at the same magnification Another of these histological abnormalities, an immature lobule, is shown in Plate I 10, and Plate I 11 shows the epithelial papillomata which were also found Table I 4 gives results which show that progesterone prevents the occurrence of all three types of abnormal develop ment provided the ocstrogen dosage is not excessive Richard son (1953) has since confirmed, on further examinauon of TABLE I 4 Occurrence of ranous types of histological abnormality in hormonally developed goat udders (from Cowie, Folley, Malpress and Richardson, 1952) No of goats in which the udder No Treatment showed Cynt al cola Fp th ไม่ pucker 8 Immature Sub Intra 1 bula AI Ducta Capsular Ibis goats I a 1 2 4 4 2 2 I 2 2 2 0 o 0 O 0 1 0 0 0 0 Hexoestrol (1 mg/day) 7 5 3 Hexoestrol (1 mg/day) + progesterone (40 mg/day) 6 6 Hexoestrol (o 25 mg/day) 2 Hexoestrol (o 25 mg/day) + 2 progesterone (100mg/day) Hexoestrol (o 25 mg/day) + progesterone (40 mg/day) 2 material from these experiments that progesteronc significantly increased the area of secretory epithelium per unit volume, that is it produced alveoli falling within the normal size range More recent experiments in our laboratory (Benson et al 1955) indicate that suitable doses of oestrogen and progesterone given to spayed virgin goats develop udders which give reasonably uniform mill yields as between differ ent animals while the incidence of appreciable asymmetry of yield between the two halves of any onc udder is much re duced as compared with goats receiving oestrogen alone Both these observations are suggestive of histological normality, an

·PLATE 18
20mm
CIOL
AN
Section of an udder half from an ovariectomized virgin goat which had recensed 10 mg
heicestrol daily for 96 days (photograph prouded by ir AC Richardson)

·16 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION the mam All workers in the field would agree that the anterior pit uitary must be regarded as the mainspring of the hormonal mechanism governing the growth of the mammae in the nor mal fem ile if only because it controls the secretion of those ovarian steroids long recognized as the essential instigators of mammary development But in addition to effects mediated by the ovary we must consider the possibility that the anterior pituitary canı indirectly influence mammary development through the other target glands the adrenal cortex and thyroid Studies of the effects of adrenalectomy on the structure of gland have given rather conflicting and by no means dramatic results and so have not contributed much in formation on the role of the adrenals in mammary growth Thus Chamorro (1946) could see no effects in the rat though we ourselves (Cowie and Folley 1947) like Trentin und Turner (1947) have observed regressive changes Other wor hers on the other hand have reported increased duct growth and budding after adrenalectomy (Reeder and Leonard, 1914, Johnston and Smithcors, 1948) As regards the mammo genc effects of steroids which have been isolated from the adrenal cortex, there is evidence from more than one labora tory (Van Heuverswyn, Folley and Gardner, 1939 Speert 1910) that deoxy corticosterone is active in this respect though contrary observations have been reported, notably by Chamorro (1945) However, it is doubtful whether dcoxycorti costerone is secreted in significant amounts by the adrenal cortex under physiological conditions As regards the ri oxygenated corticoids Flux (19546) has recently shown in our laboratory that these steroids are not only devoid of mammogenic activity in the ovariectomized virgin mouse but that they also inhubit the growth promoting effect of exogenous oestrogen on the mammary ducts Cortisone also antagonizes the mammogenic effect of endogenous oestrogen since it inhibits mammary duct growth in intact female mice On the other hand 11 deoxy corticosterone acts synergistically with ocstrogen in promoting mammary duct growth in the spayed mouse Plate I 12 illustrates these results, it shows at the same magnification whole mounts of comparable mammary glands from spayed mice which recerved the treatments in

·PLATE 110
500w
An immature lobule from a gland developed in response to oestrogen and
progesterone in an ovariectomized virgin goat note the peripheral
distribution of enlarged alveoli (from Conle Folley Malpress and
Richardson 1952)

·PLATE 19 Sections from the udders of goats at the same magnification Top Section from the udder of a normal goat in factation Middle Section from an udder developed with hexoestrol and progesterone Bottom Section from an udder developed with hexoestrol alone (from unpublished work of Mr K C Richardson to whom I am indebted for the photographs) ·ܝܝܐ DEVELOPMENT OF MAMVARY GLAND 17 dicated The two upper pictures show how cortisone markedly diminishes the mammogenic effect of administered oestrogen while the two lower ones show that deoxycorticosterone has the opposite effect However, there is reason to believe that the adrenal cortex can produce other steroids including oestro gens, progesterone and androgens which are known to possess mammogenic properties and it is worth noting in this connection that Nelson (1941) found that ACTH caused mammary growth in the spayed but not the spayed adrenalectomized rat Nevertheless it seems doubtful whether the adrenals play a sigruficant role in normal mammary development, since not only could we ourselves find marked int tion of mammary growth in the rat as a result of adrenalectomy, but we could detect no significant reduction in the marmogenic effect of an unfractionated ox anterior pituitary extract in adrenal ectomized rats (Cowie and Folley 1947) This conclusion has found a supporter in Jacobsohn (1949) on the basis of inter esting expenments on rats joined in parabiosis Similarly as regards the thyroid there is no evidence that its secretions are essential for mammary growth though there are many findings somewhat complicated and even at first sight rather conflicting which suggest that the thyroid hor mone may regulate the respective growth rates of both ducts and alveoli in response to the appropriate stimuli Recent studies by various workers on the rat and mouse lead to the general conclusion that in the former hypothyroidism in duced by thyroidectomy or by administration of anti thyroid drugs leads to enhanced development of the alveoli and often but not always of the duct system as well, in response to oestro gen and progesterone either administered experimentally or endogenously produced while the administration of thyroxine has the opposite effect In the mouse on the other hand hypo thyroidism seems to inhibit manmary development while mild hyperthyroidism stimulates it As Trentin Hurst and Turner (1948) suggest the explanation may perhaps be sought in the relative thyroid secretion rates in the two forms, that in the normal rat probably being somewhat above the optimum for mammary growth while in the mouse the opposite may be assumed to be the case This hypothesis provides an intelli gible explanation of many apparent discrepancies among the

·PLATE I 11
200
Abnormal folding of the alveolar epithelium found mainly in goat
udders developed by high dosages of oestrogen without progesterone
(from Conze Folley Malpress and Richardson 1952)

·DEVELOPMENT OF MAVRY GLAND 19 mammary des clopment in hypophysectomized animals It was from the results of these and other experiments that Turner s mammogen theory was developed To enlarge on the car! ier evidence for and against this still controversial theory is beyond the scope of this book A full discussion is ghen in the review by Folley (1952) It must sufficc to point out now that in the light of continued experiment the theory has undergone important modifications at the hands of its onginators They no longer believe that the anterior pituitary secretes two mammogens responsible respectuely for duct and abscolar de clopment the first of which unlike all other anterior pituitary hormones is a kiposd soluble substance Trentin and Turner (1948) now feel that both duct and al eolar growth can be asenbed to the acuon of a single pituitary factor or complex of factors associated with the protein fraction and they admit that the existence of a specific mammogenic hormone distinct from the six well known pstustary hormones cannot be regarded as conclusisely established at the present time It may reasonably be said that the mainmogen theory would carry more conviction iſ a specific mammogen could be separated from the pituitary in a state of reasonable chem ical and biological punty In any event as pituitary hor mones become available in an ever increasing state of purity our progress towards a deeper understanding of the role of the pituitary in mammary development must inevitably advance Some progress towards this goal may be seen in experiments recently reported by Lyons and by Nelson whuch argue rather strongly against the need to postulate the existence of specific antenor pituitary mammogens Lyons (1951) successfully sought hormone combinations which would sustain lobule ah eolar growth in virgin female rats subjected to a variety of operaove procedures and found inter alia that lobule als eolar growth could be evoked in hypophysectomized orariectomuzed rats by admom stranon of Destrone progesterone and punicd prolactin though the alveoli grew more extensively if a cruder prolactin containing ACTH and somatotrophin was used Plate I s3 shows part of the mammary gland of a rat possess ing neither ovanes nor pituitary and which was given oestrone, progesterone and prolachn Thus hormone combination pro duced quite extensive alveolar development but the complete ·18 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION experimental results in this field and fits in with the concept of a regulatory role for the pituitary thyroid mechanism in mam mary growth Our own experiments (Cowie and Folley 1947) provide evidence that anterior pituitary extracts can exert mammo genic effects that are mediated by neither ovanes nor adrenals since our unfractionated extract evoked mammary development in gonadectomized adrenalectomized rats, that is in animals which provided accessory cortical tissue was absent, possessed no known major source of steroids (steroprived rats) If then, these experiments can be accepted as decisive, the possibility that the pituitary does no more than play a so called permissive or sensitizing role or else a synergistic role in relation to the mammogenic steroids is excluded and we are left with a direct mammogenic effect as suggested for prolactin by the aforementioned intraduct injection experiments of Lyons (1942) If prolactin is indeed a direct mammogenic as well as lactogenic hormone it should be pointed out that neither our experiments nor those of Lyons exclude the possi bility of other pituitary hormones being also needed in a permissive or synergistic role, since in both cases animals possessing pituitaries were used In addition, the possibility that the pituitary growth hormone somatotrophin, is concern ed in mammary growth was suggested by the experiments of Nathanson, Shaw and Franscen (1939) and of Reece and Leonard (1941) and further evidence that this is so has rc cently come to hand Lyons Li and Johnson (1952) have shown that somatotrophin augments the mammogenic action of the hormone tnad oestrogen progesterone and prolactin in the hypophysectomized female rat (see also Lyons Li, Cole and Johnson 1953) The same group (Lyons, Johnson Cole and L1, 1955) have also shown that in the young hypophysec tomized male rat somatotrophın along with oestrogen and progesterone stimulate some degree of mammary duct growth even without prolacun The question whether the antenor pituitary secretes specific mammogenic hormones distinct from its six well authenticated protein hormones was, as is well known raised by C W Tur ner and his school many years ago, principally because they found that Ovarian steroids were incffective in promoting

·PLATE 3 12
(a)
1 cm
Second thoracic masimary glands from ovariectomized mice treated from the 21st day
of age until the 42nd day photographs at the same magnification from Flux 1953
1954)
(a) Oestrone O OZ 48 per day
(c) Destrone 001 ks per day
002 -8+ cortisone (d)
001x8+deoxy corticosterone
acetale 50 g per day
acetate 072 mng per day

·20 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION alveolar development typical of late pregnancy seemed to require somatotrophın in addition (Lyons et al 1952, 1953, 1955) The prolactin preparation could be replaced by one 12 day rat placenta daily, which suggests that in the later stages of pregnancy, at least in the rat, the placenta can assume the role of the pituitary in secreting prolactn or a substance with the same biological properties (Ray, Avenil, Lyons and Johnson, 1955) The experiments of Nelson (1951) were main ly concerned with the hormonal maintenance of oestrogen induced pseudo pregnancy with its accompanying mammary development in virgin female rats variously treated and his results also permit of the conclusion that besides oestrogen and progesterone, only well recognised pituitary hormones, pnn cipally prolactın but probably also ACTH and somatotro phın are required for complete lobule alveolar growth in the rat The design of both studies was such as to emphasize the probable dual role of prolacun in normal mammary growth the lutcotrophic effect by which progesterone sccretion is maintained and a direct 'mammogenic action They also show that oestrogen and progesterone participate in mammary growth more directly than as mere pituitary excitants Since many recent studies point to the hypothalamic con trol of pituitary function the possibility that neural and even psychological influences may exert an overnding influence on mammary development in the normal animal cannot be over looked This concept, formerly believed to be contra indicated once for all by mammary transplantation experiments such as thosc of Stricker (1929) which showed that the gland will grow in the absence of direct nervous connections must now be reconsidered in the light of the possibility that the mechan ism controlling mammary growth must be regarded as neuro endocrine in nature the endocrine phase comprising a com plicated and delicately balanced dynamic equilibrium centr ing round the anterior pituitary

·PLATE IS
(b)
(c)
1cm
Second thoracic mammary glands from ovariectomized mice treated from the 21st day
age until the 42nd day photographs at the same magnification (from Flur 1953
1954)
(ay Oestrone 002 ws per day
(c) Destrone 001 kg per day
DOZ #8+cortisone
001#8+ deoxy corticosterone
acetate 50 eB per day
acetate 071 mg per day

·PLATE 1 13 Whole mount of a mammary gland from an hypophysectomized ovariectomized rat treated with oestrogen progesterone and a prolactin preparation (from Lyons 1951) ·DEVELOPMENT OF MAMMARY GLAND 21 REFERENCES Asthood E B Geschichter C F and Rausch EO (1937) Amer 7 Anal 61 373 Benson GK Cowie A T Cox CP Flux DS and Folley SJ (1955) Endocrin 13 46 Chamorro A (1945) CR So Biol Paris 139 137 Chamorro A (1946) C R Soc Biol Paris 140 499 Corner GW (1930) Amer Physiol 95 43 Coule A T (1949) 7 Endorn 6 145 Cowse A T (1951) Collog Int CARS XXXII 1950 P 45 Cowie A T and Folley SJ (1947) Endocrinology 40 274 Cowie, A T Folley Sj Malpress F H and Richardson KC (1952) Endocran 8 64 Flux DS (1954a) 7 Endocrin. 11 223 Flux DS (19546) Endocran II 238 Folley S J (1952) in A S Parkes farshalls physiology of reproduction 3rd ed London Longmans Green & Co Chap 20 Folley S J Guthkelch A N and Zuckerman S (1939) Proc Toy Soc B 126 469 Folley S J and Malpress F H (1944) ) Erdorna. 41 Folles Sj Scott Watson, H M and Bottomley A. C (1940) 7 Physiol 98 15P Folles SJ Scott Watson H M and Bottomley A. C (1941) | Dary Res 12 24 Hammond J and Marshall FHA (1930) Proc roy Soc B 105 607 Heurerswyn J Van, Folley S J and Gardner WU (1939) Proc Soc exp Bol Nr 41 389 Huxley J S and Teissier G (1936) Nature Lond 137 780 Jacobsohn D (1949) Acta physiol scand. 17 423 Johnston RF and Southcors J F (1948) Endocrinology 43 193 Lewis AA and Turner CW (1941) 3 Dairy Sa 24 845 Lewis AA and Turner C. W (1912) Endocrinolo. 31 520 Lyons WR (1942) Proc Soc exp Bol Vr 51 308 Lyons WR (1951) Collog Int CVRS LXXII 1950 P_29 Lyons W R Johnson RE Cole R D and LCH (1955) in RW Smith O H Gaebler and CNH Long The Hypophyseal Grouth Hormone Valure and Actions New York Blahiston, Chap 26 Lyons WRL CH Cole R D and Johnson RE (1953) 3 chin. Endocrin letab 13 836 Lyons W R L C H and Johnson RE (1952) ) clin Endocrin Metab 12 937 Lyons W R and McGinty D A (1941) Proc Soc exp Bol NY Mixner J P and Twner CW (1943) Res Bull to agru Exp Sta no 378 Nathanson I T Shaw D T and Franseen CC (1939) Proc Soc exp Buol Nr 42 652 Nelson WO (1941) Anat Rec 81 (Suppl) 97 48 83

·22 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION
Nelson WO (1951) Collog Int CNRS LAXII 1950 P 19
Ray E Averill SC, Lyons W R and Johnson RE (1955) Endocrino
logy 56 359
Reece R P and Leonard SL (1941) Endocrinology 29 297
Reeder, C F and Leonard SL (1944) Proc Soc exp Biol NT 55 61
Reinche E P Meites ) Cairy, C F and Huffman, C F (1952) Proc
Book Ann Meeting Amer zet med Assoc 1952 P 325
Richardson KC (1953) 7 Endocrin 9 170
Scharf G and Lyons W R (1941) Proc Soc exp Bol NT 48 86
Silver M (1953a) 7 Endocrin 10 17
Silver M (1953b) 7 Endocrin
IO 35
Silver M (1954) ) Endocrin 10 95
Speert H (1940) Johns Hopk Hosp Bull 67 189
Stricker P (1929) R Biol Pans 102 1076
Stricker P and Grueter F (1928) C R Soc Biol Paris 99 1978
Sykes J F and Wrenn TR (1950) 3 Dairy Sci 33 194
Sykes J F and Wrenn TR (1951) J Darry Sct 34 1174
Trentin JJ De Vita J and Gardner WU (1954) Anal Rec 113 163
Trentin J J Hurst V and Turner CW (1948) Proc Soc exp Biol,
Nr 67 461
Trentin j j and Turner CW (1947) Endocrinology 46 127
Trentin j j and Turner CW (1948) Res Bull Alo agric Exp Sta
no 418
Turner CW and Schultze A B (1931) Res Bull Vo agric Exp Sla
no 157

·CHAPTER II THE INITIATION OF MILK SECRETION (LACTOGENESIS) THE BIOCHEMISTRY OF LACTOGENESIS This chapter is concerned with the initiation of mill secr ction a process known as lactogenesis Experiments involving the removal of the pituitary gland performed many years ago showed that the integnty of the hypophysis is necessary for the initiation of milk secretion (for review see Folley 1952) Other experiments involving the administra tion of pituitary extracts were even more alluminating for they proved that the hypophysis provides a positive lactogenic stimu lus The pioneer work in this latter field was that of Stricker and Grueter (1928) carried out in Bouin s laboratory at Stras bourg and this was soon followed by the general realization that anterior pituitary lactogenic extracts contain a specific hormone capable of initiating lactation in functionally com petent mammary tissue This hormone is nowadays generally called prolactin but the alternative names, mammotrophın or luteotrophın are sometimes used An interesting first hand account by Stricher (1951) of what many regard as essentially the discovery of prolactin was recently given to a conference on the physiology of milk secretion appropriately held in Strasbourg The prompt and perspicacious identification by Riddle Bates and Dyhshorn (1933) of this presumed lactogenic hor mone with a principle present in anterior-pituitary extracts, which induces enlargement and secretion of the pigeon crop gland provided the specific and convenient method of bioassay which is essential for rapid progress in the chemical punfica tion of any new hormone In the case of prolactin this went forward to such good effect that it was the first pituitary protein hormone to be isolated as a pure or nearly pure protein ·24 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION at least as judged by the cntena available at the present time In view of this it is not surprising that considerable progress has been made by now in elucidating the chemistry of prolactin The ingenious and elegant experiments of Lyons (1942), later repeated by Meites and Turner (1948), and in extensive work in our laboratory by T R Bradley (unpublished) in which localized lactation was initiated in sectors of the rabbit mammary gland by the injection of small amounts of prolactin into the appropriate teat canal neighbouring untreated sec tors remaining quite unaffected suggest that prolactin exerts a direct action on the mammary epithelium Plate II 1, taken from Lyons (1942) shows a portion of a whole mount of a mammary gland of one of his rabbits into one teat canal of which prolactin was injected The secretory hypertrophy of the alveoli of the treated sector, shown at the bottom right, contrasts with the undistended alveoli visible in the upper left of the picture However, since the rabbits used by Lyons were not hypophysectomized the possibility that other pituitary hormones of endogenous origin participated in the response was not excluded That other anterior pituitary hormones, in addition to prolactin are indeed concerned in lactogenesis is suggested by experiments carried out many years ago in the laboratories of Nelson Gaunt and Turner which showed that while systemic injections of unfractionated anterior pituitary extracts readily initiate mill secretion in hypophysectomized animals possessing the necessary mammary development, treatment with partially punfied prolactin does not do this (Gomez and Turner, 1936, Nelson and Gaunt, 1936) Thus by the criterion customarily applied to the specificity of re sponses to anterior pituitary hormones namely that these should be limited to responses elicited in hypophysectomized animals, lactogenesis cannot be considered to be a specific response to prolactin alone Successful initiation of lactation achieved by the same groups of experimenters in hypophys ectomized animals by administration of partially purified prolactin together with ACTH, adrenal cortex extracts or cortisone (eg Nelson, Gaunt and Schweizer 1943) shons that the pituitary adrenal mechanism is also involved in lactogenesis In view of thus it is evident that unul it is possible to

·PLATE !! 1
3mm
Lactogenic effect of injection of prolactin into a leat canal of a rabbit
(from Lions 1942)

·No text on this page.

·LACTOGENESIS 25 inwork with undeniably pure pituitary hormones we cannot be certain whether other hypophy sial functions may not be volved as well The observations of Meites, Trentin and Turner (1942), who reported that adrenalectomy did not enurely prevent the onset of lactation in the rat at parturition, support this concept rather than otherwise since these workers ad mitted that the secretory activity in the absence of the adrenals was very feeble In view of what I have said it is perhaps more logical, as suggested by Folley and Young (1941), to regard lactogenesis as a response to the co operative action of more than one anterior pituitary hormone rather than as a response to a single specific lactogenic hormone In other words we should think in terms of a pituitary lactogenic hormone complex It cannot be denied however, that prolactin is the limiting factor in most experimental situations involving the animal possessing a pituitary gland though, as the experiments of Reece (1939) and others show, the experimental initiation of lactation in the pseudo pregnant rat is an example of a case in which the inter vention of the pituitary adrenal mechanism in experimental lactogenesis is manifest The pseudo pregnant rat is notorious ly refractory to the lactogeni action of prolactin preparations Reece (1939) however, found it possible to initiate lantation in pseudo pregnant rats by administration of partially purified prolactin together with adrenal cortex extract More recently additional light has been thrown on the hormonal factors necessary for lactogenesis in the rat for Lyons Li Cole and Johnson (1953) have reported the expenmental initiation of lactation in hypophy sectomized ovariectomized virgin ats, possessing suitably developed mammary glands by treatment with prolactin somatotrophın and ACTH or cortisone (see also Lyons Johnson Cole and Li (1955) for hypophysecto muized male rats) Further progress in gaining insight into the mechanism of lactogenesis must come from the exploration of biochemical pathways and the groundwork for advance along these lines is now being laid Though abundant lactation does not set in until parturition or shortly thereafter there is ample cyto logical and biochemical evidence of secretory changes in the mammary gland during the second half of pregnancy Thus ·26 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION the characteristic milk protein, casein, was detected by Cutler and Lewis (1933) using immunological methods, in the bov ine udder fairly early in pregnancy Moreover, the gland at this stage can synthesize fatty acids from small molecules as shown by the isolation by Popjak and Beeckmans (1950) of labelled fatty acids from the mammae of pregnant rabbits given heavy water or 14C acetate The late Dr T H French and I had the opportunity of demonstrating, in collaboration with Dr G Popjak, that these radioactive fatty acids contain TABLE III Specific activities of the ghicende fatty acids from the mammae and lizers of rabbits killed at the 28th day of pregnancy after treatment with CH,24COON (from Popjak, Folley and French 1919) Specific activity of fatty acids uc X 10-mg C Fatty acid fraction Rabbit no 10 13 15 1 98 18 45 1 48 13 60 Volatile water soluble Volatile water insoluble Non volatile Liver fatty acids 0 73 0 72 007 0 183 O 12 099 0 146 0 58 ed the characteristic volatile short chain acids found only in milk fat and that, as shown in Table II 1, they were more radioactive than the non volatile long chain acids (Popják, Folley and French 1949) There is thus no doubt that the mammae of these pregnant rabbits were capable of synthesiz ing milk fat in accord with this, we have since shown in our laboratory (Table II 3) that mammary gland slices from preg nant rats will undoubtedly incorporate some Cinto fatty acids if incubated with unlabelled glucose + (carboxy 14C] acetate even though the activity of the tissuc in this respect may not be very great (Balmain Folley and Glascoch, 1952) Another finding which can be cited here is the striking increase in the alkaline phosphatase content of the rat mammary gland dur ·LACTOGENESIS 27 ing the secood half of pregnancy, which is illustrated in Figure II 2 drawn from the results of Folley and Greenbaum (1947) This change however, is less easy to relate to any specific syntheuc activity of the gland Parturition a aming 100 80 Enzyme acsleisy (percentage of maxlmum) 60 40 O 10 15 20 6 Pregnancy Lactation Jevolution 5 10 3 Days Fig II 2 Allaline phosphatase content of the man mary gland of the rat (from Folley and Greenbaum 1947) However despite this biochemical evidence of secretory activity in the mammary gland of the pregnant animal respiratory measurements on mammary gland slices from pregnant animals have given a picture of general metabolic quiescence contrasting with the much more active respiration hown in rutro during lactation This is illustrated by the results of Folley and French (19496) summarized in Table II 2 Thus the endogenous respiration of mammary gland slices from rats at the end of pregnancy is low - lo, having a value of only 15 and it is not increased by the addition of glucose to the medium Moreover the respiratory quotient of the tissue at this stage is below unity values between o 62 and 0 83 ·28 PHYSIOLOGY AND BIOCHEMISTRI OF LACTATION being found irrespectn e of whether or not glucose is present in the medium Mammary tissue from lactating rats on the other hand, exhibits much greater respiratory activity in utro The results in the Table show that by the first day past partum the tissue has acquired the power of utilizing glucose, in the presence of which substrate the respiratory quotient has risen to unity TABLE II 3 Respiratory metabolism of slices of rat mammary gland during pregnancy lactation and ine olution (from Folley and French, 19496) Stage Days RQ Glucose (03% Glucose (03.) Pregnancy Lactation 20 13401 15L005 o 83-001 062 +003 I 44103 40403 500+0 05 73*OOI 8 71006 45205 1 62 10 03 o 76:20 or 15 10 3L04 52404 16010 06 0 783002 22 96403 6302 33003 o 740 02 530950 0 760 03 o 64 Involution 2 By the 8th day -- loa in the presence of glucose has risen to 71 and the respiratory quotient is 1 62 at about which value it remains throughout lactation Respiratory quotients greater than unity in the presence of suitable substrates were found for lactaung mammary gland shces from the mouse, guinea pig rabbit cow goat and sheep (Folley and French 19494, 1950) This is in general agreement with results pre viously obtained in arteno venous studies on the udder of the lactating cow by Petersen and Shaw (1942) and on that of the lactating goat by Graham, Houchin Peterson and Turner (1938) and later for the perfused, isolated cow udder by Peeters and Massart (1952) The real changes in Ry, which is gencrally expressed as microlitres of oxygen consumed per milligrammc final dry ·LACTOGENESIS 29 weight per hour, are probably not so great as indicated by the results just discussed since mammary tissue from rats at the end of pregnancy contains much more metabolically inert dry matter, than mammary tissue from lactating rats The respıra tory quotients are not subject to this error, however since the dry weight cancels out in the calculation In an attempt to overcome the difficulty of finding a suitable reference standard for oxygen consumption, we attempted to calculate the total respiration of the six abdominal mammae of the rat dunng pregnancy and lactation respectively and our values support the conclusion we drew from lo, measurements namely that the onset of secretory activity at parturition is indeed associat ed with a real increase in the rate of respiration of the tissue (Folley and French 19496) Further support comes from the fact that we also found that the ratio of respiration to glycolysis (Lola) is greater for mammary slices from lactating than from pregnant rats (Folley and French, 19496) Also in harmony with this conclusion are the marked increases in the concentration of oxidative enzyme systems, succinoxi dase and cytochrome oxidase about the time of parturition which have been observed by Moore and Nelson (1952) in studies of homogenates of rabbit mammary gland Another quite stnking way in which these changes in the respiratory metabolism of rat mammary tissue at the time of initiation of lactation can be illustrated is by manometric measurements of the time course of the total gas exchange which we call the composite respiration curve of mammary gland slices incubated in Krebs saline bicarbonate in equi librium with 95 % oxygen and 5 % carbon dioxide and con taining suitable substrates Figure II 3 shows some composite respiration curves for rat mammary gland slices obtained in our laboratory (Balmain and Folley 1952) It will be seen that the metabolism of mammary slices from 20 day pregnant rats is such as to give a slow fall in pressure under these con ditions probably in the main because the respiratory quotient is less than unity and the slices therefore use oxygen at a greater rate than they evolve carbon dioxide Mammary slices from lactating rats, for which the respiratory quotient is greater than unity on the other hand give a steady increase in total pressure mainly because the slices give off carbon dioxide faster than they use up oxygen ·30 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION Some years ago reinterpreted the high respiratory quouent of lactating mammary tissue in vitro, in the presence of suitable substrates as evidence that this tissue possesses the power of effecting net fatty acid synthesis from small molecules (Folley and French, 19494 b, 1950) a conclusion which we later con firmed by isotopic assays on fatty acids isolated from mammary 10 Net gas exalutian as ji gas{mg final dry weight La Pr Hours Fig II 3 Total gas exchange of mammary gland slices from rats killed on the 14th day of lactation (La) and 20th day of pregnancy (Pr) respectively The ordinate shows net gas evolution as ul gas/mg final dry weight uptake being calculated as og and output as Co. (from Balmain and Folley 1952) slices incubated with suitably labelled substrates Experiments involving the injection of labelled substrates into the lactating goat, cow and rabbit which are discussed in Chapter V hale led to a similar conclusion We may therefore conclude that the changes in the respiratory metabolism of mammary tissue which begin at or shortly after partunton must be indicaue among other things of a rapid acceleration of a bpogcnic process already capable of proceeding at a relatively slow rate ·LACTOGENESIS 31 In support of this conclusion we have shown that, even allowing for the probable dilution effect of the greater fat content of mammary tissue at the end of pregnancy, mammary gland slices from rats killed at days 1-4 post partum incorporate much more 14C into the fatty acids, when incubated with glucose + (carboxy 14C) acetate than slices taken at the end of pregnancy Table II 3 shows results which illustrate this (Balmain, Folley and Glascoch 1952) The Table shows that the specific activi ties of mixed fatty acids isolated from mammary gland slices TABLE II 3 Specific activities of mixed fatty acids of slices of mammary gland from tals killed at the end of pregnancy or in early lactation The substrates were (carboxy C} acetale + glucose (from Balmain, Folley and Glascock 1952) WC in fatty acids Period of counts/min/mg C Experi Stage incubation Tissue at 37 Incubated Lilled at Inent no hr tissue zero time pregnancy 20th day 5-6 25 2 3 5-6 5-6 403 652 407 3 7 4 7 5 422 14 lactation and day lactation 4th day 5 3 16 146 54 taken from rats killed shortly after parturition are many times higher than those found for tissue from rats killed at the end of pregnancy It is certain also that the rates of synthesis by the mammary tissue of characteristic milk constituents other than fat undergo similar sharp increases at parturition Thus Greenbaum and Greenwood (1954) have recently shown that the levels of glutamic dehydrogenase and of glutamic aspartic transaminase in the mammary gland of the rat dramatically increase immediately before parturition and remain at this enhanced level throughout lactation declining again only ·32 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION after weaning These enzymes are believed to be concerned with protein synthesis in the cell and if this is so, then these changes are probably associated with the mception of a hugh rate of synthesis of milk proteins consequent upon the inaua tion of lactation Now, the biochemical changes observed in mammary tissue at the onset of lactation which I hase just discussed, are un doubtedly evoked by the prrutary lactogenic hormone com ples which must come into full operation at parturtion and they promise to afford a useful means of gaining further in sight into the hormonal mechanism of lactogenesis in vitro For instance the direct lactogenic action of prolactin upon the mammary epithelium so strikingly demonstrated in the rabb»t by Lyons (1942) in the experiments described earlier in thus chapter should be reproducible in tutto of the other hormonal influences operating in the normal environment of the mam +08 rolactin + Coshsone +06 +04 Costison +02 ok Net 825 evolution as a gas/mg final dry weighe 02 0$ Prolacton 00 Cassis LOF $ 2 6 9 3 Hours Fig II 4. Effects of cortisone (700 vg/ml) and of prolacun (1114 mi) on the respiration of mammary gland slices from a ratat the end of pregnancy (from Balman and Follo, 1952) ·LACTOGENESIS 33 mary gland in ino can also be reproduced in a preliminary attach upon this problem utilizing changes in the slope of the composite respiration curve as an indication of lactogenic effects, Miss Balmain and I were, however, unable to demon strate any effects of prolactin, added in vitro on the respiratory metabolism of mammary slices from 20 day pregnant rats (Balmain and Folley 1952) A typical experiment is illus trated in Figure II 4 which shows that addition of prolactın to the medium had no effect on the slope of the composite respiration curve Rat mammary tissue, at this stage has thus proved to be insensitive to the action of prolactin in zutro by any criterion so far applıcd This of course may be because the correct conditions neces to demonstrate its action have not yet been discovered On the other hand Figure II 4 also shows that cortisone, which on the basis of the in rivo work already mentioned might be expected to come into play as a result of the intervention of ACTH in lactogenesis when added to the incubation medium exerted definite effects on the composite respiration curve converting the falling pressure curve usually exhibited by mammary tissue from pregnant rats into the steadily rising one typical of secreting tissue This is the outcome one would expect if secretory changes had been initiated in the tissue by the hor mone though it must be noted that the same result might be obtained if respiration were inhibited and glycolysis increased The two hormones together had no greater effect than corti sone alone so that these experiments provided no indication of the synergism between prolactin and the pituitary adrenal axis which had been inferred from the previous experiments of others on lactogenesis in vivo Nevertheless in so far as these findings appear to suggest a role for the adrenal cortex in the induction in rat mammary tissue of certain biochemical changes similar to those seen in tissue in a secretory state they are in accord with the aforementioned findings of Reece (1939) and others in the intact pseudo pregnant rat However experiments in our laboratory on mammary slices from preg nant rats which were incubated with labelled substrates have given results difficult to reconcile with the respiratory measure ments just considered in that they suggest that cortisone in the concentrations used inhibits rather than promotes lipogenesis ·34 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION from small molecules in mammary tissue from 20 day preg nant rats Hence, although this biochemical approach to the problem of lactogenesis is promising, the results so far ob tained present certain difficulties in interpretation which it is hoped will disappear on further investigation TUE HORMONAL CONTROL OF LACTATION INDUCTION AT PARTURITION We now turn to the consideration of some of the theones which have been put forward to explain the initiation of lactation at parturition and in particular its timing Although it is generally agreed that mammary growth proper being largely complete the secretory phase begins in the mammary gland during the second half of pregnancy, nevertheless lacta tion in the ordinarily understood sense of a copious flow of normal mulh is not initiated until partunition or shortly there after The nature of the mechanism which ensures the correct timing of lactogenesis in this sense has intrigued investigators for many years and is not fully understood even yet Of the various theories n hich have been proposed space allows ment ion of only two and these can only be considered briefly The theory proposed by Nelson (1936) for the guinea pig postulated oestrogen as the agent responsible for holding lactation in check dunng the latter half of pregnancy The high body levels of oestrogen known to obtain during preg nancy, were believed 10 inhibit the secretion of prolactin by the hypophysis and also to exercise a direct inkubatory in fluence on secretory changes is the mammary gland itself if this theory be extended, as proposed by Folley and Alalpress (1917) to embrace the concept of two thresholds for opposing influences of oestrogen upon pituitary lactogesic function, 3 lower one for stimulation and a higher one for intubiuon it will be found even yet to harmonize many experimental ob servations made on other forms besides the guinea pigon which most of Nelson s observations were based Honcer in the light of recent results to be considered in more detail later it may be necessary to modify the theory stal} further to include an inlubitory role for progesteronc in the capacity of a synerg ist with oestrogen A somewhat more elaborate theory was proposed by dícitos ·LACTOGENESIS 35 and Turner (1942) on the basis of extensive investigations in Turner's laboratory of the prolactin content, as measured by a variant of the pigeon crop gland test, of the pituitary in various physiological and experimental states Briefly this theory rejects any inhibitory role for oestrogen in lactation, its authors rather see oestrogen as an indirect evocator of lacto genesis by virtue of its claimed ability even when circulating in high titres, to elicit the secretion of prolactin by the hypo physis while progesterone is regarded as the inhibiting agent operative during pregnancy with the power of nullifying or overriding the lactogenic action of oestrogen Lactogenesis is thus believed to result from a fall in the body level of pro gesterone relative to that of oestrogen which is thought to occur at parturition or shortly before Much evidence some aspects of which were later subjected to criticism (Folley 1952) was adduced in favour of this hypothesis in four papers published in 1942 and the theory has been restated with the support of additional experimental data and discussion in a more recent publication (Meites and Turner 1948) The scope of this book does not permit of discussion of this evi dence all that can be done is to state bnefly the main finding of Meites and Turner This was that treatment with oestrogen even in unphysiologically high doses caused always an in crease never a decrease in the prolactin content of the pituitary in various laboratory mammals If, however, suffi cient progesterone was injected along with oestrogen no in crease in the pituitary prolactin content resulted Incidentally the above mentioned more recent publication of Meites and Turner (1948) contains no reference to a significant paper by Atkinson and Leathem (1946) providing evidence which partially answers criticisms made a few years ago (Folley 1952) of one of the vulnerable points of the Mertes Turner theory namely, the lack of evidence for a fall in the body level of progesterone relative to that of oestrogen at parturition Using histological criteria Atkinson and Leathem could find little or no evidence of progesterone effects in the mouse on the day of parturition though signs of the presence of oestrogen were clear However despite this, the author sees no reason to modify bis previous opinion (Folley 1952) that while this ingenious ·34 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATIOV from small molecules in mammary tissue from 20 day preg nant rats Hence, although this biochemical approach to the problem of lactogenesis is promising, the results so far ob tained present certain diſhculties in interpretation which it is hoped will disappear on further invesugauon THE HORMONAL CONTROL OF LACTATION INDUCTION AT PARTU RITIO We now turn to the consideration of some of the theones which have been put forward to explain the initiation of lactation at parturition and in particular its timing Although It is generally agreed that, mammary growth proper being largely complete, the secretory phase begins in the mammary glund during the second half of pregnancy, nevertheless lacta tion in the ordinarily understood sense of a copious flow of normal mslh is not initiated until parturition or shortly there after The nature of the mechanism which ensures the correct timing of lactogenesis in this sense has iningued investigators for many year, and is not fully understood cven set of the various theories which have been propo ed space allows ment son of only two and these can only be considered bricily The theory proposed by Nelson (1936) for the guinea pig postulated oestrogen as the agent responsible for holding lactation in check during the latter half of pregnancy The high body levels of oestrogen known to obtain during preg nancy were beleved to inhibit the secretion of prolactin by the hypophysis and also to exercise a direct inhibitory in fluence on secretory changes in the mammary gland Lelf If this theory be extended as proposed by Folley and Malpress (1947), to embrace the concept of two thresholds for opposing influences of oestrogen upon pituitary lactogenic function a lower one for stimulation and a higher one for inhibition, it will be found even yet to harmonize many experimental ob sensations made on other forms besides the guinea pig on ubich most of Nelson s observations were based However, in the light of tecent results to be considered in more detail later it may be necessary to modify the theory still further to snclude an indubitory role for progesterone in the capacity of a synergist with oestrogen Asomes hat more elaborate theory was proposed by Nestes ·LACTOGENESIS 37 or termination of lactation in women the decisive inhibitory factor has been held to be the removal of the suckling stimulus, the undoubted beneficial effects of the oestrogen which is often administered being ascribed to its ability to alleviate painful breast 'engorgement' Some of those who hold that oestrogens have no inhibitory effect on lactation, at least under physiological conditions, have contended in support of their case that oestrogens are completely without effect on lactation in the absence of the TABLE II 4 Effect of progesterone on lactation in intact rats (from Folley 1942) I of young Treatment No of rats weaned (21 days) Mean wt of young at weaning g 97 42 9 38 4 94 Sesame oil (control) 4 Progesterone 10 mg daily 4 Sesame oil (control) Progesterone 15 mg daily 3 4 91 40 7 92 407 All litters reduced to 8 at birth ovary However careful consideration of the numerous papers which have been published on this subject shows that the true position is that while oestrogens will in some cases inhibit lac tation in the absence of the ovary prouded high enough doses are given, nevertheless intact animals are undoubtedly much more sensitive to the lactation inhubiting effects of oestrogen than ovariectomized ones (eg Folley and Kon 1937) This suggests either that under physiological conditions proges terone itself might be the true lactation inhibitor or alter natively that it might act synergistically with oestrogen How ever many years ago the author found that progesterone by itself was quite ineffective as an inhibitor of lactation in spayed rats (Folley, 1942), as shown in Table II 4 from which it is evident that the young of rats receiving as much as 15 mg ·36 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION theory can be regarded as perhaps the most successful attempt so far made to explain the mechanism of the initiation of lactation at parturition, the sum total of the available evidence in its favour is not even yet sufficiently unequivocal and con vincing to justify the problem being regarded as finally solved The necessity for caution is underlined by results fairly re cently reported by Mſeites and Sgouris (1953) which, taken at their face value are difficult to reconcile with the Meites Turner theory Mammary development was evoked in gonad cctomized rabbits by administration of oestrone and progester one in a favourable ratio Conunuance of this regime pre vented the initiation of lactation by prolactın in doses which were effective in the presence of either steroid alone Accord ing to the Aſeites Iurner theory prolactin should have been just as effective in evoling lactation in the presence of oestrone and progesterone as in the presence of either hormone alone More recently still, in similar experiments, Meites and Sgouris (1954) have found that whether or not lactation occurs de pends on the relative levels of prolactin on the one hand and the oestrogen progesterone combination on the other These findings appear to underline the necessity for some simplifi cation of the orginal theory and Mſeites (1954) has recently outlined his latest views on the subject I hus brings us back to the question of the nature of the hormonal influence which holds lactation in check during pregnancy Oestrogens have long been regarded as possessing the power of inhibiting lactation a concept upon which Nelson founded his theory of the mechanism of lactation initiation and which also motivated the widespread clinical use of oestrogens for presenting or inhibiting lactauon in the puerperium However some workers have expressed doubts whether the ability to inhibit lactation is a direct and specific property of oestrogen, at least under physiological conditions Those holding this view have vanously ascribed the apparent lactauon inhibiting effects of oestrogens in small animals to anorexia or other secondary toxic effects on the mother, to interference with the milk cjection reflex (see chapter IV), or to untoward effects via the milk on the young whosc growth rate is the only available measurc of lactational performance in such laboratory animals as rats As regards the prevention ·LACIOGEVESIS 39 expenments on the artificial induction of lactation in farm animals on the topic under discussion In Chapter I reference was made to lactogenic effects closed by ocstrogens under suitable conditions These have been demonstrated most spec tacularly in farm animals in which lactation has been initiated in udders grown by treatment with oestrogen pan passu with the contnuance for a ome of the oestrogen treatment Figure II 5 shows some lactations curses for cons and herfers brought into milk secretion by the subcutaneous implantation of pellets of stulboestrol (Folley and Valpress 19:14) It wall be seen that the best of these animals gave a total milh yield in a year of well over 700 gallons Experiments carried out more recently in our laboratory on the spayed virgin goat (Cowie et al 1952) indicate that it is possible to select a daily dose of oestrogen for example ! mg hexoestrol daily, wuch will cause mammary growth but at the same time tends to inhibit secretion in the sense that the unmilied udder does not become suollen with secretion in the latter stages of oestrogen treatment as often happens when a suitable louer dose is used The strong lactogenic effect of such a low dose of oestrogen is allustrated in the tho loves pictures of Plate II 6 which shows photo graphs of the udders of two ovaricciomized virgin goats in which udder growth was induced by the daily injection of 025 mg hexoestrol for 20 weeks These two udders are swollen with accumulated secretion The 1 mg duly dose of hex oestrol referred to above may be considered to have exceeded the upper of the two thresholds which Dr Vlalpress and I postulated in our double threshold theory while the o 25 mg daily dose clearly came in the lactogenic zone between the thothresholds Moreoser as the two much smaller udders in the upper pictures show the lactogenic effect of this latter dose of oestrogen was abolished completely by simultaneous administration of 100 mg progesterone darly These conclu sions about the initiation of lactation in our goats which in cidentally are in accord with findings in the rat reported by Selye (1970) depended on the gross appearance of the un milled udder milling of which was not begun unul the treat ment ended except in the case of the two goats receiving the lactogenic dose of oestrogen whose udders became so tense ·38 PHYSIOLOGI AND BIOCHEMISTRY OF LACTATION progesteronc daily grow as fast as the young of control rats We are therefore left with the alternative possibility, predicted long ago by some of the carlier workers, that progesterone and oestrogen in combination act synergistically, the combination furnishing a far more potent inhibitor of lactation than oestro gen alone Fauvet (1941) seems to have been the first to pro vide experimental cvidence for this concept when he showed that doses of oestrogen and progesterone, ineffective by them selves in combination effectively inhibited lactation in spayed female rats The ability of relatively small doses of oestrogeni in combination with progesterone to inhibit lactation in the absence of the ovary, at least in the rat, has since been re peatedly confirmed Confirmation of this concept has also been obtained in the rabbit by Meites and Sgouris (1953, 1954) in the goat (Cowie, Folley, Malpress and Richardson, 1952) and in man by Romanı and Recht (1948) It will be of interest to consider now the bearing of some 14 12 10 kg o r .00- w no 1 50 50 mg Hef r no 1 no 2 100 90mg no 3 Tall ts rmon 30 40 45 30 10 15 20 Weeks after implantation Fig 11 5 Lactation curves for boxines implanted with tablets of diethylsulboestrol The ordinate shows mean daily mulk yield (from Folley and Malpress 1914) ·LACTOGENESIS 39 expenments on the artificial induction of lactation in farm animals on the topic under discussion In Chapter I reference was made to lactogenic effects clicited by ocstrogens under suitable conditions Thcsc have been demonstrated most spec tacularly in farm animals in which lactation has been initiated in udders grown by treatment with ocstrogen pan passu with the continuance for a time of the oestrogen treatment Figure II 5 shows some lactation curves for cows and heifers brought into milk secretion by the subcutaneous implantation of pellets of sulbocstrol (Folley and Malpress 197) It will be seen that the best of these animals gave a total milk yıld in a year of well over 700 gallons Experiments carned out more recently in our laboratory on the spayed virgin goat (Cowie et al 1952) indicate that it is possible to select a daily dose of oestrogen for example 1 mg hexoestrol daily which will cause mammary growth but at the same time tends to inhibit secretion in the sense that the unmilhed udder does not become swollen with secretion in the latter stages of oestrogen treatment as often happens when a suitable, lower dose is used The strong lactogenic effect of such a low dose of oestrogen is illustrated in the two lower pictures of Plate II 6 which shows photo graphs of the udders of two ovariectomized virgin goats in which udder growth was induced by the daily injection of 0 25 mg hexoestrol for 20 weeks These two udders are swollen with accumulated secretion The 1 mg daily dose of hex oestrol referred to above may be considered to have exceeded the upper of the two thresholds which Dr Malpress and I postulated in our double threshold 'theory while the o 25 mg daily dose clearly came in the lactogenic zone between the two thresholds Moreover as the two much smaller udders in the upper pictures show the lactogenic effect of this latter dose of oestrogen was abolished completely by simultaneous administration of 100 mg progesterone daily These conclu sions about the initiation of lactation in our goats which in cidentally are in accord with findings in the rat reported by Selye (1940) depended on the gross appearance of the un milked udder mulking of which was not begun until the treat ment ended except in the case of the two goats receiving the lactogenic dose of oestrogen whose udders became so tense ·40 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION as to necessitate milking before the end of the treatment period Results which in general confirm these conclusions have since been obtained in further experiments in goats in our labora tory (Benson, Cowie, Cox, Flux and Folley, 1955) Remehe, Meites Cairy and Huffman (1952) have recently reported experiments on the artificial induction of lactation in cows in some of which truly spectacular mulk yields were obtained Treatment with oestrogen and progesterone by pellet implantation was used to induce udder growth and then the animals were given a final short term treatment with oestrogen to initiate lactation in accordance with the Meites Turner theory of lactation initiation Table II 5, taken from the American workers' paper, shows the total yields of milk TABLE II 5 Induction of lactation in cattle by treatment with stulboestrol and progesterone (results taken from Reineke, Mestes, Cary and Huffman, 1952) Description of animal Maximum Total yield in 10 months daily milk yield milk fat lb lb Іь Guernsey heifer JI Guernsey heifer J2 Guernsey heifer J3 Holstein cow (Mabel) Holstein cow (Julia) 254 247 25 9 6681 6622 6532 11330 7760 323 331 369 420 309 800 450 and fat obtained in ten months from six of their treated anı mals Their best animal, a sterile Frıcsian cow gare a peak daily yield of 80 lb and a total yield in ten months of over 1100 gallons The other five animals also gave good yields In view of these excellent results, a study has been undertaken in our laboratory of the effect of a final inggering treatment with oestrogen on the lactational performance of spayed goats in which udder growth was induced with ocstrogen and prog esterone (Benson et al 1955) Our results which are shown in

·PLATE 11 6
394
HXOZS
PG 10
THX 0 25
PG 100
383
HX 025
391
HX O25
Photographs of the udders of four goats developed by injections of
hexoestrol and progesterone The labels on each figure indicate the num
ber of the goat and the daily dose of hexoestrol (HX) and progesterone
(PG) in mg note the very large udders of goats 383 and 391 from Conte
Folley Malpress and Richardson 1952)

·No text on this page.

·LACTOGENESIS Table II 6 indicate that though the extra oestrogen caused a slightly quicker initsal rise in yield, the total yıcld over a long period was hardly affected The yield over 210 days can only be given for onc udder half since the other half was removed for histological study as soon as the yield had reached sts peak TABLE II 6 Induction of lactalis 12 olanectomized goats with hexoestrol and progesteront effect of final inggering treatment with heroestrol (results from Benson, Cowie, Cox Flux and Folley, 1955) ΛΣalk yield Whole udder over intral 29 days Pair DO Half udder over 210 days 1 A B A 2 25 5 31 3 231 3 409 211 273 15 9 134 208 134 114 134 122 92 194 136 156 128 129 254 23 8 306 18+ 7 176 22 9 Mlean 229 269 134 142 A 05 mg hexoestro} +70 mg progesterone daily for 150 days B As above then o 25 mg hexoestrol daily for 15 days These experiments provide no evidence that a short term tnggering treatment with oestrogen following prolonged treatment with a combination of oestrogen and progesterone improves the subsequent lactational performance nor do our most recent experiments on virgin heifers the results of which are given in Table II 7 (Folley and Flux unpublished} These animals were given treatments very similar to that used by Alestes and his colleagues but as can be seen their milk Sields were not spectacular The comparative success of the infrequent injections of crystal suspensions is noteworthy ·42 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION TABLE II 7 Induction of lactation in heifers uith oestrogen and progesterone (unpublished experiments of D S Flux and S J Folley 1993) 305 day Peak Name of herfer Treatment yield daily yield lb lb Winsome 31 Patty Poise Oily injections 5798 3987 24 211 181 3349 Dinky Joyce Campion 27 Tablet implants 39 16 3 7578 3773 122} 7787 3727 4128 Nell 7 Marakatoria Primrose Crystal injections 351 197 213 Injection of oily solutions 05 mg hexoestrol + 70 mg progesterone in arachis oil daily for 120 days then 10 mg hexoestrol in oil daily for 16 days Tablet implants 30 x 100 mg tablets progesterone + 1 X 100 mg tablet diethy Istilboestrol for 120 days, + 15 X 100 mg tablets sulboestrol for the last 30 days Injection of crystal suspensions 500 mg progesterone crystals at 30 day intervals over 120 days 100 mg oestradiol monobenzoate crystals on ist day and 100 mg oestradiol monobenzoate in solution in oil on goth day In considering the impact of these recent findings with farm animals upon theories of the mechanism of lactauon initiation at should be pointed out that some features of our own results with goats are in accord with the Meites Turner theory, while others are explicable on the basis of our doublethreshold' theory as are many previous results obtained on ·LACTOGENESIS 43 cons as well as goats However, the recognition of the syn ergistic action of oestrogen and progesterone in inhibiting lactation as also the aforementioned experiments of Meites and Sgouris (1953, 1954) on the rabbit necessitate some re vision of concepts I therefore i enture to advance the following tentative theory, which combines various features of previous hypotheses and seems capableof harmonizing most of the known facts regarding the initiation of lactation The new theory embodies the following postulates (1) Measurements of the prolactin content of the hypophysis do not necessarily give any indication of the rate of prolactın release and are better disregarded as largely irrelevant to the issue (2) Low circul ating levels of oestrogen activate the lactogenic function of the anterior pituitary while higher levels tend to inhibit lactation even in the absence of the ovary though it is uncer tain whether this inhibition comes into play at the level of the pituitary or of its target organ the mammary gland or both (3) Lactogenic doses of oestrogen may be deprived of their lactogenic action by suitable doses of progesterone the com bination then acting as a potent inhibitor of lactation This is the inhibiting influence which operates normally in preg nancy (4) The relative fall in the ratio of progesterone to oestrogen at parturition as demonstrated for the mouse by Atkinson and Leathem (1946) removes the inhibition which is replaced by the positive lactogenic effect of oestrogen acting unopposed REFERENCES Atkinson WB and Leathem JH (1946) Anal Rec 95 147 Balmain J H Folley S J and Glascoch R F (1952) Nature Lond 169 447 Balmain J Hand Folley SJ (1952) Arch Brochem Brophys 39 188 Benson GK Cowie ATCox C Flux D L and Folley SJ (1955) Í Endocrin 13 46 Cowie A T Folley Sj Malpress F H and Richardson KC (195) Endocris 8 64 Cuder I and Lewis JH (1933) Amer 7 Physzel 103 643 Fauvet E (1941) Arch Gnak 171 34 Folley S J (1942) Nature Lond 150 266 Folley S J (1952) in A S Parkes Marshall s Physiology of Reproduction 3rd ed London Longmans Green & Co Chap 20 ·44 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION Folley S J and French T H (1949) Brochem 7 45 117 Folley S J and French T H (19496) Biochem 7, 45 270 Folley S J and French T H (1950) Brochem Í 46 465 Folley S J and Greenbaum A L (1947) Brochem 3, 4r 261 Folley S J and hon S K (1937) Proc roy Soc B 124 476 Folley S J and Malpress F H (1944) 7 Endocrin 4 I Folley S J and Malpress F H (1947) Abstr Commun & Vilth Int Pky stol Cong P 340 Folley S J and Young FC (1941) Lancel 240 380 Gomez E T and Turner CW (1936) Proc Soc exp Biol N2 34 404 Graham 1 R jr Houchin O B, Peterson V E and Turner CW (1938) Amer Physiol, 122 150 Greenbaum A L and Greenwood, FC (1954) Biochem 7,56 625 Lyons, WR (1942) Proc Soc exp Buol Nř 51 308 Lyons, WR Johnson E Cole R D and L CH (1955) in R W Smith O H Gaebler and CNH Long The Hypophyseal Growth Hormone Nature and Actions New York Blabiston Chap 26 Lyons W R L C H Cole R D and Johnson R E (1953) 7 din Endocrin Afelab 13 836 Mertes J (1954) Rev (anad Bol 83_359 Afeites, J and Sgouris ; T (1953) Endocrinology 53 17 Meites J and Sgouris J T (1954) Endocrinology_55 530 Meites J Trentin and Turner C W (1942) Endocrinology 37 607 Meites ) and Turner CW (1942) Endocrinology 30 711 719 726 31 340 Meites J and Turner CW (1948) Res Bull Vo agric Exp Sta no 415 Moore R O and Nelson W L (1952) Arch Brochem Biophys 36 178 Nelson WO (1936) Physiol Rev 16 488 Nelson W O and Gaunt R (1936) ProC Soc exp Brol Yr 34 671 Nelson W O Gaunt R and Schweizer M (1943) Endocrinolow 33 325 Peeters Gand Massart L (1952) Nature Lond 769 627 Petersen We and Shaw Į C (1942) 7 Dairy Sa 25 708 Popjak G and Beeckmans M L (1950) Brochem 7 46 547 Popják G Folley S J and French T H (1949) Arch Brochem 23 509 Recce RP (1939) Proc Soc exp Biol V7 40 25 Reineke LPNleites J Cairy C F and Huffman CF (1952) Proc Book Ann Meeting Amer vet med Assoc 1957 P 325 Riddlc O Bates RW and Dykshorn S W (1933) Amer 7 Physiol 105 197 Roman J and Recht P (1948) Ann Endocrine 9 247 Selye H (1940) Anal Rec 78 253 Stricher P (1951) Collog Ini CNRS XHANI 19,0 p 15 Stricker P and Graeter F (1928) CR Soc Brol Parts 99 1978 ·CHAPTER III THE MAINTENANCE OF MILK SECRETION GALACTOPOIESIS It is well known that the integnty of the pituitary is essential for the maintenance of milk secretion just as it is for its initia tion This follows on the one hand from the immediate aboli tion of milk secretion which invariably follows hypophysectomy and on the other from the fact that, as we shall see later anterior pituitary extracts contain hormones capable of in (150% of FPS 32 30H 28 Controls Mean weight of young (g) 14 Operation Hypophysectoared 12 (4 of pups al 10 6 6 2 ULU 10 42 34 18 40 4 6 8 16 Days after parturition Fig III 1 Effect of hypophysectomy on lactation in the rat (from un published experiments of A T Cowe) ·46 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION creasing the rate of milk secretion The dramatic effect of hypophysectomy upon milk secretion is illustrated in Figure III i which shows the results of an unpublished experiment on lactating rats carried out by Dr AT Cowie in our laboratory The Figure shows curves representing the mean growth rates of the litters of two groups of rats the members of one of which were hypophysectomized on the fourth day of lactation and thereafter given regular injections of oxytocin to cause milk ejection so that the litters could obtain milk if it was secreted It will be seen that growth of the suchlings ceased immediately after the mothers were operated upon and they soon began to die from lack of milk It is however not yet certain whether the constellation of pituitary hormones responsible for the maintenance of estab Lashed milk secretion is identical with that concerned with its initiation If it is the same then active milk secretion could be regarded as a functional state of the morphologically com plete mammary gland which appears whenever the necessary pattern of pituitary hormones is present and unopposed by inhibiting influences a state which presumably would hold just so long as the pituitary continued to secrete the various components of the hormone complex in harmoniously balanced proportions If on the other hand the two pituitary constel lations are not identical then we must consider the possibility that prolactin exerts a trigger like action on the mammary gland which is only effective in the presence of other hypo physial influences perhaps regulating the supply of metabo lites whosc continued intervention is necessary for the main tenance of secretion On this view it is conceivable that once lactation has been initiated prolactin is no longer essential for its continuance In the light of present knowledge the first alternative seems preferable since it is in harmony with the theory proposed by Selyc (1934) according to which the release of prolactin by the hypophysis is under neural control, the hormone being believed to be reflexly secreted by the anterior hypophysis in response to the suckling stimulus regular application of which is regarded as essential for the maintenance of milk secrction The facts upon which Selyc based this theory were bnefly is follows Suckling of some of the mammary glands of a lactating ·GALACTOPOIESIS 47 rat was prevented either by surgical excision of the nipples or by covering them with collodion Provided other intact nipples belonging to the same animal were regularly suckled the unsuckled glands did not undergo the involution which in normal circumstances quickly follows weaning and at autop sy they were found to contain mulk Moreover, if the escape of mill from the rat mammary gland was prevented by ligation of the main galactophores involution was retarded and a secretory condition maintained prouded active suckling was conunued Selyes theory, the evidence for which cannot be considered in greater detail here*, thus lays emphasis on the loss of the suckling stimulus as the main cause of the regressive changes in mammary function and structure which follow weaning The fact that Williams (1945) later found it possible to mimic by prolactin administration the secretion maintain ing effects of the suckling stimulus on glands which could not be emptied of milk or even suchled because of surgical inter vention as just described not only supports Selye s theory but also suggests that continued prolactın release is an important factor in the maintenance as well as in the initiation of milk secretion It is of course likely that the secretion of other uitary hormones believed to be involved in both processes may also be susceptible of control by the suchling stimulus Thus evidence that under some circumstances suckling may cause the release of one of them ACTH has been presented by Gregoire (1947) As a result of simultaneous and independent suggestions of Bergman and Turner (1940) and of Folley and Young (1940) the term galactopoiesis has come into use to denote the ex perimental stimulation of established lactation The study of galactopoiesis is important since the hormonal mechanisms involved are probably closely related to those concerned in maintaining normal milk secretion and experimental analysis of the former will undoubtedly contribute to our understand ing of fundamental aspects of the hormonal control of normal mammary function pit PROLACTIN AND SOMATOTROPHIN Unfractionated extracts of ox anterior lobe possess con * For full discussion see Folley (1947) ·48 PHYSIOLOGY AND BIOCHEMISTRY OF L siderable galactopoietic power in lactating C single injections will evoke marked tempora mulk yield as shown in Figure III 2 (Folley and 80 76 74 72 H Milk yield (Kg) 70 65 Injections 66 group of four treated cours A group of four c ntrol cous 64 62 0 5 15 10 Days Fig III 2 Effect on the milk yield of cows of an injection of a salane extract of ox anterior pituitary (from Folley and 2 oung 1998) This Figure shows the milk yield increase exhibited by a group of four cows each of which recened a singlc subcutaneous injection of 10 mil ox antenor pituitary extract At one time It was believed that the power of anterior pituitary extracts to increase the mall yield of lactating cows was mainly if not wholly due to the prolactin present in them However the results reported long ago by the Russian workers Azimos and Krouze (1937), implied that the galactopoieuc effects of such extracts could not be quanutatirely accounted for by the prolactin present and soon afterwards Young and the author studying a series of anterior pituitary extracts found very little relationship betucen their galactopoietic potencies in lactating cons and their prolactin contents as assayed by the pigcon crop test (Folley and Young 1938 1939 1940) In actual fact the galactopoietic potency of punficd prolactin appears to be very small, at any rate as determined in single ·GALACTOPOILSIS 49 tests in cows In the experiments of Cotes, Crichton Folley and Young (1949), cows in declining lactation, known to be responsive to unfractionated ox pituitary extracts gave no significant increases in milk yield in response to single injec tions of about 1000 1 u purified prolactin (see Figure III 3) Though it may be objected that the dose used in these ex periments was not a particularly high dose per kg, neverthe less we have found that amounts of unfractionated anterior pituitary extract containing far less prolactin regularly evoke considerable galactopoietic responses in similar cows Other 10 Percentage increase in milk yield 30 mg Sot ph A1 40 mg PI 40 SPI + 30 mg Sm11 ph 15 mg 10 1 lot ph PLEY E1 Fig III 3 Effect on milk yield in the cow of injections of hormones of the anterior pituitary (this figure w constructed from the experimental results of Cotes Crichton Folley and Young 1949) Work leading to the same conclusion is reviewed by Folley (1955) As a result of our earlier investigations Young and I concluded that the galactopoietic activity of unfractionated ox pituitary extracts must be due to the action of more than one hormone and so we arrived at the concept of a pituitary galactopoietic hormone complex (Folley and Young 1941) ·50 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION As progressively more highly purified preparations of an terior pituitary hormones have become available over the years, Young and I with our collaborators, have attempted to determine the nature of the hormones, present in unfractionat ed or anterior pituitary extracts, which are responsible for their galactopoieuc activity in the earlier work with compa ratively crude extracts galactopoietic activity was often found to be associated with diabetogenic activity When, therefore Cotes Reid and Young (1949) succeeded in showing that purified pituitary growth hormone, somatotrophin, possessed diabetogenic activity, it was clearly important to find out if pure growth hormone was galactopoietic in lactaung cows Figure III 3 shows some results of experiments in which single injections of purified omatotrophin were administered to lactating cows These experiments were carried out by Cotes, Crichton Folley and Young (1949) on groups of cows at two experimental farms The responses are expressed in terms of the percentage micrca e in milk yield observed during the two days following the injection corrected for the change in yield of control cows receiving injections of salıne The histogram shows that, in accord with expectation, single injections of 30 or 60 mg punfied somatotrophin caused marked temporary increaves in null yield The magnitude of the response to these single injections of somatotrophin was such as to suggest that all or practically all of the galactopoietic activity exhibited by unfractionated ox anterior pituitary extract in single injection tests could be attributed to the growth hormone present Moreover there was no indication of a synergistic effect when growth hormone was administered together with prolactin or ACTH The probable fundamental significance of the galacto poietic activity of growth hormone is illustrated by the neat way in which its discovery, since confirmed in a number of laboratones (see Folley, 1955 for review), fulfils a prediction of Young (1947), who pointed out that galactopoiesis growth and diabetogenesis, involving as they all do the preservation of foodstuffs from oxidation, are phenomena which may be expected to owe allegiance to closely related hormonal regu lating mechanisms The finding that prolactin possesses relatively little galacto poieuc activity in lactating cows, at any rate in short term ·GALACTOPOIESIS 51 experiments, must not be taken as conflicting with the view expressed above, namely that its continued secretion is prob ably essential for the maintenance of lactation Though pro lactın does not appear to be a limiting factor in the cow in the declining phase of lactation it might well be a limiting factor in certain circumstances in other species Despite some positive results, its value for the treatment of hypogalactia in women is far from certain but in this connection it should be noted that it is often not clear whether hypogalactia should be regarded as essentially a problem of lactogenesis or of galactopoiesis In our laboratory we have recently attempted a bio chemical approach to the problem of the role of prolactin in the maintenance of a secretory state in the mammary gland In the rat, the early stage of lactation days 1-5 is a period of biochemical labılty in which, as shown in Chapter II the respiratory quotient of mammary tissue in vitro is increasing above unity and the slope of the composite respiration curve in bicarbonate buffer is changing from negative to positive We reasoned therefore that mammary tissue at this stage should be a favourable medium for demonstrating galacto poietic effects of prolactin in vitro Miss Balmain and I accord ingly investigated in preliminary experiments the effect of prolactin added to the medium on the composite respiration curve of rat mammary gland slices in early lactation (Balmain and Folley, 1952) In our experiments equilibration with prolactin significantly increased the slope of the composite Tespiration curve resulting from the metabolism of mammary gland slices taken from rats autopsied during days 1-5 post partum Figure III 4 illustrates the results of one of these experiments It shows curves representing the overall pressure changes resulting from the metabolism of mammary gland slices taken from a rat killed on the fourth day of lactation incubated in Krebs saline bicarbonate, the substrate being a mixture of acetate and glucose It will be seen that the total pressure progressively increases and that addition of prolactin significantly increases the rate of pressure rise One is probably justified in regarding the effects observed in these experiments as essentially galactopoietic rather than lactogenic in nature but some recent experiments in our laboratory by Dr TR Bradley raise doubts whether they are due to the action of ·50 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION As progressively more highly punfied preparations of an terior pituitary hormones have become available over the years, Young and I with our collaborators, have attempted to determine the nature of the hormones, present in unfractionat cd ox anterior pituitary extracts which are responsible for their galactopoietic activity In the earlier work with compa rately crude extracts galactopoietic activity was often found to be associated with diabetogenic activity When, therefore Cotes, Reid and Young (1949) succeeded in showing that purified pituitary growth hormone somatotrophın possessed diabetogenic activity, it was clearly important to find out if pure growth hormone was galactopoietic in lactating cows Figure III 3 shows some results of experiments in which single injections of purified somatotrophin were administered to lactating cows These experiments were carried out by Cotes Crichton Folley and Young (1949) on groups of cows at two experimental farms The responses are expressed in terms of the percentage increase in milk yield observed during the two days following the injection corrected for the change in yield of control cows receiving injections of salıne Thc histogram shows that, in accord with expectation, single injections of 30 or 60 mg purified somatotrophın caused marked temporary increases in milh yield The magnitude of the response to these single injections of somatotrophin was such as to suggest that all or practically all of the galactopoietic activity exhibited by unfractionated ox anterior pituitary extract in single injection tests could be attributed to the growth hormone present Morcover there was no indication of a synergistic effect when growth hormone was administered together with prolactin or ACTH The probable fundamental significance of the galacto poictic activity of growth hormone is illustrated by the neat way in which its discovery since confirmed in a number of laboratories (see Folley, 1955 for review) fulfils a prediction of Young (1947) who pointed out that galactopoiesis, growth and diabetogenesis, involving as they all do the preservation of foodstuffs from oxidation, are phenomena which may be expected to one allegiance to closely related hormonal regu lating mechanisms The finding that prolactin possesses relatively little galactopoietic activity in lactating cows, at any rate in short term

·GALACTOPOIESIS
55
as judged by the growth cures of the young was most severely
inhibited sn the latter group
QoSliam operated
Onwa Adrenalectomized cntrols
ma Adrınalectomizi-aansclomuz
Mean weight of young (2)
Operation
.
32
Days after parturition
Fig IIIS Comparison between the effects of adren
alcctomy and of adrenalectomy ovaricctomy on
lactauon in the rat (resulis from Flux 1955)
Replacement studies with adrenal corticoids in adrenal
cctomized lactating rats have given somewhat discordant
results
ough it was at first ved that the effect of the
adrenals on lactation was mediated mainly through clectrolyte
and water metabolism Gaunt Escrsole and kendall (1942)
later concluded that the critical factor for normal lactation
was a sufficiency of 11-oxygenated steroids then believed to
bc mainly concerned with protein and carbohydrate meta
bolism They arnved at this conclusion because they could
achieve complete lactation maintenance in rats adrenalecto
muzed just after parturition with 11-oxygenated steroids but
not with deoxycorticosterone In contrast Dr Cowie and the

·54 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION suggested that single injections of ACTH-protein temporanly decreased rather than increased the milk yield of lactat ing cows The availability of very active low molecular preparations of ACTH has since enabled us to show quite con clusively that this is indeed the case Figure III 5 shows the results of a recent experiment with long acting ACTH carried out in our laboratory (Flux, Folley and Rowland, 1954) The curves shown in this Figure depict the mean daily milk yields of three groups of cows, two of which received ACTH while the third served as a control group The curves for the groups of cows receiving injections of 100 or 200 1 0 ACTH on the occasions indicated by the appropnate arrows show that either dose of the hormone caused a marked though temporary depression in mill yield However although no galactopoietic responses to ACTH have been observed in cows studies of the effects of adrenal ectomy on lactation in small animals show that the pitutary adrenal axis is a critical factor in lactation maintenance just as it is in lactogenesis This is attested by numerous experiments on small animals which have shown that adrenalectomy causes a marked though not complete inhibition of lactation Paired feeding experiments on the rat though presenting difficulties in interpretation because the adrenalectomized rat has less ability to support lactation by the catabolism of body tissues (as well as to expend energy in spontaneous activity) than pair fed controls indicated that part of this decline could be attributed to chronic anorexia resulting from loss of the adrenals leaving a definite though relatively small proportion to be ascribed to the loss of the adrenals per se (Cowie and Folley 1948) However recent experiments in our laboratory (Flux, 1955) show that an ovarian secretion probably pro gesterone, contributes significantly to the partial lactation maintenance observed after adrenalectomy for when the ovaries are removed as well as the adrenals lactation is much more severely inhibited than it is when only the adrenals are removed This is shown in Figure III 6 which shows the mean growth cures of the litters of three groups of rats comprising sham-operated controls, rats adrenalectomized on the fourth day of lactation and rats from which both ovanes and adrenals were removed on the fourth day It is obvious that lactation,

·GALACTOPOIESIS
55
as judged by the growth cun es of the young was most severely
inhibited in the latter group
SOF
--Sham operated
O Adr nalectomid contris
Auronic mix-un mod
(7) Suno jo sy314 Usiw
Operation
10
12
Days after partur tion
Fig III 6 Comparison between the effects of adren
alectomy and of adrenalectory to ouanectomy on
lactauon in the rat (results from Flut 1955)
Replacement studies with adrenal corticoids in adrenal
ectomized lactating rats have given somewhat discordant
results Though it was at first believed that the effect of the
adrenals on lactation was mediated mainly through electrolyte
and water metabolism Gaunt Eversole and Kendall (1942)
later concluded that the crtical factor for normal lactation
was a sufficiency of 11-oxygenated steroids then believed to
be mainly concerned with protein and carbohydrate meta
bolism They arnved at this conclusion because they could
achieve complete lactauon maintenance in rats adrenalecto
mized just after partuntion with 11-oxygenated steroids but
not with deoxycorticosterone in contrast, Dr Cowie and the

·56 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION author have never been able to achieve complete lactation replacement in our rats with 11-oxygenated steroids alone (Cowie and Folley, 1947, Cowie, 1952) in our earlier experi ments, when scarcity of supplies limited the doses of II oxygenated steroids we were able to give, our most effective steroid was deoxycorticosterone, though even high doses of this substance did not completely restore lactation to normal Later, however, Cowie (1952) was able to show that larger doscs of cortisone were as effective in lactation maintenance as deoxycorticosterone though as emphasized above the restora tion was not complete We have thed to discover the season why the adrenalecto mized rats of Gaunt, Eversole and Kendall (1942) lactate normally on cortisone therapy while ours do not The superi ority of deoxycorticosterone in our earlier experiinents was maintained when our rats were given a ługh protein diet sath a view to providing conditions most favourable for the action of the glucocorticoids (Cowie and Folley, 1947) Cowie (1952) later found that considerable variations in the dietary intakt of sodium also failed to affect the issue appreciably Thus, despite the fact that Nagareda and Guunt (1948) found the lactation maintaining ability of deoxycorticosterone in their adrenalectomized rats to be sensitive to extreme vanauons in dietary sodium and potassium it scems hardly lilely that differences in the dictary regimes obtaining in the respective laborators could be large enough to account for these discrepancies Since, as shown in the Figure III 7 Cowic (1952) has now achieved virtually complete lactauon mainte nance in our rats by simultaneous administration of cortisone and deoxy corticosterone it scems possible that the explanation Inight be in strain differences in the nature of the above mentioned contnbuuon of the ovaries This Figure shows the mean growth curve of the litters of a group of rats adrenalecto mized on the fourth day of lactation and thercafter given corusone and deoxycorticosterone acetate by pellet implanta ton It will be seen that the latters of these rats grow as fast as those of sham operated control rats Thc discovery of the new adrenocorticoid, aldosterone, raised the question whether this substance was specially active in maintaining lactation in adrenalcctomuzed rats Cowie and Tindai (1955) found ·GALACTOPOIESIS 57 definite activity with as little as 50 mg daily but even more striking was their finding that 100 ug daily of gz chloro hydrocorusonc gave almost complete maintenance 50 امه 2G - Shani op rated Adrenalectomized -- Adrenalestom.nl + Cortisone (2 ling tablets +DCA (1-50 mg tablets O Adrenalctom.cd + cortisone 0 Adrenalsstomized + DCA 4 2 () dunok so 348m veaw 16 14 12 10 $ Operation 9 10 15 12 A Days afcer parturition Fig Illy Maintenance of lactation with adrenal corticoids in adrenalectomized rats (from Course 1952) Biochemical studies are beginning to provide some in formation about the way in which the adrenal corticoids may be involved in the regulation of normal mammary function though such explorations are us yet only in their infancy Studies on the mammary gland arginase, an enzyme which in the liver is considered to be intimately involved in either the anabolic or catabolic phases of protein metabolism or perhaps both, at one ume seemed promising but the latest results have given rise to a position less casy to evaluate Figure 111 8 shows ame results of determinations of the arginase activity of rat ·56 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION author have never been able to achieve complete lactation replacement in our rats with 11 oxygenated steroids alone (Cowie and Folley, 1947, Cowie, 1952) In our earlier expen ments, when scarcity of supplies limited the doses of 11 oxygenated steroids we were able to give, our most effective steroid was deoxycorticosterone, though even high doses of this substance did not completely restore lactation to normal Later, however, Cowie (1952) was able to show that larger doses of cortisone were as effective in lactation maintenance as deoxycorticosterone though as emphasized above the restora tion was not complete We have tried to discover the reason why the adrenalecto mized rats of Gaunt, Eversole and Kendall (1912) lactate normally on cortisone therapy while ours do not The superi ority of deoxycorticosterone in our earlier experiments was maintained when our rats were given a high protein diet with a view to providing conditions most favourable for the action of the glucocorticoids (Cowic and Folley, 1947) Cowie (1932) later found that considerable variations in the dietary intale of sodium also failed to affect the issue appreciably Thus, despite the fact that Nagareda and Gaunt (1948) found the lactation maintaining ability of deoxycorticosterone in their adrenalectomized rats to be sensitive to extreme vanations in dietary sodium and potassium it seems hardly likely that differences in the dietary regimes obtaining in the respecuve laboratories could be large enough to account for these discrepancies Since as shown in the Figure III 7, Cowie (1952) has now achieved virtually complete lactation mainte nance in our rats by simultaneous administration of cortisone and deoxycorticosterone it seems possible that the explanation might lic in strain differences in the nature of the above mentioned contrbution of the ovanes This Figure shows the mean growth curve of the litters of a group of rats adrenalecto mized on the fourth day of lactation and thercafter given cortisone and deoxycorticosterone acetate by pellct implanta tion It will be seen that the litters of these rats grow as fast as tho e of sham operated control rats The discovery of the new adrenocorticoid, aldosterone, raised the question whether this substance was specially active in maintaining lactation in adrenalectomized rats Cowie and Tindal (1952) found ·GALACTOPOIESIS 57 definite activity with as little as 50 ug daily but even more striking was their finding that 100 ug daily of gæ chloro hydrocortisone gave almost complete maintenance 0 28 GF O-Sham operated - Adrenalectomized 0-- Adrenalectomized + Cortisone (2xlImg tablets), +DCA (1x50 mg tablets) Om - Adrenalectomized + cortisone 0- Adrenalostomized+DCA Otvo Mean weight of young (8) cung 12 10 8 Operation 5 G 7 1 10 8 9 11 12 13 14 Days after parturition Fig III 7 Maintenance of lactation with adrenal corticoids in adrenalectomized rats (from Coure 1952) Biochemical studies are beginning to provide some in formation about the way in which the adrenal corticoids may be involved in the regulation of normal mammary function though such explorations are as yet only in their infancy Studies on the mammary gland arginase an enzyme which in the liver is considered to be intimately involved in either the anabolic or catabolic phases of protein metabolism or perhaps both at one time seemed promising but the latest results have given rise to a position less easy to evaluate Figure III 8 shows some results of determinations of the arginase activity of rat ·58 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATIOY mammary tissue carried out a few years ago in collaboranon with Dr A L Greenbaum (Folley and Greenbaum, 1947) It will be seen that the arginase activity of the mammarv tissue remains virtually constant at a relatively low level during pregnancy and for a few days post partum At about the fifth day post partum the enzyme content begins to nse rapidly and by the 20th day has attained a level about 5-6 times that at 1200- 1000 200 Enzyme activity (units per & moist tissue) GOO dicaning 400 Partuntion noor a Fir gnancy, Lactation revolution 5 10 SS 1005 6 20 0 Days Fug III 8 Arginase content of the rat mammary gland during pregnancy and lactation (from Folley and Greenhain 1917) partunuon decreasing sharply again after weaning In the rat in full lactation the arginase content of the mammary gland is quite notable this tissue being second only to the liver as a source of arginase in the body and having about one ninth of its activity Though the biological role of arginasc in the mammary gland is not known with certainty, as we shall sec, these results suggest a close relationship to the secretory process the same applies to the mammary gland of the mouse since the arginase content of the latter also increases as lactauon proceeds and is comparable with that of the rat (unpublished results of Follcy and Greenbaum, cited by ·GALACTOPOIESIS 59 Folley, 1949) Adrenalectomy at the fourth day of lactation largely prevents the increase in the mammary gland arginase with advancing lactation since 4s Table 111 1 shows the mammary arginase levels of adrenalectomized rats killed at the 17th day are much losser than those of pasr fed controls TABLE 1111 Effect of adrenalectomy on the arginase content of the mammary gland of the lactaling rat (from Folley and Greenbaum, 1918) No of rats Arginase content of mammary gland units/g milk free tissue Sham operation (fed ad lib) Sham operation (pair 8 1629 fed) 9 147+10 Adrenalectomized at day 4 of lactation (fed ad Irb ) 9 20–23 (Folley and Greenbaum 1948) Lactation seems also to demand an increase in the liver arginase levels at least in the rat «ince as the cures in Figure III g show the liver arginase level increases by about 50 % immediately after partuntion at which enhanced level it remains virtually constant through out lactation decreasing again only after weaning (Folley and Greenbaum 1947) Here also, as originally found by Fraenkel Conrat, Simpson and Evans (1943) for the livers of non lactating rats adrenalec tomy at the fourth day decreases the liver arginase level to about one third of that of pair fed controls The liver arginase is believed to be a component of a complicated enzyme system concerned with the deamination of amino acids so that it seems logical to interpret the increase ·60 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION in the liver arginase level observed in the rat at about the time of partunition as reflecting an increase in the rate of hepatic gluconeogenesis This would probably result from in incrcased demand for deamınızed residues which can be transported to 1001 650 6001 Arginase unitsig moist tissue 500 400 Parturition Waning 3001 Pregnancy Lactation Involution od 10 15 OO Days Fig III 9 Liver argitase levels in the rat during preg nancy and lactauon (from Folley and Greenbaum 1947) the mammary gland and there converted to lactose or mull fat or used for energy production This view tavolse the assumption that changes in the arginase level reflect changes in the rate of gluconeogenesis from protein Folley and Greenbaum (1947) likewise snterpreted the changes in the arginase levels of the mammary gland of the rat during lacta uon and after weaning as evidence that the arginase of the mammary gland plays a similar role there This scemed all the more likely since Graham, Houchin and Turner (1937), on the basis of arteno venous studies, had carlier tcported that the udder of the lactating goat produces urca It thus appeared reasonable to suppose that as the milk yield of the rat pro ·GALACTOPOIESIS 61 gressively increases the liver fails to keep pace with the everincreasing demands of the mammary glands for non nitroge nous milk precursors so that the mammary tissue is forced to realize to an increasing extent its potentialities (manifested by the presence of arginase) for effecting gluconeogenesis from protein On this view since the adrenal cortex through the action of the glucocorticoids is believed to promote gluconeo genesis the unfavourable effects of adrenalectomy upon lactation would be mediated through the resulting inhibition of gluconeogenesis from protein both in the laer and in mammary gland itself However determinations of the arginase content of the lactating mammary gland in ruminants and other herbivorous animals have shown that the mammary tissue in these species contains very little enzyme as compared with the mammac of the rat and mouse This is evident from some unpublished results on the arginase levels of mammary tissue in vanous species obtained in collaboration with Dr Green baum (see Folley 1949) which are presented in Table III 2 TABLE III 2 Arginase content of the mammary gland in canous species during lactation (unpublished results of Folley and Greenbaum 1948 cited by Folley 1949) Stage of Units of arginases Animal No of lactation animals (days) Absolute Relative Inoist tissue Rat Mouse Mouse Guinea pig Guinea pig Rabbit Rabbit Sheep Goat Cow 17 5 15 5 ΙΟ 1447 51 0 89 2 78 100 19 40 39 3 2 20 100 35 62 5 7 I 3 3 2 28 early advanced advanced 5 3 6 ΙΟ I ·62 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION In view of this finding it would seem that despite the conten tion of Graham and his collaborators that the lactating goat udder produces urea, it must be regarded as doubtful shether gluconeogenesis in the mammary gland plays any significant role in lactation in herbivorous forms In this connection it would be interesting to know how severe are the effects of adrenalectomy upon lactation in ruminants Voreover, as far as the rat itself is concerned we have found that the relative abilities of cortisone and deoxycorucosterone to maintain the tissue arginase levels in lactating rats after adrenalectomy are somewhat different from their relative lactation maintaining abilities in the same rats (Folley and Watson 1951) It there fore follows that even in the rat and mouse the only forms in which appreciable levels of arginase have so far been found in the mammary gland the biological role of arginase in this organ is still far from clear Other brochemical studies which we have made suggest that the adrenal cortex may also be concerned in the regula uon of a process for which the mammary gland exhibits outstanding capabilities, namely, lipogenesis If lactating rats are injected with (carboxy-C) acetate, the fatty acids isolated from the mammae at autopsy shortly thcreafter are strongly radioactive, the specific activity of the short chain (volatile) fraction being greater than that of the long chain (non volaule) acids Unpublished studies carried out by Dr R F Glascoch and Dr W G Duncombe in our laboratory have shown that after adrenalectomy the ratio of the specific acuyties of the volatile and non volatile fractions isolated from the mammary tissue of rats receiving radioactive acetate was higher than that of similar fractions isolated from the mammae of intact controls, suggesting that adrenalectomy had in some way disturbed the mechanism of lipogenesis from small molecules in the mammary gland Studies of the effect of adrenal corti coids in lipogenesis in mammary gland slices in titro are in harmony with this supposition, but will not be further touched upon here since lipogenesis in the mammary gland is dealt with in detail in Chapter V THE PITUTTARX TUYROID A[ECILANISHI We turn now to the role of thc pituitary thyroid mechanism ·GALACTOPOIESIS 63 in mill secretion Earlier work on the role of the thyroid gland in milk secretion led to the conclusion that while the thyroid hormone is not essential for mammary gland function nevertheless the pituitary thyroid axis may be regarded as an important regulator of the rate of milk secretion This conclu sion follows from the facts that on the one hand thyroidectomy reduces the mill yield but does not abolish secretion altogether, while on the other administration of thyroid hormone exerts a galactopoietic effect under suitable conditions The latter effect, first reported sixty years ago for the cow by Hertoghe (1896) has been confirmed and exhaustively investigated in recent years (see Blaxter 1952 for detailed review) It has also been recently demonstrated in rats by Desclin (1949) and women by Romanı Plocq and Recht (1949) and by Roche Giraud Lelong, Liardet and Coignet (1950) Galactopoietic responses in cows were obtained by Folley and Young (1938 1939) by injections of pituitary extracts rich in thyrotrophin but virtually free from prolactin and this indication of the importance of the pituitary thyroid axis as a regulator of 10 Thyroxine. Daily milk yield (Kg) 40 1 S 10 5 SO 33 to 20 25 Days Fig III 10 Increase in mulk yield of a group of four cows receiving DL-thyroxine sodium (10 mg/day) subcutaneously continuous line daily mulk production of treated cows Interrupted line daily milk production of control cows (from Folley and Ithite 1936) ·64 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION lactation is in harmony with the powerful galactopoiet action of the thyroid hormone which will now be considered The possibility of using thyroid active materials on a larg scale for increasing the milk yield of cows in declining lactatio has led to numerous experiments during the past decade o vanous aspects of the galactopoietic effect of thyroid hormone 150 100 Mean relative milk phosphatase Controls 50 Injected OL-thyroxine 8 10 *5 57 22 24 29 31 Days Fig 111 11 Decrease in the alkal ine phosphatase content of the milk of the cow after injections of DL thyroxine sodrum (10 mg/day) The results are expressed as a percentage of the mean initial value Each curic represents the mean values for four cons (from Folley and IVhite 1936) The original work of Hertoghe was lost sight of unul 1934 when the phenomenon was rediscovered by Graham (1934 4,6), who besides obtaining galactopoictic effects in cows by feeding dried thyroid gland showed that injection of synthetic thyroxine gave similar results Shortly afterwards we confirm ed and catended Graham s work in our laboratory showing that daily injection of thyroxine not only caused a considerable temporary increase in milk yield but also increased the percent ·GALACTOPOIESIS 65 ages of fat and non fatty solids in the mill (Folley and White 1936) Some of our results are shown in Figure III 10 in which the curves represent mean values for a group of cows receiving daily subcutaneous injections of 10 mg DL-thyroxine as compared with the values for a control group injected with saline We also showed that the concentration of allaline phosphatase in the milk was dramatically reduced during the treatment penod This is shown in Figure III 11 which illus trates the mean percentage drop in the milk alkaline phospha tase level This latter is a specific and sensitive response to the thyroid hormone which we later found very useful for assaying thyroid active preparations for galactopoietic potency in cons, its significance from the point of view of the mechanism of the interaction of the thyroid hormone and the mammary gland is unknown and would repay further study Studies in many laboratories of the effect of thyroid active materials on milk composition have led to the conclusion that the only changes of note besides those already mentioned are a decrease in ascorbic acid content and as shown by Chanda and Owen (1951) an increase in the organically bound phosphate The experiments of Chanda McNaught and Owen (1952) and also those of Bailey Bartlett Folley Rowland and Thompson (1951) in our Institute have estab lished that during the period of treatment with thyroid hor mone the proportion of the vitamin B, of the mill present as co carboxylase that is in the phosphorylated form, increases concomitantly with the decrease in the alkaline phosphatase content Figure III 12 shows some of our results which illus trate this These results were obtained on a group of cows receiving L thyroxine by mouth The opposite changes in the alkaline phosphatase content and in the ratio of the phosphory lated to free aneurin in the mill are clearly shown One is tempted to conclude that the unphosphorylated aneurin of the milk arises by dephosphorylation of aneurin phosphate while the mill is in the udder However since aneurin phos phate is a pyrophosphate its dephosphorylation would presum ably require the successive action of a specific pyrophos phatase and of the alkaline phosphatase While the presence of a pyrophosphatase in cows milk has been claimed this contention has not yet been satisfactorily proved Moreover ·66 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION the alkaline phosphatase, the opomum pH of which is around pH 9-10, would probably not be very acuse at the pH of mall This attractive hypothesis thus requires further proof before it can be accepted The earlier expenments on the galactopoietic effect of thvroid hormone mainly involved the feeding of odocasein (see Blaxter, 1952 for review), wluch although readily made in 10H 100 Phosphatase GO 80 Phosphatase King Armstrong units/100 ml 100 mg. L thyroxine Frå daily Per cent aneurin in phosphorylated form Combmed art, urin 60 so 40 0 3 A Weeks Fag III 12 The effect of feeding L-thyroxine on the allaline phosphatase content of the milk of the cow and on the ratio of the phosphorylated to free aneurin (from Bailey Bartlett Follo Rouland and Thompson unpublished results cited by Folley 1951) quantity and therefore cheap, possesses certain disadvantages in practice Thus, it must be standardized by biological methods often a troublesome and uncertain procedure, and some preparations possess a taste and smell which cause a proportion of cows to refuse it Morcover its administration entails a considerablc intake of non thyroxine jodine which may cause untoward effects It had come into wide use mainly because syntheuc thyroxine was unul recently scarce and expensive and, moreover, believed to be relatively inactive by the oral route in cows A few years ago, however, Chalmers Dickson, Ells and Hems (1949) described a new and improved method ·GALACTOPOIESIS 67 of synthesis of L-thyroxine which promises to make the hor mone available in quantity at a price which will compete with that of iodocasein This noteworthy advance prompted us to investigate the galactopoietic activity of L thyrowne when given to lactating cows by the oral route and we found that its potency by this mode of administration was much greater than had been supposed (Bailey Bartlett and Folley, 1949) Cont ol 35 5 mg 50 mg - 100 mg 150 mg 14 13 Kgcow/day 12 dose Start of freshnell End of treatment 10 0 10 ܘܢ 40 50 Days Fig III 13 Effect of L thyroxine given by mouth on the mill yield of groups of cows (dose levels indicated were fed daily) (from Barley Bartlett and Folley 1949) Our results are shown in Figure III 13 which shows the mean lactation curves for groups of cows receiving various daily doses of synthetic L-thyroxine by mouth It will be seen that a daily dose of 100 mg L thyroxine by mouth caused a conside rable temporary increase in milk yield, which incidentally was a little greater than the effect produced by feeding our stand ard dose of 20 g 1odocasein daily Since thyroxine is odour less and virtually tasteless and since its purity can be checked by chemical techniques thus obviatıng troublesome bioassay procedures and since moreover, its use entails a relatively small 1odine intake so that the risk of iodism is virtually ·66 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION the alkaline phosphatase, the optimum pH of which is around pH 9-10 would probably not be very active at the pH of milk This attractive hypothesis thus requires further proof before it can be accepted The earlier experiments on the galactopoietic effect of thyroid hormone mainly involved the feeding of iodocasein (see Blaxter, 1952 for review), which although readily made in 100 60 Phosphatase 80 Phosphatase King Armstrong units/100 ml 100 mg L thyroxine Fed daily Per cent aneurin in phosphorylated form Combin.d ancurin 50 GO 40 20 2 3 4 5 6 Weeks Fig III 12 The effect of feeding L-thyroxine on the alkaline phosphatase content of the milk of the cow and on the ratio of the phosphorylated to frec aneurin (from Bailey Bartlett Folley Rowland and Thompson unpublished results cited by Folley 1951) quantity and therefore cheap, possesses certain disadvantages in practice Thus, it must be standardized by biological methods often a troublesome and uncertain procedure, and some preparations possess a taste and smell which cause a proportion of cows to refuse it Moreover its administration entails a considerable intake of non thyroxine iodine which may cause untoward effects It had come into wide use mainly because synthetic thyroxine was until recently scarce and expensive and, moreover, believed to be relatively inactive by the oral route in cows A few years ago, however Chalmers, Dickson, Ells and Hems (1949) described a new and improved method ·GALACTOPOIESIS 69 inactivated in the rumen than thyroxine The first possibility was studied in an experiment in which the galactopoietic effects of equal doses, 5 mg daily, of the two substances given Beg ant g of heatment End of treats ont 3 2 0 162 S 4 6 7 8 5 Weeks {6 mg Tri dothy on 32 64 75 mg L-thy oxic per day - o o . Fig III 14 Effect of feeding L truodothyron ine on the milk yield of groups of cows the curves represent changes in the mill yield in kg/cow/day (from Bartlett Burt Folley and Rou land 1953) by subcutaneous injection were compared in lactating cows (Bartlett et al 1953) By the subcutaneous route 3 5 3 truodo L thyronine proved to be somewhat more active than thyroxine by all criteria studied – the increase in mulk yield and in the percentages of milk fat and non fatty solids the decrease in the mill alkaline phosphatase and the increase in the heart rate - though at the most its potency was no more than twice that of thyroxine To illustrate these results I have this time selected the effects on the mill phosphatase content which are shown in Figure III 15 The curves here represent the percentage changes in the milk phosphatase due to the treatment and you will note that 5 mg 3 5 3 triodo L thyronine subcutaneously daily caused a slightly larger de ·68 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION eliminated, L thyroxine is to be preferred to 1odoca ein for all purposes for which the latter has been or is intended to be used, in fact iodocasein can now be regarded as of acadenuu interest only It is worth noting that reassuring results were obtained by Leech and Bailey (1953) in a large scale expen ment in which the effects on the health and reproductie abilities of cows, of administration of L thyroxine in three suc cessive lactations, were studied Recently Roche, Lissitzkv and Michel (1952) and Gross and Patt Rivers (1953a) have independently isolated a biologically active material from the thyroid gland which has been identi fied as 3 5 3 truodo L thyronine This substance which differs from thyroxine in chemical constitution only in that its molecule contains one less atom of iodine, has been reported to be 5-7 times more active than thyroxine in various biologi, cal tests in small animals and also in humans (Gross and Pitt Rivers 1953 b, Tomich and Woollett, 1953, Lerman, 1953) Since 3 5 3 truodo L thyronine can be synthesized almost as readily us thyroxine it seemed to us important to test its activity by the galactopoietic response in cattle since of it proved several times more active in this respect than thyroxine the cost of the treatment might be considerably lowered However, in feeding experiments in lactating rows in which the effects of three doses of 3 5 3 triodo L thy ronine were compared with that of a dose of 75 mg synthetic Lrthyroxine daily we found that even the highest dose of the former 64 mg daily, had very little effect on mill secretion (Bartlett, Burt, Folley and Rowland 1959) This is evident from Figure III 14 which shows the mean mill yield responses of the various groups of cows used in this experiment Each curvc shows the increase in the milk yield of a group of cows over the mean valuc for the group before feeding began and is corrected for the change in milh yield undergone by a comparable group of untreated control cows The effects of the treatments on the milk phosphatase levels led to simular conclusions This suggested three alternative possibilitcs, (a) that 3 5 3 Inodo L-thyronine has relatively little inherent galactopoietic activity, (b) that it is not well absorbed from the digestive tract of the cow or (c) that it is more readily ·GALACTOPOIESIS 69 inactivated in the rumen than thyroxine The first possibility was studied in an expenment in which the galactopoietic effects of equal doses, 5 mg daily, of the two substances given Beant 9. of treatmet End of realne 3 2 162 4 5 7 8 O per day 0- Weeks 16 ms In odothyro SZ 64 75 mg L-thyro ine . Fig III 14 Effect of feeding L triiodothyron ine on the milk yield of groups of cows the curves represent changes in the mill yield in kg/cow/day (from Bartlett Burt Folley and Rouland 1953) by subcutaneous injection were compared in lactating cows (Bartlett et al 1953) By the subcutaneous route 3 5.3 truodo L thyronine proved to be somewhat more active than thyroxine by all criteria studied - the increase in mull yield and in the percentages of milk fat and non fatty solids' the decrease in the milk alkaline phosphatase and the increase in though at the most its potency was no more than twice that of thyroxine To illustrate these results I have this time selected the effects on the milk phosphatase content which are shown in Figure III 15 The curves here represent the percentage changes in the mill phosphatase due to the treatment and you will note that 5 mg 3 5 3 triodo L thyronine subcutaneously daily caused a slightly larger de the heart rate -- ·70 PHYSLOLOGY AND BIOCHEMISTRI OF LACTATION crease in the enzyme concentrations than the same amount of L thyroxine However, in view of the relaun ely slight activity of 3 5 3 truodo L thyronine by the oral route in cous (the only useful one for the farmer), which we believe to be due to its rapid inactivation by rumen microorganisms, thus substance clearly has no practical possibilities for use in cattle 40 Omo 5 mg L thyrus ante per day a no 5mg truodothyronine prasy 30 20 +10 DO Beginning sf syections 20 30 -404 -501 End of ang bons bu! 70) Weeks Fig III 15 Percentage change in milk phosphatase in the cow after subcutaneous injections of L-truodo thyronine (from Bartiat Burt Folley and Rowland 1953) REFERENCES Asimov G J and house, N. (1937) 3 Dairy Sa 20 -83 Bailey GL Bartlett S and Folley SJ (1947) Aature Lond 163 800 Bailey GL Bartlett Folley SJ Rowland SJ and Thompson Si unpublished routs ated by Folles S (19.1) Collog In CNR SYXUI 1950 p 81 Balmain JH and Folley S I (1952) Arch Brochem Brophis 39 188 Bartlets S Bun AW A. Folley SJ and Rowland S S (1954) 3 Endocru 10 293 ·GILACTOPOIESIS 71 Bergman A and Turner CW (1940) 7 Dairy Sc 23 1229 Daxter h La (1952) Islam and Horm 10 217 Bradley TR Folley Sj Landgrebe F Wand Mitchcu G V (1954) Beach Brooks Acta 33 49 Chalmers J R Didison CT Elks J and Homs B A (1949) J chart Sor P 3424 Chanda R Vaaught, I L and Onen EC (1952) Biochem 57 543 Chanda kasd Ouen EC (1951) Brochum 50 100 Cotes P y Crichton J A Folley SJ and loung F G (1949) Tature Land 164 992 Cotes P Aad E and Young F G (1949) Tature Lond 164 209 Cowie IT (1952) Endocrinolog) 5' 317 Cowie A T and Folley SJ (1947) } Endornan 5 24 Cowre IT and Folley SJ (1948) Endoctir 5 282 Come A T and Tindat ) S (19551 Endocrinolog> 56 619 Deschio L (1949) CR Soc Biol Park 143 1156 Flux DS (1955) Endocrin 12 57 Flux DS Folley SJ and Ronland SJ (1954) 3 Endocrin x0 333 Folley SJ (1947) Bret med Bull S 142 Folleys (1949) Bic Reo 24 316 Follcy s j (1955) in R W Smith O H Gaebler and CNH Long The Hypophyseal Croush Hormone hature and Actions le SorX Balision Chap 27 Folley Sj and Greenbaum & L (1947) Brachem 3 45 261 Folley s j and Greenbaum AL (1948: Biochem 7 43 går Folley SJ and Watson S C (1951) Proc Soc exp Biał VT 78 473 Falley s 3 and White P (1936) Preemos Soc B 120 346 Polley, s } and loung F G (1938) Proc ray Soc B 126 45 Folley s and Young F G (1939) Brochem 3 33 192 Folley s ) and yours FC (1940) Endocrin 2 26 Folley SJ and Young F G (1941) Lancet 240 380 Fraenkel Consat H Simpson M E and Exatus HN (1943) I book Chem 147 99 Gaun R. Excrsole and heada!! EC (1942) Endocrinology 37 84 Graham IV R r (19349) Sutt 7407 Graham WR Ir (1934)) Brochem 3 28 7368 Graham M R Houchin O B and Turner CW (5937) 7 brol Chem $20 29 Gregoire C (1947) 7 Endocrix 5 68 Cross J and Pitt Rivers R (19530) Biochem ) 53 645 Gross ) and Pitt Rises R (19536) Brochen 53 652 Hertoghe E (1896) Bull Acad Aled Belg (41m SETLE) 10 387 Lecch" F B and Barley GL (1953) agu SCT 43 238 Lerman J (1953) J das Endeanol felad 13 5341 Vagareda C S and Gaunt R. (1948) Anat Rec 10 723 Roche } Giraud Lelong Leardet ) and Cosgoes J (1950) Bull Acad tied Paris 734 190 Roche 3 Lassitzky S and Viche R (1952) C & Acad Sa Panas 234 997 1228

·72 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATIOV
Romani J D Plocq G and Recht P (1949) Rev Sa Med Paris
2 16
Roy A (1947) PhD Thesis University of London
Selye H (1934) Amer 7 Physiol 107 535
Tomich E G and Woollett E A (1953) Lancet 364, 726
Williams WL (1945) Anal Rec 93 171
Young F G (1947) Brit med Bull 5 155

·CHAPTER IV THE PHYSIOLOGY OF SUCHLING AND MIILLING THE MILK EJECTIOV REFLEX It has long been known that the withdrawal of milk from the mammary gland whether by the suckling young, the hand mulher or the milling machine, involves the active participa tion rather than the passive acquiescence of the lactating animal In speaking of the active participation of the mother I do not mean her behaviour pattern in promoting access of the young to her nipples which we may call nursing be haviour * I use the word participation in a limited sense to mean the role of the lactating animal in promoting the flow of milk from the mammary tissues a role which is quite unconscious The greater part of the milk present in the fully charged mammary gland, which in essence comprises the contents of the alveolt and finest ducts, only becomes available for transfer through the teat to the exterior of the gland if forcibly expressed from the alveoli into the larger ducts by the reflex contraction of effector contractile cells forming a network over the stromal surface of the alveoh This reflex the existence of which has been tacitly recognised for some time (see Folley 1947 for review), manifests itself by a sudden rise in the milk pressure in the mammary gland in response to the stimulation of sensory nerve endings in the teat that is the application of the suckling or milling stimulus This sudden reflex rise in intra mammary pressure is illustrated in Figure IV 1 The figure taken from a paper by Tgetgel (1926) shows the curve of increase of milk pressure in the udder cistern of a cow * Cowe Folley Cross Harris Jacobsohn and Richardson (1951) have proposed a system of terminology for use in lactational physiology this terminology us adopted in this chapter (see also Cowie Folley and Richardson 1954) ·74 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION between one mbling and the next We see how two successite applications of the milking stimulus at times marked by the arrows, caused sudden temporary increases in the pressure of milk in the gland cistern which soon subsided even though no milk was withdrawn The milk ejection reflex is the means by 20 Milk pressure (mm Hg) z 4 8 10 12 6 Hours after milking Fig IV 1 Vulk pressure cunc between one milling and the next (cow) reaction to the milking stimulus (from Tgets el 1926) which the active but unconscious participation of the lactating mother in the act of nursing, essential of the full yield of available milk is to be obtained, comes into play Plate IV 2 shows a famous painting by the Venetian master, Tintoretto It is called The Ongin of the Milky Way* and hangs in the National Gallery in London It seems to have been inspired by a classical myth, one sersion of which tells how Jupiter descended from Olympus to pluck the infant Hercules from the breast of Juno The picture is of interest in the present connection because it illustrates some significant points about the essential nature of the milk-ejecuon reflex It is as if Tintoretto, if not the ancients intuiusely knew that the suckling sumulus causes some response in thc lactating breast ·SUCKLING AND MILKING 75 which results in a tendency for its contents to be forcibly expelled Another important point illustrated by the painting is that the response to suckling is systemic rather than local, for the baby suckles one breast but milk is ejected from both (see Newton and Newton, 1948) In this connection we may note that it is the exception rather than the rule for milk to spurt from the nipple when the mill ejection reflex operates unless the resistance of the teat sphincter is overcome by the mouth of the suchling or the action of the teat cup of the milk ing machine The importance of this reflex for the total phenomenon of lactation is obvious If it fails to occur the end result will be the same as if secretion itself had ceased It is for this reason that it is difficult to interpret many experiments in which the effects upon lactation of the section of afferent nerves from the mam mary gland have been studied The rather elegant experiments of Ingelbrecht (1935) have often been cited He denervated the abdominal mammae of lactating rats and showed that the young died from lach of milk if they were prevented from such ling the thoracic glands while being allowed access to the ab dominal nipples If however suckling of even two of the tho racic glands was permitted then the young could obtain milk from the abdominal glands and the whole litter thrived Now there is evidence that the suchling stimulus not only evokes the mill ejection reflex but also reflexly excites the secretion by the anterior pituitary of lactogenic hormones which main tain the mammary tissues in a secretory condition It is thus not clear whether Ingelbrecht was concerned with a failure of milh secretion or with a bloch in the mill expulsion mechanism or both Partial failure of the reflex because of faulty prepara tion for milkıng may be of practical importance for if habitual it could result not only in serious malnutrition of the sucklings but also in shortening of the lactation period resulting from acceleration of involution of the mammary alveolar tissue because abnormal amounts of secretion are retained In this connection it may be noted that milling does not completely empty the alveoli of secretion as evidenced by the fact now Well known that injection of posterior pituitary extract immediately after milling enables one to obtain some more milk especially rich in fat ·76 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION Like other reflexes the milk ejection reflex can be condi tioned It is a matter of common observation that in the cow, it easily becomes conditioned to a varicty of auditory and visual stimuli, associated with milking shed routine, such as the rattling of milk buckets, washing the udder, etc Waller (1938) in his well known book, relates examples of condition ing in lactating women One woman was accustomed to drink a glass of water before putting her baby to the breast, soon the more act of filling the glass was sufficient to evoke the reflex Like other conditioned reflexes it can be inhibited by disagrecable, emotionally disturbing stimuli as has been shown experimentally by Ely and Petersen (1941) and Whittleston (1951) in the cow, Newton and Newton (1948) in woman and Cross (1955) in the rabbit It seems likely that this inhibition is central in most instances though in others it may be mainly peripheral (see Cross 19556) The probability that activation of the sympathetico adrenal system may often be responsible for the inhibition is indicated by the fact that as shown by Ely and Petersen (19.41), Cross (1953, 1955a) and others, adrenaline will block the normal reflex As we shall see later, the milk ejection response can be evoked by certain experimental procedures such as the injection of oxytocin and electrical stimulation of the supraoptico hypophy sial tract Adrenaline injection will interfere with these respon ses also For instance, Braude and Mitchell (1952) have shown that the milk ejection response to oxytocin in the lactating sow, which some claim to be especially clear cut, can be almost completely blocked by the prior injection of adrenaline In the rabbit, Cross (1953, 1955a) has shown that adrenalınc will block the mill ejection response both to oxyto cin and to electrical stimulation of the supraopticohypophysial tract These observations perhaps suggest that the inhibitory effect of adrenaline is peripheral, that is, that it abolishes the effect of the mill ejection hormone in the mammary gland itself In fact, Cross (19552) believes that the inhibitory cffcct of central stimulation on the milk ejection reflex may depend on the constnction of mammary blood vessels, thus impeding access of oxytocin to the ussues The fact that Linzell (1955) found that adrenaline did not abolish the alveolar contracting effect of topically applied oxytocin on the mammary gland

·PLATE IV 2
Repre luce i bl courtesy of the
Trusteer The National Gallery Loun

·PLATE IV 3
Milk gushing from the udder of a cow whose teats have been cannulated
after an intravenous injection of 180 1u oxytocin (from Usuell and
Piana 1952)

·SUCKLING AND MILKING 77 of the hving mouse is in accord with thus Cross (1953 19534) further showed that endogenous adrenaline released in response to electrical stimulation of sympathetic centres in the posterior hypothalamus also inhibited the milk ejection respon se to oxytocin In view of all this endence we may conclude that activation of the sympathetico adrenal system can inhibit the milk ejecuon reflex and that this inhibition is probably though not necessarily exclusi ely peripheral This mechanism is almost certainly nat molsed in all cases of inhubution since the latter may be snduced by stimuls such as embarrassment (Newton and Newton, 1948) which probably do not acuvate the sympathetico adrenal system The experiments of Cross (1953b) on the rabbit led him to conclude that the main factor in the inhibruon due to emotional disturbance is interference with the release of oxytocin by the posterior pituitary EVIDENCE FOR THE NEUROENDOCRINE REGULATION OF ALL EJECTION Let us now proceed to consider evidence that the mall ejection reflex is neuroendocrine in nature The mulk ejection reflex was once believed to consist of a purely neural arc Thus belief is however no longer held there being abundant evidence as will be shown later that the arc is a neurohormonal one The efferent component of this arc is the release from the neurohypophysis into the blood stream of a factor which as we shall see later is probably oxytocin and such as behered to evoke the contraction of the aforementioned effecior tissue associated anatomically with the mammary ah eoli lo single piece of evidence for this theory is conclusive by itself but there is now such a coherent body of concurrent and harmonious findings from various angles that the neuroendocrine theory of milk ejection can be accepted as in principle well established Nevertheless details of the afferent pathways and the precise chemical nature of the hormonal component and the idenuty of its target tissue in the mamma remain to be further investigated It has been known for over forty years ever since the pioneer work of Oct and Scott (1951) followed a little later by that of Schafer (1913) that the milk ejection reflex can be mimicked by the injection of posterior pitutary es tract Plate IV :

·78 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION from Usuelli and Piana (1952) shows how an injection of oxytocin into a lactating cow whose teats have been cannula ted causes the mill to gush forth It is forty years since Gaines (1915) described interesting experiments which today would unhesitatingly be interpreted as providing cogent evidence for a physiological role for the neurohypophysis in mill ejection Among other interesting findings, he showed that in the bitch the reflex could be inhibited by ether anaesthesia and the inhibition overcome by the injection of posterior pituitary extract These facts are illustrated in Figure IV 4 taken from Gaines (1915) which shows the milk flow curves 100 Normal 80 60 Anaesth fized with thr Milk (8) 40 A layechon of catract of posterior pituitary gland 20 6 10 12 14 16 16 Minutes Fig IV 4 Milk flow curses for a bitch nursing puppies (from Gaines 1915) for a lactating bitch obtained by weighing her puppies at intervals during suckling It will be seen that when the mother was anaesthetized the puppies could get milk at first but the flow soon stopped This represents the mill present in the larger sinuses which, like that contained in the cisterns of the bovine udder, can be drawn off without the intervention of the milk ejection reflex * If after the initial flow had ceased the bitch was given an intravenous injection of postenor pituitary extract the mill began to flow again and the puppies were able to empty her breasts Essentially simular findings in the rabbit were reported by Cross (19556) forty years later * 1e passive withdrawal (see Cowie et al 1951) ·SUCKLING AND MILKING 79 Despite his clear cut and significant results Gaines regarded the milk ejection reflex as a purely neural arc probably because hardly anything was known of neuroendocrine rela tionships at that ume so that there was no background of knowledge which would have led anyone to concerte a physiological role for the posterior lobe in this function The position remained substantially unchanged until about 1940 when Ely and Petersen (1941) performed experiments which prompted them to advance for the first time the theory that the suckling or milling stimulus causes reflex secretion by the posterior pituitary of a hormone believed by them to be oxytocin, which in turn evokes contraction of an effector tissue capable of expressing mll from the mammary aleoli Ely and Petersen (1941) showed inter alia that after section of two nerves believed to carry all the motor fibres to one half of the udder of a cow the milk ejection response could be induced by the milking stimulus or oxytocin injection in normal and denervated udder halves alike Moreover the reflex response to the milking stimulus could be blocked in both udder halves by adrenaline or by fright induced by sudden, loud noises Provided one can feel satisfied that the operative procedures had indeed cut all efferent fibres to the udder and none had regenerated at the time of the experiment, these findings argue against motor innervation of the effector contracule tissue of the udder and suggest that the terminal efferent link is hormonal in nature Later, more direct evidence that the milking stimulus evokes the liberation into the blood stream of a hormone which causes milk ejection was afforded by experiments in which it was shown that if blood taken from a cow immediately after the application of the milking stimulus is perfused through an isolated bovine udder with teats cannulated there is a more rapid and copious flow of milk than occurs when blood from a non stimulated cow is used Table IV i shows some results obtained by Peeters Massart and Coussens (1947) by means of their perfusion technique which permits the simultaneous but independent perfusion of the two separated halves of a bovine udder The milk flow from the udder half perfused with blood taken from a cow after she had been stimulated to eject her milk was in all cases greater than the flow from the ·80 PHYSIOLOGI AXD BIOCHEJISTRI OF LACTATION CON control udder-half perfused with blood from an unsumplated These results are essentially similar to thae earler reported by Petersen and Ludwich (1942) TABLE IV 1 Expulsion of mult from the time haltes of a toutil wudet perfeased respectirely with blood from the same cow before and after abplication of the milking stimulus (from Peeters Vassart and Coussens 1917) Amount of milk expelled from the udder tissue soon after circulation was established Experiment Left half perfused with Right half perfused with blood taken before milking blood taken after millor stimulus applied stupulus applied ml ml. no I 160 2 270 360 520 345 300 3 215 390 4 A number of experimenters have provided evidence that the mill ejection reflex invokes activation of the posterior lobe of the pituitary gland by demonstrating the occurrence of an antidiuretic effect of the posterior pituitary type shortly after the application of the suckling or milling stimulus to a water loaded lactating subject Such results have been obtained in the rabbit by Cross (1990), the bitch by Kalliala, Kanonen and Leppanen (1952), the cow by Peeters and Coussens (1990) and in woman by Ralliala and Kanonen (1951) Now much word, mainly biological but culminating with the chemical work of du Vigneaud who with his colleagues prepared two highly purified posterior lobe polypepudes which are biologically acuse (see du Vigneaud 1952) strongly suggests that the antidiuretic and vasopressor principles of the posterior lobe are identical Voreover, I coussens (1930) found that, in the cow mild inhabited the antıdıurcuc response ·SUCKLING AND MILLING 81 Sex without interfening with the milk ejection reflex Therefore, we may conclude that while the experiments we have been considering provide good evidence that the milking stimulus activates postenor pituitary functions it would seem improba ble that vasopressin is the natural mill-ejection hormone though, as weshall see, postenor pituitary vasopressor fractions undoubtedly possess some milk ejection activity We thought it might be interesting to approach the problem in another way It seemed possible that the milking stimulus might cause a measurable loss of oxytocic or vasopressor activity from the posterior lobe Accordingly Mrs M H I Dodd, in our laboratory, made careful measurements of the content of both activities in the posterior pituitary glands of goats some of which were autopsied just after milking while TABLE IV2 Amounts of oxytocin and rasopressin in posterior pituitaries of goals (unpublished work of Dodd cited by Folley 1952) No Age Vasopressin Oxytocin 1 u/mg dry tissue iu/mg dry tissue Non lactating goats 393 + 6 months 07-08 08 385 8 18 6 05 06 Lactating goats, unmilked for 24 hr before autopsy 316 q years 06 08 326 q 우 06 06 338 q 외 08 -07 61 08 08 Lactating goats, killed immediately after milking out 우 0 65 127 오 6 -06 165 5 08 08-10 291 3 06 08 232 4 08-10 4 06 IO 226 o 28(?) 07 of goat 18 342 2+ years 2 2 2 100 79 8 years 07 AIO 우오오오오오오 L 08 1 222 ·82 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION others had gone unmilked for some hours before autopsy (unpublished work by Dodd, 1951, cited by Folley, 1952) However no depletion of biological activity due to milling could be demonstrated As will be seen from the results given in Table IV 2 she found that the absolute amounts of oxytocic and vasopressor actrities present in the posterior lobes of goats shot immediately after normal milking were the same as those of goats unmilhed for 24 hr before autopsy Moreover, the values for a sma)] series of dry goats were indistinguishable from those of lactating animals In all cases save one the concentrations of both principles, measured in international units were cqual, as they are by definition in the international standard ox posterior pituitary powder Thus the milking stimulus caused no measurable alteration in the ratio of the concentrations of vasopressin and oxytocin in the posterior pituitary gland Similarly, Whittlestone, Bassett and Turner (1952) assaying posterior lobes from cons for mill-ejection activity by a special technique depending on the stimulation of milk flow in the lactating sow could find no unequivocal evidence of any appreciable decrease in this activity in the pars nervosa as a result of normal milking It may be that the amounts of posterior-lobe activities discharged in response to the milking stimulus are too small in relation to the amounts stored in the lobe to enable the difference to be detected by assay methods at present available In our laboratory, Dr A T Cowie has found that an intravenous injection of 1 iu oxytocin into an anaesthetized lactating goat will give a mulk ejection response approximating in magnitude and duration to that obtained during normal milling (unpublished work of Cowie 1951 cited by Folicy, 1952) His results are illustrated in Figure IV 5 which shows kymograph tracings of the risc in intra mammary pressure evoked by different in travenous doses of oxytocin into an anaesthetized goat with one teat cannulated and the cannula connected to a pressure recording device Goat posterior lobes contain 10-15 i u oxytocin (personal communication from Mrs M H I Dodd) so that the nulling stimulus may cause only a discharge of 10% or less of the amount stored in the pars nervosa The discharge may be much less cyen than tlus according to results recently reported by Dcnamur and Martinet (1953) ·Con 2 w Pitesin Los Pitesin 3 #CONSINTIAVAL on H 4. so 14.6. Pecin Stenu Pitocin MITTIT Fig IV 5 Pressure changes in the cistern of the mammary gland of a goat after intravenous injection of oxytocin (from Coure unpublished results cited by Folley 1952) 83 ·84 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION These authors obtained good milk ejection responses in anaesthetized goats receiving i mu oxytocin intravenously Maximal responses were given by io mu and, in some anı mals, just detectable effects with only o 1 mul The latest and most convincing evidence of the intervention of a hormone of the neurohypophysis in the milk ejection reflex comes from experiments involving electrical stimulation of the hypothalamus In the anaesthetized rabbit Cross and Harris (1951, 1952) have shown that clectrical stimulation of the supraoptico hypophysial tract caused ejection of mill from a cannulated teat duct Effects of similar magnitude, measured with a manometer recording on a lymograph, could be elicited by the intravenous injection of 200 mu posterior pituitary extract Rabbits in which electrolytic lesions had previously been placed in the supraoptico hypophysial tract, so that the neurohypophysis distal to the lesion had presumably degenerated, failed to exhibit the milk-ejection reflex in response to clectrical stimulation of the tract, nor could their litters obtain significant quantities of mulh in suchling tests The young could only withdraw mill from these animals if 30-200 mu posterior pituitary extract were intravenously injected immediately before suchling be gan In those cases where subsequent histological examination showed that the hypothalamıc lesions had missed the supra optico hypophysial tract, suchling by the young or electrical stimulation of the tract elicited a normal mill ejection response The long latent period, which outlasts the stimulus and the persistence of the effects aſter the stimulus has ceased are good evidence that Cross and Harris were dealing with a neuroendocnne mechanism involving the neurohypophysis Similarly, Andersson (19512) has evolcd mulk ejection responses in unanaesthetized lactating ewes and goats by electrical stimulation of hypothalamic centres in or adjacent to the supraoptic nucler Andersson concluded that these responses were undoubtedly hormonally mediated because they could be evoked dunng sacral anaesthesia or in denerv ated udder halves Moreover, he showed that blood taken from a goat immediately after clectrical stimulation evoked a prompt flow of milk from the cannulated tcats of another lactating animal on intravenous injection This indicates that ·M EX Ch opt Hip PLATE IV 6 ray photograph of the head of a boat showing electrodus implanted in the hypothalamus (photo graph by courtesy of Dr B Andursson) SY lam ·84 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION These authors obtained good milk ejection responses in anaesthetized goats receiving 1 mu oxytocin intravenously Mfaxımal responses were given by 10 mu and, in some ant mals just detectable effects with only o I mu' The latest and most convincing evidence of the intervention of a hormone of the neurohypophysis in the milk ejection reflex comes from experiments involving electrical stimulation of the hypothalamus In the anaesthetized rabbit Cross and Harris (1951 1952) have shown that electrical stimulation of the supraoptico hypophysial tract caused ejection of milk from a cannulated teat duct Effects of similar magnitude, measured with a manometer recording on a lymograph, could be elicited by the intravenous injection of 200 mu posterior pituitary extract Rabbits in which electrolytic lesions had previously been placed in the supraopticohypophysial tract, so that the neurohypophysis distal to the lesion had presumably degenerated, failed to exhibit the milk-ejection reflex in response to electrical stimulation of the tract, nor could their litters obtain significant quantities of milk in suckling tests The young could only withdraw mill from these animals af 30–200 mu posterior pituitary extract were intravenously injected immediately before suchling be gan In those cases where subsequent histological examination showed that the hypothalamic lesions had missed the supra optico hypophysial tract, suchling by the young or electrcal stimulation of the tract elicited a normal milk ejection response The long latent period, which outlasts the stimulus and the persistence of the effects after the stimulus has ceased are good evidence that Cross and Harris were dealing with a neuroendocrine mechanism involving the neurohypophysis Similarly, Andersson (19512) has evoked milk ejecuon responses in unanaesthetized lactating ewes and goats by electrical stimulation of hypothalamic centres in or adjacent to the supraoptic nuclei Andersson concluded that these responses were undoubtedly hormonally mediated because they could be evoked during sacral anaesthesia or in denery. ated udder halves Moreover, he showed that blood taken from a goat immediately after electncal stimulation evoked a prompt flow of milk from the cannulated teats of another lactating animal on intravenous injection This indica tes that ·SUCKLING AND MILLING 85 the sumulation had caused the discharge of a milk-ejection hormone into the blood strcam Andersson s cxperiments were made by the technique of Hess and Plate IV 6 shows an ray picture of the electrodes in position in the brain of one of his goats Expenments on section of the pituitary stall have not con tnbuted much to our knowledge of the mill ejection reflex In cases where stalk scctioned animals were found to rear their young it is possible that the milk ejection hormone came from that part of the neurohypophysis proximal to the point of section This might explain why the partunent neurohypo physectomized animals, described many years ago by Smith (1932) and by Houssay (1935), reared their young However in our laboratory, Dr A T Cowic has found that posterior lobectomized rats possessing functional antenor lobe tissue so that secretion was maintained would not rear their young unless milk cjection was induced two or three times daily 34 30 26 Conhyl Ilters Mean weight of young (8) Fosterior ptuitary Temare Oxytocin stopped 2 4 20 6 10 12 14 16 16 Days after parturition Fig IV 7 Growth curve of the latter of a rat neurohypophysectomized (posterior lobectomy) on the 4th day of lac tauon and injected three times daily with oxytocin (from Cour unpublushed results aited by Folley 1952) ·PLATE IV 8 200 Thick section (75 ) of part of a lobulu (rom a goat mammary gland fixed when distended showing the myocpithelial cells stained with silver (from Richardson 1949) ·SUCKLING AND MILLING 87 can be accounted for by its contamination with oxytocin, stated by the manufacturers (Parle, Davis & Co) to amount to no more than 5% Turner and Cooper (1941) suggested as alternative explana- Lions of their results either that milk ejection activity is a property both of oxytocin and vasopressin or else that the milk ejection hormone is a distinct and separate postenor lobe principle Andersson (19910) however, attempted to explain the discrepancy by assuming that a small amount of oxytocic activity is inherent in vasopressin itself and that oxytocic and inill ejection activities are related biological properties of the same posterior pituitary hormone He sugges ted that if after an injection of oxytocin or vasopressin equal amounts of either disappear from the blood in a given time the proportional destruction of the small amount of milk ejection activity associated with a relatively large amount of vasopressin would be less than of that associated with a relatively small amount of oxytocin According to this view the discrepancy arises from the fact that oxytocic activity is usually measured in vitro (by its effect in eliciting contractions of strips of uterus) while milk ejection activity is measured in vizo by a systemic response The results of Whittlestone (1952) who found that various oxytocic preparations possess mill ejection activity in proportion to their oxytocic activities while vasopressin preparations including du Vigneaud s highly purified vasopressin polypeptide exhibited more mull ejection activity than could be accounted for by their oxytocic activities, suggest that some milk ejection activity is inherent in the vasopressin molecule A similar conclusion follows from the work of Cross and van Dyke (1953) who studied the milk ejection activities of du Vigneaud s highly punfied oxytocic and vasopressor polypeptides in the rabbit I suggest that we need not attach undue importance to the discrepancy between the mill ejection and oxytocic activities of asopressin preparations for the following reason Coon (1939) has described a method for the assay of oxytocic acavity depending on the fall in blood pressure of the anaes thetised fowl When postenor pituitary extracts in which vasopressin predominates are assayed by this method the oxytocic activities found are greater than are those given by I ·86 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION by the injection of oxytocin thus allowing the young regular suckling periods (unpublished work of Cowe, 1951, cited by Folley, 1952) Figure IV 7 illustrates his results The black points represent the growth curve of the young of a group of control rats, the white circles the growth curve of the litter of a rat neurohypophysectomized on the fourth day of lacta tion and thereafter given oxytocin three times daily The vertical lines represent the amounts of milk obtained by the litter each day following the first injection of oxytocin into the mother It will be seen that the young of the neurohypophy sectomized rat grew at approximately the normal rate A significant point to be noted is the temporary, but immediate, cessation of growth of the young when oxytocin was withheld from the mother on the gth day The elegant experiments of Harris and Jacobsohn (1952) are also illuminating in connec tion with the effects of severance of the pituitary stalk upon milk ejection They showed that the mammae of hypophy sectomized rats possessing functional anterior pituitary grafts, that is grafts in which the hypophysıal portal circulation was re established, contained milk but the animals would only rear their young if regularly injected with posterior pituitary extract It is evident from what has been said that the evidence for the neuroendocrine regulation of milk ejection by a pathway involving activation of the posterior pituitary is now impressive in amount and in sum convincing NATURE OF THE MILK EJECTION HORMONE Let us now consider the nature of the milk ejection hor monc It has uniformly been found that while both the oxytocic and vasopressor fractions from posterior lobc cxtracts possess mill cjection activity, the oxytocic fraction is much more active in this respect than the pressor fraction This has given rise to the belief, now held by most workers in the field that the oxytocic principle is the natural mulk cjection hormone However, the actvity of the vasopressor preparation, Patressin has bcen found by Tumer and Cooper (1911), Cross and Har ns (1952) and Andersson (19510) to be about 20 % of that of the oxytocic preparation, Pitocin, which is much greater than

·PLATE IV 9
n
sou
Photograph of part of the surface of a small contracted alveolus (goat)
showing a myoepsthelial cell with nucleus (n) and branching processes
(from Richardson 1949)

·88 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION the conventional method depending on the contraction of uterine muscle in ztro Thus du Vigneaud (1952) has reported that the oxytocic activity of his purified vasopressor polypepti de came out three times larger by the chicken blood pressure method than by the uterine muscle assay Coon thought it possible that when present in excess, vasopressin may inter fere with the uterine muscle response in vitro so that oxytocic activities are underestimated by the in zıtro technique on the other hand the results of Strahan and Wanng (1954) indicate that the chicken blood pressure technique may over estimate oxytocic activity in some circumstances The point is that available methods for oxytocin assay scem to be subject to uncertainties at the present time In this connection assay values for highly purified oxytocin and ADH vasopressin polypeptides given in their Table I by van Dyke, Adamsons and Engel (1955) are of interest, for in the case of ADH vasopressin, assays based on the mill ejection response in the rabbit and the depression of the blood pressure in the fowl were in substantial agreement while a much lower value was given by the rat uterus method, with oxytocin, however identical values were given by all three methods The most reasonable explanation of the available evidence seems to be that milk ejection and oxytocic activities are facets of the same biological acuvity characteristic of the oxytocic polypeptide and that both activities are possessed in some small degree by the vasopressor polypeptide also It is un likely that vasopressin is the natural mulk ejection hormone because Peeters and Coussens (1950) in the cow and Cross (1950) in the rabbit found that the antidiuretic response in lactating animals could be elicited by the injection of amounts of posterior pituitary extract too small to cause milk ejection Moreover, as previously pointed out mild fright will inhabit suckling antidiuresis in lactaung cows without blocking the mill ejection reflex (Peeters and Coussens 1950) Finally in the present connection it is worth noting that du Vigneaud, Ressler, Swan, Roberts Katsoyannis and Gordon (1953) have described the synthesis of a polypepude possessing oxytocic activity and also the power to evoke mulk ejection

·PA 11 )
n
30j
Photograph of part of the surface of a small contracted alveolus (goat)
showing a myoepithelial cell with nucleus (n) and branching processes
(from Richardson 1949)

·PLATE IV 10 Thm section from thu udder of a goat showing the processes of the myoepithelial cells cut transversely and appearing in the form of oval or triangular bodies (cm) from Richardson 1949) ·SUCKLING AND MILKING 89 THE EFFECTOR CONTRACTILE TISSUE OF THE MAMMARY GLAND In conclusion, we will consider briefly the nature of the effector contractile tissue of the mammary gland This aspect of the milk ejection reflex has been considerably clanfied as a result of recent work Many authors had until even quite recently regarded smooth muscle tissue as the contractile agent, which they had supposed, on rather inadequate evi dence, to occur in the mammary gland in close association with the mammary alveoli in sufficient abundance to play an effective physiological role in squeezing milk from the alveolar tissues Seven years ago, Richardson (1949), in a careful histological study of the mamma of the goat found, however that quantitatively there is not nearly enough smooth muscle tissue surrounding the lobules to be effective in causing contraction of the alveoli individually or of the lobules as a whole Earlier histologists had figured myoepithelial cells or basket cells in close association with the mammary alveoli, as cells of ectodermal origin which by analogy with smooth muscle cells were assumed to be contractile However there was subsequently some reluctance to accept these cells as the effector contractile tissue because until Richardson (1949a) had devised a satisfactory technique for visualısıng them by their morphological characteristics quantitative distribution and concentration had been vague Richardson s studies, first on the goat (1949a) and later on the human breast (1951) have however shown conclusively that moepithelial cells are present throughout the mamma in the closest association with the duct and alveolar epithelia They lie on the epithelial side of the basement membrane and their branching processes form a network over the stromal alıeolar surface - hence the appositeness of the name basket cells Plate IV 8 for which, like the remainder of the illustrations in this chapter, I am indebted to Mr KC Richardson shows a thick section (100u) of alveolar tissue from a goat udder Thick sections are best for demonstrating myoepithel rum because fields can be chosen in which portions of the alveolar walls are shown in surface view In this figure the branching processes of the myoepithelial cells blackened with silver are clearly visible Plate IV 9 shows part of the silver impregnation ·go PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION surface of one alveolus at a higher magnification and gives an excellent impression of the myoepithelial network with the nucleus of one of the myoepithelial cells near the centre of the field It is cvident from these brilliant photomicrographs of Richardson that quantitatively these cells are sufficiently abundant - much more so than had formerly been realized – to qualify them for the collective role of effector organ in the mill ejection reflex The observations of Richardson on the occurrence of myoepithelium in the mammary gland of the goat and woman have ince been confirmed in the cat and other species by Linzell (1953) It seems probable that the smooth muscle" cells descnbed by Swanson and Turner (1941) fifteen years ago in thin sections of mammary alveolar tissue were in reality the myocpithelial cells of Richardson which, as has been pointed out, can best be studied in thich sections In thin sections it is only possible to see, as shown in Plate IV 10 the branching processes of the myoepithelial cells in cross section As regards the probable role of these cells in the mill ejection reflex, Richardson (1949a) has shown they change their configuration when the alveoli collapse as a result of milking This is illustrated in Plate IV 11 The two upper pictures show sections of alveolar tissue from the two halves of a goat udder the left hand one being from an udder half fixed in a condition of pre milking distension and the nght from an udder half fixed in a collapsed condition just after milking out The change in shape and orientation of the myoepithelial cells following milking is clearly shown in the corresponding photomicrographs set out beneath As a result of careful study of these morphological changes, Richardson (1949) concluded that the myoepithelial cells assume an onentation in relation to the folds of the alveolar epithelum in the post milking contracted 'gland which is consistent with the view that the wrinkling of the alveolar wall is due to their active contraction rather than with the idea that they passively conform to the contours of the al eoli as the latter collapse Myoepithelial cells also occur in abundance next to the strom al surface of the duct epithelium, in this case their contrac tion would tend to widen and shorten the ducts thus reducing the resistance to the passage of milk and facilitaung its escapc ·2504 <250 100 PLATE IV 11 Comparison between distended and contracted glands (goat) – Top left part of a lobule from the left half of an udder fixed while distended with milk Top right the right half of the udder from the same goat which was milked out as completely as possible before autopsy note the contracted lobules with collapsed alveoli and ducts lined with a thick folded epithelium Bottom lef Myoepithelium on the surface of distended alveoli section taken from the same left half as above Bottom right Myoepithelium on contracted alveoli section taken from the same right half as above (from Richardson 1949) ·PLATE IV 12 Longitudinal orientation of myoepithelium on an intra lobular duct (c) connected with some large clongated al coli (from Ruchurdson 1949) ·SUCKLING AND VILLING 91 from the alycolar tissue Plate IV 12 shows a thick section from a goat mammary gland which includes parts of the outer salt of a mil duct in surface VICH The longitudinal onenta uon of the silver blackened my oepithelial cells is clearly seen There can be little doubt that the effector contracule tissue of the mammary gland is the myocpithelium which must play the 174jor role in the active expulsion of milk from the alcolar tissue though there remains the possibility that vasomotor effects may also play some part The evidence though good is sull only circumstanual, however and the matter cannot be regarded as seuled beyond all possible doubt unisl it is possible to devise a technique for observing directly in living mammary tissue, the contraction of the myoepithelium experimentally saduced A step towards thus desirable end has recently beck made by Linzell (1955) who has observed under the microse pe the contraction of mammary alcol in the kving mouse in response to the local application of oxytocin and vasopressin preparations Richardson (19496) has pointed out that unul more is known of their pharmacological responses innervation and other properues it seems desirable to continue to regard the moepithelial cells and smooth muscle of the mammary gland as separate physiological entities REFERENCES Andersson B (1991a) Aria physiol scand 238 Andersson B (1958) sca physiol scand 23 24, Braude R and Michell 6 (1932) I Endocrin $ 238 Coor JM (1939) Arch Int Pharmacodyn 62 79 Cowse folley SJ Cross B A Harris Git Jacobsohn D and Richardson K. (1951) Actu z Lond' 168 421 Cowx AT Holley s and Richardson 50 (1944) Lancet 267, 601 Cross B A (1950) Aalust Lond 166 612 Cross B A (1953) 3 Endpa 9 7 Cross B A. (19554) Endoctin 12 15 Cross B A (1955.) 7 Endotra 12 29 Cross B A and Harris G W (195.) 7 Physiol 113 35P Cross B A. and Harrs G W (1952) Í Endocrin 8 148 Cros# B A and Dyke H. Byan (1953) ) Endocrin 9 332 Denamus R and Martinet (1953) C & Soc Brol Pans_247 1217 Dyke H B van Adamusons K and Engel S L (1955) Recent Progr Ely F and Petersen W E (1941) 3 Dary Sc 24 217 Folley S J (1917) Brit med Bull 5 142 Horusore Res II, 1 ·92 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION 75 85 Folley S J (1952) Recent Progi Hormone Res 7 107 Gaines W L (1915) Amer 7 Physiol 38 285 Harris G W and Jacobsohn D (1952) Proc roy Soc B 139 263 Houssay BA (1935) C R Soc Biol Paris 120 496 Ingelbrecht P (1935) C R Soc Biol Pans 120 1369 Kalliala H and Karvonen M J (1951) Ann lied Exp Fenn 29. 233 halliala H harvonen M J and Leppanen, V (1952) Ann Ved Exp Fenn 30 96 Lunzell J L (1952) 3 Anat, Lond 86 49 Linzell J L (1955) 7 Phystol 130 257 Newton M and Newton N R (1948) 1 Pedial 33 698 Ott 1 and Scott JC (1911) Proc Soc cap Biol Nr 8 48 Peeters G and Coussens R (1950) Arch Int Pharmacodyn 84 209 Peeters C Massart L and Coussens R (1947) Arch Int Pharmacodyn Petersen W E and Ludwick T M (1942) Fed Proc 1 66 Richardson AC (19499) Proc roy Soc B 136 30 Richardson AC (19496) Endocrin 6 xxv Richardson KC (1951) Collog Int CARS LXXII 1950 P 167 Schafer F A (1913) Quart I exp Physiol 6 17 Smith P E (1932) Amer Physiol 99 345 Strahan R and Waring H (1954) Austral 7 exp Blol med Sa 32 193 Swanson EW and Turner CW (1941) 7 Darry Sci 24 635 Tgetge) B (1926) Schue Arch Tierhalk 68 335 369 Turner CW and Cooper WD (1941) Endocrinolow 29 320 Usuell, F and Piana G (1952) Ru Zootec Firenze 25 69 107 140 Vigneaud V du (1952) 2èm Congr Int Brochim (4) P 20 Vigneaud V du Ressler C Swan J M Roberts C w katsoy annis PG and Gordon S (1953) 7 Amer chem Soc 75 4879 Waller H (1938) Clinical Studies in Lactation London Heinemann Whittleston WG (1951) NZ7 Sc Tech A 32 No 5 I Whittlestone WG (1952) 7 Endocrın 8 89 Whittlestone W G Bassett CG and Turner CW (1952) Dairy Sc 35 889 ·CHAPTER V THE BIOSYNTHESIS OF MILK FAT AND ITS HORMONAL CONTROL EARLIER THEORIES OF THE ORIGIN OF MILK FAT Of the three major milk constituents, casein fat and lactose most is so far known about the biosynthesis of milk fat and this chapter will be devoted to consideration of this topic The mammary gland is very active in the synthesis of fat, so much so that it is surprising that this tissue has until recently been comparatively neglected by brochemists interested in lipo genesis* Nevertheless recent studies of fat synthesis in the mammary gland have contributed materially to our know ledge of lipogenesis in general as I shall hope to show Let us begin by discussing the various views that have been held at different times about the nature of the precursors of milk fat which are brought to the mammary gland in the blood Many years ago Meigs Blatherwick and Cary (1919) advanced the theory that the precursor of milk fat was the phospholipid fraction of the blood lipids They pointed out that of this were true the lactating udder would take up more than enough phospholipid phosphorus to provide the inorganic phosphate of the milk and they believed that their experimen tal results constituted valid evidence for this theory since they found that the jugular blood of lactating cows contained more inorganic phosphate than the blood leaving the udder in the mammary vein However, subsequent investigations (Lintzel 1934 Graham Jones and Kay, 1936) in which the composition of arterial blood was compared with that of blood from the mammary vein of the lactating cow failed to provide evidence of any uptake of phospholipid phosphorus by the udder and moreover there was general agreement that arterial blood * For that matter the same remarks apply equally to the synthesis of protein and carbohydrate ·94 PHYSIOLOGI AND BIOCHEMISTRY OF LACTATION contained more inorganic phosphate than mammary venous blood The hypothesis of Meigs already thus contra indicated was finally disproved by Aten and Hevesy (1938), who in one of the earliest applications of radioactive phosphorus to bio chemical studies showed that the inorganic phosphate of the blood plasma was the direct precursor of the inorganic phos phate of the milk Micanwhile the c same arterio venous studies (Lintzel, 1934 Graham et al 1936, Maynard, McCay, Elli, Hodson and Davis, 1938) had shown that the only fraction of the blood lipids which undergoes significant diminution in passage through the lactating udder is the neutral fat fraction, that is the glycerides and possibly the cholesteryl esters, and attention accordingly became focussed on the blood glycerides as the precursors of the milk glycerides It is necessary at this juncture to point out that the glycende structure of milk fat exhibits certain characteristic features which distinguish it from fats found elsewhere in the animal body The milk glycerides particularly of herbivorous anı mals, contain considerable molar proportions of short chain saturated fatty acids, C.-C, whuch are not found in anımal depot fats It is clear that any acceptable theory of the bio synthesis of milk fat must account for the presence in milk fat of these short chain acids and not unnaturally there has been much speculation about their origin The theory that the milk fat arises directly from the blood glycerides, which has held the field until quite recently, was supported by two main lines of evidence On the one hand was the cvidence from arteno venous studies carned out on the udder of the lactating cow which has already been mentioncd All these studies showed that there is an appreciable uptake of Deutral blood fat by the lactating bovine udder and sonte authors, notably Shaw and Petersen (1938), havefurtherclaimed that the uptake of blood fat is more than enough to provide the fat of the milk These workers were thus lcd to postulate that some of the fatty acids of the blood glycerdes absorbed by the udder were parbally degraded by oxidative processes in the lactating gland, the resulung fatty acid fragments represenung the short chain acids of the milk glycerides This idea of the origin of the milk short chain fatty acids was in ·BIOSYNTHESIS OF MILK FAT 95 harmony with the theory previously advanced from quite a different viewpoint by Hilditch (1937) whose arguments were based on extensive and painstaking chemical investigation of the glyceride structure of mill fat (see also Achaya and Hill ditch, 1950) To discuss the outstanding work of Hilditch and his collaborators on the chemistry of mulk fat in sufficient detail to do justice to the way in which it has advanced this branch of chemistry is outside the scope of this chapter All that can be said in the limited space available is that these studies revealed the existence of interesting relationships between the proportion of the milk glycerides which contain only saturated fatty acids and the proportion of saturated fatty acids in the total acids relationships which were of a type characteristic of mull fats and animal depot fats alone among natural fats Briefly Hilditch and his colleagues found that wherever a short chain acyl residue occurred in a glyceride molecule from milk fat one would have expected to find an oleic residue had the glyceride been a component of a body fat These quantitative relationships led Hıldıtch to deduce that long chain fatty acids particularly oleic acid present in the blood glycerides are broken down in the udder by oxido reduction processes thus giving rise to the short chain acids of the mill fat and also to the unsaturated fatty acids of the Cio-Cle series containing a double bond in the same position as in oleic acid which occur in traces in cows milk Many observations in addition to the aforementioned arterio venous differences in blood neutral fat across the mam mary gland are in harmony with the theory of Hilditch As an example may be cited the decrease in the molar proportion of short chain acids in the milk fat, accompanied by a con comitant increase in the proportion of oleic acid caused by inanttion (Smith and Dastur, 1938) or by feeding cod liver oil (Hilditch and Thompson, 1936) However none of the evden ce that the short chain acids of the milk fat are formed by oxidauve degradation of long chain acids arising from the blood glycerides is conclusive, although there is no reason to challenge the conclusion which follows from the arterio venous studies that the lactating udder takes up appreciable quantities of neutral fat from the blood Presumably some portion at least of this blood fat gives rise to mill fat and it seems eminent ·96 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION ly possible that a proportion of the milk fatty acids, the magnu tude of which is at present unknown, enters the udder as such from the blood However, the related question whether oxidative chain shortening of acyl groups acquired in this way is an important element in the fat metabolism of the udder is much more debatable in the light of more recent evidence which has directed attention to the outstanding importance of synthetic processes in this gland In this connection the experiments of Appel Bohm, Keil and Schiller (1947) with synthetic fatty acids whose molecules contain odd numbers of carbon atoms are of interest When such unnatural acids were fed to lactating sheep no fatty acids of odd number chain length below C11 were found in the milk fat which suggests that chain shortening of natur ul fatty acids below the 10- carbon stage probably does not occur to a significant extent in the udder If so, then the short chain acıds of mill fat must mainly arise by some process other than by degradation of long chain acids SYNTHESIS OF FATTY ACIDS IN THE MAMMARY GLAND In the last few years attention has been focussed on the possibility that a large proportion of the mill fatty acids arc formed by synthesis from small molecules in the udder itself The possibility that the conversion of carbohydrate to fat can occur in the mammary gland was first suggested some cighteen years ago by Graham Houchin Peterson and Turner (1938) who studied the arterio venous differences in oxygen and carbon dioxide across the udder of the lactating goat and obtained values for the udder respiratory quotient (RO) which were greater than unity An R Q greater than unity is usually taken to imply the synthesis of oxygen poor sub stances such as fat from oxygen rich material such as carbo hydrate These results were later confirmed by Reincke, Stonecipher and Turner (1941) who morcover found that fasuing decreased the RQ of the goat udder below unity Since it was known from the previous work of Smith and Dastur (1938) on the lactating cow that fasting seriously depletes the milk fat of its short chain acids, Reinche et al deduced from their results that the short chain acids arc formed in the udder from carbohydrate ·BIOSYNTHESIS OF MILK FAT 97 In our laboratory we have shown that slices of mammary gland from lactating rats, mice guinea pigs and rabbits respire actively in the presence of glucose as sole substrate and exhibit an R Q greater than unity (Folley and French, 1949) Some of our mean values for the R Q of mammary slices from these species incubated in a medium containing glucose are shown in Table V : We interpreted these results as evidence that the mammary tissue of these species is capable of effecting net fat synthesis from carbohydrate in zitro The Table also RQ TABLE Vi Respiratory metabolism of lactating mammary gland slices in presence of glucose (03%) (from Folley and French 1949) -la Mouse 60 Rat 93 90 Rabbit 66 39 46 35 I 94 I 53 Guinea pig I 17 1 45 o 88 Sheep Goat Cow 0 86 o 80 shows that by contrast with our findings with mammary tissue from non ruminants we found that slices of lactating udder tissue from sheep goats and cows exhibited relatively little oxygen uptake in the presence of glucose alone and the RQ was below unity (Folley and French 1949 1950) This suggests that though carbohydrate might be a significant substrate for fat synthesis in the mammary gland of non, ruminants it probably plays very little part in the fatty acid metabolism of the udder of ruminants This conclusion of six years ago has been subsequently confirmed in our laboratory in experiments carried out in collaboration with Dr Judith H Balmain and Dr RF Glascock in which mammary gland slices were incubated with isotopically labelled substrates including glucose labelled uniformly with 140 Measurements ·98 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION of the isotope content of the mixed fatty acids isolated from the slices after incubation for three hours in a mixture of acetate and glucose each labelled with a different isotope (acetate 13C and SH, glucose WC) hare enabled us to draw conclusions about the relative contributions of glucose and acetate carbon to the fatty acids synthesized by the tissue during the experiment (Balmain, Folley and Glascock, 1954) A summary of our results relating to the utilization of glucose is given in Table V 2, which shows that whereas, in the presence of a mixture of acetate and glucose the latter contributes about 60 per cent of the carbon of the fatty acids synthesized by rat mammary slices, in the case of sheep udder slices the contribu tion of glucose is negligible amounting to no more than about TABLE V 2 Incorporation of glucose carbon into the fatly ands of lactating mammary gland slices incubated with CH13COONa + WC glucose (from Balmain Folley and Glascock, 1954) Glucose carbon incorporated into Mixed fatty acids of slices in 3 hr mg/100 mg per cent carbon of fatty acids synthesized during experiment coming Trom glucose 18 62 Rat Sheep 2 3 3 per cent The conclusion that carbohydrate is not an im portant source of fatty acids synthesized by the ruminant udder docs not necessarily conflict with the high R Q found for the udder of the ruminant in lio for clearly thc uulization of acetate for fat synthesis could give rise to a high R Q since acetate is just as much an oxygen rich' substance as carbohy drate The incorporation of glucose carbon into the fatty acids of mill fat has been studied in the lactating mammary gland of the rabbit in Lilo by Popják, Hunter and French (1953) they ·BIOSYNTHESIS OF MILK FAT 99 administered glucose uniformly labelled with "C to lactating rabbits and isolated radioactive fatty acids from their mill Measurements of the specific radioactivity of the volaule fraction of the mill fatty acids enabled them to conclude that about 25 per cent of the carbon of the short chain fatty acids arose from glucose dunng the six hours following the injection These findings are not in conflict with our experiments on rat mammary gland slices The concept that acetate is an important buildıng unit for fatty acid synthesis in the mammary gland has arisen only in recent years Though it appears self evident today it was not so eleven years ago when the idea first arose in our laboratory In order to preserve a degree of historical perspective it may be of interest to recall the three considerations which may be regarded as having given rise to this fruitful idea The first was the demonstration by Rıttenberg and Bloch (1945) and others made possible by the use of acetate labelled first with deuterium and later with 14C, that the liver will actively incorporate acetate into fatty acid chains The second was the accumula tion of evidence notably by the late Sir Joseph Barcroft and hus school (see Elsden and Phillipson, 1948) that in the paunch of ruminants microorganisms ferment dietary polysaccharides yielding volatile fatty acids of which the most important quantitatively appears to be acetate It is now known that much of the acetate so produced is absorbed into the blood stream giving rise to appreciable levels of acetate in the arterial blood so that acetate arising from digestion of food must be reckoned an important metabolite in the body of the ruminant Lastly there is the fact established by the chemical work of Hilditch and others that the milk fat of ruminants is distin guished even among that of herbivorous animals by a relati Vely high content of short chain acids This is shown in Table V 3 taken from Hilditch (1947), which gives the Reichert Meissl values for milk fats of vanous species The Reichert Meissl value is a measure of the steam volatile fatty acids butyric and caproic The last two of the above considera tions taken together suggested to the present author and his colleagues (see Malpress 1946) that the short chain acids of ruminant milk might be formed by successive condensation of acetate molecules and might therefore be regarded as ·100 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION TABLE V3 Short chain fatty acids (C,Co) in milk fat of various species (from Hilditch, 1947) Reichert Veissl value Ruminants Cow Buffalo Sheep Goat Camel 33-36 26-34 23-33 20-29 16 4 16 1 13 T 70 Non Ruminants Rabbit Donkey Horse Cat Mouse Man Pig Dog 44 29 14-34 17 I 2 intermediate stages in the synthesis of long chain acids by similar step wise condensation The first efforts to obtain evidence in favour of this hypo thesis of the origin of the short chain acids and thus to gain insight into the mechanism of the formation of milk fat, were not encouraging As mentioned previously fasting lowers the proportion of short chain acids in the mill fat of ruminants Dr F H Malpress and I therefore felt that the fasted goat might be a suitable preparation with which to demonstrate 1 relationship between the acetate of the blood and the short chain acıds of milk fat Accordingly, we administered sodium acetate by slow intravenous infusion to anaestheuzed fasting goats but were unable to detect any resulung increase in the depleted short chain acids of the milk fat (see Malpress, 1946) Similar negative results were reported by Mann and Shaw (1947) and by McClymont (1951) in cxperiments in whuch large quanutes of acetate were administered often over ·BIOSYNTHESIS OF VILL FAT IOI prolonged perods, to fasted lactating cous either intraven ously or by intraruminal infusion In view of the unequivocal evidence obtained by other techniques that the rumunant udder uses acetate for fatty acid synthesis, which will now be discussed, the failurc of this approach is difficult to understand and has never been satisfactonly cxplained The first positse evidence suggesting the utilization of acetate for fatty acid synthesis in the mammary gland came from respiratory measurements on mammary gland slices carned out in collaboration with the late Dr T H French in our laboratory In these experiments (Folley and French 1950), the results of which are summanzed in Table V 4 we found that lactating udder slices from sheep, goats and rows TABLE V 4 Respiratory metabolism of lactaling mammary gland slices in presence of acetate (o 02 11) (from Folley and French 1990) Qog RQ o 92 Rabbit Rat Sheep Goat Cow 68 5 3 6 1 61 44 o 82 I 17 1 17 I 12 that is from ruminants respired actively with an R. greater than unity when incubated with acetate as sole substrate and utilized considerable amounts of acetate On the other hand as the Table shows udder slices from non ruminants appeared to be more or less inert to acetate since with acetate as sole substrate the oxygen consumption was relatively low and the R O less than unity We interpreted these findings as evidence that ruminant udder tissue will effect net fat synthesis from acetate in vitro From these respiratory measurements a sharp difference seemed to emerge between the metabolism in vitro of mammary tissue from ruminants on the one hand and non ruminants on the other, the former could apparently synthe size fat from acetate but not glucose while the latter uulized ·102 PHYSIOLOGY AND BIOCHEVISTRY OF LACTATION glucose but not acetate (by itself) for lipogenesis The situation 1s, however, somewhat more complicated than this Bloch and Kramer (1948) found that glucose stimulates Lipogenesis from acetate in rat liver slices and in our laboratory we have demon strated a similar phenomenon in mammary ussue Thus an the presence of glucose, mammary gland slices from rats will uulize acetate the R Q in mixtures of acetate and glucose being at least as high as it is in presence of glucose alone Similarly in mixtures of acetate and glucose, sheep udder slices showa lugher R Q and acetate uptake than in presence of acetate alone (Folley and French, 1950) All the conclusions drawn from respiratory measuremetits, as just outlined, were later confirmed by experiments in which mammary gland slices were incubated with labelled sub strates and the mean isotope content of the mixed fatty acids, isolated from the slices at the end of the incubation, deter mined The results of our earlier isotope experiments (Bal main Folley and Glascoch 1952a) in which we uscd (carboxy 14C] acetate as labelled substrate are summarized in Table V 5 The results in this fable arc expres ed as pecific activa urs of the mixed fatty acids isolated from the slices as calcium TABLE V 5 Specific actilities (counts/min/mg C) of calcium salts of mixed fatty acids isolated from lactating mammary gland slices incubated with CH, "COONa or CH, HCOONa + glucose (results from Balmain, Folley and Glascoch, 19520) SHEEP RAT Gluca e Experiment No Experiment No Glucose + + 1 50 43 5,951 3,805 9 533 9,918 3 886 IR 2L 2R 3L 3,334 3,539 2,660 2 948 1,144 4,108 3 4 5 6 8 975 TO 749 50 58 40 3,532 3,235 1,069 8,068 742 3R ·BIOSYNTHESIS OF VILA FAT 103 salts after three hours incubation They show that rat mam mary gland incorporated hardly any acetate carbon into the fatty acids when incubated with acetate as sole substrate but there was considerable incorporation in presence of glucose Udder slices from lactating sheep on the other hand incorporated considerable amounts of acetate carbon into the fatty acids when incubated with acetate alone but here again the incorporation was greatly increased when glucose was present The mechanism of the stimulatory effect of glucose upon the utilization of acetate for lipogenesis in iito is not clear even yet It has been suggested that glucose provides a source of the energy which is necessary for lipogenesis to proceed but this would hardly apply to sheep mammary tissue which unlıke rat mammary tissue can obtain energy from the oxidation of acetate The question of the relative values of acetate and glucose as sources of carbon for lipogenesis in the mammary gland is of interest particularly in view of the species differences in mar mary metabolism already touched upon In our subsequent experiments therefore we have studied this question by in cubaung mammary slices in mixtures of acetate and glucose each labelled with different isotopes (Balmain Folley and Glascoch, 1934) The glucose was uniformly labelled with 'C and the acetate was labelled in the carboxyl group with C and in the methyl group with radioactive hydrogen (tritium) Our results are summansed in Table V6 buch shows the abundance of each isotope in the fatty acids isolated from the tissue These experiments have shown that rat mammary slices incorporate into the fatty acids in three hours about eight umes as much glucose carbon as do sheep udder slices on the other hand sheep udder slices incorporate about six times as much of either carbon atom of acetate into the fatty acids as do rat mammary gland slices Since the mammary slices from both rat and sheep had substantially the same fat content and the abundances of the isotopes in the substrates are known, it is possible from these results to calcu late the relative contrbutions of acetate and glucose carbon to the fatty acids newly synthesized during the experiment. These calculations, the results of which are given in Table V7 show that in the case of rat mammary gland roughly ·102 PHYSIOLOGY AND BIOCHEVIISTRI OF LACTATION glucose but not acetate (by itself) for lipogenesis The situation is however, somewhat more complicated than this Bloch and hramer (1918) found that glucose stimulares lipogenesis from acetate in rat liver slices and in our laboratory we have demon strated a similar phenomenon in mammary tissue Thus in the presence of glucose, mammary gland slices from rats will uulze acetate, the R Q in mixtures of acetate and glucose being at least as high as it is in presence of glucose alone Simularly in mixtures of acetate and glucose, sheep udder slices show a higher R Q and acetate uptal e than in presence of acetate alone (Folley and French, 1950) All the conclusions drawn from respiratory measurements as just outlined, were later confirmed by experiments in which mammary gland slices were incubated with labelled sub strates and the mean isotope content of the mixed fatty acids, isolated from the slices at the end of the incubation deter mined The results of our earlier isotope experiments (Bal mun rollev and Glascock, 1952a) in which we uscd (carboxy 11C) acetate as labelled substrate are summarized in Table V5 The results in this Table are expressed as specific activi ties of the mixed fatty acids isolated from the slices as calcium IABLE V 5 Specytic activities (counts/min/mg C) of calcium salts of mixed fatty acids isolated from lactating mammary gland slices incubated with CH, 1*COONa or CH, "COONa + glucose (results from Balmain, Folley and Glascock, 1952a) RAT SHEEP Glucose experiment No Experiment No Glucose + I 5951 3,805 2 50 43 40 50 3 5 886 IL IR 2L 2R 3L 3R 3,334 3,539 2 660 2,948 4,144 9,533 9,911 8,975 10 249 8 068 7 41 58 5 6 3.532 3,215 1,069 40 4 108 ·BIOSYNTHESIS OF MILK FAT 103 salts after three hours incubation They show that rat mam mary gland incorporated hardly any acetate carbon into the fatty acids when incubated with acetate as solc substrate but there was considerable incorporation in presence of glucose Udder slices from lactating sheep on the other hand, incorporated considerable amounts of acetate carbon into the fatty acids when incubated with acetate alone but here again the incorporation was greatly increased when glucose was present The mechanism of the stimulatory effect of glucose upon the utilization of acetate for lipogenesis in vitro is not clear even yet It has been suggested that glucose provides a source of the energy which is necessary for lipogenesis to proceed but this would hardly apply to sheep mammary tissue which unlike rat mammary tissue can obtain energy from the oxidation of acetate The question of the relative values of acetate and glucose as sources of carbon for lipogenesis in the mammary gland is of interest particularly in view of the species differences in mam mary metabolism already touched upon In our subsequent experiments therefore, we have studied this question by in cubating mammary slices in mixtures of acetate and glucose each labelled with different isotopes (Balmain Folley and Glascock, 1954) The glucose was uniformly labelled with 14C and the acetate was labelled in the carboxyl group with 1:and in the methyl group with radioactive hydrogen (tritium) Our results are summansed in Table V 6 which shows the abundance of each isotope in the fatty acids isolated from the tissue These experiments have shown that rat mammary slices incorporate into the fatty acids in three hours about eight times as much glucose carbon as do sheep udder slices on the other hand sheep udder slices incorporate about six times as much of either carbon atom of acetate into the fatty acids as do rat mammary gland slices Since the mammary slices from both rat and sheep had substantially the same fat content and the abundances of the isotopes in the substrates are known it is possible from these results to calcu late the relative contributions of acetate and glucose carbon to the fatty acids newly synthesized during the experiment These calculations, the results of which are given in Table V7, show that in the case of rat mammary gland roughly ·104 PHYSIOLOCY AND BIOCHEMISTRY OF LACTATION equal contributions are made by acetate and glucose in the case of sheep udder slices however, the result was quite different since about 97 per cent of the carbon of the newly TABLE V6 Relatrze incorporation of glucose and acetate carbon into the fatty acids of mammary gland slices from lactating rats and sheep the substrates were CiH, 13COONa + 14C glucose (from Balmain, Folley and Glascoch, 1954) Ratio Species Mean alb 14C (glucose carbon) counts/min/mg C Rat 307 381 124 245 110 170 223(a) Sheep 38 4 174 22 5 24 7 358 376 28(b) *H (acetate methyl carbon) counts/min/mg combustion water Rat 84 5 94 8 41 7 109 5 439 71 2 173 3(b) Sheep 421 405 501 451 502 464(a) 13C (acetate carboxyl carbon) atoms per cent excess Rat 007 005 0 055(b) 59 Sheep 0 252 O 249 0 373 0 378 o 370 o 320 0 324 (a) 79 63 502 006 0 04 TABLE V 7 Incorporation of substrate carbon into the fatty acids of lactating mammary gland slices utilizing acetale and glucose (from Balmain Folley and Glascoch, 1954) Acetate (13C) Glucosc (C) Rat II I 8 Sheep 65 02 The results are expressed as mg of substrato carbon in corporated into 100 mg fatty acid carbon in 3 hr ·BIOSYNTHESIS OF MILK FAT 105 formed fatty acids came from acetate It thus appears that ruminant mammary tissue differs from rat mammary tissue in that it can only very slowly degrade glucose to the active two carbon unit, acetyl coenzyme A, now believed to be the form in which acetatc enters the pathway of lipogenesis or else the hrebs cycle for oxidation This conclusion has interesting implications, for Duncombe and Glascoch (1953) in our Institute have recently shown that sheep mammary gland slices incubated with glucose labelled uniformly with C can produce radioactive carbon dioxide which means that sheep udder tissue can oxıdıze glucose even when present in the incubation medium as sole substrate Mammary tissue may therefore be able to oxıdıze glucose by some pathway not involving the glycolytic route and it seems likely that the direct glucose oxidation cycle (see Dickens, 1953 for review) operates in the mammary gland Evidence in favour of this suggestion for the rat mammary gland has recently been reported by Glock and McLean (1954) and Abraham Hirsch and Chaihoff (1954) The most conclusive evidence of the utilization of acetate for the synthesis of milk fat in the ruminant udder comes from experiments carried out on lactating animals in vivo Popják and Beeckmans (1950) administered deuterium or [carboxy C) acetate to pregnant rabbits and showed that fat isolated from the mammary glands of these rabbits was in each case isotopically labelled Much of the fat present in the mammary gland in late pregnancy may well be depot fat or structural lipids but the fact that in collaboration with Dr Popjah we were able to isolate short chain fatty acids from the mammary gland fat of his rabbits shows that some synthesis of true milh fat proceeds even before parturition (Popjak, Folley and French 1950) Assays for it were carried out on various fractions of these acids with results that are shown in Table V8 It will be seen that the short chain acids were highly radioactive by companson with the long chain acids and morcover they were much more radioactive than the mixed fatty acids isolated from the liver fat These results permitted three deductions first that the short chain acıds could not have ansen directly from the liver fatty acids and by implica tion from fatty acids circulating in the blood, secondly, that ·106 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION TABLE V8 Specific achizities of the glycende fally acids from the mammae and lurers of rabbits killed at the 28th day of pregnancy after treatment with CH, HCOONa (from Popjah, Folley and French, 1949) Specific activity of fatty acids uc X 10-14mg c Fatty acid fraction Rabbit no IO 13 15 073 1845 Volatile Water-soluble Volatile, water insoluble Non volatile Liver fatty acids 198 I 48 13 60 0 12 0 72 007 0 183 o 99 058 0146 they could not have been formed by the degradation of long chain acıds of the mammary gland fat and thirdly, that they probably arose by synthesis from acetate in the mammary gland itself In view of these interesting results it was decided to extend thus work to a detailed study of the secretion of truc mill fat in the lactating ruminant Accordingly 5 mc [carboxyul'C} acetate were injected intravenously into a lactating goat and milk samples taken at frequent intervals after the injection (Popjah, French and Folley, 1951) Glyceride fatty acids were isolated from the milk and separated into four fractions steam volatile acids, soluble in water, steam volaule acids, insoluble in water non volatile saturated acids, non volatile unsaturated acids The specific activity time curses for all four fractions are shown in Figure V . It will be seen that all the cures showed maxima at 3-4 hr after the injection, but the mean specific activities of the two volatie fractions at their maxima were several times greater than those of the tho non volaule fractions Moreover the specific activities of the fatty acids of the plasma fat were so much lower at a periods of the experiment than those of any fraction of the ·BIOSYNTHESIS OF MILK FAT 107 0-0 Water arbeit! }telable Our Water selubi .-- Saturated Ono lasaturated) ܝܐܙܛܕܝܗ ܕܝܬ; Jx10°jcmg c Specific acelyly 1 x 107cmg C S JO 20 40 50 Hours after injection Fig V: Specific activity time curves of glycende fatty acid fractions isolated from the mill of a goat after the intravenous injection of 5 inc of (carboxy"Cacet ate (from Popják French and Folley 1951) mill fatty acids that they cannot be shown in the same dia gram These results showed that the synthesis of fatty acids from acetate is of great importance in the fat metabolism of the goat udder and moreover that the short chain acds must anse by synthesis from 2 carbon units within the udder rather than by oxidative degradation of long chain acids as postulated by the theory of Hıldıtch Later Dr Popjak and his colleagues together with the late Dr TH French of our laboratory in continuation of this joint project isolated individual fatty acids from these crude fractions and by determination of the specific radioactivities of these pure fatty acids and of ındıvı dual carbon atoms obtained from the butyric caproic and capryllic acids by chemical degradation showed that chain elongation must take place by successive addition of two-carb on uruts to the carboxyl end of the fatty acid chain (Popjak French Hunter and Martın 1951) This provided conclusive evidence in favour of our previous contention (see Folley and French 1948 Folley, 1949) that the short chain acids of mill ·108 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION fat are intermediates in the synthesis of the long chain acids from small molecules Another important point emerged from this chemical work of Popjak et al Figure V 2, for which I am indebted to Dr G Popjah, shows the mean specific activities of all the saturated fatty acids, present in these milk 0-12 hours 15 12-48 hours toH Specific activity 1 X 10-s uc/mg C 05 29 2 4 6 8 10 12 14 16 18 18 4 6 8 10 12 14 16 18 18 Number of atoms in carbon chain Fug V 2 Specific activity of milk fatty acids after injection of 5 mc of [carboxy 'C] acetate into a lactat ing goat (figure constructed from the results of Popják French Hunter and Martın 1957) samples, from butyric to stearic together with that of the oleic acid It ill be noted that there is a sharp drop in specific activity between palmitic acid on the one hand and oleic and stearic acids on the other, which suggests that the Cis acids of milk fat, and probably all the higher ones, must be mainly formed by some mechanism other than by synthesis in the udder It seems likely that they arise chiefly from the blood glycendes as suggested by the arterio venous differences already mentioned Finally, the specific activities of the individual carbon atoms of the caproic acid suggested that the radioactive butyrate formed in the udder of the experimental goat by condensation of two acetate molecules was continually diluted by a non isotopic four carbon compound arising from ·BIOSYNTHESIS OF MILK FAT 109 the blood The blood fat of ruminants contains relatively little butyric acid but there are considerable amounts of ß hydroxy butyrate present It thus seemed possible that the four carbon compound which appears to serve as an additional substrate for lipogenesis in the goat might be 8 hydroxybutyrate This would agree with the fact that Shaw and hnodt (1941) have observed arterio venous differences in B hydroxybutyrate concentration amounting to about 25 mg/100 ml across the lactating bovine udder However, Kleiber Blach Brown, Luich Baxter and Tolbert (1954) who injected butyrate labelled with 14C into lactating cows found that butyrate carbon entered the milk fatty acids to a much smaller extent than acetate carbon About 70% of the butyrate carbon entering the organic milk constituents was found in lactose and casein The utilization of acetate labelled with 14C for fatty acid synthesis in the lactating mammary gland in ziro has also been observed by Kleiber, Smith, Blach Brown and Tolbert (1952) in the cow and by Popjah, Hunter and French (1953) in the rabbit In summary then we can say that in ruminants a propor tion of the fatty acids of the mill glycerides is synthesized in the udder from small molecules the precursors being mainly acetate and perhaps 8 hydroxybutyrate It is probable that most of the fatty acids up to and including palmitic acid are formed in this way while the oleic, stearic and higher acids probably arise mainly from the blood glycerides In the mam mary gland of non ruminants, as exemplified by the rat and rabbit a similar mechanism almost certainly obtains save that considerable quantities of glucose carbon are also used for Lipogenesis Information on the proportion of the long chain acids of ruminant milk fat arısıng from the blood glycerides would be of great interest It is being sought in this Institute at the time of writing by Duncombe and Glascoch (1954) who are feeding tritio stearic acid prepared by the tritiation of elaidic acid to lactating goats and cows Their latest prelimi Par experiments (unpublished) indicate that some 20—30% of the long chain acids originate from the blood fatty acids An interesting development in the study of the fatty acid metabolism of mammary tissue is the demonstration by Popják and Tietz (1954) and later in our laboratory by Terner (1954) ·ITO PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION that cell free homogenates of mammary gland will synthesize fatty acids from acetate The former workers have now prepared a soluble enzyme system from mammary tissue capable of effecting this synthesis and have shown that it is coenzyme A dependant (Popják and Tietz, 1955, Tietz and Popjah, 1955) THE ORIGIN OF THE GLYCEROL OF THE MILK GLYCERIDES So far we have only discussed the origin of the fatty acids of the milk fat The mammary gland, however, secretes mainly glycerides o that the source of the glycerol must also be considered Though the lactating udder absorbs glycerides from the blood the fact that it acuvely synthesizes fatty acids suggests that it does not acquire all the glycerol it needs in the form of glycerides Popjak Hunter and French (1953) in their experiments in which "C glucose was administered to lacta ting rabbits found that the glycerol of the mill glycerides was strongly labelled, its specific activity being about equal to that of the mill lactose They concluded that between 65 and 95 per cent of the glyceride glycerol was formed from glucose in the mammary gland during the six hours after the injection The experiment of Popjáh, French and Folley (1951), men tioned above, in which 5 mc of 14C labelled acetate were in jected into a lactating goat provided further evidence upon this question since the mult lactose and glyceride glycerol became labelled Figure V 3 shows the specific activity time curves for lactose and glyceride glycerol isolated from the milk of this goat (Popjak, Glascock and Folley, 1952) It will be seen that the lactose curve rises to a maximum and then declines exponentially the glycerol curve remains below the lactose curve until it in turn reaches its maximum at which point it crosses the lactose curve This is precisely the relation ship which according to Zilversmit, Entenman and Fishler (1943), should hold between the curves for a reacuon product, in this case the glycerol curve, and its immcdiatc precursor, in this case the lactose cure It is not suggested, however, that lactose is the actual precursor of glycerol it is much more likely that the immediate precursor of glycerol is glucose and that under these conditions lactose and glucose arc in rapid equilibrium so that the specific acuvues of the two sugars are ·BIOSYNTHESIS OF MILK FAT III O Lactose counts/min/mg Cx10 10 Glycerol O 6 0 18 10 16 20 22 12 Hours after injection Fig V3 Specific activity time curves for lactose and glycerol isolated from the milk of a goat which had been injected intravenously with 5 mc of [carbox) "C] acetate (from Popják Glascock and Folley 1952) the same at any moment The results shown in the Figure suggest strongly that the conversion of glucose to glycerol takes place not in the liver but in the mammary gland itself HORMONAL CONTROL OF LIPOGENESIS IN THE MAMMARY GLAND In conclusion let us discuss the hormonal control of lipo genesis in the mammary gland We will begin with insulin because so far more work has been done on the role of insulin in fatty acid synthesis than on that of any other hormone Experiments on the living animal by Drury (1940) Stetten and Boxer (1944) and others suggested that an important action of insulin is to promote the formation of fat from carbohydrate It follows that the hypoglycaemic action of insulin mıght partly be due to its action in directing carbohy drate along this pathway of metabolism Bloch and Kramer (1948) were the first to demonstrate a stimulatory effect of ·112 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION snsulin on lipogenesis in vitro They found that rat liver slices incubated with pyruvate and KC labelled acetate incorporated more acetate carbon into the fatty acids in the presence insulin than in its absence Though these authors could not confirm these results in a different strain of rat, simular and more convincing effects of insulin on lipogenesis un rat liver slices were later reported by Brady and Gurin (1950) As our group has more than once pointed out, the outstanding capabilities of mammary tissue for effecting net fat synthesis in uitro make it an eminently surtable mcdium for studying lipogenesis, and it is perhaps not surprising that we have been able to demonstrate particularly clear cut even dramatic, stimulatory effects of insulin on Bipogenesis in mammary gland slices In all our work we have used insulin free from the hyper glycaemic factor which is present in many insulin prepara uans and might confuse the issue We have found that if mammary gland slices from rats in full lactation are incubated with a mixture of acetate and glucose, addition of insulin to the medium markedly increases the R Q and also the uptake of both acetate and glucose Some of our earlier results (Balmain, French and Folley, 1950) are shown in Table V, With glucose alone as substrate similar but somewhat smaller effects were observed but insulin had no ameliorating effect on the TABLE V 9 Efect of insulin added in vitro on the utilization of acetate and glucose by lactating mammary gland slices (from Balmain, French and Follcy 1950) Number Glucosc Addition of rats man I acid RO Glucose + acciate o acid RQ 134 2 03 17 1 80 Insulin 104 tiu/ml) 10 No insulin 92 17 557 105 -27 153 ns RS KO 001 <0 oz SO001 (0001 ·BIOSYNTHESIS OF VILA FAT 113 indifference of rat mammary slices to acetate (Balmain and Folley 1931) The lowest concentration of insulin to give detectable effects was found to be in the neighbourhood of 2 mu/ml. (Balmain, Cox Folley and Vic.laught 199+) We interpreted these changes as indicating that insulin stimu lates lipogenesis from small molecules in rat mammary gland slices That this interpretation was justified was subsequently shown in experiments in which mammary gland slices were incubated with labelled substrates Table V 10 shows results TABLE V to Effect of insulin, added in vitro on lipogenesis in rat marmary gland sluces incubated with CH,13000 10 + 1C-glucose (from Balmain, Folley and Glascock 1957) Isotope content of calcum salts of mixed fatty aads after 3 hr нс H1 ISC counts/min/mg C counts/min/m3 atoms per cent combustion water Control 95 0 0 Insulin 014 (10 1u (ml) excess 381 803 202 from an expenment in which mammary gland slices from lactating rats were incubated with C-glucose together with [carboxy 13C-VH] (Balmain Folley and Glascoch 1934) The results show that insulin increases the incorporation of glucose carbon and also of both carbons of acetate into the fatty acids of the slices In companng these results with those obtained with mammary tissue from lactating ewes interesting species differences are again apparent for we have never found any stimulatory effects of insulin added in ziro upon lipogenesis in sheep mammary tissue either by respiratory methods (Balmain and Folley 1931) or by the use of isotopically labelled substrates (Balmain Folley and Glascock 1954) Results obtained with labelled substrates are shown in Table V u Udder slices from lactating enes were incubated with acetate and glucose ·114 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION TABLE V 11 Effect of insulin, added in vitro, on lipogenesis in sheep udder slices incubated wrth CH,13COONa + WC glucose (from Balmain Folley and Glascoch, 1954) Isotope content of calcium salts of mixed fatty acids after 3 hr 14C эн 130 counts/min/mg C counts/min/mg atoms per cent combustion water Control O 37 Insulin (10 iu /ml ) 485* 0 33 excess 225 501* 276 • These values are one half of those given for sheep udder half S5L in Table 7 of the original paper (Balmain et al 195+) All the values of the serics to which they belong were halted for inclusion in Table r of the onginal paper (reproduced in this book as Table V 6) so as to make thern comparable with the values for the rat labelled with different isotopes as before The results show that in no case did insulin increase the abundance of any of the isotopes in the fatty acids It will be recalled that we found that whereas under these conditions rat mammary tissue utilizes acetate and glucose to approximately equal extents for fatty acid synthesis, sheep udder slices use acetatc almost exclusively for this purpose and incorporate practically no glucose It therefore scems reasonable to suggest that the effect of insulin on lipogenesis is bound up with its effect on some metabolic system which is present in rat mammary tissue but absent from the mammary tissue of the cue In vicw of what was suid earlier it would seem that this must be the system which produces acetyl coenzyme A from glucose Our results in gencral are in harmony with the view that the effect of insulin on lipogenesis is exerted at some point in the breakdown of glucose to acetyl coenzyme A rather than on the uulizauon of acetyl coenzyme A for fatty acid synthesis It is well known that the 11-oxygenated steroids secrctcd by the adrenal cortex exert an ang insulin cffcct in certain rc spects The possibility that the adrenal cortex might play a rolc ·BIOSYNTHESIS OF MILK FAT 115 in the control of lipogenesis must therefore also be considered, since one might expect that adrenal steroids would exert an opposite effect on lipogenesis to that of insulin Evidence of such antagonistic effects was provided by Brady Lukens and Gurin (1951) who showed that pre treatment of rats with cortisone decreased the incorporation of labelled acetate carbon into the fatty acids of slices of their livers In our laboratory we have studied the effects of vanous adrenal steroids added in vztro, on lipogenesis in lactating mammary gland slices We have found that cortisone added to the medium inhibits the incorporation of both acetate and glucose carbon into the fatty acids of rat mammary gland slices in cubated with [11e 'H] acetate and WC glucose When both hormones were present cortisone partially inhibited the stimu latory effect of insulin on upogenesis (Balmain Folley and Glascoch 19526) Results of a typical experiment illustrating these facts are shown in Table V 12 In subsequent experi ments (McNaught, Glascoch Balmain and Folley 1955) TABLE V 12 Effect of cortisone and insulin on the incorporation of H and 14C into the mixed fatty acids of lactating rat mammary gland slices incubated with CH,COONa + 14C glucose (from Balmain Folley and Glascock, 1952b) 14C in the fatty acids SH in the fatty acids Addition counts/min per cent of counts/min per cent of mg C control ngH, control None 365 100 283 100 Cortisone 254 70 77 27 Insulin (001 iu /ml ) 795 218 430 152 Insulin (o on iu/ml) +cortisone (100 pg/ml) 383 118 42 103 similar inhibitory effects on lipogenesis have also been obtained with deoxycorticosterone and corticosterone though these steroids were more active than cortisone Different results ·116 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION were however given by cortisol (hydrocortisone) This steroid at the same concentration, 10opg/ml, had no inhibitory effect on lipogenesis, nor did it interfere with the stimulatory action of insulin Since there is some reason to believe that cortisol may be the natural glucocorticoid secreted by the adrenal cortex of the rat, the fact that we have found its action on lipogenesis in litro to be different from that of two other glucocorticoids, cortisone and corticosterone, is of considerable interest Sheep mammary gland slices once again have been found to behave differently from those of the rat, this time in respect of the effects of adrenal corticoids on lipogenesis in vitro as measured by the rate of incorporation of labelled acetate carbon into fatty acids (McNaught et al 1955) In sheep udder slices cortisone and cortisol at a concen tration of 100 mg/ml have been found to exert no regular cffects on lipogenesis either way, while corticosterone and deoxycorticosterone in similar concentrations strongly inhibit lipogenesis as they do in rat mammary slices These results are difficult to interpret at the present time particularly since the concentrations used are some 1000 times greater than those believed to occur in the blood stream, so that it is not yet possible to draw firm conclusions about the role of the adrenal cortex in the control of lipogenesis Clearly further work is needed upon this aspect of the subject REFERENCES Abraham S Hirsch P F and Chaikoff I L (1954) ) brol Chem 211 31 Achaya A T and Hilditch TP (1950) Pro ror So 1 137 187 Appel H Bohm H Keil W and Schiller G (1917) Hoppe Seyl z 282 2.0 Aten AH W] and Hevesy G (1938) Nature Lond 142 111 Balmain JH Cox C P Folley S J and VicNaught ML (1951) 7 Enducten II 268 Balmain J H and Folley S J (1921) Brochem 7 49 663 Balmain, j H Folley S J and Glascock RF (1952a) Brochem. 52 301 Balmain J H, Folley S J and Glascock R (19,26) Nature Lond 169 447 Balmain j H Folley S S and Glascock, R. F (199+) Biochem 7 56 234 Balmain J H French T H and Folley S J (1950) Nature Lond 165 807 ·BIOSYNTHESIS OF MILK FAT 117 Bloch i and Kramer W (1948) 3 biol Chem 173 811 Brady R O and Gurin, S (1950) Í brol Chem 186 461 Brady RO, Lukens FWD and Gurin S (1951) 3 brol Chem 193 459 Dickens F (1953) Brit med Bull 9 105 Drury DR (1940) Amer 5 Physiol 131 536 Duncombe W G and Glascock RF (1953) Biochem 755 XXI Duncombe W G and Glascock R F (1954) Brochem 7 57 x1 Elsden S R. and Phillipson A T (1948) Annu Rev Biochem 17 705 Folley S J (1949) Biol Rev 24 316 Folley s j and French T H. (1948) Brochem 7 43 lv Folley S J and French T H (1949) Brochem 3 45 117 Folley S J and French, T H (1950) Brochem 7 46 465 Clock G E and McLean P (1954) Biochem I 56 171 Graham W R jr Houchin O B Peterson V E and Turner CW (1938) Imer 7 Physiol 122 150 Graham W Rj Jones TSG and hay HD (1936) Proc roy Soc B 120 330 Hilditch T P (1937) Analyst 62 250 Hildatch T P (1947) The Chemical Constitution of Natural Fats and ed London Chapman and Hall Hilditch T P and Thompson HM (1936) Biochem 7 30 677 Kleiber M Black A. L Brown MA Luich J Baxter C F and Tolbert BM (1954) ) brol Chem 210 239 Kleber M Smith A H Black A. L Brown MA and Tolbert BM (1952) 3 brol Chem 197 371 I intzel W (1934) Z Zucht B 29 219 Malpress F H (1946) In Discussion on digestion sn the ruminant Proc R Soc Med 39 805 Mann A I and Shan I (1947) 7 Dairy Sc 30 183 McClymont GL (1951) Aust agric Res 2 158 McNaught M L Giascock, R F Balmain J H and Folley S J (1955) Brochem 60 102 Maynard L A McCay C M Ellis G H Hodson AZ and Davis GA (1938) llem Cornell agric Exp Sta Meigs E B Blatherwick N R and Cary CA (1919) 3 brol Chem 37 1 Popják G and Beeckmans ML (1950) Brochem 7 46 547 Popják G Folley SJ and French T H (1950) Arch Brochem 23 509 Popjak G French T H and Folley S J (1951) Brochem I 48 411 Popják G French T H Hunter GD and Martin AJP (1951) Biochem 3 48 612 Popják G Glascock R F and Folley SJ (1952) Brochem I 52 472 Popják G Hunter G D and French T H (1953) Brochem 7 54 238 Popják G and Tietz A (1954) Biochem 3 56 46 Popják and Tretz A. (1955) Biochem 3 60 147 Reineke E P Stonecipher W D and Turner CW (1941) Amer 7 Phystol 132 535 Rittenberg D and Bloch L. (1945) 3 biol Chem 260 417 no 211

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Shaw J C and hnodt C B (1945) brol Chem 138 287
Shaw J and Petersen W E (1938) Amer J Physiol 123 183
Smith JAB and Dastur N (1938) Biochem 7 32 1869
Stetten De Witt jr and Boxer GE (194) 3 bal Chem 156 271
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·CHAPTER VI RECENT STUDIES OF THE BIOSYNTHESIS OF LACTOSE AND MILK PROTEINS THE ORIGIN OF THE LACTOSE, OF MILK Lactose, the carbohydrate of mull is a disaccharide glu cose 4 8 galactoside(4-0-8-D-galactopyranosyl D glucopyranose), the structural formula of which (a form) is shown in F1 gure VI 1 Lactose is normally found nowhere in the body but in the mammary gland Since it is not present in blood except CH, OH CH2011 0 OH 1199 # H H 0 3 H Galactose OH H 13 (2) Glucose OH H 1 H ON H OH н CH LACTOSE Fig VI 1 when for one reason or another suckling or milling have ceased so that the gland is temporarily engorged with mull lactose must be synthesized in the gland itself In view of its intrinsic interest the problem of how lactose is formed has stimulated the imagination of many investigators but no universally acceptable and final solution has yet been reached Recent progress however encourages the hope that the resolution of this seemingly simple yet baffling enigma will soon be achieved ·120 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION There are two main possibilities to consider Lactose may be synthesized from small molecules, that is two or three carbon units, or it may be made from preformed hexose units taken from the blood It is, of course, conceivable or perhaps even probuble that the mammary gland may make lactose partly from preformed hexose molecules and partly from smal ler molecules With regard to the fir t possibility there is no conclusive evidence that lactose synthesis from small molecules cannot occur to some extent in the lactating gland Though arterio venous studies on cows (Shaw, 1946) have provided no evidence that the udder uulizes blood lactic acid or pyru vate which are the small molecular" precursors which come first to mind, they have consistently revealed an uptake of amino acids (Graham, 1937, Shaw and Petersen 1938, Reineke Peterson Houchin and Turner, 1939), some fraction of which, provided the mammary gland can carry out the necessary deamination, could provide building units for lactose synthesis The suggestion that blood amino acids might serve as „ubstrates for lactose synthesis was made nite teen years ago by Graham (1937) However the mo t probable role for amino acids in the mammary gland is to serve as inaterial for the synthesis of milk proteins and since, as we shall sce later, it has been contended that the amino acid uptake is insufficient for this purpose, it eems unlikely that there would be any to spare for lactose synthesis Furthermore, as was pointed out in Chapter III, there is no good evidence for the occurrence of gluconeogenesis in the mammary gland of any species other than the rat and mouse The possibilities are not exhausted, however, since arterio venous studies on the bovine udder have revealed uptakes of acetate (MicClymont 1951) and 8 hydroxybutyrate (Shaw and hnodt, 1941) both of which could conceivably be built into the carbon skeleton of lactose Indeed a perfusion experiment carned out by our group in collaboration with Dr G Popják of London and Professors G Peeters and L Massart of Gent has shown that the isolated bovine udder can incorporate acctatc carbon into lactose (Cowie, Duncombc, Folley, French, Glascoch, Mascart, Peeters and Popják, 1951) We added (carboxz.11C) acetate to the blood perfusing a bovinc udders half and were able to isolatc radioactive lactose from the milk ·BIOSINTHESIS OF LACTOSE AND WILL PROTEINS 121 This suggests that the udder can synthesize some lactose from small molecules but does not prove that a net synthesis of carbohydrate from acetate is possible in the isolated udder Apparently the udder incorporates acetate carbon into lactosc by some mechanism other than oxidation followed by fixation of the resulting carbon dioxide since lactose secreted by the contralateral udder half, which was simultaneously perfused with radioactive bicarbonate had no appreciable radioactivity More recent work with the perfused bovine udder carried out by Dimant Smith and Lardy (1953) strong ly suggests that lactose is formed in the udder mainly from preformed hexoses probably glucose They perfused a bovine udder with blood containing 1 14C glucose and found that in the glucose and galactose obtained by hydrolysis of the mill lactose the radioactivity was in each case mainly confined to the Cw position Since this experiment was carried out on an isolated udder the interpretation of the results was not complicated by redistribution of the label resulting from reactions in other tissues such as the liver I venture tentatively to conclude that the experimental evidence available at the present time favours the view that the mammary gland builds up lactose mainly from preformed hexose units rather than from two or three carbon units If further work should prove this to be the case, there are two principal possibilities to consider in this connection also These concern the origin of the galactose half of the lactose molecule On the one hand we must enquire whether the mammary gland uses preformed galactose which it obtains from the blood and on the other we have to consider the alternative possibility that blood glucose is the sole precursor of lactose, the galactose half of the lactose molecule being formed by rearrangement of the glucose molecule in the mammary gland The arterio venous studies of Reineke, Williamson and Turner (1941) on lactating goats have indicated that there is an appreciable arterio venous difference in plasma glyco protein across the lactating udder Now there is some evidence that the protein bound carbohydrate of the glycoprotein fraction of the blood plasma proteins consists apart from a glucosamine component of an equimolecular complex of ·122 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION mannose and galactose (Friedmann, 1949), so that there is a strong possibility that the udder may acquire galactose from the blood We have, however, no quantitative information about whether the amount of galactose taken up by the gland in the form of protein bound carbohydrate is sufficient to account for the galactose moiety of the milk lactose but a prior it seems rather unlikely that this can be so A few years ago, we found in our laboratory that mannose was the only carbohydrate besides glucose of a large number tried, which increascdaboveendogenous values theoxygen consumption and RQ of mammary gland slices from lactating rats (Folley and French 1949) This suggested that glucose and mannose arc interconvertible in the mammary gland from which it would follow that mannose could be used for any purpose for which the gland uses glucose This in turn led us to speculate upon whether some proportion at least of the mill lactosc might not be formed by molecular rearrangement in the mammary gland of the mannose galactose complex present in the plasma glycoprotein Alalpress (1951), however later reported that guinea pig mammary slices which would synthesize lactose from glucose in rutro would not form lactose in presence of mannosc and so his results do not support this suggestion As regards the alternative point of view it can be said at once that there is a considerable amount of evidence that the mammary gland can synthesize lactose from glucose alone and if this is so there seems to be no need to postulate any other precursor for lactose than the glucose of the blood The idea that the blood glucose is the precursor of the milk lactose is a comparatively old one which in more recent times has found support in the fact that in numerous studies marked arterio venous differences in blood glucose across the udders of lactating goats or cows have invariably been observed More over, it can be said that in general, procedures which lower the blood sugar tend to decrease the mll lactose concentration while procedures causing hyperglycacmja have the opposite effect (scc Folley, 1910 for review) The best cvidence in its favour, however, comes from experiments on the synthesis of lactose by mammary gland slices which were first successfully carried out by Grant (1935) For the detection and estimation of lactose Grant devised a differential fermentation method ·BIOSYNTHESIS OF LACTOSE AND VILL PROTEINS 123 involving the use of yeasts adapted to ferment lactose and galactose and if we can accept Grants method as specific for lactose, we must conclude that he was able to demonstrate a marked synthesis of lactose by slices of guinea pig mammary gland incubated with glucose as sole substrate Not only did Grant thus show that guinea pig mammary gland could form the galactose moiety of lactose from glucose but he obtained additional evidence that the provision of preformed galactose is not necessary, for he further showed (Grant 1936) that addition of galactose to the medium did not increase the amount of lactose syrithesized from glucose Grants results perhaps, should not be regarded as conclusive proof that preformed galactose is not essential since galactose might be stored in the tissue in the form of a polysaccharide In this connection it may be noted that Caputto and Trucco (1952) reported the presence of a polysacchande containing glucose and galactose in mammary gland extracts Later they identified one of the carbohydrate complexes detected by them in mamnary gland extracts as neuramın lactose a compound of lactose and neuraminic acid (Trucco and Caputto 1954) An essentially similar compound has also been identified in mammary tissue by Heyworth and Bacon (1954) A tetrose of the structure glucose galactose N acetylglucosamune galactose has been isolated from human milk by huhn Gauhe and Baer (1954) Whether either of these substances is implicated in lactose biosynthesis remains to be seen the possibility that a poly saccharide containing galactose is concerned in lactose forma tion will be referred to again later on Sıx years ago Malpress and Morrison (1950) demonstrated the synthesis of lactose from glucose by slices not only of guinea pig mammary gland, but also (see Malpress 1951) of lactating goat udder Their results are perhaps more conclusre than those of Grant because they used a rather specific colorimetric method for the determination of lactose Finally Reithel Horowitz Davidson and hittinger (1952) have succeeded, by the use of WC glucose in demonstrating for the first time the synthesis of lactose from glucose in mammary gland homogenates By means of Rethel's technique it should be possible to make further progress in the study of intermediates in lactose synthesis In fact, his later work (Katunger and Reithel 1953) ·124 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION suggests that a soluble protein extracted from gunea pig mam mae will catalyze a trans glycosidation reaction, involving glycogen or starch which yields lactose Some further reasons for the belief that glucose is the main if not the sole precursor of both halves of the lactase molecule may also be mentioned French Popjal and Malpress (1952) fed "C starch to lactating rabbits and isolated lactose fronti their milk They found that the mean specific activity of the whole lactose molecule and of the glucose and galactose obtained therefrom by acid hydrolysis were all sensıbly the same This led them to infer that both halves of the galactosc moleculc are formed equally from the same source, namely, glucose Their results suggest that if other precursors are involved to any appreciable extent they must first be con verted to glucose A simlar conclusion follows from the work of Dimant Smith and Lardy (1953) on the perfusion of the bovine udder with radioactive glucose already mentioned, for these workers also found that the glucose and galactose isolated from the lactose formed during the perfusion had equal specific activites On the other hand Barry (19524) who injected radioactive glucose into a lactating goat, found that the specific radioactivity of the galactose isolated froin the mult lactose was measurably less than that of the corre sponding glucose The next question to discuss is the mechanism of the conver sion of glucose to galactose These two hexoses differ only in the steric configuraton of the groups attached to Co, so that all that is necessary to transform glucose to galactose is a Valden inversion at thus carbon atom Nasertheless this ngly simple conversion is extremely difficult to cffect by chemical means in the laboratory and has been reported only once Some light has been thrown on this problem by studies of the converse process the conversion of galactose to glucose in adapted yeasts and also in the mammafian liver Leloir and his collaborators (Trucco Caputto Leloir and Mittelman 1948, Caputto, Leloir, Trucco Cardım and Paladını, 1949, Caputro, Leloir, Cardins and Paladışı, 1950) in a series of brilhant investigations have described an enzyınc system present in a yeast, Saccharomyces fragilis, adapted to ferment lactose, which will effect thus conversion The first step is the ·BIOSYNTHESIS OF LACTOSE AND MILK PROTEINS 125 ase as phosphorylation of galactose at Cw by an enzyme, galacto Linase which catalyzes the transfer of phosphate from ATP The a galactose 1 phosphate formed is then transformed to a glucose 1 phosphate by an enzyme called galactowalden This enzyme requires a coenzyme which the Leloir group has identified urdınc diphosphate glucose, a substance which can be regarded as glucose i phosphate combined with urdine 5 phosphate through a pyrophos phate linkage The relevance of this work to the lactose problem is obvious Should the various steps in this enzymatic conversion prove to be reversible and moreover of the enzyme system were found in mammary tissue then we would have a feasible route from glucose to galactose It is therefore, of considerable interest that Caputto and Trucco (1952) have reported the detection of galactosaldenase and its coenzyme in mammary tissue and that the presence of the coenzyme in the mammary gland has been confirmed by Rutter and Hansen (1953) These interesting findings may mean that the mam mary gland possesses the enzyme system necessary to effect the Walden inversion of glucose at C() Results obtained by Topper and Stetten (1951) on the conversion of galactose to glucose in the rat liver may also be relevant to the problem we are discussing H O L Fischer (1944-5) had suggested that the cyclic hexitol L inositol might be an intermediate in the interconversion of galactose and glucose To test this possibility Topper and Stetten ad ministered 1 14C galactose to rats and isolated labelled glycogen from their livers Chemical degradation of the glucose obtained by hydrolysis of this glycogen showed that the radioactivity resided almost exclusively in C which excluded L-inositol as an intermediate since it can be shown on theoretical grounds that this route would yield glucose labelled at C) Though the results do not entirely exclude the possibility that an isomer of L inositol muco inositol is an intermediate Topper and Stetten (1951) interpret them as favouring the view that the conversion of galactose to glucose in the liver proceeds by direct epimerization at Cie koshland (1954) has discussed this epimerization in terms of a single displacement mechanism which might be confirmed by experiments with 160 labelled water as follows ·126 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION H H,O C HO OH H 1,0* + + HO H HO The possibility was suggested by Malpress (1951), that the conversion of glucose to galactose might proceed through the cleavage of the glucose molecule to triose fragments which undergo an aldol condensation in such a way as to give the required cus configuration to the groups attached to Cia) and Ca "The localization in C) of the radioactivity of the galactose obtained from lactose secreted by the bovine udder which Dimant, Smith and Lardv (1953) perfused with 1 16C glucose, is consistent with tlus hypothesis if it be assumed that intercanversion of aldo and Leta trioses is prevented in some way for example of the first three carbon atoms of glucose remained uttached to a coenzyme If there were a single three carbon intermediate there would be some redistri bution of the radioactive carbon among other carbon atoms of the galactose molecule Honever, the fact that glycogen when synthesized in the body from galıctose in presence of DO contains no more deuterium than when glucose is the substrate (Stetten and Klein 1946) may be cited as cvidence against an aldol type of cleavage On the wholc, the evidence thus far available seems to favour the suggestion that galactose is formed from glucose in the mammary gland mainly by direct epimerization at Cras though the possibility that the conversion involves the cleavage of the hexose molecule followed by recombination, as suggested by Valpress, is not entirely excluded The detection in main marv tissue by Caputto and 'Irucco (1952), of the enzyme system, well characterized in certain galactosc adapted yeasts, which can coniert galactose i phosphate to glucose i phos phate, appears to provide the most suggestive circumstanual evidence yet available that thus mechanism may play in important role in the biosynthesis of lactose vore positive evidence will be needed however, before this conclusion can be accepted, and the work of Leloir (1951) suggests a possible way in kluch further evidence might be sought Lcloir has ·BIOSYNTHESIS OF LACTOSE AND MILK PROTEINS 127 shown that an enzyme from Saccharomyces fragilis, presumably galactowaldenase, will reversibly transform undine diphos phate glucose (UDPG) into urdine diphosphate galactose (UDPGal) so that an equilibrium is reached The reactions were formulated by Leloir as follows UDPG + Gal 1 P UDPCal + G1P (1) UDPGal + UDPG (2) Evidence in favour of this mechanism has since been provided by Trucco (1954) who showed that 14C is incorporated into UDPG when this compound together with 14C glucose I phosphate is incubated in presence of Saccharomyces fragilis extract The reversibility of the transformation of galactose i phosphate to glucose i phosphate by extracts of Lactobacillus bulgaricus has been shown by Hansen and Craine (1954) while other biological reactions which result in the formation of urdine diphosphate galactose have been studied by Kalchar Braganca, and Munch Petersen (1953) The findings of Leloir suggest that it would be interesting to carry out experi ments to find out if mammary gland extracts or homogenates will effect the interconversion of uridine diphosphate glucose and uridine diphosphate galactose If such an interconversion could be demonstrated in mammary gland preparations and of further it could be shown that mammary gland extracts contain an enzyme which will split off galactose or galactose phosphate from uridine diphosphate galactose the evidence for the participation of the galactonaldenase system in the biosynthesis of lactose would be strengthened Here would seem to be a clear indication of a direction along which further investigations might be fruitful Even if it can be shown experimentally that the mammary gland is capable of forming galactose or more probably galactose : phosphate by simple epimerization of glucose I phosphate at C) or by some other mechanism there remains die problem of how the condensation of the two hexoses is effected Some years ago the writer suggested (Folley 1949) that this condensation might be brought about by a trans glycosidation reaction analogous to that demonstrated by Hassed Doudoroff and Barker (1944) for the formation of sucrose in bacteria and which Doudoroff (1945) suggested ·128 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION might apply to sucrose formation in plants These authors have dernonstrated the ability of a bacterial phosphorylase to catalys the condensation of glucose : phosphate with fructose to give sucrose It has since been shown that sucrose phosphory lase will catalyse trans glycosidation reactions which do not involve phosphate However it must be pointed out that an analogous mechanism for the synthesis of lactose in the mam mary gland would require that mammary tissue contains 1 phosphoryla e specific for 8 galactose i pliosphate, that is an enzyme capable of catalysing the formation of the galacto sidic linhage Níoreover since the galactowaldenasc system appears to be concerned with the metabolism of a galacto e iphosphate in order to complete the theoretical cheme we are di cussing we may have to postulate the existence in the mam mary glandof a mutase of a type hitherto unknown, namely one capable of converting an a glycosidic linkage into the ß con figuration Figure VI 2, which shows this theoretical scheme GLUCOSE hexolinuse (ATO) GLUCOSE - G-P phosphoglucomutase a GLUCOSE IP Salactowaldenase (+ UOPG) GALACTOSE IP ? GLUCOSE + ß GALACTOSE 10 -? trans salactosidase LACTOSE mutasc Fig VI 2 for the biosynthesis of lactose illustrates the points just dis cussed and further shows that the energy necessary for the formation of the glycosidic bond of lactose would come from ATP It must be emphasized that, however attractive tfus hypothesis mav be, apart from the detection of the galacto waldenase system in mammary tussuc, experimental evidence in its favour is laching Reithel, Horowitz, Davidson and butunger (1952), hare synthesized | galactose i phosphate ·BIOSYNTHESIS OF LACTOSE AND WILL PROTEINS 129 but could demonstrate no appreciable formation of lactose from it when added to cell free mammary gland homogenates A further theoretical possibility possessing advantages which make it worthy of consideration is that the condensa tion mıght involve phosphate esters of both glucose and galac tose giving rise to a lactose phosphate which undergoing irreversible dephosphorylation as a final step would be continu ously removed from the reaction thus shifting the equili brium over towards synthesis In this connection it is interesting to note that McGeown and Malpress (1952) have detected by chromatographic methods, the presence of a lactose phosphate in milk Before leaving the subject of lactose it is worth while to mention yet another speculation about its mode of synthesis in the mammary gland which has been put forward by Engel hardt (1950) Doudoroff and O Neal (1945) described a reversible reaction, catalyzed by an enzyme isolated from B subtilis , between glucose and a polysaccharide levan which is built up of fructose residues This reaction which in onc direction gives a synthesis of levan, in the reverse direction Jields sucrose It is essentially a trans glycosidation reaction Engelhardt s interesting speculations concern the possibility that lactose might be formed by an analogous trans glycosida tion reaction between glucose and a polysacchande built up of galactose residues A polysaccharide composed of galactose residues galactogen has been known for a long time but until recently had only been found in the garden snail It bears some resemblance to glycogen but on hydrolysis yields only galactose which be it noted consists almost exclusively of the B epimer the form which exists in lactose The relevance of galactogen to the problem of the biosynthesis of lactose would be small were it not for the fact that in 1917 a galactogen differing slightly in properties from the galactogen of the snail was isolated from lung tissue of the bull (Wolfrom Weisblat Karabinos and Keller 1947) Since galactogen has now been found in animal tissues the question whether it occurs in the mammary gland assumes some importance As far as the writer knows no one has ever looked for galactogen in mammary tissue but it would seem to be worth while to examine this tissue for galactogen If it were found there the ·130 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION possibility that lactose could arise by a trans glycosidation reaction analogous to the levan synthesis, as formulated in Figure VI 3, would at once arise Galactogen would presuma FRUCTOSE ? FRUC - FRUC-FRUC- + (Levan) GLUCOSE VI Enzyme ( B subtils) SUCROSE GALACTOSE - GAL- GAL~ + (Galactogen) GLUCOSE Ji ? Enzyme (mammary gland) LACTOSE Fig VI 3 bly be formed from galactose a phosphate by the reversal of phosphorolysis but here again we encounter the problem of the formation of the p galactosıdıc linkage THE BIOSYNTHESIS OF MILK PROTEINS Let us turn now to consider briefly some recent work on the biosynthesis of the mill proteins The proteins of mill consist principally of the phosphoprotein casein which occurs nowhere but in milk together with relatively minor amounts of the whey proteins which can be considered as consisting of two main fractions, lactalbumin and lactoglobulin All three fractions are probably complex mixtures of proteins Since the mammary gland is a site of active synthesis of protein it is a suitable organ for studying the mechanism of protein synthesis ·BIOSYNTHESIS OF LACTOSE AND MILK PROTEINS 131 and we may anucipate that useful advances in this difficult field may well result from current studies on the mammary gland involving the use of labelled amino acids There seem to be four main possibilities to consider in respect of the mechanism of the biosynthesis of milk proteins Vilk protein may be synthesized entirely from the amino acids of the circulating blood or partly from these and partly from amino acids arising from the degradation of blood plasma protein in the mammary gland Alternatively the mill proteins might arise from blood plasma proteins by rearrangement of peptide chains involving transpeptidation reactions or they may be derived only in this manner and partly from blood amino acids Arterio venous studies carried out on the udders of lactating cows and goats have uniformly shown that there is an arterio venous difference in plasma amino acids across the lactating udder (Graham 1937 Shaw and Petersen 1938 Reneke et al 1939) The uptake is, however, small and it has been claimed, particularly by Shaw and Petersen (1938) that quantitatively it is insufficient to account for more than 40-50 per cent of the casein nitrogen of the milk It was principally because of the general acceptance of this latter belief that attention was directed to the possibility that some of the plasma proteins might be used by the udder for the synthesis of milk protein Arterio venous studies carried out by Reinehe et al (1941) led them to the conclusion that some fraction of the plasma protein probably a globulin fraction containing protein bound carbohydrate was utilized by the udder for the formation of milk protein However if plasma proteins are the sole source of the milk proteins the question arises whether the rate of regeneration of whatever fractions of the plasma protein are involved is sufficient to male good the appreciable drain on them due to the secretion from the body of considerable quantities of milk protein Such evidence as is available renders it doubtful whether this can be so Recent work has directed attention once more to the possi bility that a substantial proportion of the milk protein if not all of it is derived from the blood amino acids In the first place Bouckaert Oyaert Peeters and Sierens (1953) have ·132 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION made interesting studies of the utilization of amino acids by the perfused isolated udder of the cow Since in the course of a perfusion lasting about two hours the blood would be recircul ated through the udder at least ten times, they reasoned that if milk proteins are formed from blood amino acids it should be possible to detect a significant decrease in the anuno acid content of the perfusion blood during the course of the perfu $ion Using microbiological methods for the determination of various free amino acids they found considerable differences in the concentration of ten amino acids in the perfusion blood between the beginning and end of the perfusion (see Table Vir The uptakes were usually most marked in the case of LABLE VII Uptake of nine amino acids by the perfused, isolated bouine udder (from Bouckaert Oyaert Pecters and Sierens 1953) Left udder half Right udder half Cone at no addition to blood 158 casaminoacids added to blood Amino acid perfusion Conc at Uptak Conc at Uptake ug/ml end of during end of during perfusion perfusion perſusion perfusion wg/ml mg vg/ml mg Histidine 29 2 186 672 42 64 Isoleucine 123 96 32 4 410 Lucine 195 78 60 Lysine 149 53 80 173 544 Afethuanine 63 45 16 24 6 85 Phenylalanine 32 10 2 Bo Threonine 93 24 381 Tryptophane 72 8 138 Valine 34 5 20 ! 75 336 start of 96 448 109 ܙ ܢ ܚ ܀ ܘܶܗ ܗ 8 116 120 those amino acids which occur in the greatest concentration in the casein molecule It therefore seems reasonable to con clude that the disappcarance of these amino acids during the perfusion must have been due to their absorption by the udder This view is supported by the fact that the amounts of the various amino acids absorbed by the udder during the per ·BIOSINTHESIS OF LACTOSE AND VILL PROTEINS 133 fusion were considerably increased by the addition of a casein hydrolysate to the perfusion blood This worh appears to demonstrate that there is a selective uptake by the udder of those amino acids which occur in the greatest amounts in casein and provides good evidence that blood amino acids must be reckoned as major precursors of mill protein Recent studies with labelled amino acids have also led to a similar conclusion Barry (19526) injected (carboxy "C] lysane and (carboxy "C] tyrosine into a lactating goat and measured the specific radioactivities of these two amino acids isolated from the casein and from the plasma proteins at Vanous times after the injection He could thus comparc their decay curves with those depicting the specific radioactivities of the free lysine and tyrosinc of the blood plasma The curves for lysine, taken from Barry s paper, are shown in Figure VI 4 с.рт. 97 000 at O minuts 10 000 • Casein 1gsi 10 lysine of Hou plasma oplasma protein lysine Specif c activity of the carboxyl c bo 10 000 Hours after injection Fig VI 4 Radioactivity of the free lysine of plasma plasma protein lysine and of the casein lysine after an injection of C-lysine in a lactating goat (from Barry 1952) It will be seen that the lysine isolated from the casein of the mulk was, for some hours following the injection very much more radioactive than the lysine of the plasma protein More ·134 PHYSIOLOGY AND BIOCHEVISTRY OF LACTATION over, the curres show that the radioactivity of the lysine of the milk casein at a given time was equal to that of the free lysine of the blood plasma about 1 hours earlier This suggests that about 11 hours before a given casein molecule was secreted in the milk all its lysine had existed as free lysine in the blood plasma The curves for tyrosine behaved similarly to those for lysine and it seems reasonable to conclude from these findings that all the lysine and tyrosine of casein arises from the free lysine and tyrosine of the blood at least in the goat Taken in conjunction with the work of Peeters and his colleagues on the perfused bovine udder, Barry s results suggest that all the essential amino acids of casein are derived from the free amino acids of the blood plasma Essentially similar results for glycine, valine and lysine, all labelled with 14C have been obtained by Campbell and Work (1952) in experiments on lactating rabbits Here again the radioactivity of the milk proteins, following the injection of the labelled amino acid was much higher than that of the blood plasma proteins They further showed that when labelled plisma proteins obtained by injection of 14C glycine into a rabbit were infused into a second lactating) rabbit, the pro teins isolated from its milk were not appreciably labelled which also suggests that plasma proteins are not important precursors of milk proteins That the proteins of milk may not be derived wholly from plasma amino acids was, however, indicated by the fact that after injection of "C glycine, 14Cvaline and C-lysine, these acids could be ssolated with significantly higher specific activities from casein than from the whey proteins This suggested that the whey proteins might be denved partly from pepude fragments of plasma proteins a suggestion which is in harmony with the de tecuon by Vlorton (1950) of a transpepuduse in cow udder tissue However, it is equally possible, as Campbell and Work point out, to explain these results on the assumption that the whey proteins are synthesized mainly from the plasma amino acid pool, but also, to a small extent arise from plasma pro ten In support of this interpretation was thc later finding by Askonas, Campbell, Humphrey and Work (1954) that immunc globulin of rabbit mulk passes apparentlv unchanged from blood to milk, a finding wluch confirmed the suggestion made ·BIOSYNTHESIS OF LACTOSE AND MILL PROTEINS 135 long ago by Crowther and Rastrich (1910) Ashonas Camp bell and Work (1954) subsequently fractionated the whey proteins of the mill of a goat injected with radioactive glycine valine and lysine and found that, in the case of each acid, its specific activity at a given time after the injection was the same for casein as for B lactoglobulin indicating that both proteins were synthesized in the mammary gland from the same amino acid pool This finding, however, does not al together preclude the possibility that pre formed pepudes were used to an equal extent in the synthesis of these two proteins That this is improbable is shown by a further series of experiments in which Askonas Campbell Godin and Work (1955) found that the 14C labelled peptides obtained by partial hydrolysis of the casein and ß lactoglobulin isolated from goats milk after injection of radioactive glycine valine and lysine contained these amino acids labelled to the same extent as in the whole protein This uniforın labelling of the amino acids throughout the protein chain suggests that casein and ß lactoglobulin are synthesized wholly from free amino acids of the blood In conclusion it is worth while considering briefly what is known about the origin of the protein bound phosphorus which is present in the casein molecule in the form of phos phoserine The researches of Aten and Hevesy (1938) Barry (1952b) and of Colas, Le Bars Simonnet and Sternberg (1950), in which the radioactivities of vanous fractions of the milk phosphorus were studied after the injection of radioactive inorganic phosphate into lactating cows and goats leaves little room for doubt that the precursor of the casein phos phorus is the inorganic phosphate of the blood Simonnet and his colleagues however showed that radioactive inorgani phosphate continued to be secreted in the milk long after the casein had ceased to be radioactive This led them to put forward the view that phosphoserine is formed from a precur sor which is stored in the mammary gland They suggest that thus precursor may be phosphopyruvate from which phos phoserine could be formed by transamination It may be expected that further work on the mode of formation of milk proteins which is in progress in various laboratories may eventually provide evidence bearing on the ·136 PHYSIOLOGY AND BIOCHEMISTRY OF LACTATION rival theories of the mechanism of protein synthesis which are at present being debated The question is whether protein synthesis tahes place by the simultaneous condensation of amino acids at a "template composed of nucleic acids, as suggested by Dounce (1952), or whether the synthesis involves the formation of pepude chains as intermediates as postulated by those who support the transpepudation theory Campbell and Work (1953) discussed the possible beanng of some of their results on protein synthesis in the mammary gland on the idea that transpeptidation can occur in this organ Such an idea would be consistent with the above mentioned work of Morton (1950) and also with the results of Greenbaum and Greenwood (1954) who have shown that the cathepsin activity of rat mammary tissue incrcascs mar Ledly dunng lactation It is possible that intra cellular cat hepsins arc catalysts involved in transpeptıdation REFERENCES Ashonas BA Campbell PN Godus C and Work TS (1955) Biochem 61 105 Ashonas B A Campbell P N Humphrey J H and Work TS (1954) Brochem 7 56 597 Askonas B A Campbell P N and Work TS (1904) Biochem 3 58 326 Aten AH W jr and Heresy G (1938) Nature Lond. 142 111 Barry J M (1959a) Nature Lord 369 8,8 Barry J V (19526) 7 bol Chem 195 795 Bouchaert J HOyaert W Peeters G and Sierens G (19.3) Arch int Pharmacodyn 93 443 Campbell P N and Work TS (1952) Biochem 7 52 217 Campbell P N and Work TS (1953) Natwe Lond 171, 997 Caputto R Leloir L F Trucco RE Cardin C and Paladını AC (1949) J brol Chem 179 497 Caputto R Leloir LF Cardin C and Paladın AC (1950) 3 brol Chem 184 333 Caputto R. and Trucco R. E (19,2) Nature Lond 169 1061 Colas J Le Bars H Simonnet H and Sternberg J (1950) Ana Inst nal agron Parts 37 5 Cowic AT Duncombe HG Folley SJ French T H Glascock RF Mlassart, L. Peeters C and Popják, G (19.1) Biochem 7 Cronther C. and Raisinck H (1916) Brochen 10 434 Dimant E Smith V R. and Lardy HA. (1953) 7 biol Chenn 201 85 49 610 ·BIOSYNTHESIS or LACTOSE AND MILK PROTEINS 137 122 I 208 293 Doudorofi M (1945) Fed Proc 4 Doudoroff M and O Neal R (1945) biol Chem 159 585 Dounce A. L (1952) Enzymologia 15 25! Engelhardt VA (1950) Usp Sorrem Biol 29 60 Fischer HO L (1944 5) Haney Lect 40 156 Folley S J (1940) Biol Rr 15 421 Folley S J (1949) Biol Rev 24 316 Folley s J and French T H (1949) Brochem 1 45 117 French T H Popják G and Malpress F H (1959) Nature Lond 69 71 Friedmann R (1949) Biochem 44 117 Graham W R JT (1937) 3 brol Chem Grant GA (1935) Biochem 1 29 1909 Grant G A. (1936) Biochem 330_2027 Greenbaum A L and Greenwood FC (1954) Biochem 7 56 625 Hansen R G and Craine E1 (1954) 7 boil Chem Hassıd W 7 Doudoroff M and Barker HA (1944) 7 Amer chem Sot 66 1416 Heyworth R and Bacon J S D (1954) Brochem 5 58 XXIV Kalckar H M Braganca B and Munch Petersen (1953) Nature Lond 172 1038 Kittinger CW and Reithel FJ (1953) 3 brol Chem_205 527 koshland D jr (1954) in W D McElroy and B Glass The Ifechanism of Erme Action Baltimore Johns Hopkins Press p 608 Kuhn R Cauhe A and Baer HH (1954) Chem Bey 87 289 Leloir L F (1951) Arch Brochem Brophus 33 186 Malpress F H (1951) Collog Int CNRS LVI 1950 P 115 Malpress F H and Morrison AB (1950) Brochem 3 46 307 McClymont GL (1951) Aust 7 agric Res 2 158 McGeown M G and Míalpress F H (1952) Biochem 752 606 Morton RA (1920) Vature Lond 166 1092 Reincke E P Peterson V E Houchin O B and Turner CW (1939) Res Bull iſo agric Exp Sta No 296 Reineke E P Williamson M B and Turner CW (1941) 7 brol Chem 138 83 Reithel FJ Horowitz MG Davidson H M and hittinger GW (1952) 3 biol Chem 194, 839 Rutter W J and Hansen RG (1953) 7 brol Chem 202 323 Shaw JC (1946) Í Dairy Sa 29 183 Shaw J C and Knodt C B (1941) 7 brol Chem 138,287 Shaw JC and Petersen W E (1938) Proc Soc exp Brol NY 38 632 Stetten Dir and Klein B V (1946) brol Chem 165 157 Topper Y J and Stetten D jr (1951) 7 brol Chem 193 149 Trucco RE (1954) Mature Lond 174 1103 Trucco R E and Caputto R (1954) ) brol Chem 206 901 Trucco R E Caputto R Leloir L F and Mittelman N (1948) Arch Wolfrom M L Weisblat D I Karabinos J V and heller O (1947) Arch. Biochem Biochem 18 137

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·SUBJECT INDEX in Acetate as precursor of short chain fatty acids in ruminants milk, 99 100 incorporation of into lactose 120-121 intrarumunal infusion of in goat TOI intravenous infusion of in goat 100-101 utilization for fatty acid synthesis in mammary gland 98-116 utilization ut utro by mammary tissue of rurninants and non ruminants 101-102 Adrenal cortex relation of to anterior pituitary 16 24-25 33 53-62 role in gluconeogenesis 61 role in lactogenesis 24-25 33 role in mammary growth 6 16-17 Adrenal corticoids effect on mammary growth 6 16-17 maintenance of arginase les els by after adrenalectomy 62 role in galactopoiesis 53-57 role in lactogenesis 24-25 33 role in lipogenesis 114-116 Adrenalectomy effect on arginase content of rat mammary gland 59 62 effect on lactation 25 54-55 61-62 effect on lipogenesis in mammary gland 62 effect on morphology of mam mary gland 16-17 replacement studies after lactating rat 55-57 Adrenaline inhibitory effect of on milk ejection reflex 76-77 79 Adrenocorticotrophın (ACTH) effect on mulk yield of cow 53-54 role in galactopoiesis 47 52 53-54 role in lactogenesis 25 role in mammary alveolar growth 19-20 sce also Adrenal corticoids Aldosterone maintenance of lactation with after adrenalectomy 56-57 Allaline phosphatase content of milk effect of thyroid active substances on 65 69-70 Alveoli see Mammary alveoli Amino acids conversion of to mill fat 60 formation of milk protein from 120 130-136 possible conversion of to lactose 60 120 Aminopterin effect on mammary duct growth 11 Androgens possible role of in mammary growth 7 Aneurin, see Vitamin B Anterior lobe of pituitary see Pitui ·140 SUBJECT INDEX tary anterior lobe of Anti thyroid drugs effect of on mammary development 17 Antidiuretic hornone see Vaso pressin Arginase effect of adrenalectomy on tissue levels of 59 62 in liver 57 59 60 in mammary tissue 57-61 relation to gluconeogenesis 60 61 Arterio venous studies on lactose precursors 120-122 on milk fat precursors 94-96 108-109 on milk protein precursors 131 133 on respiratory quotient of mam mary gland 28 Ascorbic acid in milk effect of thy rozd active substances on 65 ga chlorohy drocortisone maintenance of lactation with after adrenalectomy 57 Cortisone effect on mammary grouth 16-17 effect on mammary slices in rutro 33 62 114-116 lactogenic effect of in hypophy sectomized ovariectomized animals 25 maintenance of lactation with after adrenalectomy 55-56 62 on Basket cells see Myoepithelium of mammary gland Blood plasma proteins as possible precursors of milk proteins 131-136 i deoxycorticosterone effect mammary growth 16-17 effect on mammary slices in vitro, 62 114-116 maintenance of lactation with after adrenalectomy 55-56 62 Diabetogenic activity association with galactopoiesis and growth 50 Double threshold theory of lactogenesis 34 39 42-43 Duct system of mammary gland effect of adrenalectomy on 16 effect of adrenal corticoids on gron th of 16 effect of oestrogens on growth of see Oestrogens effect of anterior pituitary hor mones on growth of 18 effect of thyroid active sub stances on 17 effect of thyroidectomy on 17 in gonadectomized animals 7 8 10, 11 16 mammogen theory of growth of 19 normal growth of 1 2 3 8 Carbohydrate conversion of to fat in udder 96-116 role of insulin in conversion of to fat 111-116 Casein biosynthesis of 130–136 detection of in udder tissue 25-26 protein bound phosphorus of 130 135 Cathepsins of mammary tissue 136 Effcctor contractele tissue of mam ·SUBJECT INDEX 141 mary gland see Myoepithelium of maminary gland Enzymncs of mammary gland allaline phosphatase 26-27 6y- 66 69 arginase see Anginase cathepsin 136 cytochrome oxidase 29 galactowaldenase 124-128 glutamic aspartic transaminase 31-32 glutamıc dehydrogenase 31-32 succinic oxidase, 29 transpeptidase 131 134 136 Equilibrium constant of relative mammary growth 3-6 11 Equipotentiality of male and female mammary gland for growth 8-9 12 Fatty acid synthesis in mammary gland from acetate 98-116 isotopic studies of 26-28 30-31 97-116 role of insulin in 111-116 Folic acid role in mammary duct growth II Galactose as lactose precursor 121-130 in carbohydrate complex of blood proteins 121-122 interconversion 17th glucose 124-127 Galactowaldenase possible role in biosynthesis of lactose 1'4-128 Gluconeogenesis 60-62 120 inhibition of by adrenalectomy 61 role of adrenal corticoids in 61 Glucose as precursor of glycerol of milk glycerides 110-111 as precursor of lactose 121-130 enzymic reaction with levan 129-130 interconversion with galactose 124-127 interconversion with mannose 122 oxidation of by direct oxida tion pathway in mammary gland 105 stipulating effect of on Lipogene sis from acetate int zitto 107- 103 utilization for fatty acid syn thesis 98 99 101-107 II- 115 Glycerides of milk 93-111 Glycerol of milk glycerides precursors of 110-111 Glycine incorporation into milk proteins 134-135 Glycolysis in mammary tissue in vitro 29 Glycoprotein of blood plasma as possible precursor of lactose I21-122 Galactogen possible role in lactose synthesis 129-130 Galactopoiesis connection with gronth and diabetogenesis 50 definition of 47 role of pituitary adrenal mech anism in 53-62 role of pituitary thyroid mech anism in, 62-70 role of prolactin in 46-52 role of somatotrophın in 50 ·142 SUBJECT INDEX Growth connection with galactopoiesis and diabetogenesis va of mammary gland see Mammary gland growth of Growth hormone of anterior pitui tary see Somatotrophin Hyperthyroidism effect on mammary development 17 Hypogalactia in women treatment of with prolactin 51 Hypoglycaemia effect on niih lactose content 122 Hypophysectomy effect on lactation 45-46 effect on lactogene is 23-25 mammary development in hypophysectomized animals 18-19 maintenance of lactation in the rat after 46 Hypothalamus control of pituitary function by lodocasein galactopoietic effect of, 66 See also Thyroid active sub stances L inositot as possible intermediate in lac tose synthesis 125 Lactalbumin 130 I actation artificial induction of 1 39-43 effect of adrenalectomy on 25 61-62 effect of adrenalectomy - ova riectomy on 54-55 effect of hypophysectomy on 4-46 inhibition of during pregnancy 34-43 localized initiation of by prolac tin 24 Lactogenesis 23-43 biochemical study of 25-43 effect of adrenalectomy on 25 clfect of hypophyscctomy on 23-25 hormonal control of at parturi tion 37-43 in hypophysectomized ovaries tomized animals 25 inhibition of by oestrogen and progesterone 31-39 43 role of anterior pituitary in 23-25 role of pituitary adrenal mecha nism in 24-25 role of prolactin in 23-25 raic of somatotrophin in 25 Lactoglobulin130 Lactose arterios enous studies of precur 30r3 of 120 biosynthesis of 119-130 cffect of hypoglycaemia on con centration of in milk 122 20 Hypothyroidism effect on mammary des clopment 17 Insulin anti insulin effect of adrenal cortex 114-11 effect of on Lipogenesis in mam mary tissue in viito 111-116 ellect of on respiration of mam mary tissue in tutto 112-116 hypoglycaemic action of 111- 112 Interncdın effect on mammary tissue in tra 52 In olution of mammary glands 47 75 ·SUBJECT INDEX 143 formation from glucose 121-130 incorporation of acetate into by isolated cow udder 120-121 nature of precursors of 119-130 neuramin lactose in marnmary gland 123 phosphate of in milk 129 possible formation of by trans glycosidation 123-124 129- 130 synthesis of from amino-acad residues 60-120 Levan enzymic reaction with glucose 129-130 Lipids of blood as precursors of milk fat 93 Lipogenesis in mammary gland 93-116 difference between ruminants and non ruminants 101-102 effect of fasting on 96 100-101 hormonal control of 111-116 isotopic study of 97-99 role of adrenal cortex in 33-34 114-116 role of insulin in 111-116 Luteotrophic action of prolactin 20 Luteotrophın see Prolactin Lysine incorporation into mull proteins 133-135 effect of prolactin on 15 19-20 24 effect of thyroid active substances on 17 effect thyroidectomy on, 17 in hypophysectomized ovanec tomized rats 19 in late pregnancy 20 mammogen theory of 18-19 Synergistic action of oestrogen and progesterone in SEE Oestrogens Progesterone Mammary alveoli effect of progesterone in prevent ing abnormal grov th of due to Ostrogen 14 histological abnormalities in due to oestrogens 13-15 measurement of mean diameter of 3 measurement of total surface area of 3 porosity index of 3 Mammary gland alkaline phosphatase activity of 65 69-70 ah eolar growth of see Mam mary als colar growth alı eolt of ste Mammary alveoli arginase activity of 57-61 arterio-venous studies of see Arteno venous studies duct system of sec Duct system of mammary gland equipotentiality of in male and female 8-9 12 fatty acad synthesis in, see Fatty acid synthesis in mammary gland galactophores ligauon of 47 gluconeogenesis see Cluconcogenesis growth of allometric 5-6 10 Mammary alveolar growth effect of adrenocorticotrophin (ACTH) on 19-20 effect of gonadectomy on 7 effect of oestrogens on 8 12-13 19-20 effect of progesterone on 8-9 19-20 ·144 SUBJECT INDEX 12 73-86 artificial induction of 12-15 quantitative method for 1 3 39-40 semi quanttative method for 2 effect of adrenal cortex on see Adrenal corticoids use of relauve growth analysis effect of anterior pituitary on for 3-7 10-11 sec Pituitary anterior lobe of Mit effect of oestrogens on see allaline phosphatase content of Oestrogens set Alkaline phosphatase con effert of progesterone on see tent of milk Progesterone glyceride fatty auds of, set isometric 5-7 Glycerides of milk pituitary thyroid mechanism withdrawal from mammary gland in 17-18 quantitative measurernent of Mill ejection hormone Measurement of rate of nature of 86-88 mammary gland develop Muk ejection reflex 73-96 ment conditionsng of 76 specific rate of 7 effect of severance of pituitary lipogenesis in see Lipogenesis stalk on 86 perfusion studies of 28 79-80 effect of vasoconstriction on 76 120-121 132-134 effector organ of 88-91 protein metabolism of 55 hormonal component of 86-88 respiratory activity of see Respi induction of by clectrical stimu ratory activity of mammary lation of supraopucohypotha gland lamic tract, 76 84-85 secretory cpithelium of see Alam inhibition of 76-77 78-79 mary alveol milling stimulus and 73 86 transplantation of 20 mimicking of by oxytocin in Mammogen theory 18-19 jection, 77-78 82-33 86-88 Mammotrophın see Prolactin neuroendocnne control of 77-88 Mannose role of mother in 73 as possible precursor of lactose suchling stimulus and 73-86 1 22 Milk fat in carbohydrate complex of biosynthesis of 26 93-116 blood proteins 122 effect of fasting on 96 100-yor spterconvertibility with glucosc cffect of thyroid acuve substances 122 on level of 64-65 69 Manometnc casurertients precursors of 93-36 of mammary gland metabolism synthesis from amino-acid resu in uro 29 ducs 60 Mieasurement of rate of malamnary Milk proteins gland de clopment biosynthesis of 120 130-136 equilibrium constant 3-6 11 Cascin 162 casern objectic techniques for 1 2 lactalbumin fraction of 130 ·SUBJECT INDEX 145 lactoglobulin fraction of 130 transpepudation in biosynthesis of 131 134, 136 whey protein 130 134-135 Milk secretion initiation of see Lactogenesis Milk yield assymetry of yield of halves of hormonally grown goat ud ders 14 effect of adrenocorticotrophin (ACTH) on 53-54 effect of oestrogens on 12-13 39-43 effect of prolactin on 48-49 effect of thyroid active substan ces on, 64-70 effect of unfractionated anterior pituitary extract on, 48-50 Milking stimulus 73-86 Myoepithelium of mammary gland 79 88-91 theory of lactogenesis 3+ 39 42-43 induction of pseudopregnancy by 20 inhubition of mammogeruc action of by glucocorticoids 16-17 inhibitory role of in lactation 34-38 39 43 lactogenic action of 35 mammary abnormalities due to 13-15 role in mammary alveolar growth 8 12-13 19-20 role in mammary duct growth I 8 11-12 stimulation of prolaction secre tion by 35 synergism with 11-deoxycorti costerone in promoting mam mary growth 16-17 synergısm with pituitary hor mones in promoting mammary growth 7 sy nergism with progesterone in lactogenic function 8-9 12- 13 15 34-38 43 triggering action of in artificial induction of lactation 40 41 43 use of crystal suspensions of in artificial induction of lactat tion 41 42 Ovarian hormones see Oestrogens Progesterone Ovariectomy effect on lactation 37 effect on lactation maintenance after adrenalectomy 54 Ovary hormones of See Oestrogens Progesterone Oxidative enzyme systems in mam mary gland see Respiratory activity of mammary gland Neuramin lactose in maintary tissue 123 Neuroendocrine reflex in regulauon of milk ejection 77-88 Nipple see Teat Non fatty solids of milk effect of thyroid active substances on 64-65 69 Nursing behaviour of mother 73 Oestrogens artificial induction of lactation by 39-43 artificial induction of mammary growth by 12-15 36 40 body feel of in relation to progesterone at partuntion 35 43 effect oa mill yield 12-13 39-43 in relation to double threshold ·144 SUBJECT INDEX artificial induction of 12-15% 39-40 effect of adrenal cortex on see Adrenal corticoids effect of anterior pituitary on see Pituitary anterior lobe of effect of oestrogens on ste Oestrogens effect of progesterone on see Progesterone isometric 5-7 pituitary thyroid mechanism in 17-18 quantitative measurement of see Measurement of rate of mammary gland develop ment specific rate of 7 lipogenesis in see Lipogenesis perfusion studies of 28 79 80 120-121 132-134 Protein metabolism of 55 respiratory activity of see Resp. ratory activity of mammary gland secretory epithelium of see Mam mary alveoli transplantation of 20 Mammogen theory 18-19 Mammotrophin see Prolactin Mannose as possible precursor of lactose 124 in carbohydrate complex of blood proteins 122 interconvertibility with glucose 122 Manometric measurements of mammary gland metabolism in vitro 29 Veasurement of rate of mammary gland development equilibrium constant 3-6 11 2 objective techniques for 1 quantitative method for 1 3 semi quantitative method for 2 12 use of relative growth analysis for 3-7 10-11 Mulk alkaline phosphatase content of sce Alkaline phosphatase con tent of milk glyceride fatty acids of see Glycendes of milk withdrawal from mammary gland 73-86 Milk ejection hormone nature of 86-88 Muk cjection reflex 73-91 conditioning of 76 effect of ses erance of pituitary stalk on 86 effect of vasoconstriction on 76 effector organ of 88-91 hormonal component of 86-88 induction of by clectrical stimu lation of supraopticohypotha lamic tract, 76 84-8, inhibition of 76-77 78-79 milking stimulus and 73-86 mimicking of by oxytocin in jection 77-,8 82-83 86-88 neuroendocrine control of 77-88 role of mother in 73 suckling sumulus and 73-86 Mulk fat biosynthesis of 26 93-116 effect of fasung on 96 100-101 effect of thyroid active substances on level of 64-65 69 precursors of 93-96 synthesis from amino-aad resi ducs 60 Velk proteins biosynthesis of 120 130-136 cascin SCE Cascin lactalbumin fraction of 130 ·SUBJECT INDEX 147 use of in artificial induction of lactation, 39-43 use of in artificial induction of mammary growth 36 40 5) nergistic action with oestrogens in lactogenic function 8-9 12-13 15 34-38 43 Prolactin effect on mammary alvcolar growth 15 19-20 24 effect on metabolism of mammary tissue in zitto 32 33 51 52 effect on milk yıld of cow 48-49 in lactogenesis 23-25 lactogeruc effect of in hypophysectomized ovariectomized animals 25 localized lactogenic effect of 24 luteotrophic effect of 20 mammogenic effect of 15 19 possible contamination of with intermedin 52 possible triggering action of in lactogenesis 46 role in lactation maintenance Respiratory activity of mammary gland in relation to galactopoiesis in utro 51 in relation to lactogenesis in vitro 26-34 in relation to lipogenesis 96-102 Respiratory measurements by arterio-venous techniques in TUO 28 96 on mammary gland slices in vitro see Respiratory activity of mammary gland on perfused isolated udder 28 120-121 Respiratory quotient of udder 8 96-98 101-102 of mammary gland slices 27-29 51 97-98 Smooth muscle in mammary gland possible role in mull ejection 88-91 Somatotrophin (STH) diabetogenic activity of 50 effect on mill yield (galactopoie tic effect) 50 lactogenic effect of in hypo physectomized ovariectomized animals 25 Specific growth rate 6-7 Steropried rats mammogenic effect of anterior pituitary extract in 18 Suckling stimulus effect of on maintenance of lac tation 46 effect of on prolaction secretion 47 effect of removal of on mill secretion 37 Teat role of teat sphincter in milk withdrawal 75 46-52 stimulation of secretion of by astrogen 35 stimulation of secretion of by suckling stimulus 47 treatment of hypogalactia by 51 Pseudo-pregnancy experimental lactogenesis during 25 induction of by oestrogen 20 Psychological influences in mammary development 20 in milk ejection reflex 76-77 88 Puberty mammary growth during 3 5 Relative growth analysis applied to quantitative study of mammary growth 3-7 10-11 ·146 SUBJECT INDEX Oxytocin assay methods for, 87-88 rolein milk ejection reflex, 76–77, 81-88 91 pituitary thyroid mechanism in mammary growth 17-18 pitutary thyroid mechanism in milk secretion 62-70 target glands of 16 effect of severance of pituitary stalk on milk ejection reflex 86 posterior lobe of antidiuretic effect of accom panying milk ejection reflux, 80 milk ejection activity of 77-88 oxytocic principle of, ste Oxy tocin Pitocin sec Oxytocin Pitressin sec Vasopressin Placenta assumption of pituitary role by no role in mamroary growth of 20 Porosity index of mammary alvcolar tusue, 3 Posterior lobe of pituitary Pituitary po.terior lobe of Posterior lobectomy maintenance of lactation after, 85 Progesterone body levels of in relation to oestrogen at parturition 35 43 in adrenal cortex 17 possible role of in lactation main tenance after adrenalectomy 54 pres ention of oestrogen induced mammary abnormalities by 13-15 role of in lactation inhibition 34-38 43 role of in mammary gronth 7 8 9 19 20 use of for induction of lactation in cow 40 41 use of Cor induction of lactation in spayed goat 40 41 Parabiosis experiments on perabiotic rats 17 Parturition mechanism of lactogenesis at 34-43 Perfusion of mammary gland 28 79-80 120-121 132-134 Phosphoserine origin of in casein molecule 135 Pigeon crop-gland stimulation by prolactin 23 assay of prolactin in 23 35 48 Pitocin, see Oxytocin Patressin see Vasopressin Patuitary anterior lobe of adrenocorticotrophin (ACTH) see Adrenocorticotrophin antı insulin effect of 53 effect of extracts of on milk yield 48-50 effect of suckling stimulus on 75 galactopoietic hormone com plex of 45-70 gonadotrophic function of 6 graſuing of 86 growth hormone of sec Soma totrophin hypothalatıc control of 20 in lactogenesis 23-43 in mammary growth 7 15-20 lactogenic hormone complex of 15–20 23-43 46 permissive role of in mam mary growth 18 pituitary adrenal mechanism in lactogenesis 24-25 SCC ·SUBJECT INDEX 147 use of in aruficial induction of lactation 39-43 use of in artificial induction of mammary growth 36 40 Synergistic action with oestrogens in lactogenic function 8-9 12-13 15 34-38 43 Prolactin effect on mammary alveolar growth 15 19-20 24 effect on metabolism of mammary tissue in euro 32 33 51 52 effect on yreld of cow 48-49 in lactogenesis 23-25 lactogenic effect of in hypophysectomized-ovancctomuzed anuals 25 localized lactogenic effect of 24 luteotrophic effect of 20 mammogenic effect of 15 19 possible contamination of with intermedın 52 possible triggering action of in lactogenesis 46 role in lactation maintenance 46-52 stimulation of secretion of by Ostrogen 35 stimulation of secretion of by suckling stuulus 47 treatment of hypogalactia by 51 Pseudo-pregnancy experimental lactogenesis during 25 induction of by oestrogen 20 Psychological influences in mammary development 20 in milh ejection reflex 76–77 88 Respiratory actavity of mammary gland in relation to galactopoiesis in miro 51 in relation to lactogenesis in vitro 26-34 in relation to lipogenesis 96-102 Respiratory measurements by arterio-venous techniques in CULO 28 96 on mammary gland slices in citto set Respiratory activity of mammary gland on perfuscd isolated udder 28 120-121 Respiratory quotient of udder 28 96-98 101-102 of mammary gland slices 27-29 51 97-98 Smooth muscle in maminary gland possible role in mulk ejection 88-91 Somatotrophin (STH) diabetogenic activity of 50 effect on muilk yield (galactopoie tic effect) 50 lactogenic effect of in hypo physectomized ovariectomized anuals 25 Specific growth rate 6-7 Steroprised rats mammogenic effect of anterior pituitary extract in 18 Suckling stimulus effect of on maintenance of lac tation 46 effect of on prolaction secretion 47 effect of removal of on mulk secretion 37 Teat role of teat sphincter in mill withdrawal 75 Puberty mammary growth during 3 5 Relatise growth analysis applied to quantitative study of mammary growth 3-7 10-11 ·148 SUBJECT INDEX Udder absorption of amino acids by 132-134 artificial induction of growth of I 12-15 39-43 effect of denervation of on milk ejection 79 fat metabolism of 95-96 respiratory quotient of 28 96- 98 101-102 urea produced by 60-62 Urea production by lactating udder 60 62 Uridine diphosphate galactose 195-127 Urdine diphosphate glucose 125-127 Terminology in lactational physiology 3 Testis effect on growth of mammary alveolar system 7 effect on growth of mammary duct system 7 Thyroid active substances comparison of galactopoietic ef fects of thyroxine with 3 5 3 truodo L thyronine 68-70 effect on alkaline phosphatase content of milh 65-66 effect on ascorbic acid content of milk 65 effect on degree of phosphoryla tion of vitamin B, in milk 65 effect on fat and non fatty solid content of milk 64-65 effect on mammary develop ment 17 galactopoietic effect of 62-70 'I ransamination in formation of the phosphose rine of casein 135 Trans glycosidation catalysis of by bacterial phos phorylase 128 possible role of in lactose for mation 124 128 reaction between glucose and levan 129-130 Transpeptıdation theory relation to milk protein synthesis 131 134 136 Tyrosine incorporation into milk proteins 133-134 Valine incorporation into milk proteins 134-135 Vasopressin mulh ejection activity of 80-82 86-88 91 Vitamin B, (Aneurin) effect of thyroxine administra tion on ratio of phosphoryla ted to free in milk 65 Walden inversion enzyne system for 124-125 in conversion of glucose to galac tose 124-125 Whey proteins 130 134-135 lactalbumin 130 Lactoglobulin 130 11 ·AUTHOR INDEX Numbers in italics refer to pages on uhich the author s name appears in a bibliography Abraham S 105 116 Achaya KT 95 116 Adamsons k 88 gr Andersson B 84 85 86 87 gr Appel H 96 116 Askonas BA 134 135 136 Astwood EB 7 21 Aten AH W jr 94 116 135 136 Atkinson WB 35 43 43 Averill SC 20 22 Azimov GJ 48 70 Boxer GE 111 118 Bradley TR 24 51 52 71 Brady RO 112 115 117 Braganca B 127 137 Braude R76 gr Brown MA 109 117 Burt AW A 68 69 70 70 Bacon J S D 123 137 Brer HH 122 137 Bailey GL 65 66 67 68 70 71 Balmain J H 26 29 30 31 32 33 43 51 52 70 97 98 102 103 104 112 113 114 115 116 116 117 Barcroft Sur Joseph 99 Barker HA 127 137 Barry GM 124 133 134 135 1,6 Bartlett S 65 66 67 68 69 70 70 Bassett E G 82 92 Bates RW 23 44 Baxter CF 109 117 Beeckmans ML 26 44 105 117 Benson GK 3 14 21 40 41 Cairy CF 15 22 40 44 Campbell PN 134 135 136 1,6 Caputto R 123 124 125 126 136 137 Cardını CE 194 136 Cary CA 93 117 Chashoff I L 105 116 Chalmers J R 66 7r Chamorro A 16 21 Chanda R65 71 Coignet J 63 77 Colas J 135 136 Cole RD 18 20 21 25 44 Coon J M 87 88 91 Cooper WD 86 87 9 Corner GW 15 r Cotes P M 48 49 50 53 71 Coussens R 79 80 88 91 9 Cowie A T 2 3 4 5 6 7 12 13 14 16 17 18 21 38 39 40 41 43 45 46 54 55 56 57 71 73 82 83 85 86 91 1,0 136 Cox, CP 3 14 1 40 41 43 113 116 Bergman AJ 47 71 Black, A L 109 117 Blatherwick NR 93 117 Blaxter K L 63 66 71 Bloch 99 102 III 117 Bohm H 96 116 Bottomley AC 12 27 Bouckaert J H 131 132 136 Craine EM 127 137 Crichton J A 48 49 50 53 71 Cross BA 73 76 77 ,8, 80 84 86 87 88 91 Crowther C Cutler 0 I 26 43 135 136 ·150 AUTHOR INDEX Dastus N N 95 96 1:8 Davidson HM, 122 128 137 Davis GK 94 127 Denamur R 82 gi Deschn L 63 71 De Vita J15 22 Dickens F 105 117 Dickson GT 66 71 Dimant E 121 124 126 137 Dodd M H I 81 82 Doudoroff M 127 129 137 Dounce AL 136 137 Drury DR 11 177 Duncombe W G 62 105 109 117 120 136 du Vigneaud V See Vigneaud V du Dyle H B van 87 88 gr Dylshorn S W23 44 105 106 107 110 111 112 113 114 115 116 116 117 1.0, 122 127 136 137 Fraenkel Conrat H 59 75 Franseen CC 18 21 French T H 26 27 28 29 30 44 97 98 101 102 105 106 107 108 109 110 112 116 117 120 122 124 136 137 Friedmann R 723 137 Elks J 66 71 Ellis G H 94 117 Elsden SR99 217 Ely F 76 79 91 Engel SL Engelhardt V A 129 137 Entenman C 110 118 Evans H M 59 71 Eiersole W J 55 56 71 Gaines W L 78 79 92 Gardner WV 25 26 27 28 Gauhe A 122 137 Gaunt R 24 44 55 56 71 Geschichter CP 72r Giraud P 63 7r Glascoch R F 26 31 43 62 97 98 102 103 104 105 rog Tro 515 113 114 115 116 116 117 120 136 Glock GE 105 117 Godin C 135 136 Gomez ET Godon S 88 92 Graham WRjr 28 44 60 62 64 71 93 94 96 117 120 131 137 Grant GA 122 123 137 Greenbaum AL 27 31 44 58 59 60 61 71 336 137 Greenwood C 31 44 136 137 Gregoire C 47 71 Gross J 68 71 Greier F 15 22 23 44 Gunn s 112 115 117 Guthkelch A N 3 21 88 91 Fautet E 38 43 Fischer HOL 125 237 Fishier C 110 18 Flux DS 3 6 12 14 16 21 40 41 42 43 53 54 55 7 Folley SJ 12 3 7 8 12 13 14 16 17 18 19 21 23 25 26, 27 28 29 30 31 32 33 34 35 37 38 39 40 41 42 43 44 47 48 49 50 51 52 53 54 56 58 59 60 61 62 63 64 65 66 67 68 69 70 70 71 73 81 82 83 85 86 95 97 98 for 102 103 104 Hammond J 15 21 Hansen RG 125 127 137 Hartis GW 73 84 86 91 Hassid y 7 127 137 Hems BA. 66 71 Hertoghe E 63 64, 7!

·- AUTHOR INDEX 151 kuhn R 123 137 Hertz R II Heuverswyn J van 16 21 Hevesy G 94 16 135 136 Heynorth R 123 137 Hilditch T P 95 99 100 107 116 117 Hirsch P F 105 116 Hodson AZ 94 117 Horowitz MG 123 128 137 Houchun O B 28 44 60 71 96 117 120 131 137 Houssay BA 85 gr Huffmann CF 15 22 40 44 Humphrey J H 134 136 Hunter GD 98 107 108 109 110 117 Hurst, V 17 22 Huxley J S 3 4 21 Landgrebe FW 5 71 Lardy HA 121 124 1.6 137 Leather JH 35 43 43 Le Bars H 135 136 Leech FB 68 71 Leloir LF 124 125 126 127 136 137 Lelong M 63 71 Leonard SL 16 18 Leppanen V 80 91 Lerman J 68 71 Lewis AA 9 13 21 Lewis J H 26 43 LICH 18 20 21 25 44 Liardet J 63 77 Lintzel W 93 94 117 Linzell J L 76 90 91 gr Lissitzky s 68 77 Ludwich TM 809 Luick J 109 117 Lukens FWD 115 117 Lyons WR 9 15 18 19 20 21 22 24 25 31 44 Ingelbrecht P 75 91 Jacobsohn D 17 21 73 86 91 Johnson, R. E 18 20 1 25 Johnston RF 26 21 Jones TSG 93 94 117 halckar HM 127 137 halliala H 80 gr Karabinos J V 129 137 Karvonen M J 80 gr Katsoyannus P G 88 92 lay HD 93 94 117 hel W 96 116 heller o 129 137 hendall E C 55 56 71 hittinger GW 123 128 137 Kleber M 109 117 Klein BV 126 137 Knodt CB 109 118 120 137 hon Sh 37 44 koshland D jr 125 137 hramer W 102 111 117 hrouze N 48 70 Malpress FH 13 14 21 34 38 39 43 44 99 100 117 122 123 124 126 129 137 Marin A I 100 117 Marshall FHA 15 21 Martin L. J P 107 108 117 Martinet J 87 91 Massart L 28 44 79 80 92 1 20 156 Maynard LA 94 117 Mergs E B 93 94 117 Meites 15 2 24 25 34 35 36 38 40 41 42 43 44 Michel R 68 70 Mitchell GM 52 77 Mitchell h. G 76 gr Mittelman N 124 137 Mixner J P 13 21 ·152 AUTHOR INDEX Moore RO 29 44 Morrison AB 123 137 Morton RK 134 136 137 Munch Petersen A 127 137 McCay CM 94 117 McClymont GL 100 117 1"0 137 McGeowrt MG 129 137 McGinty DA9 21 McLean P 105 117 McNaught VI 65 71 113 115 116 116 117 Recht P 38 44 63 72 Reece RP 18 22 25 33 44 Reeder CF 16, 22 Reid E 50 71 Reincke EP1) 22 40 44 96 117 120 121 131 137 Reithel FJ 123 128 137 Re.sler C 88 92 Richardson ho 2 3 13 14 21 22 38 39 43 73 89 90 91, 91 92 Riddle o 23 44 Rittenberg D 99 117 Roberts CW 88, 92 Roche J 63 68 71 Romani J, 38 44 63 72 Rowland S ſ 53 54 65 66 68 69 70 70 71 Roy A 53 72 Rutter Y J 125 137 Nagareda CS 56 71 Nathanson I T 18 21 Nelson H L 29 44 Nelson WO 17 ig 20 21 22 24 34 36 44 Newton 11 75 76 77 gr Newton NR 75 76 77 91 O Neal R 129 137 Ott I 77 97 Owen E C 65 77 Oyaert W 131 132 136 124 136 ? לל Scott J C Paladin AC Peeters G 28 44 79 80 88 91 92 120 131 132 134 136 Petersen, W E 28 44 76 79 80 gr 92 94 118 120 131 137 Peterson VE 28 44 96 117 120 131 137 Phillipson A T 99 117 Piana G 78 92 Pitt Rivers R 68 71 Plocq G 63 72 Popjak G 26 44 18 105 106 107, 108 109 110 111 117 118 120 124 136 137 Schafer EA 77 92 Scharf G 9 22 Schiller G 96 116 Schultze AB 732 Schweizer M 24 14 J Scott Watson H M 1 2 21 Selye H 39 44 46 47 72 Sgouru J T 36 38 43 44 Shaw DT 18 21 Shay J C 28 44 94 100 109 117 118 120 131 137 Sierens G 131 132 136 Silver M 2 5 6 7 9 10 11 12 Simpson M E 971 Simonnet H Smith AH 109 117 Smith J A B 95 96 118 Smith PE 85 92 Smith V R 121, 124 126 137 Smithcors JF 16 25 Speert H 16 22 Stemberg J 135 136 13 736 Raktrick H 135 136 Rausch Eo 7, 21 Ray E 20 22 ·AUTHOR INDEX 153 Usuelli F 78 92 van Dyke HB See Dyhe HB Stetten De Witt jr 111 118 125 126 137 Stonecipher # D 96 117 Strahan R 88 92 Stncher P 15 20 22 23 44 Swan J M 88 92 Swanson EW 90 92 Sykes JF 7 15 22 Van van Heuverswyn J See Heuvers wyn J van Vigneaud V du 8o 87 88 92 T Teissier G 3 4 Terner C 109 118 Tgetgel B 73 74, 92 Thompson HV 95 117 Thompson S Y 65 66 70 Tietz A 109 110 117 118 Tindal J S 56 71 Tolbert BM 109 117 Tomich E G 68 72 Topper I J 125 137 Trentin J J 15 16 17 19 22 25 44 Trucco R E 123 124 125 126 127 136 137 Turner CW 7 9 13 16 17 18 19 21 22 24 25 28 35 36 40 4 44 47 60 71 82 86 87 90 92 96 117 120 131 137 Waller H 76 9" Waring H 88 92 Watson, SC 62 75 Weisblat DI 129 137 White P 63 64 65 77 Whittlestone WG 76 82 8, 9° Williams WL 47 72 Williamson MB 121 131 137 Wolfrom VL 129 137 Woollett EA 68 72 Work TS 134 135 136 136 Wrenn T R 7 15 2 loung F G 25 44 47 48 49 50 53 63 71 7 118 Zilversmit, D B 110 Zuckerman S 3 27 121

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