Book - Sex and internal secretions (1961) 9

Young WC. Sex and internal secretions. (1961) 3rd Eda. Williams and Wilkins. Baltimore.

SECTION C Physiology of the Gonads and Accessory Organs

Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates

Frederick L. Hisaw, Ph.D.

The Biological Lab0Rat0Rif:S, Harvard Tniversity, Cambridge, Massachusetts

and

Frederick L. Hisaw, Jr., Ph.D.

Department Of Zoology, Oregon State College, Corvallis, Oregon

I. Introduction 556

II. Ovarian Hormones and Growth of

THE Genital Tract 558

III. Effects of Progesterone on the

Uterus 565

IV. Synergism between Estrogen and

Progesterone 567

V. Experimentally Produced Implantation Reactions 571

VI. The Cervix Uteri 572

VII. The Vagina 575

VIII. Sexual Skin 576

IX. Menstruation 578

X. The Mechanism of Menstru.^tion. . 583 XI. References 586

I. Introduction

Cyclic menstruation is the most characteristic feature of primate reproduction, and distinguishes it from the estrous cycle of lower mammals. This cardinal primate event is heralded by the bloody uterine effluent emanating from the vagina, whereas in estrus the dominant characteristic is a sudden modification in behavior featuring an intense mating drive. However, the internal secretions that regulate the various events in the menstrual and estrous cycles are the same, and this similarity is fundamentally more significant than the key descriptive differences just mentioned. Estrus comes at the peak of the growth phase of the cycle and is associated with ovulation. In

contrast, menstruation occurs in the cycle midway between times of ovulation and is not accompanied by an increase in sexual activity. From earliest times menstruation has been recognized as degenerative: the characteristic odor, and the necrotic changes in the lining of the uterus, part of which is cast off at this time, sustain this interpretation. Therefore, menstruation is at the opposite phase of the cycle from estrus. It is such an obvious event that menstrual cycles are dated from the onset of bleeding. Menstruation is not analogous to the proestrous bleeding in the dog or cow nor to the slight bleeding of primates at midpoint between menstrual periods (Hartman, 1929). The study of menstruation was at first almost entirely the province of the clinician and the material for investigation limited to w^omen. Hitschmann and Adler (1907), Meyer (1911), Schroder (1914). Novak and Te Linde (1924), and Bartelmez (1933) are among many of the earlier investigators who contributed descriptions of the cyclic changes in the human endometrium. The physiology of the menstrual cycle and attendant morphologic changes have continued to be an area of active research interest in science and medicine. Among the many more recent contributors are Bartelmez

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(1937), Latz and Reiner (1942), Haman (1942), Knaus (1950), Mazer and Israel (1951), and Crossen (1953).

The earlier concepts regarding the menstrual cycle were based primarily on the changes occurring in the human endometrium and for convenience of description the cycle was divided into four stages or periods. The first of these was the period of active menstruation, and the length of the cycle was dated from its onset. Most authors agreed that menses began by leaking of blood from superficial vessels to form lakes under the surface epithelium and that there was some sloughing of tissue after the beginning of bleeding. There was considerable disagreement as to the amount of destruction and loss of tissue; estimates of various authors ranged from very little to almost complete denudation of the surface. Bartelmez (1933) emphasized both the wide individual variability of the amount of tissue lost and differences in the stage of development of the endometria at the time of menstruation.

The second period immediately following menstruation began with regeneration of the surface epithelium, which started sometimes before menstrual bleeding had ceased and was completed in a very short time. This lieriod included the 5 to 7 days after cessation of menses, during which the endometrium grew in thickness. Frequent mitoses were recognized, especially in the glands which lengthened but remained straight and tubular.

The third ("interval") period, lasting 6 to 10 days, was characterized by a somewhat thickened endometrium, still with straight glands and showing little evidence of secretory activity. At first this was considered a quiescent period as indicated by the term "interval." However, as will be shown later, such a characterization was not justified from the physiologic viewpoint.

The fourth period, called the premenstrual period, included the 10 days or 2 weeks before menstruation. During this phase the glands continued to increase in size and became distended, coiled, or even sacculated. The glandular cells increased in height, and there was evidence of glycogen mobilization and secretion. Next, the epithelium became "frayed out" along the

outer borders, then decreased in height, indicating secretory depletion. Decidual cells appeared in the stroma at this time. The endometrium was much thickened and extremely hyperemic. At the height of this period the endometrium was approximately 5 mm. in thickness, as compared with V2 mm. toward the end of menses. The term premenstrual was usually applied to this phase but today the term progestational would seem preferable.

During and after these descriptions of the changes in the human endometrium, many attempts were made to locate the time of ovulation in the menstrual cycle. When it was found, as will be discussed later, that ovulation occurred approximately midway between two menses, and was preceded by follicular growth and followed by development of a corpus luteum, it became customary to refer to the two halves of the menstrual cycle as the follicular phase and the luteal phase. One advantage of this descriptive terminology was the emphasis it placed on the homology of the two phases of the menstrual cycle in primates with the follicular and luteal phases of the estrous cycles of lower mammals.

A theory to explain menstruation, widely adopted in 1920, was formulated from this morphologic evidence. The essentials were that menstruation occurs because the lining of the uterus, prepared for implantation of the ovum, degenerates if fertilization of the egg does not occur. This required that ovulation and corpus luteum formation precede the i^remenstrual changes in the endometrium. Subsequent research disclosed that menstrual cycles frequently occur in which ovulation does not take place and bleeding results from the breakdown of an "interval" rather than a progestational endometrium.

The discovery of anovulatory cycles not only brought about a revision of ideas regarding an explanation for menstruation but also raised questions as to what constituted a normal menstrual cycle. The length of the cycle and the amount and duration of bleeding are approximately the same regardless of whether or not ovulation has taken place. The gross features of menstruation under these two conditions are indistinguishable one from the other. However, the biologic purpose of the menstrual cycle

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PHYSIOLOGY OF GONADS

is reproduction wliicli obviously cannot l)0 fulfilled unless an ovum is made available for fertilization. Therefore, in this sense it seems quite clear that anovulatory cycles should be considered incomplete and abnormal.

The investigation of changes taking place in the uterine endometrium at various periods of the menstrual cycle in women was confronted with many difficulties, the chief one being that of obtaining normal tissue representative of specific times of the cycle. The entire uterus and both ovaries are essential for proper evaluations and it was rarely possible to meet these requirements. The material for such studies came from autopsies and surgery and tissues usually had suffered postmortem changes or the surgical condition was one involving serious pelvic disease. There have been, however, a goodly number of instances in which these difficulties were adequately overcome (Stieve, 1926, 1942, 1943, 1944; Allen, Pratt, Newell and Bland, 1930) and the clinic will continue to make important contributions (Rock and Hertig, 1942; Hertig and Rock, 1944), but quite early the need became obvious for a suitable primate that could be used as an experimental animal for research on the different aspects of the physiology of reproduction.

Since the initial observations by Corner (1923) on the menstrual cycles of captive rhesus monkeys iMacaca mulatta) , more has been learned about the physiology of reproduction of this animal than any other primate. Monkeys of this species thrive under laboratory conditions, which has made it possible to devise accurately controlled experiments on normal healthy animals and obtain reliable information on the menstrual cycle, gestation, fetal development, and the interaction of hormones concerned with regulating reproductive processes.

Other features that make the rhesus monkey such an attractive animal for these purposes are the many morphologic and physiologic attributes that are strikingly like those of the human being. Tiic modal length of their menstrual cycles is 28 days but there is wide variation (Corner, 1923; Hartman, 1932; Zuckcrman, 1937a). From an analysis of 1000 cycles recorded for some 80 females of different ages, Zuckerman

(1937a) found an average cycle length of 33.5 ± 0.6 days, and the mode 28 days with an over-all range of 9 to 200 days. Ovulation occtu's api:)roximately midway between two menstrual periods, most between the 11th and 14th days (Hartman, 1932, 1944; van Wagenen, 1945, 1947), and although these animals breed at all seasons of the year many cycles are anovulatory, especially during the hot summer months (Eckstein and Zuckerman, 1956). A method developed by Hartman for detecting the exact time of ovulation by palpation of the ovaries in the unanesthetized animal greatly facilitated the timing of events of the menstrual cycle. This procedure also made it possible to determine the age of corpora lutea with great accuracy (Corner, 1942, 1945) and correlate their develojiment and involution with corresponding changes in the endometrium (Bartelmez, 1951 ) and, in ju-egnancy, with the exact age of developing embryos ( Wislocki and Streeter, 1938; Heuser and Streeter, 1941).

The primary purpose of the present discussion is to review the results of experimental investigations of physiologic processes occurring in the female reproductive tract of lower primates during the menstrual cycle, and particularly those processes that are under hormonal control. The brief introductory presentation of basic observations could be greatly extended and we take up the discussion of endocrine problems knowing that we must return often to the work of these authors and that of others to be cited, as conclusions based on experimental data take on meaning only in terms of normal function.

II. Ovarian Hormones and Growth of the Genital Tract

The changes that are repeated in different parts of the reproductive tract with each menstrual cycle are produced by ovarian hormones, estrogens, and progesterone. The dominant hormone of the follicular phase is estradiol-17/y, which is secreted by the Graafian follicle, and in the tissues is readily transformed in i)art to estrone, an estrogenic metabolite. Progesterone, secreted by the corinis luteum, is jirimarily a hormone of the luteal phase of the cycle. However, small amounts of progesterone may appear

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ill the blood of monkeys as early as the 7th (lay and attain a concentration of 1 fxg. per ml. of serum at ovulation, whereas a maximal concentration of 10 /xg. per ml. is reached at approximately the middle of the luteal phase (Forbes, Hooker and Pfeiffer, 1950; Bryans, 1951). Also, some estrogen is present during the luteal phase, probably secreted by the corpus luteum (estrogens can be obtained from luteal tissue) or it may be partially derived from developing follicles. It is unlikely that estrogen is ever entirely absent during a normal menstrual cycle or that the presence of progesterone is completely restricted to the luteal phase.

The dependence of the reproductive tract on ovarian hormones is strikingly demonstrated by the profound atrophy that follows surgical removal of the ovaries. A progressive decrease in size of the Fallopian tubes, uterus, cervix, and vagina takes place, and usually, the involution of the uterine endometrium involves tissue loss and bleeding, commonly referred to as post castrational bleeding. Dramatic as these effects may seem, it is equally dramatic to find that these atrophic structures can be restored entirely to their original condition by the administration of ovarian hormones. Therefore, it is seen at the beginning that investigations dealing with the physiology of the female reproductive tract of primates in large measure involve a study of the independent and combined actions of estrogens and progesterone on the activities of the various structures concerned.

Much can be learned about the action of ovarian hormones by observing the changes they produce in the gross appearance of the atrophied reproductive tract of castrated animals. Daily injections of an estrogen in doses equivalent to 1000 I.U. or more for 10 days or a fortnight will restore the uterus, cervix, and vagina to a condition comparal)le with that found in a normal monkey at the close of the follicular phase of a menstrual cycle (Fig. 9.1A). If a similar castrated monkey is given 1 or 2 mg. of progesterone daily for the same length of time, very little if any change in size of the reproductive organs results. However, if first the normal condition is restored by giving estrogen and then is followed by the progesterone treatment, the size of the uterus

is maintained but that of the cervix and vagina decreases to an extent approaching that in a castrated animal (Fig. 9.1B). Such experiments show that an estrogen promotes growth of the reproductive tract whereas progesterone is comparatively ineffective when given alone. Yet progesterone can maintain the size of the uterus when administered following an estrogen treatment but it does not prevent involution of the cervix and vagina.

An additional feature of the growth-stimulating action of the ovarian hormones is brought out when estrogen and progesterone are administered concurrently. If, after repair of the reproductive tract of a castrated animal has been accomplished by a series of injections of estrogen, both estrogen (1000 I.U.) and progesterone (1 or 2 mg.) are given daily for 20 days, it will be found that the uterus is larger than when either hormone is given alone for a similar length of time, whereas the cervix and vagina have involuted and are approximately the size found in animals given only progesterone. Thus it can be demonstrated that a synergistic effect on growth of the uterus occurs when the two hormones are given simultane

FiG. 9.1. Reproductive tracts of three adolescent monkeys which were castrated and given estrogen daily for approximately 3 weeks. A shows the condition at the conclusion of the estrogen treatment, B the condition following the injection of progesterone for an additional three weeks, and C the effects of continuing the treatment with both estrogen and progesterone for a like period.

560

PHYSIOLOGY OF GONADS

ously whereas in the cervix and vagina the growth -stimulating action of estrogen is inhibited by progesterone (Fig. 9.1C). This presents a most interesting situation in which the combined actions of two hormones on three closely associated structures of the reproductive tract join in a synergistic effort in promoting the growth of the uterus although progesterone prevents estrogen from affecting growth of the cervix and vagina.

These changes in gross morphology in response to the ovarian hormones are reflected in the histology of the responding tissues. Also, the character of the response differs depending upon the physiologic nature of the tissue concerned; therefore, for the sake of clarity each will be discussed separately. The first of these to be considered is the uterus and particularly growth of the endometrium.

It has been reported (Hisaw, 1935, 1950) that growth of the endometrium, as induced by estrogen, is limited. That is, a dosage of estrogen capable of maintaining the endometrium of a castrated animal for an indefinite period without the occurrence of bleeding, stimulates rapid growth for approximately the first 2 weeks. Within this time a maximal thickness of the endometrium is attained which remains constant or may become less during the course of treatment (Fig. 9.2). Engle and Smith (1935) made similar observations. They found that the endometria of castrated monkeys receiving estrogen for 100 days or longer were thinner than endometria of animals on estrogen for a much shorter time. Also, on prolonged treatment, the stroma of the endometrium becomes dense and the lumen small whereas the size of the uterus remains about the same. In fact, they state that in

I'll,. (1.2. I'll n Ml lour i-a.-^tra(c>d monkeys wliicli were givi^i 10 /ug. estradiol daily for 10 to 78 days. A was given estrogen for 10 days, B for 30 days, C for 60 days, and D for 78 days. Depression in the endometrium of anterior wall of D is the result of a biopsy taken a year previously. (From F. L. Hisaw, in A Si/niposium on Steroid Hormones, University of Wisconsin Press, 1950.)

ESTROGEN AND PROGESTERONE

561

four experimental animals the only well developed fundus was found in the animal on the shortest treatment, i.e., 60 days.

The mitotic activity in the epithelium of the glands and surface mucosa also indicates a limited effect of estrogen. This can be demonstrated to best advantage in the endometria of castrated monkeys that have been on estrogen for different lengths of time and have received an injection of colchicine 8 hours before their uteri were removed. A comparison of the number of cells in mitosis per square centimeter of surface mucosa at 10, 30, 45, and 60 days is shown in Figure 9.3. From this it can be seen that mitotic activity approaches that in a castrated animal. Although the five points used in drawing the curve are quite inadequate for an accurate analysis of the mitotic response in the epithelial components of the endometrium, they do show that cell division is most rapid soon after the beginning of an estrogen treatment and subseciuently declines.

The loss of responsiveness of the endometrium to estrogen seems related more to the length of treatment than to dosage of hormone. An endometrium of normal thickness can be produced in 2 or 3 weeks at a dosage level of estrogen that will not maintain the growth induced for longer than about 40 days without bleeding (Hisaw, 1935; Engle and Smith, 1935; Zuckerman, 1937b). The response to a low dosage of estrogen that will prevent bleeding during the course of treatment (about 10 /xg. estradiol-17/8 daily) is one of rapid endometrial growth at first, as has been described, followed by a thinning of the endometrium. The refractoriness of the endometrium to estrogen becomes so pronounced after about 100 days of treatment that very few cell divisions are seen in the epithelium of the glands and surface mucosa. The general morphology of the endometrium retains the characteristic appearance of the follicular phase of the menstrual cycle except that the stroma is usually more dense and the cells of the glandular epithelium have large deposits of glycogen between the nucleus and the basement membrane. However, metabolically such endometria are surprisingly inactive. Although they are dependent on the presence of estrogen and may bleed within about

Mitotic Response of Uterine Lpithelium TO looo I. u. Estrogen PER D«y.

Fig. 9.3. The number of mitoses per square centimeter of surface epithelium of the endometrium in a castrated monkey and in four castrated animals given 10 /xg. estradiol daily for 10, 30, 45, and 60 days, respectively. One-tenth of actual number of mitoses is shown on the ordinate. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.)

48 hours if the treatment is stopped, the activity of their oxidative enzymes and the ratio of nucleoproteins (RNA:DNA) are about the same as in the involuted endometria of castrated animals.

The effects that accompany moderate estrogenic stimulation become exaggerated in several respects when large doses of estrogen are given for an extended period. The disparity between the area of myometrium and endometrium becomes greater as the treatment progresses (Fig. 9.4). Kaiser (1947) described the destruction of the spiraled arterioles of the endometrium in monkeys given large doses of estrogen and Hartman, Geschickter and Speert (1941) reported the reduction of the reproductive tract to the size of that of a juvenile animal by the end of 18 months during which injections of large doses of estrogen were supplemented by subcutaneous implantation of estrogen pellets. These observations not only show that the endometrium becomes unresponsive to estrogen when the treatment is prolonged but that large doses produce injurious effects.

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PHYSIOLOGY OF GONADS

Fig. 9.4. .4. i'v'< — ' ri:,,ii of the uterus of a castrated monkey which had received 1.0 mg. estradiol daily for 35 days. Compare with B which shows the effects of 1/10 this dosage (100 fig. daily) when gi\en for 185 days.

The limited response of the endometrium to estrogen is in some respects surprising in view of its remarkable growth potentialities and regenerative capacity. These qualities were dramatically demonstrated by Hartman (1944) who dissected out as carefully as possible all of the endometrium from the uterus of a monkey and wiped the uterine cavity with a rough swab and yet the undetected endometrial fragments that remained were capable of restoring the entire structure. Also, considering the enormous increase in size of the uterus during gestation, it is even more difficult to account for the rather sharp limitation of growtli under the influence of estrogen.

The increase in tonus of the uterine musculature, a known effect of estrogen, has been considered as possibly exercising a restrictive influence on growth of the endo

metrium. An attempt has been made to remove this containing influence the muscle may have by making an incision through the anterior wall of the uterus (Hisaw, 1950) . A castrated monkey was given 10 ixg. estradiol daily for 21 days at which time the operation was performed and the treatment continued with 30 ^g. estradiol daily for 40 days. The uterus was laid open by a sagittal incision from fundus to cervix and most of the endometrium was removed from the anterior wall. This caused gaping of the incised uterus and exposure of the endometrium on the posterior wall. The incision was not closed and after hemorrhage was completely controlled the uterus was returned to the abdomen.

Examination of the uterus at the conclusion of the experiment showed no indications that endometrial growth had been enhanced. The muscularis had reunited and only a few small bits of endometrium were found in the incision (Fig. 9.5). It seemed probable that the purpose of the experiment had been defeated by rapid repair of the uterus. Therefore, a similar experiment was done in which the musculature of the incised uterus was held open by suturing a wire loop into the incision. Yet the incision closed and no unusual growth of the endometrium was detected (Fig. 9.6).

Observations under these conditions are necessarily limited to those made on the uterus when it is removed at the conclusion of an experiment and comparisons must be made between uteri of different animals. Obviously, it would be more desirable if the response of an individual endometrium could be followed during the course of treatment. It is possible to meet most of these requirements under conditions afforded by utero-abdominal fistulae, exteriorized uteri, and endometrial implants in the anterior chamber of the eye. In continuing our discussion we first shall present information obtained by such techni(iues that have a bearing on the response of the endometrium to estrogen.

The surgical procedure used by Hisaw ( 19501 for preparing utero-abdominal fistulae foi' studies of the exj^erimental induction of endometrial growth by estrogen and progesterone was a modification of that used by van Wagenen and Morse (1940) for ob

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Figs. 9.5 and 9.6

The uteri of these two castrated monkeys were opened from fundus to cer\'ix by an incision through the anterior wall while the animals were receiving estrogen. Part of the endometrium of the anterior wall was removed and the incision in the myometrium was not closed.

Fig. 9.5. Estradiol, 10 fig., was given daily for 7 days; the uterus was opened and the animal continued on 30 /xg. estradiol daily for 40 days. (From F. L. Hisaw, in A Sy7nposiii7n on Steroid Hormones, University of Wisconsin Press, 1950.)

Fig. 9.6. Estradiol, 10 ^g., was given daily for 7 days; the uterus was opened and the treatment continued at a dosage of 30 /ig. estradiol daily for 20 days. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.)

serving changes in the endometrium (luring the normal menstrual cycle. This procedure makes frequent inspections possible either by hand lens or dissecting microscope, of most of the upper part of the endometrium on the anterior and posterior walls of the uterus. The elliptic slit formed by the endometrium of the two opposing walls can be located easily, the two sides pressed apart by any small smooth instrument, and the surface of the endometrium examined. Changes in thickness of the endometrium cannot be ascertained without resorting to biopsies but it is free to grow out of the opened uterus if it is so inclined. However, in such preparations the growth produced in the endometrium by daily injections of 10 fjig. estradiol for periods of 2 or 3 weeks is not sufficient to show any tendency whatever to grow out through the fistular opening or obstruct examination of the walls of the uterus. The limited growth observed in these experiments is in agreement with that obtained with estrogen on intact and incised uteri.

The cervix uteri of the rhesus monkey is sufficiently long to make it possible to bring the entire fundus to the exterior through a midal)dominal incision. Advantage of this

was taken in an attempt to exteriorize the uterus and maintain it outside the body for long enough periods to make it possible to study the growth responses of the endometrium (Hisaw, 1950). These preparations did not i)rove satisfactory in all respects but they did contribute a number of interesting observations.

The operational i^rocedure used in these experiments involved dividing the uterus transversely from fundus to cervix so that the anterior wall was deflected downward and the posterior wall upward (Fig. 9.7). The endometrium of the exteriorized uterus is difficult to maintain but with proper care it seems to retain its normal condition for at least the first few days after the uterus is opened. Small localized areas of ischemia can be seen to come and go, probably action of the coiled arteries, and there is a periodic general blanching of the endometrium associated with rhythmic contractions of the muscularis. This, however, does not seem true of the whole endometrium. A zone surrounding the internal os of the cervix tends to retain its blood-red color even during strong contractions of the uterus and the growth reactions of the endometrium in this area are of particular interest.

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PHYSIOLOGY OF GONADS

Fig. 9.7. Exteriorized uteii. The uteri were divided transversely from fundus to cervix. The anterior half is seen deflected to the right and the posterior half to the left. A and B are of the same uterus taken 13 days after exteriorization showing the "blush" and "blanch" reaction of uterine contractions. It can be seen that during blanching the endometrium of the cervix does not become ischemic. C. Uterus 18 days after exteriorization, showing response to estrogen. Ridges formed by growth of the endometrium surrounding the internal OS of the cervix can be seen at the upper edge of the photograph. D, taken 83 days after exteriorization, shows response produced by a series of injections of 10 ^ig. estradiol and 1 mg. progesterone daily. The transverse ridge is formed by the two opposed lips of endometrium derived from the area surrounding the internal os of the cervix. Growth when the two hormones are given is greater than when only estrogen is injected. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.)

The endometrium on the exposed anterior and posterior halves of the uterus underwent deterioration despite the best of care that could be given, but that surrounding the internal OS of the cervix survived and retained its capacity to grow, in one animal, for as long as 9 months. When estrogen was given this endometrium grew rapidly and within a few days stood out as large elliptic lips surrounding the internal os (Fig. 9.7). Within 2 to 3 weeks the lips appeared to reach their full size and further growth was slow or absent. When estrogen treatments were discontinued the endometrial lips underwent bleeding within a few days and were entirely lost. At no time were activities observed that could be ascribed to coiled arterioles, nor did ischemia occur during involution previous to bleeding. It seems that the response of this tissue to estrogen is like that found in other experiments but the absence of ischemia preceding bleeding is ex

ceptional. The endometrium on the anterior and posterior walls of uterine fistulae invariably showed ischemia for several hours before active bleeding following the withdrawal of estrogen.

Markee (1940) approached the problem of endometrial growth in monkeys by studying the changes that occur in bits of endometrial tissue transplanted to the anterior chamber of the eye. Such transplants retain in large measure the normal morphology of endometrial tissue and changes in their cyclic growth parallel those going on simultaneously in the uterus. So much so that if the animal has an ovulatory cycle, the ocular implants show conditions characteristic of both the follicular and luteal phases, but if ovulation fails to occur then the luteal phase is omitted. Also, the morphologic events taking place at menstruation can be seen and recorded, since the transplants regress and bleed at each menstrual period.

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These ingenious experiments will be referred to often in the course of our discussion but at present the response of endometrial transplants in the eye to estrogen is of primary interest.

Monkeys having ocular transplants were given 200 to 300 R.U. of estrone daily for about 1 to 3 months. The transplants did not grow to a certain size and then remain stationary, but instead periods of rapid growth were interrupted by periods of regression which usually involved a marked decrease in size, and if regression was extensive and rapid, bleeding ensued. It also was found that these episodes of regression in the transplants were usually accompanied by a decrease in the size of the uterus.

Comparisons between the results of these experiments and those we have discussed previously may be misleading since it seems that only 1 of the 5 animals (no. 295) used was castrated. Also, the dosage of estrogen was not sufficient to maintain the endometrium of the uterus for an indefinite period without bleeding and this also was reflected in the transplants. It seems questionable that the growth capacity of endometrial transplants in the eye can be determined unless sufficient estrogen is given to prevent bleeding in the uterus. Therefore, it would seem that these experiments contribute less to an analysis of the effects of estrogen on endometrial growth than they do to an understanding of the events that precede and accompany menstruation.

In summary, it seems clear that the outstanding effect of estrogen on the uterus of the monkey is one of growth (Allen, 1927, 1928). The involuted uterus of a castrated animal can be restored to its normal size in 2 or 3 weeks by daily injections of adequate amounts of estrogen. At this time there is an increase in vascularity, a clear-cut hyperemia as seen in rodents. There also is secretion of luminal fluid (Sturgis, 1942) but this does not distend the uterus as in the mouse and rat. This is accompanied by an increase in tissue fluid, especially in epithelial tissues (surface epithelium and glands) , and in the connective tissue of the stroma. Glycogen may be present at the basal ends of epithelial cells beneath the nuclei (Overholser and Nelson, 1936) but it apparently is not readily released under the action of estrogen

alone (Lendrum and Hisaw, 1936; Engle and Smith, 1938) . The glands of the endometrium maintain a straight tubular structure with some branching near the muscle layers. The condition produced experimentally in the monkey's uterus by short term treatments with estrogen is equivalent to that present in the normal animal at midcycle, or even a few days later if ovulation does not occur.

If, however, an estrogen treatment is continued for several months conditions develop in the uterus that are not found during the follicular phase of a normal menstrual cycle. When the daily dose of estrogen is small menstruation occurs at intervals during the treatment (Zuckerman, 1937b) and probably marks periods of endometrial regression as observed by Markee (1940) in eye transplants, but if the dosage is increased by a sufficient amount (about 10 /xg. estradiol- 17/3 daily) injections may be continued for a year or longer without bleeding. Although the size of the uterus remains within the range of normal variation as the injections are continued, the myometrium tends to increase in thickness and the endometrium becomes thinner, a condition not corrected by further increases in dosage or by prolonging the treatment. The cause responsible for the limited response of the endometrium under these conditions is not known but apparently is not a restrictive influence of the myometrium as similar responses are given when the endometrium is exposed by incising the uterus, in abdominal fistulae, and in exteriorized uteri.

III. Effects of Progesterone on the Uterus

It has been mentioned that a menstrual cycle, in which ovulation occurs, can be conveniently divided into a follicular and a luteal phase. The follicular phase extends from menstruation to ovulation and the luteal phase from ovulation to the following menstruation. It has been shown in the previous discussion that the endometrial modifications characteristic of the follicular phase of the cycle can be duplicated in a castrated monkey by the injection of estrogen. Likewise, the progestational condition characteristic of the luteal phase can be developed by giving progesterone. In fact, all

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the morphologic and physiologic features that are known for anovulatory and ovulatory cycles can be reproduced in castrated monkeys by estrogen and progesterone.

If one designs an experiment to simulate the normal cycle in a castrated monkey then estrogen should be given first to develop the conditions of the follicular phase followed by progesterone for the progestational development of the luteal phase. Experience has shown that this is the most effective procedure for the production of a progestational endometrium. Progesterone, as compared with estrogen, is a weak growth jH'omoter and although it can produce progestational changes in the atrophic endometrium of a castrated monkey when given in large doses, its action is greatly facilitated when preceded by estrogen. The first experiments in which progesterone was used for this purpose were planned on this principle (Hisaw, Meyer and Fevold, 1930; Hisaw, 1935; Engle, Smith and Shelesnyak, 1935).

The first noticeable effect of progesterone is an elongation of the epithelial cells of the surface membrane and necks of the glands. When the treatment is continued, this effect progresses down the gland towards the base. This change is followed closely by a rearrangement of the nuclei which is more pronounced in the glands than in the surface epithelium. The nuclei under the influence of estrogen in doses which reproduce the conditions of the follicular phase of a normal cvcle, are situated niostlv in the basal

Fig. 9.8. Uterus of a castratefl monkey which was given 2 mg. progesterone daily tor 113 days. The endometrium is thin but bleeding occiu\s when such treatment is stopped. The myometrimn is soft and pliable and ilir l)lood vessels are cnlarsed and have thick wails.

half of the cells, some of them touching the basement membrane. The nuclei retreat from the basement membrane when progesterone is given leaving a conspicuous clear zone. This zone is produced by intracellular deposits of glycogen. These early changes usually appear before pronounced spiraling and dilation of the glands.

Secretion begins in response to estrogenic stimulation and increases greatly as progestational changes are established. It appears first in the necks of the glands and progresses basalward. The surface epithelium takes a less conspicuous part in secretion and is usually reduced to a thin membrane when injections of progesterone are continued until a fully developed progestational endometrium is established. This progressive action of progesterone is such that it is possible to find all conditions in a single gland from active secretion and fraying in the neck region through primary swelling to an unmodified condition at the base.

When treatment is continued for 25 to 30 days at doses of about 2.0 mg. daily, the glands enter a state that has been called "secretory exhaustion" (Hisaw, 1935). This condition also is seen first in the necks of the glands and progresses toward the base. The glandular epithelium decreases in thickness, and active secretion, as judged by fraying of the cells, is absent. The glands may become narrow and straight and the endometrium may resemble that in castration atrophy. These involutionary changes become even more pronounced if the treatment is continued for several months or a year (Fig. 9.8). The endometrium by this time is extremely thin. The glands are straight, short, and narrow, and the stroma very dense. The myometrium is thick in proportion to the endometrium and the uterine blood vessels are large and have greatly thickened walls. Such uteri tend to be somewhat smaller than normal and are soft and pHable.

Thus, it is seen that when growth is produced in the endonietiiuni of a castrated monkey by giving estrogen and then continued on injections of progesterone, there follows a sequential development of all stages of the luteal phase of a normal menstiual cycle terminating in secretory exhaustion. However, this condition cannot

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567

be maintained by continuing the progesterone treatment, and involutionary processes set in and the endometrium is reduced to a thin structure. Yet, such degenerate endometria are dependent upon progesterone and will bleed within about 48 hours if the injections are stopped. It also was found that after discontinuence of progesterone daily injections of 10 /i.g. estradiol may not prevent bleeding.

IV. Synergism between Estrogen and Progesterone

There is considerable evidence that in primates progesterone under normal conditions rarely if ever produces its effects in the absence of estrogen. Large quantities of estrogen are present in human corpora lutea (Allen, Pratt, Newell and Bland, 1930) and during pregnancy the placenta secretes estrogens as well as progesterone (Diczfalusy, 1953) . This apparently is a common feature of primates, as indicated by the excretion of estrogens in the urine of pregnant chimpanzees and rhesus monkeys (Allen, Diddle, Burford and Elder, 1936; Fish, Young and Dorfman, 1941 ; Dorfman and van Wagenen, 1941). Also, correlated with this is the observation that estrogen and progesterone when given concurrently produce a greater effect on the uterus of castrated monkeys than either alone (Hisaw, Greep and Fevold, 1937; Engle, 1937; Hisaw and Greep, 1938; Engle and Smith, 1938) and that an ineffective dose of progesterone is greatly potentiated by estrogen. This synergistic effect of the two hormones on the uterus of monkeys is quite different from their action on the uteri of laboratory rodents and rabbits. In these animals the effects of progesterone can be inhibited quite easily by a surprisingly small dose of estrogen (see chapter 7).

The synergism between estrogen and progesterone in the promotion of endometrial growth can be demonstrated to best advantage under the conditions of some of the physiologic preparations that have been discussed. For instance, it was shown (Fig. 9.5) that growth of the endometrium under the influence of estrogen was not enhanced by relieving muscle tension by a midline incision through the anterior wall of the uterus. Now, if a similar operation is per

FiG. 9.9. Uterus of a castrated monkey that received 10 fig. estradiol and 1 mg. progesterone daily for 18 days, at which time the uterus was opened from fundus to cervix and most of the endometrium of the anterior wall removed. The incision was not closed and the treatment was continued for an additional 20 days. (From F. L. Hisaw, in A Symposium on Steroid Hormones, University of Wisconsin Press, 1950.)

formed on the uterus of a monkey that is receiving 10 /tg. estradiol daily and the treatment continued with the addition of a daily dose of 1 mg. progesterone, there usually follows a rapid growth of endometrial tissue out through the incision until by about 3 weeks a mass is formed which approximates the size of the entire uterus (Fig. 9.9). If this experiment is repeated and the same dosage of progesterone is given without estrogen, there is no outgrowth of the endometrium (Fig. 9.10).

A similar synergistic action can be seen in utero-abdominal fistulae. We have mentioned that estrogen does not cause excessive growth of the endometrium under these conditions. However, endometria that have reached their maximal response to estrogen will show a resumption of growth if 1 or 2 mg. progesterone are added daily to the treatment. By the 4th or 5th day lobes of blood-red endometrium begin to protrude

568

PHYSIOLOGY OF GONADS

Fig. 9.10. Uteru.s of a castrated monkey which was given 1 mg. progesteione (lail>' for 18 days following an estrogen treatment. The uterus was opened as described for Figure 9.9, and the injections of progesterone continued for 20 days. (From F. L. Hisaw, in A Syiyiposium on Steroid Hormones, University of Wisconsin Press, 1950.)

through the opening of the fistula. Within a few days tongue-like processes of endometrial tissue are thrust out of the opening with each uterine contraction and are entirely or i^artially withdrawn at each relaxation.

Such outgrowths are difficult to protect from mechanical injury and consequent tissue loss so it is not possible to determine accurately how much endometrium is produced in a given time. In one experiment an animal was kept on 10 fxg. estradiol and 2 mg. progesterone daily for 98 days and it was found that the endometrium continued to grow, but the rate seemed considerably slow^er toward the conclusion of the treatment than at the beginning. How long an endometrium would continue to grow under these conditions was not determined, but it is obvious that much more endometrial tissue was produced by the treatment than is ever found at one time in the uterus of a monkey during a normal menstrual cycle. This takes on added significance when it is compared with the endometrial response in the intact uterus of an animal given the same dosage of estrogen and progesterone for a similar length of time.

The progestational development of the endometrium, when both hormones are given, passes through the same stages as those following the injection of only progesterone; i.e., presecretory swelling of the glandular epithelium, active secretion, and

secretory exhaustion. The endometrium, however, is considerably thicker than when a comparable dose of progesterone is given alone, and secretory exhaustion may not be so pronounced by the 30th day (Fig. 9.11). The glandular epithelium in the necks of the glands may be reduced to a thin membrane scarcely thicker than the nuclei whereas some secretion is usually present in the dilated basal parts of the glands. Also dilation of the glands in the basalis is more pronounced following a 30-day estrogen-])rogesterone treatment than when the same amount of progesterone is given separately.

Secretory exhaustion appears to be the initial indication of an involutionary process that ensues when an estrogen-progesterone treatment is continued for a long time (Hisaw, 1950). When a combination of the two hormones, known to be capable of producing a large uterus with a thick, fully develojied, progestational endometrium within al)out 20 days, is given for 100 days, an astonishingly different endometrium results (Fig. 9.12). It is thin, the stroma is dense and the narrow straight glands are reduced to cords of cells in the basal area. The condition is one suggesting inactivity and atrophy.

When such dosages of estrogen and i)rogesterone are given to castrated monkeys for 200 days or a year further changes in the endometrium occur. By 200 days the epithelium of the surface mucosa and glands

Fk;. 9.11, .\ late i)r()ges1ati()iial condition produced in the endometrium of a castrated monkey by giving 10 (ig. estradiol daily for 18 days followed by 10 /xg. estradiol and 2 mg. progesterone daily for 31 davs.

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569

Fig. 9.12. The endometnuin of a castrated monkey that had received 10 /xg. estradiol and 1 mg. progesterone daily for 99 days.

is lost except for small glandular vestiges along the musciilaris at the base of the endometrium. There are no glands, coiled arteries, or large blood vessels in what one might yet call the functionalis. All that remains is a modified stroma that resembles decidual tissue (Fig. 9.13.4 and B). It is also of interest that these endometria will menstruate if the treatment is discontinued and in most if the injections of progesterone are stopped and estrogen continued, but not if estrogen is stopped and progesterone continued.

Even though in such experiments the endometrium has been under the influence of both estrogen and progesterone for a year and has undergone extremely abnormal modification, it yet is capable of responding to estrogen in a more or less characteristic way when progesterone is stopped and in

jections of estrogen continued. Apparently within about three weeks the modified endometrium is replaced, under the influence of estrogen, by one that has few glands which tend to be cystic, a mesenchymatous stroma, and no coiled arteries (Fig. 9.14).

Under similar circumstances, if estrogen is stopped and jjrogesterone is continued, the modified endometrium is lost without bleeding and there is almost no repair of the endometrium even after a period of 3 weeks. There seems to be an incompatability between the epithelial outgrowths from the mouths of the glands and the underlying stroma of the denuded surface. Consequently the epithelium crumbles away and epithelization of the raw surface is not accomplished (Fig. 9.15j. How long this condition could continue has not been determined.

570

PHYSIOLOGY OF GONADS

Fk;. 9.13. The endometrium shown m .4 is (h;i( from a castraled mdiik.N wlml, l,a,l received 10 /xg- estradiol and 2 mg. progesterone daily for 200 days. In B, jiart of ilie endometrium of a snndai animal given the same treatment for 312 days is shown at a higher magnification. The endometrium is almost entirely a modified stroma in which glandular epithelium and coiled arteries are absent. Only vestiges of glands are present in the basal area next to the myometrium.

One of the most interesting aspects of these observations is that these effects were jiroduced by dosages of estrogen and progesterone that are very probably within the range of normal physiology. From this it appears that although growth of the endometrium is greater when the two hormones are given together, due to their synergistic interaction, this does not prevent involutionary changes from setting in when the treatment is continued for a period of weeks or months. In fact, greater damage to the endometrium occurs under the simultaneous action of the two hormones than when either is given alone. Also, increasing the dose intensifies the damaging action of both estrogen and progesterone, so much so that very large doses will almost completely destroy the endometrium.

The myometrium, however, shows a different response to these treatments. Estrogen stimulates myometrial growth, which is

Fir;. 9.14. Uterus of a castrated monkey which was given 10 ixg. of estradiol and 2 mg. progesterone daily for 307 days at which time the injections of progesterone were stopped and estrogen continued for 20 days. Bleeding occurred the second day following discontinuance of progesterone. The absence of coiled arteries and the presence of cystic glands and a mesenchymetous stroma characterize the endometrium.

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Fig. 9.15. Uterus of a castrated monkey which was given 10 yug. estradiol and 2 mg. progesterone daily for 275 days at which time estrogen was stopped and progesterone was continued for 21 days. A shows the thin endometrium and dense stroma whereas B shows failure of formation of a surface epithelium following the loss of the modified functionalis presumably present at the conclusion of treatment with both hormono.^^ (see Fig. 9.13).

intensified both by cln-onic treatment and high dosage, and seems to be equally effective when it is given alone or in combination with progesterone. Progesterone also promotes growth of the muscularis but seems less effective than estrogen and differs from it by causing pronounced thickening of the walls of the arcuate blood vessels. These vascular changes extend to the coiled arteries of the endometrium, which are also affected by high dosages of estrogen. It seems remarkable that estrogen is capable of preventing the action of progesterone on the myometrial blood vessels and correcting such effects after they are produced and yet at the same time it assists in the destruction of the coiled arteries in the endometrium.

V. Experimentally Produced Implantation Reactions

Progestational endometria of the normal menstrual cycle or those produced in castrated monkeys by progesterone, if mechanically traumatized, will develop endometrial proliferations which seem identical with those found at normal implantation sites of fertilized ova (Figs. 9.16 and 9.171 (Hisaw,

1935; Hisaw, Creep and Fevold, 1937; Wislocki and Streeter, 1938; Rossman, 1940). The proliferated cells originate from the surface and glandular epithelium and grow into the surrounding stroma. The reaction spreads from the point of injury and within a few days may involve the entire inner l)ortion of the endometrium bordering the lumen. The implantation plaques on the 3rd or 4th day present a fairly homogeneous appearance but soon thereafter certain cells attain the proportions of giant cells and many are multinucleated.

The development of the plaques is most rapid during the first week, by the end of which cell division is found only in the basal half of the proliferation and evidence of regression is seen in the superficial portion adjoining the uterine lumen. After 10 days degenerative and phagocytic processes are the dominant features and by 24 days the ut'prus contains few or no ]iroliferation cells. Wislocki and Streeter ( 1938,1 found that implantation plaques during pregnancy and those experimentally induced underwent ajjl^roximately the same development arid subsequent degeneration except for modifications produced by the invading troplio

PHYSIOLOGY OF GONADS

^a.

^^--.^

tx: ^ .

Fig. 9.16. An area of the normal implantation site of a developing ovum. (From Carnegie Institution, No. C467.)

Fig. 9.17. An experimentally induced implantation reaction in a castrated monkey showing condition 6 days after mechanical traumatization of the endometrium.

blast. Rossman (1940j made an extensive morphologic study of these epithelial proliferations and concluded that they should be regarded as typical metaplasias \vith an embryotrophic function.

VI. The Cervix Uteri

The cervix uteri of the rhesus monkey is remarkable for its size and complexity. It forms a large segment that is set off from the fundus by a conspicuous constriction at the level of the internal os (Fig. 9.1). A sagittal section (Fig. 9.18) shows the cervical canal not straight but thrown into several sharp turns by colliculi that extend from its walls into the lumen. The largest of these projects from the midventral wall. The functional advantage of such tortuosity of the cervical canal is not obvious but since the cervix probably serves as a barrier between the bacterial flora of the vagina and the corpus uteri, this may be a useful adaptation.

The physiology of the cervix has received much less attention than has been given the uterus. This is regrettable in view of the consideration it must receive in practical obstetrics and gynecology, as well as the possibility that physiologically the monkey cervix may be homologous with that of the human regardless of morphologic difTerences. Recent observations indicate that this is indeed quite probable.

Fig. 9.18. Sagittal section of the cervix from a normal monkey. The vagina and the external os of the cervix are shown at the left and the entrance to the fundus is at the right.

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Fig. 9.19. Sagittal section of the cervix of a pregnant monkey showing conditions present just previous to parturition on the 154th day of gestation. The dominant features are dilation of the cervical canal and reduction of the cervical lips (shown at the left) and the coUiculi. (From Carnegie Institution, No. C713.)

Hamilton (1949) made a detailed study of the changes in the cervix of rhesus monkeys during the menstrual cycle, paying particular attention to alterations that took place in the cells of the surface epithelium of the endocervical canal and the cervical glands. It was found that heights of the cells showed consistent increases and decreases during the cycle. The peaks came on the 3rd, 13th to 15th, and 22nd days, the greatest of these being the 14th day which is approximately the time of ovulation. It also was observed that, following a peak, secretion was associated with the decline.

Attention was called b3^ Hamilton to the rather close correlation between the fluctuations in height of the cervical epithelium in monkeys and the fluctuations observed by Markee and Berg (1944) in the blood estrogens of the human menstrual cycle. It was concluded that, if similar changes in estrogen levels also occur in monkeys, one would be justified in concluding that the increase in cell height in the cervical mucosa was due to the action of estrogen and the sudden periodic drops in blood estrogen caused secretion and consequent regression. However, it is not clear how this could account for the abundant secretion of the cervical glands in

the presence of high levels of estrogen during late pregnancy (Fig. 9.19).

Much has been learned regarding the physiology of the primate cervix from experiments on castrated monkeys. The cervical mucosa is very responsive to estrogen and castration atrophy can be repaired and a normal condition maintained by daily injections of small doses. Cervical secretion may become abundant when an estrogen treatment is prolonged and especially if large doses are injected. However, the amount of secretion induced by estrogen never equals that of the last half of pregnancy, and it usually subsides if the injections are continued for several months.

Under conditions of chronic treatments with estrogen metaplastic aberrations invariably appear in the epithelium of the endocervix. This reaction was first reported in monkeys by Overholser and Allen ( 1933, 1935) and has been confirmed by many investigators (Engle and Smith, 1935; Hisaw and Lendrum, 1936; Zuckerman, 1937c (. Similar lesions may be found in the cervix uteri of women (Fluhmann, 1954). They seem especially prone to occur under conditions characterized by excessive production of estrogen, such as hyperplasia of the

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endometrium (Hellman, Rosenthal, Kistner and Gordon, 1954) and granulosa-cell tumors of the ovary. Various degrees of metaplasia may occur in the cervix during pregnancy both in the mother and newborn but Fluhmann (1954) did not find it as frequently as in nonpregnant women.

This reaction to estrogen as seen in the cervix of castrated monkeys is initiated by growth of small undifferentiated cells below the columnar mucous cells of the secretory epithelium. Fluhmann (1954) suggests that these cells are really undifferentiated cells of the cervical mucosa which have the potentiality of becoming columnar or squamous or simply undergoing multiplication and remaining as indifferent or reserve cells. These cells accumulate, in response to estrogen, to form aggregates of several cells in thickness and, although this may occur in any area of the endocervix, it is generally more pronounced below the base of the glands. As this process proceeds the columnar mucous cells are pushed outward and are finally desquamated thus exposing the underlying metaplastic cells to the lumen of the gland (Fig. 9.20).

Fig. 9.20. Al.iaph

in a castrated monkey that li.-id rci-civcd 1 nig. estriol daily for 48 days.

The cells of these lesions undergo a characteristic differentiation. When first formed they are small, cuboidal, and have spherical nuclei with dense chromatin. As they increase in number those in the center of the cellular mass become larger and acquire an eosinophilic cytoplasm. Such collections, as seen at the base of the cervical glands, may grow in height and form cone-shaped masses with the apexes protruding through the mucous epithelium into the lumen or they may remain as more or less compact structures. This difference in growth seems to have a general relation to the dosage of estrogen. Large doses cause more rapid growth and cone formation with the loss of cells from the apex either singly or in groups, whereas small doses produce slower growth and desquamated cells are seldom seen in the lumen. However, regardless of the rate of growth, the cells at the base of the lesion remain undifferentiated and continue as the principal area of cell proliferation.

Pearl formation is occasionally seen and may be quite common in animals on low dosages of estrogen. Under strong estrogenic stimulation and consequently rapid growth, these structures apparently are desquamated before they are completely formed. However, very early stages are frequently seen and may even be present in small clumps of metaplastic cells, but they are more commonly found in the larger collections at the base of the glands. Their appearance is initiated by swelling and disintegration of one or more adjacent cells that form a center around which epidermidization takes place. Further development does not proceed under the influence of esti'ogen, beyond the formation of a small central cavity.

The most conspicuous difference between the metaplastic growths produced by estrogen and true cancer of the cervix in the monkey (Hisaw and Hisaw, Jr., 1958) is that the former remain noninvasive even when the treatment is continued well over a year. They also involute when the treatiiiciit is discontinued and they do not appeal' when progesterone is given simultaneously with estrogen. When the injections of progesterone are started after metaplastic growths have been formed in response to estrogen, further growth is inhibited and

ESTROGEN AND PROGESTERONE

the keratinized cells of the lesion become vacuolated and are lost.

In contrast with the effects of estrogen on the cervix, the modifications that occur as pregnancy advances are remarkable. The cervix becomes a soft thin-walled structure, the glands increase in number, and their lumina become greatly enlarged, pressing the stroma into thin partitions between them, and the amount of mucus secreted is enormous (Fig. 9.19). Attempts at duplicating these changes in castrated animals by hormone therapy have been only partially successful. Estrogen produces a solid thickwalled cervix that tends to be larger than normal, an effect that is especially noticeable in young animals. Progesterone does not promote cervical growth and repair of the glands unless large doses are given and even then there is little if any secretion. The best results were obtained when both estrogen and progesterone were given and especially so when relaxin was added to the treatment (see chapter by Zarrow).

VII. The Vagina

The general features of the vaginal smear of rhesus monkeys have been described by several investigators (Allen, 1927; Hartman, 1932; Westman, 1932) and a detailed study of the cellular components at different times of the menstrual cycle has been made by Lopez Columbo de Allende, Shorr and Hartman (1945). The changes in the vagina of a monkey are in most respects like those found for the human being (Papanicolaou, Traut and Marchetti, 1948; Lopez Columbo de Allende and Orias, 1950). Epithelial growth and desquamation of cornified cells continue at all stages of the cycle but at various rates. The epithelium is thinnest at menstruation and gradually increases in thickness during the follicular phase, reaching a maximum at ovulation. At this time there is a well developed basal area in which numerous mitoses can be seen and from which many papillae or bulbs" extend into the underlying stroma. Above this is an intermediate zone, an interepithelial zone of cornification (so called Dierk's layer), and a heavily cornified outer zone (Fig. 9.21).

Cellular proliferation is less rapid during the luteal phase and apparently cells are

desquamated more rapidly than they are replaced. Consequently there is a decrease in the thickness of the epithelium in the luteal phase which may include an almost complete loss of the cornified zone (Davis and Hartman, 1935). The effects are probably due to progesterone because similar changes are seen following the introduction of progesterone into a treatment in which estrogen is being given.

The vaginal epithelium of a castrated monkey is remarkably sensitive to estrogen. A small daily dose of 5 to 10 /xg. estradiol will stimulate growth of an atrophic epithelium of 4 to 8 cells in thickness to one of 60 or even 80 layers thick within 3 weeks. One of the first things that is noticed as the vaginal epithelium thickens is the numerous mitotic figures in the stratum germinativum followed by a marked increase in the number of epithelial papillae along the basement membrane. This condition of rapid growth, cornification, and loss of cells into the vaginal lumen is typical of the follicular phase of the menstrual cycle and can be maintained indefinitely.

li(.. U.21. the vaginal epithelium of a castuUMJ monkej' showing growth antl cornification induced by estrogen.

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PHYSIOLOGY OF GONADS

Progesterone, in contrast with estrogen, does not produce rapid growth of the vaginal epithelium but at the same time it is not without an effect. The vaginal epithelium, weeks or months after castration, has relatively few papillae projecting from its basal border into the underlying stroma. When progesterone is given, this condition is changed but not in a spectacular way. There is very slow growth without cornification. The epithelium remains thin but the papillae become more numerous. These are mostly small epithelial buds which tend to remain solid but may show enlargement of the cells in their centers.

When estrogen and progesterone are given concurrently, the effects of estrogen on the vaginal mucosa are modified. If an estrogen treatment has continued for a sufficient time to produce full cornification and then progesterone is added, the first indication of an inhibition of estrogen is a decrease in mitotic activity. This is followed by a continuation of cornification and loss of cells faster than they are replaced ; consequently, most of the functionalis is lost and the epithelium becomes thinner. There is also a noticeable decrease in the intensity of cornification, which in the monkey is never as pronounced as in rodents, and under these conditions is quite incomplete, each cell retaining a conspicuous nucleus. Partly cornified cells may be present for several weeks

.-•^ss;^

4* ' V ^ §

Fig. 9.22. ^^•tgi^al epithelium of a pregnant monkey showing condition on the 154th day of gestation. (From Carnegie Institution, No. C713.)

when both estrogen and progesterone are given, but eventually they almost entirely disappear and the epithelium attains a condition resembling that of late pregnancy.

The inhibitory effect of progesterone on the action of estrogen is shown perhaps even better when a castrated monkey having a fully involuted reproductive tract is first given progesterone for a few days and then (>strogen is added to the treatment, or when injections of the two hormones are started at the same time. In such experiments estrogen has little effect on the vaginal mucosa even in doses that would produce marked cornification if given alone. These observations show that a fully cornified vaginal epithelium cannot be produced or maintained by estrogen when an effective dosage of progesterone is included in the treatment (Hisaw, Greep and Fevold, 1937).

Estrogens and progesterone are the dominant hormones of gestation and their simultaneous action is reflected by the changes in the vaginal epithelium. The fully cornified vagina, present at the time of ovulation, is gradually modified as pregnancy progresses into a condition strikingly like that seen in experiments when estrogen and progesterone are given concurrently. In late pregnancy the most striking feature of the thin, uncornified epithelium is the presence of numerous epithelial buds extending deeply into the underlying stroma. They may branch and rebranch and along their course there is conspicuous enlargement of the more centrally situated cells among which cavities ai^pear, enlarge, and join each other (Fig. 9.22). It seems quite probable that this process may be of considerable importance in increasing the diameter of the vagina.

VIII. Sexual Skin

A so-called sexual skin is jiresent in most catarrhine monkeys, is not found in platyriliine monkeys, and among the anthropoids occurs regularly only in the chimpanzee I l^]ckstein and Zuckcrman, 1956). Changes in the sexual skin during the menstrual cycle have been observed most extensively in the monkey (Macaca), the baboon (Papio), and the chimpanzee (Pan). The sexual skin of t!ie baboon and chimpanzee undergo jiro

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nounced swelling during the follicular phase of the cycle. A maximal size is attained by the middle of the cycle followed by a rapid regression and loss of edema which at least in the baboon is associated with a marked increase in the output of urine (Gillman, 1937a; Krohn and Zuckerman, 1937). The subsidence of the sexual skin begins approximately at the time of ovulation and remains in the reduced condition throughout the luteal phase, followed by a subsequent initiation of swelling during or soon after menstruation (Zuckerman, 1930, 1937e; Zuckerman and Parkes, 1932; Gillman and Gilbert, 1946; Young and Yerkes, 1943; Nissen and Yerkes, 1943).

A w^ell developed sexual skin is present in the monkey {Macaca mulatta) only during adolescence. With the appearance of the menstrual cycles the sexual skin undergoes a process of maturation into the adult condition in which cyclic changes in edema are absent and the most noticeable feature is a vivid red color. Such coloration is due to vascular engorgement rather than pigment (Collings, 1926) and involves the perineum, the buttocks, and may extend for various distances down the legs and over the symphysis pubis. The development and maturation of the sexual skin have been described in considerable detail by several investigators (Hartman, 1932; Zuckerman, van Wagenen and Gardiner, 1938) .

The sexual skin has been of considerable interest both as to the nature of its responsiveness to ovarian hormones and the manner in which its grossly visible changes during the menstrual cycle parallel events occurring in the reproductive tract. The sudden loss of edema at the conclusion of the follicular phase not only signals ovulation but also raises the question as to whether the loss of tissue fluid is due to a decrease in estrogen or is the direct effect of progesterone. The importance of this becomes obvious when it is considered that a similar process also goes on simultaneously in the endometrium and raises the question again as to the respective roles played by estrogen and progesterone in endometrial growth and menstruation.

That the development and edema of the sexual skin of adolescent rhesus monkeys depend on the ovaries was first demon

strated by Allen ( 1927 ) . Involution and loss of color follow castration, and the normal condition can be restored by the injection of estrogen. Also, when estrogen treatment is continued for several weeks maturation of the sexual skin occurs and a condition characteristic of that in the adult is established (Zuckerman, van Wagenen and Gardiner, 1938). The genital area loses its edema and develops a brilliant red color which is retained as long as estrogen is administered. Once this mature condition is established the response of the sexual skin to subsequent estrogen treatments is limited to a change in color.

Similar experiments have been performed on the chacma baboon, Papio porcarius (Parkes and Zuckerman, 1931; Gillman, 1937b, 1938, 1940a). The large sexual skin of these animals is very responsive to estrogen and development equal to that of the follicular phase of the menstrual cycle can be readily induced by daily injections for about 2 weeks. However, the perineal swelling of the baboon differs from the sexual skin of the genital area of the rhesus monkey in that it does not "mature" under the influence of estrogen.

When large doses of estrogen are given to a rhesus monkey a generalized edema of the skin occurs beyond the genital area. This first appears as deeply indented swellings along the sartorii from groin to knee, and next appears at the base of the tail and spreads gradually upward until it involves the entire dorsal portion of the trunk. At the same time, the skin of the face, scalp, and supraorbital ridges becomes swollen and finally the edema may extend out on the arms and down the legs to the ankles (Bachman, Collip and Selye, 1935; Hartman, Geschickter and Speert, 1941). A daily dose of 500 /xg. or more of estriol or estradiol w^ll produce this condition within 2 to 3 weeks and, when the treatment is continued for an extended period the effect tends to subside.

Progesterone has a strong inhibitory action on the effects produced by estrogen on both the genital and extragenital sexual skin of the monkey. If daily injections of progesterone are added to the treatmeiu after full development of the sexual skin has been induced by estrogen, there is a

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PHYSIOLOGY OF GONADS

noticeable loss of edema by the 4th or 5th day followed by rapid involution and reduction of the turgid folds of skin to loose, flabby wrinkles within about 10 days. When estrogen and progesterone are given concurrently to a castrated monkey from the beginning of treatment edema does not appear but the sexual skin regains its normal color. In fact, progesterone alone, like estrogen, can restore the color to the sexual skin of castrated adult monkeys (Hisaw, Greep and Fevold, 1937; Hisaw, 1942).

The interaction of estrogen and i)rogesterone on the sexual skin of rhesus monkeys can best be demonstrated by the reaction of the skin of the sexual area in adolescent animals. The most striking effect and probably the most important is the sequence of events initiated by a single dose of progesterone when given to an animal on continuous estrogen treatment. Under such treatment a full response of the sexual skin is obtained by the end of 20 days. If at this time 1 mg. progesterone is given in a single dose and the estrogen treatment continued uninterruptedly, the first indication of an effect of the luteal hormone is a slight loss of edema and color of the sexual skin on the 4th or 5th day thereafter. The sexual skin is markedly reduced by the 8th day, almost gone by the 9th, and at the end of about a fortnight regains its ability to respond to estrogen as shown by a return of color and swelling. However, the most remarkable eventuation of such treatment is menstruation which usually begins on about the 10th day (Hisaw, 1942).

Involution of the sexual skin and menstruation following a single injection of progesterone also have been produced in the baboon by Gillman (1940a). He found that 5 mg. progesterone, when given on the 8th day of a normal menstrual cycle, would cause an appreciable loss of edema of the swollen perineal sexual skin by the day after injection. This was followed by a progressive involution of the perineum until the 13th day and swelling was re-initiated by the end of the 15th day. Reduction of the sexual skin at this dosage of progesterone was not associated with menstruation. However, when the dose was increased to 20 mg. both deturgescence of the sexual skin and menstruation occurred. These effects pro

duced by progesterone in the presence of endogenous estrogen have much in common with those described above as occurring in castrated monkeys on continuous estrogen treatments.

IX. Menstruation

An experimental ai^proach to the physiology of menstruation dates from the observations of Allen (1927) that uterine bleeding would occur in castrated monkeys following the discontinuance of an estrogen treatment. He suggested that normal menstruation is due to a fluctuation in estrogen secretion and proposed the "estrogen-withdrawal" theory to account for the observed facts. This concept led to an extensive investigation of the effects of estrogens on the endometrium and of conditions that modify their action. It was soon found that in both castrated monkeys and human beings there was a quantitative relationship between the dosage of estrogen given and the maintenance of the endometrium. Bleeding occurred during treatment when the daily dose of estrogen was small, but with larger doses a point was reached at which the injections could be continued for months or even years without bleeding (Werner and Collier, 1933; Zuckerman, 1937b, d).

Estrogen also will inliihit i)ostop('rative bleeding which usually follows total castration, provided the ovaries are removed before or soon after ovulation (Hartman. 1934). With the advent of a corpus luteum and development of a progestational endometrium it becomes progressively more difficult, following castration, to prevent menstruation by injecting estrogen. Similar results are obtained when estrogen is given during a normal menstrual cycle. Small doses may not prevent the onset of menstruation, but if continued, subsequent menstrual periods are delayed (Corner, 1935). Large doses when given during the luteal phase of the cycle do not disturb the normal menstrual rhythm, but may do so if the treatment is started during the follicular phase (Zuckerman, 1935. 1936a).

Progesterone, in contrast with estrogen, will prevent menstruation from an endometrium representative of any stage of the normal cycle. It will delay onset of the next menses even when the treatment is started only

ESTROGEN AND PROGESTERONE

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a few days before the expected menstruation (Corner, 1935; Corner and Allen, 1936) . Also, the bleeding that invariably follows the discontinuance of a long treatment with estrogen can be inhibited indefinitely by giving progesterone (Hisaw, 1935; Engle, Smith and Shelesnvak, 1935; Zuckerman, 1936b).

An impression held by many of the earlier investigators was that progesterone could not produce its effects on the primate endometrium unless it w^as preceded by the action of estrogen. It is true, of course, that progesterone is a comparatively weak growth promoter and its effects can be demonstrated to best advantage on an endometrium that has been developed by estrogen. However, Hisaw, Greep and Fevold (1937) produced a progestational endometrium in a monkey that had been castrated 242 days previously by giving synthetic progesterone. Also, the endometrium of this animal was found capable of forming a decidual plaque upon traumatization. Soon afterwards Hartman and Speert (1941) observed menstruation following the withdrawal of progesterone in castrated monkeys that had not been given estrogen and more recently similar results have been reported by Eckstein ( 1950) . At the same time it has l)een found that progesterone will induce menstruation in women suffering from amenorrhea and also that uterine bleeding can l)e jirecipitated l)y similar treatment (hiring the follicular j^hase of the cycle (Zondek and Rozin, 1938; Rakoff, 1946).

These observations have been confirmed and extended by Krohn (1951; 1955) who finds that menstrual bleeding can be induced in monkeys wdth secondary amenorrhea by the injection of 5 daily doses of progesterone. Progesterone (5 mg. daily for 5 days) also precipitates uterine bleeding in castrated monkeys at intervals of about 8 days provided the treatment is started innnediately a menstrual bleeding has been induced either by removel of the ovaries or withdrawal of estrogen. The most interesting aspect of these observations is that the number of short 8-day cycles that can be obtained in this way in a castrated animal seems to be related to the size of the initial dose of estrogen used to induce withdrawal bleeding. This also applies to pro

gesterone-withdrawal bleeding, so the effect does not depend upon the particular hormone used to obtain the bleeding. It also is of interest that such conditioning of the endometrium to subsequent responses to the 5-day treatments with progesterone may last for several months on a continuous regime. It is surprising that such a series of responses cannot be initiated unless the first injection of progesterone is given within 6 days following the initial withdrawal bleeding. These observations have much in common with those of Phelps (1947) who also studied the influence of previous treatment on experimental menstruation in monkeys.

There seems to be a quantitative relationship between the dosage of progesterone given in combination with estrogen and the ability of estrogen to prevent bleeding after the injections of progesterone are stopped. It has been mentioned that once a fully developed i^rogestational reaction has been produc(Hl l)y progesterone, it is extremely difficult, if not impossible, to inhibit menstruation by giving estrogen following the withdrawal of progesterone. However, Hisaw and Greep (1938) found that progestational endometria produced ijy small doses of estrogen plus api^roximately 0.5 mg. progesterone daily for 18 to 21 days did not bleed following progesterone withdrawal when continued on 10 to 20 times the original dosage of estrogen. In fact, such endometria were brought back to a condition typical for the action of estrogen and again transformed into a presecretory progestational state without the intervention of bleeding. Similar observations were made previously by Zuckerman (1936a, 1937d).

These experimental results give grounds for some doubt as to the adequacy of the estrogen-withdrawal theory to account fully for menstruation. Not only can progesterone bring about menstruation without the intervention of estrogen but other steroid hormones are capable of pi'oducing similar effects. Desoxycorticosterone in large doses can inhibit estrogen-withdrawal bleeding in castrated monkeys (Zuckerman, 1939, 1951 ) and induce phases of uterine bleeding in rapid succession in normal monkeys (Krohn, 1951). So too can testosterone prevent estrogen-withdrawal bleeding (Hart

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man, 1937; Engle and Smith, 1939; Duncan, Allen and Hamilton, 1941) and inhibit progesterone-withdrawal bleeding as well (Engle and Smith, 1939). Testosterone also will precipitate bleeding during an estrogen treatment (Hisaw, 1943) and in normal monkeys if given early in the cycle (Krohn, 1951). Just what specific action these compounds have in common that enables them to produce these effects or whether there are different modes of action that lead to the same results is not known, but, before mentioning certain possibilities, it may be helpful to consider information regarding the influence of estrogen-progesterone interactions on menstruation.

Among the most significant observations regarding primary causes of menstruation are a few indications that there may be an intrinsic difference in the ways in which estrogen and progesterone produce their effects on the endometrium. One of the first indications of this was the discovery that a short series of injections of progesterone during treatment with estrogen will precipitate menstruation (Corner, 1937; Zuckerman, 1937d; Hisaw and Greep, 1938). This can be demonstrated by giving a castrated monkey a maintenance dose of estrogen daily for 2 or 3 weeks, then adding a daily injection of progesterone for 5 to 10 days and continuing the estrogen treatment. As a rule bleeding appears within 2 or 3 days after stopping progesterone. The most interesting point brought out by such experiments is that bleeding can occur under these conditions in the presence of an otherwise maintenance dosage of estrogen.

Perhaps the most surprising as well as most important fact brought out by subsequent experiments was the small amount of progesterone required to bring about bleeding under these conditions. It was found that only a single injection of 1 mg. was required for animals on chronic treatment with a maintenance dose of estradiol (1000 LIT.) and some bled when 0.5 mg. progesterone was given (Hisaw, 1942). The sequence of events following the injection of progesterone can be seen to best advantage in an adolescent monkey whose sexual skin also respond" to the estrogen treatment. When the 1 mg. ]irogesterone is given on the 20th day of estrogen treatment the edema of th(^

sexual skin will have attained its maximal development. The first indication of an effect of progesterone is a slight loss of edema and color of the sexual skin which appears on the 4th or 5th day and by the 9th or 10th day the edema is almost gone and the sexual skin is pale. Blood apjiears in the vaginal lavage between the 7th and 10th days, of about 70 per cent of the animals on this dosage. The sexual skin may remain markedly reduced and pale until about the 15th day after which both color and edema rapidly return. These effects can also be seen when 1 mg. progesterone is given for a series of days. However, neither the time of appearance nor loss of edema of the sexual skin is significantly hastened, and if the injections extend over no more than 5 days the time between the first injection and bleeding remains approximately the same.

Similar observations have been made by Gillman and Smyth (1939) on the South African baboon iPapio porcarius). They found that 3 mg. or more of progesterone when given in a single injection during the follicular phase of the cycle would cause the relatively enormous perineal swellings to pass rapidly through deturgescence and reach a flabby resting condition within 5 to 7 days, and after a delay of about 24 hours once again begin to swell. As much as 10 or 15 mg. in a single dose caused perineal deturgescence without bleeding, whereas 20 mg. in a single dose or a total of 15 mg. if divided into 2 or 3 injections and given at 3 or 4 day intervals, produced both deturgescence and bleeding (Gillman, 1940b). The l)aboon diff"ers from the monkey in that larger doses of progesterone are required to produce the effects and the sexual skin does not mature" on repeated treatments and lose its responsiveness; otherwise the basic physiology of the reaction in both animals seems to be the same.

The most important fact l)rought out by these experiments is that the effects of a single injection of progesterone can continue in the presence of estrogen for as long as 10 to 15 days. It is highly imi^robable that progesterone lingers in the body for so long a time (Zarrow, Shoger and Lazo-Wasem, 1954). In general it is considered the most ephemeral of the sex steroids and is probablv inactivated within at least a few hours

ESTROGEN AND PROGESTERONE

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after it is administered. It seems more lilvely that progesterone modifies the sexual skin in a way that renders it unresponsive to estrogen and that about a fortnight is required to recover the original condition.

This takes on added significance when the possibility is considered that effects similar to those seen in the sexual skin might also be going on simultaneously in the uterine endometrium. An appreciable dehydration of the endometrium occurs just previous to menstruation (van Dyke and Ch'en, 1936) and a loss of interstitial fluid before bleeding has been observed in endometrial implants in the eyes of monkeys and described in detail by Markee (1940) . This was shown by periodic regression in size and compactness of the grafts which resulted in a decrease in area of 25 to more than 75 per cent. Because cyclic changes in endometrial grafts in the eye are correlated with events of the menstrual cycle there is reason to believe that similar reactions were going on in the endometrium of the uterus.

Endometrial regression, as described by Markee, did not always lead to menstruation although it invariably preceded, accompanied, and followed menstrual bleeding. Menstruation occurred only when regression was rapid and extensive. This was seen in the endometrial grafts in the eye during a normal menstrual cycle at the time of involution of a corpus luteum and during an anovulatory cycle soon after the involution of a large follicle. It also begins soon after the last of a series of injections of estrogen or i^'ogesterone. A slow decrease in size of the ocular grafts, without concomitant bleeding, can be induced in castrated monkeys by gradual withdrawal of estrogen, and when estrogen is given in amounts that are inadequate for maintaining the endometrium for an extended period the "break through" bleeding that eventually ensues is preceded by a rapid and extensive endometrial regression. Because this reaction also occurs w^hen menstruation is induced by such an unusual procedure as spinal transection (Markee, Davis and Hinsey, 1936), it probably is a phenomenon that always precedes menstruation.

It seems from these observations that the changes in the endometrium preceding menstruation are initiated by a sudden with

drawal of a stimulus on which the endometrium at the time relies for the maintenance of a particular physiologic condition, and bleeding and tissue loss are incidents that occur during the readjustment necessary for the return to an inactive state. What this involves is only partly known, but an understanding of the initial changes in the endometrium that usher in menstruation most certainly holds the explanation of the real cause. This has been a perennial subject for discussion and many suggestions and theories have been set forth in an extensive literature to account for various aspects of menstruation. Among the more recent general discussions are those by Zuckerman (1949, 1951), Corner (19511, and Zondek ( 1954 ) .

The estrogen- withdrawal or estrogen-deprivation theory proposed by Edgar Allen has received more attention than any other. From what has been mentioned earlier it is clear that this theory can account for uterine bleeding subsequent to the discontinuance of a series of estrogen injections and also perhaps menstruation at the conclusion of an anovulatory cycle. However, it is not so obvious as to how this theory can explain the occurrence of menstruation at the close of the luteal phase of a normal cycle. Estrogen in large doses will not inhibit such bleeding, but it is postponed if progesterone is given. It is equally difficult to see how this theory is helpful in accounting for the fact that a small dose of progesterone will precipitate bleeding in the presence of a maintenance dosage of estrogen. As little as 2 /xg. progesterone will induce bleeding when applied topically to the endometrial lips of an exteriorized uterus (Fig. 9.7) in a monkey that is receiving 10 fig. estradiol daily (Hisaw, 1950).

Uterine bleeding precipitated by administering progesterone during an estrogen treatment has been explained on the grounds that progesterone in some way interferes with the action of estrogen on the endometrium. Therefore, it is assumed that an animal receiving both estrogen and progesterone is in a sense "deprived" of estrogen. That is, when the two hormones are given simultaneously, progesterone itself is capable of maintaining the endometrium without bleeding; but when it is stopped, the

582

PHYSIOLOGY OF GONADS

suggestion is that the animal is physiologically deprived of estrogen and literally deprived of progesterone (Corner, 1951). Although this view is in descriptive agreement with the observed facts the idea of the inhibitory effect of progesterone does not take into consideration the synergistic interaction of the two hormones on the endometrium.

The physiologic function of progesterone is the conversion of an estrogen-endometrium into a progestational endometrium suitable for receiving and nourishing a developing blastocyst. Such an endometrium is adapted for this specific reproductive function and accordingly its physiologic nature must be quite different from that of the follicular phase of the cycle. Indeed, it is known that these two structures (follicular and luteal phase endometrial are morphologically and biochemically unlike in a number of respects. This gradual transformation, following ovulation, occurs as progesterone becomes the dominant hormone, and consequently, as this proceeds, the endometrium progressively loses competence to respond to estrogen. However, this does not imply that estrogen is without effect in the general economy of the progestational endometrium. It has been shown in a number of ways that the action of progesterone on the primate endometrium is greatly facilitated by the presence of estrogen. In fact, it seems probable that rarely if ever does progesterone perform its function in the absence of estrogen (Hisaw, 1959; chapter by Zarrow).

After consideration of the endometrial specializations brought about by ]irogesterone, it seems rather jwintless to hark back to the follicular phase and inject the past recoi'd of accomjilishments and prerogatives that estrogen had at that time into the explanation of an entirely different hormonal situation. It seems more in keeping with the facts to state outright that menstruation following the involution of a corpus luteum or the discontinuance of progesterone, even though estrogen is present, is due to a decrease or absence of progesterone.

It also has become less certain that menstruation at the conclusion of an anovulatory cycle is really an estrogen-withdrawal bleeding. This is possible, of couisc, but at

the same time the exceedingly small amount of progesterone required to induce bleeding in the presence of estrogen makes it difficult to be sure what the situation might be. Even a negative test for progesterone in the blood, by our present methods, does not necessarily indicate the absence of a physiologically effective amount of progesterone. Zarrow, Shoger and Lazo-Wasem (1954) found that in rabbits an intramuscular injection of 40 mg. progesterone was required to produce an appreciable concentration of the hormone in the blood as determined by the Hooker-Forbes method. Yet, 0.2 mg. progesterone daily for 5 days will produce a progestational reaction in the uterus equivalent to that of the 5th day of normal pseudopregnancy. In monkeys 0.5 mg. daily when given with 10 fxg. estradiol is an adequate dosage of progesterone to induce unquestionable progestational changes in the endometrium and much less will cause bleeding. These observations indicate that the minimal effective concentration of progesterone in the blood may be less than is possible to detect by our present methods.

This also seems to hold for the human being. Estimates of secretion and metabolism of progesterone in the human being have been based primarily on the recovery of its excretory product sodium pregnanediol glucuronidate in the urine. It seems obvious that such determinations must be only general approximations because only about 20 per cent of the progesterone secreted or injected can be accounted for by the pregnanediol in the urine. Also, it is generally known that a physiologically effective dosage of progesterone does not necessarily lead to the excretion of pregnanediol (Hamblen, Cuylcr, Powell, Ashley and Baptist, 1939; Seegar, 1940). In other words, the threshold dose of progesterone for endometrial stimulation is l)elow that at which the hormone is excreted as pregnanediol. In fact, it has been suggested by some investigators that there is no quantitative relationship between the l)rogesterone present in the blood and the pregnanediol excreted in the urine (Buxton, 1940; Sommerville and Marrian, 1950; Kaufmann, Westphal and Zander, 1951).

These findings and the wide variation in the amount of prc'gnancdiol excreted during

ESTROGEN AND PROGESTERONE

58.3

a menstrual cycle (Venning and Browne, 1937) suggest that, even in the absence of ovulation, sufficient progesterone may be present to influence menstruation. There also is the possibility of progestational hormone from some extra-ovarian source, such as the suprarenal cortex. This was suggested by Zuckerman (1937b, 1941 j as a possible explanation for periodic bleeding in monkeys on a constant submaintenance dose of estrogen. This thought becomes more plausible in view of the fact that progesterone is one of the precursors in the metabolic synthesis of androgens, estrogens, and adrenal cortical steroids (Dorfman, 1956). Also, it has been shown that desoxycorticosterone acetate is converted to progesterone in vivo (Zarrow, Hisaw and Bryans, 1950). Therefore, progesterone is not restricted to ovarian luteal function but instead is of rather general occurrence in the body and the likelihood is that small amounts are a constant constituent of the blood.

Also, the amount of progesterone from extra-ovarian sources may fluctuate, as suggested by Zuckerman (1949), and consequently disturb the normal menstrual rhythm and probably cause bleeding in monkeys on a continuous submaintenance dose of estrogen. However, as to the latter, there is an alternative explanation. Castrated monkeys on a continuous treatment of 10 fxg. of estradiol daily do not show "break-through" bleeding, and a synergistic effect on growth of the uterus is seen when 0.5 mg. or more of progesterone daily is introduced into the treatment. However, the simultaneous administration of 0.25 mg. or even 0.125 mg. progesterone daily in similar exj^eriments results in bleeding between a!)out the 10th to 16th day of the combination treatment. Thus, a dosage of progesterone less than that required for synergism or prevention of bleeding when given alone, modifies the endometrium so that it can no longer be maintained by 10 fxg. estradiol daily (Hisaw, Jr., unpublished). When it is considered that the endometrium becomes increasingly dependent on estrogen during a chronic treatment, even after maximal growth is attained (Hisaw, 1942), it seems plausible that the effectiveness of a dosage of estrogen only slightly alcove the thresh

old for bleeding may be decreased sufficiently by the endogenous progesterone from extra-ovarian sources to precipitate bleeding.

Although it is obvious that the normal menstrual cycle is primarily under the control of the ovarian estrogens and progesterone, it is also equally clear that menstruation is not due to a specific hormonal action. Experimental evidence indicates that any natural or synthetic compound having the capacity for promoting growth or sustaining an existing metabolic state in the endometrium is also capable of inducing withdrawal bleeding. However, this does not imply that all compounds capable of inducing menstruation do so by the same biochemical action; in fact, there is considerable evidence that this is not so (see chapter by Villee). Yet in each instance a series of events is set in motion that leads up to active bleeding.

X. The Mechanism of Menstruation

The immediate cause and mechanism of menstruation has continued to be a topic of special interest for many years and the subject of frequent general discussions. A generalization in keeping with our present knowledge is that no gross morphologic feature of the endometrium is distinctive of menstruation. A menstruating endometrium may be representative of any stage of the follicular or luteal phase of the cycle. The most frequently discussed hypothesis regarding the mechanism of menstruation is that proposed by Markee (1940, 1946) which is based on direct observations of vascular changes in endometrial grafts in the anterior chamber of the eye of monkeys (see p. 564). The changes observed in the endometrium shortly before bleeding are, briefly, as follows. (1) There is extensive and rapid regression of the endometrium due to loss of ground substance from the stroma (Fig. 9.23). (2) The rapid regression brings about a disproportion between the length of the coiled arteries and thickness of the endometrium with the formation of additional coils. (3) The increased coiling of the arteries retards the circulation of blood through them and their branches. This stasis begins 1 to 3 davs before the onset of the

584

PHYSIOLOGY OF GONADS

Repair

Fig. 9.23. A diagram indicating correlated changes in ovary and endometrium during an ovulatory cycle of rhesus monkey. Thickness of endometrium, density of stroma, gland form, and three types of arteries are indicated. There is a gradual rise in thickness up to the time of ovulation, and a brief decline followed by development of the luteal or progestational phase with accumulation of secretion in the glands due to relaxation of the myometrium. This is followed by loss of ground substance from the stroma, which is the primary factor in the premenstrual regression of the ischemic phase. This is a prelude to extravasation and shedding of tissue. Incidentally, secretion is extruded and glands collapse. There is further regression throughout the phase of menstruation. More than the basal zone (coarse stipple) survives menstruation. During repair, thickening of the endometrium is associated with increase in ground substance in the stroma and growth in the glands. (From G. W. Bartelmez, 1957, Am. J. Obst. & Gynec, 74, 931-955, 1957, with some modification of description.)

flow, and is associated with leukocytosis in the endometrium. (4) The portion of the coiled arteries located adjacent to the muscularis constricts 4 to 24 hours before the onset of the flow. This vasoconstriction persists throughout the menstrual period except when individual coiled arteries relax and blood circulates through them for a few minutes. Markee postulated that the immediate cause of menstruation under these conditions was the injurious effect of anoxemia upon the tissues of the endometrium l)rought about by mechanical compression and constriction of the coiled arteries. Therefore, the coiled arteries and their modifications become the central feature upon which the theory is based.

Although this offers an explanation for many of the facts, it falls short in that now it is known that menstruation can occur in the absence of coiled arteries. Kaiser (1947) showed that no spiral arteries are present in

the endometrium of three species of South American monkeys known to menstruate. He also found that the coiled vessels of the endometrium could be destroyed almost completely by giving large doses of estrogen and yet bleeding followed estrogen withdrawal.

Several experimental conditions under which the coiled vessels of the endometrium are destroyed have been mentioned in the present discussion and in each instance bleeding invariably followed withdrawal of the supporting stimulus. The extremely atrophic endometrium present at the conclusion of a prolonged treatment with progesterone (Fig. 9.8) will bleed when the injections are stopped, and if estrogen injections are started immediately thereafter the endometrium that develops is normal with the exception of the absence of coiled arteries; even so, it also will bleed when the treatment is stopped. Even a more

ESTROGEN AND PROGESTERONE

58c

drastic destruction of endometrial structures occurs when both estrogen and progesterone are given for several months. Not only are the coiled arteries destroyed but also the glands and the luminal epithelium. All that remains is a modified stroma penetrated by a few small blood and lymph vessels and scattered glandular rudiments along the myometrium (Fig. 9.13). Yet, in spite of this, bleeding follows discontinuance of the treatment.

These observations prove conclusively that the spiral arteries of the endometrium do not hold the solution to the menstrual process. However, the descriptive account by Markee of the events that take place in the endometrium during the cycle remains one of the major contributions to our knowledge of the primate endometrium. Phelps (1946) also made a very careful study of the vascular changes in intraocular endometrial transplants in ovariectomized monkeys receiving estrogen and progesterone, and concluded that the primary function of the coiled arteries is concerned with vascularization of the implantation site of a developing embryo.

There also is reason for doul^ting that ischemia is a determining factor in the menstrual process. That constriction of the endometrial vessels does occur is well established, but that tissue destruction and bleeding are consequences of prolonged anoxemia may be questioned. The endometrium around the internal cervical os as seen in incised exteriorized uteri (Fig. 9.7) contains very few coiled arteries and does not take part in the periodic blushing and blanching of the fundus, but instead remains blood-red even during menstruation. Also, certain tongues of endometrium in a uterine fistula may become crowded by their neighbors to an extent of being partly or completely deprived of blood, yet they do not bleed even though their unfavorable situation leads to deterioration within a few days.

Emmel, Worthington and Allen (1941) attempted to induce menstruation in monkeys by operative ischemia. Circulation to the fundus of the uterus was interrupted by means of a tourniquet for periods of 1 to 8V4 hours, and in two instances for 19 hours. This procedure did not precijiitate uterine

bleeding nor did it hasten the onset of an expected bleeding following estrogen withdrawal. In fact, when the uterus was deprived of blood for periods longer than 3 hours impairment of the bleeding response to estrogen withdrawal was observed, and 19 hours of ischemia caused atrophy of the uterus without bleeding.

It also has been reported that a toxic substance formed in the endometrium is responsible for menstruation. This menstrual toxin is supposed to be present in the endometrium just previous to and during menstruation, and to be a substance resembling or identical with necrosin, a material found in pleural exudate following an inflammatory reaction (Smith and Smith, 1951). Zondek (1953) reports that menstrual blood, when obtained under relatively sterile conditions, is no more toxic to experimental animals than sterile tissue extracts. He also found that death of animals given injections of menstrual blood was due to bacteremia, an effect that could be prevented by giving antibiotics. Nor was he able to demonstrate a toxic substance in the premenstrual or menstrual endometrium. It might be mentioned in this connection that endometrial tissue destroyed by experimental ischemia in the experiments by Emmel, Worthington and Allen (1941), obviously did not influence menstruation nor did involuting endometrial tissue in uterine fistulae (p. 564). Therefore, the presence of a specific toxin that may induce menstruation has not been conclusively demonstrated.

Regardless of the specific cause of menstruation, the evidence shows that it can occur in the absence of coiled arteries, endometrial glands, or surface mucosa, and is unrelated to the thickness of the endometrium. This statement is based on conditions that have been experimentally induced in the monkey and they strongly indicate that menstruation, whatever the cause, is a stromal phenomenon. This view seems to be in agreement with the observations reported by Bartelmez in his elegant studies of the morphology of the endometrium of both monkeys and the human being. He emphasizes changes taking place in the connective tissue elements of the stroma and points out that much less tissue is lost at menstruation

586

PHYSIOLOGY OF GONADS

than i.< commonly thought (Bartehnez, 1957). The reduction in thickness is clue primariiy to loss of ground substance from the stroma, and conversely, the outstanding feature of repair is the increase in stromal ground substance (Fig. 9.23). ^Mitoses are rarely seen in the stroma during repair and arc not abundant enough in any phase according to Bartelmez to account for the observed increase in thickness of the endometrium. Our present knowledge indicates that an explanation of menstruation may be found in the metabolic effects induced in the stromal connective tissue of the endometrium by a sudden withdrawal of a supporting hormonal stimulus.

XI. References

Allen, E. 1927. The menstrual CA'cle in the monkey, Macacus rhesus: observations on normal animals, the effects of removal of the ovaries and the effects of injections of ovarian and placental extracts into the .spayed animals. Contr. Embrvol., Carnegie Inst. Washington, 19, 1-44.

Allen. E. 1928. Further experiments with an ovarian hormone in the ovariectomized adult monkey, Macacus rhesus, especially the degenerative phase of the experimental menstrual cycle. Am. J. Anat., 42, 467^87.

Allen, E., Diddle, A. W., Burford, T. H., .and Elder, J. H. 1936. Analyses of urine of the chimpanzee for estrogenic content during various stages of the menstrual cycle. Endocrinology, 20, 546-549.

Allen, E., Pr.\tt, J. P., Xewell, Q. U., .and Bl.and, L. J. 1930. Human tubal ova ; related early corpora lutea and uterine tubes. Contr. Embryol., Carnegie Inst. Washington, 22, 45-76.

B.ACHMAN, C, CoLLip, J. B., .\ND Selye, H. 1935. The effects of prolonged estriol administration upon the sex skin of Macaca mulatta. Proc. Roy. Soc, London, .■^er. B., 117, 16-21.

B.artel.mez, G. W. 1933. Histologic studies on the menstruating mucous membrane of the human uterus. Contr. Embryol.. Carnegie Inst. Washington, 24, 141-186.

B.artel.mez, G. W. 1937. ^Menstruation. Plnsiol. Rev.. 17, 28-72.

B.ARTELMEZ, G. W. 1951. Cyclic changes in the endometrium of the rhesus monkey {Macacus mulaltn). Contr. Embryol., Carnegie Inst. Washington. 34, 101-144.

B.ARTELMEz, G. W. 1957. The pha.scs of the menstrual cycle and their interpretation in terms of the pregnancv cycle. Am. J. Obst. & Gvnec, 74, 931-955.

BRY.AN.S, F. E. 1951. Progesterone of the lilood in the menstrual cycle of the monkey. Endocrinology, 48, 733-740.

BrxTo.N, C. L. 1940. Pregnanediol determina

tions as an aid in clinical diagnosis. Am. J. Obst. & Gynec, 40, 202-211.

CoLLiNGS, M. R. 1926. A study of the cutaneous reddening and swelling about the genitalia of the monkev, Macacus rhesus. Anat. Rec, 33, 271-287.

Corner, G. W. 1923. Ovulation and menstruation in Macacus rhesus. Contr. Embrj^oL, Carnegie Inst. Washington, 15, 73-101.

Corner, G. W. 1935. Influence of the ovarian hormones, estrin and progestin, upon the menstrual cvcle of the monkev. Am. J. Phj'siol., 113,238-250.

Corner, G. W. 1937. Experimental menstruation. Science, 85, 437-438.

Corner, G. W. 1942. The fate of the corpora lutea and the nature of the corpora aberrantia in the rhesus monkey. Contr. Embryol., Carnegie Inst. Washington, 30, 87-96.

Corner, G. W. 1945. Development, organization and breakdown of the corpus luteum in the rhesus monkey. Contr. Embrj'ol., Carnegie Inst. Washington, 31, 119-146.

Corner, G. W. 1951. Our knowledge of the menstrual cycle, 1910-1950. Lancet, 1, 919-923.

Corner, G. W., and Allen, W. M. 1936. Inhibition of menstruation by crj-stalline progesterone. Proc. Soc. Exper. Biol. & Med.. 34, 723724.

Crossex, R. J. 1953. Diseases of Women. St. Louis: C. V. Mosby Company.

Davls, M. E., and H.art^lan, C. G" 1935. Changes in vaginal epithelium during pregnancy in relation to the vaginal cvcle. J. A. M. A., 104, 279-285.

Diczfall'.sy, E. 1953. Chorionic gonadotrophin and estrogens in the human placenta. Acta endocrinol., Suppl. 12, 87-167.

DoRFMAN, R. I. 1956. Metabolism of androgens, estrogens and corticoids. Am. J. Med., 21, 679687.

DoRFMAN, R. I., AND VAN W.AGENEN, G. 1941. The

sex hormone excretion of adult female and pregnant monkevs. Surg. Gvnec. & Obst., 73, 545-548.

Duncan, P. A., Allen. E., and Ha.milton. J. B. 1941. The action of testosterone proprionate on experimental men.struation in the monkev. Endocrinology. 28, 107-111.

Eckstein, P. 1950. The induction of progesterone withdrawal bleeding in spayed monkeys. J. Endocrinol., 6, 405-411.

EcK.STEiN, P., AND ZucKERMAN, S. 1956. In Marshall's Physiology of Reproducliori. A. S. Parkes, Ed. Vol. 1, ]\ 334. London: Longmans Green & Company.

EmMEL, V. M., WORTHINGTON, R. V., AND AlLEN. E.

1941. Attempts to induce menstruation by operative ischemia in monkey's. Endocrinologv, 29, 330-335.

Engle. E. T. 1937. Problems of experimental menstruation. Cold Spring Harbor Svmposia Quant. Biol., 5, 111-114.

Engle, E. T., .and S.mith, P. E. 1935. Some uterine effects obtained in female monkeys during continued estrin administration, with especial

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reference to tlio r('i\ ix uteri. Auat. Rec, 6, 471-483.

Encle, E. T., Smith, P. E., and Shelesnyak, M. C. 1935. The role of estrin and progestin in experimental menstruation. Am. J. Obst. & Gynec. 29, 787-797.

Engle, E. T., and Smith, P. E. 1938. The endometrium of the monkey and est rone-progesterone balance. Am. J. Anat., 63, 349-365.

Engle, E. T., and Smith, P. E. 1939. Certain actions of testosterone on the endometrium of the monkey and on uterine bleeding. Endocrinology, 25^ 1-6.

Fish, W. R., Young, W. C, and Dorfman, R. I. 1941. Excretion of estrogenic and androgenic substances by female and male chimpanzees with known mating behavior records. Endocrinology, 28, 585-592.

Fluhmann, C. F. 1954. Comparative studies of squamous metaplasia of the cer\ix uteri and endometrium. Am. J. Obst. ct Gynec, 68, 1447-1462.

Forbes, T. R., Hooker, C. W., and Pfeiffer, C. A. 1950. Plasma progesterone levels and the menstrual cycle of the monkey. Proc. Soc. Exper. Biol. & Med., 73, 177-179.

GiLLMAN, J. 1937a. The cyclical changes in the vaginal smear in the baboon and its relationship to the perineal swelling. South African J. M. Sc, 2, 44-56.

GiLLMAN, J. 1937b. Experimental studies on the menstrual cycle of the baboon (Papio porcarhis). South African J. M. Sc, 2, 156-166.

GiLLMAN, J. 1938. Experimental studies on the menstrual cycle of the baboon (Papio porcarius). South African J. M. Sc, 3, 6&-71.

GiLLMAN, J. 1940a. Experimental studies on the menstrual cycle of the baboon (Papio porcarius). VI. The effect of progesterone upon the first part of the cycle in normal female baboons. Endocrinology, 26, 80-87.

GiLLMAN, J. 1940b. The effect of multiple injections of progesterone on the turgescent perineum of the baboon (Papio porcarius). Endocrinology, 26, 1072-1077.

GiLLMAN, J., AND GILBERT, C. 1946. The reproductive cycle of the chacma baboon (Papio itrsiDiis) with special reference to the problems of menstrual iriegularities as assessed by the behaviour of the sex skin. South African J. M Sc, Biol. Suppl., 11, 1-54.

GiLLMAN, J., .AND Smyth, G. S. 1939. The hormonal content of the human luteal follicle of pregnancy as determined by its effect on the perineum of the baboon. South African J. M. Sc, 4, 3&-45.

Haman, J. O. 1942. The length of the menstrual cvcle. A study of 150 normal women. Am. J. Obst. & Gynec, 43, 870-873.

Hamblen, E. C., Cuyler, W. K., Powell, N. B., Ashley, C, and B.aptist, M. 1939. Some clinical observations upon the metabolism and utilization of crystalline progesterone. Endocrinology, 25, 13-16.

Hamilton, C. E. 1949. Observations on the cervi

cal mucosa of the Rhesus monkey. Contr. EnibryoL, Carnegie Inst. Washington, 33, 81-101.

H.artman, C. G. 1929. Three types of uterine bleeding in the monkey and the homology of menstruation (Abstr.). Anat. Rec, 42, 19.

Hartman, C. G. 1932. Studies in the reproduction of the monkey, Macacus (Pithecus) rhe.S-//.S, with special reference to menstruation and piegnancy. Contr. Embryol., Carnegie Inst. Washington, 23, 1-16.

H.ARTMAN, C. G. 1934. Some attempts to influence the menstrual cvcle in the monkev. Am. J. Obst. & Gynec, 27, 564-570.

Hartman, C. G. 1937. Menstruation inhibiting action of testosterone. Proc Soc. Exper. Biol. & Med., 37, 87-89.

H.ARTMAN, C. G. 1944. Regeneration of the monkey uterus after surgical removal of the endometrium and accidental endometriosis. Western J. Surg. Obst. & Gynec, 52, 87-102.

Hartman, C. G., .and Speert, H. 1941. Action of progesterone on the genital organs of the unjirimed Rhesus monkev. Endocrinology, 29, 639-648.

H.ARTMAN, C. G., GeSCHICKTER, G. F., AND SpEERT, H.

1941. Effects of continuous estrogen administration in verv large doses. Anat. Rec, Suppl. 2, 79, 31.

Hellman, L. M., Rosenthal, A. H., Kistner, R. W., AND Gordon, R. 1954. Some factors influencing the proliferation of the leserve cells in the human cervix. Am. J. Obst. & Gvnec. 67, 899-915.

Hertig, a. T., and Rock, J. 1944. On the development of the early human ovum, with special reference to the trophoblast of the previllous stage ; a description of 7 normal and 5 pathologic human ova. Am. J. Obst. & Gvnec, 47, 149-184.

Heuser, C. H., and Streeter, G. L. 1941. Development of the macaque embryo. Contr. Embryol., Carnegie Inst. Washington, 29, 17-55.

Hisaw, F. L. 1935. The physiology of menstruation in Macacus rhesus monkevs. Am. J. Obst. & Gynec, 29, 638-659.

HiSAW% F. L. 1942. The interaction of the ovarian hormones in experimental menstruation. Endocrinology, 30, 301-308.

HiSAW, F. L. 1943. Androgens and experimental menstruation in the monkey (Macaca viulatta). Endocrinology, 33, 39-47.

HisAW, F. L. 1950. Factors influencing endometrial growth in monkeys (Macaca mulatta). In A Symposium on Steroid Hormones, E. S. Gordon, Ed., pp. 259-276. Madison: University of Wisconsin Press.

Hisaw, F. L. 1959. Endocrine adaptations of the mammalian estrous cycle and gestation. In Columbia University Symposium on Comparative Endocrinology, pp. 533-552.

Hisaw, F. L., Creep, R. O., and Fevold, H. L. 1937. Effects of progesterone on the female genital tract after castration atrophy. Proc Soc. Exper. Biol. & Med., 36, 840-842."

His.\w, F. L.. AND Creep, R. O. 1938. The inhibition of uterine bleeding with estradiol and

588

PHYSIOLOGY OF GONADS

progesterone and associated endometrial modifications. Endocrinology, 23, 1-14. His.\w, F. L., .^ND His.wv, F. L., Jr. 1958. Spontaneous carcinoma of the cervix uteii in a monkey (Macaca mulatto). Cancer, 11, 810816. HiSAW, F. L., .4ND Lendrum, F. C. 1936. Squamous metaplasia in the cervical glands of the monkey following oestrin administration. Endocrinology, 20, 228-229. HiSAW, F. L., Meyer, R. K., axd Fevold, H. L. 1930. Production of a premenstrual endometrium in castrated monkeys by ovarian hormones. Proc. Soc. Exper. Biol. & Med., 27, 400-403. HiTSCHMANN, F., AND Adler, L. 1907. Die Lehre von der Endometritus. Ztschr. Geburtsh. u. GynJik., 60, 63-86. Kaiser, I. H. 1947. Absence of coiled arterioles in the endometrium of menstruating New World monkeys. Anat. Rec, 99, 353-363. Kaufmann, C, Westphal, U., and Zander, J. 1951. Untersuchungen liber die biologische Bedeutang der Ausscheidungsprodukte des Gelbkcirperhormons. Arch. Gynak., 179, 247-299. Knaus, H. 1950. Die Physiologie der Zeugung

des Menschen. Wien: Wilhelm Maudrich. Krohn, p. L. 1951. The induction of menstrual bleeding in amenorrhoeic and normal monkeys by progesterone. J. Endocrinol., 7, 310-317. Krohn, P. L. 1955. The induction of cyclic uterine bleeding in normal and spayed rhesus monkeys by progesterone. J. Endocrinol., 12, 6985. Krohn, P. L., and Zuckerman, S. 1937. Water metabolism in relation to the menstrual cycle. J. Physiol., 88, 369-387. Latz, L. J., and Reiner, E. 1942. Further studies on the sterile and fertile periods in women. Am. J. Obst. & Gynec, 43, 74-79. Lendrum, F. C., .and Hisavv^ F. L. 1936. Cytology of the monkey endometrium under influence of follicidar and corpus luteum hormones. Proc. Soc. Exper. Biol. & Med., 34, 394-396. Lopez Columbo de Allende, I., and Orias, O. 1950. Cytology of the Human Vagina. New York: Paul B. Hoeber, Inc. Lopez Colu.mbo de Allende, I., Shorr, E., and Hartman, C. G. 1945. A comparative study of the vaginal smear cycle of the rhesus monkey and the human. Contr. Embryol., Carnegie Inst. Washington, 31, 1-26. M.arkee, J. E. 1940. Menstruation in intraocular endometi'ial tr;msplants in the I'hesus monkey. Contr. Embrvol., Carnegie Inst. Washington, 28, 219-308. Markee, J. E. 1946. Morphologic and endocrine basis foi' menstrual bleeding. In Progress in Gynecology, Meigs and Sturgis, Eds. Vol. II, pp. 37-47. New York: (hune and Stiattdii. Markee, J. E., and Berg, B. 1944. Cyclic fluctuations in blood estrogen as a possible cause of menstruation. Stanford Med. Bull., 2, 55-60. Markee, J. E., D.wis, J. H., and Hinsf.y, J. C. 1936. Uterine bleeding in spinal iii()nk(>vs. Anat. Rec, 64, 231-245.

Mazer, C, and Israel, S. L. 1951. Diagnosis and Treatment of Menstrual Disorders and Sterility. New York: Paul B. Hoeber, Inc.

Meyer, R. 1911. Uber Corpus luteum-Bildung beim Menschen. Arch. Gynak., 93, 354-404.

Nissen, H. W., and Yerkes, R. M. 1943. Reproduction in the chimpanzee: report on 49 births. Anat. Rec, 86, 567-578.

Novak, E., and Te Linde, R. W. 1924. Endometrium of menstruating uterus. J. A. M. A., 83, 900-906.

OvERHOLSER, M. D., AND Allen, E. 1933. Ovaiiau hormone and traumatic stimulation of monkey's cervix to a condition resembling early cancer. Proc Soc. Exper. Biol. & Med., 30, 1322-1326.

OvERHOLSER, M. D., AND Allen, E. 1935. Atypical growth induced in cervical epithelium of monkey by prolonged injections of ovarian hormone combined with chronic trauma. Surg. Gynec. & Obst., 60, 129-136.

OvERHOLSER, M.D.,. AND Nelson, W. 0. 1936. Migration of nuclei in uterine epithelium. A monkey following prolonged estrin injections. Proc Soc Exper. Biol. & Med., 34, 839-841.

P.APANicoL.Aou, G. N., Traut, H. F., and M.archetti, A. A. 1948. The Epithelia of Woman/s Reproductive Organs. New York: Commonwealth Fund.

Parkes, a. S., and Zuckerm.an, S. 1931. The menstrual cycle of the Primates. II. Some effects of oestrin on baboons and macaques. J. Anat., 65, 272-276.

Phelps, D. 1946. Endometrial vascular reactions and the mechanism of nidation. Am. J. Anat., 79, 167-197.

Phelps, D. H. 1947. The factor of previous treatment in experimental menstruation. J. Clin. Endocrinol., 7, 611-623.

Rakoff, a. E. 1946. Studies on high dosage progesterone therapy of amenorrhea. Am. J. Obst. & Gynec, 51, 480-491.

Rock, J., and Hertig, A. 1942. Some aspects of early human development. Am. J. Obst. & Gynec, 44, 973-983. RossM.AN, I. 1940. The decidual reaction in the rhesus monkey {Macaca mulatta). I. The epithelial proliferation. Am. J. Anat., 66, 277365. Schroder, R. 1914. Uber das Verhalten der Uterusschleimhaut um die Zeit der Menstruation. Monatsschr. Geburtsh. u. Gynak., 39, 3-21. Seeg.ar, E. G. 1940. The histologic effect of progesterone on hyperplastic endometria. Am. J. Obst. & Gynec, 39, 469-476. S.mith, O. W., and S.mith, G. V. 1951. Endocrinology and related phenomena of the human menstrual cvcle. Recent Progr. Hormone Res., 7, 209-253.

So.MMKKVILLK, I. V ., AND MaRRIAN, G. F. 1950.

I'rinary excretion of prcgnanediol in human subjects following the administration of progesterone and of pregnane-3a:20a-diol. I3iochem. J., 46, 285-289. Stieve, H. 1926. Di(> regelmassigen Verliinderungen der Muskulatur und des Bindegewebs in

ESTROGEN AND PROGESTERONE

589

der meuschlichen Gebarmutter in ihier Abhangigkeit von der Follikelreife und der Aiisbildung eines gelben Korpers, nebst Beschreibung eines menschlichen Eies im Zustand der ersten Reifteilung. Ztschr. mikroskop.-anat. Forsch, 6, 351-397.

Stieve, H. 1942. Der Einfluss von Angst und psychischer Erregung auf Bau und Funktion der weiblit'hen Geschlechtsorgane. Zeutralbl. Gynak., 66, 1698-1708.

Stieve, H. 1943. Weitere Tatsachen zur Kliirung der Frage: Wann wird das Ei aus dem Eierstock ausgestossen? Zentralbl. Gvnak., 67, 5877.

Stieve, H. 1944. Paracyclische Ovulationen. Zentralbl. Gynak., 68, 257-272.

Sturgis, S. H. 1942. Method for obtaining uterine fluid from the monkey : effect of pilocarpine, atropine, physiologic salt solution and adrenalin. Endocrinology, 31, 664-672.

VAN Dyke, H. B., and Ch'en, G. 1936. Observations on biochemistry of genital tract of female macacjue particularly during menstrual cycle. Am. J. Anat., 58, 473-499.

VAN Wagenen, G. 1945. Mating in relation to pregnancy in the monkey. Yale J. Biol. & Med., 17, 745-760.

VAN Wagenen, G. 1947. Early mating and pregnancy in the monkev. Endocrinologv, 40, 3743.

VAN W.^genen, G., and Morse, A. H. 1940. Cyclic changes in the exteriorized uterus. Endocrinology, 27, 268-273.

Venning, E. H., and Browne, J. S. L. 1937. Studies on corpus luteum function. I. The vn-inary excretion of sodium pregnandiol glucuronidate in the human men.^trual cvcle. Endocrinologv, 21,711-721.

Werner, A. A., and Collier, W. D. 1933. The effect of theelin injections on the castrated woman. J. A. M. A., 100, 633-640.

Westman, a. 1932. Studien iiber den Sexualzyklus bei Makakus-Rhesus-Affen, nebst einigen Bemerkungen liber den menstruellen Blutungs-mechanismus. Acta obst. et gynec. scandinav., 12, 282-328.

WiSLOCKi, G. B., and Streeter. G. L. 1938. On the placentation of the macaque {Macaca mulatta), from the time of implantation until the formation of the definitive placenta. Contr. Embryol., Carnegie Inst. Washington, 27, 166.

Young, W. C, and Yerkes, R. M. 1943. Factors influencing the reproductive cycle in the chimpanzee; the period of adolescent sterility and related problems. Endocrinology, 33, 121-154.

Z.ARRow, M. X., Hisaw, F. L., and Bryans, F. 1950. Conversion of desoxycosterone acetate to progesterone in vivo. Endocrinology, 46, 403-404.

Zarrow, M. X., Shoger, R. L., and Lazo-Wasem, E. A. 1954. The rate of disappearance of exogenous progesterone from the blood. J. Clin. Endocrinol., 14, 645-652.

Zondek, B. 1953. Does menstrual blood contain a specific toxin? Am. J. Obst. & Gvnec, 65, 1065-1068.

-Zondek, B. 1954. On the mechanism of uterine bleeding. Am. J. Obst. & Gynec, 68, 310-314.

Zondek. B., and Rozin, S. 1938. Production of uterine haemorrhage in the normal cycle and hi amenorrhoea through progesterone. J. Obst. & Gynaec. Brit. Emp., 45, 918-931.

Zuckerman, S. 1930. The menstrual cycle of the Primates. I. General nature and homology. Proc. Zool. Soc, London, 1930, 691-754.

Zuckerman, S. 1935. The menstrual cycles in the Primates. VIII. The estrin-vvithdrawal theory of menstruation. IX. The effect of estrin on the denervated sexual skin. Proc. Rov. Soc, London, ser. B., 118, 13-33.

Zuckerman, S. 1936a. Inhibition and induction of uterine bleeding bv means of estrone. Lancet, 2, 9-13.

Zuckerman, S. 1936b. The interrelation of estrone and progestin in the menstrual cvcle. J. PhvsioL, 86, 31-33.

Zuckerman, S. 1937a. The duration and phases of the menstrual cycle in Primates. Proc. Zool. Soc, London, ser. A., 1937, 315-329.

Zuckerman, S. 1937b. The menstrual cycle of the Primates. X. The oestrone threshold of the uterus of the rhesus monkey. XL The part played by oestrogenic hormone in the menstrual cycle. Proc. Roy. Soc, London, ser. B., 123,441-471.

ZucKER.MAN, S. 1937c Effects of prolonged oestrin-stimulation on the cervix uteri. Lancet, 1, 435-437.

Zuckerman, S. 1937d. Further observations on endocrine interaction in the menstrual cvcle. J. Physiol., 89, 49-51.

Zuckerman, S. 1937e. The duration and phases of the menstrual cycle in Primates. Proc. Zool. Soc London, ser. A., 1937, 315-329.

Zuckerman, S. 1939. The effect of sex hormones, cortin, and vasopressin on water-retention in the reproductive organs of monkeys. J. Endocrinol., 1, 147-155.

Zuckerman, S. 1941. Periodic uterine bleeding in spayed rhesus monkeys injected daily with constant threshold dose of oestrone. J. Endocrinol., 2, 263-267.

Zuckerman, S. 1949. The menstrual cvcle. Lancet, 2, 176.

Zuckerman, S. 1951. The hormonal basis of uterine bleeding. Acta endocrinol., 7, 378-388.

Zuckerman, S., and P.arkes, A. S. 1932. The menstrual cycle of the primates. V. The cycle of the baboon. Proc Zool. Soc. London, 1932, 139-191.

Zuckerman, S., van W.agenen, G., and Gardiner, R. H. 1938. The sexual skin of the rhesus monkey. Proc Zool. Soc, London, ser. A., 108, 385-401.

10

THE MAMMARY GLAND AND LACTATION

A. T. Cowie and S. J. FoUeij

NATIONAL INSTITUTE FOR RESEARCH IN DAIRYING, SHINFIELD, READING, ENGLAND

I. Introduction

I. Introduction 590

II. Development of the Mammary

Gland 591

A. Histogenesis 591

B. Normal Postnatal Development . 593

1. Methods of assessing mammary

development 593

2. Mammary development in the

nonpregnant female 594

3. Mammary growth in the male . . 595

4. Mammary development during

pregnancy 596

5. Mammary involution 598

C. Experimental Analysis of Hormonal

Influences 598

1. Ovarian hormones in the animal

with intact pituitary 598

2. Anterior pituitary hormones. . . 601

3. Metabolic hormones (corticoids,

insulin, and thyroid hormones) 604 III. Endocrine Influences in Milk Secretion 606

A. Anterior Pituitary Hormones 606

1. Initiation of secretion (laeto genesis) 606

2. Maintenance of milk secretion —

galactopoiesis 609

3. Suckling stimulus and the main tenance of lactation 611

B. Hormones of the Adrenal Corte.x . . 612

C. Ovarian Hormones 613

D. Thyroid Hormones 617

E. Parathyroid Hormone 618

F. Insulin 619

IV. Removal of Milk from the Mammary

Glands: Physiology of Suckling AND Milking 619

A. Milk-Ejection Reflex 619

B. Role of the Neurohypophysis 621

C. Milk-Ejection Hormone 622

D. Effector Contractile Mechanism of

the Mammary Gland 623

E. Inhibition of Milk Ejection 624

F. Neural Pathways of the Milk-Ejec tion Reflex 625

G. Mechanism of Suckling 626

V. Relation between the Reflexes

Concerned in the Maintenance of Milk Secretion and Milk Ejection 627 VI. Pharmacologic Blockade of the Reflexes Concerned in the Maintenance OF Milk Secretion and

Milk E.tection 630

VII. Conclusion 632

VIII. References 632

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

590

MAMMARY GLAXD AND LACTATION

591

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, 301.

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

592

PHYSIOLOGY OF GONADS

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.

MAMMARY GLAND AND LACTATION

593

PwokcTiL del

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. NORM.\L 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 re

viewed 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,

594

PHYSIOLOGY OF GONADS

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 X on 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

MAMMARY GLAXD AND LACTATION

595

olO.

rCMALC RATS

ACt$ : i - lOO DAYS

C 22 NO DAY

LOC„ CBOOY WtlCHT C>

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.

596

PHY,SI(3L0GY OF GONADS

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 L

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).

MAMMARY GLAND AND LACTATION

597

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.)

598

PHYSIOLOGY OF GONADS

5. Mam /nary 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 mav

MAMMARY GLAND AND LACTATION

599

O2 Uptake

G\

ucose

uptake

Lactic acid production.

s 12 ■Hours

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.)

be that the I'eal basis for the categories is to be found largely in differences in endogenous progesterone production by the adrenal cortex.

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

(JOO

PHYSIOLOGY OI-' GONADS

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 Richai'dson.

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 jiossible that coidd

MAMMARY GLAND AND LACTATION

601

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.)

the necessary experiments be done the human breast would show a considerable growth response to estrogen alone.

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 alveo

602

PHYSIOLOGY OF CIOXADS

lar 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 de

velopment (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

MAMMARY C5LAND AND LACTATION

603

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.)

604

PHYSIOLOGY OF GONADS

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.

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

MAMMARY GLAND AND LACTATION

605

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.

0-5 cm.

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.)

GOG

PHYSIOLOGY OF GONADS

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

Fk;. 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 modifica

MAMMARY GLAND AND LACTATION

607

tions 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

G08

PHYSIOLOGY OF GONADS

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

MAMMARY GLAXD AND LACTATION

009

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).

GIO

PHYSIOLOGY OF GONADS

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

I « 

c

-^ 4

-0

I 1

£

Z -6

z

J

E

^ 2 ^^,

TV

2 ^

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.)

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.)

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.

MAMMARY GLAND AND LACTATION

611

mq qro\Om hormone (onthmeTTc scale)

fa-25 12-5 25-0 50-0

100-0

200-0

S-«^0

'Zoo-o

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.)

C)ther 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

012

PHYSIOLOGY OF GONADS

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).

MAMM.\RY GLAND AND LACTATION

613

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 ex

614

PHYSIOLOGY OF GONADS

periments 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.)

MAMMARY GLAND AND LACTATION

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),

GIG

PHYSIOLOGY OF GONADS

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).

In 1936 one of us (Folley, 1936) reported

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.)

MAMMARY GLAND AND LACTATION

GK

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

9-9 97

o 9-3 ^ 9-1

8-9

• Guernsey

Shorthorn

8-5

•^U^ri

I L

20 40 60 80 100

Oestradiol monobenzoite (mg)

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.)

Oestradiol monobenzoate (mg) (arith. scale)

10 20 30 40 50

6-25 12-5 250 500

Oestradiol monobenzoate (mg) (log scale)

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

618

PHYSIOLOGY OF GONADS

,

/" \^

Cont-rol.

A<' / " "^ - ^*

— • DO m§.

.^\ / V- -' v;.

100 m|.

^..^-Av / V

150mg.

^^^4?^^/ . V

,.-•*.. \ vv / .^-r \ \

• — •• tva --^-^ y \ \ \

•••\-'^\ \ x- ^ .. \ ^

\. *-^ '• •■*— . \ \

• ■*•—., \ \ \ \

.... -.... "•N-:w<r:Viy: y^

Sl-art of hrcAhnc.ih \\ y' i'

hrc iXhuciil' \\ //

\v/y

\ V /

\ /

\ /

\/

V

10

50

50

Dau5

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

MAMMARY GLAND AND LACTATION

()19

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

620

PHYSIOLOGY OF GONADS

22

rs 2-5//g/ml.

20

yT

■~^

yO

i 18

- Cf

>

-o

■;;16

- i:/^

c

u=

«14

/ J3 as^g/mi.

8 12

Z' j^p^ £) 0-Vg/ml.

— 10

y rf^ ,-fP

1 3 8

si r^^ r-f^ y^ Control

o °

M J

A y^ ^cr ,^y^

4J

z

Pr( .iif^ jy^^

4

^ M^ ^r^

2

L_l 1 1 1 1 \ 1 \ 1 1 \ 1

15 30

60 90 120

Time (min)

150

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.

MAMMARY GLAND AND LACTATION

621

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

l)urely 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 Cous

622

PHYSIOLOGY OF GONADS

sens, 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

MAMMARY GLAND AND LACTATION

623

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

624

PHYSIOLOGY OF GONADS

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

MAMMARY GLAND AND LACTATION

625

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.

626

PHYSIOLOGY OF GONADS

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 some

MAMMARY GLAND AND LACTATION

627

times 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

628

PHYSIOLOGY OF GONADS

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

MAMMARY GLAND AND LACTATION

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

630

PHYSIOLOGY OF GONADS

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 per

MAMMARY GLAND AND LACTATION

631

fonnance 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 ap

peared 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

^^:f/

mm\"^>m.-Wi

■w^

.•^^:j^-^ f4kr 1"

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.)

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PHYSIOLOGY OF GONADS

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.

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.

VIII. References

Abraham, S., Cady, P., and Chaikoff, I. L. 1957. Effect of insulin in vitro on pathways of glucose utilization, other than Embden-Meyerhof, in rat mammarv gland. J. Biol. Cliem., 224, 955-962.

Ahren, K. 1959. The effect of various do.^es of estrone and progesterone on the mammary glands of castrated hypophysectomized rats injected with insulin. Acta endocrinol., 30, 435458.

Ahren, K., and Etienne, M. 1957. The development of the mammary gland in normal and castrated male rats after the age of 21 days. Acta physiol. scandinav., 41, 283-300.

Ahren, K., .\nd Etienne, M. 1958. Stimulation of mammary glands in hypophysectomized male rats treated with ovarian hormones and insulin. Acta endocrinol., 28, 89-102.

Ahren, K., and Jacoksoun, D. 1956. Mammary gland growth in hypophy-sectomized rats injected with ovarian hormones and insulin. Ada physiol. scandinav., 37, 190-203.

Ahren, K., and Jacobsohn, D. 1957. The action of cortisone on tlip mammary glands of rats

imder various states of hormonal imbalance.

Acta phy.siol. scandinav., 40, 254-274. [Al'tman, a. D.] A.abTMaH, A. JX. 1945. Hsm eneHHH b BbiMenn KopoB b nporiecce pasAOH.

Vestnik Zhivotn., 1, 85-96. Andersson, B. 1951a. Some observations on the

neurohormonal regulations of milk ejection.

Acta physiol. scandinav., 23, 1-7. Andersson, B. 1951b. The effect and localization

of electrical stimulation of certain parts of the

brain stem in sheep and goats. Acta physiol.

scandinav., 23, 8-23. Andersson, B. 1951c. Further studies on the milk

ejection mechanism in sheep and goats. Acta

physiol. scandinav., 23, 24-30. Ardran, G. M., Cowie, A. T., .-^nd Kemp, F. H.

1957. A cineradiographic study of the teat sinus during suckling in the goat. Vet. Rec, 69, 1100-1101.

Ardrax, G. M., Cowie. A. T., and Kemp, F. H.

1958. Further obser\ations on the teat sinus of the goat during suckling. Vet. Rec, 70, 808809.

Ardran, G. M., Kemp, F. H., and Lind. J. 1958a. A cineradiographic studv of bottle feeding. Brit. J. Radiol., 31, 11-22.

Ardr.\n, G. M., Kemp, F. H., .\nd Lind, J. 1958b. A cineradiographic studv of breast feeding. Brit. J. Radiol., 31, 156-162.

Ardran, G. M., and Kemp. F. H. 1959. A correlation between suckling pres-sures and the movements of the tongue. Acta pediat., 48, 261-272.

AvERiLL, S. C, R.\Y, E. W., .\ND Lyons, W. R. 1950. Maintenance of pregnancy in hypophysectomized rats with placental implants. Proc. Soc. Exper. Biol. & Med., 75, 3-6.

Bailey, G. L.. B.artlett, S, and Folley, S. J. 1949 Use of L-thyroxine by mouth for stimulating milk secretion in lactating cows. Nature, London, 163, 800.

Balinsky. B. I. 1950a. On the prenatal growth of the mammarv gland rudiment in the mouse. J. Anat., 84, 227-235.

Balin.sky. B. I. 1950b. On the doxelopmental lirocos.ses in niammary glands and other e\n(Iciinal slmctures. Tr. Roy. Soc. Edinburgh, 62, Part 1, 1-31.

Balmaix, J. H., Cox, C. P., FoLLEY, S. J., and MrNAUfiHT, M. L. 1954. The bioassay of insulin in vitro by manometric measurements on slices of mammary glands. J. Endocrinol., 11,269-276.

Balmain, J. H., AND FoLi.EY, S. J. 1951. Further ob.'^ervations on the iti vitro stimulation bv insulin of fat synthesis by lactating mammary gland slices. Biochem. J., 49, 663-670.

Balmain, J. H., Folley, S. J., and Glascock, R. F. 1952. Effects of insulin and of glycerol in vitro on the incorporation of (carboxy-C) acctale into the fatty acids of lactating mammary gland .slicrs with special refoi-(>nce to sjjc.•ics diffcnences. Biochem. J., 52, 301-306.

Bar(;.m\\n. \V., and Knoop, A. 1959. Uber die Morpliologie der Milchsekretion. Licht- und ("Icktroiii'imiikroskopische Studien an der

MAMMARY GLAND AND LACTATION

633

Milchdriise der Ratte. Ztschr. Zellforsch., 49, 344-388.

Bartlett, S., Burt, A. W. A., Folley, S. J. and Rowland, S. J. 1954. Relative galactopoietic effects of 3:5:3-triiodo-L-thyronine and L-thyroxine in lactating cows. J. Endociinol., 10, 192-20L

[Baryshxikov, L A.] BapMuiHHKOB, H. A. 1957. Pe(j|)neKTopHaH pery.TiHUHH JiaKTaunn. In npoo,neMbi OnsHO-norHii II,eHTpanbHOH HepBHott CncTeMbi, pp. 62-72. Moscow, Leningrad: Akademiya Nauk S.S.S.R. [Complete translation bv F. Lang in Dairy Science Abstracts, 21, 47-53, 1959.

Beller, F. K., Krumholz, K. H., and Zeininger, K. 1958. Vergleichende Oxytocin-Bestimmungen gemessen durch den lactagogen Effect der Milchdriise. Acta endocrinoL, 29, 1-8.

Benson, G. K. 1958. Effect of reserpine on mammary gland involution, and on other organs in the rat. Proc. Soc. Exper. Biol. & Med., 99, 550-553

Benson, G. K., Cowie. A. T., Cox, C. P., Flux, D. S., and Folley, S. J. 1955. Studies on the hormonal induction of mammary growth and lactation in the goat. II. Functional and morphological studies of hormonally developed udders with special reference to the effect of "triggering" doses of oestrogen. J. Endocrinol., 13, 46-58.

Benson, G. K., and Cowie, A. T. 1956. Lactation in the rat after hypophysial posterior lobectomy. J. Endocrinol., 14, 54-65.

Benson, G. K., Cowie, A. T., Cox, C. P., and Goldzveig, S. a. 1957. Effects of oestrone and progesterone on the mammary de\'eloi)ment in the guinea-pig. J. Endocrinol., 15, 126-144.

Benson, G. K., Cowie, A. T., Folley, S. J., and TiND.-vL, J. S. 1959. Recent developments in endocrine studies on mammary growth and lactation. In Recent Progress in the Endocrinology oj Reproduction, C. W. Lloyd, Ed., pp. 457-490. New York: Academic Press, Inc.

Benson, G. K., Cowie, A. T., and Tindal, J. S. 1958. The pituitary and the maintenance of milk secretion. Proc. Rov. Soc, London, ser. B, 149, 330-336.

Benson, G. K., and Folley, S. J. 1956. Oxytocin as stimulator for the release of prolactin from the anterior pituitary. Nature, London, 177, 700.

Benson, G. K., and Folley, S. J. 1957a. Retardation of mammary in\olution in the rat by oxytocin. J. Endocrinol., 14, xl.

Benson, G. K., and Folley, S. J. 1957b. The effect of oxytocin on mammary gland in\olution in the rat" J. Endocrinol., 16,' 189-201.

Bilek, J., AND Janovsky, M. 1956. Studium vlivu oxytocin na reflex vylucovdni mleka. Sbornik Ceskoslov. akad. zemedelskych, ved 29, 773780.

Bint.-^rningsih, Lyons, W. R., Johnson, R. E., and Li, C. H. 1957. Hormonal requirement for lactation in the hypophysectomized rat. Anat. Rec, 127, 266-267.

BiNT.^RNixGsiH, Lyons, W. R., Johnson, R. E., .-^nd Li, C. H. 1958. Hormonally induced lactation in hypophysectomized rats. Endocrinology, 63, 540-548.

Bl.^xter, K. L. 1952. Some effects of thyroxine and iodinated casein in dairy cows and their practical significance. Vitamins & Hormones, 10, 217-250.

Br.\chet, J. 1957. Biochemical Cytology. New York : Academic Press, Inc.

Bradley, T. R., and Clarke, P. M. 1956. The response of rabbit mammary glands to locally administered prolactin. J. Endocrinol., 14, 2836.

Bradley, T. R., and Cowie, A. T. 1956. The effects of hypophysectomy on the in vitro metabolism of mammary gland slices from lactating rats. J. Endocrinol., 14, 8-15.

Bradley, T. R., and Mitchell, G. M. 1957. The in vitro metabolism of mammary gland slices in the presence of posterior pituitary lobe extracts rich in melanophore-dispersing activity. J. Endocrinol., 15, 366-373.

Braude, R. 1954. Pig nutrition. In Progress in the Physiology of Farm Animals, J. Hammond, Ed., Vol. 1, Ch. 2, pp. 40-105. London: Butterworth & Company, Ltd.

Braude, R., and Mitchell, K. G. 1952. Observations on the relationship between oxytocin and adrenaline in milk ejection in the sow. J. Endocrinol., 8, 238-241.

BuscH, D. W. H. 1839. Das Gcschhi lilslol)en des Weibes in physiologischer, pai lidldgischer und therapeutischer Hinsicht. In Phy.siologie und allgemeine Pathologie des weiblichen Geschlechtslebens. Vol. I. Leipzig, F. A. Brockhaus.

Campbell, B., and Petersen, W. E. 1953. Milk "let-down" and the orgasm in the human female. Human Biol., 25, 165-168.

Canivenc, R. 1952. L'activite endocrine du placenta de la ratte. Arch, anat., 34, 105-118.

Canivenc, R., and M.-^yer, G. 1953. Nature du facteur luteotrophique de placenta de rat. Compt. rend. Soc. biol., 147, 1067-1070.

Chalmers, J. R., Dickson, G. T., Elks, J., and Hems, B. A. 1949. The synthesis of thyroxine and related substances. V. A synthesis of lthyroxine from L-tvrosine. J. Chem. Soc, 34243433.

Chen, L. T., Johnson, R. E., Lyons, W. R., Li, C. H., AND Cole, R. D. 1955. Hormonally induced mammary growth and lactation in the absence of the thvroid. Endocrinology, 57, 153-157.

Cole, H. A. 1933. The mammary gland of the mouse, during the estrous cycle, pregnancy and lactation. Proc Roy. Soc, London, ser. B., 114, 136-160.

Cooper, Sir Astley P. 1840. On the Anatomy oj the Breast. London: Longmans, Orme, Green, Brown & Longmans.

Cotes, P. M., Crichton, J. A., Folley, S. J., and Young, F. G. 1949. Galactopoietic activity

634

PHYSIOLOGY OF GONADS

of purified anterior pituitary growth hormone. Nature, London, 164, 992-993.

CowiE, A. T. 1949. The relative growth of the mammary gland in normal, gonadectomized and adrenalectomized rats. J. Endocrinol., 6, 145-157.

CowiE, A. T. 1952. Influence on the replacement value of some adrenal cortex steroids of dietary sodium and synergism of steroids in lactating and adrenalecomized rats. Endocrinology, 51, 217-225.

CowiE, A. T. 1957. The maintenance of lactation in the rat after hvpophysectomv. J. Endocrinol., 16, 135-147. "

CowiE, A. T. 1960. The hormonal control of milk secretion. In Milk: The Mammary Gland and its Secretion, S. K. Kon and A. T. Cowie, Eds., Ch. 4. New York: Academic Press, Inc.

Cowie, A. T., Cox, C. P., and N.aito, M. 1957. National In.stitute for Research in Dairying, Report 1957, p. 55.

Cowie, A. T., and Folley, S. J. 1945. Parathyroidectomy and lactation in the rat. Nature, London, 156, 719.

Cowie, A. T., and Folley, S. J. 1947a. The measurement of lactational performance in the rat in studies of the endocrine control of lactation. J. Endocrinol., 5, 9-13.

Cowie, A. T., and Folley, S. J. 1947b. Adrenalectomy and replacement therapy in lactating rats. II. Effects of deoxycorticosterone acetate on lactation in adrenalectomized rats. J. Endocrinol., 5, 14-23.

Cowie, A. T., .-^nd Folley, S. J. 1947c. Adrenalectomy and replacement therapy in lactating rats. III. Effects of deoxycorticosterone acetate and 11-oxygenated cortical steroids on lactation in adrenalectomized rats maintained on stock or high-protein diets. J. Endocrinol., 5,24-31.

Cowie, A. T., .\nd Folley, S. J. 1947d. The role of the adrenal cortex in mammary development and its relation to the mammogenic action of the anterior pituitarv. Endocrinologv. 40, 274-285.

Cowie, A. T., and Folley, S. J. 1957. Neurohypophysial hormones and the mammary gland. In The Neurohypophysis, H. Heller, Ed., pp. 183-197. London: Butterworth & Company, Lid.

Cowie, A. T., Folley, S. J., Cross, B. A., Harris, G. W., Jacobsohn, D., and Rich.^rdson, K. C. 1951. Terminology for use in lactational physiology. Nature, London, 168, 421.

Cowie, A. T., Folley, S. J., Malpress, F. H., and Richardson, K. C. 1952. Studies on the hormonal induction of mammary growth and lactation in the goat. J. Endocrinol., 8, 64-88.

Cowie, A. T., and Lyons, W. R. 1959. Mammogenesis and lactogenesis in hypophysectomizcd, ovariectomized, adrenalectomized rats. J. Endocrinol., 19, 29-32.

Cowie, A. T., and Tindal, J. S. 1955. Maintenance of lactation in adrenalectomized rats with aldosterone and 9a-halo deri\'atives of hydrocortisone. Endocrinology, 56, 612-614.

Cowie, A. T., and Tindal, J. S. 1958. Adrenalectomy in the goat: replacement therapy and the maintenance of lactation. J. Endocrinol., 16, 403-414.

Cowie, A. T., and Tindal, J. S. 1960. Effects of Iwpophysectomy of the pregnant and lactating goat. Acta endocrinol., Suppl. 51, 35, pp. 679680.

Cowie, A. T., Tindal, J. S., .\nd Benson, G. K. 1960. Pituitary grafts and milk secretion in hvpophysectomized rats. J. Endocrinol., 21, 115-123.

Cross, B. A. 1950. Suckling antidiuresis in rabbits. Nature, London, 166, 612-613.

Cross, B. A. 1953. Sympathetico-adrenal inhibition of the neurohypophysial milk-ejection mechanism. J. Endocrinol., 9, 7-18.

Cross, B. A. 1954. Milk ejection resulting from mechanical stimulation of mammary myoepithelium in the raljbit. Nature. London, 173, 450-451.

Cross, B. A. 1955a. The hypothalamus and the mechanism of sympathetico-adrenal inhibition of milk ejection. J. Endocrinol., 12, 15-28.

Cross, B. A. 1955b. Neurohormonal mechanisms in emotional inhibition of milk ejection. J. Endocrinol., 12, 29-37.

Cross, B. A. 1958. On the mechanism of labour in the rabbit. J. Endocrinol., 16, 261-276.

Cross, B. A. 1960. Neural control of lactation. In Milk: The Mammary Gland and its Secretion, S. K. Kon and A. T. Cowie, Eds., Ch. 6. New York: Academic Press, Inc.

Cross, B. A., Goodwin, R. F. W., and Silver, I. A. 1958. A histological and functional study of the mammary gland in normal and agalactic sows. J. Endocrinol., 17, 63-74.

Cross, B. A., and H.\rris, G. W. 1950. Milk ejection following electrical stimulation of the pituitary stalk in rabbits. Nature, London, 166, 994-995.

Cross, B. A., and H.\rris, G. W. 1952. The role of the neurohypophysis in the milk-ejection reflex. J. Endocrinol.," 8, 148-161.

Cross, B. A., and Silver, I. A. 1956. Milk ejection and mammarv engorgement. Proc. Rov. Soc. Med., 49, 978-979.

Dabelow, a. 1957. Die Milchdriise. In Jlandbuch der mikroskopischen Anatomie des Mcnschen, W. von MoUendorf and W. Bargmann, Eds., Vol. 3, Part 3, pp. 277-485. Berlin, Gottingen, Heidelberg: Julius Springer.

Damm, H. C, and Turner, C. W. 1958. Evidence for existence of mammogenic hormone. Proc. Soc. Exper. Biol. & Med., 99, 471-473.

Daniel, P. M., and Prichard, M. M. L. 1956. Anterior pituitary necrosis : infarction of the pars distalis produced experimentally in the rat. Quart. J. Exper. Physiol., 41, 215-219.

D.\NiEL, P. M., AND Prich.ard, M. M. L. 1957. The vas(;ular arrangements of the yiituitary gland of the sheep. Quart. J. Exiter. Phvsiol., 42, 237-248.

Daniel, P. M., and Prichard, M. M. L. 1958. The effects of pituitary stalk section in the goat. Am. J. Path., 34, 433-469.

MAMMARY GLAND AND LACTATION

635

DE JoNGH, S. E. 1932. Corpus Liiteum unci Laktation. Acta brev. neerl. Physiol., 2, 199-12L

Den..\mur, R., a'sd Martinet, J. 1953. Sensibilite de la glande mammaiie dc la chevre aux hormones posthypophysaires. Compt. rend. Soc. biol., 147, 1217-1220.

Denamur, R., and Martinet, J. 1959a. Les stimulus nerveux mammaires sont-ils necessaires a I'entretien de la lactation chez la chevre? Compt. rend. Acad. Sc, 248, 743-746.

Denamur, R., and Martinet, J. 1959b. Entretien de la lactation chez la chevre apres section de la moelle-epiniere et sympathectomie lombaire. Compt. rend. Acad. Sc, 248, 860-862.

Denamur, R., and M.\rtinet, J. 1960. Physiological mechanisms concerned in the maintenance of lactation in the goat and sheep. Nature, London, 185, 252-253.

Desclin, L. 1950. A propos du mecanisme d'action des oestrogenes sur le lobe anterieur de I'hypophvse chez le rat. Ann. endocrinoL, 11,656-659.

Desclin, L. 1952. Recherches sur le determinisme des phenomenes de secretion dans la glande mammaire du rat. Ann. endocrinoL, 13, 120-136.

Desclin, L. 1956. Hypothalamus et liberation d'hormone luteotrophicjue. Experiences de greffe hypoph.ysaire chez le rat hypophysectomise: action luteotrophique de I'ocytocine. Ann. endocrinoL, 17, 586-595.

DoNKER, J. D. 1958. Lactation studies. I. Effects upon milk ejection in the bovine of various injection treatments using oxvtocin and relaxin. J. Dairy Sc, 41, 537-544.

Donovan, B. T., and van der Werff ten Bosch, J. J. 1957. The hypothalamus and lactation in the rabbit. J. Physiol., 137, 410-420.

Eayrs, J. T., .AND Baddeley, R. M. 1956. Neural pathways in lactation. J. Anat., 90, 161-171.

Elias, J. J. 1957. Cultivation of adult mouse mammary gland in hormone-enriched synthetic medium. Science, 126, 842-844.

Ely, F., and Petersen, W. E. 1941. Factors involved in the ejection of milk. J. Diarv Sc, 24, 211-223.

Everett, J. W. 1954. Luteotrophic function of autografts of the rat hypophysis. Endocrinology, 54, 685-690.

Everett, J. W. 1956. Functional corpora lutea maintained for months by autografts of rat hypophyses. Endocrinology, 58, 786-796.

Fauvet, E. 1941a. Experimentelle Untersuchungen iiber den Laktation.svorgang. Zentralbl. Gyniik., 65, 580-584.

Fauvet, E. 1941b. Vergleichende Untersuchungen iiber die Entwickling und Funktion der Milchdriisen. VIII. Experiment elle Untersuclumgen iiber die Wnkung \ on FoUikelhormonzufuhr auf die Milchdriisen siiugender Ratten. Arcli. Gynak., 171, 342-366.

Flux, D. S. 1954a. Growth of the mammary duct system in intact and ovariectomized mice of tiie CHI strain. J. EndocrinoL, 11, 223-237.

Flux. D. S. 1954b. The effect of adrenal steroids on the growtli of the mammary glands, uteri.

thymus and adrenal glands of intact, ovariectomized and oestrone-treated ovariectomized mice. J. EndocrinoL, 11, 238-254.

Flux, D. S. 1955. The value of some steroids in replacement therapy in adrenalectomized and adrenalectomized - ovariectomized lactating rats. J. EndocrinoL, 12, 57-68.

Flux, D. S. 1958. Mammary gland growth in male mice of the CHI strain after hypophysectomy and castration. J. EndocrinoL, 17, 300306.

Flux, D. S., Folley, S. J., and Rowland, S. J. 1954. The effect of adrenocorticotrophic hormone on the yield and composition of the milk of the cow. J. EndocrinoL, 10, 333-339.

Flux, D. S., and Munford, R. E. 1957. The effect of adrenocorticotrophin on the mammaiy glands of intact mature female mice of the CHI strain. J. EndocrinoL, 14, 343-347.

Folley, S. J. 1936. The effect of oestrogenic hormones on lactation and on the phosphatase of the blood and milk of the lactating cow. Biochem.J., 30, 2262-2272.

Folley, S. J. 1940. Lactation. Biol. Rev., 15, 421-458.

Folley, S. J. 1947. The nervous svsteni and lactation. Brit. M. Bull., 5, 142-148.

Folley, S. J. 1952a. Lactation. In MarshaU's Physiology of Reproduction, 3rd ed., A. S. Parkes, Ed., Vol. II, Ch. 20. London: Longmans, Green & Company.

Folley, S. J. 1952b. Aspects of pituitary-mammary gland relationships. Recent Progr. Hormone Res., 7, 107-137.

Folley, S. J. 1954. Recherches Recentes siir la Physiologic et la Biochimie de la Secretion Lactee. Liege: Desoer; Paris: Masson et Cie.

Folley, S. J. 1955. Effects of somatotrophin and other pituitary hormones on the lactating mammary gland. In The Hypophyseal Growth Hormone, Nature and Actions, R. W. Smith, O. H. Gaebler and C. N. H. Long, Eds., pp. 473486. New York: Blakiston Company.

Folley, S. J. 1956. The Physiology and Biochemistry of Lactation. Edinburgh and London: Ohver & Boyd, Ltd.

Folley, S. J., and Co\vie, A. T. 1944. Adrenalectomy and replacement therapv in lactating rats. Yale J. Biol. & Med., 17,^67-74.

Folley, S. J., and French, T. H. 1949. The intermediary metabolism of the mammary gland.

II. Respiration and acid production of mammary tissue during pregnancy, lactation and involution in the rat. Biochem. J., 45, 270-275.

Folley, S. J., and French, T. H. 1950. Intermediary metabolism of the mammary gland.

III. Acetate metabolism of lactating mammary gland .-slices with special reference to milk fat synthesis. Biochem. J., 46, 465-473.

Folley, S. J.. .\nd Greenbaum, A. L. 1947. Changes in the argina.se and alkaline phosphatase contents of the mammary glands and liver of the rat during pregnancy, lactation and mammary involution. Biochem. J., 41, 261-269.

Folley. S. J., and Greenbaum, A. L. 1948. Effect of adrenalectomy on the argiuase levels of the liver, mammary gland and kidney in lactating rats studied bv the paired-feeding technique. Biochem. J., 43, 581-584.

FOLLEY, S. J., GUTHKELCH, A. N., AND ZUCKERMAN,

S. 1939. The mammary gland of the rhesus monkey under normal and experimental conditions. Proc. Roy. Soc, London, ser. B, 126, 469-491.

FoLLEY, S. J., AND KoN, S. K. 1938. The effect of sex hormones on lactation in the rat. Proc. Roy. Soc, London, ser. B., 124, 476-492.

FoLLEY, S. J., AND McNaught, M. L. 1958. Hormonal effects in vitro on lipogenesis in mammary tissue. Brit. M. Bull., 14, 207-211.

FoLLEY, S. J., AND McNaught, M. L. 1960. Biosynthesis of milk fat. In Milk: The Mammary Gland and its Secretion, S. K. Kon and A. T. Cowie, Eds., Ch. 12. New York: Academic Press, Inc.

FoLLEY, S. J., AND M.'VLPRESS, F. H. 1948a. Hormonal control of mammary growth. In The Hormones, G. Pincus and K. V. Thimann, Eds., Ch. XV. New York: Academic Press, Inc.

FoLLEY, S. J., AND Malpress, F. H. 1948b. Hormonal control of lactation. In The Hormones, G. Pincus and K. V. Thimann, Eds., Ch. XVI. New York: Academic Press, Inc.

FoLLEY, S. J., AND YouNG, F. G. 1941. Prolactm as a specific lactogenic hormone. Lancet, 240, 380-381.

G.-viNES, W. L. 1915. A contribution to the physiology of lactation. Am. J. Physiol., 38, 285-312.

Gaunt, R., Eversole, W. J., .-vnd Kendall, E. C. 1942. Influence of some steroid hormones on lactation in adrenalectomized rats. Endocrinology, 31, 84-88.

Glock, G. E., and McLe.\n, P. 1954. Levels of enzymes of the direct oxidative pathways of carbohydrate metabolism in mammalian tissues and tumours. Biochem. J., 56, 171-175.

Glock, G. E., and McLean, P. 1958. Pathways of glucose vitilization in mammary tissue. Proc. Roy. Soc, London, ser. B, 149, 354-362.

Glock, G. E., McLean, P., .-^nd Whitehead, J. Iv. 1956. Pathways of glucose utilization in the mammary gland of the rat. Biochim. et biophys. acta. 19, 546-547.

Gomez, E. T. 1939. The relation of the posterior hypophysis in the maintenance of lactation in hypophysectomized rats. J. Dairy Sc, 22, 488.

Gomez, E. f. 1940. Effect of posthypophyseal extract on lactation in hypophysectomized postgravid rats. J. Dairy Sc, 23, 537-538.

[Grachev, I. I.] Fpa^eB, H. H. 1953, ycjioBHbiii {)e(J)JieKc c MOJioHHOH JKe.ne3bi. Vestnik Leningrad. Universitet. No. 4., 87-99.

[Grachev. I. I.] FpaneB, H. H. 1958. Bbicmaa

HepBHaH ACHTejIbHOCTb H CeKpeUHH MO.JIOKa.

Vestnik Leningrad. Universitet. No. 21, 110 124. Graumann, \A'. 1952. Mikroskopische Anatomic

der miinnlichen Brustdriise. I. Kindheit und

Pubertiit. Ztschr. mikro-anat. Forsch., 58, 358 379. Grau.mann, W. 1953. Mikroskopische Anatomic

der miinnlichen Brustdriise. II. Mannesalter und Senium. Ztschr. mikro-anat. Forsch., 59, 523-557.

Greenbaum, a. L., and Greenwood, F. C. 1954. Some enzymic changes in the mammarj- gland of rats dvu'ing pregnancy, lactation and mammary involution. Biochem. J., 56, 625-631.

GREENB.-iUM, A. L., .-VND Sl.\ter, T. F. 1957a. Studies on the particulate components of rat mammary gland. II. Changes in the levels of the nucleic acids of the mammary glands of rats during pregnancy, lactation and mammaiy involution. Biochem. J., 66, 155-161.

Greenbaum, A. L., and Slater, T. F. 1957b. Studies on the particulate components of rat mammary gland. III. The relationship between enzyme activity and particle counts in mammary gland suspensions. Biochem. J., 66, 161166.

Gregoire, C. 1947. Factors involved in maintaining involution of the thymus during suckling. J. Endocrinol., 5, 68-87.

Griffith, D. R., and Turner, C. W. 1957. Desoxyribonucleic acid (DNA) content of mammary gland during pregnancy and lactation. Proc. Soc Exper. Biol. & Med., 95, 347-348.

Grosvenor, C. E., and Turner, C. W. 1957a. Evidence for adrenergic and cholinergic components in milk let-down reflex in lactating rats. Proc. Soc. Exper. Biol. & Med., 95, 719-722.

Grosvenor, C. E., and Turner, C. W. 1957b. Release and restoration of pituitary lactogen in response to nursing stimuli in lactating rats. Proc. Soc. Exper. Biol. & Med., 96, 723-725.

Grosvenor, C. E., and Turner, C. W. 1958a. Effects of oxytocin and blocking agents upon pituitarv lactogen discharge in lactating rats. Proc Soc. Exper. Biol. & Med., 97, 463-465.

Grosvenor, C. E., and Turner, C. W. 1958b. Effect of lactation upon thyroid secretion rate in the rat. Proc. Soc. Exper. Biol. & Med., 99, 517-519.

Grosvenor, C. E., and Turner, C. W. 1958c Pituitary lactogenic hormone concent i at ion and milk secretion in lactating rats. Endocrinologv, 63, 535-539.

Hadfield, G. 1957. The nature and origin of the mammotrophic agent present in human female urine. Lancet, 1, 1058-1061.

H.\dfield, G., and Young, S. 1958. The controlling influence of the pituitary on the growth of the normal breast. Brit. J. Surg., 46, 265-273.

Hammond, J., and Marsh.^ll, F. H. A. 1925. A'eproduction in the Rabbit. Edinlaurgh, London: Oliver & Boyd, Ltd.

H.ARDY. M. H. 1950. The development in vitro of the mammary glands of the mouse. J. Anat., 84, 388-393.

Harris, G. W. 1947. The innervation and actions of the neurohypophj'sis, an investigation using the method of remote-control stimulation. Phil. Tr. Roy. Soc, London, ser. B., 232, 385-441.

Harris, G. "w. 1955. Neural Control of the Pituitary Gland. London: Edward Arnold it Company.

Harris, G. W. 1958. The central nervous system, neurohypophysis and milk ejection. Proc. Roy. Soc, London, ser. B, 149, 336-353.

H.ARRis, G. W., AND JacobsohiX, D. 1952. Functional grafts of the anterior pituitary gland. Proc. Rov. Soc, London, ser. B, 139, 263-276.

Harris, G. W., and Pickles. V. R. 1953. Reflex stimulation of the neurohypophysis (posterior pituitary gland) and the nature of the posterior pituitary hormones. Nature, London, 172, 1049.

Hawker, R. W. 1958. Oxytocin in lactating and nonlactating women. J. Clin. Endocrinol., 18, 54 60.

Hawker, R. W., and Roberts, V. S. 1957. Oxytocin in lactating cows and goats. Brit. Vet. J., 113, 459-464.

Hawker, R. W., and Robertson, P. A. 1957. Oxytocin in human female blood. Endocrinology, 60, 652-657.

H.\ys, R. L., and Vandemark, N. L. 1953. Effect of stimulation of the reproductive organs of the cow on the release of an oxytocin-like substance. Endocrinology, 52, 634-637.

Heineman, P. G. 1919. 'Milk. Philadelphia: W. B. Saunders Company.

Hohn, E. O. 1957. The effect of oestrone on the mammary gland of adrenalectomized guineapigs. J. Endocrinol., 16, 227-230.

HoLLMANN, K. H. 1959. L'ultrastructure de la glande mammaire normale de la souris en lactation. Etude au microscope electronique. J. Ultrastructure Res., 2, 423-443.

HoussAY, B. A. 1935. Action de I'hypophysectomie sur la grossesse et la secretion lactce, chez la chienne. Compt. rend. Soc. biol., 120, 496497.

Howe, A., Richardson, K. C, and Birbeck, M. S. C. 1956. Quantitative observations on mitochondria from sections of guinea-pig mammary gland. Exper. Cell Res., 10, 194-213.

HuGGiNS, C, AND M.MNZER, K. 1957. Homional influences on mammary tumors of the rat. H. Retardation of growth of a transplanted fibroadenoma in intact female rats by steroids in the androstane series. J. Exper. Med., 105, 485-500.

HuGGiNS, C, AND Mainzer, K. 1958. In Endocrine Aspects of Breast Cancer, A. R. Currie, Ed., pp. 297-304. Edinburgh, London: E. & S. Livingstone, Ltd.

Hughes, E. S. R. 1950. The development of the mammary gland. Ann. Rov. Coll. Surgeons, England," 6, 99-119.

HuTTON, J. B. 1957. The effect of growth hormone on the yield and composition of cows' milk. J. Endocrmol., 16, 115-125.

HuTTox, J. B. 1958. Oestrogen function in established lactation in the cow. J. Endocrinol.. 17, 121-133.

Huxley, J. S.. and Teissier, G. 1936. Terminology of relative growth. Nature, London, 137, 780-781.

Ingle, D. J. 1954. Pemissibility of hormone action: a review. Acta endocrinol., 17, 172-186.

Ingleby, H., Moore, L., and Gershon-Cohen, J.

1957. Gestational breast changes. Obst. & Gynec, 10, 149-157.

Isbister, C. 1954. Clinical study of the draught reflex in human lactation. Arch. Dis. Childhood, 29, 66-72.

J.\cobsohn, D. 1958. Manunary gland growth ni relation to hormones witli metabf)lic actions. Proc. Roy. Soc, London, ser. B, 149, 325-329.

Jacobsohn, D. 1959. Thyroxin and the reaction of the mammary glands to ovarian steroids in hypophysectomized rats. J. Physiol, 148, lOPUP.

Jeffers, K. R. 1935. Cytology of the mammary gland of the albino rat. I. Pregnancy, lactation, and involution. Am. J. Anat., 56, 257-277.

Jewell, P. A. 1956. The vascular link between posterior and anterior lobes of the pituitary. J. Endocrinol., 14, xxiv.

Johnson, R. M., and Meites, J. 1955. Effects of cortisone, hydrocortisone, and ACTH on mammary growth and pituitary prolactin content of rats. Proc. Soc. Exper. Biol. & Med., 89, 455458.

Johnson, R. M., .\nd Meites, J. 1958. Effects of cortisone acetate on milk production and mammary involution in parturient rats. Endocrinology, 63, 290-294.

Kalliala, H., and K.'iRVONEN, M. J. 1951. Antidiuresis during suckling in lactating women. Ann. med. exper. et biol. Fenniae, 29, 234-241.

Kalliala, H., Karvonen, M. J., and Leppanen, V. 1952. Release of antidiuretic hormone during nursing in dog. Ann. med. exper. et biol. Fenniae, 30, 96-107.

Kirkham, W. R., and Turner, C. W. 1953. Nucleic acids of the mammary glands of rats. Proc. Soc. Exper. Biol. & Med.', 83, 123-126.

Kolbe, p. 1727. Naaukeurige en uitvoerige beschryving van de Kaap de Goede Hoop. Amsterdam. Quoted by Martiny, 1871.

Krzywanek, F. W., and Bruggemann, H. 1930. Modellversuche zur Physiologie des Saugaktes. Berl. tier. Wchnschr., 46, 710-713.

Kuramitsu, C, and Loeb, L. 1921. The effect of suckling and castration on the lactating mammary gland in rat and guinea pig. Am. J. Physiol., 61, 40-59.

L.^NDSMEER, J. M. F. 1951. Vessels of the rat's hypophysis. Acta anat., 12, 83-109.

Lauryssens, M., Peeters, G., and Doxck, A. 1956. Variations dans la teneur du coenzyme A dans la glande mammaire chez le rat. Ann. endocrinol., 17, 543-546.

Leech, F. B., and Bailey, G. L. 1953. The effect on the health of lactating cows of treatment with galactopoietic doses of thyroxine or iodinated casein. J. Agric. Sc. 43, 236-251.

Leslie, I. 1955. The nucleic acid content of tissues and cells. In The Nucleic Acids: Chemistry and Biology, E. Chargaff and J. N. Davidson, Eds., Ch. 16, pp. 1-50. New York : Academic Press, Inc.

Lewin, I. 1957. Xanthine oxidase activitv in normal and abnormal growth. Proc. Roy. Soc. Med., 50, 563-570.

LiNZELL, J. L. 1952. The silver staining of myoepithelial cells, particularly in the mammary gland, and their relation to the ejection of milk. J. Anat., 86, 49-57.

LiNZELL, J. L. 1955. Some observations on the contractile tissue of the mammary glands. J. Physiol., 130, 257-267.

LUNDAHL, W. S., MeITES, J., AND WOLTERINK, L. F.

1950. A technique for whole mount autoradiographs of rabbit mannnary glands. Science, 112,599-600.

Lyons, W. R. 1942. The direct mammotrophic action of lactogenic hormone. Proc. Soc. Exper. Biol. & Med., 51, 308-311.

Lyons, W. R. 1943. Lobulo-alveolar mammary growth induced in hypophyscctomized rats by injections of ovarian and h^'pophyseal hormones. In Essays in Biology in Honor of Herbert M. Evans, pp. 315-329. Berkeley: University of California Press.

Lyons, W. R. 1958. Hormonal synergism in mammarv growth. Proc. Rov. Soc, London, ser. B, 149, 303-325.

Lyons, W. R., Li. C. H., Cole, R. D., and Johnson, R. E. 1953. Some of the hormones required by the mammary gland in its development and functions. J. Clin. Endocrinol., 13, 836-837.

Lyons, W. R., Li, C. H., and Johnson, R. E. 1952. The enhancing effect of somatotropin on the mammary growth induced in rats with estrin, progestin and mammotropin. J. Clin. Endocrinol., 12, 937.

Lynos, W. R., Li, C. H., and Johnson, R. E. 1958. The hormonal control of mammary growth and lactation. Recent Progr. in Hormone Res. 14, 219-254.

Marshall, W. K., and Leiber.max, D. M. 1956. A rare complication of chlorpromazine treatment. Lancet, 270, 162.

Martiny, B. 1871. Die Milch, ihr Wesen und ihre Verwertlnmg, Vol. I. Danzig: Kasemann.

Martyugin, D. D.] MapTiorHH, J\. J],. 1944 IlayneHHe aKTa cocannH y Te.nHT. Trud Timiryazeva sel.-khoz. Akad., No. 31, 149-184

Masson, G. M. 1948. Effects of estradiol and progesterone on lactation. Anat. Rec, 102, 513-520.

M.\YER, G., AND Klein, M. 1948. Physiologie de la lactation. I. Les facteurs du developpement morphologi(iue du parenchyme mammaire. Ann. Nutrit. Aliment., Paris, 2, 113-157.

Mayer, G., .\nd Klein, M. 1949. Physiologie de la lactation. II. Les facteurs de I'activite fonctionelle du parenchyme mammaire. Ann. Nutrit. Aliment., Paris, 3, 667-748.

McLean, P. 1958a. Carbohydrate metabolism of mammary tissue. I. Pathways of glucose catabolism in the mammary gland. Biochim. et biophys. acta, 30, 303-315." McLean. P. 1958b. Carbohydrate metabolism of mammary tissue. II. Levels of oxidized and ri'duced diphosphopyridine nucleotide

and triphosphopyridine nucleotide in the rat mammarv gland. Biochim. et biophys. acta, 30, 316-324. McLean, P. 1959. Effect of insulin and phenazine methosulphate on pathways of glucose catabolism in the mammary gland of the rat. Nature, London, 183, 182-183. McNaught, M. L. 1956. National Institute for

Research in Dairying, Report 1956, p. 59. McNaught, M. L. 1957. National Institute for

Research in Dairying, Report 1957, p. 58. McNaught, M. L. 1958. The use of the mammary-slice bioassay method for the estimation of insulin in glucagon. J. Endocrinol., 17, xvixvii. Meites, J. 1954. Recent studies on the mechanisms controlling the initiation of lactation. Rev. canad. Biol., 13, 359-370. Meites, J. 1957a. Induction of lactation in rabbits with reserpine. Proc. Soc. Exper. Biol. & Med., 96, 728-730. Meites, J. 1957b. Effects of growth hormone on lactation and body growth of parturient rats. Proc. Soc. Exper. Biol. & Med., 96, 730-732. Meites, J. 1959. Induction and maintenance of mammary growth and lactation in rats with acet.ylcholine or epinephrine. Proc. Soc. Exper. Biol. & Med., 100, 750-754. Meites, J. 1960. Farm animals: hormonal induction of lactation and galactopoiesis. In Milk: the Mammary Gland and its Secretion, S. K. Kon and A. T. Cowie, Eds., Ch. 8. New York: Academic Press, Inc. Meites, J., and Sgouris, J. T. 1953. Can the ovarian hormones inhibit the mammary response to prolactin? Endocrinologv, 53, 1723. Meites, J., and Sgouris, J. T. 1954. Effects of altering the balance between prolactin and ovarian hormones on initiation of lactation in rabbits. Endocrinology, 55, 530-534. Meites, J., and Turner, C. W. 1942a. Studies concerning the mechanism controlling the initiation of lactation at parturition. I. Can estrogen suppress the lactogenic hormone of the pituitary? Endocrinology, 30, 711-718. Meites, J., .^nd Turner, C. W. 1942b. Studies concerning the mechanism controlling the initiation of lactation at parturition. III. Can estrogen account for the precipitous increase in the lactogen content of the pituitary following parturition? Endocrinology, 30, 726-733. Meites, J., and Turner, C. W. 1947. The induction of lactation during pregnancy in rabbits and the siH>cificilv of the lactogenic hormone. Am. J. Physiol., 150, 394-399. Meites, J., and Turner, C. W. 1948. Studies concerning the induction and maintenance of lactation. I. Tlu> mechanism controlling the initiation of ladalKni .it parturition. Res. Bull. Missouri Agric. Kxinr. S;:i., No. 415. Mercier-Parot, L. 1955. Troubles du developpement postnatal du rat apres administration de cortisone a la mere gestante ou allailante. Compt. rend. Acad. So., 240, 2259.

MAMMARY GLAND AND LACTATION

039

MixNER, J. P, AND TuRx\ER, C. W. 1943. The mammogenic hormones of the anterior pituitary. II. The lobule-alveolar growth factor. Res. Bull. Mi.ssouri Agric. Exper. Sta., No. 378.

MizuNO, H., ANV Chikamune, T. 1958. The effects of forced weaning on the respiratory activity of mouse mammary gland slice. Endocrinol. Japon., 5, 265-272.

Mizuxo, H., IiDA, K., AND Naito, M. 1955. The role of prolactin in the mammary alveolus formation. Endocrinol. Japon., 2, 163-169.

MizuNO. H., AND Naito, M. 1956. The effect of of locally administered prolactin on the nucleic acid content of the mammary gland in the rabbit. Endocrinol. Japon., 3, 227-230.

MooRE, R. 0. AND Nelson, W. L. 1952. Some oxidative enzyme systems of mammary gland tissue. Arch. Biochem., 36, 178-194.

MosiMANN, W. 1949. Zur Anatomic der Rindermilchdiiise und liber die Morphologic ihrer sezernierenden Teile. Acta anat., 8, 347-378.

MuxFORD. R. E. 1957. The effect of Cortisol acetate on oest rone-induced mammary gland growth in immature ovariectomized albino mice. J. Endocrinol., 16, 72-79.

MuNsox, P. L. 1955. Studies on the role of the parathyroids in calciimi and ijhosphorus metabolism. Ann. New York Acad. Sc, 60, 776795.

Nandi, S. 1958a. Role of somatotropin in mammogenesis and lactogenesis in C3H/HeCRGL mice. Science, 128, 772-774.

Nandi, S. 1958b. Endocrine control of mammarygland de\elopment and function in the C3H/ HeCrgl Mouse. J. Nat. Cancer Inst., 21, 10391063.

Nelson, W. O. 1937. Studies on the physiologj^ of lactation. VI. The endocrine influences concerned in the development and function of the mammary gland in the guinea pig. Am. J. Anat., 60, 341-365.

OsHiMA, M., AND Goto, T. 1955. A study of involution of the mammary gland after weaning in the albino rat. Bull. Nat. Inst. Agric. Sc, ser. G, No. 11, 81-87.

Ota, K., and Yokoyama, A. 1958. Effect of oxj^tocin administration on respiration of lactating mammary gland tissue in rats. Nature, London, 182, 1509-1510.

Patersox, J. 1844. On lactation, after-pains and the treatment of females after deli\ery. Lancet, 1, 246-247.

Peeters, G., axd Coussexs, R. 1950. The influence of the milking act on the diuresis of the lactating cow. Arch, internat. pharmacodyn., 84, 209-220.

Petersen, W. E. 1944. Lactation. Phvsiol. Rev., 24, 340-371.

Petersen, W. E. 1948. The hormonal control of lactation. Recent Progr. Hormone Res., 2, 133-158.

Pfaltz, C. R. 1949. Das embryonale und postnatale Verhalten der mannlichen Brustdriise beim Menschen. II. Das Mammarorgan im

Kindes-, Jihiglings-, Mannes- und Greisenalter. Acta anat., 8, 293-328.

Plagge, D. W. 1942. Normal development and experimental treatment of the opossum mammary gland primordium. Proc. Soc. Exper. Biol. & Med., 51, 219-220.

Pl.\tt, R., and Se.\rs, H. T. N. 1956. Reserpine in severe hypertension. Lancet, 270, 401-403.

[PopovicH, D.] nonoBHH, JX. 1958. Po.ib rnnoTajiajMyca b pery.nflmm MOTopnoft (J)yHKi;ini mojiomhoh >Kene3bi. Dokl. Akad. Nauk S.S.S.R., 121, 186-189.

Powell, J. E. 1949. Herodotus (IV, 2) Translation, Vol. 1. London: Oxford University Press.

Racadot, J. 1957. Neurosecretion et activite thyroidienne chez la chatte au cours de la gestation et de Tallaitement. Ann. endocrinol., 18, 628-634.

R.\WLINS0N, H. E., axd Pierce. G. B. 1950. Iron content as a quantitative measurement of the effect of previous pregnancies on the mammary gland of mice. Endocrinology, 46, 426-433.

Ray, E. W., Averill, S. C, Lyons, W. R., and Johnson, R. E. 1955. Rat placental hormonal activities corresponding to those of pituitary mammotropin. Eudocrinologv, 56, 359-373.

R.aynaud, a. 1947a. Effet des injections d'hormones sexuelles a la souris gravide, sur le developpement des ebauches de la glande mammaire des embryons. I. Action des substances androgenes. Ann. endocrinol., 8, 248253.

R.\YNAUD, A. 1947b. Effet des injections d'hormones sexuelles a la souris gravide, sur le developpement des ebauches de la glande mammaire des embryons. II. Action de fortes doses de substances oestrogenes. Ann. endocrinol., 8, 318-329.

R.\YNAUD, A. 1947c. Observations sur le developpement normal des ebauches de la glande mammaire des foetus males et femelles de souris. Ann. endocrinol., 8, 349-359.

Raynaud, A. 1947d. x4.ction des hormones sexuelles sur le developpement embryonnaire de la glande mammaire de la souris. Compt. rend. Soc. biol., 225, 150-152.

Raynaud, A. 1949a. Nouvelles observations sur I'appareil mammaire des foetus de souris provenant de meres ayant regu des injections de testosterone pendant la gestation. Ann. endocrinol., 10, 54-62.

Raynaud, A. 1949b. Morphologic comparee des ebauches mammaires de quelques foetus de rongeurs. Bull. Soc. zool. France, 74, 253-277.

Raynaud, A. 1952. Inhibition du developpement des ebauches mammaires de I'embryon de souris, sous I'cffet d'unc hormone oestrogene injectee directement a I'embvron. Compt. rend. Soc. bioL, 146, 544-549.

Raynaud, A. 1960. Morphogenesis of the mammary gland. In Milk: the Mammary Gland and its Secretion, S. K. Kon and A. T. Cowie, Eds., Ch. 1. New York: Academic Press, Inc.

640

PHYSIOLOGY OF GONADS

Raynald, a., and Frilley, M. 1947. Etat de de\"eloppement des ebauches mammaires et (lu cordon vaginal chez les foetus males et femelles de souris, dont les ebauches des glandes genitales ont ete detruites par une irradiation au moyen des rayons X, a I'age de treize jours. Compt. rend. Acad. Sc, 225, 13801382.

Raynaud, A., and Frilley, M. 1949. Le developpement embryonnaire de la glande mammaire de la souris apres destruction, au moyen des rayons X. des glandes genitales de 1'embryon. Bull. Soc. zool. France, 74, 156159.

R.AYNAUD. A., AND RAYNAUD, J. 1956. La production experimentale de malformations mammaires chez les foetus de souris, par Taction des hormones sexuelles. Ann. Inst. Pasteur, 90, 39-219.

R.AYN.\UD, A., .-^ND Raynaud, J. 1957. Sur les modifications precoces apportees par I'hormone oestrogene, dans la structure des ebauches mammaires des foetus de souris. Bull. Soc. zool. France. 82, 204-222.

Read, M. S., .\nd Moore, R. 0. 1958. The effect of prolactin on the metabolism of lactating guinea pig mammary tissue. Arch. Biochem. Biophys., 77, 1-12.

Reece, R. p. 1950. Effectiveness of estrogens in stimulating duct growth of the mammary glands of rats. Proc. Soc. Exper. Biol. & Med., 73, 284-285.

Reineke, E. p., Meites, J., Cairy, C. F., and Huffman, C. F. 1952. Hormonal induction of lactation in sterile cows. In Proceedings Annual Meeting American Veterinary Medical Association, 1952, pp. 325-328. Atlantic City: American Veterinary Medical Association.

RicH.^RDSON, K. C. 1949. Contractile tissues in the mammary gland, with special reference to myoepithelium in the goat. Proc. Roy. Soc. London, ser. B, 136, 30-45.

RiCH.ARDSON, K. C. 1953. Measurements of the total area of secretory epithelium in the lactating mammary gland of I he goal. J. Endocrinol., 9, 170-184.

RiCHTER. J. 1936. Der p]influss der Kastration auf die Milch- und Fleischleistung der Kiihe. Berl. tier. Wchnschr., 277-280, 293-298.

RiNOLER, I., Becker, N., and Nelson, W. L. 1954. Coenzyme A and diphosphopyridine luicleotide in guinea pig mammary tissue. .\i(li. Biochem. & Biophys., 52, 348-352.

RoHKRTsoN, P. A., AND Hawkkr. P. ^V. 1957. A siTond oxvlocin in the liNpot linlamus. Nature, London, 180, 343-344.

Salter, J., and Best, C. H. 1953. Insulin as a growth hormone. Brit. M. J., 2, 353-359.

Sawyer, C. H. 1957. Induction of lactation in the rabbit with rcserpine. Anat. Rec, 127, 362.

Sayers, G., Redgate, E. S., and Royce, P. C. 1958. Hypothalamus, adenohypophysis, and adrenal cortex. Ann. Rev. Physiol., 20", 243-274.

Scharrer, E.. and Sciiakrer, B. 1954. Hormones

])roduced bv neuroseci'etorv cells. Recent Progr. Hormone Res., 10, 183-232.

Schafer, E. a. 1898. Mechani.sm of the secretion of milk. In Text Book of Physiology, E. A. Schafer, Ed., Vol. I, pp. 662-668. Edinburgh and London: Pentland.

Selye, H. 1934. On the nervous control of lactation. Am. J. Phy.siol., 107, 535-538.

Selye, H. 1954. The effect of Cortisol upon the mammary glands. Acta endocrinol., 17, 394401.

Selye, H., Collip, J. B., and Thomson, D. L. 1934. Ner\'OUS and hormonal factors in lactation. Endocrinology, 18, 237-248.

Shaw, J. C. 1955. See Discussion, p. 486-492 in Folley, 1955.

vShaw, J. C, Chung, A. C, and Bunding, I. 1955. The effect of pituitary growth hormone and adrenocorticotropic hormone on established lactation. Endocrinology, 56, 327-334.

Shimizu, H. 1957. Biochemical studies on the mammary gland functions. I. The determination of nucleic acids in the mammary glands of rats. Tohoku J. Agric. Res., 7, 339-345.

Short, R. H. D. 1950. Alveolar epithelium in relation to growth of the lung. Phil. Tr. Roy. Soc. London, ser. B, 235, 35-86.

Silver, I. A. 1954. Myoepithelial cells in the mammarv and parotid glands. J. Phvsiol., 125, 8P-9P.

Silver, I. A. 1956. Vascular changes in the mammarv gland during engorgement with milk. J. PiiysioL, 133, 65P-66P.

Silver, M. 1953a. A ciuantitative analysis of the role of oestrogen in mammary development in the rat. J. Endocrinol., 10, 17-34.

Silver, M. 1953b. The onset of allometric mammary growth in the female hooded Norway rat. J. Endocrinol., 10, 35-45.

Smith, P. E. 1932. The nonessentiality of the posterior hvpophvsis in parturition. Am. J. Physiol., 99, 345-348.

Smith. T. C, and Richterich, B. 1958. Synergism of estrogen and progesterone on mammary gland growth in guinea pigs. Endocrinology. 63, 89-98.

Smith. V. R,, and Petersen, W. E. 1945. Negati\e ]iressure and nursing by calve.-^. J. Dairy Sc, 28, 431-433.

Smith. W. T. 1844. Lactation and after-pains, considered in relation to reflex motor action. Lancet, 1, 127-128.

SoRANUS. — Soranns' Gi/iucoldg//. transl. by 0. Timkin. 1956. Baltimon-: Johns Hopkins Press.

Speert, H. 1948. Tlio normal and experimenlal development of the mammary gland of llie rhesus monkey, with some pathological correlations. Contr. Embrvol., Carnegie Inst. Washington. 32, 9-65.

SthicivER, p., a.vd Grueter, F. 1928. Action du lobe anterieur de I'hypophyse sur la montee laiteuse. Comp. rend. Soc. biol.. 99, 1978-1980.

Stricker. p.. and Grueter, F. 1929. Recherches

MAMMARY GLAND AND LACTATION

641

experimentales sur les fonctions du lobe anterieur de I'hypophyse: influence des extraits du lobe anterieur sur I'appareil genital de la lapine et sur la montee laiteuse. Presse med., 37, 1268-127L

SuLMAN, F. G., AND WiNNiK, H. Z. 1956. Hormonal effects of chlorpromazine. Lancet, 270, 161-162.

Sykes, J. F., AND Wrenn, T. R. 1950. Hormonal development of the mammary gland of dairy heifers. J. Dairy Sc, 33, 194-204.

Sykes, J. F., and Wrenn, T. R. 1951. Hormonal development of mammary tissue in dairy heifers. J. Dairy Sc, 34, 1174-1179.

TiNDAL, J. S. 1956a. The effect of ergotamine and dihydroergotamine on lactation in the rat. J. Endocrinol., 14, 268-274.

TiND.^L, J. S. 1956b. National Institute for Research in Dairying, Report 1956, pp. 56-57.

TiNDAL, J. S. 1958. National Institute for Research in Dairying, Report 1958, pp. 48-49.

TiNDAL, J. S. 1960. A breed difference in the lactogenic response of the rabbit to reserpine. J. Endocrinol., 20, 78-81.

Tholen, H. 1949. Das Embryonale und Postnatale Verhalten der Mannlichen Brustdriise beim Menschen. I. Das mammarorgan beim Embryo und Saugling. Acta anat., 8, 201-235.

TovERUD, S. v., AND MuNSON, P. L. 1956. The influence of the parathyroids on the calcium concentration of milk. Ann. New York Acad. Sc, 64, 336.

Trentin, J. J., AND Turner, C. W. 1947. Effect of adrenalectomy on the mannnary gland of the castrated and estrogen treated castrated male rat. Endocrinology, 41, 127-134.

Trentin, J. J., and Turner, C. W. 1948. The experimental development of the mammary gland with special reference to the intei-action of the pituitary and ovarian hormones. Res. Bull. Missouri Agric Exper. Sta., No. 418.

[Tsakhabv, G. a.] II,axaeB, F. A. 1953. O npiipoAe a(j)(j)epeHTHbix ny xett pe(|).TieKca MOJiOKooTjia'm. Dokl. Akad. Nauk S.S.S.R., 93, 941-944.

[Tsakhaev, G. a.] UaxaeB, F. A. 1959. CeKpeTopHaa (j)yHKiiiiH Mo.noHHoft :xeJie3M b ycjioBiiHx neHepBauiiii rnno(})ii3a. In Bonpocw

(j[)II3HO.nOrmi CejIbCK0.\03HHCTBeHHbIX JLHBO THbix. M. G. Zaks. Ed., pp. 3-19. Vilnius, Kaunas: Akademiya Nauk Litovsk. S.S.R.

Turner, C. W. 1939. The mammary glands. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., Ch. XL Baltimore: The Williams & Wilkins Company.

Turner, C. W. 1952. The Mammary Gland. Vol. 1. The Anatomy of the Udder of Cattle and Domestic Animals. Columbia, Mo.: Lucas Bros.

Turner, C. W.. .-^nd Gomez, E. T. 1933. The normal development of the mammary gland of the male and female albino mouse. Res. Bull. Missouri Agric. Exper. Sta., No. 182.

Turner, C. W., and Gomez, E. T. 1934. The experimental development of the mammary gland. II. The male and female guinea pig. Res. Bull. Missouri Agric. Exper. Sta No 206.

Turner, C. W., and Schultze, A. B. 193L A study of the causes of the normal development of the mammary gland of the albino rat. Res Bull. Missouri Agric. Exper. Sta., No. 157.

Turner, C. W., and Slaughter, I. S. 1930. The physiological effect of pituitary extract (posterior lobe) on the lactating mammarv gland J.Dairy Sc, 13, 8-24.

[TvERSKoi, G. B.] TBepcKOH, F. B. 1953. O

npiipOAe HyBCTBIITeJILHblX ctumv.tiob c

BbmeHH, yyacTByiouiiix b pe(J)JieKTopHoft pery.nHmin ceKpei;Hn MO.noKa. Zhur. Obshchel Biol., 14, 349-359. [TvERSKoi, G. B.] TBepcKOH, F. B. 1957. O pojiH

HyBCTEHTejIbHOH IIHHepBaAHII xMOJIOMHOa

JKe.nesbi b pe({).neKTopHoii pery.nnmiH ceKpeAHii MOJioKa a mo-jiohhofo H,iipa. Zhur ObshcheT Biol., 18, 169-184.

[TvERSKoi, G. B.] TsepcKofi F. B. 1958 CeKpeuna MO.ioKa y K03 noc.Tie no.HHoft nepepe3KH cnnHHoro Moara. Dokl. Akad. Nauk. SSSR 123, 1137-1139. '

TvERSKOY, G. B. 1960. Influence of cervical sympathectomy and pituitary stalk section upon milk secretion in goats. Nature, London, 186, 782-784.

VAN W.-iGENEN, G., AND FOLLEY, S. J. 1939. The

effect of androgens on the mammary gland of the female rhesus monkev. J. Endocrinol 1, 367-372.

Vendrely, R. 1955. The deoxyribonucleic acid content of the nucleus. In The Xucleic Acids: Chemistry and Biology, E. Chargaff and J. N. Davidson, Eds., Ch. 19, pp. 155-180. New York : Academic Press, Inc.

VON Berswordt-Wallrabe, R. 1958. Versuch einer theoretischen Erklarung der Hemmung der Galaktopoese der Albinomaus durch Dienostroldiacetat. Arch. Gynak., 190, 619-637.

Walker, S. M., and M.^tthews, J. I. 1949. Observations on the effects of prepartal and postpartal estrogen and progesterone treatment on lactation in the rat. Endocrinology, 44, 8-17.

Waller, H. 1938. Clinical Studies\)i Lactation. London: William Heinemann, Ltd.

Whittlestone, W. G. 1954. Intramammary pressure changes in the lactating sow. HI. The effects of level of dose of oxytocin and the influence of rate of injection. J. Dairv Res 21, 188-193.

WiLLi.^MS, W. L. 1942. Normal and experimental mammary involution in the mouse as related to the inception and cessation of lactation. Anat. Rec, 71, 1-41.

WiLLi.-VMS, W. L. 1945. The effects of lactogenic hormone on the postparturient unsuckled mammary glands of the mouse. Anat. Rec. 93, 171-183.

642

PHYSIOLOGY OF GONADS

Williams, W. L., and Stewart, C. A. 1945. Mammarv development in the newborn human. Anat. Rec, 91, 307-308.

YoKOYAMA, A. 1956. Milk-ejection responses following administration of "tap" stimuli and posterior-pituitary extracts. Endocrinol. Japon., 3, 32-38.

[Zaks,M. G.] 3aKC, M. r. 1951. HoBbie aaHHbie o (i)yHKUHH MOTopHoro auoapaTa BbiMenu. Ix CSopHHK Ji;oKJiajiOB BTopott Bcecoio3Hofi

KoHcbepeHunii no MonoHHOMy Jlejiy, R. B. Davidov, 2d. p. 150-163. Moscow: Sel'khozgiz.

[Zaks, :\I. G.] 3aKc, M. T. 1958. <Dn.3iiononiH ABiiraTejibHoro annapara mojiomhoh a;ejie.3M cejibCKOxosHitcTBeHHbix xnBOTHbix. Moscow, Leningrad: Akademiya Nauk, S.S.S.R.

ZiETZSCHMANN, 0. 1926. Bau und Funktion der Milchdriise. In "Lehrbuch der Chemie und Physiologie der Milch, 2nd ed., W. Grimmer, Ed., p. 1-35. Berlin: Paul Parey.

Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones

Claude A. Villee, Ph.D.

Associate Professor Of Biological Chemistry, Harvard University

I. Introduction 643

II. The Biosynthesis op Steroids 643

A. Cholesterol 644

B. Progesterone 644

C. Androgens 645

D. Estrogens 647

E. Biosynthesis of Other Steroids 647

F. Interconversions of Steroids 647

G. Catabolism of Steroids (548

H. Transport, Conjugation, and Excretion 650

III. Effects of Sex Hormones on Inter mediary Metabolism 650

A. Estrogens 652

B. Androgens 659

C. Progesterone 660

IV. References 661

I. Intro<luction

The chemical structure of the sex hormones, their isohition from biologic materials, and many of their chemical properties were fully described in the previous edition of Sex and Internal Secretions (W. M. Allen, 1939; Doisy, 1939; Koch, 1939). The major steroid sex hormones were isolated and identified 20 to 30 years ago. Estrone, in fact, was crystallized from pregnancy urine by Doisy, Veler and Thayer (1929) before the true structure of the steroid nucleus was known. The isolation, identification, and chemical synthesis of estradiol, progesterone, and testosterone were accomplished during the 1930's. Additional substances with androgenic, estrogenic, or progestational activity have subsequently been isolated from urine or from tissues but these are probably metabolites of the major

sex steroids. The steroids are now routinely synthesized from cholesterol or from plant sterols. It would be possible to carry out the total synthesis of steroids from simple precursors but this is not commercially practicable.

The two decades since the previous edition have been marked by major advances in our understanding of the intermediary metabolism of steroids — the synthesis of cholesterol from two-carbon units, the conversion of cholesterol to pregnenolone and progesterone, and the derivation of corticoids, androgens, and estrogens from progesterone. These advances were made possible by the development of vastly improved methods for the isolation and identification of steroids: chromatography on paper or columns, counter-current distribution, labeling with radioactive or heavy isotopes, infrared spectroscopy, and so on. There have been concomitant increases in the information regarding the sites and mechanisms of action of these biologically important substances and the means by which they stimulate or inhibit the growth and activity of particular tissues of the body. The following discussion will attempt to present a general picture of these two fields and not an exhaustive citation of the tremendous body of relevant literature.

II. The Biosynthesis of Steroids

When the steroid hormones were first discovered it was generally believed that each endocrine gland made its characteristic steroid by some unique biosynthetic mechanism, one that was independent of those in other glands. However, there is now abundant evidence that the biosynthetic paths in the several steroid-secreting glands have many features which are similar or identical.

It is now well established that progesterone is not simply a female sex hormone produced by the corpus luteum, but a common precursor of adrenal glucocorticoids such as Cortisol and adrenal mineralocorticoids such as aldosterone, androgens, and estrogens. The adrenal cortex, ovary, testis, and placenta have in common many enzymes for the biosynthesis of steroids. Androgenic tumors of the human ovary, for example, have been shown to produce testosterone and its metabolites. The transplantation of an ovary into a castrate male mouse will result in the maintenance of the male secondary sex characters, which suggests that the normal ovary can also synthesize androgens.

A. CHOLESTEROL

The early work of Bloch (1951), Rilling, Tchen and Bloch (1958), and of Popjak (1950) showed that labeled acetate is converted to labeled cholesterol. The pattern of the labeling present in the cholesterol synthesized from acetate-1-C^^ or acetate-2-C^ as precursor led to speculations as to how the steroid nucleus is assembled. Further work (Langdon and Bloch, 1953) revealed that squalene and certain branched-chain, unsaturated fatty acids are intermediates in this synthesis. The current hypothesis, which is supported by a wealth of experimental evidence, states that two moles of acetyl coenzyme A condense to form acetoacetyl coenzyme A, which condenses with a third molecule of acetyl coenzyme A to form yS-hydroxy-^-methyl glutaric acyl coenzyme A (Fig. 11.1). The coenzyme A group is removed and the hydroxymethyl glutaric acid is reduced to mevalonic acid. Mevalonic acid, 3-hydroxy-3-methylpentano-5-lactone, is metabolized to a 5-carbon isoprenoid compound and three moles of these condense to form a 1 5-carbon hydrocarbon. The headto-head condensation of two molecules of this 15-carbon compound yields the 30

carbon equalene. This is metabolized, by way of lanosterol and the loss of three methyl groups, to cholesterol, which seems to be the common precursor of all of the steroid hormones (Tchen and Bloch, 1955; Clayton and Bloch, 1956).

The question of whether cholesterol is an obligate intermediate in the synthesis of steroid hormones has not been definitely answered. There is clear evidence that cholesterol is converted to steroids without first being degraded to small units and subsequently reassembled. Werbin and LeRoy (1954) administered cholesterol labeled with both carbon-14 and tritium (H^) to human subjects and isolated from their urine tetrahydrocortisone, tetrahydrocortisol, androsterone, etiocholanolone and 11ketoetiocholanolone. These substances, known to be metabolites of steroid hormones, were labeled with both C^^ and H^ and the labeled atoms were present in the ratio expected if they were derived directly from cholesterol. Experiments by Dorfman and his colleagues (Caspi, Rosenfeld and Dorfman, 1956) also provide evidence for the synthesis of steroids via cholesterol. Cortisol and 11-desoxycortisol were isolated from calf adrenals perfused with acetate-1C^^ and from a patient with an adrenal tumor to whom acetate- 1-C^^ had been administered. It is known that cholesterol synthesized from acetate-l-C^'* is labeled in carbon 20 but not in carbon 21. The Cortisol and 11-desoxycortisol also proved to be labeled in carbon 20 but not in carbon 21. This evidence does not, of course, exclude biosynthetic paths for the steroids other than one by way of cholesterol, but it does suggest that cholesterol is at least an important precursor of them. Direct evidence that cholesterol is synthesized from squalene in man is provided by the experiments of Eidinoff, Knoll, Marano, Kvamme, Rosenfeld and Hcllman (1958), who prepared tritiated squalene and administered it orally to human subjects. They found that the cholesterol of the blood achieved maximal specific aeti\'ity in 7 to 21 liours.

B. PROCiE.STERONE

Cholesterol undergoes an oxidative cleavage of its side chain to yield isocaproic acid and pregnenolone (Fig. 11.2). The lat

CH3C0-SC0A

STEROID SEX HORMONES

SCoA

645

CH^CO-SCoA

Acetyl CoA

CH3COCH2CO~SCoA

Acetoacetyl CoA

Sesquiterpene (15C)

COOH

► HO— C— CHI ^

CO-' SCO A

pOH-p methylglutaric acyl Co A

CHg

C-CHI

CH II CHo

Isoprene unit (5C)

CHO

I

CHo I ^

HO-C-CH^ 1 3

CHo I 2 COOH

Mevalonic acid

Squalene Lanosterol Cholesterol

(30C) (30C) (27C)

Fig. 11.1. Biogenesis of cholesterol.

ter is dehydrogenated in ring A by the enzyme 3-^-ol dehydrogenase and a spontaneous shift of the double bond from the A5 , 6 to the A4 , 5 position results in progesterone. Progesterone undergoes successive hydroxylation reactions, which require molecular oxygen and reduced triphosphopyridine nucleotide (TPNH), at carbons 17, 21, and 11. These hydroxylations yield, in succession, 17-a-hydroxy progesterone, Reichstein's compound S (ll-desoxy-17-hydroxycorticosterone), and Cortisol (17-a-hydroxvcorticosterone) .

C. ANDROGENS

17-a-Hydroxy progesterone is also the immediate precursor of androgens and estrogens. Oxidative cleavage of its side chain yields A-4-androstenedione, which undergoes reduction to testosterone (Fig. 11.2). A-4-Androstenedione may be hydroxylated at carbon 11 to yield ll-/3-hydroxy-A-4androstenedione, which is an androgen isolated from human urine. It has also been found as a metabolite of certain androgenic tumors of the adrenal cortex.

04 G

PHYSIOLOGY OF GONADS

CH

Y^ .

socaproic C

=0

c=o

r^

0^-OH

^r^

HO

HO

J

HO

Cholesterol

Pregnenolone

17-Hydr

oxy

Dehydroepi

preg

nenolone

androsterone

CH3

CH3

f3

H-C-OH 1

, f

c=o

1

1

c=o

1

^ ■

-x^V

t^

^XP""°"

r^^

^^<Y

„ijj

- \^Xaj

JJ

A -3- Ketopregnene

Progest

erone

17

-Hydroxy

20-oc-ol

y

y

progesterone

CH^OH

X

CH OH

/

c=o

1

c=o

"

Desoxy— corticosterone

CH2OH c=o

1 7- Hydroxy desoxycorticosterone (Reichstein's "S")

CH^OH HCO C=0

.JOJ

CH^OH

c=o

A -androstenedione

OH

o ' -- o

Corticosterone 18-aldo- 11- desoxy- Cortisol Testosterone

corticosterone (Kendall's

Cmpd. "F")

HCO C=0

19 -Hydroxy- ^■^■ androstenedione

.;^

Aldosterone

HO

Estradiol Fig. 11.2. Biosynthetic paths from cholesterol.

Estrone

STEROID SEX HORMONES

647

D. ESTROGENS

A-4-Androstenedione and testosterone are precursors of the estrogens. Baggett, Engel, Savard and Dorfman (1956) demonstrated the conversion of testosterone to estradiol17/? by slices of human ovary. Ryan (1958) found that the enzymes to carry out this conversion are also present in the human placenta, located in the microsomal fraction of placental homogenates. Homogenates of stallion testis convert labeled testosterone to labeled estradiol and estrone. Slices of human adrenal cortical carcinoma also have been shown to convert testosterone to estradiol and estrone, and Nathanson, Engel and Kelley (1951) found an increased urinary excretion of estradiol, estrone, and estriol following the administration of adrenocorticotrophic hormone to castrate women. Thus it seems that ovary, testis, placenta, and adrenal cortex have a similar biosynthetic mechanism for the production of estrogens and androgens. The first step in the conversion of testosterone or A-4-androstenedione to estrogens is the hydroxylation at carbon 19, again by an enzymatic process which requires molecular oxygen and TPNH. Meyer (1955) first isolated and characterized 19-hydroxy-A-4-androstene3,17-dione from a perfused calf adrenal. When this was incubated with dog placenta it was converted to estrone. The steps in the conversion of the 19-hydroxy-A-4-androstenedione to estrone appear to be the introduction of a second double bond into ring A, the elimination of carbon 19 as formaldehyde, and rearrangement to yield a phenolic ring A. The requirements for the aromatization of ring A by a microsomal fraction of human placenta were studied by Ryan (1958). West, Damast, Sarro and Pearson (1956) found that the administration of testosterone to castrated, adrenalectomized women resulted in an increased excretion of estrogen. This suggests that tissues other than adrenals and gonads, presumably the liver, can carry out this same series of reactions.

E. BIOSYNTHESIS OF OTHER STEROIDS

To complete the picture of the interrelations of the biosyntheses of steroids, it should be noted that other evidence shows

that progesterone is hydroxylated at carbon 21 to yield desoxycorticosterone and this is subsequently hydroxylated at carbon 11 to yield corticosterone. Desoxycorticosterone may undergo hydroxylation at carbon 18 and at carbon 11 to yield aldosterone, the most potent salt-retaining hormone known (Fig. 11.2).

Dehydroepiandrosterone is an androgen found in the urine of both men and women. Its rate of excretion is not decreased on castration and it seems to be synthesized only by the adrenal cortex. It has been postulated that pregnenolone is converted to 17-hydroxy pregnenolone and that this, by cleavage of the side chain between carbon 17 and carbon 20, would yield dehydroepiandrosterone.

F. INTERCONVERSIONS OF STEROIDS

The interconversion of estrone and estradiol has been shown to occur in a number of human tissues. A diphosphopyridine nucleotide-linked enzyme, estradiol- 17/3 dehydrogenase, which carries out this reaction has been prepared from human placenta and its properties have been described by Langer ancl Engel (1956). The mode of formation of estriol and its isomer, 16-epiestriol, is as yet unknown.

There are three major types of reactions which occur in the interconversions of the steroids: dehydrogenation, "hydroxylation," and the oxidative cleavage of the side chain. An example of a dehydrogenation reaction is the conversion of pregnenolone to progesterone by the enzyme 3-/3-ol dehydrogenase, which requires diphosphopyridine nucleotide (DPN) as hydrogen acceptor. This important enzyme, which is involved in the synthesis of progesterone and hence in the synthesis of all of the steroid hormones, is found in the adrenal cortex, ovary, testis, and placenta. Other dehydrogenation reactions in which DPN is the usual hydrogen acceptor are the readily reversible conversions of A-4-androstenedione ^ testosterone, estrone ^ estradiol, and progesterone

^ A-4-3-ketopregnene-20-a-ol. This latter

substance, and the enzymes producing it from progesterone, have been found by Zander (1958) in the human corpus luteum and placenta.

The oxidative reactions leading to the

PHYSIOLOGY OF GONADS

introduction of an OH group on the steroid nucleus are usually called "hydroxylations." Specific hydroxylases for the introduction of an OH group at carbons 11, 16, 17, 21, 18, and 19 have been demonstrated. All of these require molecular oxygen and a reduced pyridine nucleotide, usually TPNH. The ll-/3-hydroxylase of the adrenal cortex has been shown to be located in the mitochondria (Hayano and Dorfman, 1953) . Experiments with this enzyme system, utilizing oxygen 18, showed that the oxygen atoms are derived from gaseous oxygen and not from the oxygen in the water molecules (Hayano, Lindberg, Dorfman, Hancock and Doering, 1955). Thus this hydroxylation reaction also involves the reduction of molecular oxygen.

The oxidative cleavage of the side chains of the steroid molecule appears to involve similar hydroxylation reactions. The experiments of Solomon, Levitan and Lieberman (1956) indicate that the conversion of cholesterol to pregnenolone involves one and possibly two of these hydroxylation reactions, with the introduction of OH groups at carbons 20 and 22 before the splitting off of the isocaproic acid.

In summary, this newer knowledge of the biosynthetic paths of steroids has revealed that the differences between the several steroid-secreting glands are largely quantitative rather than qualitative. The testis, for example, produces progesterone and estrogens in addition to testosterone. The change from the secretion of estradiol by the follicle to the secretion of progesterone by the corpus luteum can be understood as a relative loss of activity of an enzyme in the path between progesterone and estradiol. If, for example, the enzyme for the 17-hydroxylation of progesterone became inactive as the follicle cells are changed into the corpus luteum, progesterone rather than estradiol would subsequently be produced.

Knowledge of these pathways also provides an explanation for certain abnormal changes in the functioning of the glands. Bongiovnnni (1953) and Jailer (1953) showed that the adrenogenital syndrome results from a loss of an enzyme or enzymes for the hydroxylation reactions at carbons 21 and 11 of progesterone, which results in an impairment in the production of Cortisol.

The pituitary, with little or no Cortisol to inhibit the secretion of adrenocorticotrophic hormone (ACTH), produces an excess of this hormone which stimulates the adrenal to produce more steroids. There is an excretion of the metabolites of progesterone and 17-hydroxy progesterone, pregnanediol and pregnanetriol respectively, but some of the 17-hydroxy progesterone is converted to androgens and is secreted in increased amount.

G. CATABOLISM OF STEROmS

Many of the steroid hormones are known to act on the pituitary to suppress its secretion of the appropriate trophic hormone, ACTH, the follicle-stimulating hormone (FSH), or luteinizing hormone (LH). It would seem that the maintenance of the proper feedback mechanism between steroid-secreting gland and pituitary requires that the steroids be continuously inactivated and catabolized. The catabolic reactions of the steroids are in general reductive in nature and involve the reduction of ketonic groups and the hydrogenation of double bonds. The reduction of a ketonic group to an OH group can lead to the production of two different stereoisomers. If the OH group projects from the steroid nucleus on the same side as the angular methyl groups at carbon 18 and carbon 19, i.e., above the plane of the four rings, it is said to have the yS-configuration and is represented by a heavy line. If the OH projects on the opposite side of the steroid nucleus, below the plane of the four rings, it is said to have the a-configuration and is represented by a dotted line. Although both isomers are possible, usually one is formed to a much greater extent than the other.

The first catabolic step is usually the reduction of the A4-3-ketone group of ring A, usually to 3aOH compounds with the hydrogen at carbon 5 attached in the /3configuration. The 5/3-configuration represents the CIS configuration of rings A and B. The elimination of the A4-3-ketone group greatly decreases the biologic activity of the steroid and increases somewhat its solubility in water. This reductive process occurs largely in the liver. Progesterone is converted by reduction of its A4-3-ketone group to pregnane-3a:20a-diol, and 17-hy

STEROID SEX HORMONES 649

EXCRETORY PRODUCTS

c=o

Progesterone

1 3

HCOH

HO H

Pregnanediol

CH,

-OH

17 Hydroxy progesterone

CH,

^ H-C-OH

■OH

HO H

Pregnanetriol

OH

Testosterone

Androsterone

HO

/

androstenedione

HO H

Etiocholanolone

Dehydroepiandrosterone

Fk;. 11.3. Excretory products of progesterone and androgen

(Iroxy progesterone is converted to pregnane3a:17a:20a-triol (Fig. 11.3). Testosterone and dehydrocpiandroesterone are both converted to A4-androstenedione and the reduction of its A4-3-ketone group results in a mixture of androsterone (3a,5a-configuration) and ctiochohmolone (3a,5/?-configuration ) .

The catabohsm of estradiol is not completely known. Estradiol, estrone, and estriol are found in the urine but they account for less than half of an administered dose of labeled estradiol. The /3-isomer, 16-epiestriol, and two other phenolic steroids, 16-ahydroxy estrone and 2-methoxyestrone, have recently been isolated from normal

650

PHYSIOLOGY OF GONADS

urine and are known to be estrogen metabolites (Marrian and Bauld, 1955).

H. TRANSPORT, CONJUGATION, AND EXCRETION

Steroids circulate in the blood in part as free steroids and in part conjugated with sulfate or glucuronic acid (c/. review by Roberts and Szego, 1953b j . The steroids are generally conjugated by the hydroxy 1 group at carbon 3 with inorganic sulfate or with glucuronic acid. In addition, either the conjugated or nonconjugated forms may be bound to certain of the plasma proteins such as the ^-globulins (Levedahl and Bernstein, 1954) . There is evidence of specific binding of certain steroids with particular proteins, e.g., the binding of Cortisol to "transcortin" (Daughaday, 1956). Between 50 and 80 per cent of the estrogens in the blood are present closely bound to plasma proteins. A similar large fraction of the other steroid hormones is bound to plasma proteins ; presumably this prevents the hormone from being filtered out of the blood as it passes through the glomerulus of the kidney. The steroids excreted in the urine are largely in the conjugated form, as sulfates or glucuronides.

The liver plays a prime role in the catabolism of the steroids. It is the major site of the reductive inactivation of the steroids and their conjugation with sulfate or glucuronic acid. These conjugated forms are more water-soluble and the conjugation probably promotes their excretion in the urine. Rather large amounts of certain steroids, notably estrogens, are found in the bile of certain species. These estrogens are free, not conjugated; the amount of estrogens present in the bile suggests that this is an important pathway by which they are excreted. It has been suggested that the bacteria of the gastrointestinal tract may degrade the steroids excreted in the bile and further that there is an "enterohepatic circulation" of steroids with reabsorption from the gut, transport in the portal system to the liver, and further degradation within the liver cells.

III. Effects of Sex Hormones on Intermediary Metabolism

The literature concerning the effects of hormones on intermediary metabolism is

voluminous and contains a number of contradictions, some of which are real and some, perhaps, are only apparent contradictions. Evidence that a hormone acts at one site does not necessarily contradict other evidence that that hormone may act on a different metabolic reaction. From the following discussion it should become evident that there may be more than one site of action, and more than one mechanism of action, of any given hormone.

The hormones are so different in their chemical structure, proteins, peptides, amino acids, and steroids, that it would seem unlikely, a priori, that they could all influence the cellular machinery by comparable means. The basic elements of an enzyme system are the protein enzyme, its cofactors and activators, and the substrates and products. A hormone might alter the over-all rate of an enzyme system by altering the amount or activity of the protein enzyme, or by altering the availability to the enzyme system of some cofactor or substrate molecule. Some of the mechanisms of hormone action which have been proposed are these. (1) The hormone may alter the rate at which enzyme molecules are produced de novo by the cell. (2) The hormone may alter the activity of a preformed enzyme molecule, i.e., it may convert an inactive form of the enzyme to an active form. (3) The hormone may alter the permeability of the cell membrane or the membrane around one of the subcellular structures within the cell and thus make substrate or cofactor more readily available to the enzyme. Or, (4) the hormone may serve as a coenzyme in the system, that is, it may be involved in some direct fashion as a partner in the reaction mediated by the enzyme. Each of these theories has been advanced to explain the mode of action of the sex hormones.

The problem of the hormonal control of metabolism has been investigated at a variety of biologic levels. The earliest experiments were done by injecting a hormone into an intact animal and subsequently measuring the amount of certain constituents of the blood, urine, or of some tissue. There are several difficulties with such experiments. All of the homeostatic mechanisms of the animal operate to keep condi

STEROID SEX HORMONES

651

tions constant and to minimize the effects of the injected hormone. In addition, there is a maze of interactions, some synergistic and some antagonistic, between the different hormones both in the endocrine gland and in the target organs, so that the true effect of the substance injected may be veiled. Our growing understanding of the interconversions of the steroid hormones warns us that an androgen, for example, may be rapidly converted into an estrogen, and the metabolic effects observed on the administration of an androgen may, at least in part, result from the estrogens produced from the injected androgen.

To eliminate some of the confusing effects of these homeostatic mechanisms some investigators remove the liver, kidneys, and other viscera before injecting the hormone under investigation. Such eviscerated preparations have been used by Levine and his colleagues in their investigations of the mode of action of insulin (c/. Levine and Goldstein, 1955).

Other investigators have incubated slices of liver, kidney, muscle, endocrine glands, or other tissues in glass vessels in a chemically defined medium and at constant temperature. Such experiments have the advantage that metabolism can be studied more directly, oxygen consumption and carbon dioxide production can be measured manonietrically, and aliquots of the incubation medium can be withdrawn for chemical and radiochemical analyses. The amounts of substrate, cofactors, and hormone present can be regulated and the interfering effects of other hormones and of other tissues are eliminated. Theoretically, working with a simpler system such as this should lead to greater insight into the physiologic and chemical events that occur when a hormone is added or deleted. The chief disadvantage of this experimental system is that it is difficult to prove that the conditions of the experiment are "physiologic." With tissue slices there is the possibility that the cut edges of the cells may introduce a sizeable artifact. Kipnis and Cori (1957) found that the rat diaphragm, as it is usually prepared for experiments in vitro, has an abnormally large extracellular space and is more permeable to certain pentoses than is the intact diaphragm.

It has been postulated that a hormone may influence the metabolism of a particular cell by altering the permeability of the cell membrane or of the membrane around one of the subcellular particles. Experiments with tissue homogenates, in which the cell membrane has been ruptured and removed, provide evidence bearing on such theories. If an identical hormone effect can be obtained in a cell-free system, and if suitable microscopic controls show that the system is indeed cell-free, the permeability theory may be ruled out.

Ideally the hormone effect should be studied in a completely defined system, with a single crystalline enzyme, known concentration of substrates and cofactors, and with known concentration of the pure hormone. Colowick, Cori and Slein (1947) reported that hexokinase extracted from diabetic muscle has a lower rate of activity than hexokinase from normal muscle and that it could be raised to the normal rate by the addition of insulin in vitro. The reality of this effect has been confirmed by some investigators and denied by others who were unable to repeat the observations. Cori has suggested that the decreased rate of hexokinase activity in the diabetic results from a labile inhibitor substance produced by the pituitary. Krahl and Bornstein (1954) have evidence that this inhibitor is a lipoprotein which is readily inactivated by oxidation.

The two hormones whose effects can be demonstrated reproducibly in an in vitro system at concentrations in the range which obtains in the tissues are epinephrine (or glucagon) and estradiol (and other estrogens) . Epinephrine or glucagon stimulates the reactivation of liver phosphorylase by increasing the concentration of adenosine3'-5'-monophosphate (Haynes, Sutherland and Rail, 1960), and estrogens stimulate an enzyme system found in endometrium, placenta, ventral i)rostate of the rat, and mammary gland. The estrogen-stimulable enzyme was originally described as a DPNlinked isocitric dehydrogenase, but the estrogen-sensitive enzyme now appears to be a transhydrogenase which transfers hydrogens from TPN to DPN (Talalay and Williams-x\shman, 1958; Yillee and Hngerman, 1958).

052

PHYSIOLOGY OF GONADS

The various tissues of the body respond in quite different degrees to the several hormones. This difference in response is especially marked with the sex hormones. Those tissues which respond dramatically to the administration of a hormone are termed the "target organs" of that hormone. Just what, at the cellular level, differentiates a target organ from the other tissues of the body is not known exactly but there is evidence that each kind of tissue is characterized by a certain pattern of enzymes. The pattern of enzymes is established, by means as yet unknown, in the course of embryonic differentiation. The enzyme glucose 6-phosphatase, which hydrolyzes glucose 6-phosphate and releases free glucose and inorganic phosphate, is present in liver but absent from skeletal muscle. Even though a given reaction in two different tissues may be mediated by what appears to be the same enzyme, the enzymes may be different and subject to different degrees of hormonal control. Henion and Sutherland (1957) showed that the phosphorylase of liver responds to glucagon but the phosphorylase of heart muscle does not. Further, the two enzymes are immunologically distinct. An antiserum to purified liver phosphorylase will not react with heart phosphorylase to form an inactive antigenantibody precipitate, but it does react in this manner with liver phosphorylase. Further, perhaps more subtle, differences between comparable enzymes from different tissues have appeared when lactic dehydrogenases from liver, heart, skeletal muscle, and other sources were tested for their rates of reaction with the several analogues of the pyridine nucleotides now available (Kaplan. Ciotti, Hamolsky and Bicbcr, 1960). p]xtension of this technique may reveal differences in response to added hormones.

In addition to these differences in the response to a hormone of the tissues of a single animal, there may be differences in the response of the comparable tissues of different species to a given dose of hormone. Estrone, estriol, and other estrogens have different potencies relative to estradiol in different species of mammals. There are slight differences in the amino acid sequences of the insulins and vasopressins from flifferent species and quite marked

differences in the chemical structure (Li and Papkoff, 1956) and physiologic activity (Knobil, Morse, Wolf and Greep, 1958) of the pituitar}^ growth hormones of cattle and swine, on the one hand, and of primates, on the other.

A. ESTROGENS

The amount or activity of certain enzymes in the target organs of estrogens has been found to vary with the amount of estrogen present. Examples of this phenomenon are /^-glucuronidase (Odell and Fishman, 1950) , fibrinolysin (Page, Glendening and Parkinson, 1951), and alkaline glycerophosphatase (Jones, Wade, and Goldberg, 1953). Kochakian (1947) reported that the amount of arginase in the rat kidney increased after the injection of estrogens. Enzyme activity is increased by other hormones as well; for example, progesterone has been found to increase the activity of phosphorylase (Zondek and Hestrin, 1947) and of adenosine triphosphatase (Jones, Wade, and Goldberg, 1952).

In most experiments the amount of enzyme present has been inferred from its activity, measured chemically or histochemically under conditions in which the amount of enzyme is rate-limiting. This does not enable one to distinguish between an actual increase in the number of molecules of enzyme present in the cell and an increase in the activity of the enzyme molecules without change in their number. A few enzymes can be measured by some other property, such as absorption at a specific wavelength, by which the actual amount of enzyme can be estimated (see review by Knox, Auerbach, and Lin, 1956). Knox and Auerbach (1955) found that the activity of the enzyme tryptophan peroxidase-oxidase (TPO) of the liver was decreased in adrenalectomized animals and increased by the administration of cortisone. Knox had shown previously that th(> administration of the substrate of the enzyme, tryptophan, would lead to an increase in the activity of the enzyme which was maximal in 6 to 10 hours. Evidence that the increased activity of enzyme following the administration of cortisone represents the synthesis of new protein molecules is supplied by experi

STEROID SEX HORMONES

653

ments in which it was found that the increase in enzyme activity is inhibited by ethionine and this inhibition is reversed by methionine. The amino acid analogue ethionine is known to inhibit protein synthesis and this inhibition of protein synthesis is overcome by methionine.

The injection of estrogen into the immature or castrate rodent produces a striking uptake of water by the uterus followed by a marked increase in its dry weight (Astwood, 1938). Holden (1939) postulated that the imbibition of water results from vasodilatation and from changes in the permeability of the blood vessels of the uterus. There is clear evidence (Mueller, 1957) that the subsequent increase in dry weight is due to an increased rate of synthesis of proteins and nucleic acids. The sex hormones and other steroids could be pictured as reacting with the protein or lipoprotein membrane around the cell or around some subcellular structure like a surface-wetting agent and in this way inducing a change in the permeability of the membrane. This might then increase the rate of entry of substances and thus alter the rate of metabolism within the cell. This theory could hardly account for the many notable specific relationships between steroid structure and biologic activity. Spaziani and Szego (1958) postulated that estrogens induce the release of histamine in the uterus and the histamine then alters the permeability of the blood vessels and produces the imbibition of water secondarily.

The uterus of the ovariectomized rat is remarkably responsive to estrogens and has been widely used as a test system. After ovariectomy, the content of ribonucleic acid of the uterus decreases to a low level and then is rapidly restored after injection of estradiol (Telfer, 1953). A single injection of 5 to 10 yu,g. of estradiol brings about (1) the hyperemia and water imbibition described previously; (2) an increased rate of over-all metabolism as reflected in increased utilization of oxygen (David, 1931; Khayyal and Scott, 1931; Kerly, 1937; MacLeod and Reynolds, 1938; Walaas, Walaas and Loken, 1952a; Roberts and Szego, 1953a) ; (3) an increased rate of glycolysis (Kerly, 1937; Carroll, 1942; Stuermer and Stein, 1952; Walaas, Walaas

and Loken, 1952b; Roberts and Szego, 1953a) ; (4) an increased rate of utilization of phosphorus (Grauer, Strickler, Wolken and Cutuly, 1950; Walaas and Walaas, 1950) ; and (5) tissue hypertrophy as reflected in increased dry weight (Astwood, 1938), increased content of ribonucleic acid and protein (Astwood, 1938; Telfer, 1953; Mueller, 1957), and finally, after about 72 hours, an increased content of desoxyribonucleic acid (Mueller, 1957).

An important series of experiments by Mueller and his colleagues revealed that estrogens injected in vivo affect the metabolism of the uterus which can be detected by subsequent incubation of the uterus in vitro with labeled substrate molecules. Mueller (1953) first showed that pretreatment with estradiol increases the rate of incorporation of glycine-2-C^'* into uterine protein. He then found that estrogen stimulation increases that rate of incorporation into protein of all other amino acids tested: alanine, serine, lysine, and tryptophan. The peak of stimulation occurred about 20 hours after the injection of estradiol. In further studies (Mueller and Herranen, 1956) it was found that estrogen increases the rate of incorporation of glycine-2-C^^ and formate-2-C^'* into protein, lipid, and the purine bases, adenine and guanine, of nucleic acids. A stimulation of cholesterol synthesis in the mouse uterus 20 hours after administration of estradiol was shown by Emmelot and Bos (1954).

In more detailed studies of the effects of estrogens on the metabolism of "one-carbon units" Herranen and Mueller (1956) found that the incorporation of serine-3-C^'* into adenine and guanine was stimulated by pretreatment with estradiol. The incorporation was greatly decreased when unlabeled formate was added to the reaction mixture to trap the one-carbon intermediate. In contrast, the incorporation of C^^02 into uridine and thymine by the surviving uterine segment was not increased by pretreatment with estradiol in vivo (Mueller, 1957).

To delineate further the site of estrogen effect on one-carbon metabohsm, Herranen and Mueller (1957) studied the effect of estrogen pretreatment on serine aldolase, the enzyme which catalyzes the equilibrium

654

PHYSIOLOGY OF GONADS

between serine and glycine plus an active one-carbon unit. They found that serine aldolase activity, measured in homogenates of rat uteri, increased 18 hours after pretreatment in vivo with estradiol. It seemed that the estrogen-induced increase in the activity of this enzyme might explain at least part of the increased rate of onecarbon metabolism following estrogen injection. They found, however, that incubation of uterine segments in tissue culture medium (Eagle, 1955) for 18 hours produced a marked increase in both the activity of serine aldolase and the incorporation of glycine-2-C^'* into protein. The addition of estradiol to Eagle's medium did not produce a greater increase than the control to which no estradiol was added. Uterine segments taken from rats pretreated with estradiol for 18 hours, with their glycine-incorporating system activated by hormonal stimulation, showed very little further stimulation on being incubated in Eagle's medium for 18 hours. With a shorter period of i^retreatment with estradiol, greater stimulation occurred on subsequent incubation in tissue culture fluid. These experiments suggest that the hormone and the incubation in tissue culture medium are affecting the same process, one which has a limited capacity to respond. When comparable experiments were performed with other labeled amino acids as substrates, similar results were obtained.

Mueller's work gave evidence that a considerable number of enzyme systems in the uterus are accelerated by the administration of estradiol — not only the enzymes for the incorporation of serine, glycine, and formate into adenine and guanine, but also the enzymes involved in the synthesis of fatty acids and cholesterol and indejX'ndent enzymes for the activation of amino acids by the formation of adenosine monoiihosphate (AMP) derivatives. The initial step in protein synthesis has been shown to be the activation of the carboxyl grou]) of the amino acid with transfer of energy from ATP, the formation of AMP -"amino acid, and the release of jiyrophosphate (Hoagland, Keller and Zamecnick, 1956). This reversible step was studied with homogenates of uterine tissue, P^--labeled ]n'rni)liosi)liate, and a variety of amino

acids (Mueller, Herranen and Jervell, 1958). Seven of the amino acids tested, leucine, tryptophan, valine, tryosine, methionine, glycine, and isoleucine, stimulated the exchange of P^^ between pyrophosphate and ATP. Pretreatment of the uteri by estradiol injected in vivo increased the activity of these three enzymes. The activating effect of mixtures of these amino acids was the sum of their individual effects, from which it was inferred that a specific enzyme is involved in the activation of each amino acid. Since estrogen stimulated the exchange reaction with each of these seven amino acids, Mueller concluded that the hormone must affect the amount of each of the amino acid-activating enzvmes in the soluble fraction of the cell.

Mueller (1957) postulated that estrogens increase the rate of many enzyme systems both by activating preformed enzyme molecules and by increasing the rate of de novo synthesis of enzyme molecules, possibly by removing membranous barriers covering the templates for enzyme synthesis. To explain why estrogens affect these enzymes in the target organs, but not comparable enzymes in other tissues, one would have to assume that embryonic differentiation results in the formation of enzymes in different tissues which, although catalyzing the same reaction, have different properties such as their responsiveness to hormonal stimulation.

As an alternative hypothesis, estrogen might affect some reaction which provides a substance required for all of these enzyme reactions. The carboxyl group of amino acids must be activated by ATP before the amino acid can be incorporated into proteins; the synthesis of both purines and pyrimidines requires ATP for the activation of the carboxyl group of certain precursors and for several other steps; the synthesis of cholesterol requires ATP for the conversion of mevalonic acid to squalene; and the synthesis of fatty acids is also an energy-requiring process. Thus if (>strogens acted in some way to increase the amount of biologically useful energy, in the form of ATP or of energy-rich thioesters such as acetyl coenzyme A, it would increase the rate of synthesis of all of these compo

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655

nents of the cell. This would occur, of course, only if the supply of ATP, rather than the amount of enzyme, substrate, or some other cofactor, were the rate-limiting factor in the synthetic processes.

When purified estrogens became available, they were tested for their effects on tissues in vitro. Estrogens added in vitro increased the utilization of oxygen by the rat uterus (Khayyal and Scott, 1931) and the rat pituitary (Victor and Andersen, 1937). The addition of estradiol- 17^ at a level of 1 fxg. per ml. of incubation medium increased the rate of utilization of oxygen and of pyruvic acid by slices of human endometrium and increased the rate at which labeled glucose and pyruvate were oxidized to C^-^Os (Hagerman and Villee, 1952, 1953a, 1953b) . In experiments with slices of human placenta similar results were obtained and it was found that estradiol increased the rate of conversion of both pyruvate-2-C^'* and acetate-l-Ci4 to C^^Os (Villee and Hagerman, 1953) . From this and other evidence it was inferred that the estrogen acted at some point in the oxidative pathway common to pyruvate and acetate, i.e., in the tricarboxylic acid cycle.

Homogenates of placenta also respond to estradiol added in vitro. With citric acid as substrate, the utilization of citric acid and oxygen and the production of a-ketoglutaric acid were increased 50 per cent by the addition of estradiol to a final concentralion of 1 fjig. per ml. (Villee and Hagerman, 1953). The homogenates were separated by differential ultracentrifugation into nuclear, mitochondrial, microsomal, and nonparticulate fractions. The estrogen-stimulable system was shown to be in the nonparticulate fraction, the material which is not sedimented by centrifugating at 57,000 X g for 60 minutes (Villee, 1955). Experiments with citric, as-aconitic, isocitric, oxalosuccinic, and a-ketoglutaric acids as substrates and with fluorocitric and transaconitic acids as inhibitors localized the estrogen-sensitive system at the oxidation of isocitric to oxalosuccinic acid, which then undergoes spontaneous decarboxylation to a-ketoglutaric acid (Villee and Gordon, 1955). Further investigations using the enzymes of the nonparticulate fraction of the human placenta revealed that, in ad

dition to isocitric acid as substrate, only DPN and a divalent cation such as Mg+ + or Mn++ were required (Villee, 1955; Gordon and Villee, 1955; Villee and Gordon, 1956). The estrogen-sensitive reaction was formulated as a DPN-linked isocitric dehydrogenase:

Isocitrate + DPN* -^ a-ketoglutarate

+ CO2 + DPXH + H*

It was found that the effect of the hormone on the enzyme can be measured by the increased rate of disappearance of citric acid, the increased rate of appearance of a-ketoglutaric acid, or by the increased rate of reduction of DPN, measured spectrophotometrically by the optical density at 340 m/x. As little as 0.001 /xg. estradiol per ml. (4 X 10~^ m) produced a measurable increase in the rate of the reaction, and there was a graded response to increasing concentrations of estrogen. The dose-response curve is typically sigmoid. This system has been used to assay the estrogen content of extracts of urine (Gordon and Villee, 1956) and of tissues (Hagerman, Wellington and Villee, 1957; Loring and Villee, 1957).

Attempts to isolate and purify the estrogen-sensitive enzyme were not very successful. By a combination of low temperature alcohol fractionation and elution from calcium phosphate gel a 20-fold purification was obtained (Hagerman and Villee, 1957). However, as the enzyme was purified it was found that an additional cofactor was required. Either uridine triphosphate (UTP) or ATP added to the system greatly increased the magnitude of the estrogen effect and, subsequently, adenosine diphosphate (ADP) was recovered from the incubation medium and identified by paper chromatography (Villee and Hagerman, 1957). Talalay and Williams-Ashman (1958) confirmed our observations and showed that the additional cofactor was triphosphopyridine nucleotide (TPN) which was required in minute amounts. This finding was confirmed by Villee and Hagerman (1958) and the estrogen-sensitive enzyme system of the placenta is now believed to be a transhydrogenase which catalyzes the transfer of hydrogen ions and electrons

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PHYSIOLOGY OF GONADS

fromTPNHtoDPN:

TPXH + DPN^ -> DPNH + TPN^

The transhydrogenation system can be coupled to glucose 6-phosphate dehydrogenase as well as to isocitric dehydrogenase (Talalay and Williams-Ashman, 1958; Villee and Hagerman, 1958) and presumably can be coupled to any TPNH-generating system.

If the estrogen-stimulable transhydrogenation reaction were readily reversible, an enzyme such as lactic dehydrogenase which requires DPN should be stimulated by estrogen if supplied with substrate amounts of TPN, catalytic amounts of DPN, and a preparation from the placenta containing the transhydrogenase. Experiments to test this prediction were made using lactic dehydrogenase and alcohol dehydrogenase of both yeast and liver (Villee, 1958a). It was not possible to demonstrate an estrogen stimulation of either enzyme system in either the forward or the reverse direction. The stimulation of the lactic dehydrogenase-DPN oxidase system of the rat uterus by estrogens administered in vivo reported by Bever, Velardo and Hisaw (1956) might be explained by the stimulation of a transhydrogenase, but it has not yet been possible to demonstrate a coupling of this transhydrogenase and lactic dehydrogenase.

The stimulating effect of a number of steroids has been tested with a system in which the transhydrogenation reaction is coupled to isocitric dehydrogenase (Villee and Gordon, 1956; Hollander, Nolan and Hollander, 1958). Estrone, equilin, equilenin, and 6-ketoestradiol have activities essentially the same as that of estradiol17 j3. Samples of 1 -methyl estrone and 2methoxy-estrone had one-half the activitj of estradiol. Estriol is only weakly estrogenic in this system; 33 fig. estriol are less active than 0.1 fig. estradiol- 17/3 (Villee, 1957a). The activities of estriol and 16epiestriol are similar, whereas 16-oxoestradiol is more active than either, with about 10 per cent as much activitv as csti'adiol17/3.

Certain analogues of stilbestrol have been shown to be anti-estrogens in vivo. When applied topically to the vagina of the rat, they prevent the cornification normally in

duced by the administration of estrogen (Barany, Morsing, Muller, Stallberg, and Stenhagen, 1955). One of these, 1,3-di-phydroxyphenylpropane, was found to be strongly anti-estrogenic in the placental system in vitro: it prevented the acceleration of the transhydrogenase-isocitric dehydrogenase system normally produced by estradiol- 17/3 (Villee and Hagerman, 1957). The inhibitory power declines as the length of the carbon chain connecting the two phenolic rings is increased and 1 , 10-di-phydroxyphenyldecane had no inhibitory action. Similar inhibitions of the estradiolsensitive system were observed with stilbestrol, estradiol-17a, and a smaller antiestrogenic effect was found with estriol (Villee, 1957a). The inhibition induced by these compounds can be overcome by adding increased amounts of estradiol-17^. When stilbestrol is added alone at low concentration, 10~' M, it has a stimulatory effect equal to that of estradiol-17^ (Glass, Loring, Spencer and Villee, 1961).

The quantitative relations between the amounts of stimulator and inhibitor suggest that this inhibition is a competitive one. It was postulated that this phenomenon involves a competition between the steroids for specific binding sites on the estrogensensitive enzyme (Villee, 1957b; Hagerman and Villee, 1957). When added alone, estriol and stilbestrol are estrogenic and increase the rate of the estrogen-sensitive enzyme. In the presence of both estradiol and estriol, the total enzyme activity observed is the sum of that due to the enzyme combined with a potent activator, estradiol- 17^, and that due to the enzyme combined with a weak activator, estriol. When the concentration of estriol is increased, some of the estradiol is displaced from the enzyme and the total activity of the enzyme system is decreased.

Two hypotheses have been proposed for the mechanism of action of estrogens on the enzyme system of the placenta. One states that the estrogen combines with an inactive form of the enzyme and converts it to an active form (Hagerman and Villee, 1957). When this theory was formulated the evidence indicated that the estrogen acted on a specific DPN-linked isocitric dehydrogenase. The theory is equally applicable if the

STEROID SEX HORMONES

657

estrogen-sensitive enzyme is a transhydrogenase, as the evidence now indicates. The results of kinetic studies with the coupled isocitric dehydrogenase-transhydrogenase system are consistent with this theory (Gordon and Villee, 1955; Villee, 1957b; Hagerman and Villee, 1957). Apparent binding constants for the enzyme-hormone complex (Gordon and Villee, 1955j and for enzyme-inhibitor complexes have been calculated (Hagerman and Villee, 1957).

The observation that estradiol and estrone, which differ in structure only by a pair of hydrogen atoms, are equally effective in stimulating the reaction suggested that the steroid might be acting in some way as a hydrogen carrier from substrate to pyridine nucleotide (Gordon and Villee, 1956). Talalay and Williams-Ashman (1958) suggested that the estrogens act as coenzymes in the transhydrogenation reaction and postulated that the reactions were:

Estrone + TPNH + H*

— Estradiol + TPN^

Estradiol + DPN+

— Estrone + DPNH + H*

Sum : TPNH

H*

- DPN^ — TPN^ + H^

DPNH

This formulation implies that the estrogen-sensitive transhydrogenation reaction is catalyzed by the estradiol-17y3 dehydrogenase characterized by Langer and Engel (1956). This enzyme was shown by Langer (1957) to use either DPN or TPN as hydrogen acceptor but it reacts more rapidly with DPN. Ryan and Engel (1953) showed that this enzyme is present in rat liver, and in human adrenal, ileum, and liver. However, no estrogen-stimulable enzyme is demonstrable in rat or human liver (Villee, 1955). The nonparticulate fraction obtained by high speed centrifugation of homogenized rabbit liver rapidly converts estradiol to estrone if DPN is present as hydrogen acceptor, but does not contain any estrogenstimulable transhydrogenation system.

It will not be possible to choose between these two hypotheses until either the estrogen-sensitive transhydrogenase and the estradiol dehydrogenase have been separated or there is conclusive proof of their

identity. Talalay, Williams-Ashman and Hurlock (1958) reported a 100-fold purification of the dehydrogenase without separation of the transhydrogenase activity and found that both activities were inhibited identically by sulfhydryl inhibitors. In contrast, Hagerman and Villee (1958) obtained partial separation of the two activities by the usual techniques of protein fractionation, and reported that a 50 per cent inhibition of transhydrogenase is obtained with p-chloromercurisulfonic acid at a concentration of 10~^ m whereas 10"^ m p-chloromercurisulfonic acid is required for a 50 per cent inhibition of the dehydrogenase. The evidence that these two activities are mediated by separate and distinct proteins has been summarized by Villee, Hagerman and Joel (1960).

The transhydrogenase present in the mitochondrial membranes of heart muscle was shown by Ball and Cooper (1957) to be inhibited by 4 X 10"^ m thyroxine. The estrogen-sensitive transhydrogenase of the placenta is also inhibited by thyroxine (Villee, 1958b). The degree of inhibition is a function of the concentration of the thyroxine and the inhibition can be overcome by increased amounts of estrogen. Suitable control experiments show that thyroxine at this concentration does not inhibit the glucose 6-phosphate dehydrogenase or isocitric dehydrogenase used as TPNH-generating systems to couple with the transhydrogenase. Triiodothyronine also inhibits the estrogen-sensitive transhydrogenase but tyrosine, diiodotyrosine and thyronine do not. The thyroxine does not seem to be inhibiting by binding the divalent cation, Mn + + or ]Mg+ + , required for activity, for the inhibition is not overcome by increasing the concentration of the cation 10-fold.

In the intact animal estrogens stimulate the growth of the tissues of certain target organs. The estrogen-sensitive enzyme has been shown to be present in many of the target organs of estrogens: in human endometrium, myometrium, placenta, mammary gland, and mammary carcinoma, in rat ventral prostate gland and uterus, and in mammotrophic-dependent transplantable tumors of the rat and mouse pituitary. In contrast, it is not demonstrable in comparable preparations from liver, heart, lung, brain, or

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PHYSIOLOGY OF GONADS

kidney. The growth of any tissue involves the utilization of energy, derived in large part from the oxidation of substrates, for the synthesis of new chemical bonds and for the reduction of substances involved in the synthesis of compounds such as fatty acids, cholesterol, purines, and pyrimidines.

The physiologic responses to estrogen action, such as water imbibition and protein and nucleic acid synthesis, are processes not directly dependent on the activity of transhydrogenase. However, all of these processes are endergonic, and one way of increasing their rate would be to increase the supply of biologically available energy by speeding up the Krebs tricarboxylic acid cycle and the flow of electrons through the electron transmitter system. Much of the oxidation of substrates by the cell produces TPNH, whereas the major fraction of the biologically useful energy of the cell comes from the oxidation of DPNH in the electron transmitter system of the cytochromes. Hormonal control of the rat of transfer of hydrogens from TPN to DPN could, at least in theory, influence the over-all rate of metabolism in the cell and secondarily influence the amount of energy available for synthetic processes. Direct evidence of this was shown in our early experiments in which the oxygen consumption of tissue slices of target organs was increased by the addition of estradiol (Hagerman and Villee, 1952; Villee and Hagerman, 1953).

This theory assumes that the supply of energy is rate-limiting for synthetic processes in these target tissues and that the activation of the estrogen-sensitive enzyme does produce a significant increase in the supply of energy. The addition of estradiol in vitro produces a significant increase in the total amount of isocitric acid dehydrogenated by the placenta (Villee, Loring and Sarner, 1958) . Slices of endometrium to which no estradiol was added in vitro utilized oxygen and metabolized substrates to carbon dioxide at rates which paralleled the levels of estradiol in the blood and urine of the patient from whom the endometrium was obtained (Hagerman and Villee, esses in these target tissues and that the 1953b). Estradiol increases the rate of synthesis of ATP by liomogenates of human

placenta (Villee, Joel, Loring and Spencer, 1960).

The reductive steps in the biosynthesis of steroids, fatty acids, purines, serine, and other substances generally require TPNH rather than DPNH as hydrogen donor. The cell ordinarily contains most of its TPN in the reduced state and most its DPN in the oxidized state (Glock and McLean, 1955). If the amount of TPN+ is ratelimiting, a transhydrogenase, by oxidizing TPN and reducing DPN, would permit further oxidation of substrates such as isocitric acid and glucose 6-phosphate, which require TPN+ as hydrogen acceptor and which are key reactions in the Krebs tricarboxylic acid cycle and the hexose monophosphate shunt, respectively. Furthermore, the experiments of Kaplan, Schwartz, Freeh and Ciotti (1956) indicate that less biologically useful energy, as ATP, is obtained when TPNH is oxidized by TPNH cytochrome c reductase than when DPNH is oxidized by DPNH cytochrome c reductase. Thus, a transhydrogenase, by transferring hydrogens from TPNH to DPN before oxidation in the cytochrome system, could increase the energy yield from a given amount of TPNH produced by isocitrate or glucose 6-phosphate oxidation. The increased amount of biologically useful energy could be used for growth, for protein and nucleic acid synthesis, for the imbibition of water, and for the other physiologic effects of estrogens.

Estrogen stimulation of the transhydrogenation reaction would tend to decrease rather than increase the amount of TPNH in the cell. Thus the estrogen-induced stimulation of the synthesis of steroids, fatty acids, proteins, and purines in the uterus can be explained more reasonably as due to an increased supply of energy rather than to an increased supply of TPNH.

The theory that estrogens stimulate transhydrogenation by acting as coenzymes which are rapidly and reversibly oxidized and reduced does not explain the pronounced estrogenic activity in vivo of stilbestrol, 17a-ethinyl estradiol, or bfsdehydrodoisynolic acid, for these substances do not contain groups that could be readily oxidized or reduced. The exact mechanism

STEROID SEX HORMONES

(359

of action of estrogens at the biochemical level remains to be elucidated, but the data available permit the formulation of a detailed working hypothesis. The notable effects of estrogens and androgens on behavior (see chapter by Young) are presumably due to some direct or indirect effect of the hormone on the central nervous system. The explanation of these phenomena in physiologic and biochemical terms remains for future investigations to provide.

B. ANDROGENS

Although there is a considerable body of literature regarding the responses at the biologic level to administered androgens and progesterone, much less is known about the site and mechanism of action of these hormones than is known about the estrogens. The review by Roberts and Szego (1953b) deals especially with the synergistic and antagonistic interactions of the several steroidal sex hormones.

The rapid growth of the capon comb following the administration of testosterone has been shown to involve a pronounced increase in the amount of mucopolysaccharide present, as measured by the content of glucosamine (Ludwig and Boas, 1950; Schiller, Benditt and Dorfman, 1952). It is not known whether the androgen acts by increasing the amount or activity of one of the enzymes involved in the synthesis of polysaccharides or whether it increases the amount or availability of some requisite cofactor. Many of the other biologic effects of androgens do not seem to involve mucopolysaccharide synthesis and the relation of these observations to the other roles of androgens remains to he determined.

Mann and Parsons (1947) found that castration of rabbits resulted in a decreased concentration of fructose in the semen. Within 2 to 3 weeks after castration the amount of fructose in the semen dropped to zero, but rapidly returned to normal following the subcutaneous implantation of a pellet of testosterone. Fructose reappeared in the semen of the castrate rat 10 hours after the injection of 10 mg. of testosterone (Rudolph and Samuels, 1949). The coagulating gland of the rat, even when trans

planted to a new site in the body, also responds by producing fructose when the host is injected with testosterone. The amount of citric acid and ergothioneine in the semen is also decreased by castration and increased by the implantation of testosterone pellets (Mann, 1955). The experiments of Hers (1956) demonstrate that fructose is produced in the seminal vesicle by the reduction of glucose to sorbitol and the subsequent oxidation of sorbitol to fructose. The reduction of glucose requires TPNH as hydrogen donor and the oxidation of sorbitol requires DPN as hydrogen acceptor. The sum of these two reactions provides for the transfer of hydrogens from TPNH to DPN. If androgens act as cofactors which are reversibly oxidized and reduced, and thus transfer hydrogens from TPNH to DPN as postulated by Talalay and Williams-Ashman (1958), one would expect that an increased amount of androgen, by providing a competing system for hydrogen transfer, would decrease rather than increase the production of fructose. The marked increases in the citric acid and ergothioneine content of semen are not readily explained by this postulated site of action of androgens.

An increase in the activity of /3-glucuronidase in the kidney has been reported following the administration of androgens (Fishman, 1951). This might be interpreted as an arlaptive increase in enzyme induced by the increased concentration of substrate, or by a direct effect of the steroid on the synthesis of the enzyme.

The respiration of slices of prostate gland of the dog is decreased by castration or by the administration of stilbestrol (Barron and Huggins, 1944). The decrease in respiration occurs with either glucose or pyruvate as substrate. The seminal vesicle of the rat responds similarly to castration. Rudolph and Samuels (1949) found that respiration of slices of seminal vesicle is decreased by castration and restored to normal values within 10 hours after the injection of testosterone. Experiments by Dr. Phillip Corfman in our laboratory with slices of prostate gland from patients with benign prostatic hypertrophy showed that oxygen utilization was reduced 50 per cent by estradiol added

660

PHYSIOLOGY OF GONADS

in vitro at a level of 1 /xg. per ml. Respiration of slices of the ventral prostate gland of the rat is decreased by castration and increased by administered testosterone (Nyden and Williams-Ashman, 1953). These workers showed that lipogenesis from acetate-l-C^* in the prostate is also significantly diminished by castration and restored to normal by administered testosterone.

The succinic dehydrogenase of the liver has been found to be increased by castration and decreased by the administration of testosterone (Kalman, 1952; Rindani, 1958), the enzyme is also inhibited by testosterone added in vitro (Kalman, 1952). In contrast, Davis, Meyer and McShan (1949) found that the succinic dehydrogenase of the prostate and seminal vesicles is decreased by castration and increased by the administration of testosterone.

An interesting example of an androgen effect on a specific target organ is the decreased size of the levator ani and other perineal muscles of the rat following castration. The administration of androgen stimulates the growth of these muscles and increases their glycogen content (Leonard, 1952). However, their succinoxidase activity is unaffected by castration or by the administration of testosterone. Courrier and Marois (1952) reported that the growth of these muscles stimulated by androgen is inhibited by cortisone. The remarkable responsiveness of these muscles to androgens in vivo gave promise that slices or homogenates of this tissue incubated with androgens might yield clues as to the mode of action of the male sex hormones. Homogenates of perineal and masseter muscles of the rat responded to androgens administered in vivo with increased oxygen consumption and ATP production iLoring, Spencer and Villee, 1961). The experiments suggested that the activity of DPNH-cytochromo r reductase in these tissues is controlled by aiKh'ogeiis.

C. PROGESTERONE

Attempts to clarify the biochemical basis of the role of progesterone have been hampered by the requirement, in most instances, for a previous stimulation of the tissue by estrogen. The work of Wade and Jones

(1956a, b) demonstrated an interesting effect of progesterone added in vitro on several aspects of metabolism in rat liver mitochondria. Progesterone, but not estradiol, testosterone, 17a-hydroxyprogesterone, or any of several other steroids tested, stimulated the adenosine triphosphatase activity of rat liver mitochondria. This stimulation is not the result of an increased permeability of the mitochondrial membrane induced by progesterone, for the stimulatory effect is also demonstrable with mitochondria that have been repeatedly frozen and thawed to break the membranes. Other experiments showed that ATP was the only substrate effective in this system ; progesterone did not activate the release of inorganic phosphate from AMP, ADP, or glycerophosphate.

In other experiments with rat liver mitochondria (Wade and Jones, 1956b), progesterone at a higher concentration (6 X lO"'* m) was found to inhibit the utilization of oxygen with one of the tricarboxylic acids or with DPNH as substrate. This inhibition is less specific and occurred with estradiol, testosterone, pregnanediol, and 17a-hydroxy progesterone, as well as with progesterone. The inhibition of respiration by high concentrations of steroids in vitro has been reported many times and with several different tissues; it seems to be relatively unspecific. Wade and Jones were able to show that progesterone inhibits the reduction of cytochrome c but accelerates the oxidation of ascorbic acid. They concluded that progesterone may perhaps uncouple oxidation from phosphorylation in a manner similar to that postulated for dinitrophenol. The site of action of this uncoupling appears to be in the oxidation-reduction path between DPNH and cytochrome c. Mueller (1953) found that progesterone added in vitro decreases the incorporation of glycine-2-C^'* into the protein of strips of rat uterus, thus counteracting the stimulatory effect of estradiol administered in vivo.

Zander (1958) reported that A4-3-ketopregnene-20-a-ol and A4-3-ketopregnene20-^-ol arc effective gestational hormones in the mouse, rabbit, and man, although somewhat less active in general than is progesterone. An enzyme in rat ovary which converts progesterone to pregnene-20-a-ol, and also catalyzes the reverse reaction, was

STEROID SEX HORMONES

661

described by Wiest (1956). The conversion occurred when slices of ovary were incubated with DPN. Wiest postulated that the progesterone-pregnene-20-a-ol system might play a role in hydrogen transfer, in a manner analogous to that postulated by Talalay and Williams-Ashman (1958) for estrone-estradiol- 17^, but his subsequent experiments ruled out this possibility, for he was unable to demonstrate any progesterone-stimulable transhydrogenation reaction.

The nature of the effect of progesterone and of estrogens on myometrium has been investigated extensively by Csapo. Csapo and Corner (1952, 1953) found that ovariectomy decreased the maximal tension of the myometrium and decreased its content of actomyosin. The administration of estradiol to the ovariectomized rabbit over a period of 7 days restored both the actomyosin content and the maximal tension of the myometrium to normal. The concentration of ATP and of creatine phosphate in the myometrium is decreased by ovariectomy but is restored by only 2 days of estrogen treatment. This suggests that the effect on intermediary metabolism occurs before the effect on protein {i.e., actomyosin) synthesis. Csapo (1956a) concluded that estrogen is a limiting substance in the synthesis of the contractile proteins of myometrium, but he could not differentiate between an effect of estrogen on some particular biosynthetic reaction and an effect of estrogen on some fundamental reaction which favors synthesis in general. He was unable to demonstrate any comparable effect of progesterone on the contractile actomyosin-ATP system of the myometrium.

Other observations provide an explanation for the well known effect of progesterone in decreasing the contractile activity of myometrium, not by any effect on the contractile system itself, but in some previous step in the excitation process. Under the domination of progesterone the myometrial cells have a decreased intracellular concentration of potassium ions and an increased concentration of sodium ions (Horvath, 1954). The change in ionic gradient across the cell membrane is believed to be responsible for the altered resting potential and the partial depolarization of the cell mem

brane which results in decreased conductivity and decreased pharmacologic reactivity of the myometrial cell. The means by which progesterone produces the changes in ionic gradients is as yet unknown. Csapo postulates that the hormone might decrease the rate of metabolism which in turn would lessen the rate of the "sodium pump" of the cell membrane. The contractile elements, the actomyosin-ATP system, are capable of full contraction but, because of the partial block in the mechanism of excitation and of propagation of impulses (Csapo, 1956b), the muscle cells cannot operate effectively; the contractile activity remains localized. Csapo (1956a) showed that the progesterone block is quickly reversible and disappears if progesterone is withdrawn for 24 hours. He concluded that the progesterone block is necessary for the continuation of pregnancy and that its withdrawal is responsible for the onset of labor.

]\Iost investigators who have speculated about the mode of action of steroids — whether they believe the effect is by activating an enzyme, by altering the permeability of a membrane, or by serving as a coenzyme in a given reaction— have emphasized the physical binding of the steroid to a protein as an essential part of the mechanism of action or a preliminary step to that action. They have in this way explained the specificities, synergisms, and antagonisms of the several steroids in terms of the formation of specific steroid-protein complexes. The differences between different target organs, e.g., those that respond to androgens and those that respond to estrogens, can be attributed to differences in the distribution of the specific proteins involved in these binding reactions. Viewed in this light, the problem of the mode of action of sex hormones becomes one aspect of the larger problem of the biochemical basis of embryonic differentiation of tissues.

IV. References

Allen, W. M. 1939. Biochemistry of the corpus luteum hormone, progesterone. In Sex and Internal Secretions. 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., pp. 901-928. Baltimore : The Wilhams & Wilkins Company.

AsTWOOD, E. B. 1938. A six-hour assay for the quantitative determination of estrogen. Endocrinology, 23, 25-31.

B.-vcGETT, B., Engel, L. L., Savard, K., .and Dorfman,

062

PHYSIOLOGY OF GONADS

R. I. 1956. The conversion of testosterone3C" to C"-estradiol-17/3 by human ovarian tissue. J. Biol. Chem., 221, 931-941. Ball, E. G., .4nd Cooper, O. 1957. Oxidation of reduced triphosphopyridine nucleotide as mediated by the transliydrogenase reaction and its inhibition by thvroxine. Proc. Nat. Acad. Sc, 43, 357-364.

B.AU\NY, E., MORSING, P., MxJLLER, W., StALLBERG,

G., AND Stenhagen, E. 1955. Inhibition of estrogen-induced proliferation of the vaginal epithelium of the rat by topical application of certain 4, 4'-hydroxy-diphenyl-alkanes and related compounds. Acta Soc. Med. Uppsala, 60, 68-74.

B.\RR0N, E. S. G., .\ND HUGGIN.S, C. 1944. The metabolism of isolated prostatic tissue. J. Urol., 51, 630-634.

Bever, a. T., Velardo, J. T., and His.w, F. L. 1956. Action of estrogens on lactic dehydrogena.se-DPNH oxidase system of rat uterus. Endocrinology, 58, 512-522.

Bloch, K. 1951. II. Use of isotopes in liormone problems: the biologic synthesis of cholesterol. Recent Progr. Hormone Res., 6, 111-129.

BoNGiovANNi, A. M. 1953. The detection of pregnanediol and pregnanetriol in the urine of patients with adrenal hyperplasia. Suppression with cortisone. Bull. John Hopkins Hosp., 92, 244-251.

Carroll, W. R. 1942. Influence of estrogen on respiration of rat uterine tissue. Proc. Soc. Exper. Biol. & Med., 49, 50-52.

Caspi, E., Rosenfeld, G., and Dorfman, R. T. 1956. Degradation of cortisol-C" and corticosterone-C" biosynthesized from acetate1-C". J. Organ. Chem., 21, 814-815.

Cl.ayton, R. B., and Bloch, K. 1956. The biologic conversion of lanosterol to cholesterol. J. Biol. Chem.. 218, 319-325.

CoLOWicK, S. P., CoRi, G. T., .AND Slein, M. W. 1947. The effect of adrenal cortex and anterior pituitary extracts and insulin on the hexokina.se reaction. J. Biol. Chem., 168, 583-596.

CouRRiER, R., AND Marois, M. 1952. Endocrinologie: relations entre la testosterone et le cortisone dans leur effets sur certains organes recepteiM's. Compt. rend. Acad. Sc, 234, 271273.

Cs.APO, A. 1956a. Progesterone Ijlock. Am. J. Anat., 98, 273.

CsAPO, A. 1956b. The mechanism of effect of the ovarian steroids. Recent Progr. Hormone Res., 12,405-431.

CsAPO, A., AND Corner, G. W. 1952. The antagonistic effects of estrogen and progesterone on the staiicase phenomenon in uterine muscle. Endocrinology, 51, 378-385.

CsAPO, A., AND Corner, G. W. 1953. The effect of estrogen on the isometric tension of rabbit uterine strips. Science, 117, 162-164.

Daughaday, W. H. 1956. E\idence for iwo corticosteroid binding systems in human plasma. J. Lab. & Clin. Med., 48, 799-800.

David, J. C. 1931. The action of estrin on the

ox.ygen consumption of the uteri of mice. J. Pharmacol. & Exper. Therap., 43, 1-11.

Davis, J. S., Meyer, R. C, and McShan, W. H. 1949. Effect of androgen and estrogen on succinic dehydrogenase and cytochrome oxidase of rat prostate and seminal vesicle. Endocrinology, 44, 1-7.

DoiSY, E. A. 1939. Biochemistry of estrogenic compounds. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., pp. 846-876. Baltimore: The Williams & Wilkins Companv.

DoiSY, E. A., Veler, CD., .\nd Th.ayer, S. A. 1929. Folliculin from urine of pregnant women. Am. J. Physiol., 90, 329-330.

E.AGLE, H. 1955. The specific amino acid requirements of a human carcinoma cell (strain HeLa) in tissue culture. J. Exper. Med., 102, 37-48.

Eidinoff, M. L., Knoll, J. E., Marano, B. J., KvAM.ME, E., Rosenfeld, R. S., and Hellman, L. 1958. Cholesterol biosynthesis: studies related to the metabolic role of squalene. J. Clin. Invest., 37, 655-659.

E.MMELOT, P., AND Bos, L. 1954. Thc influence of estrogens on the protein and lipid metabolism of the mouse uterus studied with acetate1-C". Rec. Trav. Chim., 73, 874-877.

FiSHMAN, W. H. 1951. Relationship between estrogens and enzvme activitj'. Vitamins & Hormones, 9, 213-236.

Glass, R. B., Loring. J. M., Spencer, J. M., and ViLLEE, C. A. 1961. The estrogenic properties in vitro of diethylstilbestrol and substances related to estradiol. Endocrinologv, 68, 327-333.

Glock, G. E., and McLean, P. 1955. Levels of oxidized and reduced diphosphopyridine and triphosphopyridine nucleotide in animal tissues. Biochem. J., 61, 388-390.

Gordon, E. E., .and Villee, C. A. 1955. Spectrophotometric studies of the stimulation of human placental preparations bv estradiol. J. Biol. Chem., 216, 215-224.

Gordon, E. E., and Villee, C. A. 1956. An in vitro assaj' for estradiol-17|3 and estrone. Endocrinology, 58, 150-157.

Grauer, R. C, Strickler, H. S., Wolken, J. J., and CuTULY, E. 1950. Influence of estradiol on P" uptake bv the uterus. Proc. Soc. Exper. Biol. & Med.,75, 651-654.

H.\germ.an, D. D., and Villee, C. A. 1952. Effects of estradiol on the metal)olism of human endometrium in vitro. Arch. Biocliem., 40, 481483.

Hagerman, D. D., .AND Villee, C. A. 1953a. Effects of estradiol on the metabolism of human endometrium in vitro. J. Biol. Clunn., 203, 425431.

Hagerman, D. D., AND Villee, C. A. 1953b. Effects of the menstrual cycle on the metabolism of human endometrium. Endocrinology, 53, 667-673.

HAGER.^L\x, D. D., AND ViLLEE, C. A. 1957. Estro

STEROID SEX HORMONES

663

gen sensitive isocitric dehydrogenase. J. Biol. Chem., 229, 589-597.

Hagerman, D. D., and Villee, C. A. 1958. Metabolic studies of the mechanism of action of estrogens. In Symposium on the Endocrinology of Pregnancy, Charles Lloyd, Ed., pp. 317328. New York: Academic Press, Inc.

Hagerman, D. D., Wellington, F. M., and Villee, C. A. 1957. Estrogens in marine invertebrates. Biol. Bull., 112, 180-183.

Hayano, M., and Dorf>l\n, R. I. 1953. The enzymatic C-lli3-hvdroxylation of steroids. J. Biol. Chem., 201,^175-188.

Hayano, M., Lindberg, M. C, Dorfman, R. I., Hancock, J. E. H., and Doering, W. von E. 1955. On the mechanism of the C-ll/3-hydroxylation of steroids; a study with H20^* and 02'^ Arch. Biochem., 59, 529-532.

Haynes, R. C, Sutherland, E. W., and Rall, T. W. 1960. The role of cyclic adenylic acid in hormone action. Recent Progr. Hormone Res., 16, 121-132.

Henion, W. F., and Sutherland, E. W. 1957. Immunologic differences of phosphorylases. J. Biol. Chem., 224, 477-488.

Herranen, a., and Mueller, G. C. 1956. Effect of estradiol on the metabolism of serine-3-C^* in surviving uterine segments. J. Biol. Chem., 223, 369-375.

Herranen, A. M., and Mueller, G. C. 1957. The effect of estradiol pretreatment on the serine aldolase activity of rat uteri. Biochim. et Biophys. Acta, 24, 223-224.

Hers, H. G. 1956. La mecanisme de la transformation de glucose ou fructose par les vesicles seminales. Biochim. et Biophys. Acta, 22, 202-203.

Hoagland, M. B., Keller, E. B., and Zamecnik, P. C. 1956. Enzymatic carboxyl activation of amino acids. J. Biol. Chem., 218, 345-358.

Holden, R. B. 1939. Vascular reactions of the uterus of the immature rat. Endocrinology, 25, 539-596.

Hollander, V. P., Nolan, H. M., and Hollander, N. 1958. The structural specificity of the estrogen-sensitive enzyme system in placental homogenates. J. Biol. Chem., 233, 580-582.

HoRVATH, B. 1954. Ovarian hormones and the ionic balance of uterine muscle. Proc. Nat. Acad.Sc, 40, 515-521.

Jailer, J. W. 1953. Virilism. Bull. New York Acad. Med., 29, 377-394.

Jones, H. W., Jr., W.^de, R., and Goldberg, B.

1952. Phosphate liberation by endometrium in the presence of adenosine triphosphate. Am. J. Obst. & Gynec, 64, 111&-1124.

Jones, H. W., Jr., Wade, R., and Goldberg, B.

1953. The uterus: biochemical and histochemical alkaline gh^cerophosphatase in normal endometrium, endometrial hyperplasia and adenocarcinoma. Obst. & Gynec. Surv., 8, 398-400.

Kalman, S. M. 1952. The effect of androgens on the succinoxide system of rat liver. Endocrinology, 50, 361-365.

Kaplan, N. O., Ciotti, M. M., Hamolsky, M., and Bieber, R. E. 1960. Molecular heterogeneity and evolution of enzymes. Science, 131, 392397. Kaplan, N., Schwartz, M., Frech, M.. and Ciotti, M. 1956. Phosphorylative and nonphosphorylative pathways of electron transfer in rat liver mitochondria. Proc. Nat. Acad. Sc, 42, 481-487. Kerly, M. 1937. The effect of the estrous cycle on the metabolism of isolated rat uterus. Biochem. J., 31, 1544-1552. Kh.\yyal, M. a., and Scott, C. M. 1931. The oxygen consumption of the isolated uterus of the rat and mouse. J. Physiol., 72, 13P-14P. KiPNis, D. M., and Cori, C. F. 1957. Studies of tissue permeability. III. The effect of insulin on pentose uptake by the diaphragm. J. Biol. Chem., 224, 681-693. Knobil, E., Morse, A., Wolf, R. C, and Creep, R. O. 1958. The action of bovine, porcine, and simian growth hormone preparations on the costochondral junction in the hypophysectomized rhesus monkey. Endocrinology, 62, 348-354. Knox, W. E., and Auerb.ach, V. H. 1955. The hormonal control of trvptophan peroxidase in the rat. J. Biol. Chem., 214, 307-313. Knox, W. E., Auerbach, V. H., and Lin, E. C. C. 1956. Enzymatic and metabolic adaptations in animals. Physiol. Rev., 36, 164-254. Koch, F. C. 1939. Biochemistry of androgens. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., pp. 807-845. Baltimore: The Williams & Wilkins Company. Kochakian, C. D. 1947. Effects of estrogens on the body and organ weights and the arginase and "alkaline" and "acid" phosphatases of the liver and kidney of castrated male mice. Am. J.Physiol., 151, 126-129. Krahl, M. E., and Bornstein, J. 1954. Inhibition of glucose use in muscle extracts by lipoproteins. Nature, London, 173, 949-950. L.\NGDON, R. G., AND Bloch, K. 1953. The biosvnthesis of squalene. J. Biol. Chem., 200, 129-134. Langer, L. 1957. Preparation and properties of estradiol-17/3 dehydrogenase. Thesis, Radcliffe College. L.\nger, L., and Engel, L. L. 1956. Human placental estradiol-17-/3 dehydrogenase. Fed. Proc, 15, 296; J. Biol. Chem., 233, 583-588. Leonard, S. L. 1952. A glycostatic effect of testosterone on the perineal muscles of the rat. Endocrinology, 50, 199-205. Levedahl, B. H., .\nd Bernstein, H. 1954. Testosterone binding by modified bovine serum albumin. Arch. Biochem. , 52, 353-357. Levine, R., and Goldstein, M. S. 1955. On the mechanism of action of insulin. Recent Progr. Hormone Res., 11, 343-380. f

Li, C. H., and Papkoff, H. 1956. Preparation and properties of growth hormone from human

664

PHYSIOLOGY OF GONADS

and monkey pituitary glands. Science, 124, 1293-1294.

LoRixG. J. M., Spencer. J. M., .and Villee, C. A. 1961. Some effects of androgens on intermediar\' metabolism in muscle. Endocrinology, 68, 501-506.

LoRi.vG, J. M.. AND Villee, C. A. 1957. The estrogen content of human placentas. Acta endocrinoL, 25, 371-376.

LuDwiG, A. W., AND Boas, N. F. 1950. The effects of testosterone on the connective tissue of the comb of the cockerel. Endocrinology, 46, 291298.

MacLeod, J., and Reynolds, S. R. M. 1938. Vascular, metabolic, and motility responses of uterine tissue following administration of estrin. Proc. Soc. Exper. Biol.. & Med., 37, 666-668.

Mann, T. 1955. Male sex hormone and its role in reproduction. Recent Progr. Hormone Res., 12, 353-376.

Mann, T., and Parsons, U. 1947. Effect of testicular hormone on the formation of seminal fructose. Nature, London, 160, 294.

Marrian, G. F., and Bauld. W. S. 1955. The isolation of 16-epioestriol from the urine of pregnant women. Biochem. J., 59, 136-141.

Meyer, A. S. 1955. 19-Hydroxylation of Mandrostene-3 , 17-dione and dehydroepiandrosterone by bovine adrenals. Experientia, 11, 99-102.

Mueller, G. C. 1953. Incorporation of glycine2-C" into protein by surviving uteri from estradiol-treated rats. J. Biol. Chem., 204, 77-90.

Mueller, G. C. 1957. A di.^^cussion of the mechanism of action of steroid liormones. Cancer Res., 17, 490-506.

Mueller, G. C., and Herranen, A. 1956. Metabolism of 1-carbon fragments by surviving uteri from estradiol-treated rats. J. Biol. Chem., 219, 585-594.

Mupxler, G. C, Herranen, A. M., and Jervell, K. F. 1958. Studies on the mechanism of action of estrogens. Recent Progr. Hormone Res., 14, 95-139.

Nathanson, I. T., Engel. L. L., .and Kelley, R. M. 1951. Effect of ACTH on urinary excretion of steroids in neoplastic disease. In Proceedings of the Second ACTH Conjerence, J.R. Mote, Ed., Vol. 1, p. 54.

NyDEN, S. J., AND WILLLAMS-ASHMAN, H. G. 1953.

Influence of androgens on synthetic reactions

in ventral prostate tissue. Am. J. Plivsiol.,

172,588-600. Odell, L. D., AND Flshman, W. H. 1950. Studios

on /3-glucuronidase. I. Activities in himian

endometrium. Am. J. Obst. & Gvnec, 59, 200 203. Page, E. W., Glendening, M. B., and Parkin.son.

P. 1951. Cyclic biochemical changes in tlic

human endometrium. Am. J. Obst. it Gvn(H'.,

62, 1100-1105. Pop.iAK, G. 1950. Preparation of solid samples

for assay of C". Biochem. J., 46, 560-561.

Rilling, H., Tchen, T. T., and Bloch, K. 1958. On the mechanism of srjualene biogenesis. Proc. Nat. Acad. Sc, 44, 167-173.

RiND.ANi, T. H. 1958. Influence of testosterone propionate on succinic dehydrogenase activity of male rat liver. Am. J. Phj^siol., 193, 73-74.

Roberts, S., and Szego, C. M. 1953a. The influence of steroids on uterine respiration and glycolysis. J. Biol. Chem., 201, 21-29.

Roberts, S., and Szego, C. M. 1953b. Steroid interactions in the metabolism of reproductive target organs. Physiol. Rev., 33, 593-629.

Rudolph, G. G., and S.amuels, L. T. 1949. Early effects of testosterone propionate on the seminal vesicles of castrate rats. Endocrinology, 44, 190-196.

Ryan, K. 1958. Conversion of androgens to estrogens bv human placental microsomes. Fed. Proc, 17, 1382.

Ryan, K. J., and Engel, L. L. 1953. The interconversion of estrone and estradiol-17i3 by rat liver slices. Endocrinology, 52, 277-286.

Schiller, S., Benditt, E., and Dorfman, A. 1952. Effect of testosterone and cortisone on the hexosamine content and metachromasia of chick combs. Endocrinology, 50, 504-510.

Solomon, S., Levitan, P., and Lieberman, S. 1956. Possible intermediates between cholesterol and pregneneolone in corticosteroidogenesis. Abstract. Proc. Canad. Physiol. Soc, Rev. Canad. Biol., 15, 282.

Spaziani, E., and Szego, CM. 1958. The influence of estradiol and Cortisol on uterine histamine of the ovariectomized rat. Endocrinology, 63, 669-678.

Stuermer, V. M., .AND Stein, R. J. 1952. Cytodynamic properties of the human endometrium. V. Metabolism and the enzymatic activity of the human endometrium during the menstrual cycle. Am. J. Obst. & Gynec, 63, 359-370.

Tal.alay, p., and Williams-Ashman, H. G. 1958. Activation of hydrogen transfer between pyridine nucleotides bv steroid hormones. Proc Nat. Acad. Sc, 44, 15-26.

Tal.alay, P., Willi.ams-Ashman, H. G., and HurLOCK, B. 1958. Steroid hormones as coenzymes of hydrogen transfer. Science, 127, 1060.

Tchen, T. T., and Bloch, K. 1955. In vitro conversion of squalene to lanosterol and cholesterol. J. Am. Chem. Soc, 77, 6085-6086.

Telfer, M. a. 1953. Influence of estradiol on nucleic acids, respiratory enzymes and the distribution of nitrogen in the rat uteru.*. Arch. Biochem., 44, 111-119.

Victor, J., and Andersen, D. H. 1937. Stimulation of anterior hypophysis metabolism by theelin or dihydrotheelin. Am. J. Physiol., 120, 154-166.

Villee. C. A. 1955. An estradiol-induccd stimulation of citrate utilization by placenta. J. Biol. Chem., 215, 171-182.

Villee, C. A. 1957a. Effects of estrogens and antiestiogens in vitro. Cancer Res.. 17, 507511.

STEROID SEX HORMONES

665

ViLLEE, C. A. 1957b. Role of estrogens in regulating the metabolism of the placenta and endometrium. Fertil. & Steril., 8, 156-163.

ViLLEE, C. A. 1958a. Estrogens and uterine enzymes. Ann. New York Acad. Sc, 75, 524534.

ViLLEE, C. A. 1958b. Antagonistic effects of estrogens and thyroxine on an enzyme system in vitro. In Proceedings IV International Congress Biochemistry {Vienna), p. 115. London: Pergamon Press.

ViLLEE, C. A., AND GoRDON, E. E. 1955. Further studies on the action of estradiol in vitro. J. Biol. Chem., 216, 203-214.

ViLLEE, C. A., AND GoRDON, E. E. 1956. The stimulation by estrogens of a DPN-linked isocitric dehydrogenase from human placenta. Bull. Soc. chim. belg., 65, 186-201.

ViLLEE, C. A., AND Hagerman, D. D. 1953. Effects of estradiol on the metabolism of himian placenta in vitro. J. Biol. Chem., 205, 873-882.

ViLLEE, C. A., and Hagerman, D. D. 1957. Studies on the estrogen-sensitive isocitric dehydrogenase of the human placenta. In Proceedings International Syniposiinn Enzyme Chemistry. {Japan), Vol. 1, p. 287. Tokyo: Maruzen.

ViLLEE, C. A., AND H.\GER.MAN, D. D. 1958. On the identity of the estrogen-sensitive enzyme of human placenta. J. Biol. Chem., 233, 42-48.

ViLLEE, C. A., Hagerman, D. D., and Joel, P. B. 1960. An enzyme basis for the physiologic functions of estrogens. Recent Progr. Hormone Res., 16, 49-69.

ViLLEE, C. A., Joel. P. B., Loring, J. M., .\nd Spencer, J. M. 1960. Estrogen stimulation of ATP production and protein svnthesis. Fed. Proc, 19, 53.

ViLLEE, C. A., Loring, J. M.. and Sarner, A. 1958.

Isocitric dehydrogenases of the placenta. Fed Proc, 17, 328.

Wade, R., and Jones, H. W., Jr. 1956a. Effect of progesterone on mitochondrial adenosinetriphosphatase. J. Biol. Chem., 220, 547-551.

Wade, R., and Jones, H. W., Jr. 1956b. Effect of progesterone on oxidative phosphorylation. J. Biol. Chem.. 220, 553-562.

Walaas, O., and Walaas, E. 1950. The metabolism of uterine muscle studied with radioactive phosphorus P^". Acta physiol. scandinav 21, 18-26.

Wal-aas, O.. Wal.aas, E., .\nd Loken, F. 1952a. The effect of estradiol monobenzoate on the metaboli-sin of rat uterine muscle. Acta endocrinol., 10,201-211.

Wal.'^as, 0., Walaas, E., and Loken, F. 1952b. The effect of estradiol monobenzoate on the metabolism of the rat endometrium. Acta endocrinol., 11, 61-66.

Werbin, H. and LeRoy, G. V. 1954. Cholesterol: a precursor of tetrahydrocortisone in man. J. Am. Chem. Soc, 76, 5260-5261.

West. C. D., Damast, B. L., Sarro, S. D., and Pearson, 0. H. 1956. Conversion of testosterone to estrogens in castrated, adrenalectomized human females. J. Biol. Chem., 218, 409-418.

WiEST, W. G. 1956. The metabolism of progesterone to A4-pregnen-20a-ol-3-one in eviscerated female rats. J. Biol. Chem., 221, 461467.

Zander, J. 1958. Gestagens in human pregnancy. In Proceedings Conference on Endocrinology of Reproduction, C. W. Lloyd, Ed. New York: Academic Press, Inc.

Zondek, B., and Hestrin, S. 1947. Phosphorylase activity in human endometrium. Am. J. Obst. ct Gvnec, 54, 173-175.

NUTRITIONAL EFFECTS ON ENDOCRINE SECRETIONS

James H. Leathern, Ph.D.

PROFESSOR OF ZOOLOGY, RUTGERS, THE STATE UNIVERSITY, NEW BRUNSWICK, NEW JERSEY

I. Introduction' 666 I, Introduction

II. Nature of Problems in Nutritional -j-. -, .^ ^ ,• ,• t ,

Studies 668 Despite the accumulation of many data

A. Thyroid Cxland, Nutrition, and Re- in the field of reproductive endocrinology

production ()68 during the past 20 years and the long es B. Adreiial Gland, Nutrition, and Re- ^ _^ tablished awareness of a nutritional in C. Diabetef^Mellitus, Nutrition, and ^f^f on fertility and fecundity, knowl Reproduction ()72 edge bearing on nutrition and the endocrine

D. Sterile-Obese Syndrome 673 glands subserving reproduction has ad E. Diet and the Liver 673 vanced comparatively slowly. However, re III. Hypophysis and Diet 674 markable advances have been made in each

A. Inanition 674 speciality SO that nutritional-endocrine

^roein. .. w problems should continue to be a fruitful

C. Carbohvdrate and Fat ()7() ^ „ ^i-r^x i-ii

D Vitamins 676 ^^^^^ ^^^' ^tudy. Data which have yet to

IV. Male Reproductive System (i77 be obtained eventually w'ill contribute to

A. Testis 677 the coherence one would prefer to present

1. Inanition 677 now.

2. Protein 678 'y\^q endocrinologist appreciates the deli 4 Vitamins (i8() ^^^^ balance which exists between the hy B. Influence of Nutrition on the Respon- ' pophysis and the gonads. In a sense, a simi siveness of Male Reproductive Tis- lar interdependence exists between nutrition

sues to Hormones 681 and the endocrine glands, including those

1. Testis ■ . . 681 ^j^|-^ reproductive functions. Not only does

2. feeminal vesicles and i)rostate 682 x •<• • n xi • j i -•

V. Female Reproductive System 683 nutrition influence synthesis and release of

A. Ovaries 683 hormones, but hormones in turn, through

1. Inanition 683 their regulation of the metabolism of pro 2. Protein 684 teins, carbohydrates, and fats, influence nu 3. Carbohydrate 685 trition. Thus, dietary deficiencies may create

5 Vitamins 685 endocrine imbalance, and endocrine imbal B. Influence of Nutrition on the ResiK)n- '^^ce may create demands for dietary fac siveness of Female Pe])ro<luct ive tors. It follows, therefore, that, in any conTissues to Hormones 687 sideration of this interrelationship, one must

1 . Ovary ... 687 consider not only undernutrition and lack of

2. uterus and vagina 688 -n c j \ l ^ i ^ cc i. r

3. Mammary gland 689 specific foods, but also possible effects of

C. Pregnancy. . 689 antithyroid substanccs in foods, antimetab VI. Concluding Remarks 693 olites, and overnutrition, especially for the

VII.Pkferencks (;!)4 child (Forbes, 1957).

666

NUTRITIONAL EFFECTS

667

Our understanding of the means by which hormones exert their effects is relatively slight, as is our knowledge of the biochemical mechanisms by w^hich supplements and deficiencies of vitamins and amino acids influence hormone action. Nevertheless, support for the statement that modifications of nutrition influence endocrine gland secretions or hormone action on distant target organs or tissues is provided by an enumeration of a few basic cell components requiring proteins, lipids, and vitamins. (1) Proteins combine with lipids to form lipoproteins which are essential features of the internal and external cellular membranes and interfaces. Hormones, as well as nutrition, influence cell membranes and therefore cell transport is affected. It is well known that hormones influence electrolyte and carbohydrate transfer and recently an endocrine control of amino acid transport was demonstrated (Noall, Riggs, Walker and Christensen, 1957). The effect of modifications of nutrition on the capacity of hormones to influence cell transport must await study. (2) Enzymes are proteins with chemically active surfaces and often include nonprotein groups such as vitamins. Nutritional and hormonal changes cause alterations in enzyme concentrations (Knox, Auerbach and Lin, 1956). Vitamin, mineral, and fat deficiencies favor a decrease in enzymes, whereas protein deficiencies have varied effects (Van Pilsum, Speyer and Samuels, 1957). Enzyme changes caused by hormones appear to be a consequence of metabolic adaptations. The importance of a nutritional base on which a hormone can express an effect on the enzymes of the reproductive organs can only be determined after further data have been obtained. (3) Proteins combined with nucleic acids become nucleoproteins, some of which are organized in the cytoplasm and may be templates for cellular protein synthesis. Other nucleoproteins are contained in the nucleus. Nutrition and hormones influence tissue nucleoproteins but studies involving the reproductive organs are few. How^ever, one possible cause of human infertility is low desoxyribose nucleic acid in the sperm ("Weir and Leuchtenberger, 1957).

Proteins are characteristic components of

tissues and hypophyseal hormones are protein in nature; also the major portion of gonadal dry weight is protein. Such being the case, it is important to appreciate that the protein composition of the body is in a dynamic state and that proteins from the tissues and from the diet contribute to a common metabolic pool of nitrogen. This metabolic pool contains amino acids which may be withdrawn for rebuilding tissue protein and for the formation of new protein for growth. Obviously, the character of the metabolic pool of nitrogen reflects dietary protein level and quality. A food protein which is deficient in one or more amino acids will restrict tissue protein synthesis. Hormones also influence the metabolic pool by affecting appetite as well as absorption, utilization, and excretion of foods, and thus hormones could accentuate the effect of a poor diet, or create demands beyond those normally met by an adequate diet. In addition a study of the tissues and organs of the body reveals that contributions to the metabolic pool are not uniform, thus one tissue or organ may be maintained at the expense of another. In protein deprivation in adults the liver quickly contributes increased amounts of nitrogen to the metabolic pool whereas the testis does not. On the other hand, protein contributions to the nitrogen pool by the hypophysis, and the amino acid withdrawals needed for hormone synthesis, are unknown. Data suggesting that the addition of specific nutrients to diets improves hypophyseal hormone synthesis have been presented (Leathem, 1958a).

In 1939 jNIason rightfully emphasized the need for vitamins in reproduction. Since then, much additional knowledge has been obtained. Vitamins of the B complex have been more clearly identified and a better understanding of their function has been gained. Thiamine is important for carbohydrate metabolism, pyridoxine for fat metabolism, and the conversion of tryptophan to nicotinic acid, and vitamin B12 may be involved in protein synthesis. In addition, vitamins have been found to serve as coenzymes, and folic acid to be important for estrogen action on the uterus. Tliosr. and

668

PHYSIOLOGY OF GONADS

many other findings prompt a survey of the relationships of vitamins to reproduction.

It is the intention of the author to review enough of the evidence which interrelates nutrition and reproduction to create an awareness of the problems in the area. Reviews dealing with the general subject of hormonal-nutritional interrelationships have been presented (Hertz, 1948; Samuels, 1948; Ershoff, 1952; Zubiran and GomezMont, 1953; Meites, Feng and Wilwerth, 1957; Leathem, 1958a,). Other reviews have related reproduction to nutrition with emphasis on laboratory (Mason, 1939; Guilbert, 1942; Lutwak-Mann, 1958) and farm animals (Reid, 1949; Asdell, 1949), and on protein nutrition (Leathem, 1959b, c). An encyclopedic survey of the biology of human nutrition has been made by Keys, Brozec, Hernschel, Michelsen and Taylor (1950).

II. Nature of Problems in Nutritional Studies

A. THYEOID GLAND, NUTRITION, AND REPRODUCTION

Normal development of the reproductive organs and their proper functioning in

TABLE 12.1 Ovarian response to chorionic gonadrotrophin,

as modified by thiouracil and diet

(From J. H. Leathem, in Recent Progress in

the Endocrinology of Reproduction, Academic

Press, Inc., New York, 1959.)

Diet

Ovarian Weight

Cholesterol

Total

Free

18 per cent casein .

18 tier cent +

I'hiouracil

per cent casein . . per cent + thiouracil

18 per cent gelatin . . .

mg.

87 342

59 133

59 186 27.5

% 0.41

0.20

0.47

0.22

0.66 0.26 1.56

0.18 0.12 0.23 0.15

0.22

18 per cent -f thio uracil

0.18 18

Chorionic gonadotrophin = 10 I. U. X 20 days.

adults are dependent not only on the endocrine glands composing the hypophysealgonadal axis, but on others as well. The importance of the thyroid, although not the same for all species, is readily apparent from the effects of prolonged hypo- and hyperthyroid states on reproduction. Many of these effects have been enumerated elsewhere (chapters by Albert, by Young on the ovary, and by Zarrow) , but others also are important. Thus steroid production may be altered in hypothyroid animals; certainly its metabolism is influenced. Myxedema is associated with a profound change in androgen metabolism. Endogenous production of androsterone is very low and subnormal amounts of administered testosterone are converted to androsterone. Triiodothyronine corrects this defect (Hellman, Bradlow, Zumoff, Fukushima and Gallagher, 1959). The gonads of male and female offspring of cretin rats are subnormal. The testes may contain a few spermatocytes but no spermatozoa, and Leydig cells seem to secrete little or no androgen. The ovaries may contain a few small follicles with antra, but corpora lutea are absent and ovarian lipid and cholesterol concentrations are very low. Nevertheless, the gonads are competent to respond to administered gonadotrophin with a marked increase in weight. However, administration of chorionic gonadotrophin to the hypothyroid rat stimulated follicular cyst formation rather than folliculogenesis and corpora lutea formation (Leathem, 1958b), but, for even this aberrant development, dietary protein was required (Leathem, 1959b) (Table 12.1). The relationship between hypothyroidism and ovarian function may provide a clue to a possible origin of ovarian cysts, long known to be a common cause of infertility and associated reproductive disorders. Clinical cases of untreated myxedema exhibit ovarian cysts, and rats made hypothyroid for eight months, had a higher percentage of cystic ovaries than did euthyroid rats (Janes, 1944).

The reproductive system of the adult male is less affected than that of the immature male by a decrease in thyroid function, just as the testis of the adult is less likely to reflect a change in protein nutrition which is sufficient to alter the immature rat

NUTRITIONAL EFFECTS

669

testis. On the other hand, the lack of demonstrable thyroid dysfunction in the adult male does not exclude the possibility of an effect of thyroid hormone on reproduction. The conversion of thyroxine to triiodothyronine may be hindered (Morton, 1958) . Thyroxine was found to decrease the number of active cells in the semen and to reduce motility, whereas triiodothyronine increased the number of active spermatozoa (Farris and Colton, 1958; Reed, Browning and O'Donnell, 1958j. Small dosages of thyroxine stimulated spermatogenesis in the mouse, rabbit, and ram (Maqsood, 1952) and were beneficial in normal guinea pigs and rats (Richter and Winter, 1947; Young, Rayner, Peterson and Brown, 1952).

In many species reproduction occurs despite hypothyroidism, but fecundity may be subnormal (Peterson, Webster, Rayner and Young, 1952). Feeding an antithyroid drug, thiouracil, to female rats may or may not prevent pregnancy, but it reduces the number of young per litter. Thiouracil feeding continued through lactation will decrease litter size (Leathem, 1959b). Hypothyroid guinea pigs gave birth to some live young, but the percentage approached normal only when thyroxine was administered (Hoar, Goy and Young, 1957). Pregnant euthyroid animals responded to thyroxine by delivering more living young than normal control pigs (Peterson, Webster, Ravner and Young, 1952) . The extent to which such effects are consequences of the reduction in appetite, metabolism, and absorption of food from the gut which are associated with hypothyroidism has not been determined.

Hyperthyroidism, on the other hand, will increase the appetite and enhance absorption of food from the gut, as the increased metabolism requires more calories, minerals, vitamins, choline, and methionine. In adult rats hyperthyroidism induces a marked loss in body fat and accelerates protein catabolism. The two effects, if unchecked, result in loss of body weight and death. In immature males hyperthyroidism slows gain in body weight, retards testis growth and maturation, and abolishes androgen secretion (Table 12.2). Altering dietary protein in adult animals failed to modify the thyroid hormone effects, thereby suggesting

TABLE 12.2

Effects of diet and thyroid {0.2 'per

cent) on immature rats

(From J. H. Leathem, in Recent Progress in

the Endocrinology of Reproduction, Academic

Press, Inc., New York, 1959.)

Diet

Testis Weight

Seminal

(Casein) X 30 Days

Actual

Relative

Vesicles

20 per cent

20 per cent + thyroid

6 per cent

6 per cent + thyroid

per cent

per cent -|thyroid

mg. 1G94

1090

825

245

140

95

mg./lOOgm.

1035

881 1232

650

346

261

mg. 88

9 16

7 7 6

that the metabolic demands of other tissues were making increased withdrawals from the metabolic pool of nitrogen and thus hindering testis growth. In euthyroid rats, a 6 per cent protein diet will permit testis growth in the absence of a gain in body weight, but hyperthyroidism prevents this preferential effect. Although Moore (1939) considered the effect of thyroid hormone on reproduction as possibly due to general body emaciation, the testis seems to be less responsive than the body as a whole. Adult rats fed 0.2 per cent desiccated thyroid exhibited no correlation between loss of body weight, change in testis weight, or protein composition of testis at two levels of casein and lactalbumin (Leathem, 1959b) . The testes were seemingly not influenced by the metabolic nitrogen changes which caused a loss in carcass nitrogen and an associated increase in kidney and heart nitrogen.

The mechanism of thyroid hormone action on reproduction is far from clear. As we have noted, a part of its action may be through the regulation of nutritional processes. Thyroid function is influenced by the biologic value of the dietary protein (Leathem, 1958a) and the specifie amino acids fed (Samuels, 1953). In turn, an altered thyroid function will influence the nitrogen contributions to the metabolic pool by reducing appetite and absorption from

670

PHYSIOLOGY OF GONADS

the gut, and by changing the contributions of nitrogen to the metabolic pool made by the body tissues. Hypothyroidism interferes with the refilling of body protein stores; consequently, protein needs of the reproductive organs may not be fully met (Leathern, 1953). It is consistent with this opinion that testis recovery from protein deprivation in hypothyroid rats was aided by thyroxine treatment (Horn, 1955).

Conversion of carotene to vitamin A may be prevented by hypothyroidism, suggesting that a subnormal amount of this vitamin may contribute to fetal loss. An increased intake of B vitamins might be required as hypothyroidism aggravates a vitamin B12 deficiency, and increased intake of B vitamins enhances the capacity of young rats to withstand large doses of thiouracil (Meites, 1953). Although a reduced metabolic rate might seemingly reduce vitamin requirements, more efficient metabolic activities, in the absence of hormonal stimuli, seem to occur when vitamin intake is increased (Meites, Feng and Wilwerth, 1957).

Reproduction is influenced by effects which are the opposite of a number of those just cited, i.e., effects of malnutrition on thyroid function. The need for iodine in the prevention of goiter is well known. However, certain foods prevent the utilization of iodine in the synthesis of thyroid hormone. The foods containing an antithyroid or goitrogenic agent, as tested in man, include rutabaga, cabbage, brussels sprouts, cauliflower, turnip, rape, kale, and to a lesser extent peach, pear, strawberry, spinach, and carrot (Greer and Astwood, 1948). A potent goitrogen isolated from rutabaga is L-5-vinyl-2-thiooxazolidine ((ireer, 1950, 1956). Reduced food intake will decrease the thyroid gland response to goitrogens (Gomez-Mont, Paschkis and Cantarow, 1947; Meites and Agrawala, 1949), the uptake of I^^^ (Meites, 1953), and the level of thyrotrophic hormone in laboratory animals (D'Angelo, 1951). The thyroid changes associated with malnutrition in man arc uncertain (Zubiran and Gomez-Mont, 1953). However, a decreased functioning of the gland in anorexia nervosa, followed by an increased functioning on refeeding (Poiioff, Lasche, Nodine,

Schneeberg and \'ieillard, 1954), is suggestive of a direct nutritional need.

Changes in the thyrotrophic potency of the rat hypophysis have been observed in various vitamin deficiencies. Thiamine deficiency may increase thyroid function, but vitamin A deficiency may have the opposite effect (Ershoff, 1952). The difficulties inherent in the assay of thyroid-stimulating hormone (TSH) , even by current methods, prevent one from drawing definite conclusions from the available data.

Immature animals given thyroxine are retarded in growth and do not survive. However, increasing dietary thiamine, pyridoxine, or vitamin B12 improves the ability of the young rat to withstand large dosages of thyroid substances (Meites, 1952), as does methionine (Boldt, Harper and Elvehjem, 1958). Consideration must also be given to the need for nutritional factors which play a minor role in normal metabolic states but increase in importance in stress. Thus yeast and whole liver contain antithyrotoxic substances (Drill, 1943; Ershoff, 1952; Overby, Frost and Fredrickson, 1959).

Excessive thyroid hormone will prevent maturation of the ovary and in adult rats will cause ovarian atrophy with a cessation of estrous cycles. Addition of yeast to the diet permitted estrous cycles to continue (Drill, 1943), but gonadal inhibition in the immature animal was not ]3revented. However, whole liver or its w^ater-insoluble fraction counteracted the gonadal inhibition induced by hyperthyrodism in immature rats (Ershoff, 1952). Biochemical mechanisms by which these dietary supplements can benefit rats given excessive quantities of hormone are unknown.

B. .\DREXAL GLAND, NUTRITION, AND REPRODUCTION

The problem of the relationship between adrenal steroid secretions and the reproductive system is one that still requires clarification. Furthermore, the possible influences of nutrition can only be inferred from the effects of adrenal steroids on the major metabolic systems of the body.

In the female there is a close relationshiji between the adrenal and the estrous and

NUTRITIONAL EFFECTS

iul

menstrual cycles (Zuckerman, 1953; chapter by Young on the ovary). The ovary would seem to require cortical steroids for the normal functioning of its own metabolic processes and for those which it influences peripherally. The Addisonian patient may show ovarian follicular atresia and a loss of secondary sex characteristics, and the untreated adrenalectomized rat exhibits a decrease in ovarian size and has irregular cycles (Chester Jones, 1957). The decline in size of the ovary after adrenalectomy is due to impaired sensitivity to folliclestimulating hormone (FSH) rather than to a decreased production of FSH, and the ovarian response is corrected by cortisone (Mandl, 1954).

Reproductive potential is not necessarily lost when there is adrenal insufficiency, but pregnancy is not well tolerated by women with Addison's disease. Furthermore, adrenalectomy in rats at the time of mating or 4 to 6 days after mating resulted in abortion. Improved pregnancy maintenance was obtained in adrenalectomized rats given saline or cortisone acetate (Davis and Plotz, 1954) , whereas desoxycorticosterone acetate alone extended the pregnancy period beyond normal time (Houssay, 1945). Essentially normal pregnancies were obtained in adrenalectomized rats given both cortisone acetate and desoxycorticosterone acetate (Cupps, 1955). Substitution of cortisone acetate for adrenal secretions may be incomplete because the adrenal hormone in the normal rat is primarily corticosterone and because cortisone enhances the excretion of certain amino acids and vitamins. It would be interesting to test a diet with a high vitamin content on the capacity of an adrenalectomized rat to maintain pregnancy, because improved survival of operated rats is obtained by giving vitamin Bio (Meites, 1953) or large doses of pantothenic acid, biotin, ascorbic acid, or folic acid (Ralli and Dumm, 1952; Dumm and Ralli, 1953).

An adrenal influence over protein metabolism is well known, but protein nutrition, in turn, can influence cortical steroid effectiveness. In fact, an extension of the life span of adrenalectomized rats is not obtained with adrenal steroids if the diet

lacks protein (Leathern, 1958a). A low protein diet alone will not improve survival after adrenalectomy, but better survival is obtained when the rats are given saline. When the low protein diet was supplemented with methionine, a definite improvement in life span was observed and the possibility that cortisone exerts its effect by drawing on the carcass for methionine was suggested (Aschkenasy, 1955a, b).

Reducing dietary casein to 2 per cent seriously endangers pregnancy in the rat, but the addition of progesterone permits 80 per cent of the pregnancies to be maintained. However, removal of the adrenal glands counteracts the protective action of the progesterone, only 10 per cent of the pregnancies continuing to term. Addition of methionine to the low casein diet improved pregnancy maintenance, but 1 mg. cortisone acetate plus progesterone provided the best results (Aschkenasy-Lelu and Aschkenasy, 1957; Aschkenasy and Aschkenasy-Lelu, 1957). These data emphasize the importance of nutrition in obtaining an anticipated hormone action. Further investigation might be directed toward the study of whole proteins other than casein, for the biologic value of proteins differs from normal when tested in adrenalectomized rats (Leathem, 1958a) .

As Albert has noted in his chapter, adrenalectomy has little or no effect on the testis. Gaunt and Parkins (1933) found no degenerative changes in the testes of adult rats dying of adrenal insufficiency, although an increase in the testis : body-weight ratios was noted in rats fed 18 per cent and 4 per cent protein (Aschkenasy, 1955c). If adrenalectomized rats are kept on a maintenance dosage of cortisone acetate for 20 days and fed dietary proteins of different biologic values, one finds that testis-composition of protein, lipid, and glycogen varies in the same manner as in the normal rat (Wolf and Leathem, 1955) (Table 12.3).

When the adrenal glands are intact, the influence of diet on their functional capacity and indeed on the hypophyseal- adrenal axis must be considered. Zubiran and Gomez-]\Iont (1953) showed that patients exhibiting gonadal changes associated with chronic malnutrition also exhibit adrenal

672

PHYSIOLOGY OF GOXADS

TABLE 12.3

Nutritional effects on the testes of cortisonemaintained adrenalectomized rats (From R. C. Wolf and J. H. Leathern, Endocrinology, 57, 286, 1955.)

Testes Composition

(per cent)

Treatment

Diet

Testes

Protein

Lipid

Glycogen

mg.

Corti

20 per cent

703

68.5

30.4

0.13

sone

casein

ace

tate

Control

1211

70.5

29.4

0.15

Corti

20 per cent

808

61.0

31.8

0.17

sone

wheat

ace

ghiten

tate

Control

816

62.3

32.2

0.17

Corti

20 per cent

1014

64.3

31.3

0.17

sone

peanut

ace

flour

tate

Control

699

68.5

31.3

0.12

hypofimction. Several clinical tests permitted the evaluation of subnormal adrenal function which, however, did not reach Addisonian levels. Malnutrition not onlyreduced hypophyseal adrenocorticotrophic hormone (ACTH), but also prevented an incomplete response by the adrenal glands to injected ACTH. In laboratory rodents, anterior hypophyseal function is also influenced by dietary protein and vitamin levels (Ershoff, 1952). The importance of dietary protein in the hypophyseal-adrenal system has recently been re-emphasized (Leathem, 1957; Goth, Nadashi and Slader, 1958). Furtiiermore, adrenal cortical function is affected by vitamin deficiencies (Morgan, 1951), from which it appears that pantothenic acid is essential for cortical hormone elaboration (Eisenstcin, 1957). Administration of excessive amounts of cortical steroids can induce morphologic changes which have been compared to inanition (Baker, 1952). Not only is nitrogen loss enhanced, but hyperglycemia can also be induced which, therefore, increases the need for thiamine. Cortical steroids influence the metabolism of various vitamins

(Draper and Johnson, 1953; Dhyse, Fisher, Tullner and Hertz, 1953; Aceto, Li Moli and Panebianco, 1956; Ginoulhiac and Nani, 1956). H a vitamin deficiency already exists, administration of cortisone will aggravate the condition (Meites, Feng and Wilwerth, 1957). Nevertheless, drastic effects of therapeutic doses of cortisone on reproductive function do not occur. In rare cases loss of libido has been reported in the male, but mori)hologic changes in the testis were not observed (JVladdock, Chase and Nelson, 1953). Cortisone has little if any effect on the weight of the rat testis (Moore, 1953; Aschkenasy, 1957) and does not influence testis cholesterol (Migeon, 1952). In the female, menstrual disturbances have been noted in association with cortisone therapy, with the occurrence of hot flashes (Ward, Slocumb, Policy, Lowman and Hench, 1951). However, cortisone corrected disturbances during the follicular phase, possibly by increasing FSH release (Jones, Howard and Langford, 1953). Cortisone also increased the number of follicles in the ovary of the rat (Moore, 1953), but not in the rabbit. Cortisone administration did not prevent the enhanced ovarian response to chorionic gonadotrophin seen in hypothyroid rats (Leathem, 1958b) and had little effect on mice in parabiosis ( Noumura, 1956).

In pregnant female rabbits resorption and stunting of fetuses occurred during treatment with large doses of cortisone (Courrier and Collonge, 1951). Similar effects were noted in mice (LcRoy and Domm, 1951 ; Robson and Sharaf, 1951).

Some of the metabolic derangements of human toxemia of pregnancy have been correlated with accelerated secretion of adrenal steroids creating a steroid imbalance (see chapter by Zarrow). Cortisone is reported to have a beneficial effect on some cases (Moore, Jessop, 'Donovan, Barry, Quinn and Drury, 1951). Protein inade({uacies may also be etiologic in toxemia and further (>xamination of the possibility sliould he made.

C. DIABETES MELLITUS, NUTRITION, AND REPRODUCTION

(llycosuria can be induced experimentally by starvation, overfeeding, and shifting

NUTRITIONAL EFFECTS

673

diet^ from one of high fat content to one i; which is isocaloric but high in carbohydrate (Ingle, 1948). Force feeding a high carbohydrate diet will eventually kill a rat despite insulin administration aimed at controlling glycosuria (Ingle and Nezamis, 1947). In man excessive eating leading to obesity increases insulin demand and, in many diabetics of middle age, obesity precedes the onset of diabetes. With our present knowledge we must conclude that overfeeding is wrong when glycosuria exists and that vitamin B supplements may be of value in diabetes (Meites, Feng and Wilwerth, 1957; Salvesen, 1957).

In man urinary 17-ketosteroids and androgen levels are subnormal in diabetes (Horstmann, 1950), and in the diabetic rat pituitary gonadotrophins are reduced (Shipley and Danley, 1947), but testis hyaluronidase does not change (Moore, 1948) . When hyperglycemia exists in rats, semen ■] carbohydrates increase (Mann and Lutwak-Mann, 1951).

Hypoglycemia influences the male reproductive organs. In rats tolbutamide or insulin produce lesions of the germinal epithelium which can be prevented by \' simultaneous administration of glucose. When 2 to 5 hypoglycemic comas are induced, such testis injuries increase progressively in number and frequency, and only a partial return to normal is observed a month later (Mancini, Izquierdo, Heinrich, Penhos and Gerschenfeld, 1959).

It is well known that the incidence of infertility in the pre-insulin era was high in young diabetic women. Fertility is also reduced in diabetic experimental animals, and rat estrous cycles are prolonged (Davis, Fugo and Lawrence, 1947) . Insulin is corrective (Sinden and Longwell, 1949; Ferret, Lindan and Morgans, 1950). Pregnancy in women with uncontrolled diabetes may terminate in abortion or stillbirth, possibly l)ecause toxemia of pregnancy is high (Pedersen. 1952). In rats pancreatectomy performed the 8th to 12th day of pregnancy increased the incidence of stillbirths (Hultciuist, 1950). In another experiment almost one- fourth of 163 animals with diabetes induced by alloxan on the 10th to 12th day of pregnancy died before parturition and

about 25 ])er cent of the survivors aborted (Angcrvall, 1959).

D. STERILE-OBESE SYNDROME

A sterile-obese syndrome in one colony of mice has been shown to be a recessive monogenic trait (Ingalls, Dickie and Snell, 1950). Obesity was transmitted to subsequent generations by way of ovaries that were transplanted from obese donors to nonobese recipients (Hummel, 1957). Obesity was transmitted by obese females receiving hormonal therapy and mated to obese males kept on restricted food intake (Smithberg and Runner, 1957). In addition to the investigations of the hereditary nature of the sterile-obese syndrome, the physiologic basis for the sterility has been studied in reference to the presence of germ cells, viability of ova and sperm, integrity of the ovary, and response of the uterus to estrogen (Drasher, Dickie and Lane, 1955). The data indicate that sterility in some obese males can be prevented by food restriction and that sterility in certain obese females can be corrected.

E. DIET AND THE LIVER

The concentration of hormones which reaches the target organs in the blood is the result of the rate of their production, metabolism, and excretion. How hypophyseal hormones are destroyed is not clear, but current data make it apparent that pituitary hormones have a short half-life in the circulatory system. Exerting a major control over circulating estrogen levels is the liver, with its steroid-inactivating systems. Zondek (1934) initially demonstrated that the liver could inactivate estrogens and this finding has had repeated confirmation (Cantarow, Paschkis, Rakoff and Hansen, 1943; De:\Ieio, Rakoff, Cantarow and Paschkis, 1948; Vanderlinde and Westerfield, 1950). Other steroids are also inactivated by the liver with several enzyme systems being involved; the relative concentration of these enzymes varies among species of vertebrates (Samuels, 1949).

The liver is a labile organ which readib.' responds to nutritional modifications; the induced liver changes alter the steroid-inactivating systems of this organ. Thus, inanition (Drill and Pfeiffer, 1946; Jailer,

G74

PHYSIOLOGY OF GONADS

1948) , vitamin B complex deficiency (Segaloff and Segaloff, 1944; Biskind, 1946), and protein restriction (Jailer and Seaman, 1950) all influence the capacity of the liver to detoxify steroids. Reduced protein intake is a primary factor in decreasing the effectiveness of the steroid-inactivating system (Jailer and Seaman, 1950; Vanderlinde and Westerfield, 1950). Rats fed an 8 per cent casein diet lose their capacity to inactivate estrone within 10 days. However, ascorbic acid alone or in combination with glutathione restored the estrone-inactivating system (Vasington, Parker, Headley and Vanderlinde, 1958).

Failure of steroids to be inactivated will influence the hypophyseal-gonadal axis. In turn the excess of estrogen will decrease gonadotrophin production by the hypophysis and thus reduce steroid production by the gonad. In addition nutritional modifications influence hypophyseal and possibly gonadal secretory capacity directly. Conceivably, nutritional alterations could modify the amount of steroid secreted or interfere with complete steroid synthesis by a gland.

Fatty infiltration of the liver and a general increase in fat deposition occur in fed and fasted rats after the injection of certain pituitary extracts and adrenal steroids and after the feeding of specific diets. Impaired estrogen inactivation has been associated with a fatty liver, but Szego and Barnes (1943) believe that the major influence is inanition. In fact, estrogens, especially ethinyl estradiol, interfere with fatty infiltration of the liver induced by a low protein diet (Gyorgy, Rose and Shipley, 1947) or by a choline-deficient diet (Emerson, Zamecnik and Nathanson, 1951). Stilbestrol, however, did not prevent the increase in liver fat induced by a protein-free diet (Glasser, 1957). Estrogens that are effective in preventing fatty infiltration may act by sparing methionine or choline or by inhibiting growth hormone (Flagge, Marasso and Zimmerman, 1958).

Ethionino, the antimetabolite of nu'thioniiK', Avill induce a fatty liver and inhibit hepatic protein synthesis in female, but not in male rats (Farber and Segaloff, 1955; Farber and Corban, 1958). Pretreatment of females with testosterone prevents

the ethionine effect, but this blockage of ethionine action need not be related to androgenic or progestational properties of steroids (Ranney and Drill, 1957).

III. Hypophysis and Diet

Studies involving acute and chronic starvation have shown that gonadal hypofunction during inanition is primarily due to diminished levels of circulating gonadotrophins. Because of the similarity to changes following hypophysectomy, the endocrine response to inanition has been referred to as "pseudohypophysectomy."

A. INANITION

The hypophysis has been implicated in human reproduction disturbances associated with undernutrition. Hypophyseal atrophy and a decrease in urinary gonadotrophins have been observed in chronic malnutrition (Klinefelter, Albright and Griswold, 1943; Zubiran and Gomez-]\Iont, 1953) and anorexia nervosa (Perloff, Lasche, Nodine, Schneeberg and Vieillard, 1954). Refeeding has restored urinary gonadotrophin levels in some cases, but hypoj^hyseal damage may result from severe food restriction at puberty (VoUmer, 1943; Samuels, 1948).

The influence of inanition on the reproductive organs of lal)oratory rodents is well recognized but the cft'ects on the hypophysis cannot be presented conclusively. In support of prior investigations, Mulinos and Pomerantz (1941a, b) in rats and Giroud and Desclaux (1945) in guinea pigs observed a hypophyseal atrophy following chronic underfeeding as well as a decrease in cell numbers and mitoses. In fact, refeeding after chronic starvation resulted in only a partial recovery of hypophyseal weight (Quimby, 1948). Nevertheless, complete starvation did not influence relative gland weight in female rats (Meites and Reed, 1949), and cytologic evidence (periodic acid-Schiff (PAS) test) of an estimated 3- fold increase in gonadoti'ophin content was claimed following chronic starvation (Pearse and Rinaldini, 1950). Assays of hypophyseal gonadotrophin content in chronically starved rats of both sexes have l)een reported as decreased (Mason and Wolfe, 1930; Werner,

NUTRITIONAL EFFECTS

675

1939), unchanged (,]\Iarrian and Parkes, 1929; Pomerantz and Mulinos, 1939; Maddock and Heller, 1947; Meites and Reed, 1949; Blivaiss, Hanson, Rosenzweig and McNeil, 1954), or increased (Rinaldini, 1949; Vanderlinde and Westerfield, 1950). An increase in pituitary gonadotrophin was evident when hormone content was related to milligrams of tissue (Meites and Reed, 1949). Thus, the hormone release mechanism may fail in starvation, and eventually gonadotrophin production will be reduced to a minimum (]\laddock and Heller, 1947).

Gonadectomy of fully fed rats is followed by an increase in hypophyseal gonadotrophin content. Chronic starvation, however, prevented the anticipated changes in the pituitary gland following gonadectomy in 8 of 12 female rats (Werner, 1939). On the other hand, if adult female rats were subjected to 14 days of reduced feeding 1 month after ovariectomy, no change in the elevated gonadotrophin levels was noted (Meites and Reed, 1949). In contrast, Gomez-Mont (1959) observed above normal urinary gonadotrophins in many menopausal and postmenopausal women despite undernutrition.

It is apparent that uniformity of opinion as to how starvation influences hypophyseal gonadotrophin content has not been attained. Several explanations can be given for the discrepancies. (1) There have been unfortunate variations in experimental design. IVIaddock and Heller (1947) starved rats for 12 days, whereas Rinaldini (1949) used a low calorie diet of bread and milk for 30 days. Other variations in feeding have included feeding one-half the intake required for growth (Mulinos and Pomerantz, 1941b I, regimens of full, one-half, oneciuarter, and no feeding for 7 and 14 days (Meites and Reed, 1949), and feeding inadequate amounts of a standard rat diet for 1 to 4 months (Werner, 1939). (2) Hypophyseal implants and anterior pituitary extracts should not be compared, for variable gonadotrophin production may follow implantation procedures, depending on whether necrosis or growth occurs (Maddock and Heller, 1947). (3) There has been an insufficient standardization of experimental materials. The assay animal has usually been the immature female rat, but

occasionally the immature mouse has been used, and Rinaldini (1949) used the hypophysectomized rat.

B. PROTEIN

The need for specific food elements by the hypophysis warrants consideration, for the hormones secreted by this gland are protein in nature and the amino acids for protein synthesis must be drawn from body sources. However, dietary protein levels can vary from 15 per cent to 30 per cent without influencing hypophyseal gonadotrophin content in rats (Weatherby and Reece, 1941), but diets containing 80 per cent to 90 per cent of casein increased hypophyseal gonadotrophin (TuchmannDuplessis and Aschkenasy-Lelu, 1948). Removal of protein from the diet will decrease hypophyseal gonadotrophin content in adult male rats in comparison with pair-fed and ad libitum-ied controls, but the decrease may or may not be significant in a 30-day period; luteinizing hormone (LH) seemed to be initially reduced. Extension of the period of protein depletion another 2 months resulted in a significant lowering of hy]iophyseal gonadotrophin levels (Table 12.4) (Leathern, 1958a). On the other hand, an increased FSH with no decrease in LH activity was observed in the hypophyses of adult female rats following 30 to 35 days of protein depletion (Srebnik and Nelson, 1957). The available data indicate that not only may a sex difference exist, but also that species may differ; restitution of gonadotrophin in the discharged rabbit pituitary was not influenced by in TABLE 12.4

Influence of a protein-free diet on hypophyseal

gonadotrophin content

(From J. H. Leathern, Recent Progr. Hormone

Res., 14, 141, 1958.)

Days on PFD*

Xo. of Rats

Anterior Pituitary Weight

Recipient Ovarv Weight

nig.

mg.

9

8.3

74

30

9

7.3

54

50

7

7.0

33

90

17

6.0

23

L^ntreated recipient ovarian weight = 15.4 mg.

• PFD = Protein-free diet.

67G

PHYSIOLOGY OF GONADS

adequate dietary protein (Friedman and Friedman, 1940).

When anterior pituitar}^ glands of 60gm. male rats were extracted and administered to immature female recipients, ovarian weight increased from 13.0 to 37.3 mg. After feeding 20 per cent casein or fox chow ad libitum for 14 days, the hypophyses of male rats contained almost twice as much gonadotrophin per milligram of tissue as did the hypophyses of the initial controls. Removal of protein from the diet for 14 days, however, reduced hypophyseal gonadotrophin concentration below the level of the initial controls (Leathem and Fisher, 1959).

Data on the hypophyseal hormone content as influenced by specific amino acid deficiencies have not come to the author's attention. Cytologically, however, Scott (1956) noted that an isoleucine-deficient diet depleted the pituitary gonadotrophic cells of their PAS-positive material and reduced the size of acidophilic cells. Omission of threonine, histidine, or tryptophan invoked similar effects. The changes probably represent the interference of a single amino acid deficiency with protein metabolism rather than specific effects attributable to the lack of amino acid itself. Excessive amino acid provided by injecting leucine, methionine, valine, tyrosine, or glycine caused release of gonadotrophin (Goth, Lengyel, Bencze, Saveley and Majsay, 1955).

Administration of 0.1 mg. stilbestrol for 20 days to adult male rats eliminated detectable hypophyseal gonadotrophins. Hormone levels returned during the postinjection period provided the diet contained adequate protein, whereas a protein-free diet markedly hindered the recovery of hypophyseal gonadotrophins. The gonadotrophin content of the pituitary gland correlated well with the recovery of the reproductive system, indicating that gonadotrophin production was subnormal on ]irotein-free feeding (Leathem, 1958a).

C. CAHBOHYDR.\TE .\ND FAT

Reproduction does not appear to be influenced by carbohydrates per se and hypophyseal alterations have not been noted.

Fat-deficient diets, however, do influence reproduction and the hypophysis exhibits cellular changes. Pituitary glands of female rats fed a fat-free diet contain a subnormal number of acidophiles and an increased number of basophiles (Panos and Finerty, 1953) . In male rats the feeding oi a fat-free diet increased hypophyseal basophiles, followed progessively by more castration changes (Finerty, Klein and Panos, 1957; Panos, Klein and Finerty, 1959).

D. VITAMINS

Despite the many investigations relating reproduction to vitamin requirements, relatively few have involved hypophyseal hormone estimations. Thus in 1955, Wooten, Nelson, Simpson and Evans reported the first definitive study which related pyridoxine deficiency to hypophyseal gonadotrophin content. Using the hypophysectomized rat for assay, pituitary glands from Bo-deficient rats were shown to have a 10-fold increase in FSH per milligram of tissue and a slightly increased LH content. Earlier studies had revealed that vitamin Bi-free diets decreased pituitary gonadotrophins in male rats (Evans and Simpson, 1930) and a similar effect of the folic acid antagonist, aminopterin, in the monkey was found later (Salhanick, Hisaw and Zarrow, 1952).

Male rats deficient in vitamin A exhibited a 43 per cent increase, and castrated vitamin A-deficient rats a 100 per cent increase in hypophyseal gonadotrophin potency over the normal controls (Mason and Wolfe, 1930). The increase of gonadotrophin was more marked in vitamin A-deficient male than in vitamin A-deficient female rats. Associated with the increase in hormone level was a significant increase in basophile cells (Sutton and Brief, 1939; Hodgson, Hall, Sweetman, Wiseman and Converse, 1946; Erb, Andrews, Hauge and King, 1947).

A re\-iew of the literature up to 1944 permitted Mason to suggest that the anterior hypophysis was not the instigator of reproductive disturbances in vitamin E deficiency. Nevertheless, Griesbach, Bell and Livingston (1957), in an analysis of the pituitary gland during progessive stages of

NUTRITIONAL EFFECTS

677

tocopherol deprivation, observed cytologic changes in the hypophysis which preceded testis changes. The "peripheral or FSH gonadotrophes" increased in number, size, and activity. The LH cells exhibited a hyperplasia of lesser extent, but possibly sufficient to increase LH in circulation and to cause hypertrophy of the male accessory glands. Gonadotrophic hormone content of pituitary glands from vitamin E-deficient rats may be decreased (Rowlands and Singer, 1936), unchanged (Biddulph and Meyer, 1941), or increased to a level between normal and that of the castrate, when the adult male rats were examined after 22 weeks on a deficient diet (Nelson, 1933; Drummond, Noble and Wright, 1939). Using hypophysectomized male rats as assay animals, evidence was obtained that FSH was increased in the pituitary glands of vitamin E-deficient male and female rats (P'an, Van Dyke, Kaunitz and Slanetz, 1949).

IV. Male Reproductive System

A. TESTIS

The two basic functions of the male gonads are to produce gametes and secrete steroids. Spermatogenic activity can be estimated from testis morphology and examination of semen samples. Androgen secretion can be estimated from urinary steroid levels, accessorj^ gland weight, and from analyses of accessory sex gland secretions, i.e., fructose and citric acid. In normal maturation in the rabbit, rat, boar, and bull, androgen secretion precedes spermatogenesis (Lutwak-Mann, 1958). On this basis it would appear that well fed young bulls may come into semen production 2 to 3 months sooner than poorly fed animals (Brat ton, 1957).

1. Inanition

Complete starvation will pre^•ent maturation of immature animals. Furthermore, marked undernutrition in 700 boys, 7 to 16 years of age, was associated with genital infantilism in 37 per cent and cryptorchidism in 27 per cent (Stephens, 1941). Restriction of food intake to one-half of the normal in maturing bull calves had a

marked delaying effect on the onset of seminal vesicle secretion, but a lesser delaying effect on spermatogenesis (Davies, Mann and Rowson, 1957) . Limiting the food intake to one-third of the normal did not prevent the immature rat testis from forming spermatozoa at the same time as their controls (Talbert and Hamilton, 1955). When testis maturation was prevented by inanition, a rapid growth and maturation occurred on refeeding (Ball, Barnes and Visscher, 1947; Quimby, 1948) but Schultze (1955) observed that full body size was not attained.

The reproductive organs of the adult are more resistant to changes imposed by diet than are those of the immature animal. Thus, Mann and Walton (1953) found that 23 weeks of underfeeding produced little change in sperm density and motility in mature animals although seminal vesicle function was reduced. Li the male rat testis hypofunction follows partial or complete starvation (]\Iason and Wolfe, 1930; Mulinos and Pomerantz, 1941a; Escudero, Herraiz and Mussmano, 1948), but there is no reduction in testicular nitrogen (Addis, Poo and Lew, 1936). Loss of Leydig cell function precedes cessation of spermatogenesis (Moore and Samuels, 1931) and is evident by the atrophy of the accessory sexual organs (^lulinos and Pomerantz, 1941a) and by an alteration in accessory gland secretion (Pazos and Huggins, 1945; Lutwak-]\Iann and Mann, 1950). Evidence of a tubular effect is provided by the lack of motile sperm (Reid, 1949). Severe dietary restriction is associated with the absence of spermatozoa in the seminiferous tubules and epididymis (Mason, 1933; Menze, 1941).

The human male suffering from chronic malnutrition exhibits hypogonadism. The testes atrophy and exhibit a decrease in size of the seminiferous tubules; basement membrane thickening and small Leydig cells are seen. These individuals excrete significantly subnormal amounts of 17-ketosteroids (Zubiran and Gomez-Mont, 19531. Acute starvation may also decrease urinary 17-ketosteroid and androgen levels as much as 50 per cent, with recovery evident on refeeding (Perloff, Lasche, Nodine, Schneeberg and Vieillard. 1954).

6/

PHYSIOLOGY OF GONADS

TABLE 12.5

Effect of diet on the testes of immature rats

(From J. H. Leathern, in Re-productive Phijsiology

and Protein Nutrition, Rutgers University

Press, New Brunswick, N. J., 1959.)

Sperm

Initial control . . . 20 per cent X 30

days

6 per cent X 30

days

3 per cent X 30

days

per cent X 30

days

per cent + 5 per cent liver. ,

per cent + 5 per cent yeast

G5 per cent X 30 davs

No. of Rats

Testis

Weight

mg.

mg./lOOgm.

10

329

825

10

1694

1035

16

824

890

12

380

930

10

140

346

10

112

291

10

119

296

10

1747

1040

100

50

100

2. Protein

The minimal amount of dietary protein which will support reproduction, lactation, and growth is 16.7 per cent (Goettsch, 1949) . Thus, it is not surprising that maturation of testes and accessory sex organs was prevented in immature rats (Horn, 1955) and mice (Leathern and DeFeo, 1952) when they were fed a protein-free diet for 15 to 30 days after weaning. Furthermore, supplements of 5 per cent liver to the casein-free diet had no effect. After a month, the testes, averaging 329 mg., decreased to 140 mg. in rats fed per cent casein, but the weight increased to an average of 1694 mg. and 1747 mg. in rats fed 20 per cent and 65 per cent casein, respectively (Table 12.5). Following protein depletion, there was a decrease in tubular ribonucleic acid and an increase in lipid. Accumulation of gonadal lipid in the inactive testis may be an abnormal assimilation of a degenerative nature or simply nonutilization. A diet containing 6 per cent casein permitted the formation of spermatozoa in some animals (Guilbert and Goss, 1932). When the 6 per cent casein diet was fed to immature animals for 30 days 50 per cent of the rats exhibited some spermatozoa; in addition, testis weight increased slightly and seminal

vesicle weight doubled, but body weight was not improved (Horn, 1955). Thus, as we noted earlier, the reproductive system may gain special consideration for protein allotments when supplies are limited.

Gain in testis weight in immature male rats and the biochemical composition of the immature testis are influenced by the nutritive value of the protein fed. The testes of normal immature rats contain 85 per cent water, 10.5 per cent protein, 4.5 per cent lipid, and detectable glycogen (Wolf and Leathern, 1955). Proteins of lower nutritive value (wheat gluten, peanut flour, gelatin ) may permit some increase in testis weight, but testis protein concentration decreased, percentage of water increased, and lipid and glycogen remained unchanged (Table 12.6). The enzyme ^fi-glucuronidase, which has frequently been associated with growth processes, exhibited no change in concentration as the testis matured or was jirevented from maturing by a protein-free diet (Leathem and Fisher, 1959). Not only are the weight and composition of the testis influenced by feeding proteins of varied biologic value, but the release of androgen is more markedly altered. When a 22-day-old male rat was fed a 20 per cent casein diet for 30 days, the seminal vesicle and ventral prostate weights increased 9- to 10-fold in comparison with initial control weight. Sub TABLE 12.6

Niiiritioiial effects on testis-coin position

in immature rats

(From R. C. Wolf and J. H. Leathem,

Endocrinology, 57, 286, 1955.)

20 pel' cent casein

20 per cent wheat gluten

20 per cent i)eanut flour

20 i)er cent gelatin

5 per cent casein

Fox chow

Initial control . .

No. of Rats

Final Body Weight

gm.

7

128

8

82

8

81

5

53

5

61

8

115

7

61

72.3j30.4 04.634.0

1468 1017

1257 66.1 2101

28.8

0.11 0.18 0.11 0.10

684,62.4 29.2 0.26 1515'70.3 30.3 0.15 273 72. 7:30. 10.19

NUTRITIONAL EFFECTS

679

stitution of wheat gluten and peanut flour for casein, limited the increase in the weight of the seminal vesicles to less than 100 per cent. In fact, seminal vesicle weight as related to body weight did not increase in animals fed 20 per cent wheat gluten.

The withholding of dietary protein from an immature rat for 30 days, during which time maturation occurs in the fully fed animal, did not impose a permanent damage. Refeeding of protein permitted the rapid recovery of testis weight and the appearance of spermatoza, 70 per cent of all animals having recovered in 30 days when fully fed, whereas only 25 per cent recovered when 6 per cent casein was fed. Recovery of androgen secretion was somewhat slower than that of the tubules as estimated by seminal vesicle weight.

Variations in protein quality are a reflection of amino acid patterns, and amino acid deficiencies interfere with testis maturation (Scott, 1956; Pomeranze, Piliero, Medeci and Plachta, 1959). Alterations in food intake which follow amino acid deficiencies have required forced feeding or pair-fed controls, but it is clear from what w^as found in the controls that the gonadal changes were not entirely due to inanition (Ershoff, 1952).

If the diet is varied so that caloric intake per gram is reduced to half while retaining the dietary casein level at 20 per cent, immature rat testis growth is prevented. The effect is unlike that obtained with this level of protein in the presence of adequate calories. Furthermore, the caloric restriction may increase testis glycogen (Leathem, 1959c).

Protein anabolic levels are higher in the tissues of young growing animals and the body is more dependent on dietary protein level and quality for maintenance of the metabolic nitrogen pool than in adult animals. On the other hand, body protein reserves in adult animals permit internal shifts of nitrogen to the metabolic pool and to tissues when dietary sources are reduced or endocrine imbalances are imposed. Thus, Cole, Guilbert and Goss (1932) fed a low protein diet to adult male rats for 60 to 90 days before the sperm disappeared, but the animals would not mate. Amount of semen and sperm produced by sheep have been re

TABLE 12.7

Arlult rat testes and seminal vesicles

after protein depletion

(From J. H. Leatliem, Recent Progr. Hormone

Res., 14, 141, 1958.)

Days on

No. of

Testis

H2O

Protein

Total

Seminal

PFD*

Rats

Weight

Protein

Vesical

nig.

%

%dry

gm.

mg.

Control

9

2852

85.9

66.7

0.28

1276

30

9

2600

86.0

66.1

0.24

689

50

7

2398

85.4

64.1

0.22

320

90

25

1429

85.7

69.6

0.13

168

• PFD = Protein-free diet.

lated to the dietary protein level (Popoff and Okultilschew, 1936). Removal of protein from the diet for 30 days had little effect on the adult rat testis weight, spermatogenesis, or nitrogen content (Leathem, 1954). However, seminal vesicle w^eight was reduced 50 per cent (Aschkenasy, 1954). Prolonged protein depletion was required before the testis exhibited a loss in protein and a reduction in size. A loss of spermatozoa was not observed consistently, although some testes were completely atrophic (Table 12.7). Accessory organ weight decrease reflected the disappearance of androgen (Leathem, 1958a). Interstitial cell atrophy has also been noted in rats fed a low vegetable protein (cassava) diet (Adams, Fernand and Schnieden, 1958).

Sterility may or may not be induced with diets containing 65 per cent protein (Reid. 1949; Leathem, 1959c) but a 15 to 18 per cent dietary level of a poor protein such as maize or gelatin will decrease sperm motility and increase the number of abnormal sperm. The influence of proteins having different nutritional values in support of the growth of testes from the level to which they were depressed by stilbestrol indicated that casein, lactalbumin, and wheat gluten are equally competent to support testis growth whereas gelatin is deficient. Whole proteins may have several amino acid deficiencies, but the administration of amino acid antagonists may help to identify important individual amino acids. As an example, ethionine causes severe seminiferous tubule atrophy and Leydig cell hypoplasia (Kaufman, Klavins and Kinney, 1956; Goldberg, Pfau and Ungar, 1959). Studies in man have indicated a sharp reduction in

680

PHYSIOLOGY OF GONADS

spermatozoa after 9 days on an argininedeficient diet (Holt, Albanese, Shettles, Kajdi and Wangerin, 1942).

Adequate dietary protein cannot maintain reproductive function if the diet is calorie deficient. Thus, a decrease in seminal vesicle weight could be related to a decrease in dietary calories while protein levels were constant (Rosenthal and Allison, 1956). However, the accessory gland weight loss imposed by caloric restriction could be slowed by increasing the dietary protein (Rivero-Fontan, Paschkis, West and Cantarow, 1952).

Alterations in testis function imposed by inadequate protein are corrected when protein is returned to the diet at normal levels (Aschkenasy and Dray, 1953). Nevertheless, the nutritional state of the animal as a factor influencing recovery has been demonstrated with stilbestrol-treated adult male rats. While being fed an 18 per cent casein diet, adult male rats were injected with 0.1 mg. stilbestrol daily for 20 days. Testis weight decreased from 2848 to 842 mg., spermatogenesis was abolished, and testis water and protein content were significantly reduced. Despite a reduction in food intake, pair-fed controls exhibited no effect on reproductive organs. When a protein-free diet was substituted for the normal diet during the administration of stilbestrol, atrophy of the reproductive system was observed. Cessation of hormone administration was followed by a rapid return of testicular function toward normal when 18 per cent casein was fed both during the injection period and the recovery period. Within 30 days spermatogenesis and testicular composition were fully recovered. However, when 18 per cent casein was fed in the postinjection period to rats that had received a proteinfree diet while being given stilbestrol, recovery was clearly slow. After a month, spermatozoa were observed in only 30 per cent of the testes and testis weight was subnormal. Despite the seeming similarity of response by the two nutritional groups during the injection period, the postinjection recovery on identical dietary intake revealed marked differences in rate of recoverv (Leathem, 1958a).

3. Fat

Linoleic, linolenic, and arachidonic acids are designated as essentially fatty acids, but the physiologic role of these substances is not clearly understood. Nevertheless, the male reproductive organs are influenced by dietary essential fatty acid levels. High fat diets may enhance testicular weight (Kaunitz, Slanetz, Johnson and Guilmain, 1956) whereas removal of fat from the diet resulted in a degeneration of the seminiferous tubules as evidenced by intracellular vacuolation and a reduction in spermatids and spermatozoa (Panos and Finerty, 1954). After 5 months of feeding a fatfree diet, the rat testis may be devoid of sperm (Evans, Lepkovsky and Murphy, 1934). Testis degeneration occurred despite dietary supplements of vitamins A and E and in animals whose health appeared quite normal (Ferrando, Jacques, Mabboux and Prieur, 1955; Ferrando, Jacques, Mabboux and SoUogoub, 1955).

Weanling rats fed 14 per cent arachis (peanut) oil for 15 weeks exhibited a marked impairment of spermatogenesis (Aaes-Jorgensen, Funch and Dam, 1956) and 28 per cent arachis oil induced testicular damage of such an order that 15 weeks of feeding ethyl linoleate did not restore fertility (Aaes-Jorgensen, Funch and Dam, 1957).

4. Vitamins

Testicular dysfunction as judged by failure of sperm formation or atrophy of the secondary sex organs has been observed in deprivations of thiamine, riboflavin, pyridoxine, calcium pantothenate, biotin, and vitamins A and E. One must distinguish, however, between effects of inanition associated w^ith a vitamin deficiency and a specific vitamin effect (Skelton, 1950) ; one must also consider species differences (Biskind, 1946).

There is no question but that vitamin E deficiency in the rat results in a specific and irreversible damage to the testis. Tubular damage may proceed to the point where only Sertoli cells remain and yet the interstitial cells are not influenced (Mason, 1939). Similar changes followed vitamin E deficiency in the guinea pig (Pappenheimer and SchogolefT, 1944; Curto, 1954; Ingel

NUTRITIONAL EFFECTS

681

man-Siindberg, 1954) , hamster (Mason and Mauer, 1957), and bird (Herrick, Eide and Snow, 1952; Lowe, Morton, Cunningham and Vernon, 1957). However, little or no effect of an absence of vitamin E was noted in the rabbit (Mackenzie, 1942) and mouse (Bryan and Mason, 1941), or in live stock (Blaxter and Brown, 1952), or man (Lutwak-Mann, 1958), although vitamin E is present in human testes (Dju, Mason and Filer, 1958). Treatment of low-fertility farm animals with wheat germ oil or tocopherol or the use of this vitamin clinically have provided only inconclusive results (Beckmann, 1955) . Although some positive effects have been reported in man, the results may be due in part at least to the sparing action of tocopherol toward vitamin A.

Vitamin A deficiency influences the testis but changes are closely associated with the degree of inanition. In the rat, a vitamin deficiency sufficient to cause ocular lesions did not prevent sperm formation, but a deficiency of such proportions as to cause a body weight loss did cause atrophy of the germinal epithelium (Reid, 1949). Vitamin A deficiency will induce sterility in mice (McCarthy and Cerecedo, 1952). A gross vitamin deficiency in bulls before expected breeding age prevented breeding ; adult bulls may exhibit a lower quality semen but they remain fertile (Reid, 1949). Vitamin A deficiency induces metaplastic keratinization of the epithelium lining the male accessory sex organs (Follis, 1948) and thus may influence semen.

Testis damage induced by vitamin A deficiency can be reversed, but vitamin A therapy in man for oligospermia not due to vitamin lack was without effect (Home and Maddock, 1952) .

Age of the animal and dosage are factors which influence the results obtained in male rats with administered vitamin A. Immature male rats given 250 I.U. of vitamin A per gram of body weight daily exhibited a loss of spermatocytes, an effect which was accentuated by tocopherol (Maddock, Cohen and Wolbach, 1953). Little or no effect of similar treatment was observed in adult rats. The liver is the major storage depot for vitamin A and the fact that the male rat liver is more quickly depleted and less capa

ble of storage than is the liver of the female should be considered in any attempted correlation of the vitamin and hormone levels (Booth, 1952).

Other vitamin deficiencies have been shown to influence the testis. A lack of thiamine had little effect on testis weight, but did influence the Leydig cells and prevented growth of the accessory sex organs (Pecora and Highman, 1953). A chronic lack of ascorbic acid will cause a degeneration of both Leydig cells and seminiferous tubules. The effects of vitamin deficiency on the testis has been distinguished from those due to inanition and have been related to changes in carbohydrate metabolism (Mukherjee and Banerjee, 1954; Kocen and Cavazos, 1958) . The importance of ascorbic acid in the testis as related to function is not evident, but concentrations of this vitamin are maximal at 1 week of age (Coste, Delbarre and Lacronique, 1953).

Serious anatomic and functional impairments of testes were noted in pantothenic acid deficiency (Barboriak, Krehl, Cowgill and Whedon, 1957), and development of the rat testis and seminal vesicles was retarded by a biotin deficiency (Bishop and Kosarick, 1951; Katsh, Kosarick and Alpern, 1955), but the animals did not exhibit marked alterations in other endocrine organs (Delost and Terroine, 1954). Testosterone hastened the development of vitamin deficiency and enhanced the severity of biotin deficiency in both sexes, thereby suggesting a hormone-vitamin relationship (Okey, Pencharz and Lepkovsky, 1950). On the other hand, testosterone had no effect on the tolerance of mice for aminopterin, but castration increased the tolerance (Goldin, Greenspan, Goldberg and Schoenberg 1950).

B. INFLUENCE OF NUTRITION ON THE EE SPONSIVENESS OF MALE REPRODUCTIVE

TISSUES TO HORMONES

1. Testis

a. Inanition. The testes of birds on limited food intake were more responsive to hypophyseal gonadotrophin than fully fed birds (Byerly and Burrows, 1938; Breneman, 1940). In the rat several investigators have shown that the testis will respond to gonadotrophin despite inanition (Moore and Sara

682

PHYSIOLOGY OF GONADS

TABLE 12.8

Influence of diet and pregnant mare serum (PMS)

on testes and seminal vesicles of

immature male mice

(From V. J. DeFeo and J. H. Leathern,

unpublished.)

Diet (Per cent Protein X Days Fed)

per cent X 10 per cent X 10 per cent X 20 per cent X 20

l.u.

3

3

stage of Spermatogenesis

4 1

1 5 5

Spermatids

Seminal Vesicles

mg.

2.7 4.5

2.7 3.5

uels, 1931; Funk and Funk, 1939; Meites, 1953), with a stimulation of Leydig cells, an increase in testis size, and, in 40 days, a return of spermatozoa. Underfed males injected with gonadotrophin sired litters (Mulinos and Pomerantz, 1941a, b). Improved nutrition aided by unknown liver factors enhanced the response to androgen in severe human oligospermia (Glass and Russell, 1952).

b. Protein. Feeding a protein-deficient diet to adult male rats for 60 to 90 days did not prevent stimulation of the testes and seminal vesicles after pregnant mare's serum (PMS) administration (Cole, Guilbert and Goss, 1932). As we have noted, immature animals are prevented from maturing when diets lack protein. Nevertheless, a gonadal response to injected gonadotrophin was obtained in immature mice fed a protein-free diet for 13 days; tubules and Leydig cells were stimulated and androgen was secreted (Table 12.8). Refeeding alone permitted a recovery of spermatogenesis which was not hastened by concomitant PMS (Leathem, 1959c).

The maintenance of testis weight and spermatogenic activity with testosterone propionate in hypophysectomized adult male rats is well known, but these studies have involved adequate nutrition. If hypophysectomized rats were fed a protein-free diet and injected with 0.25 mg. testosterone propionate daily, testis weight and spermatogenesis were less well maintained than in rats fed protein. Testis protein concentra

tion was also reduced. These data suggest that influences of nutrition on the testis can be direct and are not entirely mediated through hypophyeal gonadotrophin changes (Leathem, 1959b).

c. Fat. The rat fed for 20 weeks on a fatfree diet exhibits a degeneration of the seminiferous epithelium within the first weeks which progresses rapidly thereafter. Chorionic gonadotrophin or rat pituitary extract started during the 20th week failed to counteract the tubular degeneration, but testosterone propionate proved effective (Finerty, Klein and Panos, 1957). The result shows that the ineffectiveness of the gonadotrophins could not be due to the failure of androgen release (Greenberg and Ershoff, 1951).

d. Vitamins. Gonadotrophins failed to promote spermatogenesis in vitamin A(Mason, 1939) or vitamin E- (]Mason, 1933; Geller, 1933; Drummond, Noble and Wright, 1939) deficient rats, but in another experiment the atrophic accessory sex organs of vitamin A-depleted rats were stimulated (Mayer and Goodard, 1951). Lack of vitamin A favored an enhanced response to PMS when the ratios of seminal vesicle weight to body weight were computed (Meites, 1953). The failure of gonadotrophins to stimulate testis tubules suggests a specific effect of avitaminosis A and E (Mason, 1933) on the responsiveness of the germinal epithelium.

Subnormal responses of rats to PMS, as measured by relative seminal vesicle weight, were obtained when there were individual vitamin B deficiencies, but the influence was due largely to inanition (Drill and Burrill, 1944; Meites, 1953). Nevertheless, sufficient response to chorionic gonadotrophin was obtained so that fructose and citric acid levels were restored to normal. Such an effect was not observed to follow dietary correction unless an unlimited food intake was allowed (Lutwak-Mann. 1958).

2. Sc

il W.siclfx and Prosfate

a. Inanition. Although the accessory reproductive organs resppnd to direct stimulation despite an inadequate food intake (Mooi'c and Samuels. 19311, tlio increase

NUTRITIONAL EFFECTS

683

in weight may be subnormal in mice and rats (Goldsmith, Nigrelli and Ross, 1950; Kline and Dorfman, 1951a, Grayhack and Scott, 1952), or above normal in chickens (Breneman, 1940). Complete deprivation of food reduced the quantity of prostatic fluid in the dog, but exogenous androgen restored the volume, increased acid phosphatase, and induced tissue growth (Pazos and Huggins, 1945) .

6. Protein. The response of the seminal vesicles to androgen was investigated in immature rats, using weight and /5-glucuronidase as end points. Castration and 10 days on a protein-free diet preceded the 72-hour response to 0.25 mg. testosterone propionate. The lack of protein did not prevent a normal weight increase, and enzyme concentration was unchanged. If an 18 per cent diet was fed during the 3-day period that the androgen was acting, no improvement in weight response was noted, but enzyme concentration increased 100 per cent. Thus, when protein stores are depleted, the androgen response may be incomplete in the absence of dietary protein (Leathern, 1959c). Nevertheless, varied protein levels do not influence seminal vesicle weight-response when caloric intake is reduced (RiveroFontan, Paschkis, West and Cantarow, 1952).

c. Vitamins. Vitamin deficiencies do not prevent the seminal vesicles from responding to androgen. In fact, in vitamin B deficiency, testosterone restored fructose and citric acid levels to normal despite the need for thiamine in carbohydrate metabolism (Lutwak-Mann and Mann, 1950). In the male, unlike the female, the effects of folic acid deficiency in reducing responsiveness to administered androgen were largely due to inanition in both mice and rats (Goldsmith, Nigrelli and Ross, 1950; Kline and Dorfman, 1951a) , and vitamin A deficiency which leads to virtual castration does not prevent an essentially normal response of the accessory glands to testosterone propionate (Mayer and Truant, 1949). Restricting the caloric intake of vitamin Adeficient rats retarded the curative effects of vitamin A in restoring the accessory sex glands of the A-deficient animals (Mason, 1939).

V. Female Reproductive System

A. OVARIES

1. Inanition

Mammalian species generally exhibit a delay in sexual maturation when food intake is subnormal before puberty, and ovarian atrophy with associated changes in cycles if inanition is imposed on adults. In human beings a decrease in fertility and a greater incidence of menstrual irregularities were induced by war famine (Zimmer, Weill and Dubois, 1944). Ovarian atrophy with associated amenorrhea and sterility were invoked by chronic undernutrition (Stephens, 1941). The ovarian morpliologic changes were similar to those of aging. Urinary estrogens were subnormal in 22 of 25 patients exhibiting amenorrhea associated with limited food intake (Zubiran and (_lomcz-Mont, 1953).

The nutritional requirements of jM'imates other than man have been studied in female baboons. The intake of vitamins and other essential nutrients was found to be of the same order as that recommended for man. Caloric intake varied with the menstrual cycle, being least during the follicular phase and maximal during the 2 to 7 days preceding menstruation (Gilbert and Gillman, 1956). Various diets were also studied to assess their importance in maintaining the normal menstrual rhythm. The feeding of (a) maize alone, (b) assorted vegetables and fruit, or (c) maize, skimmed milk, and fat led to menstrual irregularities or to amenorrhea. The mechanism regulating ovulation was the first to be deranged. The addition of various vitamins or of animal protein did not correct the menstrual disorders. However, inclusion of ox liver in the diet did maintain the menstrual rhythmicity, but the beneficial effect could not be attributed to its protein content (Gillman and Gilbert, 1956 ) .

In lower mammals that have been studied, inanition will hinder vaginal opening, and delay puberty and ovarian maturation and functioning. In adult rats and mice ostrous cycles are interrupted and the reprorUictive system becomes atrophic when body weight loss exceeds 15 per cent. The ovaries be

684

PHYSIOLOGY OF GONADS

come smaller, ovulation fails, and large vesicular follicles decrease in number with an increase in atresia, but primary follicles show a compensatory increase (Marrian and Parkes, 1929; Mulinos and Pomerantz, 1940; Stephens and Allen, 1941; Guilbert, 1942; Bratton, 1957). The ovarian interstitial cells mav be markedly altered or absent (Huseby and Ball, 1945; Rinaldini, 1949) and the ovary may exhibit excessive luteinization (Arvy, Aschkenasy, AschkenasyLelu and Gabe, 1946) or regressing corpora lutea (Rinaldini, 1949) . However, the ovarian changes induced by inanition may be reversed by refeeding, with a return to reproductive capacity (Ball, Barnes and Visscher, 1947; Schultze, 1955). The effect of feed-level on the reproductive capacity of the ewe has been reported (El-Skukh, Nulet, Pope and Casida, 1955), but one must realize that high planes of nutrition may adversely influence fertility (Asdell, 1949). Nevertheless, additional protein and calcium added to an adequate diet extended the reproductive life span (Sherman, Pearson, Bal, McCarthy and Lanford, 1956).

2. Protein

The availability of just protein has an important influence on the female reproductive system. In immature rats ovarian maturation was prevented by feeding diets containing per cent to 1.5 per cent protein (Ryabinina, 1952) and low protein diets decreased the number of ova but without altering their ribonucleic acid (RNA) or glycogen content (Ishida, 1957). Refeeding 18 per cent protein for only 3 days was marked by the appearance of vesicular follicles and the release of estrogen in mice previously fed a protein-free diet (Leathem, 1958a). In experiments involving the opposite extreme, in which 90 per cent protein diets were used, a retardation of ovarian growth, and a delay in follicular maturation, in vaginal opening, and in the initiation of estrous cycles were noted (Aschkenasy-Lelu and Tuchmann-Duplessis, 1947; TuchniannDuplcssis and Aschkenasy-Lelu, 1948).

Adult female rats fed a protein-free diet for 30 days exhibited ovaries weighing 22 mg. compared with ovaries weighing 56 mg. from i)air-fed controls fed 18 per cent casein. Ovarian glycogen, ascorbic acid, and cho

lesterol were all influenced by protein deprivation and anestrum accompanied the ovarian changes. Furthermore, uterine weight and gl3^cogen decreased in rats fed protein-free diets (Leathem, 1959b).

In adult rats the feeding of 3.5 per cent to 5 per cent levels of protein (GuillDert and Gross, 1932) was followed by irregularity of the cycles or by their cessation. The cycles became normal when 20 to 30 per cent protein was fed (Aschkenasy-Lelu and Aschkenasy, 1947). However, abnormally high levels of casein (90 per cent) induced prolonged periods of constant estrus (Tuchmann-Duplessis and AschkenasyLelu, 1947). Nevertheless, not all species responded to protein depletion in the same manner. For example, the rabbit exhibited estrus and ovulation despite a 25 per cent body weight loss imposed by to 2 per cent protein diets (Friedman and Friedman, 1940).

Despite a normal level of protein in the diet, inadequate calories will interfere with reproductive function and induce ovarian atrophy (Escudero, Herraiz and Mussmano, 1948; Rivero-Fontan, Paschkis, West and Cantarow, 1952). Furthermore, the effects of 15 per cent and 56 per cent protein levels on estrous cycles could not be distinguished when calories were reduced 50 per cent (Lee, King and Visscher, 1952). Returning mice to full feeding after months of caloric deficiency resulted in a sharp increase in reproductive performance well above that expected for the age of the animal (Visscher, King and Lee, 1952). This type of rebound phenomenon has not been explained.

Reproductive failure assigned to dietary protein may be a reflection of protein quality as well as level. Specific amino acid deficiencies lead to cessation of estrus (White and AVhite, 1942; Berg and Rohse, 1947) and thus feeding gelatin or wheat as the protein source and at an 18 per cent level was quickly followed by an anestrum (Leathem, 1959b). Supplementation of the wheat diet with lysine corrected the reproductive abnormalities (Courrier and Raynaud, 1932), but neither lysine (Pearson, Hart and Bohstedt, 1937) nor cystine (Pearson, 1936) added to a low casein diet was beneficial. Control of food intake must be considered in studies involving amino

NUTRITIONAL EFFECTS

685

acids, for a deficiency or an excess can create an imbalance and alter appetite. Opportunity to study the amino acids in reproduction is now possible because of the work of Greenstein, Birnbaum, Winitz and Otey (1957) and Schultze (1956) , who maintained rats for two or more generations on synthetic diets containing amino acids as the only source of protein. Similarly, the amino acid needs for egg-laying in hens has been reported (Fisher, 1957). Tissue culture methods also permit the study of the nutritional requirements of embryonic gonadal tissue, the success of avian gonadal tissue in culture being judged by survival, growth, and differentiation. In experiments in which this technique was used it was found that a medium made up of amino acids as the basic nitrogen source can maintain gonadal explants very successfully, even though the choice of amino acids does not exactly correspond to the 10 essential amino acids recommended for postnatal growth (StengerHaffen and Wolff, 1957).

3. Carbohydrate

The absence of dietary carbohydrate does not appreciably affect the regularity of estrous cycles in rats provided the caloric need is met. However, the substitution of 20 per cent sucrose for corn starch induced precocious sexual maturity which was followed by sterility (Whitnah and Bogart, 1956). The ovaries contained corpora lutea, but the excessive luteinization of unruptured follicles suggested a hypophyseal disturbance. Substitution of 20 per cent lactose for corn starch had no effect. Increased amounts of lactose retarded the gain in body weight and blocked ovarian maturation, possibly because the animal could not hydrolyze adequate amounts of the disaccharide. Addition of whole liver powder to the diet counteracted the depressing action of 45 per cent lactose on the ovary (Ershoff, 1949).

4. Fat

There seems to be little doubt that dietary fat is reciuired for normal cyclic activity, successful pregnancy, and lactation, and that the requirements for essential fatty acids are lower in females than in males (Deuel, 1956).

Conception, fetal development, and par

turition can take place in animals fed a diet deficient in fatty acids (Deuel, Martin and Alfin-Slater, 1954) , despite a reduction in total carcass arachidonic acid (Kummerow. Pan and Hickman, 1952). Earlier reports indicated that a deficiency of essential fatty acids caused irregular ovulation and impaired reproduction (Burr and Burr, 1930; Maeder, 1937). The large pale ovaries lead Sherman (1941) to relate essential fatty acid deficiency to carotene metabolism. In this regard the removal of essential fatty acids from an adequate diet supplemented with vitamin A and E lead to anestrum and sterility while maintaining good health (Ferrando, Jacques, Mabboux and Prieur, 1955). Perhaps the differences in opinion regarding the effects of fatty acid deficiency can be related to the duration of the experimental period. Panos and Finerty (1953) found that growing rats placed on a fat-free diet exhibited a normal time for vaginal opening, normal ovarian weight, follicles, and corpora lutea, although interstitial cells were atrophic. However, regular estrous cycles were noted for only 20 weeks, thereafter 60 per cent of the animals exhibited irregular cycles.

A decrease in reproductive function may be invoked by adding 14 per cent arachis oil to the diet (Aaes-Jorgensen, Funch and Dam, 1956) . Increasing dietary fat by adding rape oil did not influence ovarian function but did cause the accumulation of ovarian and adrenal cholesterol (Carroll and Noble, 1952).

Essential fatty acid deficiency is associated with underdevelopment and atrophic changes of the uterine mucosa. Adding fat to a stock diet enhanced uterine weight in young animals at a more rapid pace than body weight (Umberger and Gass, 1958) .

5. Vitamins

Carotenoid pigments are present in the gonads of many vertebrates and marine invertebrates, and, in mammals, are particularly prominent in the corpus luteum. However, no progress has been made in determining either the importance of the carotenoids in the ovary or of the factors controlling their concentrations. It is well known that vitamin A deficiency induces

686

PHYSIOLOGY OF GONADS

a characteristic keratinizing metaplasia of the uterus and vagina, but estrous cycles continue despite the vaginal mucosal changes. Furthermore, ovulation occurs regularly until advanced stages of deficiency appear. The estrous cycle becomes irregular in cattle fed for a long period of time on fodder deficient in carotene. The corpora lutea fail to regress at the normal rate and ovarian follicles become atretic and cystic ( Jaskowski, Watkowski, Dobrowolska and Domanski, cited by Lutwak-Mann, 1958). The alterations in reproductive organs associated with a lack of vitamin A may be due in part to a vitamin E deficiency since the latter enhances the rate at which liver stores of vitamin A are depleted.

Definite effects of hypervitaminosis A have been observed on reproduction. Masin (1950) noted that estrus in female rats could be prolonged by administration of 37,000 I.U. of vitamin A daily. The implications, however, have not been studied. The effect of hypervitaminosis A may actually induce secondary hypovitaminoses. The displacement of vitamin K by excess A is almost certain and similar relationships appear to exist with vitamin D (Nieman and Klein Obbink, 1954).

The failure of vitamin E-deficient female rats to become pregnant is apparently due to disturbances of the implantation process rather than to the failure of ovulation. There is no direct proof of ovarian dysfunction (Blandau, Kaunitz and Slanetz, 1949). However, the ovary of the rat deficient in vitamin E may have more connective tissue and pigment, and Kaunitz (1955) showed by ovarian transplantation that some nonspecific ovarian dysfunction appears to exist (cited by Cheng, 1959 (. Vitamin E is essential for birds, but there is little evidence for a dependency in most mammals; sheep, cows, goats, and pigs have been studied. Treatment of low-fertility farm animals with tocopherol has not provided conclusive data favoring its use (Lutwak-Mann, 1958), nor has the treatment of human females been rewarded with any indication that vitamin E might be helpful in cases of abnormal cycles and habitual abortion (Beckmann, 1955).

No specific reproductive disturbances in man, the rhesus monkey, or the guinea pig

have been associated with vitamin C deficiency (Mason, 1939). Nevertheless, the high ascorbic acid content of ovarian and luteal tissue and of the adrenal cortex suggests a physiologic role in association with steroid synthesis. (3varian ascorbic acid varies with the estrous cycle, dropping sharply in the proestrum (Coste, Delbarre and Lacronique, 1953), and decreasing in resjionse to gonadotrophin (Hokfelt, 1950; Parlow, 1958). Virtually no ascorbic acid is present in bovine follicular fluid (LutwakMann, 1954) or in rat ovarian cyst fluid (Blye and Leathem, 1959). Uterine ascorbic acid decreased in immature mice treated with estrogen, but remained unchanged in rats following thiouracil administration (Leathem, 1959a). Its role in the uterus awaits elucidation.

Delayed sexual maturation and ovarian atrophy have been described when there are deficiencies of thiamine, riboflavin, pyridoxine, pantothenic acid, biotin, and B12 (Ershoff, 1952; Ullrey, Becker, Terrill and Notzold, 1955). However, as we noted when deficiencies of the vitamins were being considered, much of the impairment of reproductive function can be related to inanition rather than to a vitamin deficiency (Drill and Burrill, 1944). Pyridoxine deficiency, although not affecting structure (Morris, Dunn and Wagner, 1953) , markedly reduces the sensitivity of the ovary to administered gonadotrophin (Wooten, Nelson, Simpson and Evans, 1958) .

Bird, frog, and fish eggs contain considerable quantities of vitamins. In fact, the daily human requirements for vitamins may be contained in a hen's egg and thus it is not surprising that hatchability is decreased l)y virtually any vitamin deficiency. Lutwak-Mann (1958) has provided an excellent survey of these data with numerous references to studies of frogs and fishes. Nearly all the B vitamins are present in fish roe and the pantothenic acid concentration in cod ovaries {Gadus morrhua) exceeds most otlicr natural sources. The amount of the latter varies with the reproductive cycle, d(>creasing to its lowest level before spawning. Riboflavin and vitamin B12 , on the other h;ui(l, do not change (Braekkan, 1955).

NUTRITIONAL EFFECTS

687

B. INFLUENCE OF NUTRITION ON THE RESPONSIVENESS OF FEMALE REPRODUCTIVE TISSUES TO HORMONES

1. Ovary

a. Inanition. Marrian and Parkes (1929) were the first to show that the quiescent ovary of the underfed rat can respond to injections of anterior pituitary as evidenced by ovulation and estrous smears. Subsequently the ovaries of underfed birds, rats, and guinea pigs were found to be responsive to serum gonadotrophin (Werner, 1939; Stephens and Allen, 1941; Mulinos and Pomerantz, 1941b; Hosoda, Kaneko, Mogi and Abe, 1956). A low calorie bread-andmilk diet for 30 days did not prevent ovarian response to rat anterior pituitary or to chorionic gonadotrophin. In these animals an increase in ovarian weight with repair of interstitial tissue, as well as folhcle stimulation and corpus luteum formation, were observed (Rinaldini, 1949). Rats from which food had been withdrawn for 12 days could respond to castrated rat pituitary extract with an increase in ovarian and uterine weight (Maddock and Heller, 1947). Nevertheless, differences in the time and degree of responsiveness to administered gonadotrophin were noted in rabbits. Animals on a high plane of nutrition responded to gonadotrophin at 12 weeks, whereas rabbits on a low plane of nutrition responded at 20 weeks and fewer eggs were shed (Adams, 1953).

b. Protein. Protein or amino acid deficiencies in the rat do not prevent a response to administered gonadotrophin (Cole, Guilbert and Goss, 1932; Courrier and Raynaud, 1932) . However, the degree and type of gonadal response is influenced by the diet. Thus, immature female mice fed to 6 per cent casein for 13 days exhibited only follicular growth in response to pregnant mare serum, whereas the ovarian response in mice fed 18 per cent casein was suggestive of follicle-stimulating and strongly luteinizing actions (Table 12.9). Furthermore, the ovarian response was significantly less after 20 days of nonprotein feeding than after 10 days of depletion (Leathem, 1958a). Ovarian stimulation by a gonadotrophin involves tissue protein synthesis and thus the type of whole protein fed

could influence the responses. Yamamoto and Chow (1950) fed casein, lactalbumin, soybean, and wheat gluten at 20 per cent levels and noted that the response to gonadotrophin as estimated by tissue nitrogen was related to the nutritive value of the protein. The ovarian weight response to chorionic gonadotrophin was less in rats fed 20 per cent gelatin than those fed 20 per cent casein (Leathem, 1959b). Inasmuch as the hypophysis may influence the gonadal response to injected hormone despite the diet, hypophysectomized rats fed a protein-free diet for 5 weeks and hyophysectomized rats on a complete diet were tested for response to gonadotrophins. The response to FSH was not influenced by diet, but the protein-depleted rats were twice as sensitive to interstitial cell-stimulating hormone (ICSH), human chorionic gonadotrophin (HCG), and PMS as the normal rats (Srebnik, Nelson and Simpson, 1958). Protein-depleted, normal mice were twice as sensitive to PMS as fully fed mice (Leathem and Defeo, 1952 1 .

c. Vitamins. In the female vitamin B deficiencies do not prevent ovarian responses to gonadotrophin (Figge and Allen, 1942), but the number of studies is limited. Be deficiency in DBA mice was associated with an increased sensitivity of the ovary to gonadotrophins (Morris, Dunn and Wagner, 1953), whereas pyridoxine deficiency in the rat decreased ovarian sensitivity, especially to FSH (Wooten, Nelson, Simp TABLE 12.9

Influence of dietary protein and pregnant mare

serum {PMS) on the mouse ovary

(From J. H. Leathem, Recent Progr. Hormone

Res., 14, 141, 1958.)

Diet fPer cent Protein X Days Fed)

c-5,

•g-s

1*

k

r

1 11

c 1

I.U.

mg.

mg.

per cent X 23

1.2

4.8

per cent X 2.3

3

2.8

13

10.8

per cent X 13

1.4

4.9

per cent X 13

3

4.4

16

1

15.1

6 per cent X 13

3.2

6

7.7

6 per cent X 13

3

5.6

12

1

31.9

18 per cent X 13

5.0

10

2

51.6

18 per cent X 13

3

8.0

7

4

51.3

088

PHYSIOLOGY OF GONADS

son and Evans, 1958 j. Administration of vitamin C concomitant witli gonadotropliin has been claimed to enhance ovarian response (DiCio and Schteingart, 1942), but in another study the addition of ascorbic acid inhibited the hiteinizing and ovulating action of the gonadotrophin (Desaive, 1956).

Whether induced by vitamin deficiency or by inanition, the anestrum in rats which follows 2 to 3 weeks' feeding of a vitamin B-deficient diet has been explored as a method for the assay of gonadotrophin. Pugsley (1957) has shown that there is considerable convenience of method and a satisfactory precision of response for the assay of HCG and pregnant mare serum.

2. Uterus and Vagina

a. Inanition. Limited food intake does not prevent an increase in uterine weight after estrogen. Testosterone propionate will markedly increase uterine growth despite a 50 per cent reduction in food intake (Leathem, Nocenti and Granitsas, 1956). Furthermore, dietary manipulations involving caloric and protein levels did not prevent the uteri of spayed rats from responding to estrogen (Vanderlinde and Westerfield, 1950). More specific biochemical and physiologic responses must be measured because starvation for 4-day periods clearly interferes with deciduoma formation (DeFeo and Rothchild, 1953). A start in the direction of studying tissuecomposition changes has been made by measuring glycogen. However, no changes were noted in uterine glycogen in fasting rats (Walaas, 1952), and estrogen promoted glycogen deposition in the uteri of starved rats as well as in the uteri of fully fed rats (Bo and Atkinson, 1953).

b. Fat. Interest in the hormone content of fat from the tissues of animals treated with estrogen for the purpose of increasing body weight has raised the question of tissue hormone content. If estrogen was to be detected in tissues, an increase in dietary fat was necessary. However, the increase in dietary fat decreased the uterine response to stilbcstrol (I'mberger and Gass, 1958), thus complicating the assay.

c. Vitainins. Stimulation of the uterus by estrogen does not require tliianiinc, ribo

flavin, pyridoxine, or pantothenic acid. On the other hand, a deficiency of nicotinic acid appears to enhance the response to low doses of estrogen (Kline and Dorfman, 1951a, b). However, Bio appears to be needed for optimal oviduct response (Kline, 1955) and is required for methyl group synthesis from various one-carbon precursors including serine and glycine (Johnson, 1958).

Response of the bird oviduct to stilbestrol requires folic acid (Hertz, 1945, 1948). It was shown subsequently that stilbestrol and estrone effects in frogs, rats, and the rhesus monkey also require folic acid. A folic acid deficiency can be induced by feeding aminopterin. In this way the estrogen effects can be prevented. Aminopterin also prevents the action of progesterone in deciduoma formation, from which it may be inferred that folic acid is necessary for deciduoma formation in the rat. Increased steroid or folic acid levels can reverse the antagonist's effect (Velardo and Hisaw, 1953).

The mechanism of folic acid action is not clear. It may function in fundamental metabolic reactions linked with nucleic acid synthesis. Brown (1953) showed that desoxyribonucleic acid could be substituted for folic acid in the bird. In the rat aminopterin interferes with the increase in uterine nucleic acids, and with nitrogen and P-^- uptake by nucleic acids following estrogen. Folic acid has been implicated in the metabolism of several amino acids (Davis, Meyer and McShan, 1956).

Rats ovariectomized at weaning and maintained on a vitamin E-free diet for 6 weeks to 10 months responded to estradiol in the same manner as rats supplemented with tocopherol. This finding suggests that an intimate physiologic relationship between estradiol and vitamin E is not very probable (Kaunitz, Slanetz and Atkinson, 1949). Nevertheless, vitamin E has been re]:»orted to act synergistically with ovarian hormones in dc^ciduoma formation (Kehl, Douard and Lanfranchi. 1951 ) and to influence nucleic acid turnover (Dinning. Simc and Day, 1956).

A vitamin-hormone interrelationship is apparent when estrogen and vitamin A are considered. Vitamin A-deficient female rats present evidence of a metaplastic uterine

NUTRITIONAL EFFECTS

689

epithelium in 11 to 13 weeks, but similar changes failed to develop in ovariectomized rats. Vitamin A-deficient castrated rats quickly developed symptoms of metaplasia when estrogen alone was administered, but no adverse effect followed the administration of estrogen combined with vitamin A (Bo, 1955, 1956). The vagina is different. Its epithelium becomes cornified in vitamin A-deficient normal and castrated rats. The cornification is histologically indistinguishable from that occurring in the estrous rat and can be prevented by vitamin A. In fact, vitamin A will quantitatively inhibit the effect of estrogen on the vaginal mucosa when both are applied locally (Kahn, 1954). Conversion of ^-carotene to vitamin A is influenced by tocopherol, vitamin Bi2 , insulin, and thyroid, with evidence for and against a similar action by cortisone (Lowe and Morton, 1956; Rice and Bo, 1958). An additional vitamin-hormone relationship is suggested by the augmentation of progesterone action in rabbits given vitamin Do .

3. Mammary Gland

Inanition prevents mammary growth, but feeding above recommended requirements for maintenance and growth from birth to the first parturition also seems to interfere with mammary growth. Furthermore, steroid stimulation of the mammary gland is influenced by nutritional factors. Using the male mouse, Trentin and Turner (1941) showed that as food intake decreased, the amount of estradiol required to produce a minimal duct growth w^as proportionately increased. In the immature male rat a limited food intake prevented the growth of the mammary gland exhibited by fully fed controls. Nevertheless, the gland was competent to respond to estrogen (Reece, 1950) . Inasmuch as the glands of force-fed hypophysectomized rats did not respond to estrogen (Samuels, Reinecke and Peterson, 1941; Ahren, 1959), one can assume that, despite inanition, a hypophyseal factor was present to permit the response of the mammary gland to estrogen. However, inanition (IMeites and Reed, 1949) , but not vitamin deficiencies (Reece, Turner, Hathaway and Davis, 1937), did reduce the content of hypophyseal lactogen in the rat.

Growth of the mammary gland duct in the male rat in response to estradiol requires a minimum of 6 per cent casein. Protein levels of 3 per cent and per cent failed to support growth of the duct (Reece, 1959) .

C. PREGNANCY

The human male after attaining adulthood is confronted with the problem of maintaining the body tissues built up during the growth period. However, in the human female it has been estimated that the replacement of menstrual losses may require the synthesis of tissue equivalent to 100 per cent of her body weight (Flodin, 19531. In the event of pregnancy and in all viviparous species, the female is presented with even more formidable demands and a limitation of nutritional needs can lead to loss of the embryo or fetus. The role of nutrition at this point in reproduction has always received considerable attention and is complicated by the circumstance that many food substances influence pregnancy (Jackson, 1959). However, in many instances there is no evidence that fetal loss or malformation induced by nutritional modifications has been the consequence of an endocrine imbalance and thus limitation of the immense literature is permissible.

During the first 15 days of pregnancy, a rat may gain 50 gm. Since the fetuses and placentas are small, most of the gain is maternal and is associated with an' increase in food intake of as much as 100 calories per kilogram of body weight (Morrison, 1956). During the first 2 weeks of pregnancy, marked storage of fat and water occurs in the maternal body and the animal's positive nitrogen balance is above normal. Liver fat also increases (Shipley, Chudzik, Curtiss and Price, 1953). The increased food intake in early pregnancy may therefore provide a reserve for late fetal growth, as food intake may decline to 65 per cent of the general pregnancy level during the last 7 days (Morrison, 1956). During this last week, fetal growth is rapid. The rapid growth has been related to (1) greater demands of the fetus, (2) greater amounts of food in the maternal blood, and (31 greater permeability of the placenta. Certainly the anabolic potential of fetal tissues is high and the mother can lose weight while the

690

PHYSIOLOGY OF GONADS

fetuses gain. But it is also important to recall that there is a shift in protein, because its distribution in organs of pregnant rats differs from that in nonpregnant animals (Poo, Lew and Addis, 1939). Other changes in the maternal organism were enumerated b}^ Newton (1952) and by Souders and Morgan (1957).

A measure of nitrogen balance during pregnancy, rather than weight of young at birth, has been suggested as a means of determining a diet adequate for reproduction (Pike, Suder and Ross, 1954). After the 15th day, a retention of body protein increases, blood amino nitrogen and amino acids decrease, and urea formation decreases. These metabolic activities suggest an increase in growth hormone although the levels of this hormone have not been estimated (Beaton, Ryu and McHenry, 1955). Placental secretions have also been associated with the active anabolic state of the second half of pregnancy, because removal of the fetuses in the rat did not change the anabolic activity, whereas removal of the placentas was followed by a return to normal (Bourdel, 1957). A sharp increase in liver ribose nucleic acid has been observed during late pregnancy in mice and rats and the effect attributed to a placental secretion or to estrogen. Species differences also influence the results because only a modest change in liver RNA was observed in guinea pigs and no change occurred in cats (Campbell and Kostcrlitz, 1953; Campbell, Innes and Kosterlitz, 1953a, b).

Clinical observations have related both

TABLE 12.10

Nutrition and pregnancy in rats

(From J. H. Leathern, in Recent Progress in

the Endocrinology of Reproduction, Academic

Press, Inc., New York, 1959.)

Calories/kg. Body Weight

Fetuses, Day 20

No.

Average weight

18 per cent casein

18 per cent casein

6 per cent casein

per cent casein

18 per cent gelatin

18 per cent gelatin

200 100

250 200 200 100

8 6

gm. 6.1

3.5

toxemia of pregnancy (Pequignot, 1956) and prematurity to inadequate nutrition (Jeans, Smith and Stearns, 1955). The potential role of protein deprivation in the pathogenesis of the toxemia of pregnancy prompted studies in sheep and rats. In sheep nutritionally induced toxemia simulates the spontaneous toxemia (Parry and Taylor, 1956), but only certain aspects of toxemia were observed in the pregnant rat subjected to low protein diets. When rats were fed 5 per cent casein and mated, fluid retention was observed (Shipley, Chudzik, Curtiss and Price, 1953) and pregnancy was completed in only 48 per cent of the animals Curtiss, 1953). Gain in body weight in the adult rat and gain in fetal weight were subnormal as the result of a low protein feeding during pregnancy.

Complete removal of protein from the diet beginning at the time of mating did not prevent implantation but did induce an 86 to 100 per cent embryonic loss. The effect was not related solely to food intake (Nelson and Evans, 1953) , as we will see in what follows when the relationship between protein deficiency and the supply of estrogen and progesterone is described. Limiting protein deprivation to the first 9 to 10 days of pregnancy will also terminate a pregnancy, but when the protein was removed from the diet during only the last week of pregnancy, the maternal weight decreased without an effect on fetal or placental weight (Campbell and Kosterlitz, 1953). As would be anticipated, a successful pregnancy requires protein of good nutritional quality and the caloric intake must be adequate. Thus, an 18 per cent gelatin diet failed to maintain pregnancy when 200 calories per kilogram were fed, whereas a similar level of casein was adequate (Table 12.10). However, reducing caloric intake to 100 calories despite an otherwise adequate protein ration influenced the number and size of fetuses (Leathern, 1959b). Additional proteins should be studied and related to biochemical changes in pregnancy and to the need for specific amino acids; for example, elimination of methionine or tryptophan from the diet may or may not be followed by resorption (Sims, 1951; Kemeny, Handel, Kertesz and Sos, 1953; Albanese, Randall and Holt, 1943). Excretion of 10 amino acids was in

NUTRITIONAL EFFECTS

691

creased during normal human pregnancy (Miller, Ruttinger and Macey, 1954).

That a relationship exists, between the dietary requirements just described to the endocrine substances which participate in the control of pregnancy, is suggested by the fact that the deleterious effects of a proteinfree diet on pregnancy in rats have been counteracted by the administration of estrone and progesterone. Pregnancy was maintained in 30 per cent, 60 to 80 per cent, and per cent of protein-deficient animals by daily dosages of 0.5 /xg., 1 to 3 fig., and 6 jug. estrone, respectively. On the other hand, injection of 4 to 8 mg. progesterone alone maintained pregnancy in 70 per cent of the animals (Nelson and Evans, 1955) , and an injection of 4 mg. progesterone with 0.5 //.g. estrone provided complete replacement therapy (Nelson and Evans, 1954). Food intake did not increase. The results suggest that reproductive failure in the absence of dietary protein was due initially to lack of progesterone and secondarily to estrogen, the estrogen possibly serving as an indirect stimulation for luteotrophin secretion and release. It is well known that hypophysectomy or ovariectomy shortly after breeding will terminate a pregnancy and that replacement therapy requires both ovarian hormones. Thus, the protein-deficient state differs somewhat from the state following hypophysectomy or ovariectomy, but the factors involved are not known.

Pregnancy alters nutritional and metabolic conditions in such a way that labile protein stores of the liver and other parts of the body are influenced, but similar effects are imposed by a transplanted tumor, especially when it reaches 10 per cent of the body weight. ' Thus, transplantation of a tumor into a pregnant animal would place the fetuses in competition with the tumor for the amino acids of the metabolic pool. Under these circumstances will the pregnancy be maintained? An answer to the question may not yet be given. Nevertheless, Bly, Drevets and Migliarese (1955) observed various degrees of fetal damage in pregnant rats bearing the Walker 256 tumor, and 43 per cent fetal loss was obtained with a small hepatoma (Paschkis and Cantarow, 1958).

Essential fatty acid deficiency, at least in the initial stages, does not interfere with

development of the fetuses or parturition in the rat, but the pups may be born dead or they do not survive more than a few days (Kummerow, Pan and Hickman, 1952). A more pronounced deficiency has induced atrophic changes in the decidua, resorption of fetuses, and prolonged gestation. Death of the fetuses appears to be secondary to placental injury. Hormonal involvement, if any, when there is fatty acid deficiency and pregnancy seems not to have been investigated.

Pregnancy and lactation are major factors influencing vitamin requirements. It is not surprising, therefore, that vitamin deficiencies influence the course of a pregnancy. The subject has recently been reviewed by Lutwak-jMann (1958).

A deficiency of vitamin A does not noticeably affect early fetal development, but later in gestation placental degeneration occurs with hemorrhage and abortion. When the deficiency is moderate the pregnancy is not interrupted, but the fetuses are damaged (Warkany and Schraffenberger, 1944; Wilson, Roth and Warkany, 1953; Giroud and Martinet, 1959). In calves and pigs the abnormalities are associated with the eyes and palate (Guilbert, 1942) ; in birds skeletal abnormalities are seen (Asmundson and Kratzer, 1952). The use of hormones in an effort to counteract the effects seems to have been attempted only in the rabbit where 12.5 mg. progesterone improved reproduction impaired by vitamin A lack (Hays and Kendall, 1956). Vitamin A excess also proves highly detrimental to pregnancy, as resorption and malformations occur. Administration of excessive vitamin A on days 11 to 13 of pregnancy induced cleft palate in 90 per cent of the embryos (Giroud and Martinet, 1955) . In another experiment the effect of excessive vitamin A was augmented by cortisone (Woollam and Millen, 1957).

Vitamin E deficiency has long been known to influence pregnancy in rodents and fetal death appears to precede placental damage and involution of the corpora lutea. Gross observations of the abnormal embryos have been reported (Cheng, Chang and Bairnson, 1957). Estrogen, progesterone, and lactogen were not effective in attempts at corrective therapy (Ershoff, 1943), but estrone and progesterone markedly reduced the in

692

PHYSIOLOGY OF GONADS

cidence of congenital malformations associated with vitamin E lack (Cheng, 1959). In the test of a possible converse relationship, estradiol-induced abortion in guinea pigs was not prevented by vitamin E (Ingelman-Sundberg, 1958) .

Fat-soluble vitamins incorporated in the diet may be destroyed by oxidation of the unsaturated fatty acids. To stabilize the vitamins, the addition of diphenyl-p-phenylenediamine (DPPD) to the diet has proven successful, but recent studies show that DPPD has an adverse effect on reproduction and thus its use in rat rations is contraindicated (Draper, Goodyear, Barbee and Johnson, 1956).

Vitamin-hormone relationships in pregnancy have been studied with regard to thiamine, pyridoxine, pantothenic acid, and folic acid. Thiamine deficiency induced stillbirths, subnormal birth weights, resorption of fetuses, and loss of weight in the mother. However, as in the case of protein deficiency, pregnancy could be maintained with 0.5 fjLg. estrone and 4 mg. progesterone (Nelson and Evans, 1955). Estrone alone had some favorable effect on the maintenance of pregnancy in thiamine-deficient animals, but it was less effective in protein-deficient animals.

Fetal death and resorptions as well as serum protein and nonprotein nitrogen (NPN) changes similar to those reported for toxemia of pregnancy (Ross and Pike, 1956; Pike and Kirksey, 1959) were induced by a diet deficient in vitamin Be . Administration of 1 fxg. estrone and 4 mg. progesterone maintained pregnancy in 90 per cent of vitamin Be-deficient rats (Nelson, Lyons and Evans, 1951). However, the pyridoxinedeficient rat required both steroids to remain pregnant and in this regard resembled the hypophysectomized animal (Nelson, Lyoas and Evans, 1953). Nevertheless, a hypophyseal hormone combination which was adequate for the maintenance of pregnancy in the fully fed hypophysectomized rat (Lyons, 1951) was only partially successful when there was a deficiency of pyridoxine. An ovarian defect is suggested.

The folic acid antagonist, 4-aminopteroylglutamic acid, will rapidly induce the death of early implanted embryos in mice.

rats, and man (Thiersch, 1954j . Removal of folic acid from the diet or the addition of x-methyl folic acid will induce malformations when low doses are given and resorptions when high doses are given. Furthermore, this effect is obtained even when the folic acid deficiency is delayed until day 9 of a rat pregnancy or maintained for only a 36-hour period. A deficiency of pantothenic acid will also induce fetal resorption. The vitamin is required for hatching eggs (Gillis, Heuser and Norris, 1942). In animals deficient in folic acid or in pantothenic acid, estrone and progesterone replacement therapy did not prevent fetal loss, suggesting that the hormones cannot act (Nelson and Evans, 1956). In the above mentioned deficiencies replacement of the vitamin is effective. However, vitamins other than those specifically deleted may provide replacement, thus ascorbic acid seems to have a sparing action on calcium pantothenate (Everson, Northrop, Chung and Getty, 1954) .

Pregnancy can be interrupted by altering vitamins other than those discussed above, but the hormonal aspects have not been explored. Thus, the lack of choline, riboflavin, and Bi2 will induce fetal abnormalities and interrupt gestation (Giroud, Levy, Lefebvres and Dupuis, 1952; Dryden, Hartman and Gary, 1952; Jones, Brown, Richardson and Sinclair, 1955; Newberne and O'Dell, 1958) . Choline lack is detrimental to the placenta (Dubnov, 1958), riboflavin deficiency may impair carbohydrate use (Nelson, Arnrich and Morgan, 1957) and/or induce electrolyte disturbances (Diamant and Guggenheim, 1957) , and Bjo spares choline and may be concerned with nucleic acid synthesis (Johnson, 1958). Excessive amounts of Bio are not harmful. It is interesting to note that uterine secretions and rabbit blastocyst fluid are rich in vitamin B]2 (Lutwak-Mann, 1956), but its presence in such large amounts has not been explained.

An additional substance, lithospermin, extracted from the plant, Lathijrus odoratus, is related to hormone functioning; it is antigonadotrophic when eaten by nonpregnant animals and man. The feeding of this substance to prciiiiant rats terminated the pregnancies about the 17th day. Treatment with estrogen and progesterone was preventive (Walker and Wirtschafter, 1956). It is assumed, therefore, that lithospermin interfered with the production of these hormones. A repetition of the experiment on a species in which the hypophysis and ovaries are dispensable during much of pregnancy would be of interest.

In retrospect it has been found that a deficiency in protein and the vitamins thiamine, pyridoxine, pantothenic acid, and folic acid individually can interrupt a pregnancy. Furthermore, a combination of estrone and progesterone which is adequate to maintain pregnancy after hypophysectomy and ovariectomy, is equally effective in protein or thiamine deficiency. This suggests that the basic physiologic alteration is a deprivation of ovarian hormones. However, protein- and thiamine-deficiency states differ from each other as shown by the response to estrogen alone (thiamine deficiency is less responsive), and these states differ from hypophysectomy in which estrone alone has no effect. A pyridoxine deficiency seems to involve both ovary and hypophysis, for neither steroids nor pituitary hormones were more than partially successful in maintaining pregnancy in rats. Lastly, pantothenic acid and folic acid deficiencies may not create a steroid deficiency. What is involved is not known; many possibilities exist. Pantothenic acid, for example, participates in many chemical reactions. Furthermore, it is known that thiamine is essential for carbohydrate metabolism but not for fat metabolism whereas pyridoxine is involved in fat metabolism and in the conversion of tryptophan to nicotinic acid. It is clear, though, that much ground must be covered before the formulation of fruitful hypotheses may be anticipated.

VI. Concluding Remarks

The development, composition, and normal functioning of the reproductive system is dependent on adequate nutrition. However, the requirements are many and only gradually are data being acquired which are pertinent to the elucidation of the nutritional-gonadal relationship.

The demands for nutrient substances is not always the same. During pregnancy and lactation there is a need for supplemental feeding. A similar need exists in birds and in the many cold-blooded vertebrates in which reproduction is seasonal. Atypical endocrine states create imbalances and a need for nutrient materials which vary, unpredictably, we must acknowledge, until the numerous interrelationships have been clarified.

At many points where determination of cause and effect are possible, an indirect action of dietary factors on reproduction is indicated. No other conclusion seems possible in view of the many instances in which the effect of dietary deficiencies can be counteracted by the administration of a hormone or combination of hormones. The direct action is not immediately apparent; it probably is on the processes by which metabolic homeostasis is maintained, and is in the nature of a lowering of the responsiveness to the stimuli which normally trigger these processes into action. The processes may be those by which pituitary and gonadal hormones are produced or they may be the mechanisms by which these hormones produce their effects on the genital tracts and on the numerous other tissues on which they are known to act.

Because of the many interrelationships, some of which are antagonistic and some supportive, determination of the role of specific dietary substances is not easy. For those who work with laboratory species, the problem is further complicated by the many strain differences. For everyone, the problem is complicated by the many species differences which are the result of an evolution toward carnivorous, herbivorous, or omnivorous diets, to say nothing of the countless specific preferences within each group.

Finally, it is something of a paradox in our culture that much of our effort has been devoted to investigations of the effects of deficiencies and undernutrition rather than to the effects of excesses and overnutrition. Much evidence supports the view that in the aggregate the latter are fully as deleterious as the former, but the means by which this result is achieved are largely unknown.

VII. References

Aak.s-Jorgensen, E., Funch, J. P., and Dam, H.

1956. Role of fat in diet of rats; influence on reproduction of hydrogenated arachis oil as sole dietary fat. Brit. J. Nutrition, 10, 317.

Aaes-Jorgensen, E., Funch, J. P., and Dam, H.

1957. Role of fat in diet of rats; influence of small amount of ethyl linoleate on degeneration of spermatogenic tissue caused by hydrogenated arachis oil as sole dietary fat. Brit. J. Nutrition, 11, 298.

AcETO, G., Li Moli, S., and Panebianco, N. 1956. Influence of adrenocorticotrophin (ACTH) on the urinary excretion of thiamine. Acta vitaminol., 10, 175.

Adams, C. E. 1953. Mammalian germ cells. Ciba Foundation Symposium, 198.

Adams, C. W. M., Fernand, V. S. V., and Schnieden, H. 1958. Histochemistry of a condition resembling kwashiorkor produced in rodents by a low protein-high carbohydrate diet (cassaval). Brit. J. Exper. Path., 39, 393.

Addis, T., Poo, L. J., and Lew, W. 1936. The ciuantities of protein lost by the various organs and tissues of the body during a fast. J. Biol. Chem., 115, 111.

Ahren, K. 1959. The effect of dietary restriction on the mammary gland development produced by ovarian hormones in the rat. Acta endocrinol., 31, 137.

Albanese, a. a., Randall, R. M., .\nd Holt, L. E., Jr. 1943. The effect of tryptophan deficiency on reproduction. Science, 97, 312.

Angervall, L. 1959. Alloxan diabetes and pregnancy in the rat. Effects on offspring. Acta Endocrinol., suppl. 44, 31, 86.

Arvy, L., Aschkenasy, A., Aschkenasy-Lelu, P., AND Gabe, M. 1946. Retentissement d'un regime prolonge d'inanition proteique sur les glandes genitales du rat femelle. Compt. rend. Soc. biol., 140, 730.

Aschkenasy, A. 1954. Action de la testosterone, de la thyroxine ct do la cortisone sur revolution de I'anemie et de la leucopenie proteiprives. Le Sang, 25, 15.

Aschkenasy, A. 1955a. Influence des proteines alimentaires et de divers acides amines sur la survie et le poids somatique de i-ats surrenalectomises et de rats surrenalcctomises-castres. Ann. endocrinol., 16, 86.

Aschkenasy, A. 1955b. Influence des proteines alimentaires et de divers acides amines sur la survie et sur le poids somatique de rats surrenalcctomises et de rats surrenalectomisescastres. Ann. endocrinol.. 16, 199.

Aschkenasy, A. 1955c. Effects de la .surrenalectomie sur les poids relatifs de divers organes et tis.sus. Role du facteur temps et influence do la richnesse du regime en proteines. J. jjhvsiol., Paris, 47, 75.

Aschkenasy, A. 1957. Effets compares de I'ACTH, de la cortisone et de la delta-cortisone sur les poids de divers organes et tissus chez le rat male adulte. Ann. endocrinol., 18, 981.

Aschkenasy, A., Aschkenasy-Lelu, P. 1957. Nouvelles recerches sur le role des surrenales dans la gestation en regime pauvre en proteines. Compt. rend. Soc. biol., 151, 880.

Aschkenasy, A., and Dray, F. 1953. Action de quelques hormones sur la reprise du poids et sur la regeneration des organes apres inanition proteique experimental. Compt. rend. Soc. biol., 147, 1722.

Aschkena.sy-Lelu, p., and Aschkenasy, A. 1947. Influence du taux des protides de la ration sur le cycle oestral de la rate adulte. Perturbation du cycle avec prolongation des oestrus sous d'action d'un regime hyperprotidique. Compt. rend. Soc. biol., 141, 687.

Aschkenasy-Lelu, P., and Aschkenasy^ A. 1957. Intervention des surrenales dans la gestation en fonction de la teneur de regime en proteines. Arch. so. physioL, 11, 125.

Aschkenasy-Lelu, P., and Tuchmann-Duplessis, H. 1947. Action d'un regime hyperprotidique sur le developpement somaticjue et sexual de la jeune rate. c3ompt. rend. Soc. biol., 141, 1166.

AsDELL, S. A. 1949. Nutrition and treatment of sterility in dairy cattle: a review. J. Dairv Sc. 32, 60.

AsMUND.soN, V. S., AND Kr.\tzer, F. H. 1952. Observations on vitamin A deficiency in turkey breeding stock. Poultry Sc, 31, 71.

Baker, B. L. 1952. Comparison of histological changes induced by experimental hyperadrenalcorticalism and inanition. Recent Progr. Hormone Res., 7, 331.

Ball, Z. B., B.\rnes, R. H., and Visscher, M. B. 1947. The effects of dietary caloric restriction on maturity and senescence, with particular reference to fertility and longevitv. Am. J. Physiol., 150, 511.

Barborlak, J. J., Krehl, W. A., Cowgill, G. R.. .-AND Whedon, a. D. 1957. Effect of partial pantothenic acid deficiencj' on reproductive performance of rat. J. Nutrition, 63, 591.

BE.ATON, G. H., Ryu. M. H., and McHenry. E. W. 1955. Studies on the role of growth hormone in pregnancy. Endocrinology, 57, 748.

Beckmann, R. 1955. Vitamin E (Physiologic, pathologische Physiologie, khnische Bedeutung). Ztschr. Vitamin- Hormon- u. Fermentforsch., 7, 281.

Berg, C. P., and Rohse, W. G. 1947. Is sterility induced in growing rats on a tryptoplian deficient diet? Science. 105, 96.

BiDDULiMi, C, AND Meyer. R. K. 1941. Tho influence of vitamin E-deficiency on the endocrine glands of rats, particularly on the gonadotrophic hormone content of the pituitary gland. Am. J. Physiol., 132, 259.

Bishop, D. W., and Ko.s.\rick, E. 1951. Blot in deficicncv and crytorchidism in rats. Proc. Soc. Exper. Biol. & Med., 78, 323.

Biskind, M. S. 1946. Nutritional therapy of endocrine disturbances. Vitamins & Hormones, 4, 147.

Blandau, R. J., Kauntiz, H., and Slanetz, C. A. 1949. Ovulation, fertilization and transport of ova in old vitamin E-deficient rats. J. Nutrition, 38, 97.

Blaxter, K. L., and Brown, F. 1952. Vitamin E in the nutrition of farm animals. Nutritional Abstr. & Rev., 21, 1.

Blivaiss, B. B., Hanson, R. O., Rosenzweig, R. E., AND McNeil, K. 1954. Sexual development in female rats treated with cortisone. Proc. Soc. Exper. Biol. & Med., 86, 678.

Bly, C. G., Drevets, C., and Migliarese, J. F. 1955. Competition between fetal growth and transplanted tumor growth in pregnant rats. Fed. Proc, 14, 399.

Blye, R. p., and Leathem, J. H. 1959. Ovarian ascorbic acid in gonadotrophin-treated and normal hypothyroid rats. Fed. Proc, 18, 49.

Bo, W. J. 1955. The effect of ovariectomy on metaplastic changes in the uteri of vitamin A deficient rats. Anat. Rec, 121, 241.

Bo, W. J. 1956. The relationship between vitamin A deficiency and estrogen in producing uterine metaplasia in the rat. Anat. Rec, 124, 619.

Bo, W. J., and Atkinson, W. B. 1953. Histochemical studies on glycogen deposition in uterus of the rat. III. Effect of starvation. Proc. Soc. Exper. Biol. & Med., 83, 405.

BoLDT, R. E., Harper, A. E. and Elvehjem, C. A. 1958. The effect of amino acids on the survival of the hvperthyroid rat. Arch. Biochem., 74, 437.

Booth, V. H. 1952. Liver storage of vitamin A by male and female rats. J. Nutrition, 48, 13.

Bourdel, G. 1956. L'anabolisme gravidique: ses caracteristiques son determinisme. In Proceedings Fourth International Congress of Nutrition, p. 37.

Br.\ekkan, O. R. 1955. Role of pantothenic acid in the reproductive cycle of ovaries in fish. Nature, London, 176, 598.

BR.ATT0N, R. W. 1957. Nutrition and fertility. Cornell University Agric Exper. Sta. Bull., 924, 25.

Breneman, W. R. 1940. Limitation of food consumption as a factor influencing endocrine reactions in the chick. Endocrinolog.y, 26, 1091.

Brown, W. O. 1953. Effect of deoxypentose nucleic acid on impaired oviduct response to oestrogen in the folic acid deficient-chick. Biochim. et biophys. acta, 11, 162.

Bryan, W. L., .^nd Mason, K. E. 1941. Vitamin E deficiency in the mouse. Am. J. Physiol., 131, 263.

Burr, G. O., and Burr, M. M. 1930. On the nature and role of the fatty acids essential in nutrition. J. Biol. Chem., 86, 587.

Byerly, T. C, and Burrows, W. H. 1938. Chick testis weight response to gonadotrophic hormone. Endocrinology, 22, 366.

Campbell, R. M., Innes, I. R., and Kosterlitz, H. W. 1953a. The role of hormonal and

dietary factors in the formation of excess ribonucleic acid in the livers of pregnant rats. J. Endocrinol., 9, 52.

Campbell, R. M., Innes, I. R., and Kosterlitz, H. W. 1953b. Some dietary and hormonal effects on maternal, fetal and placental weights in the rat. J. Endocrinol., 9, 68.

Campbell, R. M., and Kosterlitz, H. W. 1953. Species differences in the deoxyribonucleic and ribonucleic acid contents of livers of nonpregnant and pregnant mice, guinea pigs and cats. J. Endocrinol., 9, 45.

Cantarow, a., Paschkis, K. E., Rakoff, A. E., and Hansen, L. P. 1943. Studies on inactivation of estradiol by the liver. Endocrinology, 33, 309.

Carroll, K. K., and Noble, R. L. 1952. Effects of feeding rape oil on some endocrine functions of the rat. Endocrinology, 51, 476.

Cheng, D. W. 1959. Effect of progesterone and estrone on the incidence of congenital malformations due to maternal vitamin E deficiency. Endocrinolog}-, 64, 270.

Cheng, D. W., Chang, L. F., and Bairnson, T. A. 1957. Gross observations on developing abnormal embryos induced by maternal vitamin E deficiency. Anat. Rec, 129, 167.

Chester Jones, I. 1957. The Adrenal Cortex. Cambridge : Cambridge University Press.

Cole, H. H., Guilbert, H. R., and Goss, H. 1932. Further considerations of the properties of the gonad-stimulating principle of mare serum. Am. J. Physiol., 102, 227.

Coste, F., Delbarre, F., .\nd Lacronique, F. 1953. Variations du taux de I'acide ascorbiques des endocrines sous I'infiuence des stimulines hypophj'saires. Taux de I'acide ascorbique des testicules du rat en fonction de I'age. Compt. rend. Soc. biol., 147, 608.

CouRRiER, R., AND CoLLONGE, A. 1951. Cortisone and gestation. J. A. M. A., 146, 493.

CouRRiER, R., AND R.^YNAUD, R. 1932. Modifications genitales chez les animaux a croissance arretee par la deficience en lysine. Compt. rend. Soc. biol., 109, 881.

Cupps, P. T. 1955. The ability of certain adrenal steroids to restore reproduction in adrenalectomized female rats. Endocrinology, 57, 1.

Curtiss, C. 1953. Effects of a low protein intake on the pregnant rat. Metabolism, 2, 344.

CuRTO, G. M. 1954. Experimental vitamin E deficiency in male guinea pigs. Acta vitaminol., 8,7.

D'Angelo, S. a. 1951. Effect of acute starvation on the thyrotrophic hormone level in the blood of rat and mouse. Endocrinology, 48, 341.

Davies, D. v., Mann, T., and Rowson, L. E. A. 1957. Effect of nutrition on the onset of male sex hormone activity and sperm formation in monozygous bull-calves. Proc. Roy. Soc. Loudon, ser. B, 147, 332.

D.wis, J. S., Meyer, R. K., and McShan, W. H. 1956. The effects of aminopterin and estrogen on the phosphate metabolism of rat uterus. Endocrinology, 59, 505.

Davis. M. E., Fugo, N. W., .'Vxd Lawrence, K. G. 1947. Effect of alloxan diabetes on reproduction in the rat. Proc. Soc. Exper. Biol. & Med., 66, 638.

Davis, M. E., and Plotz, E. J. 1954. The effects of cortisone acetate on intact and adrenalectomized rats during pregnancy. Endocrinology, 54, 384.

DeFeo, V. J., and Rothchild, I. 1953. The effects of starvation of intact young rats on deciduoma formation and the maintenance of pseudopregnancy. Anat. Rec, 115, 297.

Delost, p., and Terroine, T. 1954. Effets de la carence en biotine sur certaines glandes endocrines au cours du developpement post-natal du rat. Compt. rend. Acad. Sc, 239, 902.

DeMeig, R. H., Rakoff, A. E., Cantarow, A., and Paschkis, K. E. 1948. Mechanism of inactivation of alpha-estradiol by the rat liver in vitro. Fed. Proc, 7, 27.

Desaive, p. 1956. Du role de la vitamine C dans le fonctionnement de I'ovaire. Compt. rend. A. anat., 42, 431.

Deuel, H. J. 1956. Sex differences in fat and carbohydrate metabohsm. Internat. Ztschr. Vitaminforsch., 26, 352.

Deuel, H. J., Martin, C. R., and Alfin-Slater, R. B. 1954. The effect of fat level of the diet on general nutrition. J. Nutrition, 54, 193.

Dhyse, F. G., Fisher, G. R., Tullner, W. W., AND Hertz, R. 1953. Liver vitamin content and adrenal cortical function. Endocrinology, 53, 447.

Diamant, E. J., AND Guggenheim, K. 1957. Electrolyte metabolism is pyridoxine, riboflavin and pantothenic acid-deficient rats. Am. J. Physiol., 191, 108.

DiCio, A. v., AND Schteingart, M. 1942. The influence of ascorbic acid on the activity of gonadotrophic hormones. Endocrinology, 30, 263.

Dinning, J. S., Sime, J. T., and Day, P. L. 1956. Increased incorporation of P^- into nucleic acids by vitamin E-deficient rabbits. J. Biol. Chem., 222, 215.

Dju, M. Y., M.asgn, K. E., and Filer, L. J. 1958. Vitamin E in human tissues from birth to old age. Am. J. Clin. Nutrition, 6, 50.

Draper, H. H., Goodyear, S., Barbee, K. D., and Johnson, B. C. 1956. Tolerance of the rat for A,iV^-dipli('nvI-/^-]ihenvlenediamine. Proc. Soc. Exper. Biol. <^ Med., 93, 186.

Draper, H. H., and Johnson, B. C. 1953. Effect of cortisone on metabolism of certain B vitamins in the rat. Proc. Soc. Exper. Biol. & Med., 82, 73

Drashek, M. L., Dickie, M. M., and Lane. P. W. 1955. Physiologic differences in uteri of obese stock mice. J. Hered., 46, 209.

Drill, V. A. 1943. Interrelations between thyroid function and vitamin metabolism. Phvsiol. Rev., 23, 355.

Drill, V. A., .and Burrill, M. W. 1944. Effect of thiamine deficiency and controlled inanition on ovarian function. Endocrinology, 35, 187.

Drill, V. A., and Pfeiffer, C. A. 1946. Effect of vitamin B complex deficiency, controlled inanition and methionine on inactivation of estrogen by the liver. Endocrinology, 38, 300.

Drummond, J. C, Noble, R. L., .\nd Wright, M. D. 1939. Studies of relationship of vitamin E (tocopherols) to the endocrine system. J. Endocrinol., 1, 275.

Dryden, L. p., Hartman, A. M., and Cary, C. A. 1952. The effect of vitamin B12 deficiency upon the survival of young born to rats fed purified casein rations. J. Nutrition, 46, 281.

DuBNOv, M. V. 1958. Chohne-free diets in pregnancy. Arch. Path., 20, 68.

DuMM, M. E., AND Ralli, E. p. 1953. Factors influencing the response of adrenalectomized rats to stress. Metabolism, 2, 153.

Eisenstein, a. B. 1957. Effects of dietary factors on production of adrenal steroid hormones. Am. J. Clin. Nutrition, 5, 369.

El-Skukh, a. S., Nulet, C. V., Pope, A. L., .'^nd Casida, L. E. 1955. The effect of level of feeding on the reproductive capacity of ewe. J. Anim. Sc, 14, 919.

Emerson, W. J., Z.amecnik, P. C, and Nathanson, I. T. 1951. Effect of sex hormones on hepatic and renal lesions induced in rats by a cholinedeficient diet. Endocrinology, 48, 548.

Erb, R. E., Andrews, F. N., Hauge, S. M., and King, W. A. 1947. Observations on vitamin A deficiency in young dairy bulls. J. Dairv Sc, 30, 687."

Ershoff, B. H. 1943. Degeneration of the corpora lutea in the pregnant vitamin E-deficient rat. Anat. Rec, 87, 297.

Ershoff, B. H. 1949. Beneficial effects of liver on growth and survival of immature rats fed lactose-containing diets. Proc Soc Exper. Biol. & Med., 72, 648.

Ershoff, B. H. 1952. Nutrition and the anterior pituitary with special reference to the general adaptation svndrome. Vitamins & Hormones, 10, 79.

Escudero, p., Herraiz, M. L., .and Mussmano, E. 1948. Experimental proof of the first law of nutrition. Rev. Asoc. argent. Dietol., 6, 195.

Evans, H. M., Lepkovsky, S., and Murphy, E. 1934. Vital need of the body for certain vmsaturated fatty acids; reproduction and lactation upon fat-free diets. J. Biol. Chem., 106, 431.

Evans, H. M., and Simpson, M. E. 1930. Subnormal sex-hormone content of the hypophysis of animals with inadequate antineuritic vitamin B. Anat. Rec, 45, 216.

Everson, G., Northrop, L., Chung. N. Y„ and Getty, R. 1954. Effect of ascorbic acid on rats deprived of pantothenic acid during pregnancy. J. Nutrition, 54, 305.

Farber, "e., and Corban, M. S. 1958. Sex difference in ethionine inhibition of hepatic protein synthesis. J. Biol. Chem., 233, 625.

Farber, E., and Segaloff, a. 1955. Effect of androgens and growth and other hormones on ethionine fatty Hver in rats. J. Biol. Chem., 216,471.

Farris, E. J., AND CoLTON, S. W. 1958. Effects of L-thyroxine and biothyronine on spermatogenesis. J. Urol., 79, 863. .

Ferrando, R., Jacques, F., Mabboux, H., and Prieur, G. 1955. Influence du torteau d'arachide delipide par extraction sur la fecondite de la femelle du rat. Compt. rend. Acad. Sc, 241, 253.

Ferrando, R., Jacques, F., Mabboux, H., and SolLOGOUB, C. 1955. Influence du torteau d'arachide delipide par extraction sur la croissance et I'integrite des testicules du rat. Compt. rend. Acad. Sc, 241, 107.

Ferret, P. Lindan, O., and Morgans, M. E. 1950. Pregnancy in insulin-treated alloxan diabetic rats. J. Endocrinol., 7, 100.

FiGGE, F. H. J., AND Allen, E. 1942. Genital atrophy during pantothenic acid deficiency and responses to gonadotrophic and estrogenic hormones (Abst.). Endocrinology, 30, S1028.

Finerty, J. C, Klein, G. F., and Panos, T. C. 1957. Effects of hormonal treatment on damaged testes of fat-deficient rats. Anat. Rec, 127, 293.

Fisher, H. 1957. The development of a free amino acid diet for the maintenance of egg production in chickens. In Proceedings Fourth International Congress of Nutrition, p. 39.

Flodin, N. W. 1953. Amino acids and proteins, their place in human nutritional problems. Agric. & Food Chem., 1, 222.

FoLLis, R. H. 1948. Pathology of Nutritional Diseases. Springfield, 111.: Charles C Thomas.

Forbes, G. B. 1957. Overnutrition for child: blessing or curse. Nutritional Rev., 15, 193.

Friedman, M. H. F., .and Friedman, G. S. 1940. The relation of diet to the restitution of gonadotrophic hormone content of the discharged rabbit pituitary. Am. J. Physiol., 128, 493.

Funk, C, and Funk, I. C. 1939. Action of certain hormones as dietarv constituents. Science, 90, 443.

Gaunt, R., and Parkins, W. H. 1933. The alleged interrelationship of the adrenal cortical hormone and the gonads. Am. J. Phvsiol., 103, 511.

Geller, F. C. 1933. Vitamin E and hormone. Arch. Gynak., 156, 345.

Gilbert, C, and Gillman, J. 1956. The changing pattern of food intake and appetite during the menstrual cycle of the baboon (Papio ursinus) with a consideration of some of the controlling endocrine factors. South African J. M. Sc, 21, 75.

Gillis, M. B., Heuser, G. F., and Norris, L. C. 1942. The need for pantothenic acid and an unidentified factor in reproduction in the domestic fowl. J. Nutrition, 23, 153.

Gillman, J., and Gilbert, C. 1956. Menstrual disorders induced in the baboon (Papio ursinus) by diet with a consideration of endocrine factors underlying the menstrual disorders and body weight. South African J. M. Sc, 21, 89.

GiNOULHiAC, E., and Nani, S. 1956. Urinary excretion of thiamine, cobalamin, pantothenic acid, folic acid, and folinic acid in rats treated with prednisone. Acta vitaminol., 10, 157.

GiRouD, A., and Desclaux, p. 1945. Modifications de I'hypophyse a la suite de la sousalunentation. Ann. endocrinol., 6, 107.

GiROUD, A., Levy, G., Lefebvres, J., and Dupuis, R. 1952. Chute du taux de la riboflavine au stade oii se determinent les malformations embryonanires. Internat. Ztschr. VitaminHormon- u. Fermentforsch., 23, 490.

Giroud, a., and M.artinet, M. 1955. Malformations diverses du foetus de rat suivant les stades d 'administration de vitamine A en exces. Compt. rend. Soc bioL, 149, 1088.

Giroud, A., and M.\rtinet, M. 1959. Extension a plusierus especes de mammiferes des malformations embryonnaires par hypervitaminose A. Compt. rend. Soc biol., 153, 201.

Gl.\ss, S. J., ASD Russell, M. 1952. Improved spermatogenesis after nutritional-liver regimen with and without testosterone. Fertil. & Steril., 3, 167.

Glasser, S. R. 1957. Influence of a protein-free diet on the immediate and latent effects of stilbestrol. Am. J. Phy.siol., 189, 441.

Goettsch, M. 1949. Minimal protein requirements of the adult rat for 28-day periods of maintenance of body weight. J. Nutrition, 45, 609.

Goldberg, G. M., Pfau, A., and Ungar, H. 1959. Effect of testosterone on testicular lesions produced by f/^-ethionine in rats. Am. J. Path., 35, 649.

GoLDiN, A., Greenspan, E. M., Goldberg, B.. and Schoenberg, E. B. 1950. Studies on the mechanism of action of chemotherapeutic agents in cancer: sex difference in toxicity of the folic acid analogue, 4-aminopteroylglutamic acid. Cancer, 3, 849.

Goldsmith, E. D., Nigrelli, R. F., and Ross, L. 1950. Effects of underfeeding on testosterone action in mice. Anat. Rec, 106, 197.

GoMEZ-MoNT, F., Paschkis, K. E., and Cantarow, A. 1947. Thiouiacil action in starved rats. Endocrinology, 40, 225.

GoMEZ-MoNT, F. 1959. Endocrine changes in chronic human undernutrition. In Protein Nutrition and Reproductive Physiology, p. 58. New Brunswick : Rutgers University Press.

Goth, E., Lengyel, L., Bencze, E., S.wely, C, and Majsay, a. 1955. The role of amino acids in inchicing hormone secretion. Experentia, 11, 27.

Goth, E., N.adashi, N., .and Sl.ader, E. 1958. The effect of amino acids on pituitary-adrenal activity. Ztschr. Vitamin- Hormon- u. Fermentforsch., 9, 184.

Gr.ayhack, J. T., and Scott, W. W. 1952. The effect of general dietary deficiencie.^ and response of the prostate of the albino rat to testosterone propionate. Endocrinology', 50, 406. Greenberg, S. M., and Ershoff, B. H. 1951. Effects of chorionic gonadotrophin on sex organs of male rats deficient in essential fatty acids. Proc. Soc. Exper. Biol. & Med., 78, 552.

GrEENSTEIN, J. p., BiRNBATJM, S. M., WiNITZ, M.,

AND Otey, M. C. 1957. Quantative nutritional studies with water-soluble chemically defined diet. I. Growth, reproduction and lactation in rats. Arch. Biochem., 72, 396.

Greer, M. A. 1950. Nutrition and goiter. Physiol. Rev., 30, 513.

Greer, M. A. 1956. Isolation from rutabaga seed of progoitrin, the precursor of the naturally occurring antithyroid compound, goitrin (l-5vinyl-2-thiooxazalidone). J. Am. Chem. Soc, 78, 1260.

Greer, M. A., and Astwood, E. B. 1948. The antithyroid effect of certain foods in man as determined with radioactive iodine. Endocrinology, 43, 105.

Griesbach, W. E., Bell, M. E., and Livingston, M. 1957. Changes in the pituitary gonadotrophs of the vitamin E-deficient male rat. Endocrinology, 60, 729.

GuiLBERT, H. R. 1942. Some endocrine relationships in nutritional reproductive failure. J. Animal Sc, 1, 3.

GuiLBERT, H. R., AND Goss, H. 1932. Some effects of restricted protein intake on the estrous cycle and gestation in the rat. J. Nutrition, 5, 251.

Gyorgy, p., Rose, C. S., and Shipley, R. A. 1947. Activity of estrone as a lipotropic factor. Arch. Biochem., 12, 125.

H.AYS, R. L., AND Kendall, K. A. 1956. Beneficial effect of progesterone on pregnancy in the vitamin A deficient rabbit. J. Nutrition, 59, 337.

Hellm.an, L., Bradlow, H. L., Zumoff, B., Fukushima, d. k., and g.\llagher, t. f. 1959. Thyroid-androgen interrelations and the hypocholesteremic effect of androsterone. J. Clin. Endocrinol.. 19, 936.

Herrick, E. H., Eiue, I. M., and Snow, M. R. 1952. Vitamin E in pituitary gland function of fowls. Proc. Soc. Exper. Biol. & Med., 79, 441.

Hertz, R. 1945. The quantitative relationship between stilbestrol response and dietary "folic acid" in the chick. Endocrinology, 37, 1.

Hertz, R. 1948. The role of factors of the Bcomplex in estrogen metabolism. Recent Progr. Hormone Res., 2, 161.

Hoar, R. M., Goy, R. W., and Young, W. C. 1957. Loci of action of thyroid hormone on reproduction in the female guinea pig. Endocrinology, 60, 337.

Hodgson, R. E., Hall, S. R., Sweetman, W. J., Wiseman, H. G., and Converse, H. T. 1946. The effect of vitamin A deficiency on reproduction in dairy bulls. J. Dairy Sc, 29, 669.

HoKFELT, B. 1950. Changes in the ascorbic acid content of corpus luteum of pregnancy in rabbit after gonadotrophic stimulation. Acta physiol. scandinav., 20, 172.

Holt, L. E., Jr., Albanese, A. A., Shettles, L. B., Kajdi, C, and Wangerin, D. M. 1942. Studies of amino acid deficiency in man. Fed. Proc, 1, 116.

Horn, E. H. 1955. Nutritional and hormonal influences upon reproductive maturation, organ weight, and histochemistry of the immature male rat. Endocrinology, 57, 399.

Horne, H. W., Jr., and Maddock, C. L. 1952. Vitamin A therapy in oligospermia. Fertil. & Steril, 3, 245.

Horstmann, p. 1950. Excretion of androgens in human diabetes mellitus. Acta endocrinol., 5,261.

HosoDA, T., Kaneo, T., Mogi, K., and Abe, T. 1956. Forced ovulation in gonadotrophintreated fasting hens. Proc. Soc Exper. Biol. & Med., 92, 360.

HoussAY, B. A. 1945. Action of adrenal insufficiency during pregnancy on mother (rats) and offspring. Rev. Soc. argent, biol., 21, 316.

HuLTQUiST, G. T. 1950. Diabetes and pregnancy : an animal study. Acta path, et microbiol. scandinav., 27, 695.

Hummel, K. P. 1957. Transplantation of o\aries of the obese mouse. Anat. Rec, 128, 569.

HusEBY, R. A., AND Ball, Z. B. 1945. A study of the genesis of histologic changes produced by caloric restriction in portions of the endocrine and reproductive systems of strain "A" female mice. Anat. Rec, 92, 135.

Ingalls, a. M., Dickie, M. M., .\nd Snell, G. D. 1950. Obese, a new mutation in mouse. J. Hered., 41, 317.

Ingelman-Sundberg, a. 1954. Testicles and reproduction in vitamin E-deficient guinea pigs. Acta endocrinol., 17, 165.

Ingelman-Sundberg, A. 1958. Failure of vitamin E to prevent placental changes and abruptio placentae in guinea pigs treated with oestrogens and in women suffering from hypertension, chronic renal disease or toxaemia of pregnency. Acta endocrinol., 27, 509.

Ingle, D. J. 1948. The production of experimental glycosuria in the rat. Recent Progr. Hormone Res., 2, 229.

Ingle, D. J., and Nezamis, J. E. 1947. The effect of insulin on the tolerance of normal male rats to the overfeeding of a high carbohydrate diet. Endocrinology, 40, 353.

Ishida. K. 1957. Effect of the levels of protein in the diets on the ovaries of the young and adult rats. Tohoku J. Agric, 8, 17.

Jackson, H. 1959. Antifertility substances. Pharmacol. Rev., 11, 135.

Jailer, J. W. 1948. The effect of inanition on the inactivation of estrogen by the liver. Endocrinology, 43, 78.

Jailer, J. W., and Seaman, L. 1950. Estrogen inactivation by the liver as modified by dietary protein. Proc Soc. Exper. Biol. & Med., 73, 70.

Janes, R. G. 1944. Occurrence of follicular cysts in thyroidectomized rats treated with dietliylstilbestrol. Anat. Rec, 90, 93.

Jeans, P. C, Smith, M. B., and Stearns, G. 1955. Incidence of prematurity in relation to maternal nutrition. J. Am. Dietet. A., 31, 378.

Johnson, B. C. 1958. Studies on the mechanism of action of vitamin B12 in animal nutrition. Am. J. Clin. Nutrition, 6, 34.

Jones, C. C, Brown, S. O., Rich.ardson, L. R., and SiNCL.MR, J. G. 1955. Tissue abnormalities in newborn rats from vitamin Bi2-deficient mothers. Proc. Soc. Exper. Biol. & Med., 90, 135.

J0NE.S, G. E. S., Howard, J. E., .and Langford, H. 1953. The use of cortisone in follicular phase disturbance. Fertil. & Steril., 4, 49.

Kahn, R. H. 1954. Effect of locally appHed vitamin A and estrogen on the rat vagina. Am. J. Anat., 95, 309.

K.\TSH, S., KosARicK, E., AND Alpern, J. 1955. Effect of biotin deficiency on body and organ growth and accessory reproductive structures in male rats. Growth, 19, 45.

Kaufman, N., Klavins, J. V., and Kinney, T. D. 1956. Te.sticular damage following ethionine administration. Am. J. Path., 32, 105.

Kaunitz, H., Slanetz, C. A., and Atkinson, W. B. 1949. Estrogen response and pigmentation of the uterus in vitamin E-deficient rat. Proc. Soc. Exper. Biol. & Med.. 70, 302.

Kaunitz, H., Slanetz, C. A., Johnson, R. E.. and GuiLiuiN, J. 1956. Influence of diet compositions on caloric recjuirements, water intake and organ weights of rats during restricted food intake. J. Nutrition, 60, 221.

Kehl, R., Doxjard, T., and L.anfranchi, J. 1951. Sur Taction synergicjue de la vitamine E dans le conditionnement hormonal du deciduome traumaticiue, chez la lapine non gestante. Compt. rend. Soc. biol., 145, 1161.

Kemeny, T., Handel, M. B., Kertesz, P., and Sos, J. 1953. Effect of methionine deficiency on fertilitv in rat. Kiserletes Orvostudomanv, 5, 57.

Keys, A., Brozec, J., Hernschel, A., Michelsen. O., AND Taylor, H. L. 1950. The Biology of Human Starvation. Minneapolis: University of Minnesota Press.

Kline, I. T. 1955. Relationship of vitamin Bii to stilbestrol stimulation of the chick oviduct. Endocrinology, 57, 120.

Kline, I. T., and Dorfman, R. I. 19ola. Testosterone stimulation of seminal vesicles in pteroylglutamic acid-deficient rats. Endocrinology, 48, 34.

Kline, I. T., and Dorfman, R. I. 1951b. Estrogen stimulation of the o\iduct in vitamin-deficient chicks. Endocrinology. 48, 345.

Klinefelter, H. F., Jr., Albright, F., .and GrisWOLD, G. C. 1943. Experience with ciuantitative test for normal or decreased amounts of follicle-stimulating hormone in the urine in endocrinological diagnosis. J. Clin. Endocrinol., 3, 529.

Knox, W. E., Auerbach, V. H., .\nd Lin, C. C. 1956. Enzymatic and metabolic adaptations in animals. Physiol. Rev., 36, 164.

Kocen, B. p., .and C.wazos, L. F. 1958. Cytochemistry of male reproductive tract in scurvy

and inanition. Proc. Soc. Exper. Biol. & Med., 98, 485.

Ku.MMEROw, F. A., Pan, H. P., and Hickman, H. 1952. Effect of dietaiy fat on the reproductive performance and the mixed fatty acid composition of fat-deficient rats. J. Nutrition, 46, 489.

Leathem, J. H. 1953. Relationships between thyroid and protein metabolism. In Protein Metabolism, Hormones and Growth, p. 17. New Brunswick : Rutgers University Press.

Leathem, J. H. 1954. Hormonal and nutritional influences on male reproductive sj^stem. Anat. Rec, 118,323.

Le.athem, J. H. 1957. The adrenal and dietary protein. Arch, internat. pharmacodvn., 60, 103.

Le.athem, J. H. 1958a. Hormones and protein nutrition. Recent Progr. Hormone Res., 14, 141.

Le.athem, J. H. 19o8b. Hormonal influences on the gouadotrophin sensitive hypothyroid rat ovary. Anat. Rec, 131, 487.

Le.athem, J. H. 1959a. Some biochemical aspects of the uterus. Ann. New York Acad. Sc, 75, 463.

Le.\them, J. H. 1959b. Extragonadal factors in reproduction. In Recent Progress in the Endocrinology of Reproduction, p. 179. New York: Academic Press, Inc.

Le.athem, J. H. 1959c. The male reproductive system and protein nutrition. In Reproductive Physiology and Protein Nutrition, p. 22. New Brunswick : Rutgers University Press.

Le.\them, J. H., AND DeFeo, v. J. 1952. Responses of the mouse gonad to eciuine pituitary gouadotrophin as influenced bj' dietary protein restriction. Anat. Rec, 112, 356.

Leathem, J. H., and Fisher, C. J. 1959. Male rat reproductive system and protein nutrition. Anat. Rec, 133, 302.

Le.\them, J. H., Nocenti, M. R., and Granitsas, A. 1956. Effect of testosterone propionate and diets supplemented with methionine on body weight and organ weights of female rats. Proc Penn.sylvania Acad. Sc, 30, 38.

Lee, Y. C. P., King, J. T., and Visscher, M. B. 1952. Influence of protein and caloric intake upon certain reproductive functions and carcinogenesis in the mouse. Am. J. Phvsiol., 168,391.

LeRoy, p., and Domm, L. V. 1951. Observations on cortisone administration in normal and decapitated fetal rats. Anat. Rec, 109, 319.

Lowe, J. S., and Morton, R. A. 1956. Some aspects of vitamin A metabolism. Vitamins & Hormones, 14, 97.

Lowe, J. S., Morton, R. A., Cunningh.^m, N. F., AND Vernon, J. 1957. Vitamin A deficiencA' in domestic fowl. Biochem. J., 67, 215.

Lutwak-Mann, C. 1954. Note on the chemical composition of bovine follicular fluid. J. Agric Sc, 44, 477.

Lutwak-Mann, C. 1956. Biochemical relationships between the early rabbit embryo and its environment. In Third International Congress on Animal Reproduction, p. 50. London: Brown, Knight & Truscott. Ltd.

Lutwak-Mann, C. 1958. Dependence of gonadal function upon vitamins and other nutritional factors. Vitamins & Hormones, 16, 35.

Lutwak-Mann, C, and Manx, T. 1950. Restoration of secretory function in male accessory galnds of vitamin B-deficient rats by means of chorionic gonadotrophin. Nature, London, 165, 556.

Lyons, W. R. 1951. Maintenance of pregnancy in hypophysectomized rats. Anat. Rec, 109, 378.

Mackenzie, C. G. 1942. Cure of repeated attacks of nutritional muscular dystrophy in the rabbit by a-tocopherol. Proc. Soc. Exper. Biol. & Med., 49, 313.

Maddock, C. L., Cohen, J., and Wolb.^ch, S. R. 1953. Effect of hypervitaminosis A on the testis of the rat. Arch. Path., 56, 333.

M.\DD0CK, W. O., Chase, J. D., and Nelson, W. O. 1953. Effects of large doses of cortisone on testicular morphology and urinary gonadotrophin, estrogen, 17-ketosteroid excretion. J. Lab. & Clin. Med., 41, 608.

Maddock, W. O., and Heller, C. G. 1947. Dichotomy between hypophyseal content and amount of circulating gonadotrophins during starvation. Proc. Soc. Exper. Biol. & Med., 66, 595.

McCarthy, P. T., and Cerecedo, L. R. 1952. Vitamin A deficiency in the mouse. J. Nutrition, 46, 361.

Maeder, E. C. 1937. The effect of fat in simplified diets on the reproductive organs of the female albino rat during gestation. Anat. Rec, 70, 73.

Mancini, R. E., Izquierdo, I. A., Heinrich, J. J., Penhos, J. C, AND Gerschenfeld, H. M. 1959. Effect on albino rat testes of single and multiple hypoglycemic coma. In Tiventy-first International Congress oj Physiological Sciences, p. 174.

Mandl, a. M. 1954. The sensitivity of adrenalectomized rats to gonadotrophins. J. Endocrinol., 11, 359.

Mann, T., and Lutwak-Mann, C. 1951. Secretory function of male accessory organs of reproduction in mammals. Physiol. Rev., 31, 27.

Mann, T., and Walton, A. 1953. Effect of underfeeding on genital functions of the bull. J. Agric. Sc, 43, 343.

Maqsood, M. 1952. Thyroid functions in relation to reproduction of mammals and birds. Biol. Rev. Cambridge Phil. Soc, 27, 281.

Marrian, G. F., and Parkes, A. S. 1929. The effect of anterior pituitary preparation administered during dietary anoestrus. Proc. Roy. Soc. London, ser. B, 105, 248.

Masin, F. 1950. El efecto de la administration de dosis elevados de concentrados sobre la citologia del fluido vaginal de la rata. Acta cient. venezalana, 1, 26.

Mason, K. E. 1933. Differences in testis injury

and repair after vitamin A deficiency, vitamin E deficiency, and inanition. Am. J. Anat., 52, 153.

Mason, K. E. 1939. Relation of vitamins to sex glands. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., p. 1149. Baltimore: The Wilhams & Wilkins Company.

Mason, K. E. 1944. Physiologic action of vitamin E and its homologues. Vitamins & Hormones, 2, 107.

Mason, K. E., and Mauer, S. I. 1957. Reversible testis damage in vitamin E deficient hamster. Anat. Rec, 127, 329.

Mason, K. E., and Wolfe, J. M. 1930. The physiologic activity of the hypophysis of rats under various experimental conditions. Anat. Rec, 45, 232.

Mayer, J., and Goodard, J. W. 1951. Effects of administration of gonadotrophic hormone on vitamin A deficient rats. Proc Soc. Exper. Biol. & Med., 76, 149.

Mayer, J., and Truant, A. P. 1949. Effects of administration of testosterone on vitamin A-deficient rats. Proc. Soc. Exper. Biol. & Med., 72, 436.

Meites, J. 1952. Changes in nutritional requirements accompanying marked changes in hormone levels. Metabolism, 1, 58.

Meites, J. 1953. Relation of nutrition to endocrine-reproductive functions. Iowa State Coll. J. Sc, 28, 19.

Meites, J., and Agrawala, I. P. 1949. Effects of underfeeding on thiouracil action in rats and mice. Endocrinology, 45, 148.

Meites, J., Feng, Y. S. L., and Wilwerth, A. M. 1957. The effects of endocrine imbalances on vitamin requirements. Am. J. Clin. Nutrition, 5, 381.

Meites, J., and Reed, J. 0. 1949. Effects of restricted feed intake in intact and ovariectomized rats on pituitary lactogen and gonadotrophin. Proc. Soc. Exper. Biol. & Med., 70, 513.

Menze, W. 1941. Uber dem Einfluss des Hungerns auf die Keimdriisen. Endokrinologie, 24, 159.

Migeon, C. J. 1952. Effect of cortisone on hpids of serum, liver and testes in intact and adrenalectoniizfd lats. Proc. Soc. Exper. Biol. & Med., 80, 571.

Miller, S., Ruttinger, V., and Macey, I. G. 1954. Urinary excretion of ten amino acids by women during the reproductive cycle. J. Biol. Cliem., 209, 795.

Moore, C. K. 1939. Biology of the testis. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., p. 681. Baltimore: The Williams &: Wilkins Company.

Moore, C. R. 1953. Adrenal cortical secretions in relation to the reproductive system of rats. J. Clin. Endocrinol., 13, 330.

MooRE, C. R., AND Samuels, L. T. 1931. The action of testis hormone in correcting changes induced in the rat prostate and seminal vesicles by vitamin B deficiency or partial inanition. Am. J. Physiol., 96, 278.

Moore, H., Jessop, W. J. E., O'Donovan, D. K., Barry, A. P., Quinn, B., and Drury, M. I. 1951. Cortisone in treatment of toxaemia of pregnancy; a study of eight cases. Brit. M. J., 1, 841.

MooRE, M. 1948. Effect of alloxan diabetes on hyaluronidase level of the rat testes. Proc. Soc. Exper. Biol. & Med., 69, 391.

Morgan, A. F. 1951. The effect of vitamin deficiencies on adrenocortical function. Vitamins & Hormones, 9, 161.

Morris, H. P., Dunn, T. B., and Wagner, B. P. 1953. Influence of gonadotrophin on pyridoxine-deficient and diet-restricted female mice. J. Nat. Cancer Inst., 14, 493.

Morrison, S. D. 1956. The nitrogen balance of pregnant rats. J. Physiol., 133, 167.

Morton, J. H. 1958. Three obscure causes of female infertility. Internat. Rec. Med.. 171, 63.

MuKHERJEE, A. K., AND Banerjee, S. 1954. Studies of histologic changes in experimental scurvy. Anat. Rec, 120, 907.

MuLiNos, M. G., AND PoMERANTZ, L. 1940. Pseudohypophysectomy, a condition resembling hypophysectomy, produced by malnutrition. J. Nutrition, 19, 493.

MuLiNos, M. G., AND PoMERANTZ, L. 1941a. The reproductive organs in malnutrition; effects of chorionic gonadotrophin upon atrophic genitalia of underfed male rats. Endocrinologv, 29, 267.

MuLiNOS, M. G., AND PoMERANTZ, L. 1941b. Pituitary replacement therapj^ in pseudohypophysectomy; effects of pituitary implants upon organ weights of starved and underfed rats. Endocrinology, 29, 558.

Nelson, M. M., .-^nd Evans, H. M. 1953. Relation of dietary protein levels to reproduction in the rat. J. Nutrition, 51, 71.

Nelson, M, M., .\nd Evans, H. M. 1954. Maintenance of pregnancy in the absence of dietary protein with estrone and progesterone. Endocrinology, 55, 543.

Nelson, M. M., and Evans, H. M. 1955. Maintenance of pregnancy in absence of dietary protein with progesterone. Proc. Soc. Exper. Biol. & Med., 88, 444.

Nelson, M. M., and Ev.\ns, H. M. 1956. Failure of ovarian hormones to maintain pregnancy in rats deficient in pantothenic or pteroylglutamic acid. Proc. Soc. Exper. Biol. & Med., 91, 614.

Nelson, M. M., Lyons, W. R., and Evans, H. M. 1951. Maintenance of pregnancy in pyridoxine-deficient rats when injected with estrone and progesterone. Endocrinology, 48, 726.

Nelson, M. M.. Lyons, W. R., and Evans, H. M. 1953. Comparison of ovarian and pituitary hormone for maintenance of pregnancy in pvridoxine-deficient rats, Endocrinologv, 52, 585.

Nelson, M. R., Arnrich, L., .and Morgan, A. F. 1957. Derangement of carbohydrate metabo

lism in ribofiavin-deficient rat. Fed. Proc, 16, 394.

Nelson, W. O. 1933. Studies on anterior hypophysis. III. Anterior hypophysis in vitamin E-deficient rats, Anat, Rec, 56, 241.

Newberne, p. M., and O'Dell. B. L. 1958. Histopathology of hydrocephalus resulting from a deficiencv of B12. Proc. Soc. Exper. Biol. & Med., 97, 62.

Newton, W. H. 1952. Changes in the maternal organism during pregnancy. In MarshaU'ii Physiology 0/ Reproduction, p. 442. New York: Longmans, Green & Company.

Nieman, C, and Klein Obbink, H. J. 1954. The biochemistry and pathology of hypervitaminosis A. Vitamins & Hormones, 12, 69.

NOALL, M. W., RiGGS, T. R., W.\LKER, L. M.. AND

Christensen, H. N. 1957. Endocrine control of amino acid transfer. Science, 126, 1002.

NouMURA, T. 1956. Effects of desoxycorticosterone acetate and cortisone on ovaries of mice in parabiosis with castrated males. Jap. J. Zool., 12, 57.

Okey. R.. Pencharz, R., and Lepkovsky, S. 1950. Sex hormonal effects in incipient biotin deficiency. Am. J. Physiol., 161, 1.

Overby, L. R., Frost, D. V., and Fredrickson, R. L. 1959. The antithyrotoxic factor of liver. II. Comparative activities of defatted liver residue and various fats. J. Nutrition, 68, 251.

P'an. S. Y., Van Dyke, H. B., Kaunitz, H., and Slanetz, C. a. 1949. Effect of vitamin E deficiency on amount of gonadotrophin in the anterior pituitarj^ of rats. Proc. Soc. Exper. Biol.& Med., 72,523.

Panos, T. C, and Finerty, J. C. 1953. Effects of a fat-free diet on growing female rats, with special reference to the endocrine system. J. Nutrition, 49, 397.

Panos, T. C, and Finerty, J. C. 1954. Effects of a fat-free diet on growing male rats with special reference to the endocrine system. J. Nutrition, 54, 315.

P.\Nos, T. C, Klein, G. F., and Finerty, J. C. 1959. Effects of fat deficiency in pituitarygonad relationships. J. Nutrition, 68, 509.

P.appenheimer, a. M., .\nd Schogoleff, C. 1944. The testis in vitamin E-deficient guinea pigs. Am.J. Path., 20, 239.

P.ARLOW, A. F. 1958. A rapid bioassay method for LH and factors stimulating LH secretion. Fed. Proc, 17, 402.

Parry, H. B., and Taylor, W. H. 1956. Renal function in sheep during normal and toxaemic pregnancies. J. Physiol., 131, 383.

Paschkis, K. E., and C.\nt.\row, A. 1958. Pregnancy, tumor growth, and liver regeneration. Cancer Res., 18, 1060.

P.Azos, R., Jr., and Huggins. C. 1945. Effect of androgen on the prostate in starvation. Endocrinology, 36, 416,

Pe.\rse, a. G. E., and Rin.aldini, L. M. 1950. Histochemical determination of gonadotrophin in the rat hypophysis. Brit. J. Exper. Path., 31, 540.

Pe-ARSOX, p. B. 1936. Dietary protein in relation to the estrous cycle. Proc. Am. Soc. Anim. Prod., 282.

Pe.\rson, p. B., Hart, E. B., and Bohstedt, G. 1937. The effect of the quality of protein on the estrous cycle. J. Nutrition, 14, 329.

Pecora, L. J., AND HiGHMAN, B. 1953. Organ weights and histology of chronically thiaminedeficient rats and their pair-fed controls. J. Nutrition, 51, 219.

Peder.sen, J. 1952. Course of diabetes during pregnancy. Acta endocrinol., 9, 342.

Pequignot, E. 1956. Studies on the actual nutrition of 55 pregnant women. Bull. Inst. Nat. Hyg., 11, 107.

Perloff, W. H., Lasche, E. M., Nodine, J. H., Schneeberg, N. G., and Vieillard, C. B. 1954. The starvation state and functional hypopituitarism. J. A. M. A., 155, 1307.

Peterson, R. R., Webster, R. C., R.-vyner, B., and Young, W. C. 1952. The thyroid and reproductive performance in the adiUt female guinea pig. Endocrinology, 51, 504.

Pike, R. L., and Kirksey, A. 1959. Some effects of isonicotinic acid hydrazine-induced vitamin Bg deficiency in pregnant rats. J. Nutrition, 68,561.

Pike, R. L., Suder, H. B., and Ross, M. L. 1954. The influence of protein and energy intakes upon nitrogen retention in the pregnant rat. J. Nutrition, 52, 297.

Pl.\gge, J. C., Marasso, F. J., and Zimmerman, H. J. 1958. Estrogen inhibition of nutritional fatty liver. Metabohsm, 7, 154.

Pomer.'^ntz, L., and Mulinos, M. G. 1939. Pseudohypophysectomy produced by inanition (Abstr.). Am. J. Physiol., 126, P601.

Pomeranze, J., Piliero, S. J., Medeci, P. T., and Pl.\chta, a. 1959. Deficiency diets in young growing rats. Proc. Soc. Exper. Biol. & Med., 100, 207.

Poo, L. J., Lew, W., and Addis, T. 1939. Protein anabolism of organs and tissues during pregnancy and lactation. J. Biol. Chem., 128, 69.

PoPOFF, J. S., AND Okultilschew, G. Z. 1936. Der Einfluss der Ernahrung auf die Geschlechts-f unction. Ztschr. Zuchtung B., 1936, 221.

Pugsley, L. I. 1957. The biological assay of chorionic gonadotrophin and of the gonadotrophin of pregnant mare's scrum using dietary anoestrous adult rats. Canad. J. Biochcm. & Physiol., 35, 889.

QuiMBY, F. H. 1948. Organ weights of rats receiving hormone supplements during recovery from chronic starvation. Endocrinology, 42, 263.

Ralli, W. p., and Dumm, M. E. 1952. Nutritional fractions affecting survival in young adrenalectomized rats. Endocrinology, 5i, 135.

Ranney, R. E., and Drill, V. A. 1957. The ability of 17-ethyl-19-nortestosterone to block ethio

nine-induced fattv liver in rats. Endocrinology, 61, 476.

Reece, R. p. 1950. Effectiveness of estrogens in stimulating duct growth of the mammary glands of rats. Proc. Soc. Exper. Biol. & Med., 73, 284.

Reece, R. P. 1959. The role of nutrition in mammary gland physiology. In Reproductive Physiology and Protein Nutrition, p. 23. New Brunswick : Rutgers University Press.

Reece, R. P., Turner, C. W., Hathaway, I. L., AND D.wis, H. p. 1937. Lactogen content of pituitary glands from rats on vitamin deficient rations. Proc. Soc. Exper. Biol. & Med., 37, 293.

Reed, D. C., Browning, W. H., and O'Donnell, H. F. 1958. Male subf ertility : treatment with liothyronine (cytomel). J. Urol., 79, 868.

Reid, J. T. 1949. Relationship of nutrition to fertility in animals. J. Am. Vet. M. A., 114, 158.

Rice, E. G., and Bo, W. J. 1958. Effect of /3carotene and cortisone on vaginal epithelium of vitamin A deficient rats. Proc. Soc. Exper. Biol. & Med., 98, 877.

RiCHTER, K. M., AND WiNTER, C. A. 1947. A

ciuantative study of the effect of hyperthyroidism on genital structure and function. Am. J. Physiol., 150, 95. RiNALDiNi, L. M. 1949. Effect of chronic inanition on the gonadotrophic content of the pituitary gland. J. Endocrinol., 6, 54.

RiVERO, FONTAN, J., PaSCHKIS, K. E., WeST, E., AND

Cantarow, a. 1952. Influence of different dietary protein levels in starvation on the endocrine system of male and female rats. Endocrinology, 51, 100.

RoBSON, J. M., AND Shar.\f, A. A. 1951. Interruption of pregnancy with ACTH and cortisone. J. Physiol., 114,' lip.

Rosenthal, H. L., and Allison, J. B. 1956. Effects of caloric intake on nitrogen balance and organ composition of adult rats. Agric. & Food Chem., 4, 792.

Ross, M. L., AND Pike, R. L. 1956. The relationship of vitamin Bo to serum protein and nonprotein nitrogen in the rat during pregnancy. J. Nutrition, 60, 211.

Rowlands, I. W., and Singer, E. 1936. Gonadotrophic activity of the pituitaries of vitamin E-dcficient rats. J. Physiol., 86, 323.

Ryabinina, Z. a. 1952. Effect of protein deficieny on structure and functional state of white rat ovaries. Doklady Akad. Nauk S. S. R., 86, 877.

Salhanick, H. a., Hisaw, F. L., .^ND Zarrow, M. X. 1952. The action of estrogen and progesterone on the gonadotrophin content of the pituitary of the monkey {Macaca 7nulatta). J. Clin. Endocrinol.. 12,310.

Salvesen, H. a. 1957. Endocrine organs and undernutrition: paradoxical behavior of the pancreas. Acta endocrinol., 25, 173.

Samuels, L. T. 1948. Nutrition and Hormones. Springfield, 111.: Charles C Thomas.

Samuels, L. T. 1949. The metabolism of androgens by tissues. Recent Progr. Hormone Res., 4,65. '

Samuels, L. T. 1953. Effect of protein and amino acids of the diet on the endocrine system. In Protein Metabolism, Hormones and Growth, p. 11. New Brunswick: Rutgers Uniyersity Press.

Samuels, L. T., Reinecke, R. M., and Peterson, W. E. 1941. Relation of nutrition to mammary growth after estradiol administration to hypophysectomized rats. Proc. Soc. Exper. Biol. & Med., 46, 379.

Schultze, M. O. 1955. Effects of malnutrition in early life on subsequent growth and reproduction of rat. J. Nutrition, 56, 25.

Schultze, M. O. 1956. Reproduction of rats fed protein-free amino acid rations. J. Nutrition, 60, 35.

Scott, E. B. 1956. Histopathology of amino acid deficiencies. V. Isoleucine. Proc. Soc. Exper. Biol. & Med., 92, 134.

Segaloff, a., and Segaloff, a. 1944. The role of the yitamins of the B-complex in estrogen metabolism. Endocrinology, 34, 346.

Sherman, H. C, Pe.^rson, C. S., Bal, M. E. R., McCarthy, A., and L.\nford, C. S. 1956. Influence of protein and calcium additions to already adequate diet. Proc. Nat. Acad. Sc. U. S., 42, 682.

Sherman, W. C. 1941. The effect of certain fats and unsaturated fatty acids upon the utilization of carotene. J. Nutrition, 22, 153.

Shipley, E. G., .and Danley, K. S. 1947. Pituitary and ovarian dysfunction in experimental diabetes. Am. J. Physiol., 150, 84.

Shipley, R. A., Chudzik, E. B., Curtiss, C, and Price, J. W. 1953. Effect of lipotrophic deficient diets and steroid hormones on the pregnant rat. Metabolism, 2, 165.

Sims, F. H. 1951. Methionine and choline deficiency in the rat with special reference to the pregnant state. Brit. J. Exper. Path., 32, 481.

SiNDEN, J. A., and Longwell, B. B. 1949. Effect of alloxan diabetes on fertility' and gestation in the rat. Proc. Soc. Exper. Biol. & Med., 70, 607.

Skelton, F. R. 1950. Some specific and nonspecific effects of thiamine deficiency in the rat. Proc. Soc. Exper. Biol. & Med., 73, 516.

Smithberg, M., .^nd Runner, M. N. 1957. Pregnancy induced in genetically sterile mice. J. Hered., 48, 97.

Souders, H. J., .\ND Morgan, A. F. 1957. Weight and composition of organs during the reproductive cycle in the rat. Am. J. Physiol., 191, 1.

Srebnik, H. H.. and Nelson, M. M. 1957. Increased pituitary gonadotrophic content in protein deficient castrate rats. Anat. Rec, 127, 372.

Srebnik, H. H., Nelson, M. M., and Simpson, M. E. 1958. Response of exogenous gonadotrophins in absence of dietary protein. Proc. Soc. Exper. Biol. & Med., 99, 57.

Stenger-Haffen, K., and Wolff, E. 1957. La differenciation des organes sexues cultiyes en milieux synthetique. Role et mode d'action des hormones sexuelles. In Proceedings Fourth International Congress of Nutrition, p. 49.

Stephens, D. J. 1941. Anorexia nervosa; endocrine factors in undernutrition. J. Clin. Endocrinol., 1, 257.

Stephens, D. J., and Allen, W. M. 1941. The effect of refeeding and of the administration of a pituitarj^ extract on the ovaries of undernourished guinea pigs. Endocrinology, 28, 580.

Sutton, T. S., and Brief, B. J. 1939. Physiologic changes in the anterior hypophysis of vitamin A-deficient rats. Endocrinolog.y, 25, 302.

SzEGO, C. M., and Barnes, R. H. 1943. Dietary fatty livers in mice and sensitivity to exogenous estrogen. Endocrinology, 32, 367.

T.^lbert, G. B., and H.\milton, J. B. 1955. The effect of reduced rate of growth on maturation of the male reproductive system of the albino rat. Anat. Rec, 121, 763.

Thiersch, J. B. 1954. Effect of certain 2,4 diaminopyrimidine antagonists of folic acid on ]iregnancy and rat fetus. Proc. Soc. Exper. Biol. & Med., 87, 571.

Trentin, J. J., and Turner, C. W. 1941. Quantitative study of the effect of inanition on responsiveness of the mammaiy gland to estrogen. Endocrinology, 29, 984.

Tuchm.\nn-Duplessis, H., and Aschkenasy-Lelu, P. 1947. Modifications histologiques de I'ovaire et du tractus genital de la rate adulte soumise au regime hyperprotidique. Compt. rend. Soc. biol., 141, 689.

TUCHM.^NN-DUPLESSIS, H., AND AsCHKEN.A.SY-LeLU,

p. 1948. Basophilie et augmentation du pouvoir gonadotrope de I'hypophyse de la rate en hyperproteose. Compt. rend. Soc. biol., 142, 472.

Ullrey, D. E., Becker, D. E., Terrill, S. W., and NoTzoLD, R. A. 1955. Dietary levels of pantothenic acid and reproductive performance of female swine. J. Nutrition, 57, 401.

Umberger, E. J., AND Gass, G. H. 1958. The effect of dietary fat on the uterine weight response of immature mice to oral stilbestrol. Endocrinology, 63, 801.

Vanderlinde, R. E., and Westerfield, W. W. 1950. The inactivation of estrone by rats in relation to dietary effects on the liver. Endocrinology, 47, 265.

Van Pilsum, J. F., Speyer, J. F., and Samuels, L. T. 1957. Essential amino acid deficiency and enzyme activity. Arch. Biochem., 68, 42.

A^asington, F. D., Parker, A., Headley, W., and V.\nderlinde, R. E. 1958. Relationship of low protein diet and ascorbic acid in estrogen inactivation by liver. Endocrinology, 62, 557.

Velardo, J. T., AND HiSAW, F. L. 1953. Inhibitory effects of aminopterin on decidual development. Am. J. Physiol., 172, 535.

Visscher. M. B., King, J. T., and Lee, Y. C. P. 1952. Further studies on influence of age and diet upon reproductive senescence in strain A

female mice. Am. J. Physiol., 170, 72. VoLLMER, E. P. 1943. Relation of hypopituitarism

and starvation. Nature, London, 151, 698. W.AL.AAS, 0. 1952. Effect of estrogens on the

glycogen content of the rat uterus. Acta endo crinol., 10, 175.

\V.\LKER, D. G., AND WiRTSCHAFTER, Z. T. 1956.

Estrogenic inhibition of fetal reabsorption in lathyrism. J. Nutrition, 58, 161.

Ward, L. W., Slocumb, C. H., Polley, H. F., Lowman. E., AND Hexch, p. S. 1951. Clinical effects of cortisone administered orally to patients with rheumatoid arthritis. Proc. Staff Meet., Mayo Clin., 26, 361.

Warrant, J., and Schraffenberger, E. 1944. Congenital malformations of the eyes induced in rats by maternal vitamin A deficiency. Proc. Soc. Exper. Biol. & Med., 57, 49.

Weatherby, E. J., and Reece, R. P. 1941. Effect of a high- and low-protein ration on the gonadotrophic content of male rat pituitaries. J. Dairy Sc, 24, 508.

Weir, D. R., and Leuchtenberger, C. 1957. Low sperm desoxyribose nucleic acid as possible cause for otherwise unexplained human infertility. Fertil. & Steril.. 8, 373.

Werner, S. C. 1939. Failure of gonadotrophic function of the rat hypophysis during chronic inanition. Proc. Soc. Exper. Biol. & Med., 41, 101.

White, F. R., .and White, J. 1942. Effect of a low cystine diet on the growth of various strains of mice. J. Nat. Cancer Inst., 2, 449.

Whitnah, C. H., and Bogart, R. 1936. Reproductive capacity in female rats as affected by kinds of carbohydrate in the ration. J. Agric. Rec, 53, 527.

Wil.son, J. G., Roth, C. B., and Warkany, J. 1953. An analysis of the syndrome of malformations induced by maternal vitamin A deficiency. Effects of restoration of vitamin A at various times during gestation. Am. J. Anat., 92, 189.

Wolf, R. C, and Leathem, J. H. 1955. Hormonal and nutritional influences on the biochemical composition of the rat testis. Endocrinologv 57, 286.

WOOLLAM, D. H. M., AND MiLLEN, J. W. 1957. The effect of cortisone on the incidence of cleft palate in the offspring of female rats subjected to hypervitaminosis A. In Proceedings Fourth International Congress oj Nutrition, p. 52.

WooTEN, E., Nelson, M. M., Simpson, M. E., and Evans, H. M. 1955. Effect of pyridoxine deficiency on the gonadotrophic content of the anterior pituitary of the rat. Endocrinologv, 56, 59.

Wooten, E., Nelson, M. M.. Simpson, M. E., and Ev.ANs, H. M. 1958. Response of vitamin Bodeficient rats to hypophyseal follicle-stimulating and interstitial cell-stimulating hormones. Endocrinology, 63, 860.

Yamamoto, R. S., and Chow, B. F. 1950. Role of dietary proteins in development of sex organs of young rats under stimulus of a gonadotrophin. Fed. Proc, 9, 250.

Young, W. C, Rayner, B., Peterson, R. R., and Brown, M. M. 1952. The thyroid and reproductive performance in the adult male guinea pig. Endocrinology, 51, 12.

ZiMMER. R., Weill, J., and Dubois, H. 1944. Nutritional situation in camps of the occupied zone of France in 1941 and 1942 and its con.sequence. New England J. Med.. 230, 303.

Zondek, B. 1934. Fate of follicular hormone in the living body. Lancet, 227, 356.

ZuBiRAN, S., AND GoMEZ-MoNT, F. 1953. Endocrine di.sturbances in chronic human malnutrition. Vitamins & Hormones, 11, 97.

ZucKERMAN, S. 1953. The adrenogenital relationship. The suprarenal cortex. In Proceedings Fijth Symposium Colston Research Societij, p. 69. New York: Academic Press, Inc.