Book - Sex and internal secretions (1961) 10
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SECTION C Physiology of the Gonads and Accessory Organs
The Mammary Gland and Lactation
A. T. Cowie and S. J. Folley
National Institute For Research In Dairying, Shinfield, Reading, England
I. Introduction
This account of the hormonal control of the mammary gland is in no way intended as an exhaustive treatment of mammary gland physiology, but rather an attempted synthesis of current knowledge which it is hoped will be of interest as an exposition of the authors' conception of the present status of the subject. Since the publication of the second edition of this book, the emphasis in the field under review has tended to shift towards the development of quantitative techniques for assessing the degree of mammary development, towards attempts at a ])enetration into the interactions of hormones with the biochemical mechanisms of the mammary epithelial cells, and towards an increasing preoccupation with the interplay of nervous and endocrine influences in certain phases of lactation. The reader's acquaintance with the classical foundations of the subject as described in the second edition of this book (Turner, 1939) and in other subsequent reviews (Follcy, 1940; Petersen, 1944, 1948; Folley and Malpress, 1948a, b; Mayer and Klein. 1948, 1949; Follev, 1952a, ]9r)6; Dabelow. 1957) will therefore be assumed and used as a point of departure for the present account which can most profitably be concerned mainly with developments which have occurred since the last edition was published. Reference will freciuently be made to these reviews in which authority will be found for the many ex cathedra statements that will be made, but original sources will be cited wherever appropriate.^
As an aid to logical treatment of the subject the scheme of classification proposed by Cowie, Folley, Cross, Harris, Jacobsohn and Richardson (1951) will be followed in this chapter. Besides introducing a system of terminology in respect of the physiology of suckling or milking, these writers have put forward a classification scheme which is an extension of one previously proposed by one of the present authors (Folley, 1947). This scheme considers the phenomenon of lactation as divisible into a number of phases as follows:
[ [Milk synthesis
I Milk secretion ■! Passage of milk from I I the alveolar cells
Lactation<J [Passive withdrawal of
ij milk
JThe milk-ejection re[ Hex
Milk removal
I
As is logical and customary, discussion of lactation itself will be preceded by consideration of mammary development.
II. Development of the Mammary Gland
A. Histogenesis
References to the earlier work on the histogenesis of the mammary gland in various species will be found in Turner ( 1939,
^ Within the last 10 years there have been several symposia devoted to the problems of the physiology of lactation. The proceedings of these symposia have been published: Mecanisme physiologie de la secretion lactee. Strasbourg, 1950, Colloqvies Internationaux du Centre National de la Recherche Scientificiue. XXXII, 1951, Paris; Svmposium sur la physiologie de la lactation, Montreal, 1953, Rev. Canad. Biol., 13, No. 4. 1954; .Symposium sur la physiologie de la lactation, Brussels, 1956, Ann. endocrinol. 17, 519; A Discussion on the Physiology and Biochemistry of Lactation. London. 1958, Proc. Roy. Soc, .ser. B, 149, 1952,) and Folley (1952a). There have also been studies on the opossum (Plagge, 1942) , the mouse and certain wild rodents (Raynaud, 1949b), the rhesus monkey (Speert, 1948), and man (Williams and Stewart, 1945; Tholen, 1949; Hughes, 1950).
A question which in the last decade has been receiving attention is whether the prenatal differentiation and development of the mammary primordium is hormonally controlled. According to Balinsky (1950a, b), the mitotic index of the mammary bud in the embryo of the mouse and rabbit is lower than that of the surrounding epidermis and he concludes that differentiation of the bud is due not to cellular proliferation (growth) but to a process of aggregation ("morphogenetic movement") of epidermal cells. This author also reports that for some time after its formation, the mammary bud is cjuiescent as regards growth, thus exhibiting negative allometry compared with the whole embryo, until the sprouting of the primary duct initiates a phase of positive allometry. The cjuestion is, what is the stimulus responsible for the onset of this allometric phase? Is the growth and ramification of the duct primordium, like that of the adult duct system, due to the action of estrogen emanating from the fetal gonad or from the mother?
Hardy (1950) has shown that dift'erentiation and growth of the mammary bud of the mouse could proceed in explants from the ventral body wall of the embryo, cultured in vitro, even when no primordia were present at the time of explantation (10-day embryo). Primary and then secondary mammary ducts and a streak canal differentiated and a developmental stage similar to that in the 7-day-old mouse could be reached. Balinsky (1950b) was also able to observe the formation and growth of mammary buds in approximately their normal locations in a minority of cases in which body-wall explants of 10-day mouse embryos were cultivated in vitro. Discounting the rather remote possibility that the effects were due to minute amounts of sex hormones present in the culture media, these observations indicate that hormonal influences are not necessary for the prenatal stages of mammary develo]iment, and in accord with this Balinsky ( 1950b j found that addition of estrogens or mouse pituitary extract to the culture medium had no effect on the growth of the mammary rudiment in vitro.
On the other hand, extensive studies by Raynaud (1947c, 1949b) of the sex difference in the histogenesis of the mammary gland in the mouse, first described by Turner and Gomez (1933), indicate that the mammary rudiment is sensitive to the influence of exogenous gonadal steroids during the prenatal stages. The mammary bud in the strain of mouse studied by Raynaud shows no sex differences in development until the 15th to 16th day at which time the genital tract, hitherto indifferent, begins to differentiate. Coincident with this the mammary bud in the male becomes surrounded by a condensation of special mesenchymal cells the action of which constricts the bud at its junction with the epidermis from which it ultimately becomes completely detached (Fig. 10.1). The inguinal glands seem particularly susceptible to this influence because they exhibit this effect earlier than the thoracic glands and in some strains the second inguinal bud in the male tends to disappear completely. Sex differences in the prenatal development of the mammary rudiment in certain species of wild mouse were also described by Raynaud (1949b).
The fact that, after x-ray desti'uction of the gonad in the 13-day male mouse embryo, the mammary bud remains attached to the epidermis and the duct primordia ramify in a manner similar to the primordia in the female shows that this phenomenon of detachment of the mammary bud is due to the action of the fetal testis (Raynaud and Frilley, 1947, 1949). That the masculinizing action of the fetal testis seems to be due to the hormonal secretion of a substance having the same effect as testosterone is suggested by the fact that injection of testosterone into the pregnant mother causes the mammary buds in the female embryo to undergo the male type of development (Fig. 10.1). Here again the inguinal glands seem most sensitive because sufficiently high doses in many cases cause complete disappearance of the primordia of the second inguinal glands (Raynaud, 1947a. 1949a).
On the other hand, destruction of the fetal gonad in the female has no effect on the development of the mammary bud (Raynaud and Frilley, 1947, 1949), yet the lattW is not completely indifferent to the action of estrogen because high doses of estrogen administered to the mother, or lower doses injected early into the embryo itself inhibit the growth of the mammary bud (Raynaud. 1947b, 1952; Raynaud and Raynaud, 1956, 1957), an effect reminiscent of the well known action of excessive doses of estrogen on the adult mammary duct system (for reference see Folley, 1952a) . In pouch young of the opossum, on the other hand, Plagge (1942) found that estrogen treatment stimulated growth of the mammary duct primordia. Similarly in the fetal male mouse low doses of estrogen stimulate growth of the mammary bud (Raynaud, 1947d), but this may be an indirect effect ascribable to estrogen's antagonizing the inhibitory action of the fetal testis.
The problem of the histogenesis of the teat has also come under experimental attack. Raynaud and Frilley (1949) showed that the formation of the epithelial hood," the circular invagination of the epidermis surrounding the mammary bud which constitutes the teat anlage in the mouse, is not hormonally determined since its appearance was not prevented by the irradiation of the fetal ovary at the 13th day of life. In the male mouse the epithelial hood does not normally appear and the male is born without teats. This is undoubtedly due to the action of the fetal testis inasmuch as the teat anlagen develop in the male embryos whose testes are irradiated at 13 days (Raynaud and Frilley, 1949).
The foregoing observations jioint to an ahormonal type of development for the teat and mammary bud in the female fetus, at least in the mouse, although the mammary bud is specifically susceptible to the action of excess exogenous estrogen which can inliibit its development without affecting that of other skin gland ])rimordia. The mammary hud is a'so sus('ei)tible to the action of anch'ogen which in the normal male fetus not only dii-ects its development along charact(M-istic lines, but also suppresses the formation of the teat.
Fig. 101. Sex difference in the development of the mammaiy bud of the fetal mouse and
effect of androgen on the histogenesis of the female mammary bud. A. First inguinal gland
of female fetus (15 days, 17 hours). B. First inguinal gland of male fetus (15 days, 17 hours).
C. Second inguinal gland of female fetus (15 days, 16 hours) from a mother receiving testosterone propionate. D. First inguinal gland of female fetus from the same litter as that in C.
(From A. Ravnaud, Ann. endocrinol., 8, 248-253, 1947.)
For further information on the morphogenesis of the mammary ghmd, the reader is referred to the recent detailed accounts by Dabelow (1957) and Raynaud (1960).
B. NormaL Postnatal Development
1. Methods of Assessing Mammary Development
In the last two decades the increasing availability of the ovarian hormones in pure form and the prospect of the large scale practical application of fundamental knowledge of the hormonal control of the mammary gland to the artificial stimulation of udder growth and lactation in the cow, have together effected a demand for greater accuracy in studying and assessing the degree of mammary development. Various quantitative and objective procedures have now been evolved which allow results of developmental studies to be subjected to statistical investigation. These methods have been reviewed recently (Folley, 1956) and we need but mention them briefly.
In those species in which, save in late pregnancy, the mammae are more or less flat sheets of tissue, the classical wholemount preparations have been the basis for several quantitative studies. From such preparations the area covered by the duct systems can be measured by suitable means (e.g., as in our studies on the rat mammary gland; Cowie and Folley, 1947d), thus providing an accurate measure of duct extension. Such measurements, however, give no information on the morphologic changes within this area and so a semiquantitative scoring system to assess the degree of duct complexity has been used in conjunction with the measurements of area (see Cowie and Folley, 1947d) . More reliable and objective techniciues for measuring duct complexity were later developed in our laboratory by Silver (1953a) and Flux (1954a). Species such as the guinea pig in which the gland, even when immature, is three-dimensional demand other methods. For such cases a precise but rather tedious method has been described by Benson, Cowie, Cox and Goldzveig (1957) which involves the determination of the volume of glandular tissue from area measurements of serial sections of the gland in conjunction with semiquantitative scoring procedures for assessing the morphologic characteristics of the tissue.
Particularly applicable to the lactating gland is the procedure developed by Richardson (see Cowie, Folley, Malpress and Richardson, 1952; Richardson, 1953) for assessing the total internal surface area of the mammary alveoli. It is of interest to note in passing that this technique is based on that developed by Short (1950) for measuring the surface area of the alveoli in the lung, the similarity in the geometry of the two organs allowing ready transference of the method from one to the other.
At present these quantitative procedures have the disadvantage of being slow and time consuming, and it seems likely that their further development will involve the use of electronic scanning methods to speed up the examination of the tissues. Of recent introduction are some biochemical procedures for assessing changes in mammary development. The desoxyribonucleic acid (DNA) content of any particular type of cell is said to be remarkably constant (see Vendrely, 1955, for review) and the amount of DNA in a tissue has been used as a reference standard directly related to the number of cells present in a tissue and to provide an estimate of the number of cells formed during the developmental phases of a gland or tissue (see Leslie, 1955, for review). Studies on DNA changes which occur in the mammary gland during pregnancy and lactation have been made in the rat by Kirkham and Turner (1953), Grecnbaum and Slater (1957a), Griffith and Turner (1957), and Shimizu (1957). It should be noted, however, that some authorities have doubts as to the constancy under all conditions of the DNA content of a cell (see Brachet, 1957) and results obtained by this technique should be interpreted with some caution (see also Griffith and Turner, 1957). Other chemical methods for assessing mammary development include (a) the determination of the iron content of the gland, based on the observation that iron retention occurs in the epithelium of the mammary glands of mice (Rawlinson and Pierce, 1950) ; (b) whole-mount autoradiographs using P^(Lundahl, Meites and Wolterink, 1950) ; and (c) determination of the total content of alkaline phosphatase in the mammary gland (Huggins and Mainzer, 1957, 1958).
In view of the relative rapidity of the biochemical methods it seems likely that they will be used increasingly in the future.
A technique of clinical interest allowing the qualitative assessment of changes in mammary structure in the breast of pregnant and lactating women is the radiographic method described by Ingleby, Moore and Gershon-Cohen (1957).
To those seeking information of the microscopic anatomy of the human mammary gland we would recommend the excellent and beautifully illustrated review by Dabelow (1957), and new facts on the cytologic changes occurring during milk secretion will be found in the electron microscopic study of the rat mammary gland by Bargmann and Knoop (1959), and of the mouse mammary gland by Hollmann (1959).
Having briefly outlined the various quantitative methods of assessing mammary development we will now consider recent studies on normal mammary growth.
2. Mammary Development in the Non pregnant Female
It has been the general belief that until puberty the mammary ducts show little growth, but more precise studies in which the rate of increase in mammary gland area has been related to the increase in body size have now shown that in the monkey, rat, and mouse a phase of ra])id duct growth is initiated before puberty.
The first use of this procrdure, relative gi'owth analysis (for terminology see Huxley and Teissier, 1936), for the quantitative investigation of mammary duct growth was made by Folley, Guthkelch and Zuckerman (1939), who showed that over a wide range of body weights, the breast in the nonpregnant female rhesus monkey grows faster than the body as a whole. Subsequently, more detailed studies of the dynamics of mammary growth using relative growth
Fig. 10.2. Relative mammary gland growth in the female hooded Norway
Cowie. J. Endocrinol.. 6, 145-157, 1949.)
(From A.T.
analysis were made in the rat by Cowie (1949) and Silver (1953a, b) and in the mouse by Flux (1954a, b), and their results will now be summarized. In the rat the total mammary area increased isometrically with the body surface (a = 1.1 as compared with the theoretic value of 1.0) until the 21st to 23rd day when a phase of allometry (a = 3.0) set in. The onset of the allometric phase could be prevented by ovariectomy on the 22nd day (see Fig. 10.2). Since estrous cycles do not begin until the 35th to 42nd day in this strain of rat, it is clear that the rapid extension of the mammary ducts began well before puberty. In the immature male rat the increase of mammary area on body surface was slightly but significantly allometric; this was not altered by castration at the 22nd day. Earlier ovariectomy, i.e., when the pups were 10 days old, was followed by a phase of slightly allometric growth of the mammary glands in the fe
males (a = 1.5). With regard to the female
mouse (CHI strain) a i)hase of marked allometry in mammary duct growth set in
about the 24th day (a = 5.2) which could
also be prevented by prior ovariectomy.
It is clear that the presence of the ovary is essential for the change from isometry to allometry, but the nature of the mechanisms governing the change is still uncertain (for further discussion, see Folley, 1956).
3. Mammary Growth in the Male
The testes have apparently little effect on mammary duct extension in the rat inasmuch as the gland in the male grows isometrically or nearly so and its specific growth rate is unaffected by castration. Castration at 21 days, however, does prevent for a time development of the lobules of alveoli, first described by Turner and Schultze (1931 ) , which are characteristic of the mammary gland in the male rat. Eventually however, some alveoli do develop in the mammae of immaturely castrated male rats (Cowie and Folley, 1947d; Cowie, 1949; Ahren and Etienne, 1957) and it has been ])Ostulated that these arise from the enhanced production by the adrenal cortex of mammogenic steroids (androgens or progesterone) due to the hormone imbalance brought about by gonadectomy (see Folley, 1956)
In a recent study, Ahren and Etienne
(1957) have shown that the ducts and alveoli in the mammary gland of the male rat
are remarkable in that their epithelial lining
is unusually thick, being composed of several layers of cells. It had been previously
noted by van Wagenen and Folley (1939)
and Folley, Guthkelch and Zuckerman
(1939) that testosterone caused a thickening
of the mammary duct epithelium in the
monkey and sometimes papillomatous outgrowths of epithelium into the lumen of the
duct. It would thus seem that, although the
hormone of the testis is capable of eliciting
alveolar development, these alveoli and
ducts differ from those occurring in the female in the nature of their epithelium. It
w^as further observed by Ahren and Etienne
(1957) that in the castrated male rat the
alveoli, which eventually developed, had a
simple epithelial lining somewhat similar to
that seen in the normal female rat, suggesting that, if the adrenals are responsible, the
mammogenic steroid is more likely to be
progesterone than an androgen.
A study of considerable clinical interest is that of Pfaltz (1949) on the developmental changes in the mammary gland in the human male. The greatest development reached was at the 20th year; by the 40th year there occurred an atrophy first of the l)arenchyma and later of the connective tissue. In the second half of the fifth decade there was renewed growth of the parenchyma and connective tissues. The hormonal background of these changes and the possible relationship with prostatic hyjiertrophy are discussed by Pfaltz. (Further details of the microscopic anatomy of the mammary gland of the human male may be found in the studies by Graumann, 1952, 1953, and Dabclow, 1957.)
4. Mammary Development during Pregnancy
It has been customary to divide mammary changes during pregnancy into two phases, a phase of growth and a secretory phase. In the former there occurs hyperplasia of the mammary parenchyma whereas, in the latter, the continued increase in gland size is due to cell hypertrophy and the distension of the alveoli with secretion (see Folley, 1952a j . Although it was realized that these two phases merged gradually, recent studies have confirmed earh^ reports {e.g., those of Cole, 1933; Jeffers, 1935) that a wave of cell division occurs in the mammary gland towards the end of parturition or at the beginning of lactation. Al'tman (1945) described a doubling in number of cells per alveolus, in the mammary gland of the cow at parturition, but the statistical significance of his findings is difficult to assess. More recently, how^ever, Greenbaum and Slater (1957a) found that the DNA content of the rat mammary gland doubled between the end of pregnancy and the 3rd day of lactation, a finding which they interpret as resulting in the main from hyperplasia of the gland cells. Likewise in the mouse mammary gland, Lewin (1957) observed between parturition and the 4th day of lactation a great increase both in the DNA content of the mammary gland and in the total cell count. Studies on the factors controlling this wave of cell division are awaited with interest. Also associated with the onset of copious milk secretion is a considerable increase in cell volume and coincident ally the mitochondria elongate and may increase in diameter (Howe, Richardson and Birbeck, 1956). Cross, Goodwin and Silver (1958) have followed the histologic changes in the mammary glands of the sow, by means of a biopsy technique, at the end of pregnancy, during parturition, and at weaning. At the end of pregnancy there was a ])i'()gr('ssi\-c' distension of the alveoli, the existing hyaline eosinoi)hilic secretion within the alveoli was gradually replaced by a basophilic material, and fat globules appeared. At i)arturition the alveoli were contracted and their walls appeared folded (Fig. 10.3).
Fig. 10.3. Sections of biopsy specimens from the mammary gland of a sow before and din-ing parturition. A. Six days before parturition: the mammary alveoh are small and contain a nongranular eosinophilic secretion. B. Two days before parturition: alveoli have increased in size and fat globules are conspicuous. C. Fifteen hours before parturition: alveoli are now distended with secretion which consists of an outer zone of eosinophilic material and fat globules, and a central zone of basophilic granular secretion. D. During parturition: alveoli contracted with folded epithelium and sparse secretion. (From B. A. Cross, R. F. W. Goodwm and L A. Silver, J. Endocrinol., 17, 63-74, 1958.)
5. Mammary Involution
The involutionary changes which occur in the mammary gland after weaning in various species were described in the previous edition of this book (Turner, 1939) and in a later review by Folley (1952a). Since that time, a few further studies have appeared.
There is evidence that the course of the histologic changes in the regressing mammary gland may differ according to whether the young are weaned after lactation has reached its peak and is declining, or whether they are removed soon after parturition, when the effects of engorgement with milk seem to be more marked (see, for example, Williams, 1942, for the mouse). In rats whose young were weaned soon after parturition Silver (1956) was able to re-establish lactation provided suckling was resumed within 4 or 5 days; after that time irreversible changes in the capillary blood supply to the alveoli had set in. A further point arises from a study on the cow by Mosimann (1949) which indicates that the course of the regressive changes in a gland which has undergone one lactation only may differ from those seen in glands from muciparous animals. Oshima and Goto (1955) have used quantitative histometric methods in a study of the involuting rat mammary gland ; the values which they obtained for the percentage parenchyma 7 to 10 days after removal of the young agree quite well with tiiose reported by Benson and Folley ( 1957b) for rats weaned at the 4th day and killed 9 days later.
The biochemical changes occurring in mammary tissue during involution arc of some interest and have been studied in our laboratory by McNaught (1956, 1957). She studied mammary slices taken from rats whose young were removed at the 10th day and also slices from suckled glands, the escajie of milk from which was prevented by ligation of the galactophores, the other glands in the same animals remaining intact and serving as controls. Her results, some of whichare summarized in Figure 10.4, suggest that functional changes which may be taken as indicative of involution (decrease in oxygen up-take, respiratory quotient (R.Q.), and glucose up-take; increase in lactic acid prcxUiction ) are seen as early as
8 to 12 hours after weaning. Continued suckling without removal of milk retards the onset of these changes, but only for some hours. Injections of oxytocin into the rats after weaning (see page 607) did not retard these biochemical changes. Essentially simihii' results were independently reported by Ota and Yokoyama (1958) and Mizuno and Chikamune (i958).
C. Experimental Analysis Of Hormonal Influences
1. Ovarian Hortnones in the Animal with Intact Pituitary
We shall see later (page 602) that the mammogenic effects of the ovarian hormones are largely dependent on the integrity of the a'nterior pituitary and thus to analyze accurately the role of hormones in mammary development it is necessary to use hypophysectomized animals. Information of considerable academic and practical importance has been obtained, however, from studies in the animal with intact pituitary and these we shall now consider.
Early studies involving hormone administration pointed to the conclusion that estrogens were in general resi)onsible for the growth of the mammary (hicts, whereas progesterone was necessary for complete lobulealveolar growth (see reviews, l)y Turner, 1939; Folley and Malpress, 1948a; Folley, 1952a). The foundation for i^liis general statement is now more sure, for as a result of experimental studies over the last 10 years, what seemed to be exceptions to this generalization have been shown to be otherwise. In some species (mouse, rat, guinea \)ig, and monkey) it is true that progesterone alone, if given in sufficiently large doses, will evoke duct and alveolar development in the ovariectomized animal, but this is probably a pharmacologic rather than a physiologic effect. There are great differences in the response of the mammary ducts to estrogen and on this basis it has become usual to divide species into three broad categories (see FoUey, 1956). It is, however, necessary to add the warning that in the estrogentre.'ited spayed animal progesterone from the a(h'eiial eoiiex may synergize with the exogenous estrogen (see Folley, 1940; Trentin and 1'ui'iier, 1947; Hohn, 1957) and it maybe that the I'eal basis for the categories is to be found largely in differences in endogenous progesterone production by the adrenal cortex.
Fig. 10.4. Oxygen uptake, respiratory quotient, glucose uptake, and lactic acid production of mammary gland slices from lactating rats killed at various times after weaning (A — A) and from rats in which svickling was maintained, but in which the galactophores of certain glands were ligatured (• •) to prevent the escape of milk, the nonligatured glands (O O) acting as controls. (Courtesy of Dr. M. L. McNaught.)
The first category comprises those in which estrogens, in what are believed to be physiologic doses, evoke primarily and mainly duct growth; alveoli may appear, but only if high doses are given and the administration is prolonged. Examples of this class are the mouse, rat, rabbit, and cat. Silver (1953a), using the relative-growth technique, has obtained information on the
levels of estrogen necessary for normal mammary duct growth in the nonpregnant rat. In the young ovariectomized rat, the normal mammary growth rate was best imitated by injecting 0.1 ;u,g. estradiol dipropionate every second day (from 21 days of age) and increasing the dose step- wise with body weight. In the ovariectomized mouse, Flux (1954a) found it necessary to give 0.055 /jLg. estrone daily to attain mammarv duct growth comparable with that obser\-( . i in intact mice.
In the second category are those s]:»ecies in which estrogen in physiologic doses causes growth of the ducts and the lobule-alveoL^r system, the classical example being the guinea pig in which functional mammae can be developed after gonadectomy in either sex by estrogen alone. A recent study by Hohn (1957), however, strongly suggests that progesterone from the adrenal cortex participates in the effect. The earlier view, moreover, that complete mammary growth can be evoked in the gonadectomized guinea l)ig by estrogen alone (Turner and Gomez. 1934; Nelson, 1937.) does not find support in the recent study of Benson, Cowie, Cox and Goldzveig (1957), who, using both subjective and objective methods of assessing the degree of mammary development, found that over a wide dose range of estrone, further development of the mammary gland was obtained when jirogesterone was also administered; essentially similar conclusions have been reached by Smith and Richterich (1958).
Also in this second category are cattle and goats in which, however, the male mammary gland is not equipotential with that of the female. The early studies on these species have been reviewed at length by FoUey and Malpress (1948a) and Folley (1952a, 1956). Briefly it may be said that these studies clearly showed that estrogen alone induced extensive growth of lobule-alveolar tissue of which the functional capacity was considerable although the milk yields in general were less than those expected from similar animals after parturition. The response to estrogen treatment was, moreover, very erratic. It was generally believed that the deficiencies of this treatment could be made good if progesterone were also administered, a view supported by the observations of Mixner and Turner (1943) that the mammary gland of goats treated with estrogens, when examined histologically, showed the i)resence of cystic alv(>oli, an abnormality which tended to disappear when jirogestcrone was also administered.
When progesterone became more readily available, an extensive study of the role of estrogen and progesterone in mammary development in the goat was carried out (Cowie, Folley, ^lalpress and Richardson.
1952; Benson, Cowie, Cox, Flux and Folley, 1955). The mammary tissue was examined histologically and the procedure devised by Richardson (see page 594) used to estimate the area and "porosity" of the alveolar epithelium. The udders grown in immaturely ovariectomized virgin goats by combined treatment with estrogens and progesterone in various proportions and at different absolute dose levels were compared with udders resulting from treatment with estrogen alone. As in the earlier observations of Mixner and Turner (1943) , histologic abnormalities were noted, the more widespread being a marked deficiency of total epithelial surface, associated with the presence of cystic alveoli, in the udders of the estrogen-treated animals. The addition of progesterone prevented the appearance of many of these abnormalities and increased the surface area of the secretory epithelium. JMoreover, when estrogen and progesterone were given in a suitable ratio and absolute level the milk yields obtained were remarkably uniform as between different animals and the glandular tissue was virtually free from abnormalities.
Studies in the cow have been less extensive, but there is evidence that both estrogen and progesterone are necessary for complete normal mammary development (Sykes and Wrenn, 1950, 1951; Reineke, INIeites, Cairy and Huffman, 1952; Flux and Folley, cited by Folley, 1956; Meites, 1960).
The case for the inclusion of the monkey in the present category has been strengthened by the excellent monograph of Speert ( 1948) who has had access to more extensive material than many of the earlier workers whose results are reviewed by him (see also Folley, 1952a). The sum total of available evidence now justifies the conclusion that estrogen alone will cause virtually complete growth of the duct and lobule-alveolar systems of the monkey breast. Extensive lobulealveolar development in the monkey breast in response to estrogen is shown in Figure 10.5. The synergistic effect of estrogen and jirogesterone on the monkey breast has not yet been adequately studied, but from available evidence it does not seem to be very dramatic. If it is permissible to argue from pi'iinates to man. it seems possible that coidd the necessary experiments be done the human breast would show a considerable growth response to estrogen alone.
Fig. 10.5. Wliole mounts of breast of an ovariectomized immature female rhesus monkey before (left) and after (right) e.strogen treatment. (From H. Speert, Contr. Embrvol., Carnegie Inst. Washington, 32, 9-65, 1948.)
Finally, in the third category are those
species in which estrogen in physiologic
doses causes little or no mammary growth.
The bitch and probably the ferret seem to
belong to this class (see Folley, 1956).
There has been considerable discussion in the past regarding the ratio of progesterone to estrogen optimal for mammary growth. Only recently, however, has this question been fully investigated in any species. Benson, Cowie, Cox and Goldzveig (1957) have shown that in the guinea pig the absolute quantities of progesterone and estrogen are the crucial factors in controlling mammary growth; altering the dose levels but maintaining the ratio gave entirely different growth responses. In view of the varying ability of the different estrogens to stimulate mammary duct growth (Reece, 1950) it is essential in discussing ratios to take into consideration the nature of the estrogen used, a fact not always recognized in the past.
2. Anterior Pituitary Hormones
Soon after the discovery by Strieker and Grueter (1928, 1929) of the lactogenic effects of anterior iiituitarv extracts, it was shown that anterior i)ituitary extracts had a mammogenic effect in the ovariectomized animal and that the ovarian steroids had little or no mammogenic effect in hypophysectomized animals. C. W. Turner and his colleagues postulated that mammogenic activity of the anterior pituitary was due to specific factors which they termed "mammogens"; other workers, in particular W. R. Lyons, believed the mammogenic effect was due to prolactin. The theory of specific mammogens has been fully reviewed in the past (Trentin and Turner, 1948; Folley and Malpress, 1948a) and we do not propose to discuss it further for there is now little evidence to support it. Damm and Turner ( 1958) , while recently seeking new evidence for the existence of a specific pituitary mammogen, concur in the view expressed by Folley and Malpress (1948a) that final proof of the existence of a specific mammogen will depend on the development of l)etter assay techniques and the characterization or isolation of the active principle.
The mammogenic effects of prolactin were observed in the rabbit by Lyons (1942) who injected small quantities of prolactin directly into the galactophores of the suitably prepared mammary gland. IV'Iilk secretion occurred but Lyons also noted that the l)rolactin caused active growth of the alveolar epithelium. Recently, Mizuno, lida and Naito (1955) and Mizuno and Naito (19561 have confirmed Lyons' observations on the mammogenic effect of intracluct injections of prolactin in the rabbit both by histologic and biochemical means (DNA estimations) and there seems little doubt that the prolactin is capable of exerting a direct effect on the growth of the mammary parenchyma, at least in the rabbit whose pituitary is intact.
In the last 18 years much information on the role of the anterior pituitary in mammary growth has been obtained by Lyons and his colleagues in studies on hypophysectomized, hypophysectomized-ovariectomized, and hypophysectomized-ovariectomized-adrenalectomized (triply operated) rats of the Long-Evans strain. In 1943 Lyons showed that in the hypophysectomized-ovariectomized rat, estrogen + progesterone + prolactin induced lobulealveolar development, but the degree of development was less than that obtained in the ovariectomized rat with intact pituitary receiving estrogen and progesterone. When supplies of purified anterior-pituitary hormones became available the experiments were extended (Lyons, Li and Johnson, 1952) and it was shown that if somatotrophin (STH) was added to the hormone combination of estrogen -f progesterone + prolactin, the degree of lobulealveolar development obtained in the hypophysectomized-ovariectomized rat was much enhanced. The omission of prolactin from the hormonal tetrad prevented lobulealveolar development from occurring. In the hypophysectomized-ovariectomized-adrenalectomized rat the above hormonal tetrad could also evoke lobule-alveolar development, provided the animals were given saline to drink (Lyons, Li, Cole and Johnson, 1953). In yet more recent experiments Lyons, Li and Johnson (1958) observed that somatotrophin has a direct stimulatory effect on duct growth, but in the hypophysectomized-ovariectomized rat, the presence of estrogen is also necessary to evoke normal duct development (Fig. 10.6a, b, c) ; Likewise, in the triply operated rat, STH plus estrogen is mammogenic, but the presence of a corticoid is r('([ui]'ed to o])tain full duct development (Fig. 10.6r/). Lyons and his colleagues were able to build up the mammary glands of triply operated rats from the state of bare regressed ducts to full prolactational lobule-alveolar development by giving estrogen + STH + corticoids for a period of 10 days to obtain duct proliferation followed by a further treatment (for 10 to 20 days) with estrone + progesterone -I- STH -I- prolactin + corticoid to induce lobulealveolar development. Alilk secretion could then be induced by a third course of treatment lasting about 6 days in which only prolactin and corticoids were given (Fig. 10. 6e, /). Essentially similar results have been obtained in studies with the hooded Norway rat (Cowie and Lyons, 1959).
Studies on mammogenesis in the hypophysectomized mouse have revealed some differences in the response of the mammary gland of this species in comparison with that of the rat and indications of strain differences within the species. The mammary gland of the hypophysectomized male weanling mouse of the Strong A2G strain shows no response to the ovarian steroids alone, to prolactin, or to STH alone, but it responds with vigorous duct proliferation to combinations of estrogen + progesterone + prolactin, or of estrogen 4- progesterone + STH (Hadfield, 1957; Hadfield and Young, 1958). In the hypophysectomized male mouse of the CHI strain slight duct growth occurs in response to estrogen + jirogesterone and this is much enhanced when STH is also given; the further addition of prolactin then results in alveolar development (Flux, 1958). Extensive studies in triply operated mice of the C3H 'HeCrgl strain have been reported by Nandi (1958a, b). In this strain some duct growth was observed in triply operated animals in response to steroids alone (estrogen -I- progesterone + corticoids), but normal duct develojmient was believed to be due to the action of estrogen + STH + corticoids, a conclusion in agreement with Lyons' observations in the rat. Extensive lobuleahcohii' development could be induced by a number of hormone coml)inations, one of the most effective being estrogen + progesterone + corticoids + prolactin + STH, milk secretion occurring when the ovarian steroids were withdrawn, while the })rohictin, STH, and Cortisol were continued. A further interesting observation made by Nandi is that in the C3H/HeCrgl mouse STH can replace prolactin in the stimulation of all phases of mammary development and in the induction of milk secretion; enhanced effects were obtained, however, when prolactin and STH were given together. Nandi also considers that progesterone plays a greater role in duct development in the mouse than in the rat.
Fig. 10.6. Typical areas of whole mounts of the abdominal mammary gland of rat.s after
the following treatments: A. Untreated rat on day 31, 14 days after hypophysectomy. The
gland has regressed to a bare duct system. B. Rat hypophysectomized and ovariectomized on
day 30 and injected daily with 2 mg. somatotrophin (STH) for 7 days. Note the presence
of end clubs, r. Rat treated as in B but which received, in addition to the STH, 1 ^g. estrone.
Note profuse eiid-rhil' ] iroliferatiou. D. Rat li.\|M)]ili\s(>ctomized on day 30. ovariectomized
and adri'nali^ctoinized on day 60, and injected daily from days 60 to 69 with 1 mg. STH +
0.1 mg. DCA + 1 fig. estrone. Note again the profuse number of end buds indicative of
duct proliferation. E. Same treatment as in D followed by 10 days treatment with 5 mg.
prolactin + 2 mg. STH + 1 /xg. estrone + 2 mg. progesterone + 0.1 mg. DCA + 0.05 mg.
prednisolone acetate. Note excellent lobule-alveolar growth. F. Same treatment as in D
followed by 20 days treatment with 5 mg. prolactin + 2 mg. STH + 1 fig. estrone + 2 mg.
progesterone + 0.1 mg. DCA + 0.05 mg. prednisolone acetate; thereafter given 0.1 mg. prolactin locally over this gland and 0.1 mg. DCA + 0.1 mg. prednisolone acetate systemically for
6 days. Note fully developed lobules with ah'eoli filled with milk. (All glands at the same
magnification.) (From W. R. Lyons. C. H. Li and R. E. Johnson, Recent Progr. Hormone
Res., 14, 219-254, 1958.)
The above experiments clearly indicate
that both in the triply operated rat and
mouse, it is possible to build up the mammary gland to the full prolactational state
by injecting the known ovarian, adrenal
cortical, and anterior pituitary hormones.
There would thus seem to be no necessity
to postulate the existence of other unidentified pituitary mammogens. It must be
recognized, however, that in normal pregnancy the placenta may be an important
source of mammogenic hormones. The placenta of the rat contains a substance or substances possessing luteotrophic, mammogenic, lactogenic, and crop-sac stimulating
properties, but it is uncertain whether this
material is identical with pituitary prolactin
(Averill, Ray and Lyons, 1950; Canivenc,
1952; Canivenc and Mayer, 1953; Ray,
Averill, Lyons and Johnson, 1955). There
is also some evidence of the presence of a
somatotrophin-like principle in rat placenta
(Ray, Averill, Lyons and Johnson, 19551.
3. Metabolic Hormones {Corticoids, Insulin, and Thyroid Hormones)
We have already noted that Lyons and his colleagues were able to obtain full duct development in the triply operated rat only when corticoids were given. Early studies of the role of the adrenals in mammary development have given conflicting and uncertain results (see review by Folley, 1952a). Recent studies have not entirely clarified the position. Flux (1954b) tested a number of 11 -oxygenated corticoids, and found that not only were they devoid of mammogenic activity in the ovariectomized virgin mouse, but that they inhibited the gi'owth-promoting effects of estrogen on the mammary ducts, whereas 11-desoxycorticosterone acted synergistically with estro
gen in promoting duct growth. In subsequent
studies it was shown that injections of adrenocorticotrophin (ACTH) into intact
female mice did not influence mammary
growth (Flux and ]\lunford, 1957), but
that Cortisol acetate in low doses (12.5 /^g.
l)er day) stimulated mammary development in ovariectomized and in ovariectomized estrone-treated mice, whereas at
higher levels (25 and 50 ftg. per day) it was
without effect (Munford, 1957). In the virgin rat, on the other hand, glucocorticoids
are said to stimulate mammary growth and
to induce milk secretion (Selye, 1954; Johnson and Meites, 1955). Some light on these
conflicting results has been shed by the
studies of Ahren and Jacobsohn (1957)
who investigated the effects of cortisone on
the mammary glands of ovariectomized
and of ovariectomized-hypophysectomized
rats, both in the presence and absence of
exogenous ovarian hormones. In the hypophysectomized animals, cortisone promoted
enlargement and proliferation of the epithelial cells lining the duct walls, but normal growth and differentiation did not occur, nor did the addition of estrogen and
progesterone appreciably alter these effects ;
in rats with intact pituitaries, however,
cortisone stimulated secretion but not
mammary growth, whereas the addition of
estrogen and progesterone promoted both
growth and al)undant secretion. Ahren and
Jacobsohn concluded that "the effect elicited by cortisone in the mammary gland
should be analysed with due regard to the
endocrine state of the animal both as to its
effects on the structures of the mammary
gland and to the consequences resulting
from an eventual upset of the general metabolic equilibrium." They consider that in
circumstances optimal for mammary gland
growth and maintenance of homeostasis
the predominant actions of cortisone are enhancement of alveolar growth and stimulation of secretion, whereas under conditions
ill which the metabolic actions of cortisone
are not efficiently counteracted, gland
growth is either inhibited or an abnormal
development of certain iiianimaiy cells
may be e^■()ked.
That the general metabolic milieu may indeed profoundly influence the response of the iiuuiimarv gland to hormones has been emiiha.-^ized by the recent experiments of Jacobsohn and her colleagues. Following on the work of Salter and Best (1953) who showed that hypophysectomized rats could be made to resume body growth by the injections of long-acting insulin, Jacobsohn and her colleagues (Ahren and Jacobsohn, 1956; Ahren and Etienne, 1958; Ahren, 1959) found that treatment with estrogen and progesterone would stimulate considerable mammary duct growth in hypophysectomized-gonadectomized rats when given with suitable doses of long-acting insulin (Fig. 10.7). This growth-supporting effect of insulin could be nullified if cortisone was also administered (Ahren and Jacobsohn, 1957) but could be enhanced by giving thyroxine (Jacobsohn, 1959).
The thyroid would thus appear to be another endocrine gland whose hormones affect mammary growth intlirectly by altering the metabolic environment. Studies in this field, reviewed by Folley (1952a, 1956), indicate that in the rat some degree of hypothyroidism enhances alveolar development wdiereas in the mouse, hypothyroidism seems to inhibit mammary development. Chen, Johnson, Lyons, Li and Cole (1955) have shown that mammary growth can be induced in hypophysectomized - adrenalectomized-thyroidectomized rats by giving estrone, progesterone, prolactin, STH, and Cortisol, no replacement of the thyroid hormones being necessary.
These investigations on the effect of the metabolic environment on mammary development seem to ])e opening up new avenues of approach to the advancement of our understanding of the mechanisms of mammary growth and we would recommend.
Fig. 10.7. Whole mount preparation of .second thoracic mammary gland of : ^. Ovariectomized rats injected with estrone and progesterone. B. Hypophysectomized-ovariectomized rat injected with estrone and progesterone. C. Hypophysectomizcd-o\ariectomized rat. D. Hypophysectomized-ovariectomized rat injected with estrone, progesterone, and insulin. (From K. Ahren and D. Jacobsohn, Acta physiol. scandinav., 37, 190-203, 1956.)
to those seeking further information about this important new fiekl, the recent review by Jacobsohn (19581.
III. Endocrine Influences in Milk Secretion
A. Anterior Pituitary Hormones
1. Initiation of Secretion iLactogenesis)
The early experiments leading to the view that the anterior pituitary was not only necessary for the initiation of milk secretion, but in fact i)rovided a positive lactogenic stimulus, are now well known and the reader is referred to the reviews by Folley (1952a, 1956) and Lyons (1958) for further particulars. That pituitary prolactin can evoke milk secretion in the suitably de\-eloped mammary gland of the rabbit with intact pituitary has been amply confirmed, and the original experiments of Lyons (1942) involving the intraduct injection of prolactin have been successfully repeated by Meites and Turner (1947) and
Fig. 10.8. Liictation.'il lespon.scs in pseudoincgnant rabbit to different doses of prolactin injeclcd
intraductallv. (Fiom T. R. Bradley and P. M.
Clarke, J. Endo.ninol., 14, 28-36, 1956.)
Bradley and Clarke (1956) (Fig. 10.8). However, endogenous pituitary hormones may have participated in the response in such experiments and in the last 20 years there has been considerable discussion as to whether prolactin should be regarded as the lactogenic hormone or as a component of a lactogenic complex. This whole question has been fully discussed in recent years (see Folley, 1952a, 1956) and it now seems reasonably certain that lactogenesis is a response to the co-operative action of more than one anterior pituitary hormone, that is, to a lactogenic hormone complex of which prolactin is an important component, as first suggested by Folley and Young (1941 ) . The recent reports by Nandi (1958a, b) that STH -I- Cortisol can induce milk secretion in triply operated mice with suitably developed glands is further strong evidence against regarding prolactin as the lactogenic hormone.
Secretory activity is evident in the mammary gland during the second half of pregnancy, but abundant milk secretion does not set in until parturition or shortly thereafter. The nature of the mechanism controlling the initiation of abundant secretion has been the subject of speculation for many years. The earlier theories w^ere discussed l)y Turner ( 1939 ) in the second edition of this book, and included the theory put forward by Nelson with reference to the guinea pig, that the high levels of blood estrogen in late pregnancy suppressed the secretion or release of prolactin from the pituitary and had also a direct inhibitory cttcct on the mammary parenchyma, the fall in the levels of estrogen occurring at parturition then allowing the anterior pituitary to exert its full lactogenic effect. This concept proved inadequate to exjilain observations in other species and it was later extended by Folley and Malpress (1948b) to embrace the concept of two thresholds for oi:)posing influences of estrogen upon jiituitary lactogenic function, a lower threshold for stimulation and a higher one for inhibition. Subsequent observations on the inhibitory role of progesterone, in the pix'sence of estrogen, on milk secretion, however, necessitated further modification of the theorv. Before discussing these modifications it is convenient to refer to the ingenious theory put forward by Meites and Turner (1942a, b; 1948) which was based on their extensive investigation of the prolactin content of the pituitary in various physiologic and experimental states. According to Meites and Turner, estrogen elicits the secretion of prolactin from the anterior pituitary thereby causing lactogenesis, whereas progesterone is an inhibitory agent, operative in pregnancy, inhibiting or over-riding the lactogenic action of estrogen. The induction of lactation was thus ascribed to a fall in the body level of progesterone relative to that of estrogen heheved to occur at the time of parturition. Subsequent studies in the rabbit by jVIeites and Sgouris (1953, 1954) revealed that combinations of estrogen and progesterone could inhibit, at the mammary gland level, the lactogenic effects of exogenous prolactin. This effect was, however, relative and by increasing the prolactin or decreasing the steroids, lactogenesis ensued. Inasmuch as the theory of Meites and Turner did not take into account the eventuality that estrogen and progesterone act at the level of the mammary gland, Meites ( 1954) modified the con('ei)t, postulating that milk secretion was held in check during pregnancy first by the combined effect of estrogen and progesterone which make the mammary gland refractory to prolactin and, secondly, by a low rate of prolactin secretion. The role of progesterone in over-riding the stimulatory effect of estrogen on the pituitary he now considered to be of only minor importance. Meites also explained the continuance of lactation in pregnant animals by postulating that the initial level of prolactin was sufficiently high as a result of the suckling stimulus to overcome the inhibitory action of the ovarian hormones on the mammary gland. One of us (Folley, 1954, 1956) put forward a tentative theory, combining various features of previous hypotheses, which seemed capable of harmonizing most of the known facts regarding the initiation of milk secretion. In this it was emphasized that measurements of the prolactin content of the pituitary were not necessarily indicative of the rate of prolactin release (a recent study bv Grosvenor and Turner (1958c) lends further support to this contention) and were best considered as largely irrelevant; low circulating levels of estrogen activate the lactogenic function of the anterior pituitary whereas higher levels tend to inhibit lactation even in the absence of the ovary; lactogenic doses of estrogen may be deprived of their lactogenic action by suitable doses of progesterone, the combination then acting as a potent inhibitor of lactation, this being the influence operating in pregnancy; at parturition the relative fall in the progesterone to estrogen ratio removes the inhibition which is replaced by the positive lactogenic effect of estrogen acting unopposed.
It was observed by Gaines in 1915 that although a colostral secretion accumulated in the mammary gland during pregnancy, the initiation of copious secretion was associated with functioning of the contractile mechanisms in the udder responsible for milk ejection; later Petersen (1944) also suggested that the suckling or milking stimulus might be partly responsible for the onset of lactation. Recent studies have provided evidence that this may well be so, and these will be considered later when discussing the role of the suckling and milking stimulus in the maintenance of milk secretion (see page 611).
During the past decade a fair amount of information has been obtained about the biochemical changes which occur in mammary tissue near the time of parturition, and which are almost certainly related to lactogenesis. The earlier work has been reviewed in some detail by one of us (Folley, 1956) and need only be referred to briefly here.
Folley and French (1949), studying rat mammary gland slices incubated in media containing glucose, showed that — QOo increased from a value of about 1.3 in late pregnancy to a value of about 4.4 at day 1 of lactation, and thereafter increased still further. At the same time the R.Q. which was below unity (approximately 0.83) at the end of pregnancy, increased to unity soon after parturition, and by day 8 had reached a value of 1.62 at approximately which level it remained for the rest of the lactation period. In accord with the increased respiratory activity of the tissue about the time of parturition in the rat mammary gland, Moore and Nelson (1952) reported increases in the content of certain respiratory enzymes, succinic oxidase and cytochrome oxidase, in the guinea pig mammary gland at about this time. Greenbaum and Slater (1957b) made similar observations about mammary gland succinic oxidase in the rat. Recent work is beginning to throw light on the metabolic pathways involved in this increase in respiratory activity. Thus McLean (1958a) has adduced evidence indicating an increase in the activity of the pentose phosphate pathway in the rat mammary gland at about the time of parturition. Mammary gland slices taken from rats at various stages of the lactation cycle were incubated in media containing either glucose 1-C^^ or glucose 6-C^-^, and the amount of radioactivity appearing in the respiratory CO2 was determined. The results given in Figure 10.9 show that although the recovery of C^^'Oo from C-6 was relatively unaffected by the initiation of lactation, the C^^Oo originating from C-1 began a striking increase at the time of parturition (see also Glock, McLean and Whitehead, 1956, and Glock and McLean, 1958, from which Figure 10.9 was taken).
pregnancy
in\'oliition
Imc;. 1().<», The relative amounts of C'Oi; formed
fioin iiiilucosc 1-C'^ and glucose 6-C" by rat niani
maiy gland slices. O O, C'^Oi formed from
glucose 1-C^'. • • . C^'Oi! formed from glucose
6-C". (From G. E. Glock and P. McLean, Proc. Roy. Soc, London, ser. B, 149, 354-362, 1958.)
Despite the well known pitfalls which surround the interpretation of C-1: C-6 quotients in experiments such as these, it seems clear that lactation is associated with an increase in the metabolism of glucose by the pentose phosphate cycle, whereas the proportion going by the Embden-Meyerhof jmthway would appear to be relatively unaffected. These conclusions are supported by the fact that the levels in rat mammary tissue of two enzymes concerned in this pathway of glucose breakdown, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, show very striking increases at the time of parturition (Glock and McLean, 1954; McLean, 1958a). Other enzymes concerned in glucose breakdown whose activities in mammary tissue begin to increase at parturition are hexokinase and phosphoglucose isomerase (]\IcLean, 1958a). In connection with the glucose metabolism of rat mammary tissue it may be noted that addition of insulin to the incubation medium markedly increases the — QOo and R.Q. of rat mammary slices metabolizing glucose or glucose plus acetate (see page 619), and that this tissue only becomes sensitive to insulin just after parturition (Balmain and Folley, 1951). It is interesting to speculate which of the two above-mentioned pathways of glucose breakdown in mammary tissue resjjonds to the action of insulin. According to Abraham, Cady and Chaikoff (1957) addition of insulin in vitro increased the production by lactating rat mammary slices of C^'^Oo from glucose l-C^'*, but not from glucose 6-C^'*, which might indicate that insulin stimulates preferentially the pentose phosphate pathway. Against this, insulin increased the incorporation of both these carbon atoms (and also the 3:4 carbon atoms of glucose) into fatty acids of the slices to about the same extent. McLean (1959) believes that the stimulatory effect of insulin on the pentose jihosphate pathway in the lactating rat mammary gland is secondary to its stimulating effect on lipogenesis. The latter l)rocess generates the oxidized form of tril)hosphopyridine nucleotide (TPN) which is needed for the first two steps of the pentose phosphate cycle.
The inci-casc in the R.Q. of mammary tissue beginning at parturition observed by Folley and French (1949) was interi:)reted as indicating that this tissue assumes the power of effecting net fatty acid synthesis from ghicose at this time. Much subsequent evidence confirming this idea has been reviewed by Folley (1956). It only rt'mains to add that Ringler, Becker and Nelson (1954), Lauryssens, Peelers and Donck (1956), and Read and Moore (1958) ha^-e shown that the amount of coenzyme A in mammary tissue undergoes an increase at parturition. Moreover, the recent findings of McLean (1958b), who showed that the levels of pyridine nucleotides in the mammary gland of the rat begin to increase at parturition, reaching a high level by the end of lactation, may be significant in this connection. McLean found that although the increase in the tissue levels of diphosl^hopyridine nucleotide was almost entirely due to an increase in the oxidized form (DPN), in the case of TPN it was the reduced form (TPNH) which increased. The latter might well be used for reductive syntheses such as lipogenesis.
The rate of synthesis of milk constituents other than fat must also begin to increase at parturition, and Greenbaum and Greenwood (1954) showed that an increase in the levels of glutamic aspartic transaminase and of glutamic dehydrogenase in rat mammary tissue occurs at this time. The authors believe these enzymes are concerned in the provision of substrates for the synthesis of milk protein. It is significant in connection with milk protein synthesis that the mammary gland ribonucleic acid (RNA) in the rat undergoes a marked rise at parturition (Greenbaum and Slater, 1957a).
The above - mentioned biochemical changes in mammary tissue which occur at al)out the time of parturition are almost certainly closely related to the effect on this tissue of members of the anterior pituitary lactogenic complex, and particularly prolactin. Attempts have been made to elicit the characteristic respiratory changes, described above, in mammary slices in vitro by addition of prolactin and adrenal glucocorticoids to the incubation medium (see Folley, 1956). So far, however, definitive results luive not been obtained and it is doubt
ful whether any biochemical changes in
lactating mammary gland slices in vitro
have been demonstrated which could with
certainty be ascribed to the action of prolactin (in this connection see also Bradley
and Mitchell. 1957).
2. Maintenance of Milk Secretion — Galactopoiesis
It is well known that the removal of the pituitary of a lactating animal will end milk secretion (for references see Folley, 1952a). The cessation of milk secretion has been generally ascribed to the loss of the anterior lobe, but when the importance of the neurohypophysis in milk ejection became established (see page 621), it was clear that in the hypophysectomized animal it was necessary to distinguish between a failure in milk secretion and a failure in milk ejection, since either would lead to failure of lactation. It has now been shown in the rat that adequate oxytocin therapy ensuring the occurrence of milk ejection after hypophysectomy will not restore lactation (Cowie, 1957) and it may thus be concluded that the integrity of the anterior lobe is essential for the maintenance of milk secretion. The effect of hypophysectomy on milk secretion is dramatic, because in the rat, milk secretion virtually ceases within a day of the operation and biochemical changes in the metabolic activity of the mammary tissue can be detected within 4 to 8 hours (Bradley and Cowie, 1956). It is of interest to note that these metabolic changes are similar to those observed during mammary involution (see page 598).
Since the second edition of this book, there have been surprisingly few studies on replacement therapy in hypophysectomized lactating animals. In such studies we would stress the need for rigorous methods of assessing the efficacy of treatment. In the past the presence of milk in the gland as revealed by macroscopic or microscopic examination has been regarded as an indication of successful replacement. This, however, gives no measure of the degree of maintenance of lactation and some measure of the daily milk yield of such animals should be obtained (see also Cowie, 1957).
It is abo now obvious that oxytocin may have to be injected to ensure milk ejection; under certain circumstances, however, the neurohypophyseal tissue remaining after the removal of the posterior lol^e may be capable of releasing oxytocin and permitting milk ejection (see Benson and Cowie, 1956; Bintarningsih, Lyons, Johnson and Li. 1957, 1958).
The earliest report on the maintenance of lactation after hypophysectomy is that of Gomez (1939, 1940), who found that hypophysectomized lactating rats could rear their litters if given anterior-pituitary extract, adrenal cortical extracts, glucose, and posterior pituitary extract. These experiments are difficult to assess because they are reported only in abstract, but the use of posterior pituitary extract at a time when the role of oxytocin in milk ejection was not generally recognized is worthy of note. Recently, slight maintenance of milk secretion in hypophysectomized rats has been obtained with prolactin alone, and greater maintenance when adrenocorticotrophic hormone ( ACTH I or STH was administered with prolactin (Cowie, 1957). Similar studies were reported by Bintarningsih, Lyons, Johnson and Li (1957, 1958) (see also Lvons, Li and Johnson, 1958) in which considerable maintenance of milk secretion was obtained in hypophysectomized rats with prolactin and certain corticoids. Of related interest is the observation by Elias (1957) that Cortisol and prolactin can induce secretory activity in explants of mouse mammary gland growing on a synthetic medium. (Tissue culture techniques have been little exploited in mammary studies and further developments in this field may be expected.)
Fig. 10.10. Effect on the luilk yield of the cow
of injected hormones of the anterior pituitary.
(From the results of P. M. Cotes, J. A. Crichton,
S. J. Folley and F. G. Young, Nature, London.
164, 992-993, 1919.)
The evidence to date suggests that, in the rat, prolactin is an essential component of the hormone complex involved in the maintenance of lactation with ACTH and STH also participating, but further studies are recjuired to determine the most favorable balance of these factors.
Preliminary studies on the maintenance of lactation in the goat after hypophysectomy suggest that both prolactin and STH are important in the initiation and maintenance of milk secretioii (Cowie and Tindal, 1960). Our knowledge of the process in other species is derived from studies on the effect of exogenous anterior pituitary hormones on established lactation in intact animals— galactopoietic effects (for reference see Folley, 1952a, 1956). In the cow, considerable increase in milk yield can be obtained by injecting STH (Cotes, Crichton, Folley and Young, 1949), whereas prolactin has a negligible galactopoietic effect (Fig. 10.10; for discussion see also Folley, 1955). Recently the precise relationship between the dose of STH (ox) and the lactational response in the cow was established in our laboratory by Hutton (1957) who observed a highly significant linear relationship between log doses of STH (single injection) and the increase in milk yield obtained (Fig. 10.11 ) ; increases in fat yield relative to the yield of nonfatty solids also occurred. In the lactating rat, on the other hand, STH has no galactopoietic effect (Meites, 1957b; Cowie, Cox and Naito, 1957), whereas prolactin has (Johnson and Meites, 1958). Such studies must be interpreted with caution as endogenous pituitary hormones were present ; nevertheless, it seems reasonable to conclude that STH is likely to be an impoi'tant factor in the maintenance of lactation in the row.
Fig. 10. IL Effect of graded doses of growth hormone on milk yield of row. Upper curve, doses plotted on arithmetic scale. Lower curve, doses plotted on logarithmic scale. (From J. B. Hutton, J. Endocrinol., 16, 115-125, 1957.)
Other hormones of the anterior pituitary in all probability influence milk secretion through their target glands and these will be dealt with later.
3. Suckling Stimulus and the Maintenance of Lactation
It has been long believed that regular milking is an important factor in maintaining lactation and that if milk is allowed to accumulate in the gland, as occurs at weaning, atrophy of the alveolar epithelium and glandular involution occur. Evidence in support of this concept was obtained in studies showing that ligature or occlusion of
the main ducts of some of the mammae of a lactating animal resulted in atrophy of the glands concerned although the other glands were suckled normally (Kuramitsu and Loeb, 1921; Hammond and Marshall, 1925; Fauvet, 1941a). Studies by Selye and his colleagues, however, revealed that such occluded glands did not atrophy as quickly as did glands of animals in which the suckling stimulus was no longer maintained (Selye, 1934; Selye, Collip and Thomson, 1934) and it was postulated that the suckling stimulus evoked from the anterior pituitary the secretion of prolactin which maintained the secretory activity of the gland. This theory has been widely accepted although it has been suggested that a complex of hormones rather than prolactin alone is released (Folley, 1947). Williams (1945) showed that prolactin could in fact maintain the integrity of the mammary gland in the unsuckled mouse thus mimicking the effects of the suckhng stimulus; other supporting evidence has been reviewed by Folley (1952a). Recent studies in goats, however, have shown that milk secretion may continue more or less at the normal level after complete denervation of the udder (Tverskoi, 1958; Denamur and Martinet, 1959a, b, 1960) and it may be that in some species the suckling or milking stimulus is loss important in the maintenance of milk secretion.
Milk secretion is essentially a continuous process whereas the suckling or milking stimulus is intermittent ; indeed the milking stimulus may be of remarkably brief duration (in the cow about 10 minutes in all per 24 hours) and it is therefore likely that the stimulus triggers off the release of sufficient galactopoietic complex to maintain mammary function for some hours. Grosvenor and Turner (1957b) reported that suckling causes a rapid drop in the prolactin content of the pituitary in the rat, and that the prenursing level of prolactin in the pituitary is not fully regained some 9 hours later. It is difficult, however, to relate pituitary levels of prolactin to the rate of its secretion into the circulation and, although these observations are interesting, further advances are unlikely until a method of assay for blood prolactin becomes available and the "half-life" of prolactin in circulation is known.
The experiments of Gregoire (1947) on the maintenance of involution of the thymus during nursing suggests that the suckling stimulus releases ACTH which, as we have seen, is galactopoietic in the rat; thus, so far as the rat is concerned, there would appear to be good evidence that the suckling stimulus releases at least two known important components of the galactopoietic complex.
The milking and suckling stimulus is also responsible for eliciting the milk-ejection reflex and the relation between the two reflexes will be discussed later in this chapter (sec ])age 619 1.
B. Hormones Of The Adrenal Cortex
Adrenalectomy results in a marked inhibition of milk secretion and the early experiments in this field were reviewed by Turner in 1939. Since then, however, purified adrenal steroids have become available enabling further analysis to be made of the role of the adrenal cortex in lactation.
Gaunt, Eversole and Kendall (1942) considered that in the rat the defect in milk secretion after adrenalectomy could be repaired by the administration of the adrenal steroids most closely concerned with carbohydrate metabolism, whereas we came to the somewhat opposing view that the defect was best remedied by those hormones primarily concerned with electrolyte metabolism (Folley and Cowie, 1944; Cowie and Folley, 1947b, c). The reasons for these differing observations are not yet entirely clear. Virtually complete restoration of milk secretion was subsequently obtained in our strain of rat by the combined administration of desoxycorticosterone acetate (DCA) and cortisone, or with the halogenated steroids, 9a-chlorocortisol and 9afluorocortisol (Cowie, 1952; Cowie and Tindal, 1955; Cowie and Tindal, unpublished; see also Table 10.1). It would therefore seem that both glucocorticoid and mineralocorticoid activity was necessary to maintain the intensity of milk secretion at its normal level. The interesting observation was made by Flux (1955» and later confirmed by Cowie and Tindal (unpublished) that the ovaries contribute to the maintenance of lactation after adrenalectomy, a contribution which could be simulated in the adrenalectomized-ovariectomized rat by the administration of 3 mg. progesterone daily. The differences in the size of the ovarian contribution may partly accoimt for the apparent differences in various strains of rat of the relative importance of mineralo- and glucocorticoids in sustaining milk secretion after adrenalectomy. The only other species in which the maintenance of lactation after adrenalectomy has been studied is the goat in which, as in the rat, lactation can be maintained with cortisone and desoxycorticosterone, the latter being apparently the more critical steroid (Cowie and Tindal. 1958; Figs. 10.12a, b).
There have been several studies on the effects of corticoids and adrenocortieotrophin on lactation in the intact animal. ACTH and the corticoids depress lactation in the intact cow (Fig. 10.10) (Cotes, Crichton, Folley and Young, 1949; Flux, Folley and Rowland, 1954; Shaw, Chung and Bunding, 1955; Shaw, 1955), whereas in the rat ACTH and cortisone have been reported as exhibiting galactopoietic effects (Meites, private communication; Johnson and Meites, 1958). With larger doses of cortisone, however, an inhibition of milk secretion in the rat has been reported (MercierParot, 1955).
The main function of the cortical steroids in lactation is still uncertain. They may act in a "supporting" or "permissive" manner (see Ingle, 1954), maintaining the alveolar cells in a state responsive to the galacto])oictic complex, or they may act by maintaining the necessary levels of milk precursors in the blood.
Biochemical studies are, however, Ix'ginning to add to our information on the role of the corticoids in lactation. In the rat, adrenalectomy prevents the increase in liver and mammary gland arginase which occurs during normal lactation and it has been suggested that this depression of arginase activity interferes with deamination of amino acids, and thereby inhibits any increase in gluconeogenesis from protein and thus starves the mammary gland of nonnitrogenous milk precursors (Folley and Greenbaum, 1947, 1948). As there is little arginase in the mammary gland of other species {e.g., rabbit, cow, goat, sheep), this mechanism may not have general validity (for further discussion see Folley, 1956). Other biochemical studies have suggested that the steroids of the adrenal cortex may be concerned in mammary lipogenesis, but the results so far have been conflicting and no firm conclusions can as yet be drawn (see Folley, 1956).
C. Ovarian Hormones
There is no evidence that ovariectomy has any deleterious effect on lactation (Kuramitsu and Loeb, 1921; de Jongh, 1932; Folley and Kon, 1938; Flux, 1955); neither is there evidence for the belief, once
TABLE 10.1
Replacement therapy in lactating rats
adrenalectomized on the fourth
day of lactation
(From A. T. Cowie and S. J. Folley,
J. Endocrinol., 5, 9-13, 1947.)
Treatment
Number of
Litters
Number
of Pups
per
Litter
Litter-growth
Index* gm. + S.E.
Control
Adrenalectomy
Adrenalectomy + cortisone + DC A (tablet implantsf)
8
9
7
8
8
8
15.6 + 0.5
7.5 ± 0.6 14.9 ± 0.6
(Above results from Cowie, 1952)
Control
6
8
14.5 ± 0.8
Adrenalectomy
6
8
6.2 ± 0.4
Adrenalectomy + chloro
5
8
13.1 ± 0.5
cortisol (100 Mg per
day)
(Above results from Cowie and Tindal, 1955)
Control
8
12
17.7 ± 0.8
Adrenalectomy
8
12
7.5 ± 0.5
Adrenalectomy + ovari
5
12
3.6 ± 0.5
ectomy
Adrenalectomy + ovari
7
12
14.5 ± 0.7
ectomy + fiuorocorti
sol (200 Mg per day)
(Above results from Cowie and Tindal, unpublished)
- The litter-growth index is defined as the mean
daily gain in weight per litter over the 5-day period from the 6th to the 11th days.
t 2 X 11 mg. tablets cortisone giving mean daily absorption of 850 ^ig., and 1 X 50 mg. tablet DCA giving mean daily absorption of 360 ng.
widely held, that ovariectomy increases and prolongs lactation in the nonpregnant cow (see Richter, 1936).
Although the integrity of the ovary is not essential for the maintenance of lactation, there can be no doubt that ovarian hormones, in certain circumstances, profoundly influence milk secretion. Estrogens have long been regarded as possessing the power to inhibit lactation, a concept on which Nelson based his theory of the mechanism of lactation initiation (see page 606 1 . Some workers, however, have expressed doubts that the effect is primarily on milk secretion, and have suggested that in experiments on laboratory animals the apparent failure in milk secretion could be a secondary effect due to either a toxic action of the estrogen causing an anorexia in the mother, interference with milk ejection, or disturbance of maternal behavior or to toxic effects on the young, whose growth rate serves as a measure of lactational performance, through estrogens being excreted in milk. The evidence to date shows that in the intact rat estrogens even in very low doses inhibit milk secretion, their action depending on the presence of the ovary ; the ovarian factor concerned appears to be progesterone, estrogen and progesterone acting locally on the mammary gland and rendering it refractory to the lactogenic complex. In the ovariectomized rat much larger doses of estrogen are necessary to inhibit lactation, and the evidence is not entirely
Body
Goat 478
weight ^^L
- .) 45 L
Plasma Na (m-equiv./l.) ^^^^
Plasma K (m-equiv./l
Milk K 40 (m-equiv./l.) 30
Milk Na ,
(m-equiv./l.)
Solids-notfat (%)
Yield of solids-notfat (g) Fat (%)
Milk yield (kg)
Goat died-*
5 15 25 4 14 24 Mgr. Apr.
Fig. 10.12i4. Effect of replaconi(>nt therapy with (losoxycoiticostcM-oiu c-ortisone aoetate (CA) on milk yield, milk composition, and concent
(DCA) and tion of Na and K in milk and blood plasma of the goat after adrenalectomy. Duration of replacement therapy (pellet implantation) indicated by horizontal lines; the names of steroids and their mean daily absorption rates are given adjacent to the lines. Note in Figure 12.4 the considerable maintenance of milk vield with DCA alone. See also Figure 12/?. (From A. T. Cowic and J. S. Tindal. J. Endocrinol., 16, 403-414, 1958.)
6L
Goat 515
Body 5Q _ weight —
(kg) 40 150 Plasma Na ^ ^. / /I \ ^40 —
(m-equiv./l) —
130
Plasma K (m-equiv./l)
Milk K (m-equiv./l.)
Milk Na (m-equiv./l.)
Solids-not- ^ H
fat {%) 7 U
Yield of 200 solids-not- —
fat (g) 100 Fat (- ^
Fat yield
Milk yield (kg)
13 23 2 12 22 2 12 22
Oct. Nov Dec.
Fig. 12B.
11 21 31 10 20
Jan. Feb
conclusive that there is a true inhibition of milk secretion (see Cowie, 1960). In the cow estrogen in sufficient doses depresses milk yield, but its mode of action has not been fully elucidated. In women, estrogens are used clinically to suppress unwanted lactation, but as the suckling stimulus is also removed about the same time, the role of the estrogen is difficult to assess (see Meites and Turner, 1942a).
It has been well established that progesterone by itself has no effect on milk secretion (see Folley, 1952a), save in the adrenalectomized animal (see page 612), and so it would appear that the physiologic inhibition of lactation is effected Ijy estrogen and progesterone acting synergistically as first demonstrated by Fauvet (1941b) and confirmed by others including Masson (1948), Walker and Matthews (1949), Cowie, FoUey, Malpress and Richarcl.son (1952J,, and Meites and Sgouris (1954). There is clear evidence that the estrogenprogesterone combination acts at least partly on the mammary parenchyma (Desclin, 1952; Meites and Sgouris, 1953) but the mechanism of the action is unknown. The hormonal interplay and complex endocrine interactions in the process of lactation inhibition with estrogen has recently been discussed at length by von Berswordt-Wallrabe (1958).
Lactogenic effects of estrogens have already been mentioned; these have been demonstrated most strikingly in cows and goats, in which milk secretion has been induced in udders being developed by exogenous estrogen. These experiments have been reviewed in some detail by Folley and Malpress (1948b) and Folley (1956).^ It is generally assumed that estrogens act by stimulating the production of lactogenic and galactopoietic factors by the anterior pituitary. In experiments on the ovariectomized goat we have shown (Cowie, Folley, Malpress and Richardson, 1952; Benson, Cowie, Cox, Flux and Folley, 1955) that it is possible to select a daily dose of estrogen which will induce mammary growth but relatively little secretion in the sense that the udder does not become tense and distended as will happen when a lower dose of estrogen is given — an observation we may quote in support of the "double-threshold" theory of estrogen action. The lactogenic effect of the lower dose of estrogen could be abolished, however, by administering progesterone simultaneously with the estrogen (Fig. 10.13), an observation in accord with those of other workers on the rabbit and rat (see above).
Fig. 10.13. Photographs of goat uddois dovelopcd by daily injections of hoxoostiol (HX)
with and without progesterone (PG). The hibels indicate the daily dose in mg. of each
substance. (Results from A. T. Cowie, S. J. Folley, F. H. Malpre.ss and K. C. Ricliardson,
J. Endocrinol., 8, 64-88, 1952.)
In 1936 one of us (Folley, 1936) reported that certain dose levels of estrogen in the lactating cow produced long-lasting changes in milk composition characterized by increases in the percentages of fat and nonfatty solids. This was regarded as an example of galactopoiesis and was termed the "enrichment" effect. The effect, however, w^as somewhat erratic and it has recently been re-investigated by Hiitton (1958) who confirmed and extended the earlier observations. Hutton found that galactopoietic responses (Figs. 10.14 and 10.15) were obtained only within a restricted dose range, the limits of which were affected by the stage of pregnancy and the breed of the cow. Hutton further concluded that in the normal cow changes in milk composition and yield associated with advancing pregnancy were probably determined by the progressive rise of blood estrogen levels.
D. Thyroid Hormones
Studies on the effect of removal of the thyroids on milk secretion have been reviewed by one of us (Folley, 1952a) ; the evidence strongly suggests that the thyroid glands are not essential for milk secretion, but in their absence the intensity and duration of lactation is reduced. Histologic and cytologic studies of the thyroid of the lactating cat suggest that there is a considerable outpouring of the thyroid secretion in the early stages of lactation (Racadot, 1957), and Grosvenor and Turner (1958b) have reported that the thyroid secretion rate is higher in lactating than in nonlactating rats.
Since the last edition of this l)ook, a great volume of experimental results has been published on the use of thyroid-active materials for increasing the milk yield of cows. These experiments have been extensively reviewed by Blaxter (1952) and Meites (1960) and we need here only touch on the salient points.
In the early studies i^reparations of dried thyroid gland were fed to cows or injections of DL-thyroxine were given, but the use on a large scale of thyroid-active materials for increasing the milk yield of cows only became feasible when it was shown that certain iodinated proteins exhibited thyroidlike activitv when given in the feed. Al
Fig. 10.14. Effect of graded doses of estradiol benzoate on percentage of nonfatty solids in milk from cows of three breeds. (From J. B. Hutton, J. Endocrinol., 17, 121-133, 1958.)
Fig. 10.15. Effect of graded doses of estradiol
benzoate on fat content of cows' milk. Upper curve,
doses plotted on arithmetic scale. Lower curve,
doses plotted on logarithmic scale. (From J. B.
Hutton, J. Endocrinol., 17, 121-133, 1958.)
though these materials were readily made
and were economical for large-scale use, they
possessed several disadvantages. Their activity was difficult to assay and standardize,
they were frequently unpalatable, and their
administration entailed a considerable intake of iodine which could be undesirable.
Nevertheless, a large number of experiments
were carried out all over the world with
this type of material. In 1949, however, a
new and improved method for the synthesis
of L-thyroxine was developed (Chalmers,
Dickson, Elks and Hems, 1949) and thyroxine became available in large quantities.
It was then shown jjy Bailey, Bartlett and
Folley (1949) that this material was ealac
Fig. 10.16. Effect of L-thyroxine given in the feed on the milk yield of groups of cows
(the indicated dose levels were fed daily). (From G. L. Bailey, S. Bartlett and S. J. Folley,
Nature, London, 163, 800. 1949.)
topoietic when ]ed to lactating cows in daily doses of about 100 mg. (Fig. 10.16). It had, moreover, none of the drawbacks of the iodinated proteins, its purity could be checked chemically, it was odorless and tasteless. AVith the introduction of synthetic thyroxine, iodinated proteins have become obsolete as galactopoietic agents.
The more recently isolated 3:5:3-triiodo-L-thyronine, reported to be 5 to 7 times more active than thyroxine in various biologic tests in small animals and also in man, has little or no effect on the milk yield when fed to cows, but is somewhat more active than thyroxine in promoting galactopoiesis when administered subcutaneously, which suggests that the material is inactivated in the gut, probably in the rumen f Bartlett, Burt, Folley and Rowland, 1954).
The extensive experiments on galactopoiesis in dairy cattle with thyroxine and thyroid-active substances have made it possible to reach reasonably firm conclusions as to the practical value of the procedure. There is great variability in the response to treatment; in general a better response is ol)taincd during the decline of lactation than at the peak and end of lactation. The use of thyroid-active substances
in animals undergoing their first, second, or third lactation is of doubtful benefit because the boost in yield is largely cancelled out by a shortening of the lactation period. Short-term administration at suitable times can result in considerable galactopoiesis, but this is frequently followed by marked falls in yield when the administration of thyroid-active material ends. The administration of thyroid-active materials to dairy cows, if carried out with due care, has no ill effects on the health and reproductive abilities of the cows (see Leech and Bailey, 1953) , but because of the rather small net gain in yield (about 3 per cent) the practical application of the procedure seems to be limited.
The mode of action of thyroxine and thyroid-active substances on milk secretion is uncertain. It is tmlikely that it is a specific effect on the alveolar cells; rather is it probably related to the effects of the thyroid hormone on the general metabolic rate.
E. Parathyrom Hormone
The early studies on the influence of the parathyroid glands on milk secretion indicated, as might be expected from their role in calcium metabolism, that the parathyroids were important in the maintenance of secretion (see review by Folley, 1952a). Indeed in the rat, we demonstrated that the severe impairment of milk secretion previously observed in "thyroidectomized" rats was due not to the removal of the thyroids, but to the simultaneous ablation of the l)arathyroids (Cowie and Folley, 1945). This observation has since been confirmed and extended by Munson and his colleagues (Munson, 1955) who demonstrated an influence on the calcium-concentrating mechanism of the mammary glands. Within 24 hours of parathyroidectomy the concentration of calcium in the milk of the lactating rat was increased markedly despite a greatly depressed level of calcium in the serum; there was also a decrease in water content of the milk, but this did not entirely account for the increase in calcium content since the calcium content expressed as mg. per gm. milk solids was significantly higher after parathyroidectomy (Toverud and Munson, 1956). Further studies in this field are awaited with interest.
F. Insulin
Early experiments (see review by Folley, 1952a) indicated that the endocrine pancreas might influence mammary function in two ways; indirectly by way of the general intermediary metabolism by which the supply of milk precursors may be regulated, and directly through its role in the carbohydrate metabolism of the mammary gland itself.
Most recent studies have been concerned with the effect of insulin on mammary tissue in vitro. Mammary gland slices from lactating rats actively synthesize fat from small molecules, glucose, and glucose plus acetate, but not from acetate alone (Folley and French, 1950). The addition of insulin to the incubation medium very markedly increases the R.Q. (see Table 10.2) and glucose uptake of the tissue slices and experiments with isotopes show that the rate of fat synthesis is increased (Balmain, Folley and Glascock, 1952). Mammary gland slices from lactating sheep, on the other hand, can utilize acetate alone but not glucose alone for fat synthesis (Folley and French, 1950) and sheep tissue is not re
TABLE 10.2
Effect of different substrates and of insulin on the
respiratory quotient (R.Q.) of lactating mammary
gland slices from various species
(From S. J. Follev and M. L. McNaught, Brit.
M. BulL, 14, 207-211, 1958.)
Respiratory Quotients
Anlrml
Substrate
Without
insulin
With
insulin
Mouse
Glucose
1.90
2.14
Glucose + acetate
1.46
2.14
Rat
Glucose
1.57
1.80
Acetate
0.82
Glucose + acetate
1.53
2.03
Guinea pig
Glucose
1.17
Rabbit
Glucose
1.30
_
Acetate
0.92
Glucose -t- acetate
1.24
1.67
Sheep
Glucose
Acetate
0.88
1.09
1.09
Glucose + acetate
1.52
1.50
Goat
Glucose
0.86
Acetate
1.17
—
Cow
Glucose
0.84
_
Acetate
1.12
—
sponsive to insulin in vitro. This clear-cut species difference is interesting and underlines the need for further study. It is of passing interest to note that the response in vitro of rat mammary tissue to insulin has been made the basis of a highly specific in vitro bio-assay for insulin (Fig. 10.17) (Balmain, Cox, Folley and McNaught, 1954; McNaught, 1958)!
Further references and discussion on the role of insulin in mammary function and lipogenesis will be found in the reviews by Folley (1956), and Folley and McNaught (1958, 1960).
IV. Removal of Milk from the Mammary Glands: Physiology of Suckling and Milking
A. Milk-Ejection Reflex
Since the second edition of this book, there have been major advances in our knowledge of the physiology of milk removal. In the mammary gland the greater
yT
Fig. 10.17. Effect of various concentrations of
insulin on the respiratory metabolism of slices
of rat mammarj' glands. (From J. H. Balmain, C. P.
Cox, S J. Folley and M. L. McNaught, J. Endocrinol., 11, 269-276, 1954.)
portion of the milk secreted by the alveohir cells in the intervals between suckling or milking remains within the alveoli and the fine ducts. Only a small portion passes into the larger ducts and cisterns or sinuses from which it can be immediately removed by suckling, milking, or cannulation; its removal requires no maternal participation and has been termed passive withdrawal (see Cowie, Folley, Cross, Harris, Jacobsohn and Richardson, 1951, and page 612). The larger portion of the milk in the alveoli and fine ducts becomes available only with the active participation of the mother and requires the reflex contraction of special cells (see page 623) surrounding the alveoli in response to the milking or suckling stimulus to eject the milk from the alveoli and fine ducts into the cistern and sinuses of the gland. The occurrence of this reflex has long been known, although its true nature has only recently been generally recognized.^
-H. K. Waller {Clinical Slujlits un Lnrfallon, London: Heinemann, 1938), and later one of us (S. J. Folley, Physiology and Biochemistry of Lactation, London and Edinburgh: Oliver & Boyd, 1956) have drawn attention to the fact that the theme of the "milk-ejection reflex" was the inspiration of a paiming by II Tintoretto entitled "The Origin of the Milky Way" which hangs in the 111 the past it has been termed the "draught" in lactating women (see Isbister, 1954) and the "let-down" of milk in the cow. The latter term is particularly misleading since it implies the release of some restraint, whereas there is, in fact, an active and forceful expulsion of milk from the alveoli and we have, therefore, urged that this term be no longer used in scientific literature and that it be replaced by the term "milk ejection" (Folley, 1947; Cowie, Folley, Cross, Harris, Jacobsohn and Richardson, 1951), a term, incidentally, which was used by Gaines in 1915 in his classical researches on the phenomenon (see below j.
The true nature of the milk removal process was for many years not recognized, probably because it was assumed that the mammary gland could not contain all the milk obtainable at a milking, and this assumption made it necessary to postulate a very active secretion of milk during suckling or milking. Even as late as 1926 two phases of milk secretion were described in the cow ; the first phase was one of slow secretion occurring between milkings, the second phase was one of very active secretion occurring in response to the milking stimulus when a volume of milk about equal to that produced in the first phase was secreted in a matter of a few minutes (Zietzschmann, 1926). That some physiologic mechanism
National Gallery, London. Both authors point out tliat the picture shows evidence of a considerable intuiti^■e understanding of the physiologic nature of the milk-ejection reflex. Thus, it illustrates, first, that the application of the suckling stimulus causes a considerable increase in intranianiinai >• jiressure resulting, in this instance, in a sjnni cii' milk from the nipples, and second, that ihv Muklmg stimulus applied to one nipple gives rise to a systemic rather than a localized effect, for the milk is forcibly ejected from the suckled and unsuckled breasts ahke. The same theme was also treatetl by Rubens in a picture called "The Birth of the Milky Way" which can be seen in the Prado Museum, Madrid. This picture differs from Tintoretto's in one important detail, the stream of milk coming only from one breast.
The forcible ejection of milk from the nipple has doubtless been the subject of many statues. An example known to the authors is the fountain in the Sfiuare at Palos Verdes, near Los Angeles, California. The center piece of this fountain has a nude female torso at each of its four corners from whose nipples spurt streams of water.
was involved in the discharge of preformed milk from the mammary gland had, however, been recognized. Schafer (1898) considered that milk discharge was aided by contraction of plain muscle w^ithin the gland and pressure on the alveoli produced by vasodilation.
The first full investigation of the physiology of milk removal was that by Gaines in 1915. Unfortunately, his remarkably accurate observations and perspicacious conclusions aroused little general interest and were almost wholly overlooked for more than quarter of a century. It is now of interest to recall the more important of Gaines' observations. First, he made a clear distinction between milk ejection and milk secretion — "Milk secretion, in the sense of the formation of the milk constituents, is one thing; the ejection of the milk from the gland after it is formed is quite another thing. The one is probably continuous; the other, certainly discontinuous." Secondly, he concluded that "Nursing, milking and the insertion of a cannula in the teat, excite a reflex contraction of the gland musculature and expression of milk. There is a latent period of 35 to 65 seconds. . . . Removal of milk from the gland is dependent on this reflex, and it may be completely inhibited l)y anaesthesia. The conduction in the reflex arc is dependent upon the psychic condition of the mother." He also observed that the increased flow of milk following the latent period after stimulation was associated wath a steep rise in pressure within the gland cistern and that the reflex could be conditioned. Thirdly, with reference to the gland capacity, he reported that "the indication is that practically the entire quantity of milk obtained at any one time is present as such in the udder at the beginning of milking." Lastl3^ he confirmed earlier observations that injections of posterior pituitary extract caused a flow of milk in the lactating animal and he postulated that "pituitrin has a muscular action on the active mammary gland causing a constriction of the milk ducts and alveoli with a consequent expression of milk. This action holds, also, on the excised gland in the absence of any true secretory action." Gaines regarded the milk-ejection reflex as a purely neural arc although he emphasized that the effect was "very similar to that produced by pituitrin." All that is required to bring these views of milk ejection in line with present day concepts is to recognize that the reflex arc is neurohormonal in character, the efferent component of which is a hormone released from the neurohypophysis. When Gaines was carrying out these experiments hardly anything was known of neuro-endocrine relationships and there was no background of knowledge to lead anyone to conceive that the effects of the posterior pituitary extract might represent a physiologic rather than a pharmacologic effect. In 1930 Turner and Slaughter hinted at a possible physiologic role of the posterior pituitary in milk ejection and, as we have noted (page 610), Gomez (1939) used posterior pituitary extract in replacement therapy given to hypophysectomized lactating rats. It was not until 1941, however, that the role of the posterior pituitary in milk ejection was seriously postulated by Ely and Petersen (1941) who, having shown in the cow that milk ejection occurred in the mammary gland to which all efferent nerve fibers had been cut, suggested that the reflex was neurohormonal, the hormonal component being derived from the posterior pituitary, and being, in all likelihood, oxytocin. The neurohormonal theory of Ely and Petersen and the subsequent work of Petersen and his colleagues (see reviews by Petersen, 1948; and Harris, 1958), unlike the earlier work of Gaines, aroused wide interest and its practical applications permitted rationalization of milking techniques in the cowshed thereby improving milk yields. Despite the attractiveness of the concept, however, a further 10 years were to elapse before unequivocal evidence of the correctness of the theory was forthcoming and this evidence we shall now briefly review.
B. Role of the Neurohypophysis
The first reliable indication that the suckling or milking stimulus does in fact cause an outpouring of neurohypophyseal hormones were the observations that inhibition of diuresis occurred following the application of the milking or suckling stimulus (Cross, 1950; Peeters and Coussens, 1950; Kalliala and Karvoncn, 1951; Kalliala, Karvonen and Leppanen, 1952). It was also shown that electrical stimulation of the nerve paths to the posterior pituitary resulted in milk ejection (Cross and Harris, 1950, 1952; Andersson, 1951a, b, c; Popovich, 1958 », and that when lesions were placed in these tracts the milk-ejection reflex was abolished (Cross and Harris, 1952) .
Further evidence was adduced when it was found that removal of the posterior pituitary immediately abolished the milkejection reflex in the lactating rat, and that it was necessary to inject such animals several times a day with oxytocin if their litters were to be reared (Cowie, quoted by Folley, 1952b). Earlier workers had claimed that the posterior lolie was not essential for lactation (Smith, 1932; Houssay, 1935), but an explanation of these discordant conclusions was provided when it was shown that the impairment of the reflex after removal of the posterior lobe is not permanent and that the reflex re-establishes itself after some weeks, presumably because the remaining portions of the neurohypophysis take over the functions of the posterior lobe (Benson and Cowie, 1956). That the neurohypophysis participates in milk ejection would now appear to be beyond question.
The discovery of the role of the neurohypophyseal hormones in milk ejection has provided an explanation of some longstanding clinical observations on what has been termed the natural "sympathy" between the uterus and the breasts. Thus the beneficial effects of the suckling stimulus and the occurrence of the "draught" {i.e., milk ejection) in causing uterine contraction after parturition were emphasized over a century ago by both Smith (1844) and Patcrson (1844). 0})servations have also been made on the I'cciprocal process of stimuli arising from the reproductive organs apparently causing milk ejection. In domestic animals two such examples were mentioned by Martiny (1871). According to Herodotus, the Scythians milk their mares thus: "They take l)lowpipes of bone, very like flutes, and put them into the genitals of the mares and blow with their mouths, others milk. And they say that the I'cason why thoy do so is this, that when the marc's \-cins ai'c filled with air, the udder cometh down" (translation by Powell, 1949). Kolbe (1727) described a similar procedure of blowing air into the vagina used by the Hottentots when milking cows which were normally suckled by calves and in which, presumably, milk ejection did not occur in response to hand nnlking. A drawing depicting this procedure from Kolbe's book was recently published in the Ciba Zeitschrift (No. 84^ 1957) along with a photograph of African natives still using the method!-^
In 1839, Busch described the occurrence of milk ejection, the milk actually spurting from the nipple, in a lactating woman during coitus. A satisfactory explanation of these curious observations is now forthcoming. Harris (1947) suggested that coitus might cause the liberation of oxytocin from the neurohypophysis and, within the next few years it was demonstrated that stimulation of the reproductive organs evoked milk ejection in the cow (Hays and VanDemark. 1953) and reports confirmatory of Busch's long forgotten observations also appeared (Harris and Pickles, 1953; Campliell and Petersen, 1953).^
C. Milk-Ejectiox Hormone
There is much circumstantial evidence to confirm the belief that the milk-ejection hormone is oxytocin (see Cowie and Policy. 1957). Attemi)ts, however, to demonstrate oxytocin in the blood after application of the milking stimulus have given rather inconclusive results. Early claims that the hormone could be demonstrated in blood are
^ A similar drawing, also apparently from Kolbe '.•< book, has been used in the campaign for clean milk production! Heineman (1919) discussing sanitary l^recautions in the cowshed says of the picture "another picture shows a nude Hottentot milking a cow while another one is liolding the tail of the cow to prevent its dropping into the open pail. This ])icture might well serve as a model to some modern producers who do not take such precautions and calmly lift the tail out of the milk with their hands wlicn it hnjipens to switch into the pail."
' W(- h;i\(' hi'cii able to find only one painting illustrating this plienomenon. It is a picture by a contemporary French painter, Andre Masson, entitled "Le Viol" and painted in 1939. It illustrates in Masson 's personal idiom the act of rape and it is interesting to note that a stream of milk is depicted as being I'orcibly (\iected from one breast of the of doiil)tful validity, because the milk-ejection effect observed may have been due to 5-hydroxytryptamine (see Linzell, 1955), and more recent attempts to assay the level of oxytocin in the blood have not been entirely satisfactory or conclusive. There seem to be other polypeptide substances in blood which possess oxytocic activity, although the thiogly collate inactivation test indicates that these are different from oxytocin (Robertson and Hawker, 1957), and no marked changes in the blood oxytocic activity associated with suckling or milking have been detected (Hawker and Roberts, 1957; Hawker, 1958). However, it would seem doubtful whether the present assay techniques are sufficiently sensitive and specific to detect changes in blood oxytocin of the magnitude likely to be associated with milking or suckling. In the lactating cow the intravenous injection of 0.05 to 2.0 I.U. oxytocin will cause milk ejection (Bilek and .Tanovsk>% 1956; Donker, 1958), in the goat 0.01 to 1 I.U. (Cowie, cited by Folley, 1952b; Denamur and Martinet, 1953), in the sow 0.2 to 1.0 I.U. (Braude, 1954; Whittlestone, 1954; Cross, Goodwin and Silver, 1958) in the rabbit 0.05 I.U. (Cross, 1955b) , and in the lactating woman 0.01 I.U. (Beller, Krumholz and Zeininger, 1958) . If these (loses give any indication of the quantity of endogenous oxytocin released, then the concentration in the peripheral blood is likely to be very small ; indeed Cross, Goodwin and Silver (1958) calculated that a threshold dose (10 mU.) of oxytocin in the sow w^ould give a plasma concentration of about 1 (U,U. per ml, and until it can be shown that the assay techniques are sufficiently sensitive to detect the changes in oxytocin concentration produced by intravenous injections of "physiologic" doses of oxytocin, no great reliance can be placed on the results of assays.
Attempts have been made to demonstrate alterations in the hormone content of the neural lobe following the suckling or milking stimulus. In the goat and cow no detectable changes have been reported, but in the smaller species (dog, cat, rat, guinea pig) decreases have been described (see Cowie and Folley, 1957). It is likely that in many species the amount released is small relative to the total hormone content of the gland and within the limits of error of the assay.
D. Effector Contractile Mechanism of the Mammary Gland
In the last 10 years considerable research has been devoted to a study of the effector contractile tissue in the mammary gland; this work has recently been reviewed in some detail (see Folley, 1956) and only the salient features need be mentioned here.
Although earlier histologists had from time to time figured myoepithelial or "basket" cells in close association with the mammary alveoli, the morphology and distribution of the cells remained vague until Richardson (1949) published a detailed and illuminating description (Fig. 10.18). His beautiful observations have since been confirmed and supplemented by Linzell (1952) and Silver (1954). Richardson also disposed of the oft repeated view that smooth-muscle fibers around the alveoli played an iml)ortant role in milk ejection. From a study of the general orientation of the myoepithelial cells and the precise relationship between these cells and the folds in the secretory epithelium from contracted glands, Richardson considered it reasonable to regard the myoepithelium as the contractile tissue in the mammary gland which responds to oxytocin causing contraction of the alveoli and widening of the ducts. The evidence adduced by Richardson, although good, was nevertheless circumstantial, and it was desirable that attempts be made to visualize the contraction of the myoepithelial cells in response to oxj^tocin. In this connection it is of interest to recall that Gaines (1915) reported that when a drop of pituitrin was placed on the cut surface of the mammary gland from a lactating guinea pig, minute white dots appeared within a few seconds beneath the pituitrin and slowly swelled to tiny milky rivulets streaming beautifully through the clear liquid. Much later the local effects of posterior pituitary extract on the mammary gland were studied by Zaks (1951) in the living mouse, when it was reported that it caused contraction of the alveoli and expansion of the ducts. These observations were considerablv extended bv Linzell
Fig. 10.18. Surface view of contracted alveoli (of goat) showing myoepithelial cells.
(Courtesy of K. C. Richardson.)
Fig. 10.19. Recording of pressure changes witliin
a galactophore of a forcibly restrained lactating
rabbit. The litter was allowed to suckle the noncannulated mammary glands but obtained only
8 gm. milk, there being only a slight rise in the
milk pressure probably associated with a slight
contraction of the myoepithelium in response to
mechanical stimulation. When 5 mU. oxytocin were
injected (5P) there was a rapid milk ejection
response which could be inhibited by injecting 1
yug. adrenaline (lA) just before the oxytocin. After
a few minutes 5 mU. oxytocin were again effective
and the litter obtained 44 gm. milk when they were
allowed to suckle. A more complete milk ejection
respon.so was obtained with 50 mU. oxytocin (50P)
and the young obtained a further 59 gm. milk.
Anesthesia did not enhance the milk-ejection response to 50 mU. oxytocin. During emotional inhibition of milk ejection the mammary gland thus
remains responsive to oxytocin. (From B. A. Cross,
J. Endocrinol., 12, 29-37, 1955.)
(19ooi who studied the local effects of liighly purified oxytocin and vasopressin and a number of other drugs on the mammnry gland, and confirmed that oxytocin and vasopressin produced alveolar contraction and widening of the ducts. Although in these experiments the myoepithelial cells themselves could not be visualized, nevertheless the effects observed leave little doubt that the effector mechanism was the niyoei)ithelium.
The myoepithelium is responsive to stimuli other than those arising from the presence of neurohypophyseal hormones in the blood inasmuch as partial milk ejection may occur in response to local mechanical stimulation of the mammary gland (Cross, 1954; Yokoyama, 1956; see also Fig. 10.191. These observations may explain the recent reports by Tverskoi (1958) and Denamuiand Martinet (1959a, b) that milk yields can be maintained in goats in the absence of the milk-ejection reflex.
E. Inhibition of Milk Ejection
(laines (1915) stressed that the conduction in the milk-ejection reflex pathway was dei)endent on the psychic condition of the mother. Many years later Ely and Petersen (1941) confirmed this and, having shown that injections of adrenaline blocked the milk-ejection reflex, postulated that the increased blood level of adrenaline in emotionally disturbed cows interfered with the action of oxytocin. In the last few years, the nature of the inhibitory mechanisms has been more fully investigated. Braude and Mitchell (1952) showed in the sow that adrenaline exerts at least part of its inhibitory effect at the level of the mammary gland and that, whereas the injection of adrenaline before the injection of oxytocin blocked milk ejection, less inhibition occurred if both were given together. Cross (1953, 1955a) confirmed these observations in the rabbit and demonstrated that electrical stimulation of the posterior hypothalamus (sympathetic centers) inhibited the milk-ejection response to injected oxytocin, an effect which was abolished after adrenalectomy. Cross concluded from his experiments that any central stimulation causing sympathetico-adrenal activity inhibits the milk-ejection response and that the effect appears to depend on a constriction of the mammary blood vessels resulting from the release of adrenaline and excitation of the sympathetic fibers to the mammary glands. Whereas such a mechanism could account for the emotional disturbance of the reflex. Cross was careful to point out that there was no direct proof that this was so and he later demonstrated (Cross, 1955b) that in rabbits in which emotional inhibition of milk ejection was present, milk ejection could be effected by the injection of oxytocin (Fig. 10.19). In such cases there was clearly no peripheral inhibitory effect of milk ejection. Cross concluded that the main factor in emotional disturbance of the milkejection reflex is a partial or complete inhibition of oxytocin release from the posterior pituitary gland. At present nothing is known of the nature of this central inhibitory mechanism.^
^ A curious form of the suckling stimulus is illustrated in carvings which siumount the main door of the church of Sainte Croix in Bordeaux. The carvings illustrate penances prescribed for wrong doers who have committed one of the seven deadly sins. The penance for indulgence in the sin of luxiu y is the application to the breasts of serpents or toads.
Inhibition of the milk ejection reflex may also occur when the mammary gland becomes engorged with secretion to such an extent that the capillary circulation is so reduced that oxytocin can no longer reach the myoepithelium (Cross and Silver, 1956; Cross, Goodwin and Silver, 1958).
F. Neural Pathways of the Milk-Ejection Reflex
Interpretation of some of the earlier studies on neural pathways is difficult because investigators did not realize that, although the milk ejection reflex normally occurs in response to the suckling stimulus, it can become conditioned and can then occur in response to visual or auditory stimuli associated with the act of nursing. In such cases an apparent lack of effect on milk ejection of section of nerves or nerve tracts would not necessarily imply that the nerves normally carrying the stimuli arising from the suckling had not been cut. Studies on the effects of hemisection of the spinal cord in a few goats led Tsakhaev (1953) to the conclusion that the apparent pathway used by the milk-ejection stimulus was uncrossed. More recently pathways within the spinal cord have been investigated by Eayrs and Baddeley (1956) who found inter alia that lactation in the rat was inhibited by lesions to the lateral funiculi, and by section of the dorsal roots of nerves supplying the segments in which the suckled nipples were situated. With few exceptions hemisection of the spinal cord abolished lactation when the only nipples available for suckling were on the same side as the lesion, but not when the contralateral nipples were available. It was concluded that the pathway used by the suckling stimulus enters the central nervous system by the dorsal routes and ascends the cord deep in the lateral funiculus of the same side. Inasmuch as in these experiments lactation was assessed from the growth curve of the pups, it is not always clear whether the failure of lactation was due to a cessation of milk secretion or to loss of the milk-ejection reflex. It was noted, however, that injections of oxytocin in some
It may be questioned whether this unusual form of the suckling stimulus would not inhibit rather than evoke the milk-ejection reflex.
cases restored lactation for up to 2 days
after it had ceased as a result of lesions of
the cord which would suggest a primary
interference with milk ejection. In the goat,
Andersson (1951b) considered that stimuli may reach the hypothalamus by way of
the medial lemniscus in the medulla, but
little definite information is available concerning the pathways used by the stimuli
to reach the hypothalamus and there is here
scope for further investigations. (For further discussion see review by Cross, 1960.)
From the hyopthalamus there is little doubt
that the route to the posterior lobe is by
way of the hypothalamo-hypophyseal tract
which receives nerve fibers from the cells in
the hypothalamic nuclei, and in the main
from the paraventricular and supra-optic
nuclei. It was generally assumed that the
posterior lobe hormones were secreted in
the posterior lobe from the pituicytes in response to stimuli passing down the hypothalamo-hypophyseal tract. In the last decade, however, much evidence has come to
light which suggests that the so-called posterior lobe hormones are in fact elaborated
in the cells of the hypothalamic nuclei and
are then transported down the axones as a
neurosecretion and stored in the posterior
lobe (see Scharrer and Scharrer, 1954).
Before leaving the neural pathways of the milk-ejection reflex, brief reference must be made to the recent discovery by Soviet physiologists that there is also a purely nervous reflex (segmental in nature) involved in the ejection of milk. It is said that within a few seconds of the application of the milking stimulus, reflex contraction of the smooth muscle in the mammary ducts occurs, causing a flow of milk from the ducts into the cistern. This reflex contraction of the smooth muscle is also believed to occur in response to stimuli arising within the gland between milkings thus aiding the redistribution of milk in the udder. This purely nervous reflex is stated to occur some 30 to 60 seconds before the reflex ejection of milk from the alveoli by oxytocin (for further details sec review by Baryshnikov, 1957). The conditioned reflexes associated with suckling and milking have been the subject of numerous investigations l)y Grachev (see Grachev, 1953, 1958) ; these and other Russian researches into the motor apparatus of the udder have been fully reviewed by Zaks (1958).
G. Mechanism Of Suckling
In the past, various theories have been put forward as to how the suckling obtains milk from its mother's mammary gland. In the human infant some considered that the lips formed an airtight seal around the nipple and areola thus allowing the child to suck, whereas others believed that compression of the lacteal sinuses between the gums aided the expulsion of the milk (see Ardran, Kemp and Lind, 1958a, b for review) . In the calf the act of suckling was studied by Krzywanek and Briiggemann (1930) who described how the base of the teat was pinched off between upper and lower jaws and the teat compressed from its base towards its tip by a stripping action of the tongue. Smith and Petersen (1945) on the other hand, concluded that the calf wrapped its tongue round the teat and obtained milk by suction.
Much misunderstanding about the nature of the act of suckling has arisen because the occurrence of milk ejection was overlooked or its significance was not appreciated. As a result, the idea became prevalent that success or failure in obtaining milk could be reckoned solely in terms of the power behind the baby's suction. This erroneous concept was vigorously attacked by Waller (1938), who pointed out that once the "draught" had occurred the milk at times flowed so freely from the breast that the baby had to break off and turn its head to avoid choking. A similar observation had been made by Sir Astley Cooper in 1840 who in describing the "draught" in nursing women wrote, "If the nipple be not immediately caught by the child, the milk escapes from it, and the child when it receives the nipple is almost choked l)y the rapid and abundant flow of the fluid; if it lets go its hold, the milk spurts into the infant's eyes." An even earlier comment was made by Soranus, a writer on paediatrics in the cai'ly half of the second century A.D., that it was unwise to allow the infant to fall asleep at the breast since the milk sometimes flowed without suckling and the infant choked. It must thus be emphasized that once milk ejection has occurred the milk in the gland cisterns or sinuses is under considerable pressure and the suckling has merely to overcome the resistance of the sphincters in the nipple or teat to obtain the milk.
Recently the use of cineradiograjihy has allowed a more accurate analysis of the mechanism of suckling. Studies by Ardran, Kemp and Lind (1958b) have shown that the human infant sucks the nipple to the back of the mouth and forms a "teat" from the mother's breast; when the jaw is raised this teat is compressed between the upper gum and the tip of the tongue resting on the lower gum, the tongue is then applied to the lower surface of the "teat" from before backwards pressing it against the hard palate. Suction may assist the flow of milk so expressed from the nipple, but is only of secondary importance. Studies by Ardran, Cowie and Kemp (1957, 1958) in the goat have extended these observations, because it was possible in this species to follow the withdrawal, from the udder, of milk made radiopaque with barium sulfate. As with the infant, the neck of the teat was obliterated between the tongue and the palate of the kid and the contents of the teat sinus were displaced into the mouth cavity by a suitable movement of the tongue; while the first mouthful w^as being displaced into the pharynx, the jaw and tongue were lowered to allow the refilling of the teat sinus. The normal method of obtaining milk is, therefore, for the suckling to occlude the neck of the teat and then to expel the contents of the teat sinus by exerting positive pressure on the teat (120 mm. Hg in the goat), so forcing the contents through the teat canal or nipple orifices into the mouth cavity, a process which may be aided by negative pressure created at the tip of the teat. Human infants, goat kids, and calves can obtain milk through rubber teats by suction alone provided the orifice is large enough (see Krzywanek and Briiggemann, 1930; Martyugin, 1944; Ardran, Kemp and Lind, 1958a) , but this procedure occurs only w^hen the structure of the rubber teat is such that the suckling is unable to ol)literate the neck of the teat and cannot, therefore, strip the contents of the teat by positive pressure.
V. Relation between the Reflexes Concerned in the Maintenance of Milk Secretion and Milk Ejection
We have seen that the suckling or milking stimulus is responsible for initiating the reflex concerned wath the maintenance of milk secretion and also the milk-ejection reflex; the question now arises as to what extent their arcs share common paths. It would seem logical to assume that a common path to the hypothalamus exists and parts of this, as we have seen, have been partially elucidated. Although the hypothalamo-hypophyseal nerve tracts provide an obvious link between hypothalamus and the posterior lobe, the connections between the hypothalamus and anterior pituitary are still a matter of some controversy. The possible avenues of communication to the anterior lobe are neural and vascular and these may be subdivided into central and peripheral neural connections and into portal and systemic vascular connections. The various experimental findings relating to these routes have recently been critically discussed by Sayers, Redgate and Royce (1958), and by Greep and Everett in their chapters in this book, and it is clear that at present no definite conclusions can be reached concerning their relative importance. So far as the specific question of maintenance of milk secretion is concerned, the experiments of Harris and Jacobsohn (1952), which showed that pituitary grafts maintained lactation when implanted adjacent to the median eminence in hypophysectomized rats, were consistent with the existence of a hormonal transmitter, passing by w^ay of the hypophyseal portal system. On the other hand, transplantation studies by Desclin (1950, 1956) and Everett ( 1954, 1956) have revealed that in the rat the anterior lobe can spontaneously secrete prolactin in situations remote from the median eminence, and Donovan and van der Werff ten Bosch (1957) have reported that milk secretion continued in rabbits in wiiich the pituitary portal vessels had been completely destroyed, although there was, however, an inferred change in milk composition. Evidence has recentlv been obtained which has confirmed that pituitary tissue grafted under the kidney capsule in rats apparently secretes prolactin and will give slight maintenance of milk secretion in hypophysectomized animals, this maintenance being considerably enhanced if ACTH or STH is also administered (Cowie, Tindal and Benson, 1960). It would thus seem that the cells of the anterior lobe have the ability when isolated from the hypophyseal portal system to secrete prolactin, but the experiments cited above allow no conclusions to be drawn regarding the route by which the galactopoietic function of the pituitary is normally controlled.
Recent reports that bilateral cervical sympathectomy in the lactating goat causes a fall in the milk yield suggest that the galactopoietic functions of the anterior lobe may be influenced by the sympathetic nervous system (Tsakhaev, 1959; Tverskoy, 1960) . Declines in milk yield also occur after section of the pituitary stalk in the goat, but it is not clear in such cases whether the effects are due to the interruption of nervous or vascular pathways within the stalk (Tsakhaev, 1959; Tverskoy, 1960). In these studies on stalk section the cut ends of the pituitary stalk were not separated by a plastic plate, so some restoration of the hyl^ophyseal portal system may have occurred. Further experiments on the effects of section of the pituitary stalk on lactation in which restoration of the hypophyseal portal is prevented by the insertion of a plate are being conducted in our laboratory and also in the Soviet Union. Another possible mode of communication between hypothalamus and anterior pituitary has been investigated by Benson and Folley (1956, 1957a, b) who have suggested that the oxytocin released from the neurohypophysis in response to the suckling stimulus may directly act on the cells of the anterior lobe and stimulate the release of the galactopoietic complex. The careful anatomic researches of Landsmeer (1951), Daniel and Prichard (1956, 1957, 1958) and Jewell (1956) have demonstrated in several species the existence of direct vascular connections from the neurohylK)physis to the anterior lobe so that the neurohypophyseal hormones liberated into the blood stream would in fact be carried
direct to the anterior pituitary cells in very high concentrations. Clearly such a concept would provide a simple explanation of how the hormonal integration, coordination, and maintenance of mammary function is achieved. It has already been noted (see page 607) that a connection between milk ejection and the onset of copious lactation has been suggested. There is considerable evidence that oxytocin is liberated during parturition in sufficient quantities to cause contraction of the alveoli and milk ejection (see Harris, 1955; Cross, 1958; Cross, Goodwin and Silver, 1958) ; if, therefore, oxytocin can release the lactogenic and galatopoietic complexes from the anterior pituitary, a simple explanation of the mechanism triggering off the onset of copious milk secretion, before the application of the milking stimulus, is available.
We must now consider what experimental evidence there is to support this rather attractive theory. First, Benson and Folley (1956, 1957a, b) demonstrated that regular injections of oxytocin can retard mammary regression after weaning in a similar fashion to injections of prolactin (see page 610), and they have shown that the presence of the pituitary is essential for oxytocin to elicit this effect. Synthetic oxytocin proved equally effective, thus discounting the possibility of a contaminant in natural oxytocin being concerned (Fig. 10.20) . These experiments have so far only been carried out in rats, but they strongly suggest that oxytocin can elicit the secretion of prolactin. In agreement with this concept are several observations that regular injections of oxytocin have galactopoietic effects in lactating cows and that oxytocin has luteotrophic effects in rats (see review by Benson, Cowie and Tindal, 1958) . There is, moreover, some evidence that the suckling stimulus may cause the release of vasopressin or the antidiuretic hormone (ADH) from the neurohypoi)hysis (see page 621), and it has been shown that ADH or some material closely associated with it may cause the secretion of ACTH from the anterior lobe (see review by Benson, Cowie and Tindal, 1958) ; so there are some grounds for supposing that the hormones of the posterior lobe evoke the secretion of several components of the
Fig. 10.20. Sections from abdominal mammary gland of rats from wliuli Ur- pups were removed on the fourth day of lactation and which received thereafter for 9 daj^s: A. LO I.U. synthetic oxytocin three times daily. B. Saline daily. Note the maintenance of gland structure in A. (Courtesy of Dr. G. K. Benson.)
galactopoietic complex from the anterior lobe. It was hoped to gain further evidence on this point by studies on hypophysectomized rats bearing pituitary homografts under the kidney capsule (see Benson, Cowie, Folley and Tindal, 1959) . As already noted, such grafts secrete prolactin and will give a slight maintenance of milk secretion, but these grafts will not maintain normal milk secretion even when such animals are injected with oxytocin and ADH (Cowie, Tindal and Benson, 1960). It must, therefore, be assumed that if these posterior pituitary hormones are responsible for the release of the galactopoietic complex, some other hypothalamic factor is also necessary to maintain the anterior lobe in a responsive condition. Everett (1956) suggested that the hypothalamus by way of its neurovascular connections with the anterior lobe, normally exerts a partial inhibitory effect on prolactin secretion. It may thus be that when the anterior lobe is removed from hypothalamic influence, the synthetic activities of its cells are centered on prolactin production to the detriment of the other components of the galactopoietic complex, so that these are no longer available for release in response to neurohypophyseal hormones. There is need, however, for experimentation in other species.
The theory that the release of the galactopoietic complex is effected by the hormones of the posterior lobe secreted in response to the suckling stimulus is attractive in that it appears to afford a simple explanation of the hormonal integration of mammary function, but it must be pointed out that the observations on the maintenance of mammary structure after weaning by injections of oxytocin do not prove that prolactin or the galactopoietic complex is released in response to oxytocin under normal conditions of milking or suckling, and more research, particularly in species other than the rat, is necessary. Grosvenor and Turner (1958a) injected oxytocin into anesthetized lactating rats and, on the basis of assays of the pituitary content of prolactin, considered that oxytocin caused no significant release of prolactin. They had previously shown that there was an immediate fall in the pituitary content of prolactin after nursing (Grosvcnor and Turner, 1957b) and therefore concluded that their findings were contrary to the hypothesis that oxytocin is a hormonal link in the discharge of prolactin. This, however, cannot be regarded as conclusive because of the difficulties of relating pituitary content of a hormone to blood levels of the hormone and also the difficulty of determining the physiologic dose of oxytocin, for if the oxytocin is carried directly from the neurohypophysis into the anterior lobe, then the concentration in the blood reaching the anterior lobe may be relatively great (see also Cowie and Folley, 1957).
Other theories of the reflex maintenance of milk secretion have been put forward. In 1953 Tverskoi, observing that repeated injections of oxytocin were galactopoietic in the goat, suggested that alveolar contraction stimulated sensory nerve endings in the alveolar walls which reflcxly caused the release of prolactin. It is obvious that his observations could be explained on the basis of the Benson-Folley theory of direct pituitary stimulation by oxytocin. This possibility was indeed considered by Tverskoi. but rejected on the grounds that oxytocin did not affect the prolactin content of the pituitary (Meites and Turner, 1948). In 1957 Tverskoi found it necessary to revise his theory, having found that full lactation could be maintained in the goat after complete and repeated denervation of the udder provided oxytocin was regularly given to evoke milk ejection. He then suggested that alveolar contraction stimulates the synthetic activities of the mammary epithelium causing an uptake of prolactin from the blood, the fall in the blood prolactin level then stimulating the further production of prolactin by the anterior lobe. Although these latter observations of Tverskoi might again be explained on the basis of direct pituitary stimulation by exogenous oxytocin, more recent studies on goats have cast doubts on the validity of such an explanation. Tverskoi (1958) and Denannir and Martinet (1959a, b, 1960) have shown that lactating goats will continue to lactate, giving nonnal or onlv niodcratelv reduced milk yields after section of all nervous connections between the udder and brain (cord section, radicotomy, bilateral sympathectomy) and without their receiving oxytocin and in the absence of conditioned milkejection reflexes. It has already been noted that milk ejection in such animals may result from mechanical stimulation of the myoepithelial cells by udder massage (see page 624) , but the release of the galactopoietic complex from the anterior pituitary would seem in these goats to have been independent of neurohormonal reflex activities. AVhether in such animals the release is spontaneous or dependent on the level of hormones in the blood as suggested by Tverskoi (1957) is a matter for further research.
VI. Pharmacologic Blockade of the Reflexes Concerned in the Maintenance of Milk Secretion and Milk Ejection
Various attempts have been made to investigate the mechanism controlling release of anterior pituitary hormones by the use of dibenamine, atropine, and other drugs. In reviewing such experiments, Harris (1955) concluded that there was no convincing evidence of the participation of adrenergic, cholinergic, or histaminergic agents in the control of gonadotrophic and adrenocorticotrophic hormone release. Recently Grosvenor and Turner (1957a) reported that various ergot alkaloids, dibenamine, and atropine blocked milk ejection in the rat; the ergot alkaloids doing so within 10 minutes of administration, the atropine and dibenamine within 2 to 4 hours. Inasmuch as milk ejection occurred in response to exogenous oxytocin, it was concluded that these drugs acted centrally, and the presence of adrenergic and cholinergic links in the neurohormone arc was postulated to be responsible for the discharge of oxytocin. Later, on the basis of assays of jntuitary prolactin after nursing in druginjected lactating rats, it was suggested that cholinergic and adrenergic links are iinohcd in the reflex resi)onsible for prolactin release (Grosvenor and Turner, 1958a). Ergot alkaloids, however, administered in our laboratory to lactating rats had no significant effect on the lactational performance as judged by the growth of the litters in comparison with the growth of litters of pair-fed control rats, showing that apparent inhibitory effects of the alkaloids on lactation were due to depressed food intake of the mothers (Tindal, 1956a). Inasmuch as growth of the litter depends on efficient milk secretion and milk ejection, Tindal's observations seem to throw doubt on the importance of the adrenergic link in these reflexes. On the other hand, IVIeites (1959) has reported that adrenaline and acetylcholine can induce or maintain mammary development and milk secretion in suitably prepared rats, observations which could be interpreted as supporting the presence of adrenergic and cholinergic links as postulated by Grosvenor and Turner (1958a).
There have been clinical reports of women developing galactorrhoea after treatment with trancjuilizing drugs {e.g., Sulman and Winnik, 1956; Marshall and Leiberman, 1956; Piatt and Sears, 19561 and interesting observations have recently appeared on the lactogenic effects of reserpine in animals. Milk secretion has been initiated both in virgin rabbits after suitable estrogen priming and in the pseudopregnant rabbit by reserpine (Sawyer, 1957; Meites, 1957a). On the other hand, in our laboratory Tindal (1956b, 1958) had been unable to detect any mammogenic or lactogenic effects with chlorpromazine or reserpine in rabbits (Dutch breed), rats, or goats, nor did reserpine stimulate the crop-sac when injected into pigeons. Recently, using New Zealand White rabbits, Tindal (1960) has induced milk secretion with reserpine. The reason for these contradictory results is not entirely clear, although breed differences in the response would appear to exist in the rabbit. In our laboratory, Benson (1958) has shown that reserpine is strikingly active in retarding mammary involution in the lactating rat after weaning, the effect being of such a magnitude as has so far only been equalled by a combination of prolactin and STH (Fig. 10.21). It has been tentatively suggested that the tranquilizing drugs may remove .some hypothalamic restraining mechanism on the release of jn'olactin and probably of other anterior-pituitary hormones (Sulman and Winnik, 1956; Benson, Cowie and Tindal, 1958), an effect which, if confirmed, may throw light on the behavior of pituitary transplants in sites remote from the median eminence.
Fig. 10.2L Sections from the abdominal mammary gland of rats from whichthe pujis were removed on the fourth day of lactation and which received thereafter for 9 days: A 100 fj.g. reserpine daily. B. Sahne dailJ^ Note the retardation of involution effected by reserpine. (Courtesy of Dr. G. K. Benson.)
VII. Conclusion
Any reader familiar with the chajiter on the mammary gland in the previous edition of this book cannot fail to note the main directions in which the subject has advanced in the intervening two decades. These reflect, as they are bound to do, the road taken by the science of endocrinology itself, a road leading to greater biochemical understanding on the one hand and to ever closer rapprochement with neurophysiology on the other.
The mammary gland offers unique opportunities of studying the biochemical mechanisms of hormone action because it is an organ with quite exceptional synthetic capabilities, an organ which is perhaps the most comprehensive hormone target in the mammalian body. Biochemists are entering this promising field in increasing numbers and we may expect to reap the fruits of their labors in the future.
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