Book - Sex and internal secretions (1961) 12
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SECTION C Physiology of the Gonads and Accessory Organs
Nutritional Effects on Endocrine Secretions
James H. Leathern, Ph.D.
Professor Of Zoology, Rutgers, The State University, New Brunswick, New Jersey
I. Introduction
production ()68 during the past 20 years and the long established awareness of a nutritional in on fertility and fecundity, knowledge bearing on nutrition and the endocrine
D. Sce one would prefer to present now.
endocrinologist appreciates the delicate balance which exists between the hypophysis and the gonads. In a sense, a similar interdependence exists between nutrition and the endocrine glands, including those reproductive functions. Not only does nutrition influence synthesis and release of hormones, but hormones in turn, through their regulation of the metabolism of proteins, carbohydrates, and fats, influence nutrition. Thus, dietary deficiencies may create endocrine imbalance, and endocrine imbalace 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 consider not only undernutrition and lack of specific foods, but also possible effects of antithyroid substances in foods, antimetabolites, and overnutrition, especially for the child (Forbes, 1957).
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 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 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 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. Adrenal 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 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
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
Glycosuria can be induced experimentally by starvation, overfeeding, and shifting 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, 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, 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 inadequate dietary protein (Friedman and Friedman, 1940).
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.
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 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).
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 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; Ingelman-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 capable 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 Eesponsiveness 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
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 concentration 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 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 become 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 cholesterol 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 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 parturition 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 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).
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, Simpson 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).
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
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, riboflavin, 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 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 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 increased 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 incidence 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.
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