|
|
Line 2,641: |
Line 2,641: |
| Zondek, B., and Hestrin, S. 1947. Phosphorylase | | Zondek, B., and Hestrin, S. 1947. Phosphorylase |
| activity in human endometrium. Am. J. Obst. | | activity in human endometrium. Am. J. Obst. |
| ct Gvnec, 54, 173-175. | | ct Gvnec, 54, 173-175. |
| | |
| | |
| | |
| =Nutritional Effects on Endocrine Secretions=
| |
| | |
| James H. Leathern, Ph.D.
| |
| | |
| Professor Of Zoology, Rutgers, The State University, New Brunswick, New Jersey
| |
| | |
| | |
| | |
| I. Introduction' 666 I, Introduction
| |
| | |
| II. Nature of Problems in Nutritional -j-. -, .^ ^ ,• ,• t ,
| |
| | |
| Studies 668 Despite the accumulation of many data
| |
| | |
| A. Thyroid Cxland, Nutrition, and Re- in the field of reproductive endocrinology
| |
| | |
| production ()68 during the past 20 years and the long es
| |
| B. Adreiial Gland, Nutrition, and Re- ^ _^ tablished awareness of a nutritional in
| |
| C. Diabetef^Mellitus, Nutrition, and '' ^f^f on fertility and fecundity, knowl
| |
| Reproduction ()72 edge bearing on nutrition and the endocrine
| |
| | |
| D. Sterile-Obese Syndrome 673 glands subserving reproduction has ad
| |
| E. Diet and the Liver 673 vanced comparatively slowly. However, re
| |
| III. Hypophysis and Diet 674 markable advances have been made in each
| |
| | |
| A. Inanition 674 speciality SO that nutritional-endocrine
| |
| | |
| ^roein. .. w problems should continue to be a fruitful
| |
| | |
| C. Carbohvdrate and Fat ()7() ^ „ ^i-r^x i-ii
| |
| | |
| D Vitamins 676 ^^^^^ ^^^' ^tudy. Data which have yet to
| |
| | |
| IV. Male Reproductive System (i77 be obtained eventually w'ill contribute to
| |
| | |
| A. Testis 677 the coherence one would prefer to present
| |
| | |
| 1. Inanition 677 now.
| |
| | |
| 2. Protein 678 'y\^q endocrinologist appreciates the deli
| |
| 4 Vitamins (i8() ^^^^ balance which exists between the hy
| |
| B. Influence of Nutrition on the Respon- ' pophysis and the gonads. In a sense, a simi
| |
| siveness of Male Reproductive Tis- lar interdependence exists between nutrition
| |
| | |
| sues to Hormones 681 and the endocrine glands, including those
| |
| | |
| 1. Testis ■ . . 681 ^j^|-^ reproductive functions. Not only does
| |
| | |
| 2. feeminal vesicles and i)rostate 682 x •<• • n xi • j i -•
| |
| | |
| V. Female Reproductive System 683 nutrition influence synthesis and release of
| |
| | |
| A. Ovaries 683 hormones, but hormones in turn, through
| |
| | |
| 1. Inanition 683 their regulation of the metabolism of pro
| |
| 2. Protein 684 teins, carbohydrates, and fats, influence nu
| |
| 3. Carbohydrate 685 trition. Thus, dietary deficiencies may create
| |
| | |
| 5 Vitamins 685 endocrine imbalance, and endocrine imbal
| |
| B. Influence of Nutrition on the ResiK)n- '^^ce may create demands for dietary fac
| |
| siveness of Female Pe])ro<luct ive tors. It follows, therefore, that, in any conTissues to Hormones 687 sideration of this interrelationship, one must
| |
| | |
| 1 . Ovary ... 687 consider not only undernutrition and lack of
| |
| | |
| 2. uterus and vagina 688 -n c j \ l ^ i ^ cc i. r
| |
| | |
| 3. Mammary gland 689 specific foods, but also possible effects of
| |
| | |
| C. Pregnancy. . 689 antithyroid substanccs in foods, antimetab
| |
| VI. Concluding Remarks 693 olites, and overnutrition, especially for the
| |
| | |
| VII.Pkferencks (;!)4 child (Forbes, 1957).
| |
| | |
| 666
| |
| | |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| 667
| |
| | |
| | |
| | |
| Our understanding of the means by which
| |
| hormones exert their effects is relatively
| |
| slight, as is our knowledge of the biochemical mechanisms by w^hich supplements and
| |
| deficiencies of vitamins and amino acids
| |
| influence hormone action. Nevertheless,
| |
| support for the statement that modifications
| |
| of nutrition influence endocrine gland secretions or hormone action on distant target organs or tissues is provided by an
| |
| enumeration of a few basic cell components
| |
| requiring proteins, lipids, and vitamins.
| |
| (1) Proteins combine with lipids to form
| |
| lipoproteins which are essential features
| |
| of the internal and external cellular membranes and interfaces. Hormones, as well as
| |
| nutrition, influence cell membranes and
| |
| therefore cell transport is affected. It is well
| |
| known that hormones influence electrolyte
| |
| and carbohydrate transfer and recently an
| |
| endocrine control of amino acid transport
| |
| was demonstrated (Noall, Riggs, Walker
| |
| and Christensen, 1957). The effect of modifications of nutrition on the capacity of hormones to influence cell transport must await
| |
| study. (2) Enzymes are proteins with chemically active surfaces and often include nonprotein groups such as vitamins. Nutritional
| |
| and hormonal changes cause alterations in
| |
| enzyme concentrations (Knox, Auerbach
| |
| and Lin, 1956). Vitamin, mineral, and fat
| |
| deficiencies favor a decrease in enzymes,
| |
| whereas protein deficiencies have varied effects (Van Pilsum, Speyer and Samuels,
| |
| 1957). Enzyme changes caused by hormones
| |
| appear to be a consequence of metabolic
| |
| adaptations. The importance of a nutritional base on which a hormone can express
| |
| an effect on the enzymes of the reproductive
| |
| organs can only be determined after further
| |
| data have been obtained. (3) Proteins combined with nucleic acids become nucleoproteins, some of which are organized in the
| |
| cytoplasm and may be templates for cellular protein synthesis. Other nucleoproteins
| |
| are contained in the nucleus. Nutrition and
| |
| hormones influence tissue nucleoproteins
| |
| but studies involving the reproductive organs are few. How^ever, one possible cause
| |
| of human infertility is low desoxyribose
| |
| nucleic acid in the sperm ("Weir and Leuchtenberger, 1957).
| |
| | |
| Proteins are characteristic components of
| |
| | |
| | |
| | |
| tissues and hypophyseal hormones are protein in nature; also the major portion of
| |
| gonadal dry weight is protein. Such being
| |
| the case, it is important to appreciate that
| |
| the protein composition of the body is in
| |
| a dynamic state and that proteins from the
| |
| tissues and from the diet contribute to a
| |
| common metabolic pool of nitrogen. This
| |
| metabolic pool contains amino acids which
| |
| may be withdrawn for rebuilding tissue
| |
| protein and for the formation of new protein
| |
| for growth. Obviously, the character of the
| |
| metabolic pool of nitrogen reflects dietary
| |
| protein level and quality. A food protein
| |
| which is deficient in one or more amino
| |
| acids will restrict tissue protein synthesis.
| |
| Hormones also influence the metabolic pool
| |
| by affecting appetite as well as absorption,
| |
| utilization, and excretion of foods, and thus
| |
| hormones could accentuate the effect of a
| |
| poor diet, or create demands beyond those
| |
| normally met by an adequate diet. In addition a study of the tissues and organs of
| |
| the body reveals that contributions to the
| |
| metabolic pool are not uniform, thus one
| |
| tissue or organ may be maintained at the
| |
| expense of another. In protein deprivation
| |
| in adults the liver quickly contributes increased amounts of nitrogen to the metabolic
| |
| pool whereas the testis does not. On the
| |
| other hand, protein contributions to the
| |
| nitrogen pool by the hypophysis, and the
| |
| amino acid withdrawals needed for hormone
| |
| synthesis, are unknown. Data suggesting
| |
| that the addition of specific nutrients to
| |
| diets improves hypophyseal hormone synthesis have been presented (Leathem,
| |
| 1958a).
| |
| | |
| In 1939 jNIason rightfully emphasized the
| |
| need for vitamins in reproduction. Since
| |
| then, much additional knowledge has been
| |
| obtained. Vitamins of the B complex have
| |
| been more clearly identified and a better
| |
| understanding of their function has been
| |
| gained. Thiamine is important for carbohydrate metabolism, pyridoxine for fat metabolism, and the conversion of tryptophan
| |
| to nicotinic acid, and vitamin B12 may be
| |
| involved in protein synthesis. In addition,
| |
| vitamins have been found to serve as coenzymes, and folic acid to be important for
| |
| estrogen action on the uterus. Tliosr. and
| |
| | |
| | |
| | |
| 668
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GONADS
| |
| | |
| | |
| | |
| many other findings prompt a survey of the
| |
| relationships of vitamins to reproduction.
| |
| | |
| It is the intention of the author to review
| |
| enough of the evidence which interrelates
| |
| nutrition and reproduction to create an
| |
| awareness of the problems in the area.
| |
| Reviews dealing with the general subject
| |
| of hormonal-nutritional interrelationships
| |
| have been presented (Hertz, 1948; Samuels,
| |
| 1948; Ershoff, 1952; Zubiran and GomezMont, 1953; Meites, Feng and Wilwerth,
| |
| 1957; Leathem, 1958a,). Other reviews have
| |
| related reproduction to nutrition with emphasis on laboratory (Mason, 1939; Guilbert, 1942; Lutwak-Mann, 1958) and farm
| |
| animals (Reid, 1949; Asdell, 1949), and on
| |
| protein nutrition (Leathem, 1959b, c). An
| |
| encyclopedic survey of the biology of human nutrition has been made by Keys,
| |
| Brozec, Hernschel, Michelsen and Taylor
| |
| (1950).
| |
| | |
| II. Nature of Problems in Nutritional
| |
| Studies
| |
| | |
| A. THYEOID GLAND, NUTRITION, AND
| |
| REPRODUCTION
| |
| | |
| Normal development of the reproductive
| |
| organs and their proper functioning in
| |
| | |
| TABLE 12.1
| |
| Ovarian response to chorionic gonadrotrophin,
| |
| | |
| as modified by thiouracil and diet
| |
| | |
| (From J. H. Leathem, in Recent Progress in
| |
| | |
| the Endocrinology of Reproduction, Academic
| |
| | |
| Press, Inc., New York, 1959.)
| |
| | |
| | |
| | |
| Diet
| |
| | |
| | |
| Ovarian Weight
| |
| | |
| | |
| Cholesterol
| |
| | |
| | |
| Total
| |
| | |
| | |
| Free
| |
| | |
| | |
| 18 per cent casein .
| |
| | |
| 18 tier cent +
| |
| | |
| I'hiouracil
| |
| | |
| per cent casein . .
| |
| per cent + thiouracil
| |
| | |
| 18 per cent gelatin . . .
| |
| | |
| | |
| mg.
| |
| | |
| 87
| |
| 342
| |
| | |
| 59
| |
| 133
| |
| | |
| 59
| |
| 186
| |
| 27.5
| |
| | |
| | |
| %
| |
| 0.41
| |
| | |
| 0.20
| |
| | |
| 0.47
| |
| | |
| 0.22
| |
| | |
| 0.66
| |
| 0.26
| |
| 1.56
| |
| | |
| | |
| 0.18
| |
| 0.12
| |
| 0.23
| |
| 0.15
| |
| | |
| 0.22
| |
| | |
| | |
| 18 per cent -f
| |
| thio uracil
| |
| | |
| | |
| 0.18
| |
| 18
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| Chorionic gonadotrophin = 10 I. U. X 20 days.
| |
| | |
| | |
| | |
| adults are dependent not only on the endocrine glands composing the hypophysealgonadal axis, but on others as well. The
| |
| importance of the thyroid, although not
| |
| the same for all species, is readily apparent
| |
| from the effects of prolonged hypo- and
| |
| hyperthyroid states on reproduction. Many
| |
| of these effects have been enumerated elsewhere (chapters by Albert, by Young on the
| |
| ovary, and by Zarrow) , but others also are
| |
| important. Thus steroid production may be
| |
| altered in hypothyroid animals; certainly
| |
| its metabolism is influenced. Myxedema is
| |
| associated with a profound change in androgen metabolism. Endogenous production of
| |
| androsterone is very low and subnormal
| |
| amounts of administered testosterone are
| |
| converted to androsterone. Triiodothyronine
| |
| corrects this defect (Hellman, Bradlow,
| |
| Zumoff, Fukushima and Gallagher, 1959).
| |
| The gonads of male and female offspring of
| |
| cretin rats are subnormal. The testes may
| |
| contain a few spermatocytes but no spermatozoa, and Leydig cells seem to secrete
| |
| little or no androgen. The ovaries may contain a few small follicles with antra, but
| |
| corpora lutea are absent and ovarian lipid
| |
| and cholesterol concentrations are very
| |
| low. Nevertheless, the gonads are competent to respond to administered gonadotrophin with a marked increase in weight.
| |
| However, administration of chorionic gonadotrophin to the hypothyroid rat stimulated follicular cyst formation rather than
| |
| folliculogenesis and corpora lutea formation
| |
| (Leathem, 1958b), but, for even this aberrant development, dietary protein was required (Leathem, 1959b) (Table 12.1). The
| |
| relationship between hypothyroidism and
| |
| ovarian function may provide a clue to a
| |
| possible origin of ovarian cysts, long known
| |
| to be a common cause of infertility and associated reproductive disorders. Clinical
| |
| cases of untreated myxedema exhibit ovarian cysts, and rats made hypothyroid for
| |
| eight months, had a higher percentage of
| |
| cystic ovaries than did euthyroid rats
| |
| (Janes, 1944).
| |
| | |
| The reproductive system of the adult
| |
| male is less affected than that of the immature male by a decrease in thyroid function, just as the testis of the adult is less
| |
| likely to reflect a change in protein nutrition
| |
| which is sufficient to alter the immature rat
| |
| | |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| 669
| |
| | |
| | |
| | |
| testis. On the other hand, the lack of
| |
| demonstrable thyroid dysfunction in the
| |
| adult male does not exclude the possibility
| |
| of an effect of thyroid hormone on reproduction. The conversion of thyroxine to triiodothyronine may be hindered (Morton,
| |
| 1958) . Thyroxine was found to decrease the
| |
| number of active cells in the semen and to
| |
| reduce motility, whereas triiodothyronine
| |
| increased the number of active spermatozoa
| |
| (Farris and Colton, 1958; Reed, Browning
| |
| and O'Donnell, 1958j. Small dosages of thyroxine stimulated spermatogenesis in the
| |
| mouse, rabbit, and ram (Maqsood, 1952)
| |
| and were beneficial in normal guinea pigs
| |
| and rats (Richter and Winter, 1947; Young,
| |
| Rayner, Peterson and Brown, 1952).
| |
| | |
| In many species reproduction occurs despite hypothyroidism, but fecundity may
| |
| be subnormal (Peterson, Webster, Rayner
| |
| and Young, 1952). Feeding an antithyroid
| |
| drug, thiouracil, to female rats may or may
| |
| not prevent pregnancy, but it reduces the
| |
| number of young per litter. Thiouracil feeding continued through lactation will decrease litter size (Leathem, 1959b). Hypothyroid guinea pigs gave birth to some live
| |
| young, but the percentage approached normal only when thyroxine was administered
| |
| (Hoar, Goy and Young, 1957). Pregnant
| |
| euthyroid animals responded to thyroxine
| |
| by delivering more living young than normal control pigs (Peterson, Webster, Ravner and Young, 1952) . The extent to which
| |
| such effects are consequences of the reduction in appetite, metabolism, and absorption of food from the gut which are associated with hypothyroidism has not been
| |
| determined.
| |
| | |
| Hyperthyroidism, on the other hand, will
| |
| increase the appetite and enhance absorption of food from the gut, as the increased
| |
| metabolism requires more calories, minerals,
| |
| vitamins, choline, and methionine. In adult
| |
| rats hyperthyroidism induces a marked loss
| |
| in body fat and accelerates protein catabolism. The two effects, if unchecked, result
| |
| in loss of body weight and death. In immature males hyperthyroidism slows gain
| |
| in body weight, retards testis growth and
| |
| maturation, and abolishes androgen secretion (Table 12.2). Altering dietary protein
| |
| in adult animals failed to modify the thyroid hormone effects, thereby suggesting
| |
| | |
| | |
| | |
| TABLE 12.2
| |
| | |
| Effects of diet and thyroid {0.2 'per
| |
| | |
| cent) on immature rats
| |
| | |
| (From J. H. Leathem, in Recent Progress in
| |
| | |
| the Endocrinology of Reproduction, Academic
| |
| | |
| Press, Inc., New York, 1959.)
| |
| | |
| | |
| | |
| Diet
| |
| | |
| | |
| Testis Weight
| |
| | |
| | |
| Seminal
| |
| | |
| | |
| (Casein) X 30 Days
| |
| | |
| | |
| Actual
| |
| | |
| | |
| Relative
| |
| | |
| | |
| Vesicles
| |
| | |
| | |
| 20 per cent
| |
| | |
| 20 per cent +
| |
| thyroid
| |
| | |
| 6 per cent
| |
| | |
| 6 per cent +
| |
| thyroid
| |
| | |
| per cent
| |
| | |
| per cent -|thyroid
| |
| | |
| | |
| mg.
| |
| 1G94
| |
| | |
| 1090
| |
| | |
| 825
| |
| | |
| 245
| |
| | |
| 140
| |
| | |
| 95
| |
| | |
| | |
| mg./lOOgm.
| |
| | |
| 1035
| |
| | |
| 881
| |
| 1232
| |
| | |
| 650
| |
| | |
| 346
| |
| | |
| 261
| |
| | |
| | |
| mg.
| |
| 88
| |
| | |
| 9
| |
| 16
| |
| | |
| 7
| |
| 7
| |
| 6
| |
| | |
| | |
| | |
| that the metabolic demands of other tissues
| |
| were making increased withdrawals from
| |
| the metabolic pool of nitrogen and thus
| |
| hindering testis growth. In euthyroid rats, a
| |
| 6 per cent protein diet will permit testis
| |
| growth in the absence of a gain in body
| |
| weight, but hyperthyroidism prevents this
| |
| preferential effect. Although Moore (1939)
| |
| considered the effect of thyroid hormone on
| |
| reproduction as possibly due to general body
| |
| emaciation, the testis seems to be less responsive than the body as a whole. Adult
| |
| rats fed 0.2 per cent desiccated thyroid
| |
| exhibited no correlation between loss of
| |
| body weight, change in testis weight, or
| |
| protein composition of testis at two levels
| |
| of casein and lactalbumin (Leathem,
| |
| 1959b) . The testes were seemingly not influenced by the metabolic nitrogen changes
| |
| which caused a loss in carcass nitrogen
| |
| and an associated increase in kidney and
| |
| heart nitrogen.
| |
| | |
| The mechanism of thyroid hormone action on reproduction is far from clear. As
| |
| we have noted, a part of its action may be
| |
| through the regulation of nutritional processes. Thyroid function is influenced by
| |
| the biologic value of the dietary protein
| |
| (Leathem, 1958a) and the specifie amino
| |
| acids fed (Samuels, 1953). In turn, an
| |
| altered thyroid function will influence the
| |
| nitrogen contributions to the metabolic pool
| |
| by reducing appetite and absorption from
| |
| | |
| | |
| | |
| 670
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GONADS
| |
| | |
| | |
| | |
| the gut, and by changing the contributions
| |
| of nitrogen to the metabolic pool made by
| |
| the body tissues. Hypothyroidism interferes
| |
| with the refilling of body protein stores;
| |
| consequently, protein needs of the reproductive organs may not be fully met (Leathern, 1953). It is consistent with this
| |
| opinion that testis recovery from protein
| |
| deprivation in hypothyroid rats was aided
| |
| by thyroxine treatment (Horn, 1955).
| |
| | |
| Conversion of carotene to vitamin A may
| |
| be prevented by hypothyroidism, suggesting
| |
| that a subnormal amount of this vitamin
| |
| may contribute to fetal loss. An increased
| |
| intake of B vitamins might be required as
| |
| hypothyroidism aggravates a vitamin B12
| |
| deficiency, and increased intake of B vitamins enhances the capacity of young rats to
| |
| withstand large doses of thiouracil (Meites,
| |
| 1953). Although a reduced metabolic rate
| |
| might seemingly reduce vitamin requirements, more efficient metabolic activities,
| |
| in the absence of hormonal stimuli, seem to
| |
| occur when vitamin intake is increased
| |
| (Meites, Feng and Wilwerth, 1957).
| |
| | |
| Reproduction is influenced by effects
| |
| which are the opposite of a number of
| |
| those just cited, i.e., effects of malnutrition
| |
| on thyroid function. The need for iodine in
| |
| the prevention of goiter is well known.
| |
| However, certain foods prevent the utilization of iodine in the synthesis of thyroid
| |
| hormone. The foods containing an antithyroid or goitrogenic agent, as tested in
| |
| man, include rutabaga, cabbage, brussels
| |
| sprouts, cauliflower, turnip, rape, kale, and
| |
| to a lesser extent peach, pear, strawberry,
| |
| spinach, and carrot (Greer and Astwood,
| |
| 1948). A potent goitrogen isolated from
| |
| rutabaga is L-5-vinyl-2-thiooxazolidine
| |
| ((ireer, 1950, 1956). Reduced food intake
| |
| will decrease the thyroid gland response to
| |
| goitrogens (Gomez-Mont, Paschkis and
| |
| Cantarow, 1947; Meites and Agrawala,
| |
| 1949), the uptake of I^^^ (Meites, 1953),
| |
| and the level of thyrotrophic hormone in
| |
| laboratory animals (D'Angelo, 1951). The
| |
| thyroid changes associated with malnutrition in man arc uncertain (Zubiran and
| |
| Gomez-Mont, 1953). However, a decreased
| |
| functioning of the gland in anorexia nervosa, followed by an increased functioning
| |
| on refeeding (Poiioff, Lasche, Nodine,
| |
| | |
| | |
| | |
| Schneeberg and \'ieillard, 1954), is suggestive of a direct nutritional need.
| |
| | |
| Changes in the thyrotrophic potency of
| |
| the rat hypophysis have been observed in
| |
| various vitamin deficiencies. Thiamine deficiency may increase thyroid function, but
| |
| vitamin A deficiency may have the opposite
| |
| effect (Ershoff, 1952). The difficulties inherent in the assay of thyroid-stimulating
| |
| hormone (TSH) , even by current methods,
| |
| prevent one from drawing definite conclusions from the available data.
| |
| | |
| Immature animals given thyroxine are
| |
| retarded in growth and do not survive. However, increasing dietary thiamine, pyridoxine, or vitamin B12 improves the ability of
| |
| the young rat to withstand large dosages
| |
| of thyroid substances (Meites, 1952), as
| |
| does methionine (Boldt, Harper and Elvehjem, 1958). Consideration must also be
| |
| given to the need for nutritional factors
| |
| which play a minor role in normal metabolic
| |
| states but increase in importance in stress.
| |
| Thus yeast and whole liver contain antithyrotoxic substances (Drill, 1943; Ershoff,
| |
| 1952; Overby, Frost and Fredrickson,
| |
| 1959).
| |
| | |
| Excessive thyroid hormone will prevent
| |
| maturation of the ovary and in adult rats
| |
| will cause ovarian atrophy with a cessation
| |
| of estrous cycles. Addition of yeast to the
| |
| diet permitted estrous cycles to continue
| |
| (Drill, 1943), but gonadal inhibition in the
| |
| immature animal was not ]3revented. However, whole liver or its w^ater-insoluble fraction counteracted the gonadal inhibition induced by hyperthyrodism in immature
| |
| rats (Ershoff, 1952). Biochemical mechanisms by which these dietary supplements
| |
| can benefit rats given excessive quantities
| |
| of hormone are unknown.
| |
| | |
| B. .\DREXAL GLAND, NUTRITION, AND
| |
| REPRODUCTION
| |
| | |
| The problem of the relationship between
| |
| adrenal steroid secretions and the reproductive system is one that still requires
| |
| clarification. Furthermore, the possible influences of nutrition can only be inferred
| |
| from the effects of adrenal steroids on the
| |
| major metabolic systems of the body.
| |
| | |
| In the female there is a close relationshiji
| |
| between the adrenal and the estrous and
| |
| | |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| iul
| |
| | |
| | |
| | |
| menstrual cycles (Zuckerman, 1953; chapter by Young on the ovary). The ovary
| |
| would seem to require cortical steroids for
| |
| the normal functioning of its own metabolic
| |
| processes and for those which it influences
| |
| peripherally. The Addisonian patient may
| |
| show ovarian follicular atresia and a loss of
| |
| secondary sex characteristics, and the untreated adrenalectomized rat exhibits a
| |
| decrease in ovarian size and has irregular
| |
| cycles (Chester Jones, 1957). The decline
| |
| in size of the ovary after adrenalectomy
| |
| is due to impaired sensitivity to folliclestimulating hormone (FSH) rather than to
| |
| a decreased production of FSH, and the
| |
| ovarian response is corrected by cortisone
| |
| (Mandl, 1954).
| |
| | |
| Reproductive potential is not necessarily
| |
| lost when there is adrenal insufficiency, but
| |
| pregnancy is not well tolerated by women
| |
| with Addison's disease. Furthermore, adrenalectomy in rats at the time of mating or
| |
| 4 to 6 days after mating resulted in abortion. Improved pregnancy maintenance was
| |
| obtained in adrenalectomized rats given
| |
| saline or cortisone acetate (Davis and Plotz,
| |
| 1954) , whereas desoxycorticosterone acetate
| |
| alone extended the pregnancy period beyond
| |
| normal time (Houssay, 1945). Essentially
| |
| normal pregnancies were obtained in adrenalectomized rats given both cortisone
| |
| acetate and desoxycorticosterone acetate
| |
| (Cupps, 1955). Substitution of cortisone
| |
| acetate for adrenal secretions may be incomplete because the adrenal hormone in
| |
| the normal rat is primarily corticosterone
| |
| and because cortisone enhances the excretion of certain amino acids and vitamins. It
| |
| would be interesting to test a diet with a
| |
| high vitamin content on the capacity of an
| |
| adrenalectomized rat to maintain pregnancy, because improved survival of operated rats is obtained by giving vitamin Bio
| |
| (Meites, 1953) or large doses of pantothenic acid, biotin, ascorbic acid, or folic
| |
| acid (Ralli and Dumm, 1952; Dumm and
| |
| Ralli, 1953).
| |
| | |
| An adrenal influence over protein metabolism is well known, but protein nutrition,
| |
| in turn, can influence cortical steroid effectiveness. In fact, an extension of the life
| |
| span of adrenalectomized rats is not obtained with adrenal steroids if the diet
| |
| | |
| | |
| | |
| lacks protein (Leathern, 1958a). A low
| |
| protein diet alone will not improve survival after adrenalectomy, but better survival is obtained when the rats are given
| |
| saline. When the low protein diet was supplemented with methionine, a definite improvement in life span was observed and the
| |
| possibility that cortisone exerts its effect by
| |
| drawing on the carcass for methionine was
| |
| suggested (Aschkenasy, 1955a, b).
| |
| | |
| Reducing dietary casein to 2 per cent
| |
| seriously endangers pregnancy in the rat,
| |
| but the addition of progesterone permits
| |
| 80 per cent of the pregnancies to be maintained. However, removal of the adrenal
| |
| glands counteracts the protective action of
| |
| the progesterone, only 10 per cent of the
| |
| pregnancies continuing to term. Addition
| |
| of methionine to the low casein diet improved pregnancy maintenance, but 1 mg.
| |
| cortisone acetate plus progesterone provided the best results (Aschkenasy-Lelu
| |
| and Aschkenasy, 1957; Aschkenasy and
| |
| Aschkenasy-Lelu, 1957). These data emphasize the importance of nutrition in obtaining an anticipated hormone action. Further investigation might be directed toward
| |
| the study of whole proteins other than casein, for the biologic value of proteins differs from normal when tested in adrenalectomized rats (Leathem, 1958a) .
| |
| | |
| As Albert has noted in his chapter, adrenalectomy has little or no effect on the
| |
| testis. Gaunt and Parkins (1933) found no
| |
| degenerative changes in the testes of adult
| |
| rats dying of adrenal insufficiency, although
| |
| an increase in the testis : body-weight ratios
| |
| was noted in rats fed 18 per cent and 4 per
| |
| cent protein (Aschkenasy, 1955c). If adrenalectomized rats are kept on a maintenance dosage of cortisone acetate for 20
| |
| days and fed dietary proteins of different
| |
| biologic values, one finds that testis-composition of protein, lipid, and glycogen varies in the same manner as in the normal
| |
| rat (Wolf and Leathem, 1955) (Table 12.3).
| |
| | |
| When the adrenal glands are intact, the
| |
| influence of diet on their functional capacity and indeed on the hypophyseal- adrenal
| |
| axis must be considered. Zubiran and Gomez-]\Iont (1953) showed that patients exhibiting gonadal changes associated with
| |
| chronic malnutrition also exhibit adrenal
| |
| | |
| | |
| | |
| 672
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GOXADS
| |
| | |
| | |
| | |
| TABLE 12.3
| |
| | |
| Nutritional effects on the testes of cortisonemaintained adrenalectomized rats
| |
| (From R. C. Wolf and J. H. Leathern,
| |
| Endocrinology, 57, 286, 1955.)
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| Testes Composition
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| (per cent)
| |
| | |
| | |
| Treatment
| |
| | |
| | |
| Diet
| |
| | |
| | |
| Testes
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| Protein
| |
| | |
| | |
| Lipid
| |
| | |
| | |
| Glycogen
| |
| | |
| | |
| | |
| | |
| | |
| | |
| mg.
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| Corti
| |
| | |
| 20 per cent
| |
| | |
| | |
| 703
| |
| | |
| | |
| 68.5
| |
| | |
| | |
| 30.4
| |
| | |
| | |
| 0.13
| |
| | |
| | |
| sone
| |
| | |
| | |
| casein
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| ace
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| tate
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| Control
| |
| | |
| | |
| | |
| | |
| 1211
| |
| | |
| | |
| 70.5
| |
| | |
| | |
| 29.4
| |
| | |
| | |
| 0.15
| |
| | |
| | |
| Corti
| |
| | |
| 20 per cent
| |
| | |
| | |
| 808
| |
| | |
| | |
| 61.0
| |
| | |
| | |
| 31.8
| |
| | |
| | |
| 0.17
| |
| | |
| | |
| sone
| |
| | |
| | |
| wheat
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| ace
| |
| | |
| ghiten
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| tate
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| Control
| |
| | |
| | |
| | |
| | |
| 816
| |
| | |
| | |
| 62.3
| |
| | |
| | |
| 32.2
| |
| | |
| | |
| 0.17
| |
| | |
| | |
| Corti
| |
| | |
| 20 per cent
| |
| | |
| | |
| 1014
| |
| | |
| | |
| 64.3
| |
| | |
| | |
| 31.3
| |
| | |
| | |
| 0.17
| |
| | |
| | |
| sone
| |
| | |
| | |
| peanut
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| ace
| |
| | |
| flour
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| tate
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| Control
| |
| | |
| | |
| | |
| | |
| 699
| |
| | |
| | |
| 68.5
| |
| | |
| | |
| 31.3
| |
| | |
| | |
| 0.12
| |
| | |
| | |
| | |
| hypofimction. Several clinical tests permitted the evaluation of subnormal adrenal
| |
| function which, however, did not reach
| |
| Addisonian levels. Malnutrition not onlyreduced hypophyseal adrenocorticotrophic
| |
| hormone (ACTH), but also prevented an
| |
| incomplete response by the adrenal glands
| |
| to injected ACTH. In laboratory rodents,
| |
| anterior hypophyseal function is also influenced by dietary protein and vitamin
| |
| levels (Ershoff, 1952). The importance of
| |
| dietary protein in the hypophyseal-adrenal
| |
| system has recently been re-emphasized
| |
| (Leathem, 1957; Goth, Nadashi and Slader,
| |
| 1958). Furtiiermore, adrenal cortical function is affected by vitamin deficiencies
| |
| (Morgan, 1951), from which it appears
| |
| that pantothenic acid is essential for cortical hormone elaboration (Eisenstcin, 1957).
| |
| Administration of excessive amounts of
| |
| cortical steroids can induce morphologic
| |
| changes which have been compared to inanition (Baker, 1952). Not only is nitrogen
| |
| loss enhanced, but hyperglycemia can also
| |
| be induced which, therefore, increases the
| |
| need for thiamine. Cortical steroids influence the metabolism of various vitamins
| |
| | |
| | |
| | |
| (Draper and Johnson, 1953; Dhyse, Fisher,
| |
| Tullner and Hertz, 1953; Aceto, Li Moli
| |
| and Panebianco, 1956; Ginoulhiac and
| |
| Nani, 1956). H a vitamin deficiency already
| |
| exists, administration of cortisone will aggravate the condition (Meites, Feng and
| |
| Wilwerth, 1957). Nevertheless, drastic effects of therapeutic doses of cortisone on
| |
| reproductive function do not occur. In rare
| |
| cases loss of libido has been reported in
| |
| the male, but mori)hologic changes in the
| |
| testis were not observed (JVladdock, Chase
| |
| and Nelson, 1953). Cortisone has little if
| |
| any effect on the weight of the rat testis
| |
| (Moore, 1953; Aschkenasy, 1957) and does
| |
| not influence testis cholesterol (Migeon,
| |
| 1952). In the female, menstrual disturbances have been noted in association with
| |
| cortisone therapy, with the occurrence of
| |
| hot flashes (Ward, Slocumb, Policy, Lowman and Hench, 1951). However, cortisone
| |
| corrected disturbances during the follicular
| |
| phase, possibly by increasing FSH release
| |
| (Jones, Howard and Langford, 1953). Cortisone also increased the number of follicles
| |
| in the ovary of the rat (Moore, 1953), but
| |
| not in the rabbit. Cortisone administration
| |
| did not prevent the enhanced ovarian response to chorionic gonadotrophin seen in
| |
| hypothyroid rats (Leathem, 1958b) and
| |
| had little effect on mice in parabiosis ( Noumura, 1956).
| |
| | |
| In pregnant female rabbits resorption and
| |
| stunting of fetuses occurred during treatment with large doses of cortisone (Courrier
| |
| and Collonge, 1951). Similar effects were
| |
| noted in mice (LcRoy and Domm, 1951 ;
| |
| Robson and Sharaf, 1951).
| |
| | |
| Some of the metabolic derangements of
| |
| human toxemia of pregnancy have been
| |
| correlated with accelerated secretion of
| |
| adrenal steroids creating a steroid imbalance (see chapter by Zarrow). Cortisone is
| |
| reported to have a beneficial effect on some
| |
| cases (Moore, Jessop, 'Donovan, Barry,
| |
| Quinn and Drury, 1951). Protein inade({uacies may also be etiologic in toxemia and
| |
| further (>xamination of the possibility
| |
| sliould he made.
| |
| | |
| C. DIABETES MELLITUS, NUTRITION, AND
| |
| REPRODUCTION
| |
| | |
| (llycosuria can be induced experimentally
| |
| by starvation, overfeeding, and shifting
| |
| | |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| 673
| |
| | |
| | |
| | |
| diet^ from one of high fat content to one
| |
| i; which is isocaloric but high in carbohydrate
| |
| (Ingle, 1948). Force feeding a high carbohydrate diet will eventually kill a rat despite insulin administration aimed at controlling glycosuria (Ingle and Nezamis,
| |
| 1947). In man excessive eating leading to
| |
| obesity increases insulin demand and, in
| |
| many diabetics of middle age, obesity precedes the onset of diabetes. With our present
| |
| knowledge we must conclude that overfeeding is wrong when glycosuria exists and that
| |
| vitamin B supplements may be of value
| |
| in diabetes (Meites, Feng and Wilwerth,
| |
| 1957; Salvesen, 1957).
| |
| | |
| In man urinary 17-ketosteroids and androgen levels are subnormal in diabetes
| |
| (Horstmann, 1950), and in the diabetic rat
| |
| pituitary gonadotrophins are reduced
| |
| (Shipley and Danley, 1947), but testis hyaluronidase does not change (Moore, 1948) .
| |
| When hyperglycemia exists in rats, semen
| |
| ■] carbohydrates increase (Mann and Lutwak-Mann, 1951).
| |
| | |
| Hypoglycemia influences the male reproductive organs. In rats tolbutamide or
| |
| insulin produce lesions of the germinal
| |
| epithelium which can be prevented by
| |
| \' simultaneous administration of glucose.
| |
| When 2 to 5 hypoglycemic comas are induced, such testis injuries increase progressively in number and frequency, and
| |
| only a partial return to normal is observed
| |
| a month later (Mancini, Izquierdo, Heinrich, Penhos and Gerschenfeld, 1959).
| |
| | |
| It is well known that the incidence of
| |
| infertility in the pre-insulin era was high
| |
| in young diabetic women. Fertility is also
| |
| reduced in diabetic experimental animals,
| |
| and rat estrous cycles are prolonged (Davis,
| |
| Fugo and Lawrence, 1947) . Insulin is corrective (Sinden and Longwell, 1949; Ferret,
| |
| Lindan and Morgans, 1950). Pregnancy in
| |
| women with uncontrolled diabetes may
| |
| terminate in abortion or stillbirth, possibly
| |
| l)ecause toxemia of pregnancy is high (Pedersen. 1952). In rats pancreatectomy performed the 8th to 12th day of pregnancy
| |
| increased the incidence of stillbirths (Hultciuist, 1950). In another experiment almost
| |
| one- fourth of 163 animals with diabetes
| |
| induced by alloxan on the 10th to 12th day
| |
| of pregnancy died before parturition and
| |
| | |
| | |
| | |
| about 25 ])er cent of the survivors aborted
| |
| (Angcrvall, 1959).
| |
| | |
| D. STERILE-OBESE SYNDROME
| |
| | |
| A sterile-obese syndrome in one colony of
| |
| mice has been shown to be a recessive monogenic trait (Ingalls, Dickie and Snell, 1950).
| |
| Obesity was transmitted to subsequent generations by way of ovaries that were transplanted from obese donors to nonobese recipients (Hummel, 1957). Obesity was
| |
| transmitted by obese females receiving hormonal therapy and mated to obese males
| |
| kept on restricted food intake (Smithberg
| |
| and Runner, 1957). In addition to the investigations of the hereditary nature of the
| |
| sterile-obese syndrome, the physiologic
| |
| basis for the sterility has been studied in
| |
| reference to the presence of germ cells, viability of ova and sperm, integrity of the
| |
| ovary, and response of the uterus to estrogen (Drasher, Dickie and Lane, 1955). The
| |
| data indicate that sterility in some obese
| |
| males can be prevented by food restriction
| |
| and that sterility in certain obese females
| |
| can be corrected.
| |
| | |
| E. DIET AND THE LIVER
| |
| | |
| The concentration of hormones which
| |
| reaches the target organs in the blood is the
| |
| result of the rate of their production, metabolism, and excretion. How hypophyseal
| |
| hormones are destroyed is not clear, but
| |
| current data make it apparent that pituitary hormones have a short half-life in the
| |
| circulatory system. Exerting a major control over circulating estrogen levels is the
| |
| liver, with its steroid-inactivating systems.
| |
| Zondek (1934) initially demonstrated that
| |
| the liver could inactivate estrogens and this
| |
| finding has had repeated confirmation
| |
| (Cantarow, Paschkis, Rakoff and Hansen,
| |
| 1943; De:\Ieio, Rakoff, Cantarow and
| |
| Paschkis, 1948; Vanderlinde and Westerfield, 1950). Other steroids are also inactivated by the liver with several enzyme
| |
| systems being involved; the relative concentration of these enzymes varies among
| |
| species of vertebrates (Samuels, 1949).
| |
| | |
| The liver is a labile organ which readib.'
| |
| responds to nutritional modifications; the
| |
| induced liver changes alter the steroid-inactivating systems of this organ. Thus, inanition (Drill and Pfeiffer, 1946; Jailer,
| |
| | |
| | |
| | |
| G74
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GONADS
| |
| | |
| | |
| | |
| 1948) , vitamin B complex deficiency (Segaloff and Segaloff, 1944; Biskind, 1946), and
| |
| protein restriction (Jailer and Seaman,
| |
| 1950) all influence the capacity of the liver
| |
| to detoxify steroids. Reduced protein intake
| |
| is a primary factor in decreasing the effectiveness of the steroid-inactivating system
| |
| (Jailer and Seaman, 1950; Vanderlinde and
| |
| Westerfield, 1950). Rats fed an 8 per cent
| |
| casein diet lose their capacity to inactivate
| |
| estrone within 10 days. However, ascorbic
| |
| acid alone or in combination with glutathione restored the estrone-inactivating system (Vasington, Parker, Headley and Vanderlinde, 1958).
| |
| | |
| Failure of steroids to be inactivated will
| |
| influence the hypophyseal-gonadal axis. In
| |
| turn the excess of estrogen will decrease
| |
| gonadotrophin production by the hypophysis and thus reduce steroid production by
| |
| the gonad. In addition nutritional modifications influence hypophyseal and possibly
| |
| gonadal secretory capacity directly. Conceivably, nutritional alterations could
| |
| modify the amount of steroid secreted or
| |
| interfere with complete steroid synthesis
| |
| by a gland.
| |
| | |
| Fatty infiltration of the liver and a
| |
| general increase in fat deposition occur in
| |
| fed and fasted rats after the injection of
| |
| certain pituitary extracts and adrenal steroids and after the feeding of specific diets.
| |
| Impaired estrogen inactivation has been
| |
| associated with a fatty liver, but Szego and
| |
| Barnes (1943) believe that the major influence is inanition. In fact, estrogens, especially ethinyl estradiol, interfere with
| |
| fatty infiltration of the liver induced by a
| |
| low protein diet (Gyorgy, Rose and Shipley, 1947) or by a choline-deficient diet
| |
| (Emerson, Zamecnik and Nathanson, 1951).
| |
| Stilbestrol, however, did not prevent the increase in liver fat induced by a protein-free
| |
| diet (Glasser, 1957). Estrogens that are
| |
| effective in preventing fatty infiltration may
| |
| act by sparing methionine or choline or by
| |
| inhibiting growth hormone (Flagge, Marasso and Zimmerman, 1958).
| |
| | |
| Ethionino, the antimetabolite of nu'thioniiK', Avill induce a fatty liver and inhibit hepatic protein synthesis in female,
| |
| but not in male rats (Farber and Segaloff,
| |
| 1955; Farber and Corban, 1958). Pretreatment of females with testosterone prevents
| |
| | |
| | |
| | |
| the ethionine effect, but this blockage of
| |
| ethionine action need not be related to
| |
| androgenic or progestational properties of
| |
| steroids (Ranney and Drill, 1957).
| |
| | |
| III. Hypophysis and Diet
| |
| | |
| Studies involving acute and chronic starvation have shown that gonadal hypofunction during inanition is primarily due to
| |
| diminished levels of circulating gonadotrophins. Because of the similarity to
| |
| changes following hypophysectomy, the
| |
| endocrine response to inanition has been
| |
| referred to as "pseudohypophysectomy."
| |
| | |
| A. INANITION
| |
| | |
| The hypophysis has been implicated in
| |
| human reproduction disturbances associated with undernutrition. Hypophyseal
| |
| atrophy and a decrease in urinary gonadotrophins have been observed in chronic
| |
| malnutrition (Klinefelter, Albright and
| |
| Griswold, 1943; Zubiran and Gomez-]\Iont,
| |
| 1953) and anorexia nervosa (Perloff,
| |
| Lasche, Nodine, Schneeberg and Vieillard,
| |
| 1954). Refeeding has restored urinary
| |
| gonadotrophin levels in some cases, but
| |
| hypoj^hyseal damage may result from severe
| |
| food restriction at puberty (VoUmer, 1943;
| |
| Samuels, 1948).
| |
| | |
| The influence of inanition on the reproductive organs of lal)oratory rodents
| |
| is well recognized but the cft'ects on the
| |
| hypophysis cannot be presented conclusively. In support of prior investigations,
| |
| Mulinos and Pomerantz (1941a, b) in rats
| |
| and Giroud and Desclaux (1945) in guinea
| |
| pigs observed a hypophyseal atrophy following chronic underfeeding as well as a
| |
| decrease in cell numbers and mitoses. In
| |
| fact, refeeding after chronic starvation
| |
| resulted in only a partial recovery of hypophyseal weight (Quimby, 1948). Nevertheless, complete starvation did not influence relative gland weight in female rats
| |
| (Meites and Reed, 1949), and cytologic
| |
| evidence (periodic acid-Schiff (PAS) test)
| |
| of an estimated 3- fold increase in gonadoti'ophin content was claimed following
| |
| chronic starvation (Pearse and Rinaldini,
| |
| 1950). Assays of hypophyseal gonadotrophin content in chronically starved rats
| |
| of both sexes have l)een reported as decreased (Mason and Wolfe, 1930; Werner,
| |
| | |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| 675
| |
| | |
| | |
| | |
| 1939), unchanged (,]\Iarrian and Parkes,
| |
| 1929; Pomerantz and Mulinos, 1939; Maddock and Heller, 1947; Meites and Reed,
| |
| 1949; Blivaiss, Hanson, Rosenzweig and
| |
| McNeil, 1954), or increased (Rinaldini,
| |
| 1949; Vanderlinde and Westerfield, 1950).
| |
| An increase in pituitary gonadotrophin was
| |
| evident when hormone content was related
| |
| to milligrams of tissue (Meites and Reed,
| |
| 1949). Thus, the hormone release mechanism may fail in starvation, and eventually
| |
| gonadotrophin production will be reduced
| |
| to a minimum (]\laddock and Heller, 1947).
| |
| | |
| Gonadectomy of fully fed rats is followed
| |
| by an increase in hypophyseal gonadotrophin content. Chronic starvation, however, prevented the anticipated changes in
| |
| the pituitary gland following gonadectomy
| |
| in 8 of 12 female rats (Werner, 1939). On
| |
| the other hand, if adult female rats were
| |
| subjected to 14 days of reduced feeding
| |
| 1 month after ovariectomy, no change in
| |
| the elevated gonadotrophin levels was noted
| |
| (Meites and Reed, 1949). In contrast,
| |
| Gomez-Mont (1959) observed above normal
| |
| urinary gonadotrophins in many menopausal and postmenopausal women despite
| |
| undernutrition.
| |
| | |
| It is apparent that uniformity of opinion
| |
| as to how starvation influences hypophyseal
| |
| gonadotrophin content has not been attained. Several explanations can be given for
| |
| the discrepancies. (1) There have been unfortunate variations in experimental design.
| |
| IVIaddock and Heller (1947) starved rats
| |
| for 12 days, whereas Rinaldini (1949) used
| |
| a low calorie diet of bread and milk for
| |
| 30 days. Other variations in feeding have
| |
| included feeding one-half the intake required for growth (Mulinos and Pomerantz,
| |
| 1941b I, regimens of full, one-half, oneciuarter, and no feeding for 7 and 14 days
| |
| (Meites and Reed, 1949), and feeding inadequate amounts of a standard rat diet
| |
| for 1 to 4 months (Werner, 1939). (2)
| |
| Hypophyseal implants and anterior pituitary extracts should not be compared, for
| |
| variable gonadotrophin production may follow implantation procedures, depending on
| |
| whether necrosis or growth occurs (Maddock and Heller, 1947). (3) There has been
| |
| an insufficient standardization of experimental materials. The assay animal has
| |
| usually been the immature female rat, but
| |
| | |
| | |
| | |
| occasionally the immature mouse has been
| |
| used, and Rinaldini (1949) used the hypophysectomized rat.
| |
| | |
| B. PROTEIN
| |
| | |
| The need for specific food elements by
| |
| the hypophysis warrants consideration,
| |
| for the hormones secreted by this gland
| |
| are protein in nature and the amino acids
| |
| for protein synthesis must be drawn from
| |
| body sources. However, dietary protein
| |
| levels can vary from 15 per cent to 30 per
| |
| cent without influencing hypophyseal gonadotrophin content in rats (Weatherby and
| |
| Reece, 1941), but diets containing 80 per
| |
| cent to 90 per cent of casein increased
| |
| hypophyseal gonadotrophin (TuchmannDuplessis and Aschkenasy-Lelu, 1948). Removal of protein from the diet will decrease
| |
| hypophyseal gonadotrophin content in
| |
| adult male rats in comparison with pair-fed
| |
| and ad libitum-ied controls, but the decrease may or may not be significant in a
| |
| 30-day period; luteinizing hormone (LH)
| |
| seemed to be initially reduced. Extension of
| |
| the period of protein depletion another 2
| |
| months resulted in a significant lowering of
| |
| hy]iophyseal gonadotrophin levels (Table
| |
| 12.4) (Leathern, 1958a). On the other
| |
| hand, an increased FSH with no decrease
| |
| in LH activity was observed in the hypophyses of adult female rats following
| |
| 30 to 35 days of protein depletion (Srebnik
| |
| and Nelson, 1957). The available data indicate that not only may a sex difference
| |
| exist, but also that species may differ; restitution of gonadotrophin in the discharged
| |
| rabbit pituitary was not influenced by in
| |
| TABLE 12.4
| |
| | |
| Influence of a protein-free diet on hypophyseal
| |
| | |
| gonadotrophin content
| |
| | |
| (From J. H. Leathern, Recent Progr. Hormone
| |
| | |
| Res., 14, 141, 1958.)
| |
| | |
| | |
| | |
| Days on PFD*
| |
| | |
| | |
| Xo. of Rats
| |
| | |
| | |
| Anterior
| |
| Pituitary
| |
| Weight
| |
| | |
| | |
| Recipient
| |
| Ovarv
| |
| Weight
| |
| | |
| | |
| | |
| | |
| | |
| | |
| nig.
| |
| | |
| | |
| mg.
| |
| | |
| | |
| | |
| | |
| | |
| 9
| |
| | |
| | |
| 8.3
| |
| | |
| | |
| 74
| |
| | |
| | |
| 30
| |
| | |
| | |
| 9
| |
| | |
| | |
| 7.3
| |
| | |
| | |
| 54
| |
| | |
| | |
| 50
| |
| | |
| | |
| 7
| |
| | |
| | |
| 7.0
| |
| | |
| | |
| 33
| |
| | |
| | |
| 90
| |
| | |
| | |
| 17
| |
| | |
| | |
| 6.0
| |
| | |
| | |
| 23
| |
| | |
| | |
| | |
| L^ntreated recipient ovarian weight = 15.4 mg.
| |
| * PFD = Protein-free diet.
| |
| | |
| | |
| | |
| 67G
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GONADS
| |
| | |
| | |
| | |
| adequate dietary protein (Friedman and
| |
| Friedman, 1940).
| |
| | |
| When anterior pituitar}^ glands of 60gm. male rats were extracted and administered to immature female recipients, ovarian
| |
| weight increased from 13.0 to 37.3 mg.
| |
| After feeding 20 per cent casein or fox chow
| |
| ad libitum for 14 days, the hypophyses of
| |
| male rats contained almost twice as much
| |
| gonadotrophin per milligram of tissue as
| |
| did the hypophyses of the initial controls.
| |
| Removal of protein from the diet for 14
| |
| days, however, reduced hypophyseal gonadotrophin concentration below the level
| |
| of the initial controls (Leathem and Fisher,
| |
| 1959).
| |
| | |
| Data on the hypophyseal hormone content as influenced by specific amino acid
| |
| deficiencies have not come to the author's
| |
| attention. Cytologically, however, Scott
| |
| (1956) noted that an isoleucine-deficient
| |
| diet depleted the pituitary gonadotrophic
| |
| cells of their PAS-positive material and reduced the size of acidophilic cells. Omission
| |
| of threonine, histidine, or tryptophan invoked similar effects. The changes probably represent the interference of a single
| |
| amino acid deficiency with protein metabolism rather than specific effects attributable to the lack of amino acid itself. Excessive amino acid provided by injecting
| |
| leucine, methionine, valine, tyrosine, or
| |
| glycine caused release of gonadotrophin
| |
| (Goth, Lengyel, Bencze, Saveley and Majsay, 1955).
| |
| | |
| Administration of 0.1 mg. stilbestrol for
| |
| 20 days to adult male rats eliminated detectable hypophyseal gonadotrophins. Hormone levels returned during the postinjection period provided the diet contained
| |
| adequate protein, whereas a protein-free diet
| |
| markedly hindered the recovery of hypophyseal gonadotrophins. The gonadotrophin
| |
| content of the pituitary gland correlated
| |
| well with the recovery of the reproductive
| |
| system, indicating that gonadotrophin production was subnormal on ]irotein-free feeding (Leathem, 1958a).
| |
| | |
| C. CAHBOHYDR.\TE .\ND FAT
| |
| | |
| Reproduction does not appear to be influenced by carbohydrates per se and hypophyseal alterations have not been noted.
| |
| | |
| | |
| | |
| Fat-deficient diets, however, do influence
| |
| reproduction and the hypophysis exhibits
| |
| cellular changes. Pituitary glands of female rats fed a fat-free diet contain a subnormal number of acidophiles and an increased number of basophiles (Panos and
| |
| Finerty, 1953) . In male rats the feeding oi
| |
| a fat-free diet increased hypophyseal basophiles, followed progessively by more castration changes (Finerty, Klein and Panos,
| |
| 1957; Panos, Klein and Finerty, 1959).
| |
| | |
| D. VITAMINS
| |
| | |
| Despite the many investigations relating
| |
| reproduction to vitamin requirements, relatively few have involved hypophyseal hormone estimations. Thus in 1955, Wooten,
| |
| Nelson, Simpson and Evans reported the
| |
| first definitive study which related pyridoxine deficiency to hypophyseal gonadotrophin content. Using the hypophysectomized rat for assay, pituitary glands
| |
| from Bo-deficient rats were shown to have
| |
| a 10-fold increase in FSH per milligram of
| |
| tissue and a slightly increased LH content.
| |
| Earlier studies had revealed that vitamin
| |
| Bi-free diets decreased pituitary gonadotrophins in male rats (Evans and Simpson,
| |
| 1930) and a similar effect of the folic acid
| |
| antagonist, aminopterin, in the monkey was
| |
| found later (Salhanick, Hisaw and Zarrow, 1952).
| |
| | |
| Male rats deficient in vitamin A exhibited
| |
| a 43 per cent increase, and castrated vitamin
| |
| A-deficient rats a 100 per cent increase in
| |
| hypophyseal gonadotrophin potency over
| |
| the normal controls (Mason and Wolfe,
| |
| 1930). The increase of gonadotrophin was
| |
| more marked in vitamin A-deficient male
| |
| than in vitamin A-deficient female rats.
| |
| Associated with the increase in hormone
| |
| level was a significant increase in basophile
| |
| cells (Sutton and Brief, 1939; Hodgson,
| |
| Hall, Sweetman, Wiseman and Converse,
| |
| 1946; Erb, Andrews, Hauge and King,
| |
| 1947).
| |
| | |
| A re\-iew of the literature up to 1944 permitted Mason to suggest that the anterior
| |
| hypophysis was not the instigator of reproductive disturbances in vitamin E deficiency. Nevertheless, Griesbach, Bell and
| |
| Livingston (1957), in an analysis of the
| |
| pituitary gland during progessive stages of
| |
| | |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| 677
| |
| | |
| | |
| | |
| tocopherol deprivation, observed cytologic
| |
| changes in the hypophysis which preceded
| |
| testis changes. The "peripheral or FSH
| |
| gonadotrophes" increased in number, size,
| |
| and activity. The LH cells exhibited a
| |
| hyperplasia of lesser extent, but possibly
| |
| sufficient to increase LH in circulation and
| |
| to cause hypertrophy of the male accessory
| |
| glands. Gonadotrophic hormone content of
| |
| pituitary glands from vitamin E-deficient
| |
| rats may be decreased (Rowlands and
| |
| Singer, 1936), unchanged (Biddulph and
| |
| Meyer, 1941), or increased to a level between normal and that of the castrate, when
| |
| the adult male rats were examined after 22
| |
| weeks on a deficient diet (Nelson, 1933;
| |
| Drummond, Noble and Wright, 1939).
| |
| Using hypophysectomized male rats as assay animals, evidence was obtained that
| |
| FSH was increased in the pituitary glands
| |
| of vitamin E-deficient male and female rats
| |
| (P'an, Van Dyke, Kaunitz and Slanetz,
| |
| 1949).
| |
| | |
| IV. Male Reproductive System
| |
| | |
| A. TESTIS
| |
| | |
| The two basic functions of the male
| |
| gonads are to produce gametes and secrete
| |
| steroids. Spermatogenic activity can be
| |
| estimated from testis morphology and examination of semen samples. Androgen secretion can be estimated from urinary steroid levels, accessorj^ gland weight, and
| |
| from analyses of accessory sex gland secretions, i.e., fructose and citric acid. In normal maturation in the rabbit, rat, boar,
| |
| and bull, androgen secretion precedes spermatogenesis (Lutwak-Mann, 1958). On this
| |
| basis it would appear that well fed young
| |
| bulls may come into semen production 2 to
| |
| 3 months sooner than poorly fed animals
| |
| (Brat ton, 1957).
| |
| | |
| 1. Inanition
| |
| | |
| Complete starvation will pre^•ent maturation of immature animals. Furthermore,
| |
| marked undernutrition in 700 boys, 7 to 16
| |
| years of age, was associated with genital
| |
| infantilism in 37 per cent and cryptorchidism in 27 per cent (Stephens, 1941).
| |
| Restriction of food intake to one-half of the
| |
| normal in maturing bull calves had a
| |
| | |
| | |
| | |
| marked delaying effect on the onset of
| |
| seminal vesicle secretion, but a lesser delaying effect on spermatogenesis (Davies,
| |
| Mann and Rowson, 1957) . Limiting the food
| |
| intake to one-third of the normal did not
| |
| prevent the immature rat testis from forming spermatozoa at the same time as their
| |
| controls (Talbert and Hamilton, 1955).
| |
| When testis maturation was prevented by
| |
| inanition, a rapid growth and maturation
| |
| occurred on refeeding (Ball, Barnes and
| |
| Visscher, 1947; Quimby, 1948) but Schultze
| |
| (1955) observed that full body size was not
| |
| attained.
| |
| | |
| The reproductive organs of the adult are
| |
| more resistant to changes imposed by diet
| |
| than are those of the immature animal.
| |
| Thus, Mann and Walton (1953) found that
| |
| 23 weeks of underfeeding produced little
| |
| change in sperm density and motility in mature animals although seminal vesicle function was reduced. Li the male rat testis
| |
| hypofunction follows partial or complete
| |
| starvation (]\Iason and Wolfe, 1930; Mulinos and Pomerantz, 1941a; Escudero, Herraiz and Mussmano, 1948), but there is no
| |
| reduction in testicular nitrogen (Addis, Poo
| |
| and Lew, 1936). Loss of Leydig cell function precedes cessation of spermatogenesis
| |
| (Moore and Samuels, 1931) and is evident
| |
| by the atrophy of the accessory sexual organs (^lulinos and Pomerantz, 1941a) and
| |
| by an alteration in accessory gland secretion
| |
| (Pazos and Huggins, 1945; Lutwak-]\Iann
| |
| and Mann, 1950). Evidence of a tubular
| |
| effect is provided by the lack of motile
| |
| sperm (Reid, 1949). Severe dietary restriction is associated with the absence of
| |
| spermatozoa in the seminiferous tubules and
| |
| epididymis (Mason, 1933; Menze, 1941).
| |
| | |
| The human male suffering from chronic
| |
| malnutrition exhibits hypogonadism. The
| |
| testes atrophy and exhibit a decrease in size
| |
| of the seminiferous tubules; basement membrane thickening and small Leydig cells
| |
| are seen. These individuals excrete significantly subnormal amounts of 17-ketosteroids (Zubiran and Gomez-Mont, 19531.
| |
| Acute starvation may also decrease urinary
| |
| 17-ketosteroid and androgen levels as much
| |
| as 50 per cent, with recovery evident on refeeding (Perloff, Lasche, Nodine, Schneeberg and Vieillard. 1954).
| |
| | |
| | |
| | |
| 6/
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GONADS
| |
| | |
| | |
| | |
| TABLE 12.5
| |
| | |
| Effect of diet on the testes of immature rats
| |
| | |
| (From J. H. Leathern, in Re-productive Phijsiology
| |
| | |
| and Protein Nutrition, Rutgers University
| |
| | |
| Press, New Brunswick, N. J., 1959.)
| |
| | |
| | |
| | |
| Sperm
| |
| | |
| | |
| | |
| Initial control . . .
| |
| 20 per cent X 30
| |
| | |
| days
| |
| | |
| 6 per cent X 30
| |
| | |
| days
| |
| | |
| 3 per cent X 30
| |
| | |
| days
| |
| | |
| per cent X 30
| |
| | |
| days
| |
| | |
| per cent + 5
| |
| per cent liver. ,
| |
| | |
| per cent + 5
| |
| per cent yeast
| |
| | |
| G5 per cent X 30
| |
| davs
| |
| | |
| | |
| | |
| No. of
| |
| Rats
| |
| | |
| | |
| Testis
| |
| | |
| | |
| Weight
| |
| | |
| | |
| | |
| | |
| mg.
| |
| | |
| | |
| mg./lOOgm.
| |
| | |
| | |
| 10
| |
| | |
| | |
| 329
| |
| | |
| | |
| 825
| |
| | |
| | |
| 10
| |
| | |
| | |
| 1694
| |
| | |
| | |
| 1035
| |
| | |
| | |
| 16
| |
| | |
| | |
| 824
| |
| | |
| | |
| 890
| |
| | |
| | |
| 12
| |
| | |
| | |
| 380
| |
| | |
| | |
| 930
| |
| | |
| | |
| 10
| |
| | |
| | |
| 140
| |
| | |
| | |
| 346
| |
| | |
| | |
| 10
| |
| | |
| | |
| 112
| |
| | |
| | |
| 291
| |
| | |
| | |
| 10
| |
| | |
| | |
| 119
| |
| | |
| | |
| 296
| |
| | |
| | |
| 10
| |
| | |
| | |
| 1747
| |
| | |
| | |
| 1040
| |
| | |
| | |
| | |
| | |
| | |
| 100
| |
| | |
| 50
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| 100
| |
| | |
| | |
| | |
| 2. Protein
| |
| | |
| The minimal amount of dietary protein
| |
| which will support reproduction, lactation,
| |
| and growth is 16.7 per cent (Goettsch, 1949) .
| |
| Thus, it is not surprising that maturation
| |
| of testes and accessory sex organs was prevented in immature rats (Horn, 1955) and
| |
| mice (Leathern and DeFeo, 1952) when
| |
| they were fed a protein-free diet for 15 to
| |
| 30 days after weaning. Furthermore, supplements of 5 per cent liver to the casein-free
| |
| diet had no effect. After a month, the testes,
| |
| averaging 329 mg., decreased to 140 mg. in
| |
| rats fed per cent casein, but the weight
| |
| increased to an average of 1694 mg. and
| |
| 1747 mg. in rats fed 20 per cent and 65 per
| |
| cent casein, respectively (Table 12.5). Following protein depletion, there was a decrease in tubular ribonucleic acid and an increase in lipid. Accumulation of gonadal
| |
| lipid in the inactive testis may be an abnormal assimilation of a degenerative nature or simply nonutilization. A diet containing 6 per cent casein permitted the
| |
| formation of spermatozoa in some animals
| |
| (Guilbert and Goss, 1932). When the 6 per
| |
| cent casein diet was fed to immature animals for 30 days 50 per cent of the rats
| |
| exhibited some spermatozoa; in addition,
| |
| testis weight increased slightly and seminal
| |
| | |
| | |
| | |
| vesicle weight doubled, but body weight was
| |
| not improved (Horn, 1955). Thus, as we
| |
| noted earlier, the reproductive system may
| |
| gain special consideration for protein allotments when supplies are limited.
| |
| | |
| Gain in testis weight in immature male
| |
| rats and the biochemical composition of the
| |
| immature testis are influenced by the nutritive value of the protein fed. The testes of
| |
| normal immature rats contain 85 per cent
| |
| water, 10.5 per cent protein, 4.5 per cent
| |
| lipid, and detectable glycogen (Wolf and
| |
| Leathern, 1955). Proteins of lower nutritive
| |
| value (wheat gluten, peanut flour, gelatin ) may permit some increase in testis
| |
| weight, but testis protein concentration decreased, percentage of water increased, and
| |
| lipid and glycogen remained unchanged
| |
| (Table 12.6). The enzyme ^fi-glucuronidase,
| |
| which has frequently been associated with
| |
| growth processes, exhibited no change in
| |
| concentration as the testis matured or was
| |
| jirevented from maturing by a protein-free
| |
| diet (Leathem and Fisher, 1959). Not only
| |
| are the weight and composition of the testis
| |
| influenced by feeding proteins of varied biologic value, but the release of androgen is
| |
| more markedly altered. When a 22-day-old
| |
| male rat was fed a 20 per cent casein diet
| |
| for 30 days, the seminal vesicle and ventral
| |
| prostate weights increased 9- to 10-fold in
| |
| comparison with initial control weight. Sub
| |
| TABLE 12.6
| |
| | |
| Niiiritioiial effects on testis-coin position
| |
| | |
| in immature rats
| |
| | |
| (From R. C. Wolf and J. H. Leathem,
| |
| | |
| Endocrinology, 57, 286, 1955.)
| |
| | |
| | |
| | |
| 20 pel' cent casein
| |
| | |
| 20 per cent wheat
| |
| gluten
| |
| | |
| 20 per cent i)eanut flour
| |
| | |
| 20 i)er cent gelatin
| |
| | |
| 5 per cent casein
| |
| | |
| Fox chow
| |
| | |
| Initial control . .
| |
| | |
| | |
| | |
| No. of
| |
| Rats
| |
| | |
| | |
| Final
| |
| Body
| |
| Weight
| |
| | |
| | |
| | |
| | |
| gm.
| |
| | |
| | |
| 7
| |
| | |
| | |
| 128
| |
| | |
| | |
| 8
| |
| | |
| | |
| 82
| |
| | |
| | |
| 8
| |
| | |
| | |
| 81
| |
| | |
| | |
| 5
| |
| | |
| | |
| 53
| |
| | |
| | |
| 5
| |
| | |
| | |
| 61
| |
| | |
| | |
| 8
| |
| | |
| | |
| 115
| |
| | |
| | |
| 7
| |
| | |
| | |
| 61
| |
| | |
| | |
| | |
| 72.3j30.4
| |
| 04.634.0
| |
| | |
| | |
| | |
| 1468
| |
| 1017
| |
| | |
| 1257 66.1
| |
| 2101
| |
| | |
| | |
| | |
| 28.8
| |
| | |
| | |
| | |
| 0.11
| |
| 0.18
| |
| 0.11
| |
| 0.10
| |
| | |
| | |
| | |
| 684,62.4 29.2 0.26
| |
| 1515'70.3 30.3 0.15
| |
| 273 72. 7:30. 10.19
| |
| | |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| 679
| |
| | |
| | |
| | |
| stitution of wheat gluten and peanut flour
| |
| for casein, limited the increase in the weight
| |
| of the seminal vesicles to less than 100 per
| |
| cent. In fact, seminal vesicle weight as related to body weight did not increase in
| |
| animals fed 20 per cent wheat gluten.
| |
| | |
| The withholding of dietary protein from
| |
| an immature rat for 30 days, during which
| |
| time maturation occurs in the fully fed animal, did not impose a permanent damage.
| |
| Refeeding of protein permitted the rapid recovery of testis weight and the appearance
| |
| of spermatoza, 70 per cent of all animals
| |
| having recovered in 30 days when fully fed,
| |
| whereas only 25 per cent recovered when
| |
| 6 per cent casein was fed. Recovery of
| |
| androgen secretion was somewhat slower
| |
| than that of the tubules as estimated by
| |
| seminal vesicle weight.
| |
| | |
| Variations in protein quality are a reflection of amino acid patterns, and amino
| |
| acid deficiencies interfere with testis maturation (Scott, 1956; Pomeranze, Piliero,
| |
| Medeci and Plachta, 1959). Alterations in
| |
| food intake which follow amino acid deficiencies have required forced feeding or
| |
| pair-fed controls, but it is clear from what
| |
| w^as found in the controls that the gonadal
| |
| changes were not entirely due to inanition
| |
| (Ershoff, 1952).
| |
| | |
| If the diet is varied so that caloric intake
| |
| per gram is reduced to half while retaining
| |
| the dietary casein level at 20 per cent, immature rat testis growth is prevented. The
| |
| effect is unlike that obtained with this level
| |
| of protein in the presence of adequate calories. Furthermore, the caloric restriction
| |
| may increase testis glycogen (Leathem,
| |
| 1959c).
| |
| | |
| Protein anabolic levels are higher in the
| |
| tissues of young growing animals and the
| |
| body is more dependent on dietary protein
| |
| level and quality for maintenance of the
| |
| metabolic nitrogen pool than in adult animals. On the other hand, body protein reserves in adult animals permit internal
| |
| shifts of nitrogen to the metabolic pool and
| |
| to tissues when dietary sources are reduced
| |
| or endocrine imbalances are imposed. Thus,
| |
| Cole, Guilbert and Goss (1932) fed a low
| |
| protein diet to adult male rats for 60 to 90
| |
| days before the sperm disappeared, but the
| |
| animals would not mate. Amount of semen
| |
| and sperm produced by sheep have been re
| |
| | |
| | |
| TABLE 12.7
| |
| | |
| Arlult rat testes and seminal vesicles
| |
| | |
| after protein depletion
| |
| | |
| (From J. H. Leatliem, Recent Progr. Hormone
| |
| | |
| Res., 14, 141, 1958.)
| |
| | |
| | |
| | |
| Days on
| |
| | |
| | |
| No. of
| |
| | |
| | |
| Testis
| |
| | |
| | |
| H2O
| |
| | |
| | |
| Protein
| |
| | |
| | |
| Total
| |
| | |
| | |
| Seminal
| |
| | |
| | |
| PFD*
| |
| | |
| | |
| Rats
| |
| | |
| | |
| Weight
| |
| | |
| | |
| Protein
| |
| | |
| | |
| Vesical
| |
| | |
| | |
| | |
| | |
| | |
| | |
| nig.
| |
| | |
| | |
| %
| |
| | |
| | |
| %dry
| |
| | |
| | |
| gm.
| |
| | |
| | |
| mg.
| |
| | |
| | |
| Control
| |
| | |
| | |
| 9
| |
| | |
| | |
| 2852
| |
| | |
| | |
| 85.9
| |
| | |
| | |
| 66.7
| |
| | |
| | |
| 0.28
| |
| | |
| | |
| 1276
| |
| | |
| | |
| 30
| |
| | |
| | |
| 9
| |
| | |
| | |
| 2600
| |
| | |
| | |
| 86.0
| |
| | |
| | |
| 66.1
| |
| | |
| | |
| 0.24
| |
| | |
| | |
| 689
| |
| | |
| | |
| 50
| |
| | |
| | |
| 7
| |
| | |
| | |
| 2398
| |
| | |
| | |
| 85.4
| |
| | |
| | |
| 64.1
| |
| | |
| | |
| 0.22
| |
| | |
| | |
| 320
| |
| | |
| | |
| 90
| |
| | |
| | |
| 25
| |
| | |
| | |
| 1429
| |
| | |
| | |
| 85.7
| |
| | |
| | |
| 69.6
| |
| | |
| | |
| 0.13
| |
| | |
| | |
| 168
| |
| | |
| | |
| | |
| * PFD = Protein-free diet.
| |
| | |
| lated to the dietary protein level (Popoff
| |
| and Okultilschew, 1936). Removal of protein from the diet for 30 days had little
| |
| effect on the adult rat testis weight, spermatogenesis, or nitrogen content (Leathem,
| |
| 1954). However, seminal vesicle w^eight was
| |
| reduced 50 per cent (Aschkenasy, 1954).
| |
| Prolonged protein depletion was required
| |
| before the testis exhibited a loss in protein
| |
| and a reduction in size. A loss of spermatozoa was not observed consistently, although some testes were completely atrophic
| |
| (Table 12.7). Accessory organ weight decrease reflected the disappearance of androgen (Leathem, 1958a). Interstitial cell atrophy has also been noted in rats fed a low
| |
| vegetable protein (cassava) diet (Adams,
| |
| Fernand and Schnieden, 1958).
| |
| | |
| Sterility may or may not be induced with
| |
| diets containing 65 per cent protein (Reid.
| |
| 1949; Leathem, 1959c) but a 15 to 18 per
| |
| cent dietary level of a poor protein such
| |
| as maize or gelatin will decrease sperm motility and increase the number of abnormal
| |
| sperm. The influence of proteins having different nutritional values in support of the
| |
| growth of testes from the level to which
| |
| they were depressed by stilbestrol indicated
| |
| that casein, lactalbumin, and wheat gluten
| |
| are equally competent to support testis
| |
| growth whereas gelatin is deficient. Whole
| |
| proteins may have several amino acid deficiencies, but the administration of amino
| |
| acid antagonists may help to identify important individual amino acids. As an example, ethionine causes severe seminiferous
| |
| tubule atrophy and Leydig cell hypoplasia
| |
| (Kaufman, Klavins and Kinney, 1956;
| |
| Goldberg, Pfau and Ungar, 1959). Studies
| |
| in man have indicated a sharp reduction in
| |
| | |
| | |
| | |
| 680
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GONADS
| |
| | |
| | |
| | |
| spermatozoa after 9 days on an argininedeficient diet (Holt, Albanese, Shettles,
| |
| Kajdi and Wangerin, 1942).
| |
| | |
| Adequate dietary protein cannot maintain reproductive function if the diet is
| |
| calorie deficient. Thus, a decrease in seminal
| |
| vesicle weight could be related to a decrease
| |
| in dietary calories while protein levels were
| |
| constant (Rosenthal and Allison, 1956).
| |
| However, the accessory gland weight loss
| |
| imposed by caloric restriction could be
| |
| slowed by increasing the dietary protein
| |
| (Rivero-Fontan, Paschkis, West and Cantarow, 1952).
| |
| | |
| Alterations in testis function imposed by
| |
| inadequate protein are corrected when protein is returned to the diet at normal levels
| |
| (Aschkenasy and Dray, 1953). Nevertheless, the nutritional state of the animal as
| |
| a factor influencing recovery has been demonstrated with stilbestrol-treated adult male
| |
| rats. While being fed an 18 per cent casein
| |
| diet, adult male rats were injected with 0.1
| |
| mg. stilbestrol daily for 20 days. Testis
| |
| weight decreased from 2848 to 842 mg.,
| |
| spermatogenesis was abolished, and testis
| |
| water and protein content were significantly
| |
| reduced. Despite a reduction in food intake,
| |
| pair-fed controls exhibited no effect on reproductive organs. When a protein-free diet
| |
| was substituted for the normal diet during
| |
| the administration of stilbestrol, atrophy of
| |
| the reproductive system was observed. Cessation of hormone administration was followed by a rapid return of testicular function toward normal when 18 per cent casein
| |
| was fed both during the injection period
| |
| and the recovery period. Within 30 days
| |
| spermatogenesis and testicular composition
| |
| were fully recovered. However, when 18
| |
| per cent casein was fed in the postinjection
| |
| period to rats that had received a proteinfree diet while being given stilbestrol, recovery was clearly slow. After a month,
| |
| spermatozoa were observed in only 30 per
| |
| cent of the testes and testis weight was subnormal. Despite the seeming similarity of
| |
| response by the two nutritional groups during the injection period, the postinjection recovery on identical dietary intake revealed
| |
| marked differences in rate of recoverv (Leathem, 1958a).
| |
| | |
| | |
| | |
| 3. Fat
| |
| | |
| Linoleic, linolenic, and arachidonic acids
| |
| are designated as essentially fatty acids, but
| |
| the physiologic role of these substances is
| |
| not clearly understood. Nevertheless, the
| |
| male reproductive organs are influenced by
| |
| dietary essential fatty acid levels. High
| |
| fat diets may enhance testicular weight
| |
| (Kaunitz, Slanetz, Johnson and Guilmain,
| |
| 1956) whereas removal of fat from the diet
| |
| resulted in a degeneration of the seminiferous tubules as evidenced by intracellular
| |
| vacuolation and a reduction in spermatids
| |
| and spermatozoa (Panos and Finerty,
| |
| 1954). After 5 months of feeding a fatfree diet, the rat testis may be devoid of
| |
| sperm (Evans, Lepkovsky and Murphy,
| |
| 1934). Testis degeneration occurred despite
| |
| dietary supplements of vitamins A and E
| |
| and in animals whose health appeared quite
| |
| normal (Ferrando, Jacques, Mabboux and
| |
| Prieur, 1955; Ferrando, Jacques, Mabboux
| |
| and SoUogoub, 1955).
| |
| | |
| Weanling rats fed 14 per cent arachis (peanut) oil for 15 weeks exhibited a marked
| |
| impairment of spermatogenesis (Aaes-Jorgensen, Funch and Dam, 1956) and 28
| |
| per cent arachis oil induced testicular damage of such an order that 15 weeks of feeding
| |
| ethyl linoleate did not restore fertility
| |
| (Aaes-Jorgensen, Funch and Dam, 1957).
| |
| | |
| 4. Vitamins
| |
| | |
| Testicular dysfunction as judged by failure of sperm formation or atrophy of the
| |
| secondary sex organs has been observed in
| |
| deprivations of thiamine, riboflavin, pyridoxine, calcium pantothenate, biotin, and
| |
| vitamins A and E. One must distinguish,
| |
| however, between effects of inanition associated w^ith a vitamin deficiency and a
| |
| specific vitamin effect (Skelton, 1950) ; one
| |
| must also consider species differences (Biskind, 1946).
| |
| | |
| There is no question but that vitamin E
| |
| deficiency in the rat results in a specific
| |
| and irreversible damage to the testis. Tubular damage may proceed to the point where
| |
| only Sertoli cells remain and yet the interstitial cells are not influenced (Mason,
| |
| 1939). Similar changes followed vitamin E
| |
| deficiency in the guinea pig (Pappenheimer
| |
| and SchogolefT, 1944; Curto, 1954; Ingel
| |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| 681
| |
| | |
| | |
| | |
| man-Siindberg, 1954) , hamster (Mason and
| |
| Mauer, 1957), and bird (Herrick, Eide and
| |
| Snow, 1952; Lowe, Morton, Cunningham
| |
| and Vernon, 1957). However, little or no
| |
| effect of an absence of vitamin E was noted
| |
| in the rabbit (Mackenzie, 1942) and mouse
| |
| (Bryan and Mason, 1941), or in live stock
| |
| (Blaxter and Brown, 1952), or man (Lutwak-Mann, 1958), although vitamin E is
| |
| present in human testes (Dju, Mason and
| |
| Filer, 1958). Treatment of low-fertility
| |
| farm animals with wheat germ oil or tocopherol or the use of this vitamin clinically
| |
| have provided only inconclusive results
| |
| (Beckmann, 1955) . Although some positive
| |
| effects have been reported in man, the results may be due in part at least to the sparing action of tocopherol toward vitamin A.
| |
| | |
| Vitamin A deficiency influences the testis
| |
| but changes are closely associated with the
| |
| degree of inanition. In the rat, a vitamin deficiency sufficient to cause ocular lesions
| |
| did not prevent sperm formation, but a deficiency of such proportions as to cause a
| |
| body weight loss did cause atrophy of the
| |
| germinal epithelium (Reid, 1949). Vitamin
| |
| A deficiency will induce sterility in mice
| |
| (McCarthy and Cerecedo, 1952). A gross
| |
| vitamin deficiency in bulls before expected
| |
| breeding age prevented breeding ; adult bulls
| |
| may exhibit a lower quality semen but they
| |
| remain fertile (Reid, 1949). Vitamin A deficiency induces metaplastic keratinization
| |
| of the epithelium lining the male accessory
| |
| sex organs (Follis, 1948) and thus may influence semen.
| |
| | |
| Testis damage induced by vitamin A deficiency can be reversed, but vitamin A
| |
| therapy in man for oligospermia not due to
| |
| vitamin lack was without effect (Home
| |
| and Maddock, 1952) .
| |
| | |
| Age of the animal and dosage are factors
| |
| which influence the results obtained in male
| |
| rats with administered vitamin A. Immature
| |
| male rats given 250 I.U. of vitamin A per
| |
| gram of body weight daily exhibited a loss
| |
| of spermatocytes, an effect which was accentuated by tocopherol (Maddock, Cohen
| |
| and Wolbach, 1953). Little or no effect of
| |
| similar treatment was observed in adult
| |
| rats. The liver is the major storage depot
| |
| for vitamin A and the fact that the male rat
| |
| liver is more quickly depleted and less capa
| |
| | |
| | |
| ble of storage than is the liver of the female
| |
| should be considered in any attempted correlation of the vitamin and hormone levels
| |
| (Booth, 1952).
| |
| | |
| Other vitamin deficiencies have been
| |
| shown to influence the testis. A lack of
| |
| thiamine had little effect on testis weight,
| |
| but did influence the Leydig cells and prevented growth of the accessory sex organs
| |
| (Pecora and Highman, 1953). A chronic
| |
| lack of ascorbic acid will cause a degeneration of both Leydig cells and seminiferous
| |
| tubules. The effects of vitamin deficiency on
| |
| the testis has been distinguished from those
| |
| due to inanition and have been related to
| |
| changes in carbohydrate metabolism (Mukherjee and Banerjee, 1954; Kocen and
| |
| Cavazos, 1958) . The importance of ascorbic
| |
| acid in the testis as related to function is
| |
| not evident, but concentrations of this vitamin are maximal at 1 week of age (Coste,
| |
| Delbarre and Lacronique, 1953).
| |
| | |
| Serious anatomic and functional impairments of testes were noted in pantothenic
| |
| acid deficiency (Barboriak, Krehl, Cowgill
| |
| and Whedon, 1957), and development of
| |
| the rat testis and seminal vesicles was retarded by a biotin deficiency (Bishop and
| |
| Kosarick, 1951; Katsh, Kosarick and Alpern, 1955), but the animals did not exhibit
| |
| marked alterations in other endocrine organs (Delost and Terroine, 1954). Testosterone hastened the development of vitamin
| |
| deficiency and enhanced the severity of biotin deficiency in both sexes, thereby suggesting a hormone-vitamin relationship
| |
| (Okey, Pencharz and Lepkovsky, 1950). On
| |
| the other hand, testosterone had no effect on
| |
| the tolerance of mice for aminopterin, but
| |
| castration increased the tolerance (Goldin,
| |
| Greenspan, Goldberg and Schoenberg 1950).
| |
| | |
| B. INFLUENCE OF NUTRITION ON THE EE
| |
| SPONSIVENESS OF MALE REPRODUCTIVE
| |
| | |
| TISSUES TO HORMONES
| |
| | |
| 1. Testis
| |
| | |
| a. Inanition. The testes of birds on limited
| |
| food intake were more responsive to hypophyseal gonadotrophin than fully fed birds
| |
| (Byerly and Burrows, 1938; Breneman,
| |
| 1940). In the rat several investigators have
| |
| shown that the testis will respond to gonadotrophin despite inanition (Moore and Sara
| |
| | |
| | |
| 682
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GONADS
| |
| | |
| | |
| | |
| TABLE 12.8
| |
| | |
| Influence of diet and pregnant mare serum (PMS)
| |
| | |
| on testes and seminal vesicles of
| |
| | |
| immature male mice
| |
| | |
| (From V. J. DeFeo and J. H. Leathern,
| |
| | |
| unpublished.)
| |
| | |
| | |
| | |
| Diet (Per cent
| |
| Protein X Days Fed)
| |
| | |
| | |
| | |
| per cent X 10
| |
| per cent X 10
| |
| per cent X 20
| |
| per cent X 20
| |
| | |
| | |
| | |
| l.u.
| |
| | |
| 3
| |
| | |
| 3
| |
| | |
| | |
| | |
| stage of
| |
| Spermatogenesis
| |
| | |
| | |
| | |
| 4
| |
| 1
| |
| | |
| 1 5
| |
| 5
| |
| | |
| | |
| | |
| Spermatids
| |
| | |
| | |
| | |
| Seminal
| |
| Vesicles
| |
| | |
| | |
| | |
| mg.
| |
| | |
| 2.7
| |
| 4.5
| |
| | |
| 2.7
| |
| 3.5
| |
| | |
| | |
| | |
| uels, 1931; Funk and Funk, 1939; Meites,
| |
| 1953), with a stimulation of Leydig cells,
| |
| an increase in testis size, and, in 40 days,
| |
| a return of spermatozoa. Underfed males injected with gonadotrophin sired litters (Mulinos and Pomerantz, 1941a, b). Improved
| |
| nutrition aided by unknown liver factors
| |
| enhanced the response to androgen in severe human oligospermia (Glass and Russell, 1952).
| |
| | |
| b. Protein. Feeding a protein-deficient
| |
| diet to adult male rats for 60 to 90 days
| |
| did not prevent stimulation of the testes and
| |
| seminal vesicles after pregnant mare's serum
| |
| (PMS) administration (Cole, Guilbert and
| |
| Goss, 1932). As we have noted, immature
| |
| animals are prevented from maturing when
| |
| diets lack protein. Nevertheless, a gonadal
| |
| response to injected gonadotrophin was obtained in immature mice fed a protein-free
| |
| diet for 13 days; tubules and Leydig cells
| |
| were stimulated and androgen was secreted
| |
| (Table 12.8). Refeeding alone permitted a
| |
| recovery of spermatogenesis which was not
| |
| hastened by concomitant PMS (Leathem,
| |
| 1959c).
| |
| | |
| The maintenance of testis weight and
| |
| spermatogenic activity with testosterone
| |
| propionate in hypophysectomized adult
| |
| male rats is well known, but these studies
| |
| have involved adequate nutrition. If hypophysectomized rats were fed a protein-free
| |
| diet and injected with 0.25 mg. testosterone
| |
| propionate daily, testis weight and spermatogenesis were less well maintained than
| |
| in rats fed protein. Testis protein concentra
| |
| | |
| | |
| tion was also reduced. These data suggest
| |
| that influences of nutrition on the testis can
| |
| be direct and are not entirely mediated
| |
| through hypophyeal gonadotrophin changes
| |
| (Leathem, 1959b).
| |
| | |
| c. Fat. The rat fed for 20 weeks on a fatfree diet exhibits a degeneration of the
| |
| seminiferous epithelium within the first
| |
| weeks which progresses rapidly thereafter.
| |
| Chorionic gonadotrophin or rat pituitary extract started during the 20th week failed to
| |
| counteract the tubular degeneration, but
| |
| testosterone propionate proved effective
| |
| (Finerty, Klein and Panos, 1957). The result shows that the ineffectiveness of the
| |
| gonadotrophins could not be due to the failure of androgen release (Greenberg and Ershoff, 1951).
| |
| | |
| d. Vitamins. Gonadotrophins failed to
| |
| promote spermatogenesis in vitamin A(Mason, 1939) or vitamin E- (]Mason,
| |
| 1933; Geller, 1933; Drummond, Noble and
| |
| Wright, 1939) deficient rats, but in another
| |
| experiment the atrophic accessory sex organs of vitamin A-depleted rats were stimulated (Mayer and Goodard, 1951). Lack
| |
| of vitamin A favored an enhanced response
| |
| to PMS when the ratios of seminal vesicle
| |
| weight to body weight were computed
| |
| (Meites, 1953). The failure of gonadotrophins to stimulate testis tubules suggests a
| |
| specific effect of avitaminosis A and E
| |
| (Mason, 1933) on the responsiveness of the
| |
| germinal epithelium.
| |
| | |
| Subnormal responses of rats to PMS, as
| |
| measured by relative seminal vesicle weight,
| |
| were obtained when there were individual
| |
| vitamin B deficiencies, but the influence was
| |
| due largely to inanition (Drill and Burrill,
| |
| 1944; Meites, 1953). Nevertheless, sufficient
| |
| response to chorionic gonadotrophin was obtained so that fructose and citric acid levels
| |
| were restored to normal. Such an effect was
| |
| not observed to follow dietary correction unless an unlimited food intake was allowed
| |
| (Lutwak-Mann. 1958).
| |
| | |
| | |
| | |
| 2. Sc
| |
| | |
| | |
| | |
| il W.siclfx and Prosfate
| |
| | |
| | |
| | |
| a. Inanition. Although the accessory reproductive organs resppnd to direct stimulation despite an inadequate food intake
| |
| (Mooi'c and Samuels. 19311, tlio increase
| |
| | |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| 683
| |
| | |
| | |
| | |
| in weight may be subnormal in mice and
| |
| rats (Goldsmith, Nigrelli and Ross, 1950;
| |
| Kline and Dorfman, 1951a, Grayhack and
| |
| Scott, 1952), or above normal in chickens
| |
| (Breneman, 1940). Complete deprivation
| |
| of food reduced the quantity of prostatic
| |
| fluid in the dog, but exogenous androgen restored the volume, increased acid phosphatase, and induced tissue growth (Pazos and
| |
| Huggins, 1945) .
| |
| | |
| 6. Protein. The response of the seminal
| |
| vesicles to androgen was investigated in immature rats, using weight and /5-glucuronidase as end points. Castration and 10 days
| |
| on a protein-free diet preceded the 72-hour
| |
| response to 0.25 mg. testosterone propionate.
| |
| The lack of protein did not prevent a normal weight increase, and enzyme concentration was unchanged. If an 18 per cent diet
| |
| was fed during the 3-day period that the
| |
| androgen was acting, no improvement in
| |
| weight response was noted, but enzyme concentration increased 100 per cent. Thus,
| |
| when protein stores are depleted, the androgen response may be incomplete in the absence of dietary protein (Leathern, 1959c).
| |
| Nevertheless, varied protein levels do not
| |
| influence seminal vesicle weight-response
| |
| when caloric intake is reduced (RiveroFontan, Paschkis, West and Cantarow,
| |
| 1952).
| |
| | |
| c. Vitamins. Vitamin deficiencies do not
| |
| prevent the seminal vesicles from responding to androgen. In fact, in vitamin B deficiency, testosterone restored fructose and
| |
| citric acid levels to normal despite the need
| |
| for thiamine in carbohydrate metabolism
| |
| (Lutwak-Mann and Mann, 1950). In the
| |
| male, unlike the female, the effects of folic
| |
| acid deficiency in reducing responsiveness
| |
| to administered androgen were largely due
| |
| to inanition in both mice and rats (Goldsmith, Nigrelli and Ross, 1950; Kline and
| |
| Dorfman, 1951a) , and vitamin A deficiency
| |
| which leads to virtual castration does not
| |
| prevent an essentially normal response of
| |
| the accessory glands to testosterone propionate (Mayer and Truant, 1949). Restricting the caloric intake of vitamin Adeficient rats retarded the curative effects
| |
| of vitamin A in restoring the accessory sex
| |
| glands of the A-deficient animals (Mason,
| |
| 1939).
| |
| | |
| | |
| | |
| V. Female Reproductive System
| |
| | |
| A. OVARIES
| |
| | |
| 1. Inanition
| |
| | |
| Mammalian species generally exhibit a
| |
| delay in sexual maturation when food intake is subnormal before puberty, and
| |
| ovarian atrophy with associated changes
| |
| in cycles if inanition is imposed on adults.
| |
| In human beings a decrease in fertility and
| |
| a greater incidence of menstrual irregularities were induced by war famine (Zimmer,
| |
| Weill and Dubois, 1944). Ovarian atrophy
| |
| with associated amenorrhea and sterility
| |
| were invoked by chronic undernutrition
| |
| (Stephens, 1941). The ovarian morpliologic
| |
| changes were similar to those of aging.
| |
| Urinary estrogens were subnormal in 22 of
| |
| 25 patients exhibiting amenorrhea associated with limited food intake (Zubiran and
| |
| (_lomcz-Mont, 1953).
| |
| | |
| The nutritional requirements of jM'imates
| |
| other than man have been studied in female
| |
| baboons. The intake of vitamins and other
| |
| essential nutrients was found to be of the
| |
| same order as that recommended for man.
| |
| Caloric intake varied with the menstrual
| |
| cycle, being least during the follicular phase
| |
| and maximal during the 2 to 7 days preceding menstruation (Gilbert and Gillman,
| |
| 1956). Various diets were also studied to
| |
| assess their importance in maintaining the
| |
| normal menstrual rhythm. The feeding of
| |
| (a) maize alone, (b) assorted vegetables
| |
| and fruit, or (c) maize, skimmed milk, and
| |
| fat led to menstrual irregularities or to
| |
| amenorrhea. The mechanism regulating ovulation was the first to be deranged. The
| |
| addition of various vitamins or of animal
| |
| protein did not correct the menstrual disorders. However, inclusion of ox liver in
| |
| the diet did maintain the menstrual rhythmicity, but the beneficial effect could not
| |
| be attributed to its protein content (Gillman and Gilbert, 1956 ) .
| |
| | |
| In lower mammals that have been studied,
| |
| inanition will hinder vaginal opening, and
| |
| delay puberty and ovarian maturation and
| |
| functioning. In adult rats and mice ostrous
| |
| cycles are interrupted and the reprorUictive
| |
| system becomes atrophic when body weight
| |
| loss exceeds 15 per cent. The ovaries be
| |
| | |
| | |
| 684
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GONADS
| |
| | |
| | |
| | |
| come smaller, ovulation fails, and large
| |
| vesicular follicles decrease in number with
| |
| an increase in atresia, but primary follicles
| |
| show a compensatory increase (Marrian
| |
| and Parkes, 1929; Mulinos and Pomerantz,
| |
| 1940; Stephens and Allen, 1941; Guilbert,
| |
| 1942; Bratton, 1957). The ovarian interstitial cells mav be markedly altered or absent
| |
| (Huseby and Ball, 1945; Rinaldini, 1949)
| |
| and the ovary may exhibit excessive luteinization (Arvy, Aschkenasy, AschkenasyLelu and Gabe, 1946) or regressing corpora
| |
| lutea (Rinaldini, 1949) . However, the ovarian changes induced by inanition may be
| |
| reversed by refeeding, with a return to reproductive capacity (Ball, Barnes and Visscher, 1947; Schultze, 1955). The effect of
| |
| feed-level on the reproductive capacity of
| |
| the ewe has been reported (El-Skukh, Nulet,
| |
| Pope and Casida, 1955), but one must realize that high planes of nutrition may adversely influence fertility (Asdell, 1949).
| |
| Nevertheless, additional protein and calcium added to an adequate diet extended
| |
| the reproductive life span (Sherman, Pearson, Bal, McCarthy and Lanford, 1956).
| |
| | |
| 2. Protein
| |
| | |
| The availability of just protein has an
| |
| important influence on the female reproductive system. In immature rats ovarian
| |
| maturation was prevented by feeding diets
| |
| containing per cent to 1.5 per cent protein
| |
| (Ryabinina, 1952) and low protein diets decreased the number of ova but without altering their ribonucleic acid (RNA) or glycogen content (Ishida, 1957). Refeeding 18
| |
| per cent protein for only 3 days was marked
| |
| by the appearance of vesicular follicles and
| |
| the release of estrogen in mice previously fed
| |
| a protein-free diet (Leathem, 1958a). In
| |
| experiments involving the opposite extreme,
| |
| in which 90 per cent protein diets were used,
| |
| a retardation of ovarian growth, and a delay in follicular maturation, in vaginal
| |
| opening, and in the initiation of estrous
| |
| cycles were noted (Aschkenasy-Lelu and
| |
| Tuchmann-Duplessis, 1947; TuchniannDuplcssis and Aschkenasy-Lelu, 1948).
| |
| | |
| Adult female rats fed a protein-free diet
| |
| for 30 days exhibited ovaries weighing 22
| |
| mg. compared with ovaries weighing 56 mg.
| |
| from i)air-fed controls fed 18 per cent casein.
| |
| Ovarian glycogen, ascorbic acid, and cho
| |
| | |
| | |
| lesterol were all influenced by protein
| |
| deprivation and anestrum accompanied
| |
| the ovarian changes. Furthermore, uterine
| |
| weight and gl3^cogen decreased in rats fed
| |
| protein-free diets (Leathem, 1959b).
| |
| | |
| In adult rats the feeding of 3.5 per cent
| |
| to 5 per cent levels of protein (GuillDert and
| |
| Gross, 1932) was followed by irregularity
| |
| of the cycles or by their cessation. The
| |
| cycles became normal when 20 to 30 per cent
| |
| protein was fed (Aschkenasy-Lelu and
| |
| Aschkenasy, 1947). However, abnormally
| |
| high levels of casein (90 per cent) induced prolonged periods of constant estrus
| |
| (Tuchmann-Duplessis and AschkenasyLelu, 1947). Nevertheless, not all species
| |
| responded to protein depletion in the same
| |
| manner. For example, the rabbit exhibited
| |
| estrus and ovulation despite a 25 per cent
| |
| body weight loss imposed by to 2 per cent
| |
| protein diets (Friedman and Friedman,
| |
| 1940).
| |
| | |
| Despite a normal level of protein in the
| |
| diet, inadequate calories will interfere with
| |
| reproductive function and induce ovarian
| |
| atrophy (Escudero, Herraiz and Mussmano,
| |
| 1948; Rivero-Fontan, Paschkis, West and
| |
| Cantarow, 1952). Furthermore, the effects
| |
| of 15 per cent and 56 per cent protein levels
| |
| on estrous cycles could not be distinguished
| |
| when calories were reduced 50 per cent (Lee,
| |
| King and Visscher, 1952). Returning mice
| |
| to full feeding after months of caloric deficiency resulted in a sharp increase in reproductive performance well above that expected for the age of the animal (Visscher,
| |
| King and Lee, 1952). This type of rebound
| |
| phenomenon has not been explained.
| |
| | |
| Reproductive failure assigned to dietary
| |
| protein may be a reflection of protein
| |
| quality as well as level. Specific amino acid
| |
| deficiencies lead to cessation of estrus
| |
| (White and AVhite, 1942; Berg and Rohse,
| |
| 1947) and thus feeding gelatin or wheat
| |
| as the protein source and at an 18 per cent
| |
| level was quickly followed by an anestrum
| |
| (Leathem, 1959b). Supplementation of the
| |
| wheat diet with lysine corrected the reproductive abnormalities (Courrier and
| |
| Raynaud, 1932), but neither lysine (Pearson, Hart and Bohstedt, 1937) nor cystine
| |
| (Pearson, 1936) added to a low casein diet
| |
| was beneficial. Control of food intake must
| |
| be considered in studies involving amino
| |
| | |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| 685
| |
| | |
| | |
| | |
| acids, for a deficiency or an excess can create an imbalance and alter appetite. Opportunity to study the amino acids in reproduction is now possible because of the work of
| |
| Greenstein, Birnbaum, Winitz and Otey
| |
| (1957) and Schultze (1956) , who maintained
| |
| rats for two or more generations on synthetic diets containing amino acids as the
| |
| only source of protein. Similarly, the amino
| |
| acid needs for egg-laying in hens has been
| |
| reported (Fisher, 1957). Tissue culture
| |
| methods also permit the study of the nutritional requirements of embryonic gonadal
| |
| tissue, the success of avian gonadal tissue in
| |
| culture being judged by survival, growth,
| |
| and differentiation. In experiments in which
| |
| this technique was used it was found that a
| |
| medium made up of amino acids as the basic
| |
| nitrogen source can maintain gonadal explants very successfully, even though the
| |
| choice of amino acids does not exactly correspond to the 10 essential amino acids recommended for postnatal growth (StengerHaffen and Wolff, 1957).
| |
| | |
| 3. Carbohydrate
| |
| | |
| The absence of dietary carbohydrate does
| |
| not appreciably affect the regularity of
| |
| estrous cycles in rats provided the caloric
| |
| need is met. However, the substitution of
| |
| 20 per cent sucrose for corn starch induced
| |
| precocious sexual maturity which was followed by sterility (Whitnah and Bogart,
| |
| 1956). The ovaries contained corpora lutea,
| |
| but the excessive luteinization of unruptured
| |
| follicles suggested a hypophyseal disturbance. Substitution of 20 per cent lactose for
| |
| corn starch had no effect. Increased amounts
| |
| of lactose retarded the gain in body weight
| |
| and blocked ovarian maturation, possibly
| |
| because the animal could not hydrolyze adequate amounts of the disaccharide. Addition of whole liver powder to the diet
| |
| counteracted the depressing action of 45 per
| |
| cent lactose on the ovary (Ershoff, 1949).
| |
| | |
| 4. Fat
| |
| | |
| There seems to be little doubt that dietary
| |
| fat is reciuired for normal cyclic activity,
| |
| successful pregnancy, and lactation, and
| |
| that the requirements for essential fatty
| |
| acids are lower in females than in males
| |
| (Deuel, 1956).
| |
| | |
| Conception, fetal development, and par
| |
| | |
| | |
| turition can take place in animals fed a
| |
| diet deficient in fatty acids (Deuel, Martin
| |
| and Alfin-Slater, 1954) , despite a reduction
| |
| in total carcass arachidonic acid (Kummerow. Pan and Hickman, 1952). Earlier
| |
| reports indicated that a deficiency of essential fatty acids caused irregular ovulation and impaired reproduction (Burr and
| |
| Burr, 1930; Maeder, 1937). The large pale
| |
| ovaries lead Sherman (1941) to relate essential fatty acid deficiency to carotene
| |
| metabolism. In this regard the removal of
| |
| essential fatty acids from an adequate diet
| |
| supplemented with vitamin A and E lead
| |
| to anestrum and sterility while maintaining
| |
| good health (Ferrando, Jacques, Mabboux
| |
| and Prieur, 1955). Perhaps the differences
| |
| in opinion regarding the effects of fatty acid
| |
| deficiency can be related to the duration of
| |
| the experimental period. Panos and Finerty
| |
| (1953) found that growing rats placed on
| |
| a fat-free diet exhibited a normal time for
| |
| vaginal opening, normal ovarian weight,
| |
| follicles, and corpora lutea, although interstitial cells were atrophic. However, regular estrous cycles were noted for only 20
| |
| weeks, thereafter 60 per cent of the animals
| |
| exhibited irregular cycles.
| |
| | |
| A decrease in reproductive function may
| |
| be invoked by adding 14 per cent arachis
| |
| oil to the diet (Aaes-Jorgensen, Funch and
| |
| Dam, 1956) . Increasing dietary fat by adding rape oil did not influence ovarian function but did cause the accumulation of
| |
| ovarian and adrenal cholesterol (Carroll
| |
| and Noble, 1952).
| |
| | |
| Essential fatty acid deficiency is associated with underdevelopment and
| |
| atrophic changes of the uterine mucosa.
| |
| Adding fat to a stock diet enhanced uterine
| |
| weight in young animals at a more rapid
| |
| pace than body weight (Umberger and
| |
| Gass, 1958) .
| |
| | |
| 5. Vitamins
| |
| | |
| Carotenoid pigments are present in the
| |
| gonads of many vertebrates and marine invertebrates, and, in mammals, are particularly prominent in the corpus luteum.
| |
| However, no progress has been made in
| |
| determining either the importance of the
| |
| carotenoids in the ovary or of the factors
| |
| controlling their concentrations. It is well
| |
| known that vitamin A deficiency induces
| |
| | |
| | |
| | |
| 686
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GONADS
| |
| | |
| | |
| | |
| a characteristic keratinizing metaplasia of
| |
| the uterus and vagina, but estrous cycles
| |
| continue despite the vaginal mucosal
| |
| changes. Furthermore, ovulation occurs
| |
| regularly until advanced stages of deficiency appear. The estrous cycle becomes
| |
| irregular in cattle fed for a long period
| |
| of time on fodder deficient in carotene. The
| |
| corpora lutea fail to regress at the normal
| |
| rate and ovarian follicles become atretic and
| |
| cystic ( Jaskowski, Watkowski, Dobrowolska and Domanski, cited by Lutwak-Mann,
| |
| 1958). The alterations in reproductive organs associated with a lack of vitamin A
| |
| may be due in part to a vitamin E deficiency
| |
| since the latter enhances the rate at which
| |
| liver stores of vitamin A are depleted.
| |
| | |
| Definite effects of hypervitaminosis A
| |
| have been observed on reproduction. Masin
| |
| (1950) noted that estrus in female rats
| |
| could be prolonged by administration of
| |
| 37,000 I.U. of vitamin A daily. The implications, however, have not been studied.
| |
| The effect of hypervitaminosis A may actually induce secondary hypovitaminoses.
| |
| The displacement of vitamin K by excess
| |
| A is almost certain and similar relationships
| |
| appear to exist with vitamin D (Nieman
| |
| and Klein Obbink, 1954).
| |
| | |
| The failure of vitamin E-deficient female rats to become pregnant is apparently
| |
| due to disturbances of the implantation
| |
| process rather than to the failure of ovulation. There is no direct proof of ovarian
| |
| dysfunction (Blandau, Kaunitz and Slanetz,
| |
| 1949). However, the ovary of the rat deficient in vitamin E may have more connective tissue and pigment, and Kaunitz
| |
| (1955) showed by ovarian transplantation
| |
| that some nonspecific ovarian dysfunction
| |
| appears to exist (cited by Cheng, 1959 (.
| |
| Vitamin E is essential for birds, but there
| |
| is little evidence for a dependency in most
| |
| mammals; sheep, cows, goats, and pigs have
| |
| been studied. Treatment of low-fertility
| |
| farm animals with tocopherol has not provided conclusive data favoring its use (Lutwak-Mann, 1958), nor has the treatment
| |
| of human females been rewarded with any
| |
| indication that vitamin E might be helpful
| |
| in cases of abnormal cycles and habitual
| |
| abortion (Beckmann, 1955).
| |
| | |
| No specific reproductive disturbances in
| |
| man, the rhesus monkey, or the guinea pig
| |
| | |
| | |
| | |
| have been associated with vitamin C deficiency (Mason, 1939). Nevertheless, the
| |
| high ascorbic acid content of ovarian and
| |
| luteal tissue and of the adrenal cortex suggests a physiologic role in association with
| |
| steroid synthesis. (3varian ascorbic acid
| |
| varies with the estrous cycle, dropping
| |
| sharply in the proestrum (Coste, Delbarre
| |
| and Lacronique, 1953), and decreasing in
| |
| resjionse to gonadotrophin (Hokfelt, 1950;
| |
| Parlow, 1958). Virtually no ascorbic acid is
| |
| present in bovine follicular fluid (LutwakMann, 1954) or in rat ovarian cyst fluid
| |
| (Blye and Leathem, 1959). Uterine ascorbic
| |
| acid decreased in immature mice treated
| |
| with estrogen, but remained unchanged
| |
| in rats following thiouracil administration
| |
| (Leathem, 1959a). Its role in the uterus
| |
| awaits elucidation.
| |
| | |
| Delayed sexual maturation and ovarian
| |
| atrophy have been described when there
| |
| are deficiencies of thiamine, riboflavin, pyridoxine, pantothenic acid, biotin, and B12
| |
| (Ershoff, 1952; Ullrey, Becker, Terrill and
| |
| Notzold, 1955). However, as we noted when
| |
| deficiencies of the vitamins were being considered, much of the impairment of reproductive function can be related to inanition
| |
| rather than to a vitamin deficiency (Drill
| |
| and Burrill, 1944). Pyridoxine deficiency,
| |
| although not affecting structure (Morris,
| |
| Dunn and Wagner, 1953) , markedly reduces
| |
| the sensitivity of the ovary to administered
| |
| gonadotrophin (Wooten, Nelson, Simpson
| |
| and Evans, 1958) .
| |
| | |
| Bird, frog, and fish eggs contain considerable quantities of vitamins. In fact, the
| |
| daily human requirements for vitamins may
| |
| be contained in a hen's egg and thus it is
| |
| not surprising that hatchability is decreased
| |
| l)y virtually any vitamin deficiency. Lutwak-Mann (1958) has provided an excellent
| |
| survey of these data with numerous references to studies of frogs and fishes. Nearly
| |
| all the B vitamins are present in fish roe
| |
| and the pantothenic acid concentration in
| |
| cod ovaries {Gadus morrhua) exceeds most
| |
| otlicr natural sources. The amount of the
| |
| latter varies with the reproductive cycle,
| |
| d(>creasing to its lowest level before spawning. Riboflavin and vitamin B12 , on the
| |
| other h;ui(l, do not change (Braekkan,
| |
| 1955).
| |
| | |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| 687
| |
| | |
| | |
| | |
| B. INFLUENCE OF NUTRITION ON THE RESPONSIVENESS OF FEMALE REPRODUCTIVE TISSUES
| |
| TO HORMONES
| |
| | |
| 1. Ovary
| |
| | |
| a. Inanition. Marrian and Parkes (1929)
| |
| were the first to show that the quiescent
| |
| ovary of the underfed rat can respond to
| |
| injections of anterior pituitary as evidenced
| |
| by ovulation and estrous smears. Subsequently the ovaries of underfed birds, rats,
| |
| and guinea pigs were found to be responsive
| |
| to serum gonadotrophin (Werner, 1939;
| |
| Stephens and Allen, 1941; Mulinos and
| |
| Pomerantz, 1941b; Hosoda, Kaneko, Mogi
| |
| and Abe, 1956). A low calorie bread-andmilk diet for 30 days did not prevent ovarian response to rat anterior pituitary or to
| |
| chorionic gonadotrophin. In these animals
| |
| an increase in ovarian weight with repair
| |
| of interstitial tissue, as well as folhcle
| |
| stimulation and corpus luteum formation,
| |
| were observed (Rinaldini, 1949). Rats from
| |
| which food had been withdrawn for 12
| |
| days could respond to castrated rat pituitary extract with an increase in ovarian
| |
| and uterine weight (Maddock and Heller,
| |
| 1947). Nevertheless, differences in the time
| |
| and degree of responsiveness to administered gonadotrophin were noted in rabbits.
| |
| Animals on a high plane of nutrition responded to gonadotrophin at 12 weeks,
| |
| whereas rabbits on a low plane of nutrition
| |
| responded at 20 weeks and fewer eggs were
| |
| shed (Adams, 1953).
| |
| | |
| b. Protein. Protein or amino acid deficiencies in the rat do not prevent a response
| |
| to administered gonadotrophin (Cole, Guilbert and Goss, 1932; Courrier and Raynaud,
| |
| 1932) . However, the degree and type of
| |
| gonadal response is influenced by the diet.
| |
| Thus, immature female mice fed to 6 per
| |
| cent casein for 13 days exhibited only follicular growth in response to pregnant mare
| |
| serum, whereas the ovarian response in
| |
| mice fed 18 per cent casein was suggestive
| |
| of follicle-stimulating and strongly luteinizing actions (Table 12.9). Furthermore,
| |
| the ovarian response was significantly less
| |
| after 20 days of nonprotein feeding than
| |
| after 10 days of depletion (Leathem,
| |
| 1958a). Ovarian stimulation by a gonadotrophin involves tissue protein synthesis
| |
| and thus the type of whole protein fed
| |
| | |
| | |
| | |
| could influence the responses. Yamamoto
| |
| and Chow (1950) fed casein, lactalbumin,
| |
| soybean, and wheat gluten at 20 per cent
| |
| levels and noted that the response to gonadotrophin as estimated by tissue nitrogen
| |
| was related to the nutritive value of the
| |
| protein. The ovarian weight response to
| |
| chorionic gonadotrophin was less in rats
| |
| fed 20 per cent gelatin than those fed 20
| |
| per cent casein (Leathem, 1959b). Inasmuch
| |
| as the hypophysis may influence the gonadal response to injected hormone despite
| |
| the diet, hypophysectomized rats fed a protein-free diet for 5 weeks and hyophysectomized rats on a complete diet were tested
| |
| for response to gonadotrophins. The response to FSH was not influenced by diet,
| |
| but the protein-depleted rats were twice
| |
| as sensitive to interstitial cell-stimulating
| |
| hormone (ICSH), human chorionic gonadotrophin (HCG), and PMS as the normal
| |
| rats (Srebnik, Nelson and Simpson, 1958).
| |
| Protein-depleted, normal mice were twice as
| |
| sensitive to PMS as fully fed mice (Leathem and Defeo, 1952 1 .
| |
| | |
| c. Vitamins. In the female vitamin B
| |
| deficiencies do not prevent ovarian responses to gonadotrophin (Figge and Allen,
| |
| 1942), but the number of studies is limited.
| |
| Be deficiency in DBA mice was associated
| |
| with an increased sensitivity of the ovary
| |
| to gonadotrophins (Morris, Dunn and Wagner, 1953), whereas pyridoxine deficiency
| |
| in the rat decreased ovarian sensitivity,
| |
| especially to FSH (Wooten, Nelson, Simp
| |
| TABLE 12.9
| |
| | |
| Influence of dietary protein and pregnant mare
| |
| | |
| serum {PMS) on the mouse ovary
| |
| | |
| (From J. H. Leathem, Recent Progr. Hormone
| |
| | |
| Res., 14, 141, 1958.)
| |
| | |
| | |
| | |
| Diet fPer cent
| |
| Protein X Days Fed)
| |
| | |
| | |
| | |
| | |
| c-5,
| |
| | |
| •g-s
| |
| | |
| 1*
| |
| | |
| | |
| k
| |
| | |
| r
| |
| | |
| | |
| 1
| |
| 11
| |
| | |
| | |
| c
| |
| 1
| |
| | |
| | |
| | |
| | |
| I.U.
| |
| | |
| | |
| mg.
| |
| | |
| | |
| | |
| | |
| | |
| | |
| mg.
| |
| | |
| | |
| per cent X 23
| |
| | |
| | |
| | |
| | |
| | |
| 1.2
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| 4.8
| |
| | |
| | |
| per cent X 2.3
| |
| | |
| | |
| 3
| |
| | |
| | |
| 2.8
| |
| | |
| | |
| 13
| |
| | |
| | |
| | |
| | |
| | |
| 10.8
| |
| | |
| | |
| per cent X 13
| |
| | |
| | |
| | |
| | |
| | |
| 1.4
| |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| | |
| 4.9
| |
| | |
| | |
| per cent X 13
| |
| | |
| | |
| 3
| |
| | |
| | |
| 4.4
| |
| | |
| | |
| 16
| |
| | |
| | |
| 1
| |
| | |
| | |
| 15.1
| |
| | |
| | |
| 6 per cent X 13
| |
| | |
| | |
| | |
| | |
| | |
| 3.2
| |
| | |
| | |
| 6
| |
| | |
| | |
| | |
| | |
| | |
| 7.7
| |
| | |
| | |
| 6 per cent X 13
| |
| | |
| | |
| 3
| |
| | |
| | |
| 5.6
| |
| | |
| | |
| 12
| |
| | |
| | |
| 1
| |
| | |
| | |
| 31.9
| |
| | |
| | |
| 18 per cent X 13
| |
| | |
| | |
| | |
| | |
| | |
| 5.0
| |
| | |
| | |
| 10
| |
| | |
| | |
| 2
| |
| | |
| | |
| 51.6
| |
| | |
| | |
| 18 per cent X 13
| |
| | |
| | |
| 3
| |
| | |
| | |
| 8.0
| |
| | |
| | |
| 7
| |
| | |
| | |
| 4
| |
| | |
| | |
| 51.3
| |
| | |
| | |
| | |
| 088
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GONADS
| |
| | |
| | |
| | |
| son and Evans, 1958 j. Administration of
| |
| vitamin C concomitant witli gonadotropliin
| |
| has been claimed to enhance ovarian response (DiCio and Schteingart, 1942), but
| |
| in another study the addition of ascorbic
| |
| acid inhibited the hiteinizing and ovulating
| |
| action of the gonadotrophin (Desaive,
| |
| 1956).
| |
| | |
| Whether induced by vitamin deficiency
| |
| or by inanition, the anestrum in rats which
| |
| follows 2 to 3 weeks' feeding of a vitamin
| |
| B-deficient diet has been explored as a
| |
| method for the assay of gonadotrophin.
| |
| Pugsley (1957) has shown that there is
| |
| considerable convenience of method and a
| |
| satisfactory precision of response for the
| |
| assay of HCG and pregnant mare serum.
| |
| | |
| 2. Uterus and Vagina
| |
| | |
| a. Inanition. Limited food intake does
| |
| not prevent an increase in uterine weight
| |
| after estrogen. Testosterone propionate will
| |
| markedly increase uterine growth despite
| |
| a 50 per cent reduction in food intake
| |
| (Leathem, Nocenti and Granitsas, 1956).
| |
| Furthermore, dietary manipulations involving caloric and protein levels did not prevent the uteri of spayed rats from responding to estrogen (Vanderlinde and
| |
| Westerfield, 1950). More specific biochemical and physiologic responses must be
| |
| measured because starvation for 4-day periods clearly interferes with deciduoma formation (DeFeo and Rothchild, 1953). A
| |
| start in the direction of studying tissuecomposition changes has been made by
| |
| measuring glycogen. However, no changes
| |
| were noted in uterine glycogen in fasting
| |
| rats (Walaas, 1952), and estrogen promoted
| |
| glycogen deposition in the uteri of starved
| |
| rats as well as in the uteri of fully fed rats
| |
| (Bo and Atkinson, 1953).
| |
| | |
| b. Fat. Interest in the hormone content of
| |
| fat from the tissues of animals treated with
| |
| estrogen for the purpose of increasing body
| |
| weight has raised the question of tissue hormone content. If estrogen was to be detected in tissues, an increase in dietary fat
| |
| was necessary. However, the increase in
| |
| dietary fat decreased the uterine response to
| |
| stilbcstrol (I'mberger and Gass, 1958), thus
| |
| complicating the assay.
| |
| | |
| c. Vitainins. Stimulation of the uterus
| |
| by estrogen does not require tliianiinc, ribo
| |
| | |
| | |
| flavin, pyridoxine, or pantothenic acid. On
| |
| the other hand, a deficiency of nicotinic acid
| |
| appears to enhance the response to low
| |
| doses of estrogen (Kline and Dorfman,
| |
| 1951a, b). However, Bio appears to be
| |
| needed for optimal oviduct response (Kline,
| |
| 1955) and is required for methyl group synthesis from various one-carbon precursors
| |
| including serine and glycine (Johnson,
| |
| 1958).
| |
| | |
| Response of the bird oviduct to stilbestrol
| |
| requires folic acid (Hertz, 1945, 1948). It
| |
| was shown subsequently that stilbestrol and
| |
| estrone effects in frogs, rats, and the rhesus
| |
| monkey also require folic acid. A folic acid
| |
| deficiency can be induced by feeding aminopterin. In this way the estrogen effects
| |
| can be prevented. Aminopterin also prevents
| |
| the action of progesterone in deciduoma
| |
| formation, from which it may be inferred
| |
| that folic acid is necessary for deciduoma
| |
| formation in the rat. Increased steroid or
| |
| folic acid levels can reverse the antagonist's
| |
| effect (Velardo and Hisaw, 1953).
| |
| | |
| The mechanism of folic acid action is not
| |
| clear. It may function in fundamental metabolic reactions linked with nucleic acid
| |
| synthesis. Brown (1953) showed that
| |
| desoxyribonucleic acid could be substituted
| |
| for folic acid in the bird. In the rat aminopterin interferes with the increase in
| |
| uterine nucleic acids, and with nitrogen
| |
| and P-^- uptake by nucleic acids following
| |
| estrogen. Folic acid has been implicated in
| |
| the metabolism of several amino acids
| |
| (Davis, Meyer and McShan, 1956).
| |
| | |
| Rats ovariectomized at weaning and
| |
| maintained on a vitamin E-free diet for
| |
| 6 weeks to 10 months responded to estradiol
| |
| in the same manner as rats supplemented
| |
| with tocopherol. This finding suggests that
| |
| an intimate physiologic relationship between estradiol and vitamin E is not very
| |
| probable (Kaunitz, Slanetz and Atkinson,
| |
| 1949). Nevertheless, vitamin E has been
| |
| re]:»orted to act synergistically with ovarian
| |
| hormones in dc^ciduoma formation (Kehl,
| |
| Douard and Lanfranchi. 1951 ) and to influence nucleic acid turnover (Dinning.
| |
| Simc and Day, 1956).
| |
| | |
| A vitamin-hormone interrelationship is
| |
| apparent when estrogen and vitamin A are
| |
| considered. Vitamin A-deficient female rats
| |
| present evidence of a metaplastic uterine
| |
| | |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| 689
| |
| | |
| | |
| | |
| epithelium in 11 to 13 weeks, but similar
| |
| changes failed to develop in ovariectomized
| |
| rats. Vitamin A-deficient castrated rats
| |
| quickly developed symptoms of metaplasia
| |
| when estrogen alone was administered, but
| |
| no adverse effect followed the administration of estrogen combined with vitamin A
| |
| (Bo, 1955, 1956). The vagina is different.
| |
| Its epithelium becomes cornified in vitamin
| |
| A-deficient normal and castrated rats. The
| |
| cornification is histologically indistinguishable from that occurring in the estrous
| |
| rat and can be prevented by vitamin A.
| |
| In fact, vitamin A will quantitatively inhibit the effect of estrogen on the vaginal
| |
| mucosa when both are applied locally
| |
| (Kahn, 1954). Conversion of ^-carotene to
| |
| vitamin A is influenced by tocopherol, vitamin Bi2 , insulin, and thyroid, with evidence
| |
| for and against a similar action by cortisone
| |
| (Lowe and Morton, 1956; Rice and Bo,
| |
| 1958). An additional vitamin-hormone relationship is suggested by the augmentation
| |
| of progesterone action in rabbits given vitamin Do .
| |
| | |
| 3. Mammary Gland
| |
| | |
| Inanition prevents mammary growth, but
| |
| feeding above recommended requirements
| |
| for maintenance and growth from birth
| |
| to the first parturition also seems to interfere with mammary growth. Furthermore,
| |
| steroid stimulation of the mammary gland
| |
| is influenced by nutritional factors. Using
| |
| the male mouse, Trentin and Turner (1941)
| |
| showed that as food intake decreased, the
| |
| amount of estradiol required to produce a
| |
| minimal duct growth w^as proportionately
| |
| increased. In the immature male rat a
| |
| limited food intake prevented the growth of
| |
| the mammary gland exhibited by fully fed
| |
| controls. Nevertheless, the gland was competent to respond to estrogen (Reece, 1950) .
| |
| Inasmuch as the glands of force-fed hypophysectomized rats did not respond to estrogen (Samuels, Reinecke and Peterson,
| |
| 1941; Ahren, 1959), one can assume that,
| |
| despite inanition, a hypophyseal factor was
| |
| present to permit the response of the mammary gland to estrogen. However, inanition
| |
| (IMeites and Reed, 1949) , but not vitamin
| |
| deficiencies (Reece, Turner, Hathaway and
| |
| Davis, 1937), did reduce the content of
| |
| hypophyseal lactogen in the rat.
| |
| | |
| | |
| | |
| Growth of the mammary gland duct in
| |
| the male rat in response to estradiol requires a minimum of 6 per cent casein. Protein levels of 3 per cent and per cent failed
| |
| to support growth of the duct (Reece, 1959) .
| |
| | |
| C. PREGNANCY
| |
| | |
| The human male after attaining adulthood is confronted with the problem of
| |
| maintaining the body tissues built up during
| |
| the growth period. However, in the human
| |
| female it has been estimated that the replacement of menstrual losses may require
| |
| the synthesis of tissue equivalent to 100
| |
| per cent of her body weight (Flodin, 19531.
| |
| In the event of pregnancy and in all viviparous species, the female is presented with
| |
| even more formidable demands and a limitation of nutritional needs can lead to loss
| |
| of the embryo or fetus. The role of nutrition
| |
| at this point in reproduction has always received considerable attention and is complicated by the circumstance that many food
| |
| substances influence pregnancy (Jackson,
| |
| 1959). However, in many instances there is
| |
| no evidence that fetal loss or malformation
| |
| induced by nutritional modifications has
| |
| been the consequence of an endocrine imbalance and thus limitation of the immense
| |
| literature is permissible.
| |
| | |
| During the first 15 days of pregnancy, a
| |
| rat may gain 50 gm. Since the fetuses and
| |
| placentas are small, most of the gain is maternal and is associated with an' increase
| |
| in food intake of as much as 100 calories
| |
| per kilogram of body weight (Morrison,
| |
| 1956). During the first 2 weeks of pregnancy, marked storage of fat and water occurs in the maternal body and the animal's
| |
| positive nitrogen balance is above normal.
| |
| Liver fat also increases (Shipley, Chudzik,
| |
| Curtiss and Price, 1953). The increased food
| |
| intake in early pregnancy may therefore
| |
| provide a reserve for late fetal growth, as
| |
| food intake may decline to 65 per cent of
| |
| the general pregnancy level during the last
| |
| 7 days (Morrison, 1956). During this last
| |
| week, fetal growth is rapid. The rapid
| |
| growth has been related to (1) greater demands of the fetus, (2) greater amounts of
| |
| food in the maternal blood, and (31 greater
| |
| permeability of the placenta. Certainly the
| |
| anabolic potential of fetal tissues is high
| |
| and the mother can lose weight while the
| |
| | |
| | |
| | |
| 690
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GONADS
| |
| | |
| | |
| | |
| fetuses gain. But it is also important to
| |
| recall that there is a shift in protein, because
| |
| its distribution in organs of pregnant rats
| |
| differs from that in nonpregnant animals
| |
| (Poo, Lew and Addis, 1939). Other changes
| |
| in the maternal organism were enumerated
| |
| b}^ Newton (1952) and by Souders and
| |
| Morgan (1957).
| |
| | |
| A measure of nitrogen balance during
| |
| pregnancy, rather than weight of young at
| |
| birth, has been suggested as a means of
| |
| determining a diet adequate for reproduction (Pike, Suder and Ross, 1954). After
| |
| the 15th day, a retention of body protein
| |
| increases, blood amino nitrogen and amino
| |
| acids decrease, and urea formation decreases. These metabolic activities suggest
| |
| an increase in growth hormone although the
| |
| levels of this hormone have not been estimated (Beaton, Ryu and McHenry, 1955).
| |
| Placental secretions have also been associated with the active anabolic state of the
| |
| second half of pregnancy, because removal
| |
| of the fetuses in the rat did not change the
| |
| anabolic activity, whereas removal of the
| |
| placentas was followed by a return to normal (Bourdel, 1957). A sharp increase in
| |
| liver ribose nucleic acid has been observed
| |
| during late pregnancy in mice and rats and
| |
| the effect attributed to a placental secretion
| |
| or to estrogen. Species differences also influence the results because only a modest
| |
| change in liver RNA was observed in guinea
| |
| pigs and no change occurred in cats (Campbell and Kostcrlitz, 1953; Campbell, Innes
| |
| and Kosterlitz, 1953a, b).
| |
| | |
| Clinical observations have related both
| |
| | |
| TABLE 12.10
| |
| | |
| Nutrition and pregnancy in rats
| |
| | |
| (From J. H. Leathern, in Recent Progress in
| |
| | |
| the Endocrinology of Reproduction, Academic
| |
| | |
| Press, Inc., New York, 1959.)
| |
| | |
| | |
| | |
| | |
| | |
| Calories/kg.
| |
| Body Weight
| |
| | |
| | |
| Fetuses, Day 20
| |
| | |
| | |
| | |
| | |
| No.
| |
| | |
| | |
| Average
| |
| weight
| |
| | |
| | |
| 18 per cent casein
| |
| | |
| 18 per cent casein
| |
| | |
| 6 per cent casein
| |
| | |
| per cent casein
| |
| | |
| 18 per cent gelatin
| |
| | |
| 18 per cent gelatin
| |
| | |
| | |
| 200
| |
| 100
| |
| | |
| 250
| |
| 200
| |
| 200
| |
| 100
| |
| | |
| | |
| 8
| |
| 6
| |
| | |
| | |
| | |
| | |
| | |
| | |
| gm.
| |
| 6.1
| |
| | |
| 3.5
| |
| | |
| | |
| | |
| toxemia of pregnancy (Pequignot, 1956)
| |
| and prematurity to inadequate nutrition
| |
| (Jeans, Smith and Stearns, 1955). The potential role of protein deprivation in the
| |
| pathogenesis of the toxemia of pregnancy
| |
| prompted studies in sheep and rats. In sheep
| |
| nutritionally induced toxemia simulates the
| |
| spontaneous toxemia (Parry and Taylor,
| |
| 1956), but only certain aspects of toxemia
| |
| were observed in the pregnant rat subjected
| |
| to low protein diets. When rats were fed 5
| |
| per cent casein and mated, fluid retention
| |
| was observed (Shipley, Chudzik, Curtiss
| |
| and Price, 1953) and pregnancy was completed in only 48 per cent of the animals
| |
| Curtiss, 1953). Gain in body weight in the
| |
| adult rat and gain in fetal weight were subnormal as the result of a low protein feeding
| |
| during pregnancy.
| |
| | |
| Complete removal of protein from the
| |
| diet beginning at the time of mating did not
| |
| prevent implantation but did induce an 86
| |
| to 100 per cent embryonic loss. The effect
| |
| was not related solely to food intake (Nelson and Evans, 1953) , as we will see in what
| |
| follows when the relationship between protein deficiency and the supply of estrogen
| |
| and progesterone is described. Limiting protein deprivation to the first 9 to 10 days of
| |
| pregnancy will also terminate a pregnancy,
| |
| but when the protein was removed from the
| |
| diet during only the last week of pregnancy,
| |
| the maternal weight decreased without an
| |
| effect on fetal or placental weight (Campbell and Kosterlitz, 1953). As would be anticipated, a successful pregnancy requires
| |
| protein of good nutritional quality and the
| |
| caloric intake must be adequate. Thus, an
| |
| 18 per cent gelatin diet failed to maintain
| |
| pregnancy when 200 calories per kilogram
| |
| were fed, whereas a similar level of casein
| |
| was adequate (Table 12.10). However, reducing caloric intake to 100 calories despite
| |
| an otherwise adequate protein ration influenced the number and size of fetuses
| |
| (Leathern, 1959b). Additional proteins
| |
| should be studied and related to biochemical changes in pregnancy and to the need for
| |
| specific amino acids; for example, elimination of methionine or tryptophan from the
| |
| diet may or may not be followed by resorption (Sims, 1951; Kemeny, Handel, Kertesz
| |
| and Sos, 1953; Albanese, Randall and Holt,
| |
| 1943). Excretion of 10 amino acids was in
| |
| | |
| | |
| NUTRITIONAL EFFECTS
| |
| | |
| | |
| | |
| 691
| |
| | |
| | |
| | |
| creased during normal human pregnancy
| |
| (Miller, Ruttinger and Macey, 1954).
| |
| | |
| That a relationship exists, between the
| |
| dietary requirements just described to the
| |
| endocrine substances which participate in
| |
| the control of pregnancy, is suggested by the
| |
| fact that the deleterious effects of a proteinfree diet on pregnancy in rats have been
| |
| counteracted by the administration of estrone and progesterone. Pregnancy was
| |
| maintained in 30 per cent, 60 to 80 per cent,
| |
| and per cent of protein-deficient animals
| |
| by daily dosages of 0.5 /xg., 1 to 3 fig., and 6
| |
| jug. estrone, respectively. On the other hand,
| |
| injection of 4 to 8 mg. progesterone alone
| |
| maintained pregnancy in 70 per cent of the
| |
| animals (Nelson and Evans, 1955) , and an
| |
| injection of 4 mg. progesterone with 0.5 //.g.
| |
| estrone provided complete replacement
| |
| therapy (Nelson and Evans, 1954). Food
| |
| intake did not increase. The results suggest
| |
| that reproductive failure in the absence of
| |
| dietary protein was due initially to lack of
| |
| progesterone and secondarily to estrogen,
| |
| the estrogen possibly serving as an indirect
| |
| stimulation for luteotrophin secretion and
| |
| release. It is well known that hypophysectomy or ovariectomy shortly after breeding
| |
| will terminate a pregnancy and that replacement therapy requires both ovarian
| |
| hormones. Thus, the protein-deficient state
| |
| differs somewhat from the state following
| |
| hypophysectomy or ovariectomy, but the
| |
| factors involved are not known.
| |
| | |
| Pregnancy alters nutritional and metabolic conditions in such a way that labile
| |
| protein stores of the liver and other parts of
| |
| the body are influenced, but similar effects
| |
| are imposed by a transplanted tumor, especially when it reaches 10 per cent of the
| |
| body weight. ' Thus, transplantation of a
| |
| tumor into a pregnant animal would place
| |
| the fetuses in competition with the tumor for
| |
| the amino acids of the metabolic pool. Under
| |
| these circumstances will the pregnancy be
| |
| maintained? An answer to the question may
| |
| not yet be given. Nevertheless, Bly, Drevets
| |
| and Migliarese (1955) observed various degrees of fetal damage in pregnant rats bearing the Walker 256 tumor, and 43 per cent
| |
| fetal loss was obtained with a small hepatoma (Paschkis and Cantarow, 1958).
| |
| | |
| Essential fatty acid deficiency, at least
| |
| in the initial stages, does not interfere with
| |
| | |
| | |
| | |
| development of the fetuses or parturition in
| |
| the rat, but the pups may be born dead or
| |
| they do not survive more than a few days
| |
| (Kummerow, Pan and Hickman, 1952). A
| |
| more pronounced deficiency has induced
| |
| atrophic changes in the decidua, resorption
| |
| of fetuses, and prolonged gestation. Death
| |
| of the fetuses appears to be secondary to
| |
| placental injury. Hormonal involvement, if
| |
| any, when there is fatty acid deficiency and
| |
| pregnancy seems not to have been investigated.
| |
| | |
| Pregnancy and lactation are major factors influencing vitamin requirements. It is
| |
| not surprising, therefore, that vitamin deficiencies influence the course of a pregnancy. The subject has recently been reviewed by Lutwak-jMann (1958).
| |
| | |
| A deficiency of vitamin A does not noticeably affect early fetal development, but
| |
| later in gestation placental degeneration occurs with hemorrhage and abortion. When
| |
| the deficiency is moderate the pregnancy is
| |
| not interrupted, but the fetuses are damaged
| |
| (Warkany and Schraffenberger, 1944; Wilson, Roth and Warkany, 1953; Giroud and
| |
| Martinet, 1959). In calves and pigs the
| |
| abnormalities are associated with the eyes
| |
| and palate (Guilbert, 1942) ; in birds skeletal abnormalities are seen (Asmundson and
| |
| Kratzer, 1952). The use of hormones in an
| |
| effort to counteract the effects seems to have
| |
| been attempted only in the rabbit where
| |
| 12.5 mg. progesterone improved reproduction impaired by vitamin A lack (Hays and
| |
| Kendall, 1956). Vitamin A excess also
| |
| proves highly detrimental to pregnancy, as
| |
| resorption and malformations occur. Administration of excessive vitamin A on days
| |
| 11 to 13 of pregnancy induced cleft palate in
| |
| 90 per cent of the embryos (Giroud and
| |
| Martinet, 1955) . In another experiment the
| |
| effect of excessive vitamin A was augmented
| |
| by cortisone (Woollam and Millen, 1957).
| |
| | |
| Vitamin E deficiency has long been known
| |
| to influence pregnancy in rodents and fetal
| |
| death appears to precede placental damage
| |
| and involution of the corpora lutea. Gross
| |
| observations of the abnormal embryos have
| |
| been reported (Cheng, Chang and Bairnson, 1957). Estrogen, progesterone, and lactogen were not effective in attempts at corrective therapy (Ershoff, 1943), but estrone
| |
| and progesterone markedly reduced the in
| |
| | |
| | |
| 692
| |
| | |
| | |
| | |
| PHYSIOLOGY OF GONADS
| |
| | |
| | |
| | |
| cidence of congenital malformations associated with vitamin E lack (Cheng, 1959).
| |
| In the test of a possible converse relationship, estradiol-induced abortion in guinea
| |
| pigs was not prevented by vitamin E (Ingelman-Sundberg, 1958) .
| |
| | |
| Fat-soluble vitamins incorporated in the
| |
| diet may be destroyed by oxidation of the
| |
| unsaturated fatty acids. To stabilize the
| |
| vitamins, the addition of diphenyl-p-phenylenediamine (DPPD) to the diet has
| |
| proven successful, but recent studies show
| |
| that DPPD has an adverse effect on reproduction and thus its use in rat rations
| |
| is contraindicated (Draper, Goodyear, Barbee and Johnson, 1956).
| |
| | |
| Vitamin-hormone relationships in pregnancy have been studied with regard to
| |
| thiamine, pyridoxine, pantothenic acid, and
| |
| folic acid. Thiamine deficiency induced stillbirths, subnormal birth weights, resorption
| |
| of fetuses, and loss of weight in the mother.
| |
| However, as in the case of protein deficiency, pregnancy could be maintained with
| |
| 0.5 fjLg. estrone and 4 mg. progesterone (Nelson and Evans, 1955). Estrone alone had
| |
| some favorable effect on the maintenance
| |
| of pregnancy in thiamine-deficient animals,
| |
| but it was less effective in protein-deficient
| |
| animals.
| |
| | |
| Fetal death and resorptions as well as
| |
| serum protein and nonprotein nitrogen
| |
| (NPN) changes similar to those reported
| |
| for toxemia of pregnancy (Ross and Pike,
| |
| 1956; Pike and Kirksey, 1959) were induced
| |
| by a diet deficient in vitamin Be . Administration of 1 fxg. estrone and 4 mg. progesterone maintained pregnancy in 90 per cent of
| |
| vitamin Be-deficient rats (Nelson, Lyons
| |
| and Evans, 1951). However, the pyridoxinedeficient rat required both steroids to remain pregnant and in this regard resembled
| |
| the hypophysectomized animal (Nelson,
| |
| Lyoas and Evans, 1953). Nevertheless, a
| |
| hypophyseal hormone combination which
| |
| was adequate for the maintenance of pregnancy in the fully fed hypophysectomized
| |
| rat (Lyons, 1951) was only partially successful when there was a deficiency of pyridoxine. An ovarian defect is suggested.
| |
| | |
| The folic acid antagonist, 4-aminopteroylglutamic acid, will rapidly induce the
| |
| death of early implanted embryos in mice.
| |
| | |
| | |
| | |
| rats, and man (Thiersch, 1954j . Removal
| |
| of folic acid from the diet or the addition
| |
| of x-methyl folic acid will induce malformations when low doses are given and resorptions when high doses are given. Furthermore, this effect is obtained even when
| |
| the folic acid deficiency is delayed until
| |
| day 9 of a rat pregnancy or maintained for
| |
| only a 36-hour period. A deficiency of
| |
| pantothenic acid will also induce fetal resorption. The vitamin is required for hatching eggs (Gillis, Heuser and Norris, 1942).
| |
| In animals deficient in folic acid or in
| |
| pantothenic acid, estrone and progesterone
| |
| replacement therapy did not prevent fetal
| |
| loss, suggesting that the hormones cannot
| |
| act (Nelson and Evans, 1956). In the above
| |
| mentioned deficiencies replacement of the
| |
| vitamin is effective. However, vitamins
| |
| other than those specifically deleted may
| |
| provide replacement, thus ascorbic acid
| |
| seems to have a sparing action on calcium
| |
| pantothenate (Everson, Northrop, Chung
| |
| and Getty, 1954) .
| |
| | |
| Pregnancy can be interrupted by altering
| |
| vitamins other than those discussed above,
| |
| but the hormonal aspects have not been
| |
| explored. Thus, the lack of choline, riboflavin, and Bi2 will induce fetal abnormalities and interrupt gestation (Giroud, Levy,
| |
| Lefebvres and Dupuis, 1952; Dryden, Hartman and Gary, 1952; Jones, Brown, Richardson and Sinclair, 1955; Newberne and
| |
| O'Dell, 1958) . Choline lack is detrimental to
| |
| the placenta (Dubnov, 1958), riboflavin
| |
| deficiency may impair carbohydrate use
| |
| (Nelson, Arnrich and Morgan, 1957) and/or
| |
| induce electrolyte disturbances (Diamant
| |
| and Guggenheim, 1957) , and Bjo spares choline and may be concerned with nucleic
| |
| acid synthesis (Johnson, 1958). Excessive
| |
| amounts of Bio are not harmful. It is interesting to note that uterine secretions and
| |
| rabbit blastocyst fluid are rich in vitamin
| |
| B]2 (Lutwak-Mann, 1956), but its presence
| |
| in such large amounts has not been explained.
| |
| | |
| An additional substance, lithospermin, extracted from the plant, Lathijrus odoratus,
| |
| is related to hormone functioning; it is antigonadotrophic when eaten by nonpregnant
| |
| animals and man. The feeding of this substance to prciiiiant rats terminated the pregnancies about the 17th day. Treatment with
| |
| estrogen and progesterone was preventive
| |
| (Walker and Wirtschafter, 1956). It is assumed, therefore, that lithospermin interfered with the production of these hormones.
| |
| A repetition of the experiment on a species
| |
| in which the hypophysis and ovaries are
| |
| dispensable during much of pregnancy
| |
| would be of interest.
| |
| | |
| In retrospect it has been found that a
| |
| deficiency in protein and the vitamins
| |
| thiamine, pyridoxine, pantothenic acid, and
| |
| folic acid individually can interrupt a pregnancy. Furthermore, a combination of estrone and progesterone which is adequate to
| |
| maintain pregnancy after hypophysectomy
| |
| and ovariectomy, is equally effective in protein or thiamine deficiency. This suggests
| |
| that the basic physiologic alteration is a
| |
| deprivation of ovarian hormones. However,
| |
| protein- and thiamine-deficiency states differ from each other as shown by the response to estrogen alone (thiamine deficiency is less responsive), and these states
| |
| differ from hypophysectomy in which estrone alone has no effect. A pyridoxine deficiency seems to involve both ovary and
| |
| hypophysis, for neither steroids nor pituitary hormones were more than partially
| |
| successful in maintaining pregnancy in rats.
| |
| Lastly, pantothenic acid and folic acid
| |
| deficiencies may not create a steroid deficiency. What is involved is not known;
| |
| many possibilities exist. Pantothenic acid,
| |
| for example, participates in many chemical
| |
| reactions. Furthermore, it is known that
| |
| thiamine is essential for carbohydrate metabolism but not for fat metabolism whereas
| |
| pyridoxine is involved in fat metabolism
| |
| and in the conversion of tryptophan to nicotinic acid. It is clear, though, that much
| |
| ground must be covered before the formulation of fruitful hypotheses may be anticipated.
| |
| | |
| VI. Concluding Remarks
| |
| | |
| The development, composition, and normal functioning of the reproductive system
| |
| is dependent on adequate nutrition. However, the requirements are many and only
| |
| gradually are data being acquired which are
| |
| pertinent to the elucidation of the nutritional-gonadal relationship.
| |
| | |
| | |
| | |
| The demands for nutrient substances is
| |
| not always the same. During pregnancy and
| |
| lactation there is a need for supplemental
| |
| feeding. A similar need exists in birds and
| |
| in the many cold-blooded vertebrates in
| |
| which reproduction is seasonal. Atypical endocrine states create imbalances and a need
| |
| for nutrient materials which vary, unpredictably, we must acknowledge, until the
| |
| numerous interrelationships have been clarified.
| |
| | |
| At many points where determination of
| |
| cause and effect are possible, an indirect
| |
| action of dietary factors on reproduction is
| |
| indicated. No other conclusion seems possible in view of the many instances in which
| |
| the effect of dietary deficiencies can be
| |
| counteracted by the administration of a
| |
| hormone or combination of hormones. The
| |
| direct action is not immediately apparent;
| |
| it probably is on the processes by which
| |
| metabolic homeostasis is maintained, and is
| |
| in the nature of a lowering of the responsiveness to the stimuli which normally trigger these processes into action. The processes
| |
| may be those by which pituitary and gonadal hormones are produced or they may be
| |
| the mechanisms by which these hormones
| |
| produce their effects on the genital tracts
| |
| and on the numerous other tissues on which
| |
| they are known to act.
| |
| | |
| Because of the many interrelationships,
| |
| some of which are antagonistic and some
| |
| supportive, determination of the role of specific dietary substances is not easy. For
| |
| those who work with laboratory species,
| |
| the problem is further complicated by the
| |
| many strain differences. For everyone, the
| |
| problem is complicated by the many species
| |
| differences which are the result of an evolution toward carnivorous, herbivorous, or
| |
| omnivorous diets, to say nothing of the
| |
| countless specific preferences within each
| |
| group.
| |
| | |
| Finally, it is something of a paradox in our
| |
| culture that much of our effort has been devoted to investigations of the effects of deficiencies and undernutrition rather than
| |
| to the effects of excesses and overnutrition.
| |
| Much evidence supports the view that in
| |
| the aggregate the latter are fully as deleterious as the former, but the means by which this result is achieved are largely unknown.
| |
| | |
| | |
| | |
| VII. References
| |
| | |
| Aak.s-Jorgensen, E., Funch, J. P., and Dam, H.
| |
| | |
| 1956. Role of fat in diet of rats; influence
| |
| on reproduction of hydrogenated arachis oil
| |
| as sole dietary fat. Brit. J. Nutrition, 10, 317.
| |
| | |
| Aaes-Jorgensen, E., Funch, J. P., and Dam, H.
| |
| | |
| 1957. Role of fat in diet of rats; influence of
| |
| small amount of ethyl linoleate on degeneration of spermatogenic tissue caused by hydrogenated arachis oil as sole dietary fat. Brit. J.
| |
| Nutrition, 11, 298.
| |
| | |
| AcETO, G., Li Moli, S., and Panebianco, N. 1956.
| |
| Influence of adrenocorticotrophin (ACTH) on
| |
| the urinary excretion of thiamine. Acta vitaminol., 10, 175.
| |
| | |
| Adams, C. E. 1953. Mammalian germ cells. Ciba
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| Foundation Symposium, 198.
| |
| | |
| Adams, C. W. M., Fernand, V. S. V., and Schnieden,
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| H. 1958. Histochemistry of a condition resembling kwashiorkor produced in rodents by
| |
| a low protein-high carbohydrate diet (cassaval). Brit. J. Exper. Path., 39, 393.
| |
| | |
| Addis, T., Poo, L. J., and Lew, W. 1936. The
| |
| ciuantities of protein lost by the various organs
| |
| and tissues of the body during a fast. J. Biol.
| |
| Chem., 115, 111.
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| | |
| Ahren, K. 1959. The effect of dietary restriction
| |
| on the mammary gland development produced
| |
| by ovarian hormones in the rat. Acta endocrinol., 31, 137.
| |
| | |
| Albanese, a. a., Randall, R. M., .\nd Holt, L. E.,
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| Jr. 1943. The effect of tryptophan deficiency
| |
| on reproduction. Science, 97, 312.
| |
| | |
| Angervall, L. 1959. Alloxan diabetes and pregnancy in the rat. Effects on offspring. Acta
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| Endocrinol., suppl. 44, 31, 86.
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| Arvy, L., Aschkenasy, A., Aschkenasy-Lelu, P.,
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| AND Gabe, M. 1946. Retentissement d'un regime prolonge d'inanition proteique sur les
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| Aschkenasy, A. 1954. Action de la testosterone, de la thyroxine ct do la cortisone sur
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| Aschkenasy, A. 1955a. Influence des proteines
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| Aschkenasy, A. 1955b. Influence des proteines
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| Aschkenasy, A. 1955c. Effects de la .surrenalectomie sur les poids relatifs de divers organes
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| Aschkenasy, A. 1957. Effets compares de
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| sur les poids de divers organes et tissus chez
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| le rat male adulte. Ann. endocrinol., 18, 981.
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| Aschkenasy, A., Aschkenasy-Lelu, P. 1957.
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| Nouvelles recerches sur le role des surrenales
| |
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| Aschkenasy, A., and Dray, F. 1953. Action de
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| Aschkena.sy-Lelu, p., and Aschkenasy, A. 1947.
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| |
| le cycle oestral de la rate adulte. Perturbation
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| |
| rend. Soc. biol., 141, 687.
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| | |
| Aschkenasy-Lelu, P., and Aschkenasy^ A. 1957.
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| Intervention des surrenales dans la gestation
| |
| en fonction de la teneur de regime en proteines.
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| Aschkenasy-Lelu, P., and Tuchmann-Duplessis,
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| AsDELL, S. A. 1949. Nutrition and treatment of
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| AsMUND.soN, V. S., AND Kr.\tzer, F. H. 1952. Observations on vitamin A deficiency in turkey
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| Baker, B. L. 1952. Comparison of histological
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| Ball, Z. B., B.\rnes, R. H., and Visscher, M. B.
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| Barborlak, J. J., Krehl, W. A., Cowgill, G. R..
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| BE.ATON, G. H., Ryu. M. H., and McHenry. E. W.
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| Beckmann, R. 1955. Vitamin E (Physiologic,
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| Berg, C. P., and Rohse, W. G. 1947. Is sterility
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| Bishop, D. W., and Ko.s.\rick, E. 1951. Blot in
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| Biskind, M. S. 1946. Nutritional therapy of endocrine disturbances. Vitamins & Hormones,
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| Blandau, R. J., Kauntiz, H., and Slanetz, C. A.
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| Blaxter, K. L., and Brown, F. 1952. Vitamin E
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| Blivaiss, B. B., Hanson, R. O., Rosenzweig, R. E.,
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| Bly, C. G., Drevets, C., and Migliarese, J. F.
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| transplanted tumor growth in pregnant rats.
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| Blye, R. p., and Leathem, J. H. 1959. Ovarian
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| Bo, W. J. 1955. The effect of ovariectomy on
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| Bo, W. J. 1956. The relationship between vitamin A deficiency and estrogen in producing
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| Booth, V. H. 1952. Liver storage of vitamin A
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| Bourdel, G. 1956. L'anabolisme gravidique: ses
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| Br.\ekkan, O. R. 1955. Role of pantothenic acid
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
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| |
| | |
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| |
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| |
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| |
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| |
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| |
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| | |
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| |
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| | |
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| |
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| |
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| |
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| | |
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| |
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| |
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| |
| | |
| Drill, V. A., and Pfeiffer, C. A. 1946. Effect of
| |
| vitamin B complex deficiency, controlled inanition and methionine on inactivation of
| |
| estrogen by the liver. Endocrinology, 38, 300.
| |
| | |
| Drummond, J. C, Noble, R. L., .\nd Wright, M. D.
| |
| 1939. Studies of relationship of vitamin E
| |
| (tocopherols) to the endocrine system. J.
| |
| Endocrinol., 1, 275.
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Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones
Claude A. Villee, Ph.D.
Associate Professor Of Biological Chemistry, Harvard University
I. Introduction 643
II. The Biosynthesis op Steroids 643
A. Cholesterol 644
B. Progesterone 644
C. Androgens 645
D. Estrogens 647
E. Biosynthesis of Other Steroids 647
F. Interconversions of Steroids 647
G. Catabolism of Steroids (548
H. Transport, Conjugation, and Excretion 650
III. Effects of Sex Hormones on Inter
mediary Metabolism 650
A. Estrogens 652
B. Androgens 659
C. Progesterone 660
IV. References 661
I. Intro<luction
The chemical structure of the sex hormones, their isohition from biologic materials, and many of their chemical properties were fully described in the previous
edition of Sex and Internal Secretions (W.
M. Allen, 1939; Doisy, 1939; Koch, 1939).
The major steroid sex hormones were isolated and identified 20 to 30 years ago.
Estrone, in fact, was crystallized from pregnancy urine by Doisy, Veler and Thayer
(1929) before the true structure of the steroid nucleus was known. The isolation, identification, and chemical synthesis of estradiol, progesterone, and testosterone were
accomplished during the 1930's. Additional
substances with androgenic, estrogenic, or
progestational activity have subsequently
been isolated from urine or from tissues but
these are probably metabolites of the major
sex steroids. The steroids are now routinely
synthesized from cholesterol or from plant
sterols. It would be possible to carry out
the total synthesis of steroids from simple
precursors but this is not commercially
practicable.
The two decades since the previous edition have been marked by major advances
in our understanding of the intermediary
metabolism of steroids — the synthesis of
cholesterol from two-carbon units, the conversion of cholesterol to pregnenolone and
progesterone, and the derivation of corticoids, androgens, and estrogens from progesterone. These advances were made possible
by the development of vastly improved
methods for the isolation and identification
of steroids: chromatography on paper or
columns, counter-current distribution, labeling with radioactive or heavy isotopes,
infrared spectroscopy, and so on. There have
been concomitant increases in the information regarding the sites and mechanisms of
action of these biologically important substances and the means by which they stimulate or inhibit the growth and activity of
particular tissues of the body. The following
discussion will attempt to present a general
picture of these two fields and not an exhaustive citation of the tremendous body of
relevant literature.
II. The Biosynthesis of Steroids
When the steroid hormones were first discovered it was generally believed that each endocrine gland made its characteristic
steroid by some unique biosynthetic mechanism, one that was independent of those in
other glands. However, there is now abundant evidence that the biosynthetic paths
in the several steroid-secreting glands have
many features which are similar or identical.
It is now well established that progesterone is not simply a female sex hormone
produced by the corpus luteum, but a common precursor of adrenal glucocorticoids
such as Cortisol and adrenal mineralocorticoids such as aldosterone, androgens, and
estrogens. The adrenal cortex, ovary, testis,
and placenta have in common many enzymes for the biosynthesis of steroids. Androgenic tumors of the human ovary, for example, have been shown to produce
testosterone and its metabolites. The transplantation of an ovary into a castrate male
mouse will result in the maintenance of the
male secondary sex characters, which suggests that the normal ovary can also synthesize androgens.
A. CHOLESTEROL
The early work of Bloch (1951), Rilling,
Tchen and Bloch (1958), and of Popjak
(1950) showed that labeled acetate is converted to labeled cholesterol. The pattern of
the labeling present in the cholesterol synthesized from acetate-1-C^^ or acetate-2-C^
as precursor led to speculations as to how
the steroid nucleus is assembled. Further
work (Langdon and Bloch, 1953) revealed
that squalene and certain branched-chain,
unsaturated fatty acids are intermediates
in this synthesis. The current hypothesis,
which is supported by a wealth of experimental evidence, states that two moles of
acetyl coenzyme A condense to form acetoacetyl coenzyme A, which condenses with a
third molecule of acetyl coenzyme A to form
yS-hydroxy-^-methyl glutaric acyl coenzyme
A (Fig. 11.1). The coenzyme A group is
removed and the hydroxymethyl glutaric
acid is reduced to mevalonic acid. Mevalonic
acid, 3-hydroxy-3-methylpentano-5-lactone,
is metabolized to a 5-carbon isoprenoid
compound and three moles of these condense
to form a 1 5-carbon hydrocarbon. The headto-head condensation of two molecules of
this 15-carbon compound yields the 30
carbon equalene. This is metabolized, by
way of lanosterol and the loss of three
methyl groups, to cholesterol, which seems
to be the common precursor of all of the
steroid hormones (Tchen and Bloch, 1955;
Clayton and Bloch, 1956).
The question of whether cholesterol is
an obligate intermediate in the synthesis of
steroid hormones has not been definitely
answered. There is clear evidence that cholesterol is converted to steroids without first
being degraded to small units and subsequently reassembled. Werbin and LeRoy
(1954) administered cholesterol labeled
with both carbon-14 and tritium (H^) to
human subjects and isolated from their
urine tetrahydrocortisone, tetrahydrocortisol, androsterone, etiocholanolone and 11ketoetiocholanolone. These substances,
known to be metabolites of steroid hormones, were labeled with both C^^ and H^
and the labeled atoms were present in the
ratio expected if they were derived directly
from cholesterol. Experiments by Dorfman
and his colleagues (Caspi, Rosenfeld and
Dorfman, 1956) also provide evidence for
the synthesis of steroids via cholesterol.
Cortisol and 11-desoxycortisol were isolated
from calf adrenals perfused with acetate-1C^^ and from a patient with an adrenal tumor to whom acetate- 1-C^^ had been administered. It is known that cholesterol
synthesized from acetate-l-C^'* is labeled in
carbon 20 but not in carbon 21. The Cortisol
and 11-desoxycortisol also proved to be labeled in carbon 20 but not in carbon 21.
This evidence does not, of course, exclude
biosynthetic paths for the steroids other
than one by way of cholesterol, but it does
suggest that cholesterol is at least an important precursor of them. Direct evidence
that cholesterol is synthesized from squalene
in man is provided by the experiments of
Eidinoff, Knoll, Marano, Kvamme, Rosenfeld and Hcllman (1958), who prepared tritiated squalene and administered it orally
to human subjects. They found that the
cholesterol of the blood achieved maximal
specific aeti\'ity in 7 to 21 liours.
B. PROCiE.STERONE
Cholesterol undergoes an oxidative cleavage of its side chain to yield isocaproic
acid and pregnenolone (Fig. 11.2). The lat
CH3C0-SC0A
STEROID SEX HORMONES
SCoA
645
CH^CO-SCoA
Acetyl CoA
CH3COCH2CO~SCoA
Acetoacetyl CoA
Sesquiterpene
(15C)
COOH
► HO— C— CHI ^
CO-' SCO A
pOH-p methylglutaric acyl Co A
CHg
C-CHI
CH
II
CHo
Isoprene unit
(5C)
CHO
I
CHo
I ^
HO-C-CH^
1 3
CHo
I 2
COOH
Mevalonic acid
Squalene Lanosterol Cholesterol
(30C) (30C) (27C)
Fig. 11.1. Biogenesis of cholesterol.
ter is dehydrogenated in ring A by the
enzyme 3-^-ol dehydrogenase and a spontaneous shift of the double bond from the
A5 , 6 to the A4 , 5 position results in progesterone. Progesterone undergoes successive
hydroxylation reactions, which require molecular oxygen and reduced triphosphopyridine nucleotide (TPNH), at carbons 17,
21, and 11. These hydroxylations yield,
in succession, 17-a-hydroxy progesterone,
Reichstein's compound S (ll-desoxy-17-hydroxycorticosterone), and Cortisol (17-a-hydroxvcorticosterone) .
C. ANDROGENS
17-a-Hydroxy progesterone is also the immediate precursor of androgens and estrogens. Oxidative cleavage of its side chain
yields A-4-androstenedione, which undergoes reduction to testosterone (Fig. 11.2).
A-4-Androstenedione may be hydroxylated
at carbon 11 to yield ll-/3-hydroxy-A-4androstenedione, which is an androgen isolated from human urine. It has also been
found as a metabolite of certain androgenic
tumors of the adrenal cortex.
04 G
PHYSIOLOGY OF GONADS
CH
Y^ .
socaproic C
=0
c=o
r^
0^-OH
^r^
HO
HO
J
HO
Cholesterol
Pregnenolone
17-Hydr
oxy
Dehydroepi
preg
nenolone
androsterone
CH3
CH3
f3
H-C-OH
1
, f
c=o
1
1
c=o
1
^ ■
-x^V
t^
^XP""°"
r^^
^^<Y
„ijj
- \^Xaj
JJ
A -3- Ketopregnene
Progest
erone
17
-Hydroxy
20-oc-ol
y
y
progesterone
CH^OH
X
CH OH
/
c=o
1
c=o
"
Desoxy—
corticosterone
CH2OH
c=o
1 7- Hydroxy desoxycorticosterone
(Reichstein's "S")
CH^OH
HCO C=0
.JOJ
CH^OH
c=o
A -androstenedione
OH
o ' -- o
Corticosterone 18-aldo- 11- desoxy- Cortisol Testosterone
corticosterone (Kendall's
Cmpd. "F")
HCO C=0
19 -Hydroxy- ^■^■
androstenedione
.;^
Aldosterone
HO
Estradiol
Fig. 11.2. Biosynthetic paths from cholesterol.
Estrone
STEROID SEX HORMONES
647
D. ESTROGENS
A-4-Androstenedione and testosterone are
precursors of the estrogens. Baggett, Engel,
Savard and Dorfman (1956) demonstrated
the conversion of testosterone to estradiol17/? by slices of human ovary. Ryan (1958)
found that the enzymes to carry out this
conversion are also present in the human
placenta, located in the microsomal fraction
of placental homogenates. Homogenates of
stallion testis convert labeled testosterone
to labeled estradiol and estrone. Slices of
human adrenal cortical carcinoma also have
been shown to convert testosterone to estradiol and estrone, and Nathanson, Engel
and Kelley (1951) found an increased urinary excretion of estradiol, estrone, and estriol following the administration of adrenocorticotrophic hormone to castrate women.
Thus it seems that ovary, testis, placenta,
and adrenal cortex have a similar biosynthetic mechanism for the production of estrogens and androgens. The first step in
the conversion of testosterone or A-4-androstenedione to estrogens is the hydroxylation
at carbon 19, again by an enzymatic process
which requires molecular oxygen and
TPNH. Meyer (1955) first isolated and
characterized 19-hydroxy-A-4-androstene3,17-dione from a perfused calf adrenal.
When this was incubated with dog placenta
it was converted to estrone. The steps in the
conversion of the 19-hydroxy-A-4-androstenedione to estrone appear to be the introduction of a second double bond into ring
A, the elimination of carbon 19 as formaldehyde, and rearrangement to yield a
phenolic ring A. The requirements for the
aromatization of ring A by a microsomal
fraction of human placenta were studied by
Ryan (1958). West, Damast, Sarro and
Pearson (1956) found that the administration of testosterone to castrated, adrenalectomized women resulted in an increased
excretion of estrogen. This suggests that
tissues other than adrenals and gonads, presumably the liver, can carry out this same
series of reactions.
E. BIOSYNTHESIS OF OTHER STEROIDS
To complete the picture of the interrelations of the biosyntheses of steroids, it
should be noted that other evidence shows
that progesterone is hydroxylated at carbon
21 to yield desoxycorticosterone and this is
subsequently hydroxylated at carbon 11 to
yield corticosterone. Desoxycorticosterone
may undergo hydroxylation at carbon 18
and at carbon 11 to yield aldosterone, the
most potent salt-retaining hormone known
(Fig. 11.2).
Dehydroepiandrosterone is an androgen
found in the urine of both men and women.
Its rate of excretion is not decreased on
castration and it seems to be synthesized
only by the adrenal cortex. It has been
postulated that pregnenolone is converted
to 17-hydroxy pregnenolone and that this,
by cleavage of the side chain between carbon 17 and carbon 20, would yield dehydroepiandrosterone.
F. INTERCONVERSIONS OF STEROIDS
The interconversion of estrone and estradiol has been shown to occur in a number
of human tissues. A diphosphopyridine nucleotide-linked enzyme, estradiol- 17/3 dehydrogenase, which carries out this reaction
has been prepared from human placenta
and its properties have been described by
Langer ancl Engel (1956). The mode of
formation of estriol and its isomer, 16-epiestriol, is as yet unknown.
There are three major types of reactions
which occur in the interconversions of the
steroids: dehydrogenation, "hydroxylation,"
and the oxidative cleavage of the side chain.
An example of a dehydrogenation reaction
is the conversion of pregnenolone to progesterone by the enzyme 3-/3-ol dehydrogenase,
which requires diphosphopyridine nucleotide (DPN) as hydrogen acceptor. This important enzyme, which is involved in the
synthesis of progesterone and hence in the
synthesis of all of the steroid hormones, is
found in the adrenal cortex, ovary, testis,
and placenta. Other dehydrogenation reactions in which DPN is the usual hydrogen
acceptor are the readily reversible conversions of A-4-androstenedione ^ testosterone, estrone ^ estradiol, and progesterone
- ^ A-4-3-ketopregnene-20-a-ol. This latter
substance, and the enzymes producing it
from progesterone, have been found by Zander (1958) in the human corpus luteum and
placenta.
The oxidative reactions leading to the
PHYSIOLOGY OF GONADS
introduction of an OH group on the steroid
nucleus are usually called "hydroxylations."
Specific hydroxylases for the introduction
of an OH group at carbons 11, 16, 17, 21, 18,
and 19 have been demonstrated. All of these
require molecular oxygen and a reduced
pyridine nucleotide, usually TPNH. The
ll-/3-hydroxylase of the adrenal cortex has
been shown to be located in the mitochondria (Hayano and Dorfman, 1953) . Experiments with this enzyme system, utilizing
oxygen 18, showed that the oxygen atoms
are derived from gaseous oxygen and not
from the oxygen in the water molecules
(Hayano, Lindberg, Dorfman, Hancock and
Doering, 1955). Thus this hydroxylation reaction also involves the reduction of molecular oxygen.
The oxidative cleavage of the side chains
of the steroid molecule appears to involve
similar hydroxylation reactions. The experiments of Solomon, Levitan and Lieberman
(1956) indicate that the conversion of cholesterol to pregnenolone involves one and
possibly two of these hydroxylation reactions, with the introduction of OH groups
at carbons 20 and 22 before the splitting off
of the isocaproic acid.
In summary, this newer knowledge of the
biosynthetic paths of steroids has revealed
that the differences between the several
steroid-secreting glands are largely quantitative rather than qualitative. The testis,
for example, produces progesterone and
estrogens in addition to testosterone. The
change from the secretion of estradiol by
the follicle to the secretion of progesterone
by the corpus luteum can be understood as
a relative loss of activity of an enzyme in
the path between progesterone and estradiol.
If, for example, the enzyme for the 17-hydroxylation of progesterone became inactive
as the follicle cells are changed into the
corpus luteum, progesterone rather than
estradiol would subsequently be produced.
Knowledge of these pathways also provides an explanation for certain abnormal
changes in the functioning of the glands.
Bongiovnnni (1953) and Jailer (1953)
showed that the adrenogenital syndrome
results from a loss of an enzyme or enzymes
for the hydroxylation reactions at carbons
21 and 11 of progesterone, which results in
an impairment in the production of Cortisol.
The pituitary, with little or no Cortisol to
inhibit the secretion of adrenocorticotrophic
hormone (ACTH), produces an excess of
this hormone which stimulates the adrenal
to produce more steroids. There is an excretion of the metabolites of progesterone
and 17-hydroxy progesterone, pregnanediol
and pregnanetriol respectively, but some of
the 17-hydroxy progesterone is converted to
androgens and is secreted in increased
amount.
G. CATABOLISM OF STEROmS
Many of the steroid hormones are known
to act on the pituitary to suppress its secretion of the appropriate trophic hormone,
ACTH, the follicle-stimulating hormone
(FSH), or luteinizing hormone (LH). It
would seem that the maintenance of the
proper feedback mechanism between steroid-secreting gland and pituitary requires
that the steroids be continuously inactivated
and catabolized. The catabolic reactions of
the steroids are in general reductive in nature and involve the reduction of ketonic
groups and the hydrogenation of double
bonds. The reduction of a ketonic group to
an OH group can lead to the production of
two different stereoisomers. If the OH group
projects from the steroid nucleus on the
same side as the angular methyl groups
at carbon 18 and carbon 19, i.e., above the
plane of the four rings, it is said to have
the yS-configuration and is represented by a
heavy line. If the OH projects on the opposite side of the steroid nucleus, below
the plane of the four rings, it is said to
have the a-configuration and is represented
by a dotted line. Although both isomers are
possible, usually one is formed to a much
greater extent than the other.
The first catabolic step is usually the
reduction of the A4-3-ketone group of ring
A, usually to 3aOH compounds with the
hydrogen at carbon 5 attached in the /3configuration. The 5/3-configuration represents the CIS configuration of rings A and B.
The elimination of the A4-3-ketone group
greatly decreases the biologic activity of
the steroid and increases somewhat its solubility in water. This reductive process occurs largely in the liver. Progesterone is
converted by reduction of its A4-3-ketone
group to pregnane-3a:20a-diol, and 17-hy
STEROID SEX HORMONES 649
EXCRETORY PRODUCTS
c=o
Progesterone
1 3
HCOH
HO H
Pregnanediol
CH,
-OH
17 Hydroxy progesterone
CH,
^ H-C-OH
■OH
HO H
Pregnanetriol
OH
Testosterone
Androsterone
HO
/
androstenedione
HO H
Etiocholanolone
Dehydroepiandrosterone
Fk;. 11.3. Excretory products of progesterone and androgen
(Iroxy progesterone is converted to pregnane3a:17a:20a-triol (Fig. 11.3). Testosterone
and dehydrocpiandroesterone are both converted to A4-androstenedione and the reduction of its A4-3-ketone group results in a
mixture of androsterone (3a,5a-configuration) and ctiochohmolone (3a,5/?-configuration ) .
The catabohsm of estradiol is not completely known. Estradiol, estrone, and estriol are found in the urine but they account
for less than half of an administered dose of
labeled estradiol. The /3-isomer, 16-epiestriol, and two other phenolic steroids, 16-ahydroxy estrone and 2-methoxyestrone,
have recently been isolated from normal
650
PHYSIOLOGY OF GONADS
urine and are known to be estrogen metabolites (Marrian and Bauld, 1955).
H. TRANSPORT, CONJUGATION, AND EXCRETION
Steroids circulate in the blood in part as
free steroids and in part conjugated with
sulfate or glucuronic acid (c/. review by
Roberts and Szego, 1953b j . The steroids are
generally conjugated by the hydroxy 1 group
at carbon 3 with inorganic sulfate or with
glucuronic acid. In addition, either the conjugated or nonconjugated forms may be
bound to certain of the plasma proteins such
as the ^-globulins (Levedahl and Bernstein,
1954) . There is evidence of specific binding
of certain steroids with particular proteins,
e.g., the binding of Cortisol to "transcortin"
(Daughaday, 1956). Between 50 and 80 per
cent of the estrogens in the blood are present closely bound to plasma proteins. A
similar large fraction of the other steroid
hormones is bound to plasma proteins ; presumably this prevents the hormone from being filtered out of the blood as it passes
through the glomerulus of the kidney. The
steroids excreted in the urine are largely in
the conjugated form, as sulfates or glucuronides.
The liver plays a prime role in the catabolism of the steroids. It is the major site
of the reductive inactivation of the steroids
and their conjugation with sulfate or glucuronic acid. These conjugated forms are more
water-soluble and the conjugation probably
promotes their excretion in the urine. Rather
large amounts of certain steroids, notably
estrogens, are found in the bile of certain
species. These estrogens are free, not conjugated; the amount of estrogens present
in the bile suggests that this is an important pathway by which they are excreted. It has been suggested that the bacteria of the gastrointestinal tract may
degrade the steroids excreted in the bile and
further that there is an "enterohepatic circulation" of steroids with reabsorption
from the gut, transport in the portal system
to the liver, and further degradation within
the liver cells.
III. Effects of Sex Hormones on
Intermediary Metabolism
The literature concerning the effects of
hormones on intermediary metabolism is
voluminous and contains a number of contradictions, some of which are real and
some, perhaps, are only apparent contradictions. Evidence that a hormone acts at one
site does not necessarily contradict other
evidence that that hormone may act on a
different metabolic reaction. From the following discussion it should become evident
that there may be more than one site of
action, and more than one mechanism of
action, of any given hormone.
The hormones are so different in their
chemical structure, proteins, peptides,
amino acids, and steroids, that it would
seem unlikely, a priori, that they could all
influence the cellular machinery by comparable means. The basic elements of an
enzyme system are the protein enzyme, its
cofactors and activators, and the substrates
and products. A hormone might alter the
over-all rate of an enzyme system by altering the amount or activity of the protein
enzyme, or by altering the availability to
the enzyme system of some cofactor or substrate molecule. Some of the mechanisms
of hormone action which have been proposed are these. (1) The hormone may alter
the rate at which enzyme molecules are
produced de novo by the cell. (2) The hormone may alter the activity of a preformed
enzyme molecule, i.e., it may convert an
inactive form of the enzyme to an active
form. (3) The hormone may alter the permeability of the cell membrane or the
membrane around one of the subcellular
structures within the cell and thus make
substrate or cofactor more readily available
to the enzyme. Or, (4) the hormone may
serve as a coenzyme in the system, that is,
it may be involved in some direct fashion
as a partner in the reaction mediated by the
enzyme. Each of these theories has been
advanced to explain the mode of action of
the sex hormones.
The problem of the hormonal control of
metabolism has been investigated at a variety of biologic levels. The earliest experiments were done by injecting a hormone
into an intact animal and subsequently
measuring the amount of certain constituents of the blood, urine, or of some tissue.
There are several difficulties with such experiments. All of the homeostatic mechanisms of the animal operate to keep condi
STEROID SEX HORMONES
651
tions constant and to minimize the effects
of the injected hormone. In addition, there
is a maze of interactions, some synergistic
and some antagonistic, between the different
hormones both in the endocrine gland and
in the target organs, so that the true effect
of the substance injected may be veiled. Our
growing understanding of the interconversions of the steroid hormones warns us that
an androgen, for example, may be rapidly
converted into an estrogen, and the metabolic effects observed on the administration
of an androgen may, at least in part, result
from the estrogens produced from the injected androgen.
To eliminate some of the confusing effects
of these homeostatic mechanisms some investigators remove the liver, kidneys, and
other viscera before injecting the hormone
under investigation. Such eviscerated preparations have been used by Levine and his
colleagues in their investigations of the
mode of action of insulin (c/. Levine and
Goldstein, 1955).
Other investigators have incubated slices
of liver, kidney, muscle, endocrine glands,
or other tissues in glass vessels in a chemically defined medium and at constant temperature. Such experiments have the advantage that metabolism can be studied
more directly, oxygen consumption and carbon dioxide production can be measured
manonietrically, and aliquots of the incubation medium can be withdrawn for chemical
and radiochemical analyses. The amounts of
substrate, cofactors, and hormone present
can be regulated and the interfering effects
of other hormones and of other tissues are
eliminated. Theoretically, working with a
simpler system such as this should lead to
greater insight into the physiologic and
chemical events that occur when a hormone
is added or deleted. The chief disadvantage
of this experimental system is that it is
difficult to prove that the conditions of the
experiment are "physiologic." With tissue
slices there is the possibility that the cut
edges of the cells may introduce a sizeable
artifact. Kipnis and Cori (1957) found that
the rat diaphragm, as it is usually prepared
for experiments in vitro, has an abnormally
large extracellular space and is more permeable to certain pentoses than is the intact
diaphragm.
It has been postulated that a hormone
may influence the metabolism of a particular cell by altering the permeability of the
cell membrane or of the membrane around
one of the subcellular particles. Experiments
with tissue homogenates, in which the cell
membrane has been ruptured and removed,
provide evidence bearing on such theories.
If an identical hormone effect can be obtained in a cell-free system, and if suitable
microscopic controls show that the system is indeed cell-free, the permeability
theory may be ruled out.
Ideally the hormone effect should be
studied in a completely defined system,
with a single crystalline enzyme, known
concentration of substrates and cofactors,
and with known concentration of the pure
hormone. Colowick, Cori and Slein (1947)
reported that hexokinase extracted from
diabetic muscle has a lower rate of activity
than hexokinase from normal muscle and
that it could be raised to the normal rate
by the addition of insulin in vitro. The
reality of this effect has been confirmed by
some investigators and denied by others
who were unable to repeat the observations.
Cori has suggested that the decreased rate
of hexokinase activity in the diabetic results from a labile inhibitor substance produced by the pituitary. Krahl and Bornstein
(1954) have evidence that this inhibitor is
a lipoprotein which is readily inactivated
by oxidation.
The two hormones whose effects can be
demonstrated reproducibly in an in vitro
system at concentrations in the range which
obtains in the tissues are epinephrine (or
glucagon) and estradiol (and other estrogens) . Epinephrine or glucagon stimulates
the reactivation of liver phosphorylase by
increasing the concentration of adenosine3'-5'-monophosphate (Haynes, Sutherland
and Rail, 1960), and estrogens stimulate an
enzyme system found in endometrium,
placenta, ventral i)rostate of the rat, and
mammary gland. The estrogen-stimulable
enzyme was originally described as a DPNlinked isocitric dehydrogenase, but the estrogen-sensitive enzyme now appears to be
a transhydrogenase which transfers hydrogens from TPN to DPN (Talalay and Williams-x\shman, 1958; Yillee and Hngerman,
1958).
052
PHYSIOLOGY OF GONADS
The various tissues of the body respond in
quite different degrees to the several hormones. This difference in response is especially marked with the sex hormones.
Those tissues which respond dramatically
to the administration of a hormone are
termed the "target organs" of that hormone. Just what, at the cellular level, differentiates a target organ from the other
tissues of the body is not known exactly
but there is evidence that each kind of tissue is characterized by a certain pattern of
enzymes. The pattern of enzymes is established, by means as yet unknown, in the
course of embryonic differentiation. The
enzyme glucose 6-phosphatase, which hydrolyzes glucose 6-phosphate and releases
free glucose and inorganic phosphate, is
present in liver but absent from skeletal
muscle. Even though a given reaction in
two different tissues may be mediated by
what appears to be the same enzyme, the
enzymes may be different and subject to
different degrees of hormonal control. Henion and Sutherland (1957) showed that the
phosphorylase of liver responds to glucagon
but the phosphorylase of heart muscle does
not. Further, the two enzymes are immunologically distinct. An antiserum to purified
liver phosphorylase will not react with heart
phosphorylase to form an inactive antigenantibody precipitate, but it does react in
this manner with liver phosphorylase. Further, perhaps more subtle, differences between comparable enzymes from different
tissues have appeared when lactic dehydrogenases from liver, heart, skeletal muscle,
and other sources were tested for their rates
of reaction with the several analogues of the
pyridine nucleotides now available (Kaplan. Ciotti, Hamolsky and Bicbcr, 1960).
p]xtension of this technique may reveal differences in response to added hormones.
In addition to these differences in the response to a hormone of the tissues of a
single animal, there may be differences in
the response of the comparable tissues of
different species to a given dose of hormone.
Estrone, estriol, and other estrogens have
different potencies relative to estradiol in
different species of mammals. There are
slight differences in the amino acid sequences of the insulins and vasopressins
from flifferent species and quite marked
differences in the chemical structure (Li and
Papkoff, 1956) and physiologic activity
(Knobil, Morse, Wolf and Greep, 1958)
of the pituitar}^ growth hormones of cattle
and swine, on the one hand, and of primates, on the other.
A. ESTROGENS
The amount or activity of certain enzymes in the target organs of estrogens
has been found to vary with the amount of
estrogen present. Examples of this phenomenon are /^-glucuronidase (Odell and
Fishman, 1950) , fibrinolysin (Page, Glendening and Parkinson, 1951), and alkaline
glycerophosphatase (Jones, Wade, and
Goldberg, 1953). Kochakian (1947) reported that the amount of arginase in the
rat kidney increased after the injection of
estrogens. Enzyme activity is increased by
other hormones as well; for example, progesterone has been found to increase the
activity of phosphorylase (Zondek and
Hestrin, 1947) and of adenosine triphosphatase (Jones, Wade, and Goldberg, 1952).
In most experiments the amount of enzyme present has been inferred from its
activity, measured chemically or histochemically under conditions in which the
amount of enzyme is rate-limiting. This
does not enable one to distinguish between
an actual increase in the number of molecules of enzyme present in the cell and an
increase in the activity of the enzyme molecules without change in their number. A
few enzymes can be measured by some
other property, such as absorption at a
specific wavelength, by which the actual
amount of enzyme can be estimated (see
review by Knox, Auerbach, and Lin, 1956).
Knox and Auerbach (1955) found that the
activity of the enzyme tryptophan peroxidase-oxidase (TPO) of the liver was
decreased in adrenalectomized animals and
increased by the administration of cortisone. Knox had shown previously that
th(> administration of the substrate of
the enzyme, tryptophan, would lead
to an increase in the activity of the enzyme which was maximal in 6 to 10
hours. Evidence that the increased activity
of enzyme following the administration of
cortisone represents the synthesis of new
protein molecules is supplied by experi
STEROID SEX HORMONES
653
ments in which it was found that the increase in enzyme activity is inhibited by
ethionine and this inhibition is reversed
by methionine. The amino acid analogue
ethionine is known to inhibit protein synthesis and this inhibition of protein synthesis is overcome by methionine.
The injection of estrogen into the immature or castrate rodent produces a striking uptake of water by the uterus followed
by a marked increase in its dry weight
(Astwood, 1938). Holden (1939) postulated that the imbibition of water results
from vasodilatation and from changes in the
permeability of the blood vessels of the
uterus. There is clear evidence (Mueller,
1957) that the subsequent increase in dry
weight is due to an increased rate of synthesis of proteins and nucleic acids. The
sex hormones and other steroids could be
pictured as reacting with the protein or
lipoprotein membrane around the cell or
around some subcellular structure like a
surface-wetting agent and in this way inducing a change in the permeability of the
membrane. This might then increase the
rate of entry of substances and thus alter
the rate of metabolism within the cell.
This theory could hardly account for the
many notable specific relationships between
steroid structure and biologic activity.
Spaziani and Szego (1958) postulated that
estrogens induce the release of histamine in
the uterus and the histamine then alters the
permeability of the blood vessels and produces the imbibition of water secondarily.
The uterus of the ovariectomized rat is
remarkably responsive to estrogens and
has been widely used as a test system.
After ovariectomy, the content of ribonucleic acid of the uterus decreases to a
low level and then is rapidly restored after
injection of estradiol (Telfer, 1953). A
single injection of 5 to 10 yu,g. of estradiol
brings about (1) the hyperemia and water
imbibition described previously; (2) an
increased rate of over-all metabolism as
reflected in increased utilization of oxygen
(David, 1931; Khayyal and Scott, 1931;
Kerly, 1937; MacLeod and Reynolds, 1938;
Walaas, Walaas and Loken, 1952a; Roberts
and Szego, 1953a) ; (3) an increased rate
of glycolysis (Kerly, 1937; Carroll, 1942;
Stuermer and Stein, 1952; Walaas, Walaas
and Loken, 1952b; Roberts and Szego,
1953a) ; (4) an increased rate of utilization
of phosphorus (Grauer, Strickler, Wolken
and Cutuly, 1950; Walaas and Walaas,
1950) ; and (5) tissue hypertrophy as reflected in increased dry weight (Astwood,
1938), increased content of ribonucleic acid
and protein (Astwood, 1938; Telfer, 1953;
Mueller, 1957), and finally, after about
72 hours, an increased content of desoxyribonucleic acid (Mueller, 1957).
An important series of experiments by
Mueller and his colleagues revealed that
estrogens injected in vivo affect the metabolism of the uterus which can be detected
by subsequent incubation of the uterus in
vitro with labeled substrate molecules.
Mueller (1953) first showed that pretreatment with estradiol increases the rate
of incorporation of glycine-2-C^'* into uterine protein. He then found that estrogen
stimulation increases that rate of incorporation into protein of all other amino acids
tested: alanine, serine, lysine, and tryptophan. The peak of stimulation occurred
about 20 hours after the injection of estradiol. In further studies (Mueller and Herranen, 1956) it was found that estrogen
increases the rate of incorporation of glycine-2-C^^ and formate-2-C^'* into protein,
lipid, and the purine bases, adenine and
guanine, of nucleic acids. A stimulation of
cholesterol synthesis in the mouse uterus
20 hours after administration of estradiol
was shown by Emmelot and Bos (1954).
In more detailed studies of the effects of
estrogens on the metabolism of "one-carbon
units" Herranen and Mueller (1956) found
that the incorporation of serine-3-C^'* into
adenine and guanine was stimulated by
pretreatment with estradiol. The incorporation was greatly decreased when unlabeled
formate was added to the reaction mixture
to trap the one-carbon intermediate. In
contrast, the incorporation of C^^02 into
uridine and thymine by the surviving uterine segment was not increased by pretreatment with estradiol in vivo (Mueller,
1957).
To delineate further the site of estrogen
effect on one-carbon metabohsm, Herranen
and Mueller (1957) studied the effect of
estrogen pretreatment on serine aldolase,
the enzyme which catalyzes the equilibrium
654
PHYSIOLOGY OF GONADS
between serine and glycine plus an active
one-carbon unit. They found that serine
aldolase activity, measured in homogenates of rat uteri, increased 18 hours after
pretreatment in vivo with estradiol. It
seemed that the estrogen-induced increase
in the activity of this enzyme might explain
at least part of the increased rate of onecarbon metabolism following estrogen injection. They found, however, that incubation of uterine segments in tissue culture
medium (Eagle, 1955) for 18 hours produced a marked increase in both the activity
of serine aldolase and the incorporation of
glycine-2-C^'* into protein. The addition of
estradiol to Eagle's medium did not produce
a greater increase than the control to
which no estradiol was added. Uterine segments taken from rats pretreated with estradiol for 18 hours, with their glycine-incorporating system activated by hormonal
stimulation, showed very little further
stimulation on being incubated in Eagle's
medium for 18 hours. With a shorter period
of i^retreatment with estradiol, greater stimulation occurred on subsequent incubation
in tissue culture fluid. These experiments
suggest that the hormone and the incubation in tissue culture medium are affecting
the same process, one which has a limited
capacity to respond. When comparable experiments were performed with other
labeled amino acids as substrates, similar
results were obtained.
Mueller's work gave evidence that a considerable number of enzyme systems in the
uterus are accelerated by the administration
of estradiol — not only the enzymes for the
incorporation of serine, glycine, and formate
into adenine and guanine, but also the enzymes involved in the synthesis of fatty
acids and cholesterol and indejX'ndent enzymes for the activation of amino acids by
the formation of adenosine monoiihosphate
(AMP) derivatives. The initial step in
protein synthesis has been shown to be the
activation of the carboxyl grou]) of the
amino acid with transfer of energy from
ATP, the formation of AMP -"amino
acid, and the release of jiyrophosphate
(Hoagland, Keller and Zamecnick, 1956).
This reversible step was studied with homogenates of uterine tissue, P^--labeled
]n'rni)liosi)liate, and a variety of amino
acids (Mueller, Herranen and Jervell,
1958). Seven of the amino acids tested,
leucine, tryptophan, valine, tryosine, methionine, glycine, and isoleucine, stimulated the
exchange of P^^ between pyrophosphate and
ATP. Pretreatment of the uteri by estradiol
injected in vivo increased the activity
of these three enzymes. The activating
effect of mixtures of these amino acids
was the sum of their individual effects,
from which it was inferred that a specific
enzyme is involved in the activation of
each amino acid. Since estrogen stimulated the exchange reaction with each of
these seven amino acids, Mueller concluded that the hormone must affect the
amount of each of the amino acid-activating enzvmes in the soluble fraction of the
cell.
Mueller (1957) postulated that estrogens
increase the rate of many enzyme systems
both by activating preformed enzyme molecules and by increasing the rate of de novo
synthesis of enzyme molecules, possibly by
removing membranous barriers covering the
templates for enzyme synthesis. To explain
why estrogens affect these enzymes in the
target organs, but not comparable enzymes
in other tissues, one would have to assume
that embryonic differentiation results in
the formation of enzymes in different tissues
which, although catalyzing the same reaction, have different properties such as
their responsiveness to hormonal stimulation.
As an alternative hypothesis, estrogen
might affect some reaction which provides
a substance required for all of these enzyme reactions. The carboxyl group of
amino acids must be activated by ATP before the amino acid can be incorporated
into proteins; the synthesis of both purines
and pyrimidines requires ATP for the
activation of the carboxyl group of certain
precursors and for several other steps; the
synthesis of cholesterol requires ATP for
the conversion of mevalonic acid to
squalene; and the synthesis of fatty acids
is also an energy-requiring process. Thus if
(>strogens acted in some way to increase the
amount of biologically useful energy, in
the form of ATP or of energy-rich thioesters
such as acetyl coenzyme A, it would increase
the rate of synthesis of all of these compo
STEROID SEX HORMONES
655
nents of the cell. This would occur, of course,
only if the supply of ATP, rather than the
amount of enzyme, substrate, or some other
cofactor, were the rate-limiting factor in the
synthetic processes.
When purified estrogens became available, they were tested for their effects on
tissues in vitro. Estrogens added in vitro increased the utilization of oxygen by the rat
uterus (Khayyal and Scott, 1931) and the
rat pituitary (Victor and Andersen, 1937).
The addition of estradiol- 17^ at a level of
1 fxg. per ml. of incubation medium increased
the rate of utilization of oxygen and of
pyruvic acid by slices of human endometrium and increased the rate at which labeled glucose and pyruvate were oxidized to
C^-^Os (Hagerman and Villee, 1952, 1953a,
1953b) . In experiments with slices of human
placenta similar results were obtained and
it was found that estradiol increased the
rate of conversion of both pyruvate-2-C^'*
and acetate-l-Ci4 to C^^Os (Villee and
Hagerman, 1953) . From this and other evidence it was inferred that the estrogen acted
at some point in the oxidative pathway
common to pyruvate and acetate, i.e., in the
tricarboxylic acid cycle.
Homogenates of placenta also respond to
estradiol added in vitro. With citric acid as
substrate, the utilization of citric acid and
oxygen and the production of a-ketoglutaric
acid were increased 50 per cent by the
addition of estradiol to a final concentralion of 1 fjig. per ml. (Villee and Hagerman,
1953). The homogenates were separated by
differential ultracentrifugation into nuclear,
mitochondrial, microsomal, and nonparticulate fractions. The estrogen-stimulable system was shown to be in the nonparticulate
fraction, the material which is not sedimented by centrifugating at 57,000 X g
for 60 minutes (Villee, 1955). Experiments
with citric, as-aconitic, isocitric, oxalosuccinic, and a-ketoglutaric acids as substrates and with fluorocitric and transaconitic acids as inhibitors localized the
estrogen-sensitive system at the oxidation
of isocitric to oxalosuccinic acid, which then
undergoes spontaneous decarboxylation to
a-ketoglutaric acid (Villee and Gordon,
1955). Further investigations using the enzymes of the nonparticulate fraction of
the human placenta revealed that, in ad
dition to isocitric acid as substrate, only
DPN and a divalent cation such as Mg+ +
or Mn++ were required (Villee, 1955; Gordon and Villee, 1955; Villee and Gordon,
1956). The estrogen-sensitive reaction was
formulated as a DPN-linked isocitric dehydrogenase:
Isocitrate + DPN* -^ a-ketoglutarate
+ CO2 + DPXH + H*
It was found that the effect of the hormone on the enzyme can be measured by
the increased rate of disappearance of citric
acid, the increased rate of appearance of
a-ketoglutaric acid, or by the increased
rate of reduction of DPN, measured spectrophotometrically by the optical density at
340 m/x. As little as 0.001 /xg. estradiol per
ml. (4 X 10~^ m) produced a measurable
increase in the rate of the reaction, and
there was a graded response to increasing
concentrations of estrogen. The dose-response curve is typically sigmoid. This system has been used to assay the estrogen
content of extracts of urine (Gordon and
Villee, 1956) and of tissues (Hagerman,
Wellington and Villee, 1957; Loring and
Villee, 1957).
Attempts to isolate and purify the estrogen-sensitive enzyme were not very successful. By a combination of low temperature
alcohol fractionation and elution from calcium phosphate gel a 20-fold purification
was obtained (Hagerman and Villee, 1957).
However, as the enzyme was purified it was
found that an additional cofactor was required. Either uridine triphosphate (UTP)
or ATP added to the system greatly
increased the magnitude of the estrogen effect and, subsequently, adenosine diphosphate (ADP) was recovered from the incubation medium and identified by paper
chromatography (Villee and Hagerman,
1957). Talalay and Williams-Ashman
(1958) confirmed our observations and
showed that the additional cofactor was
triphosphopyridine nucleotide (TPN) which
was required in minute amounts. This finding was confirmed by Villee and Hagerman
(1958) and the estrogen-sensitive enzyme
system of the placenta is now believed to be
a transhydrogenase which catalyzes the
transfer of hydrogen ions and electrons
656
PHYSIOLOGY OF GONADS
fromTPNHtoDPN:
TPXH + DPN^ -> DPNH + TPN^
The transhydrogenation system can be
coupled to glucose 6-phosphate dehydrogenase as well as to isocitric dehydrogenase
(Talalay and Williams-Ashman, 1958; Villee and Hagerman, 1958) and presumably
can be coupled to any TPNH-generating
system.
If the estrogen-stimulable transhydrogenation reaction were readily reversible, an
enzyme such as lactic dehydrogenase which
requires DPN should be stimulated by
estrogen if supplied with substrate amounts
of TPN, catalytic amounts of DPN, and
a preparation from the placenta containing
the transhydrogenase. Experiments to test
this prediction were made using lactic dehydrogenase and alcohol dehydrogenase of
both yeast and liver (Villee, 1958a). It was
not possible to demonstrate an estrogen
stimulation of either enzyme system in
either the forward or the reverse direction.
The stimulation of the lactic dehydrogenase-DPN oxidase system of the rat uterus
by estrogens administered in vivo reported
by Bever, Velardo and Hisaw (1956)
might be explained by the stimulation of
a transhydrogenase, but it has not yet been
possible to demonstrate a coupling of this
transhydrogenase and lactic dehydrogenase.
The stimulating effect of a number of
steroids has been tested with a system in
which the transhydrogenation reaction is
coupled to isocitric dehydrogenase (Villee
and Gordon, 1956; Hollander, Nolan and
Hollander, 1958). Estrone, equilin, equilenin, and 6-ketoestradiol have activities
essentially the same as that of estradiol17 j3. Samples of 1 -methyl estrone and 2methoxy-estrone had one-half the activitj
of estradiol. Estriol is only weakly estrogenic in this system; 33 fig. estriol are less
active than 0.1 fig. estradiol- 17/3 (Villee,
1957a). The activities of estriol and 16epiestriol are similar, whereas 16-oxoestradiol is more active than either, with about
10 per cent as much activitv as csti'adiol17/3.
Certain analogues of stilbestrol have been
shown to be anti-estrogens in vivo. When
applied topically to the vagina of the rat,
they prevent the cornification normally in
duced by the administration of estrogen
(Barany, Morsing, Muller, Stallberg, and
Stenhagen, 1955). One of these, 1,3-di-phydroxyphenylpropane, was found to be
strongly anti-estrogenic in the placental
system in vitro: it prevented the acceleration of the transhydrogenase-isocitric dehydrogenase system normally produced by
estradiol- 17/3 (Villee and Hagerman, 1957).
The inhibitory power declines as the length
of the carbon chain connecting the two
phenolic rings is increased and 1 , 10-di-phydroxyphenyldecane had no inhibitory action. Similar inhibitions of the estradiolsensitive system were observed with stilbestrol, estradiol-17a, and a smaller antiestrogenic effect was found with estriol
(Villee, 1957a). The inhibition induced
by these compounds can be overcome by
adding increased amounts of estradiol-17^.
When stilbestrol is added alone at low concentration, 10~' M, it has a stimulatory effect equal to that of estradiol-17^ (Glass,
Loring, Spencer and Villee, 1961).
The quantitative relations between the
amounts of stimulator and inhibitor suggest
that this inhibition is a competitive one. It
was postulated that this phenomenon involves a competition between the steroids
for specific binding sites on the estrogensensitive enzyme (Villee, 1957b; Hagerman
and Villee, 1957). When added alone, estriol
and stilbestrol are estrogenic and increase
the rate of the estrogen-sensitive enzyme.
In the presence of both estradiol and estriol,
the total enzyme activity observed is the
sum of that due to the enzyme combined
with a potent activator, estradiol- 17^, and
that due to the enzyme combined with a
weak activator, estriol. When the concentration of estriol is increased, some of the
estradiol is displaced from the enzyme and
the total activity of the enzyme system is
decreased.
Two hypotheses have been proposed for
the mechanism of action of estrogens on the
enzyme system of the placenta. One states
that the estrogen combines with an inactive
form of the enzyme and converts it to an
active form (Hagerman and Villee, 1957).
When this theory was formulated the evidence indicated that the estrogen acted on
a specific DPN-linked isocitric dehydrogenase. The theory is equally applicable if the
STEROID SEX HORMONES
657
estrogen-sensitive enzyme is a transhydrogenase, as the evidence now indicates. The
results of kinetic studies with the coupled
isocitric dehydrogenase-transhydrogenase
system are consistent with this theory
(Gordon and Villee, 1955; Villee, 1957b;
Hagerman and Villee, 1957). Apparent
binding constants for the enzyme-hormone
complex (Gordon and Villee, 1955j and for
enzyme-inhibitor complexes have been calculated (Hagerman and Villee, 1957).
The observation that estradiol and estrone, which differ in structure only by a
pair of hydrogen atoms, are equally effective in stimulating the reaction suggested
that the steroid might be acting in some way
as a hydrogen carrier from substrate to
pyridine nucleotide (Gordon and Villee,
1956). Talalay and Williams-Ashman
(1958) suggested that the estrogens act as
coenzymes in the transhydrogenation reaction and postulated that the reactions were:
Estrone + TPNH + H*
— Estradiol + TPN^
Estradiol + DPN+
— Estrone + DPNH + H*
Sum : TPNH
H*
- DPN^
— TPN^ + H^
DPNH
This formulation implies that the estrogen-sensitive transhydrogenation reaction
is catalyzed by the estradiol-17y3 dehydrogenase characterized by Langer and Engel
(1956). This enzyme was shown by Langer
(1957) to use either DPN or TPN as hydrogen acceptor but it reacts more rapidly
with DPN. Ryan and Engel (1953) showed
that this enzyme is present in rat liver, and
in human adrenal, ileum, and liver. However, no estrogen-stimulable enzyme is
demonstrable in rat or human liver (Villee,
1955). The nonparticulate fraction obtained
by high speed centrifugation of homogenized rabbit liver rapidly converts estradiol
to estrone if DPN is present as hydrogen
acceptor, but does not contain any estrogenstimulable transhydrogenation system.
It will not be possible to choose between
these two hypotheses until either the estrogen-sensitive transhydrogenase and the
estradiol dehydrogenase have been separated or there is conclusive proof of their
identity. Talalay, Williams-Ashman and
Hurlock (1958) reported a 100-fold purification of the dehydrogenase without separation of the transhydrogenase activity
and found that both activities were inhibited identically by sulfhydryl inhibitors.
In contrast, Hagerman and Villee (1958)
obtained partial separation of the two activities by the usual techniques of protein
fractionation, and reported that a 50 per
cent inhibition of transhydrogenase is obtained with p-chloromercurisulfonic acid at
a concentration of 10~^ m whereas 10"^ m
p-chloromercurisulfonic acid is required for
a 50 per cent inhibition of the dehydrogenase. The evidence that these two activities are mediated by separate and distinct proteins has been summarized by
Villee, Hagerman and Joel (1960).
The transhydrogenase present in the
mitochondrial membranes of heart muscle
was shown by Ball and Cooper (1957) to be
inhibited by 4 X 10"^ m thyroxine. The
estrogen-sensitive transhydrogenase of the
placenta is also inhibited by thyroxine (Villee, 1958b). The degree of inhibition is a
function of the concentration of the thyroxine and the inhibition can be overcome by
increased amounts of estrogen. Suitable control experiments show that thyroxine at
this concentration does not inhibit the glucose 6-phosphate dehydrogenase or isocitric
dehydrogenase used as TPNH-generating
systems to couple with the transhydrogenase. Triiodothyronine also inhibits the
estrogen-sensitive transhydrogenase but
tyrosine, diiodotyrosine and thyronine do
not. The thyroxine does not seem to be
inhibiting by binding the divalent cation,
Mn + + or ]Mg+ + , required for activity, for
the inhibition is not overcome by increasing
the concentration of the cation 10-fold.
In the intact animal estrogens stimulate
the growth of the tissues of certain target
organs. The estrogen-sensitive enzyme has
been shown to be present in many of the
target organs of estrogens: in human endometrium, myometrium, placenta, mammary
gland, and mammary carcinoma, in rat ventral prostate gland and uterus, and in mammotrophic-dependent transplantable tumors
of the rat and mouse pituitary. In contrast,
it is not demonstrable in comparable preparations from liver, heart, lung, brain, or
658
PHYSIOLOGY OF GONADS
kidney. The growth of any tissue involves
the utilization of energy, derived in large
part from the oxidation of substrates, for
the synthesis of new chemical bonds and for
the reduction of substances involved in the
synthesis of compounds such as fatty acids,
cholesterol, purines, and pyrimidines.
The physiologic responses to estrogen
action, such as water imbibition and protein
and nucleic acid synthesis, are processes not
directly dependent on the activity of transhydrogenase. However, all of these processes
are endergonic, and one way of increasing
their rate would be to increase the supply of
biologically available energy by speeding
up the Krebs tricarboxylic acid cycle and
the flow of electrons through the electron
transmitter system. Much of the oxidation
of substrates by the cell produces TPNH,
whereas the major fraction of the biologically useful energy of the cell comes from the
oxidation of DPNH in the electron transmitter system of the cytochromes. Hormonal
control of the rat of transfer of hydrogens
from TPN to DPN could, at least in theory,
influence the over-all rate of metabolism in
the cell and secondarily influence the
amount of energy available for synthetic
processes. Direct evidence of this was shown
in our early experiments in which the oxygen consumption of tissue slices of target
organs was increased by the addition of
estradiol (Hagerman and Villee, 1952; Villee and Hagerman, 1953).
This theory assumes that the supply of
energy is rate-limiting for synthetic processes in these target tissues and that the
activation of the estrogen-sensitive enzyme
does produce a significant increase in the
supply of energy. The addition of estradiol
in vitro produces a significant increase in
the total amount of isocitric acid dehydrogenated by the placenta (Villee, Loring
and Sarner, 1958) . Slices of endometrium to
which no estradiol was added in vitro
utilized oxygen and metabolized substrates
to carbon dioxide at rates which paralleled
the levels of estradiol in the blood and urine
of the patient from whom the endometrium
was obtained (Hagerman and Villee,
esses in these target tissues and that the
1953b). Estradiol increases the rate of synthesis of ATP by liomogenates of human
placenta (Villee, Joel, Loring and Spencer,
1960).
The reductive steps in the biosynthesis of
steroids, fatty acids, purines, serine, and
other substances generally require TPNH
rather than DPNH as hydrogen donor. The
cell ordinarily contains most of its TPN
in the reduced state and most its DPN in
the oxidized state (Glock and McLean,
1955). If the amount of TPN+ is ratelimiting, a transhydrogenase, by oxidizing
TPN and reducing DPN, would permit
further oxidation of substrates such as isocitric acid and glucose 6-phosphate, which
require TPN+ as hydrogen acceptor and
which are key reactions in the Krebs tricarboxylic acid cycle and the hexose monophosphate shunt, respectively. Furthermore,
the experiments of Kaplan, Schwartz, Freeh
and Ciotti (1956) indicate that less biologically useful energy, as ATP, is obtained
when TPNH is oxidized by TPNH cytochrome c reductase than when DPNH is
oxidized by DPNH cytochrome c reductase.
Thus, a transhydrogenase, by transferring
hydrogens from TPNH to DPN before
oxidation in the cytochrome system, could
increase the energy yield from a given
amount of TPNH produced by isocitrate or
glucose 6-phosphate oxidation. The increased amount of biologically useful energy
could be used for growth, for protein and
nucleic acid synthesis, for the imbibition of
water, and for the other physiologic effects
of estrogens.
Estrogen stimulation of the transhydrogenation reaction would tend to decrease
rather than increase the amount of TPNH
in the cell. Thus the estrogen-induced stimulation of the synthesis of steroids, fatty
acids, proteins, and purines in the uterus
can be explained more reasonably as due to
an increased supply of energy rather than
to an increased supply of TPNH.
The theory that estrogens stimulate transhydrogenation by acting as coenzymes
which are rapidly and reversibly oxidized
and reduced does not explain the pronounced estrogenic activity in vivo of stilbestrol, 17a-ethinyl estradiol, or bfsdehydrodoisynolic acid, for these substances do
not contain groups that could be readily
oxidized or reduced. The exact mechanism
STEROID SEX HORMONES
(359
of action of estrogens at the biochemical
level remains to be elucidated, but the
data available permit the formulation of a
detailed working hypothesis. The notable
effects of estrogens and androgens on behavior (see chapter by Young) are presumably due to some direct or indirect effect of the hormone on the central nervous
system. The explanation of these phenomena
in physiologic and biochemical terms remains for future investigations to provide.
B. ANDROGENS
Although there is a considerable body of
literature regarding the responses at the biologic level to administered androgens and
progesterone, much less is known about the
site and mechanism of action of these hormones than is known about the estrogens.
The review by Roberts and Szego (1953b)
deals especially with the synergistic and
antagonistic interactions of the several
steroidal sex hormones.
The rapid growth of the capon comb following the administration of testosterone
has been shown to involve a pronounced
increase in the amount of mucopolysaccharide present, as measured by the content
of glucosamine (Ludwig and Boas, 1950;
Schiller, Benditt and Dorfman, 1952). It
is not known whether the androgen acts by
increasing the amount or activity of one of
the enzymes involved in the synthesis of
polysaccharides or whether it increases the
amount or availability of some requisite
cofactor. Many of the other biologic effects
of androgens do not seem to involve
mucopolysaccharide synthesis and the
relation of these observations to the
other roles of androgens remains to he determined.
Mann and Parsons (1947) found that
castration of rabbits resulted in a decreased
concentration of fructose in the semen.
Within 2 to 3 weeks after castration the
amount of fructose in the semen dropped
to zero, but rapidly returned to normal following the subcutaneous implantation of a
pellet of testosterone. Fructose reappeared
in the semen of the castrate rat 10 hours
after the injection of 10 mg. of testosterone
(Rudolph and Samuels, 1949). The coagulating gland of the rat, even when trans
planted to a new site in the body, also responds by producing fructose when the host
is injected with testosterone. The amount
of citric acid and ergothioneine in the semen
is also decreased by castration and increased by the implantation of testosterone
pellets (Mann, 1955). The experiments of
Hers (1956) demonstrate that fructose
is produced in the seminal vesicle by
the reduction of glucose to sorbitol and
the subsequent oxidation of sorbitol to
fructose. The reduction of glucose requires TPNH as hydrogen donor and the
oxidation of sorbitol requires DPN as
hydrogen acceptor. The sum of these two
reactions provides for the transfer of hydrogens from TPNH to DPN. If androgens
act as cofactors which are reversibly oxidized and reduced, and thus transfer hydrogens from TPNH to DPN as postulated
by Talalay and Williams-Ashman (1958),
one would expect that an increased amount
of androgen, by providing a competing system for hydrogen transfer, would decrease
rather than increase the production of fructose. The marked increases in the citric
acid and ergothioneine content of semen
are not readily explained by this postulated
site of action of androgens.
An increase in the activity of /3-glucuronidase in the kidney has been reported
following the administration of androgens
(Fishman, 1951). This might be interpreted
as an arlaptive increase in enzyme induced
by the increased concentration of substrate,
or by a direct effect of the steroid on the
synthesis of the enzyme.
The respiration of slices of prostate gland
of the dog is decreased by castration or by
the administration of stilbestrol (Barron
and Huggins, 1944). The decrease in respiration occurs with either glucose or pyruvate
as substrate. The seminal vesicle of the rat
responds similarly to castration. Rudolph
and Samuels (1949) found that respiration
of slices of seminal vesicle is decreased by
castration and restored to normal values
within 10 hours after the injection of testosterone. Experiments by Dr. Phillip Corfman in our laboratory with slices of prostate
gland from patients with benign prostatic
hypertrophy showed that oxygen utilization
was reduced 50 per cent by estradiol added
660
PHYSIOLOGY OF GONADS
in vitro at a level of 1 /xg. per ml. Respiration of slices of the ventral prostate gland
of the rat is decreased by castration and increased by administered testosterone (Nyden and Williams-Ashman, 1953). These
workers showed that lipogenesis from acetate-l-C^* in the prostate is also significantly diminished by castration and
restored to normal by administered testosterone.
The succinic dehydrogenase of the liver
has been found to be increased by castration
and decreased by the administration of testosterone (Kalman, 1952; Rindani, 1958),
the enzyme is also inhibited by testosterone
added in vitro (Kalman, 1952). In contrast,
Davis, Meyer and McShan (1949) found
that the succinic dehydrogenase of the
prostate and seminal vesicles is decreased
by castration and increased by the administration of testosterone.
An interesting example of an androgen
effect on a specific target organ is the decreased size of the levator ani and
other perineal muscles of the rat following castration. The administration of
androgen stimulates the growth of these
muscles and increases their glycogen content
(Leonard, 1952). However, their succinoxidase activity is unaffected by castration
or by the administration of testosterone.
Courrier and Marois (1952) reported that
the growth of these muscles stimulated by
androgen is inhibited by cortisone. The
remarkable responsiveness of these muscles
to androgens in vivo gave promise that
slices or homogenates of this tissue incubated with androgens might yield clues as to
the mode of action of the male sex hormones. Homogenates of perineal and masseter muscles of the rat responded to androgens administered in vivo with increased
oxygen consumption and ATP production
iLoring, Spencer and Villee, 1961). The experiments suggested that the activity of
DPNH-cytochromo r reductase in these
tissues is controlled by aiKh'ogeiis.
C. PROGESTERONE
Attempts to clarify the biochemical basis
of the role of progesterone have been hampered by the requirement, in most instances,
for a previous stimulation of the tissue by
estrogen. The work of Wade and Jones
(1956a, b) demonstrated an interesting effect of progesterone added in vitro on several aspects of metabolism in rat liver mitochondria. Progesterone, but not estradiol,
testosterone, 17a-hydroxyprogesterone, or
any of several other steroids tested, stimulated the adenosine triphosphatase activity
of rat liver mitochondria. This stimulation
is not the result of an increased permeability
of the mitochondrial membrane induced by
progesterone, for the stimulatory effect is
also demonstrable with mitochondria that
have been repeatedly frozen and thawed to
break the membranes. Other experiments
showed that ATP was the only substrate
effective in this system ; progesterone did not
activate the release of inorganic phosphate
from AMP, ADP, or glycerophosphate.
In other experiments with rat liver mitochondria (Wade and Jones, 1956b), progesterone at a higher concentration (6 X lO"'*
m) was found to inhibit the utilization of
oxygen with one of the tricarboxylic acids
or with DPNH as substrate. This inhibition
is less specific and occurred with estradiol,
testosterone, pregnanediol, and 17a-hydroxy progesterone, as well as with progesterone. The inhibition of respiration by high
concentrations of steroids in vitro has been
reported many times and with several different tissues; it seems to be relatively unspecific. Wade and Jones were able to show
that progesterone inhibits the reduction of
cytochrome c but accelerates the oxidation
of ascorbic acid. They concluded that progesterone may perhaps uncouple oxidation
from phosphorylation in a manner similar
to that postulated for dinitrophenol. The
site of action of this uncoupling appears to
be in the oxidation-reduction path between
DPNH and cytochrome c. Mueller (1953)
found that progesterone added in vitro decreases the incorporation of glycine-2-C^'*
into the protein of strips of rat uterus, thus
counteracting the stimulatory effect of estradiol administered in vivo.
Zander (1958) reported that A4-3-ketopregnene-20-a-ol and A4-3-ketopregnene20-^-ol arc effective gestational hormones
in the mouse, rabbit, and man, although
somewhat less active in general than is
progesterone. An enzyme in rat ovary which
converts progesterone to pregnene-20-a-ol,
and also catalyzes the reverse reaction, was
STEROID SEX HORMONES
661
described by Wiest (1956). The conversion
occurred when slices of ovary were incubated with DPN. Wiest postulated that
the progesterone-pregnene-20-a-ol system
might play a role in hydrogen transfer, in
a manner analogous to that postulated by
Talalay and Williams-Ashman (1958) for
estrone-estradiol- 17^, but his subsequent
experiments ruled out this possibility, for
he was unable to demonstrate any progesterone-stimulable transhydrogenation reaction.
The nature of the effect of progesterone
and of estrogens on myometrium has been
investigated extensively by Csapo. Csapo
and Corner (1952, 1953) found that ovariectomy decreased the maximal tension of
the myometrium and decreased its content
of actomyosin. The administration of estradiol to the ovariectomized rabbit over a
period of 7 days restored both the actomyosin content and the maximal tension of the
myometrium to normal. The concentration
of ATP and of creatine phosphate in the
myometrium is decreased by ovariectomy
but is restored by only 2 days of estrogen
treatment. This suggests that the effect on
intermediary metabolism occurs before the
effect on protein {i.e., actomyosin) synthesis. Csapo (1956a) concluded that estrogen
is a limiting substance in the synthesis of
the contractile proteins of myometrium, but
he could not differentiate between an effect
of estrogen on some particular biosynthetic
reaction and an effect of estrogen on some
fundamental reaction which favors synthesis in general. He was unable to demonstrate
any comparable effect of progesterone on
the contractile actomyosin-ATP system of
the myometrium.
Other observations provide an explanation for the well known effect of progesterone in decreasing the contractile activity of
myometrium, not by any effect on the contractile system itself, but in some previous
step in the excitation process. Under the
domination of progesterone the myometrial
cells have a decreased intracellular concentration of potassium ions and an increased
concentration of sodium ions (Horvath,
1954). The change in ionic gradient across
the cell membrane is believed to be responsible for the altered resting potential and
the partial depolarization of the cell mem
brane which results in decreased conductivity and decreased pharmacologic reactivity
of the myometrial cell. The means by which
progesterone produces the changes in ionic
gradients is as yet unknown. Csapo postulates that the hormone might decrease the
rate of metabolism which in turn would
lessen the rate of the "sodium pump" of the
cell membrane. The contractile elements,
the actomyosin-ATP system, are capable of
full contraction but, because of the partial
block in the mechanism of excitation and of
propagation of impulses (Csapo, 1956b),
the muscle cells cannot operate effectively;
the contractile activity remains localized.
Csapo (1956a) showed that the progesterone
block is quickly reversible and disappears if
progesterone is withdrawn for 24 hours. He
concluded that the progesterone block is
necessary for the continuation of pregnancy
and that its withdrawal is responsible for
the onset of labor.
]\Iost investigators who have speculated
about the mode of action of steroids —
whether they believe the effect is by activating an enzyme, by altering the permeability of a membrane, or by serving as a
coenzyme in a given reaction— have emphasized the physical binding of the steroid to
a protein as an essential part of the mechanism of action or a preliminary step to
that action. They have in this way explained
the specificities, synergisms, and antagonisms of the several steroids in terms of the
formation of specific steroid-protein complexes. The differences between different
target organs, e.g., those that respond to
androgens and those that respond to estrogens, can be attributed to differences in the
distribution of the specific proteins involved
in these binding reactions. Viewed in this
light, the problem of the mode of action of
sex hormones becomes one aspect of the
larger problem of the biochemical basis of
embryonic differentiation of tissues.
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PHYSIOLOGY OF GONADS
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