Book - Sex and internal secretions (1961) 11

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.

IV. References

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

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

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

062

PHYSIOLOGY OF GONADS

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

H.\germ.an, D. D., and Villee, C. A. 1952. Effects of estradiol on the metal)olism of human endometrium in vitro. Arch. Biocliem., 40, 481483.

Hagerman, D. D., .AND Villee, C. A. 1953a. Effects of estradiol on the metabolism of human endometrium in vitro. J. Biol. Clunn., 203, 425431.

Hagerman, D. D., AND Villee, C. A. 1953b. Effects of the menstrual cycle on the metabolism of human endometrium. Endocrinology, 53, 667-673.

HAGER.^L\x, D. D., AND ViLLEE, C. A. 1957. Estro

STEROID SEX HORMONES

663

gen sensitive isocitric dehydrogenase. J. Biol. Chem., 229, 589-597.

Hagerman, D. D., and Villee, C. A. 1958. Metabolic studies of the mechanism of action of estrogens. In Symposium on the Endocrinology of Pregnancy, Charles Lloyd, Ed., pp. 317328. New York: Academic Press, Inc.

Hagerman, D. D., Wellington, F. M., and Villee, C. A. 1957. Estrogens in marine invertebrates. Biol. Bull., 112, 180-183.

Hayano, M., and Dorf>l\n, R. I. 1953. The enzymatic C-lli3-hvdroxylation of steroids. J. Biol. Chem., 201,^175-188.

Hayano, M., Lindberg, M. C, Dorfman, R. I., Hancock, J. E. H., and Doering, W. von E. 1955. On the mechanism of the C-ll/3-hydroxylation of steroids; a study with H20^* and 02'^ Arch. Biochem., 59, 529-532.

Haynes, R. C, Sutherland, E. W., and Rall, T. W. 1960. The role of cyclic adenylic acid in hormone action. Recent Progr. Hormone Res., 16, 121-132.

Henion, W. F., and Sutherland, E. W. 1957. Immunologic differences of phosphorylases. J. Biol. Chem., 224, 477-488.

Herranen, a., and Mueller, G. C. 1956. Effect of estradiol on the metabolism of serine-3-C^* in surviving uterine segments. J. Biol. Chem., 223, 369-375.

Herranen, A. M., and Mueller, G. C. 1957. The effect of estradiol pretreatment on the serine aldolase activity of rat uteri. Biochim. et Biophys. Acta, 24, 223-224.

Hers, H. G. 1956. La mecanisme de la transformation de glucose ou fructose par les vesicles seminales. Biochim. et Biophys. Acta, 22, 202-203.

Hoagland, M. B., Keller, E. B., and Zamecnik, P. C. 1956. Enzymatic carboxyl activation of amino acids. J. Biol. Chem., 218, 345-358.

Holden, R. B. 1939. Vascular reactions of the uterus of the immature rat. Endocrinology, 25, 539-596.

Hollander, V. P., Nolan, H. M., and Hollander, N. 1958. The structural specificity of the estrogen-sensitive enzyme system in placental homogenates. J. Biol. Chem., 233, 580-582.

HoRVATH, B. 1954. Ovarian hormones and the ionic balance of uterine muscle. Proc. Nat. Acad.Sc, 40, 515-521.

Jailer, J. W. 1953. Virilism. Bull. New York Acad. Med., 29, 377-394.

Jones, H. W., Jr., W.^de, R., and Goldberg, B.

1952. Phosphate liberation by endometrium in the presence of adenosine triphosphate. Am. J. Obst. & Gynec, 64, 111&-1124.

Jones, H. W., Jr., Wade, R., and Goldberg, B.

1953. The uterus: biochemical and histochemical alkaline gh^cerophosphatase in normal endometrium, endometrial hyperplasia and adenocarcinoma. Obst. & Gynec. Surv., 8, 398-400.

Kalman, S. M. 1952. The effect of androgens on the succinoxide system of rat liver. Endocrinology, 50, 361-365.

Kaplan, N. O., Ciotti, M. M., Hamolsky, M., and Bieber, R. E. 1960. Molecular heterogeneity and evolution of enzymes. Science, 131, 392397. Kaplan, N., Schwartz, M., Frech, M.. and Ciotti, M. 1956. Phosphorylative and nonphosphorylative pathways of electron transfer in rat liver mitochondria. Proc. Nat. Acad. Sc, 42, 481-487. Kerly, M. 1937. The effect of the estrous cycle on the metabolism of isolated rat uterus. Biochem. J., 31, 1544-1552. Kh.\yyal, M. a., and Scott, C. M. 1931. The oxygen consumption of the isolated uterus of the rat and mouse. J. Physiol., 72, 13P-14P. KiPNis, D. M., and Cori, C. F. 1957. Studies of tissue permeability. III. The effect of insulin on pentose uptake by the diaphragm. J. Biol. Chem., 224, 681-693. Knobil, E., Morse, A., Wolf, R. C, and Creep, R. O. 1958. The action of bovine, porcine, and simian growth hormone preparations on the costochondral junction in the hypophysectomized rhesus monkey. Endocrinology, 62, 348-354. Knox, W. E., and Auerb.ach, V. H. 1955. The hormonal control of trvptophan peroxidase in the rat. J. Biol. Chem., 214, 307-313. Knox, W. E., Auerbach, V. H., and Lin, E. C. C. 1956. Enzymatic and metabolic adaptations in animals. Physiol. Rev., 36, 164-254. Koch, F. C. 1939. Biochemistry of androgens. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., pp. 807-845. Baltimore: The Williams & Wilkins Company. Kochakian, C. D. 1947. Effects of estrogens on the body and organ weights and the arginase and "alkaline" and "acid" phosphatases of the liver and kidney of castrated male mice. Am. J.Physiol., 151, 126-129. Krahl, M. E., and Bornstein, J. 1954. Inhibition of glucose use in muscle extracts by lipoproteins. Nature, London, 173, 949-950. L.\NGDON, R. G., AND Bloch, K. 1953. The biosvnthesis of squalene. J. Biol. Chem., 200, 129-134. Langer, L. 1957. Preparation and properties of estradiol-17/3 dehydrogenase. Thesis, Radcliffe College. L.\nger, L., and Engel, L. L. 1956. Human placental estradiol-17-/3 dehydrogenase. Fed. Proc, 15, 296; J. Biol. Chem., 233, 583-588. Leonard, S. L. 1952. A glycostatic effect of testosterone on the perineal muscles of the rat. Endocrinology, 50, 199-205. Levedahl, B. H., .\nd Bernstein, H. 1954. Testosterone binding by modified bovine serum albumin. Arch. Biochem. , 52, 353-357. Levine, R., and Goldstein, M. S. 1955. On the mechanism of action of insulin. Recent Progr. Hormone Res., 11, 343-380. f

Li, C. H., and Papkoff, H. 1956. Preparation and properties of growth hormone from human

664

PHYSIOLOGY OF GONADS

and monkey pituitary glands. Science, 124, 1293-1294.

LoRixG. J. M., Spencer. J. M., .and Villee, C. A. 1961. Some effects of androgens on intermediar\' metabolism in muscle. Endocrinology, 68, 501-506.

LoRi.vG, J. M.. AND Villee, C. A. 1957. The estrogen content of human placentas. Acta endocrinoL, 25, 371-376.

LuDwiG, A. W., AND Boas, N. F. 1950. The effects of testosterone on the connective tissue of the comb of the cockerel. Endocrinology, 46, 291298.

MacLeod, J., and Reynolds, S. R. M. 1938. Vascular, metabolic, and motility responses of uterine tissue following administration of estrin. Proc. Soc. Exper. Biol.. & Med., 37, 666-668.

Mann, T. 1955. Male sex hormone and its role in reproduction. Recent Progr. Hormone Res., 12, 353-376.

Mann, T., and Parsons, U. 1947. Effect of testicular hormone on the formation of seminal fructose. Nature, London, 160, 294.

Marrian, G. F., and Bauld. W. S. 1955. The isolation of 16-epioestriol from the urine of pregnant women. Biochem. J., 59, 136-141.

Meyer, A. S. 1955. 19-Hydroxylation of Mandrostene-3 , 17-dione and dehydroepiandrosterone by bovine adrenals. Experientia, 11, 99-102.

Mueller, G. C. 1953. Incorporation of glycine2-C" into protein by surviving uteri from estradiol-treated rats. J. Biol. Chem., 204, 77-90.

Mueller, G. C. 1957. A di.^^cussion of the mechanism of action of steroid liormones. Cancer Res., 17, 490-506.

Mueller, G. C., and Herranen, A. 1956. Metabolism of 1-carbon fragments by surviving uteri from estradiol-treated rats. J. Biol. Chem., 219, 585-594.

Mupxler, G. C, Herranen, A. M., and Jervell, K. F. 1958. Studies on the mechanism of action of estrogens. Recent Progr. Hormone Res., 14, 95-139.

Nathanson, I. T., Engel. L. L., .and Kelley, R. M. 1951. Effect of ACTH on urinary excretion of steroids in neoplastic disease. In Proceedings of the Second ACTH Conjerence, J.R. Mote, Ed., Vol. 1, p. 54.

NyDEN, S. J., AND WILLLAMS-ASHMAN, H. G. 1953.

Influence of androgens on synthetic reactions

in ventral prostate tissue. Am. J. Plivsiol.,

172,588-600. Odell, L. D., AND Flshman, W. H. 1950. Studios

on /3-glucuronidase. I. Activities in himian

endometrium. Am. J. Obst. & Gvnec, 59, 200 203. Page, E. W., Glendening, M. B., and Parkin.son.

P. 1951. Cyclic biochemical changes in tlic

human endometrium. Am. J. Obst. it Gvn(H'.,

62, 1100-1105. Pop.iAK, G. 1950. Preparation of solid samples

for assay of C". Biochem. J., 46, 560-561.

Rilling, H., Tchen, T. T., and Bloch, K. 1958. On the mechanism of srjualene biogenesis. Proc. Nat. Acad. Sc, 44, 167-173.

RiND.ANi, T. H. 1958. Influence of testosterone propionate on succinic dehydrogenase activity of male rat liver. Am. J. Phj^siol., 193, 73-74.

Roberts, S., and Szego, C. M. 1953a. The influence of steroids on uterine respiration and glycolysis. J. Biol. Chem., 201, 21-29.

Roberts, S., and Szego, C. M. 1953b. Steroid interactions in the metabolism of reproductive target organs. Physiol. Rev., 33, 593-629.

Rudolph, G. G., and S.amuels, L. T. 1949. Early effects of testosterone propionate on the seminal vesicles of castrate rats. Endocrinology, 44, 190-196.

Ryan, K. 1958. Conversion of androgens to estrogens bv human placental microsomes. Fed. Proc, 17, 1382.

Ryan, K. J., and Engel, L. L. 1953. The interconversion of estrone and estradiol-17i3 by rat liver slices. Endocrinology, 52, 277-286.

Schiller, S., Benditt, E., and Dorfman, A. 1952. Effect of testosterone and cortisone on the hexosamine content and metachromasia of chick combs. Endocrinology, 50, 504-510.

Solomon, S., Levitan, P., and Lieberman, S. 1956. Possible intermediates between cholesterol and pregneneolone in corticosteroidogenesis. Abstract. Proc. Canad. Physiol. Soc, Rev. Canad. Biol., 15, 282.

Spaziani, E., and Szego, CM. 1958. The influence of estradiol and Cortisol on uterine histamine of the ovariectomized rat. Endocrinology, 63, 669-678.

Stuermer, V. M., .AND Stein, R. J. 1952. Cytodynamic properties of the human endometrium. V. Metabolism and the enzymatic activity of the human endometrium during the menstrual cycle. Am. J. Obst. & Gynec, 63, 359-370.

Tal.alay, p., and Williams-Ashman, H. G. 1958. Activation of hydrogen transfer between pyridine nucleotides bv steroid hormones. Proc Nat. Acad. Sc, 44, 15-26.

Tal.alay, P., Willi.ams-Ashman, H. G., and HurLOCK, B. 1958. Steroid hormones as coenzymes of hydrogen transfer. Science, 127, 1060.

Tchen, T. T., and Bloch, K. 1955. In vitro conversion of squalene to lanosterol and cholesterol. J. Am. Chem. Soc, 77, 6085-6086.

Telfer, M. a. 1953. Influence of estradiol on nucleic acids, respiratory enzymes and the distribution of nitrogen in the rat uteru.*. Arch. Biochem., 44, 111-119.

Victor, J., and Andersen, D. H. 1937. Stimulation of anterior hypophysis metabolism by theelin or dihydrotheelin. Am. J. Physiol., 120, 154-166.

Villee. C. A. 1955. An estradiol-induccd stimulation of citrate utilization by placenta. J. Biol. Chem., 215, 171-182.

Villee, C. A. 1957a. Effects of estrogens and antiestiogens in vitro. Cancer Res.. 17, 507511.

STEROID SEX HORMONES

665

ViLLEE, C. A. 1957b. Role of estrogens in regulating the metabolism of the placenta and endometrium. Fertil. & Steril., 8, 156-163.

ViLLEE, C. A. 1958a. Estrogens and uterine enzymes. Ann. New York Acad. Sc, 75, 524534.

ViLLEE, C. A. 1958b. Antagonistic effects of estrogens and thyroxine on an enzyme system in vitro. In Proceedings IV International Congress Biochemistry {Vienna), p. 115. London: Pergamon Press.

ViLLEE, C. A., AND GoRDON, E. E. 1955. Further studies on the action of estradiol in vitro. J. Biol. Chem., 216, 203-214.

ViLLEE, C. A., AND GoRDON, E. E. 1956. The stimulation by estrogens of a DPN-linked isocitric dehydrogenase from human placenta. Bull. Soc. chim. belg., 65, 186-201.

ViLLEE, C. A., AND Hagerman, D. D. 1953. Effects of estradiol on the metabolism of himian placenta in vitro. J. Biol. Chem., 205, 873-882.

ViLLEE, C. A., and Hagerman, D. D. 1957. Studies on the estrogen-sensitive isocitric dehydrogenase of the human placenta. In Proceedings International Syniposiinn Enzyme Chemistry. {Japan), Vol. 1, p. 287. Tokyo: Maruzen.

ViLLEE, C. A., AND H.\GER.MAN, D. D. 1958. On the identity of the estrogen-sensitive enzyme of human placenta. J. Biol. Chem., 233, 42-48.

ViLLEE, C. A., Hagerman, D. D., and Joel, P. B. 1960. An enzyme basis for the physiologic functions of estrogens. Recent Progr. Hormone Res., 16, 49-69.

ViLLEE, C. A., Joel. P. B., Loring, J. M., .\nd Spencer, J. M. 1960. Estrogen stimulation of ATP production and protein svnthesis. Fed. Proc, 19, 53.

ViLLEE, C. A., Loring, J. M.. and Sarner, A. 1958.

Isocitric dehydrogenases of the placenta. Fed Proc, 17, 328.

Wade, R., and Jones, H. W., Jr. 1956a. Effect of progesterone on mitochondrial adenosinetriphosphatase. J. Biol. Chem., 220, 547-551.

Wade, R., and Jones, H. W., Jr. 1956b. Effect of progesterone on oxidative phosphorylation. J. Biol. Chem.. 220, 553-562.

Walaas, O., and Walaas, E. 1950. The metabolism of uterine muscle studied with radioactive phosphorus P^". Acta physiol. scandinav 21, 18-26.

Wal-aas, O.. Wal.aas, E., .\nd Loken, F. 1952a. The effect of estradiol monobenzoate on the metaboli-sin of rat uterine muscle. Acta endocrinol., 10,201-211.

Wal.'^as, 0., Walaas, E., and Loken, F. 1952b. The effect of estradiol monobenzoate on the metabolism of the rat endometrium. Acta endocrinol., 11, 61-66.

Werbin, H. and LeRoy, G. V. 1954. Cholesterol: a precursor of tetrahydrocortisone in man. J. Am. Chem. Soc, 76, 5260-5261.

West. C. D., Damast, B. L., Sarro, S. D., and Pearson, 0. H. 1956. Conversion of testosterone to estrogens in castrated, adrenalectomized human females. J. Biol. Chem., 218, 409-418.

WiEST, W. G. 1956. The metabolism of progesterone to A4-pregnen-20a-ol-3-one in eviscerated female rats. J. Biol. Chem., 221, 461467.

Zander, J. 1958. Gestagens in human pregnancy. In Proceedings Conference on Endocrinology of Reproduction, C. W. Lloyd, Ed. New York: Academic Press, Inc.

Zondek, B., and Hestrin, S. 1947. Phosphorylase activity in human endometrium. Am. J. Obst. ct Gvnec, 54, 173-175.

NUTRITIONAL EFFECTS ON ENDOCRINE SECRETIONS

James H. Leathern, Ph.D.

PROFESSOR OF ZOOLOGY, RUTGERS, THE STATE UNIVERSITY, NEW BRUNSWICK, NEW JERSEY

I. Introduction' 666 I, Introduction

II. Nature of Problems in Nutritional -j-. -, .^ ^ ,• ,• t ,

Studies 668 Despite the accumulation of many data

A. Thyroid Cxland, Nutrition, and Re- in the field of reproductive endocrinology

production ()68 during the past 20 years and the long es B. Adreiial Gland, Nutrition, and Re- ^ _^ tablished awareness of a nutritional in C. Diabetef^Mellitus, Nutrition, and ^f^f on fertility and fecundity, knowl Reproduction ()72 edge bearing on nutrition and the endocrine

D. Sterile-Obese Syndrome 673 glands subserving reproduction has ad E. Diet and the Liver 673 vanced comparatively slowly. However, re III. Hypophysis and Diet 674 markable advances have been made in each

A. Inanition 674 speciality SO that nutritional-endocrine

^roein. .. w problems should continue to be a fruitful

C. Carbohvdrate and Fat ()7() ^ „ ^i-r^x i-ii

D Vitamins 676 ^^^^^ ^^^' ^tudy. Data which have yet to

IV. Male Reproductive System (i77 be obtained eventually w'ill contribute to

A. Testis 677 the coherence one would prefer to present

1. Inanition 677 now.

2. Protein 678 'y\^q endocrinologist appreciates the deli 4 Vitamins (i8() ^^^^ balance which exists between the hy B. Influence of Nutrition on the Respon- ' pophysis and the gonads. In a sense, a simi siveness of Male Reproductive Tis- lar interdependence exists between nutrition

sues to Hormones 681 and the endocrine glands, including those

1. Testis ■ . . 681 ^j^|-^ reproductive functions. Not only does

2. feeminal vesicles and i)rostate 682 x •<• • n xi • j i -•

V. Female Reproductive System 683 nutrition influence synthesis and release of

A. Ovaries 683 hormones, but hormones in turn, through

1. Inanition 683 their regulation of the metabolism of pro 2. Protein 684 teins, carbohydrates, and fats, influence nu 3. Carbohydrate 685 trition. Thus, dietary deficiencies may create

5 Vitamins 685 endocrine imbalance, and endocrine imbal B. Influence of Nutrition on the ResiK)n- '^^ce may create demands for dietary fac siveness of Female Pe])ro<luct ive tors. It follows, therefore, that, in any conTissues to Hormones 687 sideration of this interrelationship, one must

1 . Ovary ... 687 consider not only undernutrition and lack of

2. uterus and vagina 688 -n c j \ l ^ i ^ cc i. r

3. Mammary gland 689 specific foods, but also possible effects of

C. Pregnancy. . 689 antithyroid substanccs in foods, antimetab VI. Concluding Remarks 693 olites, and overnutrition, especially for the

VII.Pkferencks (;!)4 child (Forbes, 1957).

666

NUTRITIONAL EFFECTS

667

Our understanding of the means by which hormones exert their effects is relatively slight, as is our knowledge of the biochemical mechanisms by w^hich supplements and deficiencies of vitamins and amino acids influence hormone action. Nevertheless, support for the statement that modifications of nutrition influence endocrine gland secretions or hormone action on distant target organs or tissues is provided by an enumeration of a few basic cell components requiring proteins, lipids, and vitamins. (1) Proteins combine with lipids to form lipoproteins which are essential features of the internal and external cellular membranes and interfaces. Hormones, as well as nutrition, influence cell membranes and therefore cell transport is affected. It is well known that hormones influence electrolyte and carbohydrate transfer and recently an endocrine control of amino acid transport was demonstrated (Noall, Riggs, Walker and Christensen, 1957). The effect of modifications of nutrition on the capacity of hormones to influence cell transport must await study. (2) Enzymes are proteins with chemically active surfaces and often include nonprotein groups such as vitamins. Nutritional and hormonal changes cause alterations in enzyme concentrations (Knox, Auerbach and Lin, 1956). Vitamin, mineral, and fat deficiencies favor a decrease in enzymes, whereas protein deficiencies have varied effects (Van Pilsum, Speyer and Samuels, 1957). Enzyme changes caused by hormones appear to be a consequence of metabolic adaptations. The importance of a nutritional base on which a hormone can express an effect on the enzymes of the reproductive organs can only be determined after further data have been obtained. (3) Proteins combined with nucleic acids become nucleoproteins, some of which are organized in the cytoplasm and may be templates for cellular protein synthesis. Other nucleoproteins are contained in the nucleus. Nutrition and hormones influence tissue nucleoproteins but studies involving the reproductive organs are few. How^ever, one possible cause of human infertility is low desoxyribose nucleic acid in the sperm ("Weir and Leuchtenberger, 1957).

Proteins are characteristic components of

tissues and hypophyseal hormones are protein in nature; also the major portion of gonadal dry weight is protein. Such being the case, it is important to appreciate that the protein composition of the body is in a dynamic state and that proteins from the tissues and from the diet contribute to a common metabolic pool of nitrogen. This metabolic pool contains amino acids which may be withdrawn for rebuilding tissue protein and for the formation of new protein for growth. Obviously, the character of the metabolic pool of nitrogen reflects dietary protein level and quality. A food protein which is deficient in one or more amino acids will restrict tissue protein synthesis. Hormones also influence the metabolic pool by affecting appetite as well as absorption, utilization, and excretion of foods, and thus hormones could accentuate the effect of a poor diet, or create demands beyond those normally met by an adequate diet. In addition a study of the tissues and organs of the body reveals that contributions to the metabolic pool are not uniform, thus one tissue or organ may be maintained at the expense of another. In protein deprivation in adults the liver quickly contributes increased amounts of nitrogen to the metabolic pool whereas the testis does not. On the other hand, protein contributions to the nitrogen pool by the hypophysis, and the amino acid withdrawals needed for hormone synthesis, are unknown. Data suggesting that the addition of specific nutrients to diets improves hypophyseal hormone synthesis have been presented (Leathem, 1958a).

In 1939 jNIason rightfully emphasized the need for vitamins in reproduction. Since then, much additional knowledge has been obtained. Vitamins of the B complex have been more clearly identified and a better understanding of their function has been gained. Thiamine is important for carbohydrate metabolism, pyridoxine for fat metabolism, and the conversion of tryptophan to nicotinic acid, and vitamin B12 may be involved in protein synthesis. In addition, vitamins have been found to serve as coenzymes, and folic acid to be important for estrogen action on the uterus. Tliosr. and

668

PHYSIOLOGY OF GONADS

many other findings prompt a survey of the relationships of vitamins to reproduction.

It is the intention of the author to review enough of the evidence which interrelates nutrition and reproduction to create an awareness of the problems in the area. Reviews dealing with the general subject of hormonal-nutritional interrelationships have been presented (Hertz, 1948; Samuels, 1948; Ershoff, 1952; Zubiran and GomezMont, 1953; Meites, Feng and Wilwerth, 1957; Leathem, 1958a,). Other reviews have related reproduction to nutrition with emphasis on laboratory (Mason, 1939; Guilbert, 1942; Lutwak-Mann, 1958) and farm animals (Reid, 1949; Asdell, 1949), and on protein nutrition (Leathem, 1959b, c). An encyclopedic survey of the biology of human nutrition has been made by Keys, Brozec, Hernschel, Michelsen and Taylor (1950).

II. Nature of Problems in Nutritional Studies

A. THYEOID GLAND, NUTRITION, AND REPRODUCTION

Normal development of the reproductive organs and their proper functioning in

TABLE 12.1 Ovarian response to chorionic gonadrotrophin,

as modified by thiouracil and diet

(From J. H. Leathem, in Recent Progress in

the Endocrinology of Reproduction, Academic

Press, Inc., New York, 1959.)

Diet

Ovarian Weight

Cholesterol

Total

Free

18 per cent casein .

18 tier cent +

I'hiouracil

per cent casein . . per cent + thiouracil

18 per cent gelatin . . .

mg.

87 342

59 133

59 186 27.5

% 0.41

0.20

0.47

0.22

0.66 0.26 1.56

0.18 0.12 0.23 0.15

0.22

18 per cent -f thio uracil

0.18 18

Chorionic gonadotrophin = 10 I. U. X 20 days.

adults are dependent not only on the endocrine glands composing the hypophysealgonadal axis, but on others as well. The importance of the thyroid, although not the same for all species, is readily apparent from the effects of prolonged hypo- and hyperthyroid states on reproduction. Many of these effects have been enumerated elsewhere (chapters by Albert, by Young on the ovary, and by Zarrow) , but others also are important. Thus steroid production may be altered in hypothyroid animals; certainly its metabolism is influenced. Myxedema is associated with a profound change in androgen metabolism. Endogenous production of androsterone is very low and subnormal amounts of administered testosterone are converted to androsterone. Triiodothyronine corrects this defect (Hellman, Bradlow, Zumoff, Fukushima and Gallagher, 1959). The gonads of male and female offspring of cretin rats are subnormal. The testes may contain a few spermatocytes but no spermatozoa, and Leydig cells seem to secrete little or no androgen. The ovaries may contain a few small follicles with antra, but corpora lutea are absent and ovarian lipid and cholesterol concentrations are very low. Nevertheless, the gonads are competent to respond to administered gonadotrophin with a marked increase in weight. However, administration of chorionic gonadotrophin to the hypothyroid rat stimulated follicular cyst formation rather than folliculogenesis and corpora lutea formation (Leathem, 1958b), but, for even this aberrant development, dietary protein was required (Leathem, 1959b) (Table 12.1). The relationship between hypothyroidism and ovarian function may provide a clue to a possible origin of ovarian cysts, long known to be a common cause of infertility and associated reproductive disorders. Clinical cases of untreated myxedema exhibit ovarian cysts, and rats made hypothyroid for eight months, had a higher percentage of cystic ovaries than did euthyroid rats (Janes, 1944).

The reproductive system of the adult male is less affected than that of the immature male by a decrease in thyroid function, just as the testis of the adult is less likely to reflect a change in protein nutrition which is sufficient to alter the immature rat

NUTRITIONAL EFFECTS

669

testis. On the other hand, the lack of demonstrable thyroid dysfunction in the adult male does not exclude the possibility of an effect of thyroid hormone on reproduction. The conversion of thyroxine to triiodothyronine may be hindered (Morton, 1958) . Thyroxine was found to decrease the number of active cells in the semen and to reduce motility, whereas triiodothyronine increased the number of active spermatozoa (Farris and Colton, 1958; Reed, Browning and O'Donnell, 1958j. Small dosages of thyroxine stimulated spermatogenesis in the mouse, rabbit, and ram (Maqsood, 1952) and were beneficial in normal guinea pigs and rats (Richter and Winter, 1947; Young, Rayner, Peterson and Brown, 1952).

In many species reproduction occurs despite hypothyroidism, but fecundity may be subnormal (Peterson, Webster, Rayner and Young, 1952). Feeding an antithyroid drug, thiouracil, to female rats may or may not prevent pregnancy, but it reduces the number of young per litter. Thiouracil feeding continued through lactation will decrease litter size (Leathem, 1959b). Hypothyroid guinea pigs gave birth to some live young, but the percentage approached normal only when thyroxine was administered (Hoar, Goy and Young, 1957). Pregnant euthyroid animals responded to thyroxine by delivering more living young than normal control pigs (Peterson, Webster, Ravner and Young, 1952) . The extent to which such effects are consequences of the reduction in appetite, metabolism, and absorption of food from the gut which are associated with hypothyroidism has not been determined.

Hyperthyroidism, on the other hand, will increase the appetite and enhance absorption of food from the gut, as the increased metabolism requires more calories, minerals, vitamins, choline, and methionine. In adult rats hyperthyroidism induces a marked loss in body fat and accelerates protein catabolism. The two effects, if unchecked, result in loss of body weight and death. In immature males hyperthyroidism slows gain in body weight, retards testis growth and maturation, and abolishes androgen secretion (Table 12.2). Altering dietary protein in adult animals failed to modify the thyroid hormone effects, thereby suggesting

TABLE 12.2

Effects of diet and thyroid {0.2 'per

cent) on immature rats

(From J. H. Leathem, in Recent Progress in

the Endocrinology of Reproduction, Academic

Press, Inc., New York, 1959.)

Diet

Testis Weight

Seminal

(Casein) X 30 Days

Actual

Relative

Vesicles

20 per cent

20 per cent + thyroid

6 per cent

6 per cent + thyroid

per cent

per cent -|thyroid

mg. 1G94

1090

825

245

140

95

mg./lOOgm.

1035

881 1232

650

346

261

mg. 88

9 16

7 7 6

that the metabolic demands of other tissues were making increased withdrawals from the metabolic pool of nitrogen and thus hindering testis growth. In euthyroid rats, a 6 per cent protein diet will permit testis growth in the absence of a gain in body weight, but hyperthyroidism prevents this preferential effect. Although Moore (1939) considered the effect of thyroid hormone on reproduction as possibly due to general body emaciation, the testis seems to be less responsive than the body as a whole. Adult rats fed 0.2 per cent desiccated thyroid exhibited no correlation between loss of body weight, change in testis weight, or protein composition of testis at two levels of casein and lactalbumin (Leathem, 1959b) . The testes were seemingly not influenced by the metabolic nitrogen changes which caused a loss in carcass nitrogen and an associated increase in kidney and heart nitrogen.

The mechanism of thyroid hormone action on reproduction is far from clear. As we have noted, a part of its action may be through the regulation of nutritional processes. Thyroid function is influenced by the biologic value of the dietary protein (Leathem, 1958a) and the specifie amino acids fed (Samuels, 1953). In turn, an altered thyroid function will influence the nitrogen contributions to the metabolic pool by reducing appetite and absorption from

670

PHYSIOLOGY OF GONADS

the gut, and by changing the contributions of nitrogen to the metabolic pool made by the body tissues. Hypothyroidism interferes with the refilling of body protein stores; consequently, protein needs of the reproductive organs may not be fully met (Leathern, 1953). It is consistent with this opinion that testis recovery from protein deprivation in hypothyroid rats was aided by thyroxine treatment (Horn, 1955).

Conversion of carotene to vitamin A may be prevented by hypothyroidism, suggesting that a subnormal amount of this vitamin may contribute to fetal loss. An increased intake of B vitamins might be required as hypothyroidism aggravates a vitamin B12 deficiency, and increased intake of B vitamins enhances the capacity of young rats to withstand large doses of thiouracil (Meites, 1953). Although a reduced metabolic rate might seemingly reduce vitamin requirements, more efficient metabolic activities, in the absence of hormonal stimuli, seem to occur when vitamin intake is increased (Meites, Feng and Wilwerth, 1957).

Reproduction is influenced by effects which are the opposite of a number of those just cited, i.e., effects of malnutrition on thyroid function. The need for iodine in the prevention of goiter is well known. However, certain foods prevent the utilization of iodine in the synthesis of thyroid hormone. The foods containing an antithyroid or goitrogenic agent, as tested in man, include rutabaga, cabbage, brussels sprouts, cauliflower, turnip, rape, kale, and to a lesser extent peach, pear, strawberry, spinach, and carrot (Greer and Astwood, 1948). A potent goitrogen isolated from rutabaga is L-5-vinyl-2-thiooxazolidine ((ireer, 1950, 1956). Reduced food intake will decrease the thyroid gland response to goitrogens (Gomez-Mont, Paschkis and Cantarow, 1947; Meites and Agrawala, 1949), the uptake of I^^^ (Meites, 1953), and the level of thyrotrophic hormone in laboratory animals (D'Angelo, 1951). The thyroid changes associated with malnutrition in man arc uncertain (Zubiran and Gomez-Mont, 1953). However, a decreased functioning of the gland in anorexia nervosa, followed by an increased functioning on refeeding (Poiioff, Lasche, Nodine,

Schneeberg and \'ieillard, 1954), is suggestive of a direct nutritional need.

Changes in the thyrotrophic potency of the rat hypophysis have been observed in various vitamin deficiencies. Thiamine deficiency may increase thyroid function, but vitamin A deficiency may have the opposite effect (Ershoff, 1952). The difficulties inherent in the assay of thyroid-stimulating hormone (TSH) , even by current methods, prevent one from drawing definite conclusions from the available data.

Immature animals given thyroxine are retarded in growth and do not survive. However, increasing dietary thiamine, pyridoxine, or vitamin B12 improves the ability of the young rat to withstand large dosages of thyroid substances (Meites, 1952), as does methionine (Boldt, Harper and Elvehjem, 1958). Consideration must also be given to the need for nutritional factors which play a minor role in normal metabolic states but increase in importance in stress. Thus yeast and whole liver contain antithyrotoxic substances (Drill, 1943; Ershoff, 1952; Overby, Frost and Fredrickson, 1959).

Excessive thyroid hormone will prevent maturation of the ovary and in adult rats will cause ovarian atrophy with a cessation of estrous cycles. Addition of yeast to the diet permitted estrous cycles to continue (Drill, 1943), but gonadal inhibition in the immature animal was not ]3revented. However, whole liver or its w^ater-insoluble fraction counteracted the gonadal inhibition induced by hyperthyrodism in immature rats (Ershoff, 1952). Biochemical mechanisms by which these dietary supplements can benefit rats given excessive quantities of hormone are unknown.

B. .\DREXAL GLAND, NUTRITION, AND REPRODUCTION

The problem of the relationship between adrenal steroid secretions and the reproductive system is one that still requires clarification. Furthermore, the possible influences of nutrition can only be inferred from the effects of adrenal steroids on the major metabolic systems of the body.

In the female there is a close relationshiji between the adrenal and the estrous and

NUTRITIONAL EFFECTS

iul

menstrual cycles (Zuckerman, 1953; chapter by Young on the ovary). The ovary would seem to require cortical steroids for the normal functioning of its own metabolic processes and for those which it influences peripherally. The Addisonian patient may show ovarian follicular atresia and a loss of secondary sex characteristics, and the untreated adrenalectomized rat exhibits a decrease in ovarian size and has irregular cycles (Chester Jones, 1957). The decline in size of the ovary after adrenalectomy is due to impaired sensitivity to folliclestimulating hormone (FSH) rather than to a decreased production of FSH, and the ovarian response is corrected by cortisone (Mandl, 1954).

Reproductive potential is not necessarily lost when there is adrenal insufficiency, but pregnancy is not well tolerated by women with Addison's disease. Furthermore, adrenalectomy in rats at the time of mating or 4 to 6 days after mating resulted in abortion. Improved pregnancy maintenance was obtained in adrenalectomized rats given saline or cortisone acetate (Davis and Plotz, 1954) , whereas desoxycorticosterone acetate alone extended the pregnancy period beyond normal time (Houssay, 1945). Essentially normal pregnancies were obtained in adrenalectomized rats given both cortisone acetate and desoxycorticosterone acetate (Cupps, 1955). Substitution of cortisone acetate for adrenal secretions may be incomplete because the adrenal hormone in the normal rat is primarily corticosterone and because cortisone enhances the excretion of certain amino acids and vitamins. It would be interesting to test a diet with a high vitamin content on the capacity of an adrenalectomized rat to maintain pregnancy, because improved survival of operated rats is obtained by giving vitamin Bio (Meites, 1953) or large doses of pantothenic acid, biotin, ascorbic acid, or folic acid (Ralli and Dumm, 1952; Dumm and Ralli, 1953).

An adrenal influence over protein metabolism is well known, but protein nutrition, in turn, can influence cortical steroid effectiveness. In fact, an extension of the life span of adrenalectomized rats is not obtained with adrenal steroids if the diet

lacks protein (Leathern, 1958a). A low protein diet alone will not improve survival after adrenalectomy, but better survival is obtained when the rats are given saline. When the low protein diet was supplemented with methionine, a definite improvement in life span was observed and the possibility that cortisone exerts its effect by drawing on the carcass for methionine was suggested (Aschkenasy, 1955a, b).

Reducing dietary casein to 2 per cent seriously endangers pregnancy in the rat, but the addition of progesterone permits 80 per cent of the pregnancies to be maintained. However, removal of the adrenal glands counteracts the protective action of the progesterone, only 10 per cent of the pregnancies continuing to term. Addition of methionine to the low casein diet improved pregnancy maintenance, but 1 mg. cortisone acetate plus progesterone provided the best results (Aschkenasy-Lelu and Aschkenasy, 1957; Aschkenasy and Aschkenasy-Lelu, 1957). These data emphasize the importance of nutrition in obtaining an anticipated hormone action. Further investigation might be directed toward the study of whole proteins other than casein, for the biologic value of proteins differs from normal when tested in adrenalectomized rats (Leathem, 1958a) .

As Albert has noted in his chapter, adrenalectomy has little or no effect on the testis. Gaunt and Parkins (1933) found no degenerative changes in the testes of adult rats dying of adrenal insufficiency, although an increase in the testis : body-weight ratios was noted in rats fed 18 per cent and 4 per cent protein (Aschkenasy, 1955c). If adrenalectomized rats are kept on a maintenance dosage of cortisone acetate for 20 days and fed dietary proteins of different biologic values, one finds that testis-composition of protein, lipid, and glycogen varies in the same manner as in the normal rat (Wolf and Leathem, 1955) (Table 12.3).

When the adrenal glands are intact, the influence of diet on their functional capacity and indeed on the hypophyseal- adrenal axis must be considered. Zubiran and Gomez-]\Iont (1953) showed that patients exhibiting gonadal changes associated with chronic malnutrition also exhibit adrenal

672

PHYSIOLOGY OF GOXADS

TABLE 12.3

Nutritional effects on the testes of cortisonemaintained adrenalectomized rats (From R. C. Wolf and J. H. Leathern, Endocrinology, 57, 286, 1955.)

Testes Composition

(per cent)

Treatment

Diet

Testes

Protein

Lipid

Glycogen

mg.

Corti

20 per cent

703

68.5

30.4

0.13

sone

casein

ace

tate

Control

1211

70.5

29.4

0.15

Corti

20 per cent

808

61.0

31.8

0.17

sone

wheat

ace

ghiten

tate

Control

816

62.3

32.2

0.17

Corti

20 per cent

1014

64.3

31.3

0.17

sone

peanut

ace

flour

tate

Control

699

68.5

31.3

0.12

hypofimction. Several clinical tests permitted the evaluation of subnormal adrenal function which, however, did not reach Addisonian levels. Malnutrition not onlyreduced hypophyseal adrenocorticotrophic hormone (ACTH), but also prevented an incomplete response by the adrenal glands to injected ACTH. In laboratory rodents, anterior hypophyseal function is also influenced by dietary protein and vitamin levels (Ershoff, 1952). The importance of dietary protein in the hypophyseal-adrenal system has recently been re-emphasized (Leathem, 1957; Goth, Nadashi and Slader, 1958). Furtiiermore, adrenal cortical function is affected by vitamin deficiencies (Morgan, 1951), from which it appears that pantothenic acid is essential for cortical hormone elaboration (Eisenstcin, 1957). Administration of excessive amounts of cortical steroids can induce morphologic changes which have been compared to inanition (Baker, 1952). Not only is nitrogen loss enhanced, but hyperglycemia can also be induced which, therefore, increases the need for thiamine. Cortical steroids influence the metabolism of various vitamins

(Draper and Johnson, 1953; Dhyse, Fisher, Tullner and Hertz, 1953; Aceto, Li Moli and Panebianco, 1956; Ginoulhiac and Nani, 1956). H a vitamin deficiency already exists, administration of cortisone will aggravate the condition (Meites, Feng and Wilwerth, 1957). Nevertheless, drastic effects of therapeutic doses of cortisone on reproductive function do not occur. In rare cases loss of libido has been reported in the male, but mori)hologic changes in the testis were not observed (JVladdock, Chase and Nelson, 1953). Cortisone has little if any effect on the weight of the rat testis (Moore, 1953; Aschkenasy, 1957) and does not influence testis cholesterol (Migeon, 1952). In the female, menstrual disturbances have been noted in association with cortisone therapy, with the occurrence of hot flashes (Ward, Slocumb, Policy, Lowman and Hench, 1951). However, cortisone corrected disturbances during the follicular phase, possibly by increasing FSH release (Jones, Howard and Langford, 1953). Cortisone also increased the number of follicles in the ovary of the rat (Moore, 1953), but not in the rabbit. Cortisone administration did not prevent the enhanced ovarian response to chorionic gonadotrophin seen in hypothyroid rats (Leathem, 1958b) and had little effect on mice in parabiosis ( Noumura, 1956).

In pregnant female rabbits resorption and stunting of fetuses occurred during treatment with large doses of cortisone (Courrier and Collonge, 1951). Similar effects were noted in mice (LcRoy and Domm, 1951 ; Robson and Sharaf, 1951).

Some of the metabolic derangements of human toxemia of pregnancy have been correlated with accelerated secretion of adrenal steroids creating a steroid imbalance (see chapter by Zarrow). Cortisone is reported to have a beneficial effect on some cases (Moore, Jessop, 'Donovan, Barry, Quinn and Drury, 1951). Protein inade({uacies may also be etiologic in toxemia and further (>xamination of the possibility sliould he made.

C. DIABETES MELLITUS, NUTRITION, AND REPRODUCTION

(llycosuria can be induced experimentally by starvation, overfeeding, and shifting

NUTRITIONAL EFFECTS

673

diet^ from one of high fat content to one i; which is isocaloric but high in carbohydrate (Ingle, 1948). Force feeding a high carbohydrate diet will eventually kill a rat despite insulin administration aimed at controlling glycosuria (Ingle and Nezamis, 1947). In man excessive eating leading to obesity increases insulin demand and, in many diabetics of middle age, obesity precedes the onset of diabetes. With our present knowledge we must conclude that overfeeding is wrong when glycosuria exists and that vitamin B supplements may be of value in diabetes (Meites, Feng and Wilwerth, 1957; Salvesen, 1957).

In man urinary 17-ketosteroids and androgen levels are subnormal in diabetes (Horstmann, 1950), and in the diabetic rat pituitary gonadotrophins are reduced (Shipley and Danley, 1947), but testis hyaluronidase does not change (Moore, 1948) . When hyperglycemia exists in rats, semen ■] carbohydrates increase (Mann and Lutwak-Mann, 1951).

Hypoglycemia influences the male reproductive organs. In rats tolbutamide or insulin produce lesions of the germinal epithelium which can be prevented by \' simultaneous administration of glucose. When 2 to 5 hypoglycemic comas are induced, such testis injuries increase progressively in number and frequency, and only a partial return to normal is observed a month later (Mancini, Izquierdo, Heinrich, Penhos and Gerschenfeld, 1959).

It is well known that the incidence of infertility in the pre-insulin era was high in young diabetic women. Fertility is also reduced in diabetic experimental animals, and rat estrous cycles are prolonged (Davis, Fugo and Lawrence, 1947) . Insulin is corrective (Sinden and Longwell, 1949; Ferret, Lindan and Morgans, 1950). Pregnancy in women with uncontrolled diabetes may terminate in abortion or stillbirth, possibly l)ecause toxemia of pregnancy is high (Pedersen. 1952). In rats pancreatectomy performed the 8th to 12th day of pregnancy increased the incidence of stillbirths (Hultciuist, 1950). In another experiment almost one- fourth of 163 animals with diabetes induced by alloxan on the 10th to 12th day of pregnancy died before parturition and

about 25 ])er cent of the survivors aborted (Angcrvall, 1959).

D. STERILE-OBESE SYNDROME

A sterile-obese syndrome in one colony of mice has been shown to be a recessive monogenic trait (Ingalls, Dickie and Snell, 1950). Obesity was transmitted to subsequent generations by way of ovaries that were transplanted from obese donors to nonobese recipients (Hummel, 1957). Obesity was transmitted by obese females receiving hormonal therapy and mated to obese males kept on restricted food intake (Smithberg and Runner, 1957). In addition to the investigations of the hereditary nature of the sterile-obese syndrome, the physiologic basis for the sterility has been studied in reference to the presence of germ cells, viability of ova and sperm, integrity of the ovary, and response of the uterus to estrogen (Drasher, Dickie and Lane, 1955). The data indicate that sterility in some obese males can be prevented by food restriction and that sterility in certain obese females can be corrected.

E. DIET AND THE LIVER

The concentration of hormones which reaches the target organs in the blood is the result of the rate of their production, metabolism, and excretion. How hypophyseal hormones are destroyed is not clear, but current data make it apparent that pituitary hormones have a short half-life in the circulatory system. Exerting a major control over circulating estrogen levels is the liver, with its steroid-inactivating systems. Zondek (1934) initially demonstrated that the liver could inactivate estrogens and this finding has had repeated confirmation (Cantarow, Paschkis, Rakoff and Hansen, 1943; De:\Ieio, Rakoff, Cantarow and Paschkis, 1948; Vanderlinde and Westerfield, 1950). Other steroids are also inactivated by the liver with several enzyme systems being involved; the relative concentration of these enzymes varies among species of vertebrates (Samuels, 1949).

The liver is a labile organ which readib.' responds to nutritional modifications; the induced liver changes alter the steroid-inactivating systems of this organ. Thus, inanition (Drill and Pfeiffer, 1946; Jailer,

G74

PHYSIOLOGY OF GONADS

1948) , vitamin B complex deficiency (Segaloff and Segaloff, 1944; Biskind, 1946), and protein restriction (Jailer and Seaman, 1950) all influence the capacity of the liver to detoxify steroids. Reduced protein intake is a primary factor in decreasing the effectiveness of the steroid-inactivating system (Jailer and Seaman, 1950; Vanderlinde and Westerfield, 1950). Rats fed an 8 per cent casein diet lose their capacity to inactivate estrone within 10 days. However, ascorbic acid alone or in combination with glutathione restored the estrone-inactivating system (Vasington, Parker, Headley and Vanderlinde, 1958).

Failure of steroids to be inactivated will influence the hypophyseal-gonadal axis. In turn the excess of estrogen will decrease gonadotrophin production by the hypophysis and thus reduce steroid production by the gonad. In addition nutritional modifications influence hypophyseal and possibly gonadal secretory capacity directly. Conceivably, nutritional alterations could modify the amount of steroid secreted or interfere with complete steroid synthesis by a gland.

Fatty infiltration of the liver and a general increase in fat deposition occur in fed and fasted rats after the injection of certain pituitary extracts and adrenal steroids and after the feeding of specific diets. Impaired estrogen inactivation has been associated with a fatty liver, but Szego and Barnes (1943) believe that the major influence is inanition. In fact, estrogens, especially ethinyl estradiol, interfere with fatty infiltration of the liver induced by a low protein diet (Gyorgy, Rose and Shipley, 1947) or by a choline-deficient diet (Emerson, Zamecnik and Nathanson, 1951). Stilbestrol, however, did not prevent the increase in liver fat induced by a protein-free diet (Glasser, 1957). Estrogens that are effective in preventing fatty infiltration may act by sparing methionine or choline or by inhibiting growth hormone (Flagge, Marasso and Zimmerman, 1958).

Ethionino, the antimetabolite of nu'thioniiK', Avill induce a fatty liver and inhibit hepatic protein synthesis in female, but not in male rats (Farber and Segaloff, 1955; Farber and Corban, 1958). Pretreatment of females with testosterone prevents

the ethionine effect, but this blockage of ethionine action need not be related to androgenic or progestational properties of steroids (Ranney and Drill, 1957).

III. Hypophysis and Diet

Studies involving acute and chronic starvation have shown that gonadal hypofunction during inanition is primarily due to diminished levels of circulating gonadotrophins. Because of the similarity to changes following hypophysectomy, the endocrine response to inanition has been referred to as "pseudohypophysectomy."

A. INANITION

The hypophysis has been implicated in human reproduction disturbances associated with undernutrition. Hypophyseal atrophy and a decrease in urinary gonadotrophins have been observed in chronic malnutrition (Klinefelter, Albright and Griswold, 1943; Zubiran and Gomez-]\Iont, 1953) and anorexia nervosa (Perloff, Lasche, Nodine, Schneeberg and Vieillard, 1954). Refeeding has restored urinary gonadotrophin levels in some cases, but hypoj^hyseal damage may result from severe food restriction at puberty (VoUmer, 1943; Samuels, 1948).

The influence of inanition on the reproductive organs of lal)oratory rodents is well recognized but the cft'ects on the hypophysis cannot be presented conclusively. In support of prior investigations, Mulinos and Pomerantz (1941a, b) in rats and Giroud and Desclaux (1945) in guinea pigs observed a hypophyseal atrophy following chronic underfeeding as well as a decrease in cell numbers and mitoses. In fact, refeeding after chronic starvation resulted in only a partial recovery of hypophyseal weight (Quimby, 1948). Nevertheless, complete starvation did not influence relative gland weight in female rats (Meites and Reed, 1949), and cytologic evidence (periodic acid-Schiff (PAS) test) of an estimated 3- fold increase in gonadoti'ophin content was claimed following chronic starvation (Pearse and Rinaldini, 1950). Assays of hypophyseal gonadotrophin content in chronically starved rats of both sexes have l)een reported as decreased (Mason and Wolfe, 1930; Werner,

NUTRITIONAL EFFECTS

675

1939), unchanged (,]\Iarrian and Parkes, 1929; Pomerantz and Mulinos, 1939; Maddock and Heller, 1947; Meites and Reed, 1949; Blivaiss, Hanson, Rosenzweig and McNeil, 1954), or increased (Rinaldini, 1949; Vanderlinde and Westerfield, 1950). An increase in pituitary gonadotrophin was evident when hormone content was related to milligrams of tissue (Meites and Reed, 1949). Thus, the hormone release mechanism may fail in starvation, and eventually gonadotrophin production will be reduced to a minimum (]\laddock and Heller, 1947).

Gonadectomy of fully fed rats is followed by an increase in hypophyseal gonadotrophin content. Chronic starvation, however, prevented the anticipated changes in the pituitary gland following gonadectomy in 8 of 12 female rats (Werner, 1939). On the other hand, if adult female rats were subjected to 14 days of reduced feeding 1 month after ovariectomy, no change in the elevated gonadotrophin levels was noted (Meites and Reed, 1949). In contrast, Gomez-Mont (1959) observed above normal urinary gonadotrophins in many menopausal and postmenopausal women despite undernutrition.

It is apparent that uniformity of opinion as to how starvation influences hypophyseal gonadotrophin content has not been attained. Several explanations can be given for the discrepancies. (1) There have been unfortunate variations in experimental design. IVIaddock and Heller (1947) starved rats for 12 days, whereas Rinaldini (1949) used a low calorie diet of bread and milk for 30 days. Other variations in feeding have included feeding one-half the intake required for growth (Mulinos and Pomerantz, 1941b I, regimens of full, one-half, oneciuarter, and no feeding for 7 and 14 days (Meites and Reed, 1949), and feeding inadequate amounts of a standard rat diet for 1 to 4 months (Werner, 1939). (2) Hypophyseal implants and anterior pituitary extracts should not be compared, for variable gonadotrophin production may follow implantation procedures, depending on whether necrosis or growth occurs (Maddock and Heller, 1947). (3) There has been an insufficient standardization of experimental materials. The assay animal has usually been the immature female rat, but

occasionally the immature mouse has been used, and Rinaldini (1949) used the hypophysectomized rat.

B. PROTEIN

The need for specific food elements by the hypophysis warrants consideration, for the hormones secreted by this gland are protein in nature and the amino acids for protein synthesis must be drawn from body sources. However, dietary protein levels can vary from 15 per cent to 30 per cent without influencing hypophyseal gonadotrophin content in rats (Weatherby and Reece, 1941), but diets containing 80 per cent to 90 per cent of casein increased hypophyseal gonadotrophin (TuchmannDuplessis and Aschkenasy-Lelu, 1948). Removal of protein from the diet will decrease hypophyseal gonadotrophin content in adult male rats in comparison with pair-fed and ad libitum-ied controls, but the decrease may or may not be significant in a 30-day period; luteinizing hormone (LH) seemed to be initially reduced. Extension of the period of protein depletion another 2 months resulted in a significant lowering of hy]iophyseal gonadotrophin levels (Table 12.4) (Leathern, 1958a). On the other hand, an increased FSH with no decrease in LH activity was observed in the hypophyses of adult female rats following 30 to 35 days of protein depletion (Srebnik and Nelson, 1957). The available data indicate that not only may a sex difference exist, but also that species may differ; restitution of gonadotrophin in the discharged rabbit pituitary was not influenced by in TABLE 12.4

Influence of a protein-free diet on hypophyseal

gonadotrophin content

(From J. H. Leathern, Recent Progr. Hormone

Res., 14, 141, 1958.)

Days on PFD*

Xo. of Rats

Anterior Pituitary Weight

Recipient Ovarv Weight

nig.

mg.

9

8.3

74

30

9

7.3

54

50

7

7.0

33

90

17

6.0

23

L^ntreated recipient ovarian weight = 15.4 mg.

• PFD = Protein-free diet.

67G

PHYSIOLOGY OF GONADS

adequate dietary protein (Friedman and Friedman, 1940).

When anterior pituitar}^ glands of 60gm. male rats were extracted and administered to immature female recipients, ovarian weight increased from 13.0 to 37.3 mg. After feeding 20 per cent casein or fox chow ad libitum for 14 days, the hypophyses of male rats contained almost twice as much gonadotrophin per milligram of tissue as did the hypophyses of the initial controls. Removal of protein from the diet for 14 days, however, reduced hypophyseal gonadotrophin concentration below the level of the initial controls (Leathem and Fisher, 1959).

Data on the hypophyseal hormone content as influenced by specific amino acid deficiencies have not come to the author's attention. Cytologically, however, Scott (1956) noted that an isoleucine-deficient diet depleted the pituitary gonadotrophic cells of their PAS-positive material and reduced the size of acidophilic cells. Omission of threonine, histidine, or tryptophan invoked similar effects. The changes probably represent the interference of a single amino acid deficiency with protein metabolism rather than specific effects attributable to the lack of amino acid itself. Excessive amino acid provided by injecting leucine, methionine, valine, tyrosine, or glycine caused release of gonadotrophin (Goth, Lengyel, Bencze, Saveley and Majsay, 1955).

Administration of 0.1 mg. stilbestrol for 20 days to adult male rats eliminated detectable hypophyseal gonadotrophins. Hormone levels returned during the postinjection period provided the diet contained adequate protein, whereas a protein-free diet markedly hindered the recovery of hypophyseal gonadotrophins. The gonadotrophin content of the pituitary gland correlated well with the recovery of the reproductive system, indicating that gonadotrophin production was subnormal on ]irotein-free feeding (Leathem, 1958a).

C. CAHBOHYDR.\TE .\ND FAT

Reproduction does not appear to be influenced by carbohydrates per se and hypophyseal alterations have not been noted.

Fat-deficient diets, however, do influence reproduction and the hypophysis exhibits cellular changes. Pituitary glands of female rats fed a fat-free diet contain a subnormal number of acidophiles and an increased number of basophiles (Panos and Finerty, 1953) . In male rats the feeding oi a fat-free diet increased hypophyseal basophiles, followed progessively by more castration changes (Finerty, Klein and Panos, 1957; Panos, Klein and Finerty, 1959).

D. VITAMINS

Despite the many investigations relating reproduction to vitamin requirements, relatively few have involved hypophyseal hormone estimations. Thus in 1955, Wooten, Nelson, Simpson and Evans reported the first definitive study which related pyridoxine deficiency to hypophyseal gonadotrophin content. Using the hypophysectomized rat for assay, pituitary glands from Bo-deficient rats were shown to have a 10-fold increase in FSH per milligram of tissue and a slightly increased LH content. Earlier studies had revealed that vitamin Bi-free diets decreased pituitary gonadotrophins in male rats (Evans and Simpson, 1930) and a similar effect of the folic acid antagonist, aminopterin, in the monkey was found later (Salhanick, Hisaw and Zarrow, 1952).

Male rats deficient in vitamin A exhibited a 43 per cent increase, and castrated vitamin A-deficient rats a 100 per cent increase in hypophyseal gonadotrophin potency over the normal controls (Mason and Wolfe, 1930). The increase of gonadotrophin was more marked in vitamin A-deficient male than in vitamin A-deficient female rats. Associated with the increase in hormone level was a significant increase in basophile cells (Sutton and Brief, 1939; Hodgson, Hall, Sweetman, Wiseman and Converse, 1946; Erb, Andrews, Hauge and King, 1947).

A re\-iew of the literature up to 1944 permitted Mason to suggest that the anterior hypophysis was not the instigator of reproductive disturbances in vitamin E deficiency. Nevertheless, Griesbach, Bell and Livingston (1957), in an analysis of the pituitary gland during progessive stages of

NUTRITIONAL EFFECTS

677

tocopherol deprivation, observed cytologic changes in the hypophysis which preceded testis changes. The "peripheral or FSH gonadotrophes" increased in number, size, and activity. The LH cells exhibited a hyperplasia of lesser extent, but possibly sufficient to increase LH in circulation and to cause hypertrophy of the male accessory glands. Gonadotrophic hormone content of pituitary glands from vitamin E-deficient rats may be decreased (Rowlands and Singer, 1936), unchanged (Biddulph and Meyer, 1941), or increased to a level between normal and that of the castrate, when the adult male rats were examined after 22 weeks on a deficient diet (Nelson, 1933; Drummond, Noble and Wright, 1939). Using hypophysectomized male rats as assay animals, evidence was obtained that FSH was increased in the pituitary glands of vitamin E-deficient male and female rats (P'an, Van Dyke, Kaunitz and Slanetz, 1949).

IV. Male Reproductive System

A. TESTIS

The two basic functions of the male gonads are to produce gametes and secrete steroids. Spermatogenic activity can be estimated from testis morphology and examination of semen samples. Androgen secretion can be estimated from urinary steroid levels, accessorj^ gland weight, and from analyses of accessory sex gland secretions, i.e., fructose and citric acid. In normal maturation in the rabbit, rat, boar, and bull, androgen secretion precedes spermatogenesis (Lutwak-Mann, 1958). On this basis it would appear that well fed young bulls may come into semen production 2 to 3 months sooner than poorly fed animals (Brat ton, 1957).

1. Inanition

Complete starvation will pre^•ent maturation of immature animals. Furthermore, marked undernutrition in 700 boys, 7 to 16 years of age, was associated with genital infantilism in 37 per cent and cryptorchidism in 27 per cent (Stephens, 1941). Restriction of food intake to one-half of the normal in maturing bull calves had a

marked delaying effect on the onset of seminal vesicle secretion, but a lesser delaying effect on spermatogenesis (Davies, Mann and Rowson, 1957) . Limiting the food intake to one-third of the normal did not prevent the immature rat testis from forming spermatozoa at the same time as their controls (Talbert and Hamilton, 1955). When testis maturation was prevented by inanition, a rapid growth and maturation occurred on refeeding (Ball, Barnes and Visscher, 1947; Quimby, 1948) but Schultze (1955) observed that full body size was not attained.

The reproductive organs of the adult are more resistant to changes imposed by diet than are those of the immature animal. Thus, Mann and Walton (1953) found that 23 weeks of underfeeding produced little change in sperm density and motility in mature animals although seminal vesicle function was reduced. Li the male rat testis hypofunction follows partial or complete starvation (]\Iason and Wolfe, 1930; Mulinos and Pomerantz, 1941a; Escudero, Herraiz and Mussmano, 1948), but there is no reduction in testicular nitrogen (Addis, Poo and Lew, 1936). Loss of Leydig cell function precedes cessation of spermatogenesis (Moore and Samuels, 1931) and is evident by the atrophy of the accessory sexual organs (^lulinos and Pomerantz, 1941a) and by an alteration in accessory gland secretion (Pazos and Huggins, 1945; Lutwak-]\Iann and Mann, 1950). Evidence of a tubular effect is provided by the lack of motile sperm (Reid, 1949). Severe dietary restriction is associated with the absence of spermatozoa in the seminiferous tubules and epididymis (Mason, 1933; Menze, 1941).

The human male suffering from chronic malnutrition exhibits hypogonadism. The testes atrophy and exhibit a decrease in size of the seminiferous tubules; basement membrane thickening and small Leydig cells are seen. These individuals excrete significantly subnormal amounts of 17-ketosteroids (Zubiran and Gomez-Mont, 19531. Acute starvation may also decrease urinary 17-ketosteroid and androgen levels as much as 50 per cent, with recovery evident on refeeding (Perloff, Lasche, Nodine, Schneeberg and Vieillard. 1954).

6/

PHYSIOLOGY OF GONADS

TABLE 12.5

Effect of diet on the testes of immature rats

(From J. H. Leathern, in Re-productive Phijsiology

and Protein Nutrition, Rutgers University

Press, New Brunswick, N. J., 1959.)

Sperm

Initial control . . . 20 per cent X 30

days

6 per cent X 30

days

3 per cent X 30

days

per cent X 30

days

per cent + 5 per cent liver. ,

per cent + 5 per cent yeast

G5 per cent X 30 davs

No. of Rats

Testis

Weight

mg.

mg./lOOgm.

10

329

825

10

1694

1035

16

824

890

12

380

930

10

140

346

10

112

291

10

119

296

10

1747

1040

100

50

100

2. Protein

The minimal amount of dietary protein which will support reproduction, lactation, and growth is 16.7 per cent (Goettsch, 1949) . Thus, it is not surprising that maturation of testes and accessory sex organs was prevented in immature rats (Horn, 1955) and mice (Leathern and DeFeo, 1952) when they were fed a protein-free diet for 15 to 30 days after weaning. Furthermore, supplements of 5 per cent liver to the casein-free diet had no effect. After a month, the testes, averaging 329 mg., decreased to 140 mg. in rats fed per cent casein, but the weight increased to an average of 1694 mg. and 1747 mg. in rats fed 20 per cent and 65 per cent casein, respectively (Table 12.5). Following protein depletion, there was a decrease in tubular ribonucleic acid and an increase in lipid. Accumulation of gonadal lipid in the inactive testis may be an abnormal assimilation of a degenerative nature or simply nonutilization. A diet containing 6 per cent casein permitted the formation of spermatozoa in some animals (Guilbert and Goss, 1932). When the 6 per cent casein diet was fed to immature animals for 30 days 50 per cent of the rats exhibited some spermatozoa; in addition, testis weight increased slightly and seminal

vesicle weight doubled, but body weight was not improved (Horn, 1955). Thus, as we noted earlier, the reproductive system may gain special consideration for protein allotments when supplies are limited.

Gain in testis weight in immature male rats and the biochemical composition of the immature testis are influenced by the nutritive value of the protein fed. The testes of normal immature rats contain 85 per cent water, 10.5 per cent protein, 4.5 per cent lipid, and detectable glycogen (Wolf and Leathern, 1955). Proteins of lower nutritive value (wheat gluten, peanut flour, gelatin ) may permit some increase in testis weight, but testis protein concentration decreased, percentage of water increased, and lipid and glycogen remained unchanged (Table 12.6). The enzyme ^fi-glucuronidase, which has frequently been associated with growth processes, exhibited no change in concentration as the testis matured or was jirevented from maturing by a protein-free diet (Leathem and Fisher, 1959). Not only are the weight and composition of the testis influenced by feeding proteins of varied biologic value, but the release of androgen is more markedly altered. When a 22-day-old male rat was fed a 20 per cent casein diet for 30 days, the seminal vesicle and ventral prostate weights increased 9- to 10-fold in comparison with initial control weight. Sub TABLE 12.6

Niiiritioiial effects on testis-coin position

in immature rats

(From R. C. Wolf and J. H. Leathem,

Endocrinology, 57, 286, 1955.)

20 pel' cent casein

20 per cent wheat gluten

20 per cent i)eanut flour

20 i)er cent gelatin

5 per cent casein

Fox chow

Initial control . .

No. of Rats

Final Body Weight

gm.

7

128

8

82

8

81

5

53

5

61

8

115

7

61

72.3j30.4 04.634.0

1468 1017

1257 66.1 2101

28.8

0.11 0.18 0.11 0.10

684,62.4 29.2 0.26 1515'70.3 30.3 0.15 273 72. 7:30. 10.19

NUTRITIONAL EFFECTS

679

stitution of wheat gluten and peanut flour for casein, limited the increase in the weight of the seminal vesicles to less than 100 per cent. In fact, seminal vesicle weight as related to body weight did not increase in animals fed 20 per cent wheat gluten.

The withholding of dietary protein from an immature rat for 30 days, during which time maturation occurs in the fully fed animal, did not impose a permanent damage. Refeeding of protein permitted the rapid recovery of testis weight and the appearance of spermatoza, 70 per cent of all animals having recovered in 30 days when fully fed, whereas only 25 per cent recovered when 6 per cent casein was fed. Recovery of androgen secretion was somewhat slower than that of the tubules as estimated by seminal vesicle weight.

Variations in protein quality are a reflection of amino acid patterns, and amino acid deficiencies interfere with testis maturation (Scott, 1956; Pomeranze, Piliero, Medeci and Plachta, 1959). Alterations in food intake which follow amino acid deficiencies have required forced feeding or pair-fed controls, but it is clear from what w^as found in the controls that the gonadal changes were not entirely due to inanition (Ershoff, 1952).

If the diet is varied so that caloric intake per gram is reduced to half while retaining the dietary casein level at 20 per cent, immature rat testis growth is prevented. The effect is unlike that obtained with this level of protein in the presence of adequate calories. Furthermore, the caloric restriction may increase testis glycogen (Leathem, 1959c).

Protein anabolic levels are higher in the tissues of young growing animals and the body is more dependent on dietary protein level and quality for maintenance of the metabolic nitrogen pool than in adult animals. On the other hand, body protein reserves in adult animals permit internal shifts of nitrogen to the metabolic pool and to tissues when dietary sources are reduced or endocrine imbalances are imposed. Thus, Cole, Guilbert and Goss (1932) fed a low protein diet to adult male rats for 60 to 90 days before the sperm disappeared, but the animals would not mate. Amount of semen and sperm produced by sheep have been re

TABLE 12.7

Arlult rat testes and seminal vesicles

after protein depletion

(From J. H. Leatliem, Recent Progr. Hormone

Res., 14, 141, 1958.)

Days on

No. of

Testis

H2O

Protein

Total

Seminal

PFD*

Rats

Weight

Protein

Vesical

nig.

%

%dry

gm.

mg.

Control

9

2852

85.9

66.7

0.28

1276

30

9

2600

86.0

66.1

0.24

689

50

7

2398

85.4

64.1

0.22

320

90

25

1429

85.7

69.6

0.13

168

• PFD = Protein-free diet.

lated to the dietary protein level (Popoff and Okultilschew, 1936). Removal of protein from the diet for 30 days had little effect on the adult rat testis weight, spermatogenesis, or nitrogen content (Leathem, 1954). However, seminal vesicle w^eight was reduced 50 per cent (Aschkenasy, 1954). Prolonged protein depletion was required before the testis exhibited a loss in protein and a reduction in size. A loss of spermatozoa was not observed consistently, although some testes were completely atrophic (Table 12.7). Accessory organ weight decrease reflected the disappearance of androgen (Leathem, 1958a). Interstitial cell atrophy has also been noted in rats fed a low vegetable protein (cassava) diet (Adams, Fernand and Schnieden, 1958).

Sterility may or may not be induced with diets containing 65 per cent protein (Reid. 1949; Leathem, 1959c) but a 15 to 18 per cent dietary level of a poor protein such as maize or gelatin will decrease sperm motility and increase the number of abnormal sperm. The influence of proteins having different nutritional values in support of the growth of testes from the level to which they were depressed by stilbestrol indicated that casein, lactalbumin, and wheat gluten are equally competent to support testis growth whereas gelatin is deficient. Whole proteins may have several amino acid deficiencies, but the administration of amino acid antagonists may help to identify important individual amino acids. As an example, ethionine causes severe seminiferous tubule atrophy and Leydig cell hypoplasia (Kaufman, Klavins and Kinney, 1956; Goldberg, Pfau and Ungar, 1959). Studies in man have indicated a sharp reduction in

680

PHYSIOLOGY OF GONADS

spermatozoa after 9 days on an argininedeficient diet (Holt, Albanese, Shettles, Kajdi and Wangerin, 1942).

Adequate dietary protein cannot maintain reproductive function if the diet is calorie deficient. Thus, a decrease in seminal vesicle weight could be related to a decrease in dietary calories while protein levels were constant (Rosenthal and Allison, 1956). However, the accessory gland weight loss imposed by caloric restriction could be slowed by increasing the dietary protein (Rivero-Fontan, Paschkis, West and Cantarow, 1952).

Alterations in testis function imposed by inadequate protein are corrected when protein is returned to the diet at normal levels (Aschkenasy and Dray, 1953). Nevertheless, the nutritional state of the animal as a factor influencing recovery has been demonstrated with stilbestrol-treated adult male rats. While being fed an 18 per cent casein diet, adult male rats were injected with 0.1 mg. stilbestrol daily for 20 days. Testis weight decreased from 2848 to 842 mg., spermatogenesis was abolished, and testis water and protein content were significantly reduced. Despite a reduction in food intake, pair-fed controls exhibited no effect on reproductive organs. When a protein-free diet was substituted for the normal diet during the administration of stilbestrol, atrophy of the reproductive system was observed. Cessation of hormone administration was followed by a rapid return of testicular function toward normal when 18 per cent casein was fed both during the injection period and the recovery period. Within 30 days spermatogenesis and testicular composition were fully recovered. However, when 18 per cent casein was fed in the postinjection period to rats that had received a proteinfree diet while being given stilbestrol, recovery was clearly slow. After a month, spermatozoa were observed in only 30 per cent of the testes and testis weight was subnormal. Despite the seeming similarity of response by the two nutritional groups during the injection period, the postinjection recovery on identical dietary intake revealed marked differences in rate of recoverv (Leathem, 1958a).

3. Fat

Linoleic, linolenic, and arachidonic acids are designated as essentially fatty acids, but the physiologic role of these substances is not clearly understood. Nevertheless, the male reproductive organs are influenced by dietary essential fatty acid levels. High fat diets may enhance testicular weight (Kaunitz, Slanetz, Johnson and Guilmain, 1956) whereas removal of fat from the diet resulted in a degeneration of the seminiferous tubules as evidenced by intracellular vacuolation and a reduction in spermatids and spermatozoa (Panos and Finerty, 1954). After 5 months of feeding a fatfree diet, the rat testis may be devoid of sperm (Evans, Lepkovsky and Murphy, 1934). Testis degeneration occurred despite dietary supplements of vitamins A and E and in animals whose health appeared quite normal (Ferrando, Jacques, Mabboux and Prieur, 1955; Ferrando, Jacques, Mabboux and SoUogoub, 1955).

Weanling rats fed 14 per cent arachis (peanut) oil for 15 weeks exhibited a marked impairment of spermatogenesis (Aaes-Jorgensen, Funch and Dam, 1956) and 28 per cent arachis oil induced testicular damage of such an order that 15 weeks of feeding ethyl linoleate did not restore fertility (Aaes-Jorgensen, Funch and Dam, 1957).

4. Vitamins

Testicular dysfunction as judged by failure of sperm formation or atrophy of the secondary sex organs has been observed in deprivations of thiamine, riboflavin, pyridoxine, calcium pantothenate, biotin, and vitamins A and E. One must distinguish, however, between effects of inanition associated w^ith a vitamin deficiency and a specific vitamin effect (Skelton, 1950) ; one must also consider species differences (Biskind, 1946).

There is no question but that vitamin E deficiency in the rat results in a specific and irreversible damage to the testis. Tubular damage may proceed to the point where only Sertoli cells remain and yet the interstitial cells are not influenced (Mason, 1939). Similar changes followed vitamin E deficiency in the guinea pig (Pappenheimer and SchogolefT, 1944; Curto, 1954; Ingel

NUTRITIONAL EFFECTS

681

man-Siindberg, 1954) , hamster (Mason and Mauer, 1957), and bird (Herrick, Eide and Snow, 1952; Lowe, Morton, Cunningham and Vernon, 1957). However, little or no effect of an absence of vitamin E was noted in the rabbit (Mackenzie, 1942) and mouse (Bryan and Mason, 1941), or in live stock (Blaxter and Brown, 1952), or man (Lutwak-Mann, 1958), although vitamin E is present in human testes (Dju, Mason and Filer, 1958). Treatment of low-fertility farm animals with wheat germ oil or tocopherol or the use of this vitamin clinically have provided only inconclusive results (Beckmann, 1955) . Although some positive effects have been reported in man, the results may be due in part at least to the sparing action of tocopherol toward vitamin A.

Vitamin A deficiency influences the testis but changes are closely associated with the degree of inanition. In the rat, a vitamin deficiency sufficient to cause ocular lesions did not prevent sperm formation, but a deficiency of such proportions as to cause a body weight loss did cause atrophy of the germinal epithelium (Reid, 1949). Vitamin A deficiency will induce sterility in mice (McCarthy and Cerecedo, 1952). A gross vitamin deficiency in bulls before expected breeding age prevented breeding ; adult bulls may exhibit a lower quality semen but they remain fertile (Reid, 1949). Vitamin A deficiency induces metaplastic keratinization of the epithelium lining the male accessory sex organs (Follis, 1948) and thus may influence semen.

Testis damage induced by vitamin A deficiency can be reversed, but vitamin A therapy in man for oligospermia not due to vitamin lack was without effect (Home and Maddock, 1952) .

Age of the animal and dosage are factors which influence the results obtained in male rats with administered vitamin A. Immature male rats given 250 I.U. of vitamin A per gram of body weight daily exhibited a loss of spermatocytes, an effect which was accentuated by tocopherol (Maddock, Cohen and Wolbach, 1953). Little or no effect of similar treatment was observed in adult rats. The liver is the major storage depot for vitamin A and the fact that the male rat liver is more quickly depleted and less capa

ble of storage than is the liver of the female should be considered in any attempted correlation of the vitamin and hormone levels (Booth, 1952).

Other vitamin deficiencies have been shown to influence the testis. A lack of thiamine had little effect on testis weight, but did influence the Leydig cells and prevented growth of the accessory sex organs (Pecora and Highman, 1953). A chronic lack of ascorbic acid will cause a degeneration of both Leydig cells and seminiferous tubules. The effects of vitamin deficiency on the testis has been distinguished from those due to inanition and have been related to changes in carbohydrate metabolism (Mukherjee and Banerjee, 1954; Kocen and Cavazos, 1958) . The importance of ascorbic acid in the testis as related to function is not evident, but concentrations of this vitamin are maximal at 1 week of age (Coste, Delbarre and Lacronique, 1953).

Serious anatomic and functional impairments of testes were noted in pantothenic acid deficiency (Barboriak, Krehl, Cowgill and Whedon, 1957), and development of the rat testis and seminal vesicles was retarded by a biotin deficiency (Bishop and Kosarick, 1951; Katsh, Kosarick and Alpern, 1955), but the animals did not exhibit marked alterations in other endocrine organs (Delost and Terroine, 1954). Testosterone hastened the development of vitamin deficiency and enhanced the severity of biotin deficiency in both sexes, thereby suggesting a hormone-vitamin relationship (Okey, Pencharz and Lepkovsky, 1950). On the other hand, testosterone had no effect on the tolerance of mice for aminopterin, but castration increased the tolerance (Goldin, Greenspan, Goldberg and Schoenberg 1950).

B. INFLUENCE OF NUTRITION ON THE EE SPONSIVENESS OF MALE REPRODUCTIVE

TISSUES TO HORMONES

1. Testis

a. Inanition. The testes of birds on limited food intake were more responsive to hypophyseal gonadotrophin than fully fed birds (Byerly and Burrows, 1938; Breneman, 1940). In the rat several investigators have shown that the testis will respond to gonadotrophin despite inanition (Moore and Sara

682

PHYSIOLOGY OF GONADS

TABLE 12.8

Influence of diet and pregnant mare serum (PMS)

on testes and seminal vesicles of

immature male mice

(From V. J. DeFeo and J. H. Leathern,

unpublished.)

Diet (Per cent Protein X Days Fed)

per cent X 10 per cent X 10 per cent X 20 per cent X 20

l.u.

3

3

stage of Spermatogenesis

4 1

1 5 5

Spermatids

Seminal Vesicles

mg.

2.7 4.5

2.7 3.5

uels, 1931; Funk and Funk, 1939; Meites, 1953), with a stimulation of Leydig cells, an increase in testis size, and, in 40 days, a return of spermatozoa. Underfed males injected with gonadotrophin sired litters (Mulinos and Pomerantz, 1941a, b). Improved nutrition aided by unknown liver factors enhanced the response to androgen in severe human oligospermia (Glass and Russell, 1952).

b. Protein. Feeding a protein-deficient diet to adult male rats for 60 to 90 days did not prevent stimulation of the testes and seminal vesicles after pregnant mare's serum (PMS) administration (Cole, Guilbert and Goss, 1932). As we have noted, immature animals are prevented from maturing when diets lack protein. Nevertheless, a gonadal response to injected gonadotrophin was obtained in immature mice fed a protein-free diet for 13 days; tubules and Leydig cells were stimulated and androgen was secreted (Table 12.8). Refeeding alone permitted a recovery of spermatogenesis which was not hastened by concomitant PMS (Leathem, 1959c).

The maintenance of testis weight and spermatogenic activity with testosterone propionate in hypophysectomized adult male rats is well known, but these studies have involved adequate nutrition. If hypophysectomized rats were fed a protein-free diet and injected with 0.25 mg. testosterone propionate daily, testis weight and spermatogenesis were less well maintained than in rats fed protein. Testis protein concentra

tion was also reduced. These data suggest that influences of nutrition on the testis can be direct and are not entirely mediated through hypophyeal gonadotrophin changes (Leathem, 1959b).

c. Fat. The rat fed for 20 weeks on a fatfree diet exhibits a degeneration of the seminiferous epithelium within the first weeks which progresses rapidly thereafter. Chorionic gonadotrophin or rat pituitary extract started during the 20th week failed to counteract the tubular degeneration, but testosterone propionate proved effective (Finerty, Klein and Panos, 1957). The result shows that the ineffectiveness of the gonadotrophins could not be due to the failure of androgen release (Greenberg and Ershoff, 1951).

d. Vitamins. Gonadotrophins failed to promote spermatogenesis in vitamin A(Mason, 1939) or vitamin E- (]Mason, 1933; Geller, 1933; Drummond, Noble and Wright, 1939) deficient rats, but in another experiment the atrophic accessory sex organs of vitamin A-depleted rats were stimulated (Mayer and Goodard, 1951). Lack of vitamin A favored an enhanced response to PMS when the ratios of seminal vesicle weight to body weight were computed (Meites, 1953). The failure of gonadotrophins to stimulate testis tubules suggests a specific effect of avitaminosis A and E (Mason, 1933) on the responsiveness of the germinal epithelium.

Subnormal responses of rats to PMS, as measured by relative seminal vesicle weight, were obtained when there were individual vitamin B deficiencies, but the influence was due largely to inanition (Drill and Burrill, 1944; Meites, 1953). Nevertheless, sufficient response to chorionic gonadotrophin was obtained so that fructose and citric acid levels were restored to normal. Such an effect was not observed to follow dietary correction unless an unlimited food intake was allowed (Lutwak-Mann. 1958).

2. Sc

il W.siclfx and Prosfate

a. Inanition. Although the accessory reproductive organs resppnd to direct stimulation despite an inadequate food intake (Mooi'c and Samuels. 19311, tlio increase

NUTRITIONAL EFFECTS

683

in weight may be subnormal in mice and rats (Goldsmith, Nigrelli and Ross, 1950; Kline and Dorfman, 1951a, Grayhack and Scott, 1952), or above normal in chickens (Breneman, 1940). Complete deprivation of food reduced the quantity of prostatic fluid in the dog, but exogenous androgen restored the volume, increased acid phosphatase, and induced tissue growth (Pazos and Huggins, 1945) .

6. Protein. The response of the seminal vesicles to androgen was investigated in immature rats, using weight and /5-glucuronidase as end points. Castration and 10 days on a protein-free diet preceded the 72-hour response to 0.25 mg. testosterone propionate. The lack of protein did not prevent a normal weight increase, and enzyme concentration was unchanged. If an 18 per cent diet was fed during the 3-day period that the androgen was acting, no improvement in weight response was noted, but enzyme concentration increased 100 per cent. Thus, when protein stores are depleted, the androgen response may be incomplete in the absence of dietary protein (Leathern, 1959c). Nevertheless, varied protein levels do not influence seminal vesicle weight-response when caloric intake is reduced (RiveroFontan, Paschkis, West and Cantarow, 1952).

c. Vitamins. Vitamin deficiencies do not prevent the seminal vesicles from responding to androgen. In fact, in vitamin B deficiency, testosterone restored fructose and citric acid levels to normal despite the need for thiamine in carbohydrate metabolism (Lutwak-Mann and Mann, 1950). In the male, unlike the female, the effects of folic acid deficiency in reducing responsiveness to administered androgen were largely due to inanition in both mice and rats (Goldsmith, Nigrelli and Ross, 1950; Kline and Dorfman, 1951a) , and vitamin A deficiency which leads to virtual castration does not prevent an essentially normal response of the accessory glands to testosterone propionate (Mayer and Truant, 1949). Restricting the caloric intake of vitamin Adeficient rats retarded the curative effects of vitamin A in restoring the accessory sex glands of the A-deficient animals (Mason, 1939).

V. Female Reproductive System

A. OVARIES

1. Inanition

Mammalian species generally exhibit a delay in sexual maturation when food intake is subnormal before puberty, and ovarian atrophy with associated changes in cycles if inanition is imposed on adults. In human beings a decrease in fertility and a greater incidence of menstrual irregularities were induced by war famine (Zimmer, Weill and Dubois, 1944). Ovarian atrophy with associated amenorrhea and sterility were invoked by chronic undernutrition (Stephens, 1941). The ovarian morpliologic changes were similar to those of aging. Urinary estrogens were subnormal in 22 of 25 patients exhibiting amenorrhea associated with limited food intake (Zubiran and (_lomcz-Mont, 1953).

The nutritional requirements of jM'imates other than man have been studied in female baboons. The intake of vitamins and other essential nutrients was found to be of the same order as that recommended for man. Caloric intake varied with the menstrual cycle, being least during the follicular phase and maximal during the 2 to 7 days preceding menstruation (Gilbert and Gillman, 1956). Various diets were also studied to assess their importance in maintaining the normal menstrual rhythm. The feeding of (a) maize alone, (b) assorted vegetables and fruit, or (c) maize, skimmed milk, and fat led to menstrual irregularities or to amenorrhea. The mechanism regulating ovulation was the first to be deranged. The addition of various vitamins or of animal protein did not correct the menstrual disorders. However, inclusion of ox liver in the diet did maintain the menstrual rhythmicity, but the beneficial effect could not be attributed to its protein content (Gillman and Gilbert, 1956 ) .

In lower mammals that have been studied, inanition will hinder vaginal opening, and delay puberty and ovarian maturation and functioning. In adult rats and mice ostrous cycles are interrupted and the reprorUictive system becomes atrophic when body weight loss exceeds 15 per cent. The ovaries be

684

PHYSIOLOGY OF GONADS

come smaller, ovulation fails, and large vesicular follicles decrease in number with an increase in atresia, but primary follicles show a compensatory increase (Marrian and Parkes, 1929; Mulinos and Pomerantz, 1940; Stephens and Allen, 1941; Guilbert, 1942; Bratton, 1957). The ovarian interstitial cells mav be markedly altered or absent (Huseby and Ball, 1945; Rinaldini, 1949) and the ovary may exhibit excessive luteinization (Arvy, Aschkenasy, AschkenasyLelu and Gabe, 1946) or regressing corpora lutea (Rinaldini, 1949) . However, the ovarian changes induced by inanition may be reversed by refeeding, with a return to reproductive capacity (Ball, Barnes and Visscher, 1947; Schultze, 1955). The effect of feed-level on the reproductive capacity of the ewe has been reported (El-Skukh, Nulet, Pope and Casida, 1955), but one must realize that high planes of nutrition may adversely influence fertility (Asdell, 1949). Nevertheless, additional protein and calcium added to an adequate diet extended the reproductive life span (Sherman, Pearson, Bal, McCarthy and Lanford, 1956).

2. Protein

The availability of just protein has an important influence on the female reproductive system. In immature rats ovarian maturation was prevented by feeding diets containing per cent to 1.5 per cent protein (Ryabinina, 1952) and low protein diets decreased the number of ova but without altering their ribonucleic acid (RNA) or glycogen content (Ishida, 1957). Refeeding 18 per cent protein for only 3 days was marked by the appearance of vesicular follicles and the release of estrogen in mice previously fed a protein-free diet (Leathem, 1958a). In experiments involving the opposite extreme, in which 90 per cent protein diets were used, a retardation of ovarian growth, and a delay in follicular maturation, in vaginal opening, and in the initiation of estrous cycles were noted (Aschkenasy-Lelu and Tuchmann-Duplessis, 1947; TuchniannDuplcssis and Aschkenasy-Lelu, 1948).

Adult female rats fed a protein-free diet for 30 days exhibited ovaries weighing 22 mg. compared with ovaries weighing 56 mg. from i)air-fed controls fed 18 per cent casein. Ovarian glycogen, ascorbic acid, and cho

lesterol were all influenced by protein deprivation and anestrum accompanied the ovarian changes. Furthermore, uterine weight and gl3^cogen decreased in rats fed protein-free diets (Leathem, 1959b).

In adult rats the feeding of 3.5 per cent to 5 per cent levels of protein (GuillDert and Gross, 1932) was followed by irregularity of the cycles or by their cessation. The cycles became normal when 20 to 30 per cent protein was fed (Aschkenasy-Lelu and Aschkenasy, 1947). However, abnormally high levels of casein (90 per cent) induced prolonged periods of constant estrus (Tuchmann-Duplessis and AschkenasyLelu, 1947). Nevertheless, not all species responded to protein depletion in the same manner. For example, the rabbit exhibited estrus and ovulation despite a 25 per cent body weight loss imposed by to 2 per cent protein diets (Friedman and Friedman, 1940).

Despite a normal level of protein in the diet, inadequate calories will interfere with reproductive function and induce ovarian atrophy (Escudero, Herraiz and Mussmano, 1948; Rivero-Fontan, Paschkis, West and Cantarow, 1952). Furthermore, the effects of 15 per cent and 56 per cent protein levels on estrous cycles could not be distinguished when calories were reduced 50 per cent (Lee, King and Visscher, 1952). Returning mice to full feeding after months of caloric deficiency resulted in a sharp increase in reproductive performance well above that expected for the age of the animal (Visscher, King and Lee, 1952). This type of rebound phenomenon has not been explained.

Reproductive failure assigned to dietary protein may be a reflection of protein quality as well as level. Specific amino acid deficiencies lead to cessation of estrus (White and AVhite, 1942; Berg and Rohse, 1947) and thus feeding gelatin or wheat as the protein source and at an 18 per cent level was quickly followed by an anestrum (Leathem, 1959b). Supplementation of the wheat diet with lysine corrected the reproductive abnormalities (Courrier and Raynaud, 1932), but neither lysine (Pearson, Hart and Bohstedt, 1937) nor cystine (Pearson, 1936) added to a low casein diet was beneficial. Control of food intake must be considered in studies involving amino

NUTRITIONAL EFFECTS

685

acids, for a deficiency or an excess can create an imbalance and alter appetite. Opportunity to study the amino acids in reproduction is now possible because of the work of Greenstein, Birnbaum, Winitz and Otey (1957) and Schultze (1956) , who maintained rats for two or more generations on synthetic diets containing amino acids as the only source of protein. Similarly, the amino acid needs for egg-laying in hens has been reported (Fisher, 1957). Tissue culture methods also permit the study of the nutritional requirements of embryonic gonadal tissue, the success of avian gonadal tissue in culture being judged by survival, growth, and differentiation. In experiments in which this technique was used it was found that a medium made up of amino acids as the basic nitrogen source can maintain gonadal explants very successfully, even though the choice of amino acids does not exactly correspond to the 10 essential amino acids recommended for postnatal growth (StengerHaffen and Wolff, 1957).

3. Carbohydrate

The absence of dietary carbohydrate does not appreciably affect the regularity of estrous cycles in rats provided the caloric need is met. However, the substitution of 20 per cent sucrose for corn starch induced precocious sexual maturity which was followed by sterility (Whitnah and Bogart, 1956). The ovaries contained corpora lutea, but the excessive luteinization of unruptured follicles suggested a hypophyseal disturbance. Substitution of 20 per cent lactose for corn starch had no effect. Increased amounts of lactose retarded the gain in body weight and blocked ovarian maturation, possibly because the animal could not hydrolyze adequate amounts of the disaccharide. Addition of whole liver powder to the diet counteracted the depressing action of 45 per cent lactose on the ovary (Ershoff, 1949).

4. Fat

There seems to be little doubt that dietary fat is reciuired for normal cyclic activity, successful pregnancy, and lactation, and that the requirements for essential fatty acids are lower in females than in males (Deuel, 1956).

Conception, fetal development, and par

turition can take place in animals fed a diet deficient in fatty acids (Deuel, Martin and Alfin-Slater, 1954) , despite a reduction in total carcass arachidonic acid (Kummerow. Pan and Hickman, 1952). Earlier reports indicated that a deficiency of essential fatty acids caused irregular ovulation and impaired reproduction (Burr and Burr, 1930; Maeder, 1937). The large pale ovaries lead Sherman (1941) to relate essential fatty acid deficiency to carotene metabolism. In this regard the removal of essential fatty acids from an adequate diet supplemented with vitamin A and E lead to anestrum and sterility while maintaining good health (Ferrando, Jacques, Mabboux and Prieur, 1955). Perhaps the differences in opinion regarding the effects of fatty acid deficiency can be related to the duration of the experimental period. Panos and Finerty (1953) found that growing rats placed on a fat-free diet exhibited a normal time for vaginal opening, normal ovarian weight, follicles, and corpora lutea, although interstitial cells were atrophic. However, regular estrous cycles were noted for only 20 weeks, thereafter 60 per cent of the animals exhibited irregular cycles.

A decrease in reproductive function may be invoked by adding 14 per cent arachis oil to the diet (Aaes-Jorgensen, Funch and Dam, 1956) . Increasing dietary fat by adding rape oil did not influence ovarian function but did cause the accumulation of ovarian and adrenal cholesterol (Carroll and Noble, 1952).

Essential fatty acid deficiency is associated with underdevelopment and atrophic changes of the uterine mucosa. Adding fat to a stock diet enhanced uterine weight in young animals at a more rapid pace than body weight (Umberger and Gass, 1958) .

5. Vitamins

Carotenoid pigments are present in the gonads of many vertebrates and marine invertebrates, and, in mammals, are particularly prominent in the corpus luteum. However, no progress has been made in determining either the importance of the carotenoids in the ovary or of the factors controlling their concentrations. It is well known that vitamin A deficiency induces

686

PHYSIOLOGY OF GONADS

a characteristic keratinizing metaplasia of the uterus and vagina, but estrous cycles continue despite the vaginal mucosal changes. Furthermore, ovulation occurs regularly until advanced stages of deficiency appear. The estrous cycle becomes irregular in cattle fed for a long period of time on fodder deficient in carotene. The corpora lutea fail to regress at the normal rate and ovarian follicles become atretic and cystic ( Jaskowski, Watkowski, Dobrowolska and Domanski, cited by Lutwak-Mann, 1958). The alterations in reproductive organs associated with a lack of vitamin A may be due in part to a vitamin E deficiency since the latter enhances the rate at which liver stores of vitamin A are depleted.

Definite effects of hypervitaminosis A have been observed on reproduction. Masin (1950) noted that estrus in female rats could be prolonged by administration of 37,000 I.U. of vitamin A daily. The implications, however, have not been studied. The effect of hypervitaminosis A may actually induce secondary hypovitaminoses. The displacement of vitamin K by excess A is almost certain and similar relationships appear to exist with vitamin D (Nieman and Klein Obbink, 1954).

The failure of vitamin E-deficient female rats to become pregnant is apparently due to disturbances of the implantation process rather than to the failure of ovulation. There is no direct proof of ovarian dysfunction (Blandau, Kaunitz and Slanetz, 1949). However, the ovary of the rat deficient in vitamin E may have more connective tissue and pigment, and Kaunitz (1955) showed by ovarian transplantation that some nonspecific ovarian dysfunction appears to exist (cited by Cheng, 1959 (. Vitamin E is essential for birds, but there is little evidence for a dependency in most mammals; sheep, cows, goats, and pigs have been studied. Treatment of low-fertility farm animals with tocopherol has not provided conclusive data favoring its use (Lutwak-Mann, 1958), nor has the treatment of human females been rewarded with any indication that vitamin E might be helpful in cases of abnormal cycles and habitual abortion (Beckmann, 1955).

No specific reproductive disturbances in man, the rhesus monkey, or the guinea pig

have been associated with vitamin C deficiency (Mason, 1939). Nevertheless, the high ascorbic acid content of ovarian and luteal tissue and of the adrenal cortex suggests a physiologic role in association with steroid synthesis. (3varian ascorbic acid varies with the estrous cycle, dropping sharply in the proestrum (Coste, Delbarre and Lacronique, 1953), and decreasing in resjionse to gonadotrophin (Hokfelt, 1950; Parlow, 1958). Virtually no ascorbic acid is present in bovine follicular fluid (LutwakMann, 1954) or in rat ovarian cyst fluid (Blye and Leathem, 1959). Uterine ascorbic acid decreased in immature mice treated with estrogen, but remained unchanged in rats following thiouracil administration (Leathem, 1959a). Its role in the uterus awaits elucidation.

Delayed sexual maturation and ovarian atrophy have been described when there are deficiencies of thiamine, riboflavin, pyridoxine, pantothenic acid, biotin, and B12 (Ershoff, 1952; Ullrey, Becker, Terrill and Notzold, 1955). However, as we noted when deficiencies of the vitamins were being considered, much of the impairment of reproductive function can be related to inanition rather than to a vitamin deficiency (Drill and Burrill, 1944). Pyridoxine deficiency, although not affecting structure (Morris, Dunn and Wagner, 1953) , markedly reduces the sensitivity of the ovary to administered gonadotrophin (Wooten, Nelson, Simpson and Evans, 1958) .

Bird, frog, and fish eggs contain considerable quantities of vitamins. In fact, the daily human requirements for vitamins may be contained in a hen's egg and thus it is not surprising that hatchability is decreased l)y virtually any vitamin deficiency. Lutwak-Mann (1958) has provided an excellent survey of these data with numerous references to studies of frogs and fishes. Nearly all the B vitamins are present in fish roe and the pantothenic acid concentration in cod ovaries {Gadus morrhua) exceeds most otlicr natural sources. The amount of the latter varies with the reproductive cycle, d(>creasing to its lowest level before spawning. Riboflavin and vitamin B12 , on the other h;ui(l, do not change (Braekkan, 1955).

NUTRITIONAL EFFECTS

687

B. INFLUENCE OF NUTRITION ON THE RESPONSIVENESS OF FEMALE REPRODUCTIVE TISSUES TO HORMONES

1. Ovary

a. Inanition. Marrian and Parkes (1929) were the first to show that the quiescent ovary of the underfed rat can respond to injections of anterior pituitary as evidenced by ovulation and estrous smears. Subsequently the ovaries of underfed birds, rats, and guinea pigs were found to be responsive to serum gonadotrophin (Werner, 1939; Stephens and Allen, 1941; Mulinos and Pomerantz, 1941b; Hosoda, Kaneko, Mogi and Abe, 1956). A low calorie bread-andmilk diet for 30 days did not prevent ovarian response to rat anterior pituitary or to chorionic gonadotrophin. In these animals an increase in ovarian weight with repair of interstitial tissue, as well as folhcle stimulation and corpus luteum formation, were observed (Rinaldini, 1949). Rats from which food had been withdrawn for 12 days could respond to castrated rat pituitary extract with an increase in ovarian and uterine weight (Maddock and Heller, 1947). Nevertheless, differences in the time and degree of responsiveness to administered gonadotrophin were noted in rabbits. Animals on a high plane of nutrition responded to gonadotrophin at 12 weeks, whereas rabbits on a low plane of nutrition responded at 20 weeks and fewer eggs were shed (Adams, 1953).

b. Protein. Protein or amino acid deficiencies in the rat do not prevent a response to administered gonadotrophin (Cole, Guilbert and Goss, 1932; Courrier and Raynaud, 1932) . However, the degree and type of gonadal response is influenced by the diet. Thus, immature female mice fed to 6 per cent casein for 13 days exhibited only follicular growth in response to pregnant mare serum, whereas the ovarian response in mice fed 18 per cent casein was suggestive of follicle-stimulating and strongly luteinizing actions (Table 12.9). Furthermore, the ovarian response was significantly less after 20 days of nonprotein feeding than after 10 days of depletion (Leathem, 1958a). Ovarian stimulation by a gonadotrophin involves tissue protein synthesis and thus the type of whole protein fed

could influence the responses. Yamamoto and Chow (1950) fed casein, lactalbumin, soybean, and wheat gluten at 20 per cent levels and noted that the response to gonadotrophin as estimated by tissue nitrogen was related to the nutritive value of the protein. The ovarian weight response to chorionic gonadotrophin was less in rats fed 20 per cent gelatin than those fed 20 per cent casein (Leathem, 1959b). Inasmuch as the hypophysis may influence the gonadal response to injected hormone despite the diet, hypophysectomized rats fed a protein-free diet for 5 weeks and hyophysectomized rats on a complete diet were tested for response to gonadotrophins. The response to FSH was not influenced by diet, but the protein-depleted rats were twice as sensitive to interstitial cell-stimulating hormone (ICSH), human chorionic gonadotrophin (HCG), and PMS as the normal rats (Srebnik, Nelson and Simpson, 1958). Protein-depleted, normal mice were twice as sensitive to PMS as fully fed mice (Leathem and Defeo, 1952 1 .

c. Vitamins. In the female vitamin B deficiencies do not prevent ovarian responses to gonadotrophin (Figge and Allen, 1942), but the number of studies is limited. Be deficiency in DBA mice was associated with an increased sensitivity of the ovary to gonadotrophins (Morris, Dunn and Wagner, 1953), whereas pyridoxine deficiency in the rat decreased ovarian sensitivity, especially to FSH (Wooten, Nelson, Simp TABLE 12.9

Influence of dietary protein and pregnant mare

serum {PMS) on the mouse ovary

(From J. H. Leathem, Recent Progr. Hormone

Res., 14, 141, 1958.)

Diet fPer cent Protein X Days Fed)

c-5,

•g-s

1*

k

r

1 11

c 1

I.U.

mg.

mg.

per cent X 23

1.2

4.8

per cent X 2.3

3

2.8

13

10.8

per cent X 13

1.4

4.9

per cent X 13

3

4.4

16

1

15.1

6 per cent X 13

3.2

6

7.7

6 per cent X 13

3

5.6

12

1

31.9

18 per cent X 13

5.0

10

2

51.6

18 per cent X 13

3

8.0

7

4

51.3

088

PHYSIOLOGY OF GONADS

son and Evans, 1958 j. Administration of vitamin C concomitant witli gonadotropliin has been claimed to enhance ovarian response (DiCio and Schteingart, 1942), but in another study the addition of ascorbic acid inhibited the hiteinizing and ovulating action of the gonadotrophin (Desaive, 1956).

Whether induced by vitamin deficiency or by inanition, the anestrum in rats which follows 2 to 3 weeks' feeding of a vitamin B-deficient diet has been explored as a method for the assay of gonadotrophin. Pugsley (1957) has shown that there is considerable convenience of method and a satisfactory precision of response for the assay of HCG and pregnant mare serum.

2. Uterus and Vagina

a. Inanition. Limited food intake does not prevent an increase in uterine weight after estrogen. Testosterone propionate will markedly increase uterine growth despite a 50 per cent reduction in food intake (Leathem, Nocenti and Granitsas, 1956). Furthermore, dietary manipulations involving caloric and protein levels did not prevent the uteri of spayed rats from responding to estrogen (Vanderlinde and Westerfield, 1950). More specific biochemical and physiologic responses must be measured because starvation for 4-day periods clearly interferes with deciduoma formation (DeFeo and Rothchild, 1953). A start in the direction of studying tissuecomposition changes has been made by measuring glycogen. However, no changes were noted in uterine glycogen in fasting rats (Walaas, 1952), and estrogen promoted glycogen deposition in the uteri of starved rats as well as in the uteri of fully fed rats (Bo and Atkinson, 1953).

b. Fat. Interest in the hormone content of fat from the tissues of animals treated with estrogen for the purpose of increasing body weight has raised the question of tissue hormone content. If estrogen was to be detected in tissues, an increase in dietary fat was necessary. However, the increase in dietary fat decreased the uterine response to stilbcstrol (I'mberger and Gass, 1958), thus complicating the assay.

c. Vitainins. Stimulation of the uterus by estrogen does not require tliianiinc, ribo

flavin, pyridoxine, or pantothenic acid. On the other hand, a deficiency of nicotinic acid appears to enhance the response to low doses of estrogen (Kline and Dorfman, 1951a, b). However, Bio appears to be needed for optimal oviduct response (Kline, 1955) and is required for methyl group synthesis from various one-carbon precursors including serine and glycine (Johnson, 1958).

Response of the bird oviduct to stilbestrol requires folic acid (Hertz, 1945, 1948). It was shown subsequently that stilbestrol and estrone effects in frogs, rats, and the rhesus monkey also require folic acid. A folic acid deficiency can be induced by feeding aminopterin. In this way the estrogen effects can be prevented. Aminopterin also prevents the action of progesterone in deciduoma formation, from which it may be inferred that folic acid is necessary for deciduoma formation in the rat. Increased steroid or folic acid levels can reverse the antagonist's effect (Velardo and Hisaw, 1953).

The mechanism of folic acid action is not clear. It may function in fundamental metabolic reactions linked with nucleic acid synthesis. Brown (1953) showed that desoxyribonucleic acid could be substituted for folic acid in the bird. In the rat aminopterin interferes with the increase in uterine nucleic acids, and with nitrogen and P-^- uptake by nucleic acids following estrogen. Folic acid has been implicated in the metabolism of several amino acids (Davis, Meyer and McShan, 1956).

Rats ovariectomized at weaning and maintained on a vitamin E-free diet for 6 weeks to 10 months responded to estradiol in the same manner as rats supplemented with tocopherol. This finding suggests that an intimate physiologic relationship between estradiol and vitamin E is not very probable (Kaunitz, Slanetz and Atkinson, 1949). Nevertheless, vitamin E has been re]:»orted to act synergistically with ovarian hormones in dc^ciduoma formation (Kehl, Douard and Lanfranchi. 1951 ) and to influence nucleic acid turnover (Dinning. Simc and Day, 1956).

A vitamin-hormone interrelationship is apparent when estrogen and vitamin A are considered. Vitamin A-deficient female rats present evidence of a metaplastic uterine

NUTRITIONAL EFFECTS

689

epithelium in 11 to 13 weeks, but similar changes failed to develop in ovariectomized rats. Vitamin A-deficient castrated rats quickly developed symptoms of metaplasia when estrogen alone was administered, but no adverse effect followed the administration of estrogen combined with vitamin A (Bo, 1955, 1956). The vagina is different. Its epithelium becomes cornified in vitamin A-deficient normal and castrated rats. The cornification is histologically indistinguishable from that occurring in the estrous rat and can be prevented by vitamin A. In fact, vitamin A will quantitatively inhibit the effect of estrogen on the vaginal mucosa when both are applied locally (Kahn, 1954). Conversion of ^-carotene to vitamin A is influenced by tocopherol, vitamin Bi2 , insulin, and thyroid, with evidence for and against a similar action by cortisone (Lowe and Morton, 1956; Rice and Bo, 1958). An additional vitamin-hormone relationship is suggested by the augmentation of progesterone action in rabbits given vitamin Do .

3. Mammary Gland

Inanition prevents mammary growth, but feeding above recommended requirements for maintenance and growth from birth to the first parturition also seems to interfere with mammary growth. Furthermore, steroid stimulation of the mammary gland is influenced by nutritional factors. Using the male mouse, Trentin and Turner (1941) showed that as food intake decreased, the amount of estradiol required to produce a minimal duct growth w^as proportionately increased. In the immature male rat a limited food intake prevented the growth of the mammary gland exhibited by fully fed controls. Nevertheless, the gland was competent to respond to estrogen (Reece, 1950) . Inasmuch as the glands of force-fed hypophysectomized rats did not respond to estrogen (Samuels, Reinecke and Peterson, 1941; Ahren, 1959), one can assume that, despite inanition, a hypophyseal factor was present to permit the response of the mammary gland to estrogen. However, inanition (IMeites and Reed, 1949) , but not vitamin deficiencies (Reece, Turner, Hathaway and Davis, 1937), did reduce the content of hypophyseal lactogen in the rat.

Growth of the mammary gland duct in the male rat in response to estradiol requires a minimum of 6 per cent casein. Protein levels of 3 per cent and per cent failed to support growth of the duct (Reece, 1959) .

C. PREGNANCY

The human male after attaining adulthood is confronted with the problem of maintaining the body tissues built up during the growth period. However, in the human female it has been estimated that the replacement of menstrual losses may require the synthesis of tissue equivalent to 100 per cent of her body weight (Flodin, 19531. In the event of pregnancy and in all viviparous species, the female is presented with even more formidable demands and a limitation of nutritional needs can lead to loss of the embryo or fetus. The role of nutrition at this point in reproduction has always received considerable attention and is complicated by the circumstance that many food substances influence pregnancy (Jackson, 1959). However, in many instances there is no evidence that fetal loss or malformation induced by nutritional modifications has been the consequence of an endocrine imbalance and thus limitation of the immense literature is permissible.

During the first 15 days of pregnancy, a rat may gain 50 gm. Since the fetuses and placentas are small, most of the gain is maternal and is associated with an' increase in food intake of as much as 100 calories per kilogram of body weight (Morrison, 1956). During the first 2 weeks of pregnancy, marked storage of fat and water occurs in the maternal body and the animal's positive nitrogen balance is above normal. Liver fat also increases (Shipley, Chudzik, Curtiss and Price, 1953). The increased food intake in early pregnancy may therefore provide a reserve for late fetal growth, as food intake may decline to 65 per cent of the general pregnancy level during the last 7 days (Morrison, 1956). During this last week, fetal growth is rapid. The rapid growth has been related to (1) greater demands of the fetus, (2) greater amounts of food in the maternal blood, and (31 greater permeability of the placenta. Certainly the anabolic potential of fetal tissues is high and the mother can lose weight while the

690

PHYSIOLOGY OF GONADS

fetuses gain. But it is also important to recall that there is a shift in protein, because its distribution in organs of pregnant rats differs from that in nonpregnant animals (Poo, Lew and Addis, 1939). Other changes in the maternal organism were enumerated b}^ Newton (1952) and by Souders and Morgan (1957).

A measure of nitrogen balance during pregnancy, rather than weight of young at birth, has been suggested as a means of determining a diet adequate for reproduction (Pike, Suder and Ross, 1954). After the 15th day, a retention of body protein increases, blood amino nitrogen and amino acids decrease, and urea formation decreases. These metabolic activities suggest an increase in growth hormone although the levels of this hormone have not been estimated (Beaton, Ryu and McHenry, 1955). Placental secretions have also been associated with the active anabolic state of the second half of pregnancy, because removal of the fetuses in the rat did not change the anabolic activity, whereas removal of the placentas was followed by a return to normal (Bourdel, 1957). A sharp increase in liver ribose nucleic acid has been observed during late pregnancy in mice and rats and the effect attributed to a placental secretion or to estrogen. Species differences also influence the results because only a modest change in liver RNA was observed in guinea pigs and no change occurred in cats (Campbell and Kostcrlitz, 1953; Campbell, Innes and Kosterlitz, 1953a, b).

Clinical observations have related both

TABLE 12.10

Nutrition and pregnancy in rats

(From J. H. Leathern, in Recent Progress in

the Endocrinology of Reproduction, Academic

Press, Inc., New York, 1959.)

Calories/kg. Body Weight

Fetuses, Day 20

No.

Average weight

18 per cent casein

18 per cent casein

6 per cent casein

per cent casein

18 per cent gelatin

18 per cent gelatin

200 100

250 200 200 100

8 6

gm. 6.1

3.5

toxemia of pregnancy (Pequignot, 1956) and prematurity to inadequate nutrition (Jeans, Smith and Stearns, 1955). The potential role of protein deprivation in the pathogenesis of the toxemia of pregnancy prompted studies in sheep and rats. In sheep nutritionally induced toxemia simulates the spontaneous toxemia (Parry and Taylor, 1956), but only certain aspects of toxemia were observed in the pregnant rat subjected to low protein diets. When rats were fed 5 per cent casein and mated, fluid retention was observed (Shipley, Chudzik, Curtiss and Price, 1953) and pregnancy was completed in only 48 per cent of the animals Curtiss, 1953). Gain in body weight in the adult rat and gain in fetal weight were subnormal as the result of a low protein feeding during pregnancy.

Complete removal of protein from the diet beginning at the time of mating did not prevent implantation but did induce an 86 to 100 per cent embryonic loss. The effect was not related solely to food intake (Nelson and Evans, 1953) , as we will see in what follows when the relationship between protein deficiency and the supply of estrogen and progesterone is described. Limiting protein deprivation to the first 9 to 10 days of pregnancy will also terminate a pregnancy, but when the protein was removed from the diet during only the last week of pregnancy, the maternal weight decreased without an effect on fetal or placental weight (Campbell and Kosterlitz, 1953). As would be anticipated, a successful pregnancy requires protein of good nutritional quality and the caloric intake must be adequate. Thus, an 18 per cent gelatin diet failed to maintain pregnancy when 200 calories per kilogram were fed, whereas a similar level of casein was adequate (Table 12.10). However, reducing caloric intake to 100 calories despite an otherwise adequate protein ration influenced the number and size of fetuses (Leathern, 1959b). Additional proteins should be studied and related to biochemical changes in pregnancy and to the need for specific amino acids; for example, elimination of methionine or tryptophan from the diet may or may not be followed by resorption (Sims, 1951; Kemeny, Handel, Kertesz and Sos, 1953; Albanese, Randall and Holt, 1943). Excretion of 10 amino acids was in

NUTRITIONAL EFFECTS

691

creased during normal human pregnancy (Miller, Ruttinger and Macey, 1954).

That a relationship exists, between the dietary requirements just described to the endocrine substances which participate in the control of pregnancy, is suggested by the fact that the deleterious effects of a proteinfree diet on pregnancy in rats have been counteracted by the administration of estrone and progesterone. Pregnancy was maintained in 30 per cent, 60 to 80 per cent, and per cent of protein-deficient animals by daily dosages of 0.5 /xg., 1 to 3 fig., and 6 jug. estrone, respectively. On the other hand, injection of 4 to 8 mg. progesterone alone maintained pregnancy in 70 per cent of the animals (Nelson and Evans, 1955) , and an injection of 4 mg. progesterone with 0.5 //.g. estrone provided complete replacement therapy (Nelson and Evans, 1954). Food intake did not increase. The results suggest that reproductive failure in the absence of dietary protein was due initially to lack of progesterone and secondarily to estrogen, the estrogen possibly serving as an indirect stimulation for luteotrophin secretion and release. It is well known that hypophysectomy or ovariectomy shortly after breeding will terminate a pregnancy and that replacement therapy requires both ovarian hormones. Thus, the protein-deficient state differs somewhat from the state following hypophysectomy or ovariectomy, but the factors involved are not known.

Pregnancy alters nutritional and metabolic conditions in such a way that labile protein stores of the liver and other parts of the body are influenced, but similar effects are imposed by a transplanted tumor, especially when it reaches 10 per cent of the body weight. ' Thus, transplantation of a tumor into a pregnant animal would place the fetuses in competition with the tumor for the amino acids of the metabolic pool. Under these circumstances will the pregnancy be maintained? An answer to the question may not yet be given. Nevertheless, Bly, Drevets and Migliarese (1955) observed various degrees of fetal damage in pregnant rats bearing the Walker 256 tumor, and 43 per cent fetal loss was obtained with a small hepatoma (Paschkis and Cantarow, 1958).

Essential fatty acid deficiency, at least in the initial stages, does not interfere with

development of the fetuses or parturition in the rat, but the pups may be born dead or they do not survive more than a few days (Kummerow, Pan and Hickman, 1952). A more pronounced deficiency has induced atrophic changes in the decidua, resorption of fetuses, and prolonged gestation. Death of the fetuses appears to be secondary to placental injury. Hormonal involvement, if any, when there is fatty acid deficiency and pregnancy seems not to have been investigated.

Pregnancy and lactation are major factors influencing vitamin requirements. It is not surprising, therefore, that vitamin deficiencies influence the course of a pregnancy. The subject has recently been reviewed by Lutwak-jMann (1958).

A deficiency of vitamin A does not noticeably affect early fetal development, but later in gestation placental degeneration occurs with hemorrhage and abortion. When the deficiency is moderate the pregnancy is not interrupted, but the fetuses are damaged (Warkany and Schraffenberger, 1944; Wilson, Roth and Warkany, 1953; Giroud and Martinet, 1959). In calves and pigs the abnormalities are associated with the eyes and palate (Guilbert, 1942) ; in birds skeletal abnormalities are seen (Asmundson and Kratzer, 1952). The use of hormones in an effort to counteract the effects seems to have been attempted only in the rabbit where 12.5 mg. progesterone improved reproduction impaired by vitamin A lack (Hays and Kendall, 1956). Vitamin A excess also proves highly detrimental to pregnancy, as resorption and malformations occur. Administration of excessive vitamin A on days 11 to 13 of pregnancy induced cleft palate in 90 per cent of the embryos (Giroud and Martinet, 1955) . In another experiment the effect of excessive vitamin A was augmented by cortisone (Woollam and Millen, 1957).

Vitamin E deficiency has long been known to influence pregnancy in rodents and fetal death appears to precede placental damage and involution of the corpora lutea. Gross observations of the abnormal embryos have been reported (Cheng, Chang and Bairnson, 1957). Estrogen, progesterone, and lactogen were not effective in attempts at corrective therapy (Ershoff, 1943), but estrone and progesterone markedly reduced the in

692

PHYSIOLOGY OF GONADS

cidence of congenital malformations associated with vitamin E lack (Cheng, 1959). In the test of a possible converse relationship, estradiol-induced abortion in guinea pigs was not prevented by vitamin E (Ingelman-Sundberg, 1958) .

Fat-soluble vitamins incorporated in the diet may be destroyed by oxidation of the unsaturated fatty acids. To stabilize the vitamins, the addition of diphenyl-p-phenylenediamine (DPPD) to the diet has proven successful, but recent studies show that DPPD has an adverse effect on reproduction and thus its use in rat rations is contraindicated (Draper, Goodyear, Barbee and Johnson, 1956).

Vitamin-hormone relationships in pregnancy have been studied with regard to thiamine, pyridoxine, pantothenic acid, and folic acid. Thiamine deficiency induced stillbirths, subnormal birth weights, resorption of fetuses, and loss of weight in the mother. However, as in the case of protein deficiency, pregnancy could be maintained with 0.5 fjLg. estrone and 4 mg. progesterone (Nelson and Evans, 1955). Estrone alone had some favorable effect on the maintenance of pregnancy in thiamine-deficient animals, but it was less effective in protein-deficient animals.

Fetal death and resorptions as well as serum protein and nonprotein nitrogen (NPN) changes similar to those reported for toxemia of pregnancy (Ross and Pike, 1956; Pike and Kirksey, 1959) were induced by a diet deficient in vitamin Be . Administration of 1 fxg. estrone and 4 mg. progesterone maintained pregnancy in 90 per cent of vitamin Be-deficient rats (Nelson, Lyons and Evans, 1951). However, the pyridoxinedeficient rat required both steroids to remain pregnant and in this regard resembled the hypophysectomized animal (Nelson, Lyoas and Evans, 1953). Nevertheless, a hypophyseal hormone combination which was adequate for the maintenance of pregnancy in the fully fed hypophysectomized rat (Lyons, 1951) was only partially successful when there was a deficiency of pyridoxine. An ovarian defect is suggested.

The folic acid antagonist, 4-aminopteroylglutamic acid, will rapidly induce the death of early implanted embryos in mice.

rats, and man (Thiersch, 1954j . Removal of folic acid from the diet or the addition of x-methyl folic acid will induce malformations when low doses are given and resorptions when high doses are given. Furthermore, this effect is obtained even when the folic acid deficiency is delayed until day 9 of a rat pregnancy or maintained for only a 36-hour period. A deficiency of pantothenic acid will also induce fetal resorption. The vitamin is required for hatching eggs (Gillis, Heuser and Norris, 1942). In animals deficient in folic acid or in pantothenic acid, estrone and progesterone replacement therapy did not prevent fetal loss, suggesting that the hormones cannot act (Nelson and Evans, 1956). In the above mentioned deficiencies replacement of the vitamin is effective. However, vitamins other than those specifically deleted may provide replacement, thus ascorbic acid seems to have a sparing action on calcium pantothenate (Everson, Northrop, Chung and Getty, 1954) .

Pregnancy can be interrupted by altering vitamins other than those discussed above, but the hormonal aspects have not been explored. Thus, the lack of choline, riboflavin, and Bi2 will induce fetal abnormalities and interrupt gestation (Giroud, Levy, Lefebvres and Dupuis, 1952; Dryden, Hartman and Gary, 1952; Jones, Brown, Richardson and Sinclair, 1955; Newberne and O'Dell, 1958) . Choline lack is detrimental to the placenta (Dubnov, 1958), riboflavin deficiency may impair carbohydrate use (Nelson, Arnrich and Morgan, 1957) and/or induce electrolyte disturbances (Diamant and Guggenheim, 1957) , and Bjo spares choline and may be concerned with nucleic acid synthesis (Johnson, 1958). Excessive amounts of Bio are not harmful. It is interesting to note that uterine secretions and rabbit blastocyst fluid are rich in vitamin B]2 (Lutwak-Mann, 1956), but its presence in such large amounts has not been explained.

An additional substance, lithospermin, extracted from the plant, Lathijrus odoratus, is related to hormone functioning; it is antigonadotrophic when eaten by nonpregnant animals and man. The feeding of this substance to prciiiiant rats terminated the pregnancies about the 17th day. Treatment with estrogen and progesterone was preventive (Walker and Wirtschafter, 1956). It is assumed, therefore, that lithospermin interfered with the production of these hormones. A repetition of the experiment on a species in which the hypophysis and ovaries are dispensable during much of pregnancy would be of interest.

In retrospect it has been found that a deficiency in protein and the vitamins thiamine, pyridoxine, pantothenic acid, and folic acid individually can interrupt a pregnancy. Furthermore, a combination of estrone and progesterone which is adequate to maintain pregnancy after hypophysectomy and ovariectomy, is equally effective in protein or thiamine deficiency. This suggests that the basic physiologic alteration is a deprivation of ovarian hormones. However, protein- and thiamine-deficiency states differ from each other as shown by the response to estrogen alone (thiamine deficiency is less responsive), and these states differ from hypophysectomy in which estrone alone has no effect. A pyridoxine deficiency seems to involve both ovary and hypophysis, for neither steroids nor pituitary hormones were more than partially successful in maintaining pregnancy in rats. Lastly, pantothenic acid and folic acid deficiencies may not create a steroid deficiency. What is involved is not known; many possibilities exist. Pantothenic acid, for example, participates in many chemical reactions. Furthermore, it is known that thiamine is essential for carbohydrate metabolism but not for fat metabolism whereas pyridoxine is involved in fat metabolism and in the conversion of tryptophan to nicotinic acid. It is clear, though, that much ground must be covered before the formulation of fruitful hypotheses may be anticipated.

VI. Concluding Remarks

The development, composition, and normal functioning of the reproductive system is dependent on adequate nutrition. However, the requirements are many and only gradually are data being acquired which are pertinent to the elucidation of the nutritional-gonadal relationship.

The demands for nutrient substances is not always the same. During pregnancy and lactation there is a need for supplemental feeding. A similar need exists in birds and in the many cold-blooded vertebrates in which reproduction is seasonal. Atypical endocrine states create imbalances and a need for nutrient materials which vary, unpredictably, we must acknowledge, until the numerous interrelationships have been clarified.

At many points where determination of cause and effect are possible, an indirect action of dietary factors on reproduction is indicated. No other conclusion seems possible in view of the many instances in which the effect of dietary deficiencies can be counteracted by the administration of a hormone or combination of hormones. The direct action is not immediately apparent; it probably is on the processes by which metabolic homeostasis is maintained, and is in the nature of a lowering of the responsiveness to the stimuli which normally trigger these processes into action. The processes may be those by which pituitary and gonadal hormones are produced or they may be the mechanisms by which these hormones produce their effects on the genital tracts and on the numerous other tissues on which they are known to act.

Because of the many interrelationships, some of which are antagonistic and some supportive, determination of the role of specific dietary substances is not easy. For those who work with laboratory species, the problem is further complicated by the many strain differences. For everyone, the problem is complicated by the many species differences which are the result of an evolution toward carnivorous, herbivorous, or omnivorous diets, to say nothing of the countless specific preferences within each group.

Finally, it is something of a paradox in our culture that much of our effort has been devoted to investigations of the effects of deficiencies and undernutrition rather than to the effects of excesses and overnutrition. Much evidence supports the view that in the aggregate the latter are fully as deleterious as the former, but the means by which this result is achieved are largely unknown.

VII. References

Aak.s-Jorgensen, E., Funch, J. P., and Dam, H.

1956. Role of fat in diet of rats; influence on reproduction of hydrogenated arachis oil as sole dietary fat. Brit. J. Nutrition, 10, 317.

Aaes-Jorgensen, E., Funch, J. P., and Dam, H.

1957. Role of fat in diet of rats; influence of small amount of ethyl linoleate on degeneration of spermatogenic tissue caused by hydrogenated arachis oil as sole dietary fat. Brit. J. Nutrition, 11, 298.

AcETO, G., Li Moli, S., and Panebianco, N. 1956. Influence of adrenocorticotrophin (ACTH) on the urinary excretion of thiamine. Acta vitaminol., 10, 175.

Adams, C. E. 1953. Mammalian germ cells. Ciba Foundation Symposium, 198.

Adams, C. W. M., Fernand, V. S. V., and Schnieden, H. 1958. Histochemistry of a condition resembling kwashiorkor produced in rodents by a low protein-high carbohydrate diet (cassaval). Brit. J. Exper. Path., 39, 393.

Addis, T., Poo, L. J., and Lew, W. 1936. The ciuantities of protein lost by the various organs and tissues of the body during a fast. J. Biol. Chem., 115, 111.

Ahren, K. 1959. The effect of dietary restriction on the mammary gland development produced by ovarian hormones in the rat. Acta endocrinol., 31, 137.

Albanese, a. a., Randall, R. M., .\nd Holt, L. E., Jr. 1943. The effect of tryptophan deficiency on reproduction. Science, 97, 312.

Angervall, L. 1959. Alloxan diabetes and pregnancy in the rat. Effects on offspring. Acta Endocrinol., suppl. 44, 31, 86.

Arvy, L., Aschkenasy, A., Aschkenasy-Lelu, P., AND Gabe, M. 1946. Retentissement d'un regime prolonge d'inanition proteique sur les glandes genitales du rat femelle. Compt. rend. Soc. biol., 140, 730.

Aschkenasy, A. 1954. Action de la testosterone, de la thyroxine ct do la cortisone sur revolution de I'anemie et de la leucopenie proteiprives. Le Sang, 25, 15.

Aschkenasy, A. 1955a. Influence des proteines alimentaires et de divers acides amines sur la survie et le poids somatique de i-ats surrenalectomises et de rats surrenalcctomises-castres. Ann. endocrinol., 16, 86.

Aschkenasy, A. 1955b. Influence des proteines alimentaires et de divers acides amines sur la survie et sur le poids somatique de rats surrenalcctomises et de rats surrenalectomisescastres. Ann. endocrinol.. 16, 199.

Aschkenasy, A. 1955c. Effects de la .surrenalectomie sur les poids relatifs de divers organes et tis.sus. Role du facteur temps et influence do la richnesse du regime en proteines. J. jjhvsiol., Paris, 47, 75.

Aschkenasy, A. 1957. Effets compares de I'ACTH, de la cortisone et de la delta-cortisone sur les poids de divers organes et tissus chez le rat male adulte. Ann. endocrinol., 18, 981.

Aschkenasy, A., Aschkenasy-Lelu, P. 1957. Nouvelles recerches sur le role des surrenales dans la gestation en regime pauvre en proteines. Compt. rend. Soc. biol., 151, 880.

Aschkenasy, A., and Dray, F. 1953. Action de quelques hormones sur la reprise du poids et sur la regeneration des organes apres inanition proteique experimental. Compt. rend. Soc. biol., 147, 1722.

Aschkena.sy-Lelu, p., and Aschkenasy, A. 1947. Influence du taux des protides de la ration sur le cycle oestral de la rate adulte. Perturbation du cycle avec prolongation des oestrus sous d'action d'un regime hyperprotidique. Compt. rend. Soc. biol., 141, 687.

Aschkenasy-Lelu, P., and Aschkenasy^ A. 1957. Intervention des surrenales dans la gestation en fonction de la teneur de regime en proteines. Arch. so. physioL, 11, 125.

Aschkenasy-Lelu, P., and Tuchmann-Duplessis, H. 1947. Action d'un regime hyperprotidique sur le developpement somaticjue et sexual de la jeune rate. c3ompt. rend. Soc. biol., 141, 1166.

AsDELL, S. A. 1949. Nutrition and treatment of sterility in dairy cattle: a review. J. Dairv Sc. 32, 60.

AsMUND.soN, V. S., AND Kr.\tzer, F. H. 1952. Observations on vitamin A deficiency in turkey breeding stock. Poultry Sc, 31, 71.

Baker, B. L. 1952. Comparison of histological changes induced by experimental hyperadrenalcorticalism and inanition. Recent Progr. Hormone Res., 7, 331.

Ball, Z. B., B.\rnes, R. H., and Visscher, M. B. 1947. The effects of dietary caloric restriction on maturity and senescence, with particular reference to fertility and longevitv. Am. J. Physiol., 150, 511.

Barborlak, J. J., Krehl, W. A., Cowgill, G. R.. .-AND Whedon, a. D. 1957. Effect of partial pantothenic acid deficiencj' on reproductive performance of rat. J. Nutrition, 63, 591.

BE.ATON, G. H., Ryu. M. H., and McHenry. E. W. 1955. Studies on the role of growth hormone in pregnancy. Endocrinology, 57, 748.

Beckmann, R. 1955. Vitamin E (Physiologic, pathologische Physiologie, khnische Bedeutung). Ztschr. Vitamin- Hormon- u. Fermentforsch., 7, 281.

Berg, C. P., and Rohse, W. G. 1947. Is sterility induced in growing rats on a tryptoplian deficient diet? Science. 105, 96.

BiDDULiMi, C, AND Meyer. R. K. 1941. Tho influence of vitamin E-deficiency on the endocrine glands of rats, particularly on the gonadotrophic hormone content of the pituitary gland. Am. J. Physiol., 132, 259.

Bishop, D. W., and Ko.s.\rick, E. 1951. Blot in deficicncv and crytorchidism in rats. Proc. Soc. Exper. Biol. & Med., 78, 323.

Biskind, M. S. 1946. Nutritional therapy of endocrine disturbances. Vitamins & Hormones, 4, 147.

Blandau, R. J., Kauntiz, H., and Slanetz, C. A. 1949. Ovulation, fertilization and transport of ova in old vitamin E-deficient rats. J. Nutrition, 38, 97.

Blaxter, K. L., and Brown, F. 1952. Vitamin E in the nutrition of farm animals. Nutritional Abstr. & Rev., 21, 1.

Blivaiss, B. B., Hanson, R. O., Rosenzweig, R. E., AND McNeil, K. 1954. Sexual development in female rats treated with cortisone. Proc. Soc. Exper. Biol. & Med., 86, 678.

Bly, C. G., Drevets, C., and Migliarese, J. F. 1955. Competition between fetal growth and transplanted tumor growth in pregnant rats. Fed. Proc, 14, 399.

Blye, R. p., and Leathem, J. H. 1959. Ovarian ascorbic acid in gonadotrophin-treated and normal hypothyroid rats. Fed. Proc, 18, 49.

Bo, W. J. 1955. The effect of ovariectomy on metaplastic changes in the uteri of vitamin A deficient rats. Anat. Rec, 121, 241.

Bo, W. J. 1956. The relationship between vitamin A deficiency and estrogen in producing uterine metaplasia in the rat. Anat. Rec, 124, 619.

Bo, W. J., and Atkinson, W. B. 1953. Histochemical studies on glycogen deposition in uterus of the rat. III. Effect of starvation. Proc. Soc. Exper. Biol. & Med., 83, 405.

BoLDT, R. E., Harper, A. E. and Elvehjem, C. A. 1958. The effect of amino acids on the survival of the hvperthyroid rat. Arch. Biochem., 74, 437.

Booth, V. H. 1952. Liver storage of vitamin A by male and female rats. J. Nutrition, 48, 13.

Bourdel, G. 1956. L'anabolisme gravidique: ses caracteristiques son determinisme. In Proceedings Fourth International Congress of Nutrition, p. 37.

Br.\ekkan, O. R. 1955. Role of pantothenic acid in the reproductive cycle of ovaries in fish. Nature, London, 176, 598.

BR.ATT0N, R. W. 1957. Nutrition and fertility. Cornell University Agric Exper. Sta. Bull., 924, 25.

Breneman, W. R. 1940. Limitation of food consumption as a factor influencing endocrine reactions in the chick. Endocrinolog.y, 26, 1091.

Brown, W. O. 1953. Effect of deoxypentose nucleic acid on impaired oviduct response to oestrogen in the folic acid deficient-chick. Biochim. et biophys. acta, 11, 162.

Bryan, W. L., .^nd Mason, K. E. 1941. Vitamin E deficiency in the mouse. Am. J. Physiol., 131, 263.

Burr, G. O., and Burr, M. M. 1930. On the nature and role of the fatty acids essential in nutrition. J. Biol. Chem., 86, 587.

Byerly, T. C, and Burrows, W. H. 1938. Chick testis weight response to gonadotrophic hormone. Endocrinology, 22, 366.

Campbell, R. M., Innes, I. R., and Kosterlitz, H. W. 1953a. The role of hormonal and

dietary factors in the formation of excess ribonucleic acid in the livers of pregnant rats. J. Endocrinol., 9, 52.

Campbell, R. M., Innes, I. R., and Kosterlitz, H. W. 1953b. Some dietary and hormonal effects on maternal, fetal and placental weights in the rat. J. Endocrinol., 9, 68.

Campbell, R. M., and Kosterlitz, H. W. 1953. Species differences in the deoxyribonucleic and ribonucleic acid contents of livers of nonpregnant and pregnant mice, guinea pigs and cats. J. Endocrinol., 9, 45.

Cantarow, a., Paschkis, K. E., Rakoff, A. E., and Hansen, L. P. 1943. Studies on inactivation of estradiol by the liver. Endocrinology, 33, 309.

Carroll, K. K., and Noble, R. L. 1952. Effects of feeding rape oil on some endocrine functions of the rat. Endocrinology, 51, 476.

Cheng, D. W. 1959. Effect of progesterone and estrone on the incidence of congenital malformations due to maternal vitamin E deficiency. Endocrinolog}-, 64, 270.

Cheng, D. W., Chang, L. F., and Bairnson, T. A. 1957. Gross observations on developing abnormal embryos induced by maternal vitamin E deficiency. Anat. Rec, 129, 167.

Chester Jones, I. 1957. The Adrenal Cortex. Cambridge : Cambridge University Press.

Cole, H. H., Guilbert, H. R., and Goss, H. 1932. Further considerations of the properties of the gonad-stimulating principle of mare serum. Am. J. Physiol., 102, 227.

Coste, F., Delbarre, F., .\nd Lacronique, F. 1953. Variations du taux de I'acide ascorbiques des endocrines sous I'infiuence des stimulines hypophj'saires. Taux de I'acide ascorbique des testicules du rat en fonction de I'age. Compt. rend. Soc. biol., 147, 608.

CouRRiER, R., AND CoLLONGE, A. 1951. Cortisone and gestation. J. A. M. A., 146, 493.

CouRRiER, R., AND R.^YNAUD, R. 1932. Modifications genitales chez les animaux a croissance arretee par la deficience en lysine. Compt. rend. Soc. biol., 109, 881.

Cupps, P. T. 1955. The ability of certain adrenal steroids to restore reproduction in adrenalectomized female rats. Endocrinology, 57, 1.

Curtiss, C. 1953. Effects of a low protein intake on the pregnant rat. Metabolism, 2, 344.

CuRTO, G. M. 1954. Experimental vitamin E deficiency in male guinea pigs. Acta vitaminol., 8,7.

D'Angelo, S. a. 1951. Effect of acute starvation on the thyrotrophic hormone level in the blood of rat and mouse. Endocrinology, 48, 341.

Davies, D. v., Mann, T., and Rowson, L. E. A. 1957. Effect of nutrition on the onset of male sex hormone activity and sperm formation in monozygous bull-calves. Proc. Roy. Soc. Loudon, ser. B, 147, 332.

D.wis, J. S., Meyer, R. K., and McShan, W. H. 1956. The effects of aminopterin and estrogen on the phosphate metabolism of rat uterus. Endocrinology, 59, 505.

Davis. M. E., Fugo, N. W., .'Vxd Lawrence, K. G. 1947. Effect of alloxan diabetes on reproduction in the rat. Proc. Soc. Exper. Biol. & Med., 66, 638.

Davis, M. E., and Plotz, E. J. 1954. The effects of cortisone acetate on intact and adrenalectomized rats during pregnancy. Endocrinology, 54, 384.

DeFeo, V. J., and Rothchild, I. 1953. The effects of starvation of intact young rats on deciduoma formation and the maintenance of pseudopregnancy. Anat. Rec, 115, 297.

Delost, p., and Terroine, T. 1954. Effets de la carence en biotine sur certaines glandes endocrines au cours du developpement post-natal du rat. Compt. rend. Acad. Sc, 239, 902.

DeMeig, R. H., Rakoff, A. E., Cantarow, A., and Paschkis, K. E. 1948. Mechanism of inactivation of alpha-estradiol by the rat liver in vitro. Fed. Proc, 7, 27.

Desaive, p. 1956. Du role de la vitamine C dans le fonctionnement de I'ovaire. Compt. rend. A. anat., 42, 431.

Deuel, H. J. 1956. Sex differences in fat and carbohydrate metabohsm. Internat. Ztschr. Vitaminforsch., 26, 352.

Deuel, H. J., Martin, C. R., and Alfin-Slater, R. B. 1954. The effect of fat level of the diet on general nutrition. J. Nutrition, 54, 193.

Dhyse, F. G., Fisher, G. R., Tullner, W. W., AND Hertz, R. 1953. Liver vitamin content and adrenal cortical function. Endocrinology, 53, 447.

Diamant, E. J., AND Guggenheim, K. 1957. Electrolyte metabolism is pyridoxine, riboflavin and pantothenic acid-deficient rats. Am. J. Physiol., 191, 108.

DiCio, A. v., AND Schteingart, M. 1942. The influence of ascorbic acid on the activity of gonadotrophic hormones. Endocrinology, 30, 263.

Dinning, J. S., Sime, J. T., and Day, P. L. 1956. Increased incorporation of P^- into nucleic acids by vitamin E-deficient rabbits. J. Biol. Chem., 222, 215.

Dju, M. Y., M.asgn, K. E., and Filer, L. J. 1958. Vitamin E in human tissues from birth to old age. Am. J. Clin. Nutrition, 6, 50.

Draper, H. H., Goodyear, S., Barbee, K. D., and Johnson, B. C. 1956. Tolerance of the rat for A,iV^-dipli('nvI-/^-]ihenvlenediamine. Proc. Soc. Exper. Biol. <^ Med., 93, 186.

Draper, H. H., and Johnson, B. C. 1953. Effect of cortisone on metabolism of certain B vitamins in the rat. Proc. Soc. Exper. Biol. & Med., 82, 73

Drashek, M. L., Dickie, M. M., and Lane. P. W. 1955. Physiologic differences in uteri of obese stock mice. J. Hered., 46, 209.

Drill, V. A. 1943. Interrelations between thyroid function and vitamin metabolism. Phvsiol. Rev., 23, 355.

Drill, V. A., .and Burrill, M. W. 1944. Effect of thiamine deficiency and controlled inanition on ovarian function. Endocrinology, 35, 187.

Drill, V. A., and Pfeiffer, C. A. 1946. Effect of vitamin B complex deficiency, controlled inanition and methionine on inactivation of estrogen by the liver. Endocrinology, 38, 300.

Drummond, J. C, Noble, R. L., .\nd Wright, M. D. 1939. Studies of relationship of vitamin E (tocopherols) to the endocrine system. J. Endocrinol., 1, 275.

Dryden, L. p., Hartman, A. M., and Cary, C. A. 1952. The effect of vitamin B12 deficiency upon the survival of young born to rats fed purified casein rations. J. Nutrition, 46, 281.

DuBNOv, M. V. 1958. Chohne-free diets in pregnancy. Arch. Path., 20, 68.

DuMM, M. E., AND Ralli, E. p. 1953. Factors influencing the response of adrenalectomized rats to stress. Metabolism, 2, 153.

Eisenstein, a. B. 1957. Effects of dietary factors on production of adrenal steroid hormones. Am. J. Clin. Nutrition, 5, 369.

El-Skukh, a. S., Nulet, C. V., Pope, A. L., .'^nd Casida, L. E. 1955. The effect of level of feeding on the reproductive capacity of ewe. J. Anim. Sc, 14, 919.

Emerson, W. J., Z.amecnik, P. C, and Nathanson, I. T. 1951. Effect of sex hormones on hepatic and renal lesions induced in rats by a cholinedeficient diet. Endocrinology, 48, 548.

Erb, R. E., Andrews, F. N., Hauge, S. M., and King, W. A. 1947. Observations on vitamin A deficiency in young dairy bulls. J. Dairv Sc, 30, 687."

Ershoff, B. H. 1943. Degeneration of the corpora lutea in the pregnant vitamin E-deficient rat. Anat. Rec, 87, 297.

Ershoff, B. H. 1949. Beneficial effects of liver on growth and survival of immature rats fed lactose-containing diets. Proc Soc Exper. Biol. & Med., 72, 648.

Ershoff, B. H. 1952. Nutrition and the anterior pituitary with special reference to the general adaptation svndrome. Vitamins & Hormones, 10, 79.

Escudero, p., Herraiz, M. L., .and Mussmano, E. 1948. Experimental proof of the first law of nutrition. Rev. Asoc. argent. Dietol., 6, 195.

Evans, H. M., Lepkovsky, S., and Murphy, E. 1934. Vital need of the body for certain vmsaturated fatty acids; reproduction and lactation upon fat-free diets. J. Biol. Chem., 106, 431.

Evans, H. M., and Simpson, M. E. 1930. Subnormal sex-hormone content of the hypophysis of animals with inadequate antineuritic vitamin B. Anat. Rec, 45, 216.

Everson, G., Northrop, L., Chung. N. Y„ and Getty, R. 1954. Effect of ascorbic acid on rats deprived of pantothenic acid during pregnancy. J. Nutrition, 54, 305.

Farber, "e., and Corban, M. S. 1958. Sex difference in ethionine inhibition of hepatic protein synthesis. J. Biol. Chem., 233, 625.

Farber, E., and Segaloff, a. 1955. Effect of androgens and growth and other hormones on ethionine fatty Hver in rats. J. Biol. Chem., 216,471.

Farris, E. J., AND CoLTON, S. W. 1958. Effects of L-thyroxine and biothyronine on spermatogenesis. J. Urol., 79, 863. .

Ferrando, R., Jacques, F., Mabboux, H., and Prieur, G. 1955. Influence du torteau d'arachide delipide par extraction sur la fecondite de la femelle du rat. Compt. rend. Acad. Sc, 241, 253.

Ferrando, R., Jacques, F., Mabboux, H., and SolLOGOUB, C. 1955. Influence du torteau d'arachide delipide par extraction sur la croissance et I'integrite des testicules du rat. Compt. rend. Acad. Sc, 241, 107.

Ferret, P. Lindan, O., and Morgans, M. E. 1950. Pregnancy in insulin-treated alloxan diabetic rats. J. Endocrinol., 7, 100.

FiGGE, F. H. J., AND Allen, E. 1942. Genital atrophy during pantothenic acid deficiency and responses to gonadotrophic and estrogenic hormones (Abst.). Endocrinology, 30, S1028.

Finerty, J. C, Klein, G. F., and Panos, T. C. 1957. Effects of hormonal treatment on damaged testes of fat-deficient rats. Anat. Rec, 127, 293.

Fisher, H. 1957. The development of a free amino acid diet for the maintenance of egg production in chickens. In Proceedings Fourth International Congress of Nutrition, p. 39.

Flodin, N. W. 1953. Amino acids and proteins, their place in human nutritional problems. Agric. & Food Chem., 1, 222.

FoLLis, R. H. 1948. Pathology of Nutritional Diseases. Springfield, 111.: Charles C Thomas.

Forbes, G. B. 1957. Overnutrition for child: blessing or curse. Nutritional Rev., 15, 193.

Friedman, M. H. F., .and Friedman, G. S. 1940. The relation of diet to the restitution of gonadotrophic hormone content of the discharged rabbit pituitary. Am. J. Physiol., 128, 493.

Funk, C, and Funk, I. C. 1939. Action of certain hormones as dietarv constituents. Science, 90, 443.

Gaunt, R., and Parkins, W. H. 1933. The alleged interrelationship of the adrenal cortical hormone and the gonads. Am. J. Phvsiol., 103, 511.

Geller, F. C. 1933. Vitamin E and hormone. Arch. Gynak., 156, 345.

Gilbert, C, and Gillman, J. 1956. The changing pattern of food intake and appetite during the menstrual cycle of the baboon (Papio ursinus) with a consideration of some of the controlling endocrine factors. South African J. M. Sc, 21, 75.

Gillis, M. B., Heuser, G. F., and Norris, L. C. 1942. The need for pantothenic acid and an unidentified factor in reproduction in the domestic fowl. J. Nutrition, 23, 153.

Gillman, J., and Gilbert, C. 1956. Menstrual disorders induced in the baboon (Papio ursinus) by diet with a consideration of endocrine factors underlying the menstrual disorders and body weight. South African J. M. Sc, 21, 89.

GiNOULHiAC, E., and Nani, S. 1956. Urinary excretion of thiamine, cobalamin, pantothenic acid, folic acid, and folinic acid in rats treated with prednisone. Acta vitaminol., 10, 157.

GiRouD, A., and Desclaux, p. 1945. Modifications de I'hypophyse a la suite de la sousalunentation. Ann. endocrinol., 6, 107.

GiROUD, A., Levy, G., Lefebvres, J., and Dupuis, R. 1952. Chute du taux de la riboflavine au stade oii se determinent les malformations embryonanires. Internat. Ztschr. VitaminHormon- u. Fermentforsch., 23, 490.

Giroud, a., and M.artinet, M. 1955. Malformations diverses du foetus de rat suivant les stades d 'administration de vitamine A en exces. Compt. rend. Soc bioL, 149, 1088.

Giroud, A., and M.\rtinet, M. 1959. Extension a plusierus especes de mammiferes des malformations embryonnaires par hypervitaminose A. Compt. rend. Soc biol., 153, 201.

Gl.\ss, S. J., ASD Russell, M. 1952. Improved spermatogenesis after nutritional-liver regimen with and without testosterone. Fertil. & Steril., 3, 167.

Glasser, S. R. 1957. Influence of a protein-free diet on the immediate and latent effects of stilbestrol. Am. J. Phy.siol., 189, 441.

Goettsch, M. 1949. Minimal protein requirements of the adult rat for 28-day periods of maintenance of body weight. J. Nutrition, 45, 609.

Goldberg, G. M., Pfau, A., and Ungar, H. 1959. Effect of testosterone on testicular lesions produced by f/^-ethionine in rats. Am. J. Path., 35, 649.

GoLDiN, A., Greenspan, E. M., Goldberg, B.. and Schoenberg, E. B. 1950. Studies on the mechanism of action of chemotherapeutic agents in cancer: sex difference in toxicity of the folic acid analogue, 4-aminopteroylglutamic acid. Cancer, 3, 849.

Goldsmith, E. D., Nigrelli, R. F., and Ross, L. 1950. Effects of underfeeding on testosterone action in mice. Anat. Rec, 106, 197.

GoMEZ-MoNT, F., Paschkis, K. E., and Cantarow, A. 1947. Thiouiacil action in starved rats. Endocrinology, 40, 225.

GoMEZ-MoNT, F. 1959. Endocrine changes in chronic human undernutrition. In Protein Nutrition and Reproductive Physiology, p. 58. New Brunswick : Rutgers University Press.

Goth, E., Lengyel, L., Bencze, E., S.wely, C, and Majsay, a. 1955. The role of amino acids in inchicing hormone secretion. Experentia, 11, 27.

Goth, E., N.adashi, N., .and Sl.ader, E. 1958. The effect of amino acids on pituitary-adrenal activity. Ztschr. Vitamin- Hormon- u. Fermentforsch., 9, 184.

Gr.ayhack, J. T., and Scott, W. W. 1952. The effect of general dietary deficiencie.^ and response of the prostate of the albino rat to testosterone propionate. Endocrinology', 50, 406. Greenberg, S. M., and Ershoff, B. H. 1951. Effects of chorionic gonadotrophin on sex organs of male rats deficient in essential fatty acids. Proc. Soc. Exper. Biol. & Med., 78, 552.

GrEENSTEIN, J. p., BiRNBATJM, S. M., WiNITZ, M.,

AND Otey, M. C. 1957. Quantative nutritional studies with water-soluble chemically defined diet. I. Growth, reproduction and lactation in rats. Arch. Biochem., 72, 396.

Greer, M. A. 1950. Nutrition and goiter. Physiol. Rev., 30, 513.

Greer, M. A. 1956. Isolation from rutabaga seed of progoitrin, the precursor of the naturally occurring antithyroid compound, goitrin (l-5vinyl-2-thiooxazalidone). J. Am. Chem. Soc, 78, 1260.

Greer, M. A., and Astwood, E. B. 1948. The antithyroid effect of certain foods in man as determined with radioactive iodine. Endocrinology, 43, 105.

Griesbach, W. E., Bell, M. E., and Livingston, M. 1957. Changes in the pituitary gonadotrophs of the vitamin E-deficient male rat. Endocrinology, 60, 729.

GuiLBERT, H. R. 1942. Some endocrine relationships in nutritional reproductive failure. J. Animal Sc, 1, 3.

GuiLBERT, H. R., AND Goss, H. 1932. Some effects of restricted protein intake on the estrous cycle and gestation in the rat. J. Nutrition, 5, 251.

Gyorgy, p., Rose, C. S., and Shipley, R. A. 1947. Activity of estrone as a lipotropic factor. Arch. Biochem., 12, 125.

H.AYS, R. L., AND Kendall, K. A. 1956. Beneficial effect of progesterone on pregnancy in the vitamin A deficient rabbit. J. Nutrition, 59, 337.

Hellm.an, L., Bradlow, H. L., Zumoff, B., Fukushima, d. k., and g.\llagher, t. f. 1959. Thyroid-androgen interrelations and the hypocholesteremic effect of androsterone. J. Clin. Endocrinol.. 19, 936.

Herrick, E. H., Eiue, I. M., and Snow, M. R. 1952. Vitamin E in pituitary gland function of fowls. Proc. Soc. Exper. Biol. & Med., 79, 441.

Hertz, R. 1945. The quantitative relationship between stilbestrol response and dietary "folic acid" in the chick. Endocrinology, 37, 1.

Hertz, R. 1948. The role of factors of the Bcomplex in estrogen metabolism. Recent Progr. Hormone Res., 2, 161.

Hoar, R. M., Goy, R. W., and Young, W. C. 1957. Loci of action of thyroid hormone on reproduction in the female guinea pig. Endocrinology, 60, 337.

Hodgson, R. E., Hall, S. R., Sweetman, W. J., Wiseman, H. G., and Converse, H. T. 1946. The effect of vitamin A deficiency on reproduction in dairy bulls. J. Dairy Sc, 29, 669.

HoKFELT, B. 1950. Changes in the ascorbic acid content of corpus luteum of pregnancy in rabbit after gonadotrophic stimulation. Acta physiol. scandinav., 20, 172.

Holt, L. E., Jr., Albanese, A. A., Shettles, L. B., Kajdi, C, and Wangerin, D. M. 1942. Studies of amino acid deficiency in man. Fed. Proc, 1, 116.

Horn, E. H. 1955. Nutritional and hormonal influences upon reproductive maturation, organ weight, and histochemistry of the immature male rat. Endocrinology, 57, 399.

Horne, H. W., Jr., and Maddock, C. L. 1952. Vitamin A therapy in oligospermia. Fertil. & Steril, 3, 245.

Horstmann, p. 1950. Excretion of androgens in human diabetes mellitus. Acta endocrinol., 5,261.

HosoDA, T., Kaneo, T., Mogi, K., and Abe, T. 1956. Forced ovulation in gonadotrophintreated fasting hens. Proc. Soc Exper. Biol. & Med., 92, 360.

HoussAY, B. A. 1945. Action of adrenal insufficiency during pregnancy on mother (rats) and offspring. Rev. Soc. argent, biol., 21, 316.

HuLTQUiST, G. T. 1950. Diabetes and pregnancy : an animal study. Acta path, et microbiol. scandinav., 27, 695.

Hummel, K. P. 1957. Transplantation of o\aries of the obese mouse. Anat. Rec, 128, 569.

HusEBY, R. A., AND Ball, Z. B. 1945. A study of the genesis of histologic changes produced by caloric restriction in portions of the endocrine and reproductive systems of strain "A" female mice. Anat. Rec, 92, 135.

Ingalls, a. M., Dickie, M. M., .\nd Snell, G. D. 1950. Obese, a new mutation in mouse. J. Hered., 41, 317.

Ingelman-Sundberg, a. 1954. Testicles and reproduction in vitamin E-deficient guinea pigs. Acta endocrinol., 17, 165.

Ingelman-Sundberg, A. 1958. Failure of vitamin E to prevent placental changes and abruptio placentae in guinea pigs treated with oestrogens and in women suffering from hypertension, chronic renal disease or toxaemia of pregnency. Acta endocrinol., 27, 509.

Ingle, D. J. 1948. The production of experimental glycosuria in the rat. Recent Progr. Hormone Res., 2, 229.

Ingle, D. J., and Nezamis, J. E. 1947. The effect of insulin on the tolerance of normal male rats to the overfeeding of a high carbohydrate diet. Endocrinology, 40, 353.

Ishida. K. 1957. Effect of the levels of protein in the diets on the ovaries of the young and adult rats. Tohoku J. Agric, 8, 17.

Jackson, H. 1959. Antifertility substances. Pharmacol. Rev., 11, 135.

Jailer, J. W. 1948. The effect of inanition on the inactivation of estrogen by the liver. Endocrinology, 43, 78.

Jailer, J. W., and Seaman, L. 1950. Estrogen inactivation by the liver as modified by dietary protein. Proc Soc. Exper. Biol. & Med., 73, 70.

Janes, R. G. 1944. Occurrence of follicular cysts in thyroidectomized rats treated with dietliylstilbestrol. Anat. Rec, 90, 93.

Jeans, P. C, Smith, M. B., and Stearns, G. 1955. Incidence of prematurity in relation to maternal nutrition. J. Am. Dietet. A., 31, 378.

Johnson, B. C. 1958. Studies on the mechanism of action of vitamin B12 in animal nutrition. Am. J. Clin. Nutrition, 6, 34.

Jones, C. C, Brown, S. O., Rich.ardson, L. R., and SiNCL.MR, J. G. 1955. Tissue abnormalities in newborn rats from vitamin Bi2-deficient mothers. Proc. Soc. Exper. Biol. & Med., 90, 135.

J0NE.S, G. E. S., Howard, J. E., .and Langford, H. 1953. The use of cortisone in follicular phase disturbance. Fertil. & Steril., 4, 49.

Kahn, R. H. 1954. Effect of locally appHed vitamin A and estrogen on the rat vagina. Am. J. Anat., 95, 309.

K.\TSH, S., KosARicK, E., AND Alpern, J. 1955. Effect of biotin deficiency on body and organ growth and accessory reproductive structures in male rats. Growth, 19, 45.

Kaufman, N., Klavins, J. V., and Kinney, T. D. 1956. Te.sticular damage following ethionine administration. Am. J. Path., 32, 105.

Kaunitz, H., Slanetz, C. A., and Atkinson, W. B. 1949. Estrogen response and pigmentation of the uterus in vitamin E-deficient rat. Proc. Soc. Exper. Biol. & Med.. 70, 302.

Kaunitz, H., Slanetz, C. A., Johnson, R. E.. and GuiLiuiN, J. 1956. Influence of diet compositions on caloric recjuirements, water intake and organ weights of rats during restricted food intake. J. Nutrition, 60, 221.

Kehl, R., Doxjard, T., and L.anfranchi, J. 1951. Sur Taction synergicjue de la vitamine E dans le conditionnement hormonal du deciduome traumaticiue, chez la lapine non gestante. Compt. rend. Soc. biol., 145, 1161.

Kemeny, T., Handel, M. B., Kertesz, P., and Sos, J. 1953. Effect of methionine deficiency on fertilitv in rat. Kiserletes Orvostudomanv, 5, 57.

Keys, A., Brozec, J., Hernschel, A., Michelsen. O., AND Taylor, H. L. 1950. The Biology of Human Starvation. Minneapolis: University of Minnesota Press.

Kline, I. T. 1955. Relationship of vitamin Bii to stilbestrol stimulation of the chick oviduct. Endocrinology, 57, 120.

Kline, I. T., and Dorfman, R. I. 19ola. Testosterone stimulation of seminal vesicles in pteroylglutamic acid-deficient rats. Endocrinology, 48, 34.

Kline, I. T., and Dorfman, R. I. 1951b. Estrogen stimulation of the o\iduct in vitamin-deficient chicks. Endocrinology. 48, 345.

Klinefelter, H. F., Jr., Albright, F., .and GrisWOLD, G. C. 1943. Experience with ciuantitative test for normal or decreased amounts of follicle-stimulating hormone in the urine in endocrinological diagnosis. J. Clin. Endocrinol., 3, 529.

Knox, W. E., Auerbach, V. H., .\nd Lin, C. C. 1956. Enzymatic and metabolic adaptations in animals. Physiol. Rev., 36, 164.

Kocen, B. p., .and C.wazos, L. F. 1958. Cytochemistry of male reproductive tract in scurvy

and inanition. Proc. Soc. Exper. Biol. & Med., 98, 485.

Ku.MMEROw, F. A., Pan, H. P., and Hickman, H. 1952. Effect of dietaiy fat on the reproductive performance and the mixed fatty acid composition of fat-deficient rats. J. Nutrition, 46, 489.

Leathem, J. H. 1953. Relationships between thyroid and protein metabolism. In Protein Metabolism, Hormones and Growth, p. 17. New Brunswick : Rutgers University Press.

Leathem, J. H. 1954. Hormonal and nutritional influences on male reproductive sj^stem. Anat. Rec, 118,323.

Le.athem, J. H. 1957. The adrenal and dietary protein. Arch, internat. pharmacodvn., 60, 103.

Le.athem, J. H. 1958a. Hormones and protein nutrition. Recent Progr. Hormone Res., 14, 141.

Le.athem, J. H. 19o8b. Hormonal influences on the gouadotrophin sensitive hypothyroid rat ovary. Anat. Rec, 131, 487.

Le.athem, J. H. 1959a. Some biochemical aspects of the uterus. Ann. New York Acad. Sc, 75, 463.

Le.\them, J. H. 1959b. Extragonadal factors in reproduction. In Recent Progress in the Endocrinology of Reproduction, p. 179. New York: Academic Press, Inc.

Le.athem, J. H. 1959c. The male reproductive system and protein nutrition. In Reproductive Physiology and Protein Nutrition, p. 22. New Brunswick : Rutgers University Press.

Le.\them, J. H., AND DeFeo, v. J. 1952. Responses of the mouse gonad to eciuine pituitary gouadotrophin as influenced bj' dietary protein restriction. Anat. Rec, 112, 356.

Leathem, J. H., and Fisher, C. J. 1959. Male rat reproductive system and protein nutrition. Anat. Rec, 133, 302.

Le.\them, J. H., Nocenti, M. R., and Granitsas, A. 1956. Effect of testosterone propionate and diets supplemented with methionine on body weight and organ weights of female rats. Proc Penn.sylvania Acad. Sc, 30, 38.

Lee, Y. C. P., King, J. T., and Visscher, M. B. 1952. Influence of protein and caloric intake upon certain reproductive functions and carcinogenesis in the mouse. Am. J. Phvsiol., 168,391.

LeRoy, p., and Domm, L. V. 1951. Observations on cortisone administration in normal and decapitated fetal rats. Anat. Rec, 109, 319.

Lowe, J. S., and Morton, R. A. 1956. Some aspects of vitamin A metabolism. Vitamins & Hormones, 14, 97.

Lowe, J. S., Morton, R. A., Cunningh.^m, N. F., AND Vernon, J. 1957. Vitamin A deficiencA' in domestic fowl. Biochem. J., 67, 215.

Lutwak-Mann, C. 1954. Note on the chemical composition of bovine follicular fluid. J. Agric Sc, 44, 477.

Lutwak-Mann, C. 1956. Biochemical relationships between the early rabbit embryo and its environment. In Third International Congress on Animal Reproduction, p. 50. London: Brown, Knight & Truscott. Ltd.

Lutwak-Mann, C. 1958. Dependence of gonadal function upon vitamins and other nutritional factors. Vitamins & Hormones, 16, 35.

Lutwak-Mann, C, and Manx, T. 1950. Restoration of secretory function in male accessory galnds of vitamin B-deficient rats by means of chorionic gonadotrophin. Nature, London, 165, 556.

Lyons, W. R. 1951. Maintenance of pregnancy in hypophysectomized rats. Anat. Rec, 109, 378.

Mackenzie, C. G. 1942. Cure of repeated attacks of nutritional muscular dystrophy in the rabbit by a-tocopherol. Proc. Soc. Exper. Biol. & Med., 49, 313.

Maddock, C. L., Cohen, J., and Wolb.^ch, S. R. 1953. Effect of hypervitaminosis A on the testis of the rat. Arch. Path., 56, 333.

M.\DD0CK, W. O., Chase, J. D., and Nelson, W. O. 1953. Effects of large doses of cortisone on testicular morphology and urinary gonadotrophin, estrogen, 17-ketosteroid excretion. J. Lab. & Clin. Med., 41, 608.

Maddock, W. O., and Heller, C. G. 1947. Dichotomy between hypophyseal content and amount of circulating gonadotrophins during starvation. Proc. Soc. Exper. Biol. & Med., 66, 595.

McCarthy, P. T., and Cerecedo, L. R. 1952. Vitamin A deficiency in the mouse. J. Nutrition, 46, 361.

Maeder, E. C. 1937. The effect of fat in simplified diets on the reproductive organs of the female albino rat during gestation. Anat. Rec, 70, 73.

Mancini, R. E., Izquierdo, I. A., Heinrich, J. J., Penhos, J. C, AND Gerschenfeld, H. M. 1959. Effect on albino rat testes of single and multiple hypoglycemic coma. In Tiventy-first International Congress oj Physiological Sciences, p. 174.

Mandl, a. M. 1954. The sensitivity of adrenalectomized rats to gonadotrophins. J. Endocrinol., 11, 359.

Mann, T., and Lutwak-Mann, C. 1951. Secretory function of male accessory organs of reproduction in mammals. Physiol. Rev., 31, 27.

Mann, T., and Walton, A. 1953. Effect of underfeeding on genital functions of the bull. J. Agric. Sc, 43, 343.

Maqsood, M. 1952. Thyroid functions in relation to reproduction of mammals and birds. Biol. Rev. Cambridge Phil. Soc, 27, 281.

Marrian, G. F., and Parkes, A. S. 1929. The effect of anterior pituitary preparation administered during dietary anoestrus. Proc. Roy. Soc. London, ser. B, 105, 248.

Masin, F. 1950. El efecto de la administration de dosis elevados de concentrados sobre la citologia del fluido vaginal de la rata. Acta cient. venezalana, 1, 26.

Mason, K. E. 1933. Differences in testis injury

and repair after vitamin A deficiency, vitamin E deficiency, and inanition. Am. J. Anat., 52, 153.

Mason, K. E. 1939. Relation of vitamins to sex glands. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., p. 1149. Baltimore: The Wilhams & Wilkins Company.

Mason, K. E. 1944. Physiologic action of vitamin E and its homologues. Vitamins & Hormones, 2, 107.

Mason, K. E., and Mauer, S. I. 1957. Reversible testis damage in vitamin E deficient hamster. Anat. Rec, 127, 329.

Mason, K. E., and Wolfe, J. M. 1930. The physiologic activity of the hypophysis of rats under various experimental conditions. Anat. Rec, 45, 232.

Mayer, J., and Goodard, J. W. 1951. Effects of administration of gonadotrophic hormone on vitamin A deficient rats. Proc Soc. Exper. Biol. & Med., 76, 149.

Mayer, J., and Truant, A. P. 1949. Effects of administration of testosterone on vitamin A-deficient rats. Proc. Soc. Exper. Biol. & Med., 72, 436.

Meites, J. 1952. Changes in nutritional requirements accompanying marked changes in hormone levels. Metabolism, 1, 58.

Meites, J. 1953. Relation of nutrition to endocrine-reproductive functions. Iowa State Coll. J. Sc, 28, 19.

Meites, J., and Agrawala, I. P. 1949. Effects of underfeeding on thiouracil action in rats and mice. Endocrinology, 45, 148.

Meites, J., Feng, Y. S. L., and Wilwerth, A. M. 1957. The effects of endocrine imbalances on vitamin requirements. Am. J. Clin. Nutrition, 5, 381.

Meites, J., and Reed, J. 0. 1949. Effects of restricted feed intake in intact and ovariectomized rats on pituitary lactogen and gonadotrophin. Proc. Soc. Exper. Biol. & Med., 70, 513.

Menze, W. 1941. Uber dem Einfluss des Hungerns auf die Keimdriisen. Endokrinologie, 24, 159.

Migeon, C. J. 1952. Effect of cortisone on hpids of serum, liver and testes in intact and adrenalectoniizfd lats. Proc. Soc. Exper. Biol. & Med., 80, 571.

Miller, S., Ruttinger, V., and Macey, I. G. 1954. Urinary excretion of ten amino acids by women during the reproductive cycle. J. Biol. Cliem., 209, 795.

Moore, C. K. 1939. Biology of the testis. In Sex and Internal Secretions, 2nd ed., E. Allen, C. H. Danforth and E. A. Doisy, Eds., p. 681. Baltimore: The Williams &: Wilkins Company.

Moore, C. R. 1953. Adrenal cortical secretions in relation to the reproductive system of rats. J. Clin. Endocrinol., 13, 330.

MooRE, C. R., AND Samuels, L. T. 1931. The action of testis hormone in correcting changes induced in the rat prostate and seminal vesicles by vitamin B deficiency or partial inanition. Am. J. Physiol., 96, 278.

Moore, H., Jessop, W. J. E., O'Donovan, D. K., Barry, A. P., Quinn, B., and Drury, M. I. 1951. Cortisone in treatment of toxaemia of pregnancy; a study of eight cases. Brit. M. J., 1, 841.

MooRE, M. 1948. Effect of alloxan diabetes on hyaluronidase level of the rat testes. Proc. Soc. Exper. Biol. & Med., 69, 391.

Morgan, A. F. 1951. The effect of vitamin deficiencies on adrenocortical function. Vitamins & Hormones, 9, 161.

Morris, H. P., Dunn, T. B., and Wagner, B. P. 1953. Influence of gonadotrophin on pyridoxine-deficient and diet-restricted female mice. J. Nat. Cancer Inst., 14, 493.

Morrison, S. D. 1956. The nitrogen balance of pregnant rats. J. Physiol., 133, 167.

Morton, J. H. 1958. Three obscure causes of female infertility. Internat. Rec. Med.. 171, 63.

MuKHERJEE, A. K., AND Banerjee, S. 1954. Studies of histologic changes in experimental scurvy. Anat. Rec, 120, 907.

MuLiNos, M. G., AND PoMERANTZ, L. 1940. Pseudohypophysectomy, a condition resembling hypophysectomy, produced by malnutrition. J. Nutrition, 19, 493.

MuLiNos, M. G., AND PoMERANTZ, L. 1941a. The reproductive organs in malnutrition; effects of chorionic gonadotrophin upon atrophic genitalia of underfed male rats. Endocrinologv, 29, 267.

MuLiNOS, M. G., AND PoMERANTZ, L. 1941b. Pituitary replacement therapj^ in pseudohypophysectomy; effects of pituitary implants upon organ weights of starved and underfed rats. Endocrinology, 29, 558.

Nelson, M. M., .-^nd Evans, H. M. 1953. Relation of dietary protein levels to reproduction in the rat. J. Nutrition, 51, 71.

Nelson, M, M., .\nd Evans, H. M. 1954. Maintenance of pregnancy in the absence of dietary protein with estrone and progesterone. Endocrinology, 55, 543.

Nelson, M. M., and Evans, H. M. 1955. Maintenance of pregnancy in absence of dietary protein with progesterone. Proc. Soc. Exper. Biol. & Med., 88, 444.

Nelson, M. M., and Ev.\ns, H. M. 1956. Failure of ovarian hormones to maintain pregnancy in rats deficient in pantothenic or pteroylglutamic acid. Proc. Soc. Exper. Biol. & Med., 91, 614.

Nelson, M. M., Lyons, W. R., and Evans, H. M. 1951. Maintenance of pregnancy in pyridoxine-deficient rats when injected with estrone and progesterone. Endocrinology, 48, 726.

Nelson, M. M.. Lyons, W. R., and Evans, H. M. 1953. Comparison of ovarian and pituitary hormone for maintenance of pregnancy in pvridoxine-deficient rats, Endocrinologv, 52, 585.

Nelson, M. R., Arnrich, L., .and Morgan, A. F. 1957. Derangement of carbohydrate metabo

lism in ribofiavin-deficient rat. Fed. Proc, 16, 394.

Nelson, W. O. 1933. Studies on anterior hypophysis. III. Anterior hypophysis in vitamin E-deficient rats, Anat, Rec, 56, 241.

Newberne, p. M., and O'Dell. B. L. 1958. Histopathology of hydrocephalus resulting from a deficiencv of B12. Proc. Soc. Exper. Biol. & Med., 97, 62.

Newton, W. H. 1952. Changes in the maternal organism during pregnancy. In MarshaU'ii Physiology 0/ Reproduction, p. 442. New York: Longmans, Green & Company.

Nieman, C, and Klein Obbink, H. J. 1954. The biochemistry and pathology of hypervitaminosis A. Vitamins & Hormones, 12, 69.

NOALL, M. W., RiGGS, T. R., W.\LKER, L. M.. AND

Christensen, H. N. 1957. Endocrine control of amino acid transfer. Science, 126, 1002.

NouMURA, T. 1956. Effects of desoxycorticosterone acetate and cortisone on ovaries of mice in parabiosis with castrated males. Jap. J. Zool., 12, 57.

Okey. R.. Pencharz, R., and Lepkovsky, S. 1950. Sex hormonal effects in incipient biotin deficiency. Am. J. Physiol., 161, 1.

Overby, L. R., Frost, D. V., and Fredrickson, R. L. 1959. The antithyrotoxic factor of liver. II. Comparative activities of defatted liver residue and various fats. J. Nutrition, 68, 251.

P'an. S. Y., Van Dyke, H. B., Kaunitz, H., and Slanetz, C. a. 1949. Effect of vitamin E deficiency on amount of gonadotrophin in the anterior pituitarj^ of rats. Proc. Soc. Exper. Biol.& Med., 72,523.

Panos, T. C, and Finerty, J. C. 1953. Effects of a fat-free diet on growing female rats, with special reference to the endocrine system. J. Nutrition, 49, 397.

Panos, T. C, and Finerty, J. C. 1954. Effects of a fat-free diet on growing male rats with special reference to the endocrine system. J. Nutrition, 54, 315.

P.\Nos, T. C, Klein, G. F., and Finerty, J. C. 1959. Effects of fat deficiency in pituitarygonad relationships. J. Nutrition, 68, 509.

P.appenheimer, a. M., .\nd Schogoleff, C. 1944. The testis in vitamin E-deficient guinea pigs. Am.J. Path., 20, 239.

P.ARLOW, A. F. 1958. A rapid bioassay method for LH and factors stimulating LH secretion. Fed. Proc, 17, 402.

Parry, H. B., and Taylor, W. H. 1956. Renal function in sheep during normal and toxaemic pregnancies. J. Physiol., 131, 383.

Paschkis, K. E., and C.\nt.\row, A. 1958. Pregnancy, tumor growth, and liver regeneration. Cancer Res., 18, 1060.

P.Azos, R., Jr., and Huggins. C. 1945. Effect of androgen on the prostate in starvation. Endocrinology, 36, 416,

Pe.\rse, a. G. E., and Rin.aldini, L. M. 1950. Histochemical determination of gonadotrophin in the rat hypophysis. Brit. J. Exper. Path., 31, 540.

Pe-ARSOX, p. B. 1936. Dietary protein in relation to the estrous cycle. Proc. Am. Soc. Anim. Prod., 282.

Pe.\rson, p. B., Hart, E. B., and Bohstedt, G. 1937. The effect of the quality of protein on the estrous cycle. J. Nutrition, 14, 329.

Pecora, L. J., AND HiGHMAN, B. 1953. Organ weights and histology of chronically thiaminedeficient rats and their pair-fed controls. J. Nutrition, 51, 219.

Peder.sen, J. 1952. Course of diabetes during pregnancy. Acta endocrinol., 9, 342.

Pequignot, E. 1956. Studies on the actual nutrition of 55 pregnant women. Bull. Inst. Nat. Hyg., 11, 107.

Perloff, W. H., Lasche, E. M., Nodine, J. H., Schneeberg, N. G., and Vieillard, C. B. 1954. The starvation state and functional hypopituitarism. J. A. M. A., 155, 1307.

Peterson, R. R., Webster, R. C., R.-vyner, B., and Young, W. C. 1952. The thyroid and reproductive performance in the adiUt female guinea pig. Endocrinology, 51, 504.

Pike, R. L., and Kirksey, A. 1959. Some effects of isonicotinic acid hydrazine-induced vitamin Bg deficiency in pregnant rats. J. Nutrition, 68,561.

Pike, R. L., Suder, H. B., and Ross, M. L. 1954. The influence of protein and energy intakes upon nitrogen retention in the pregnant rat. J. Nutrition, 52, 297.

Pl.\gge, J. C., Marasso, F. J., and Zimmerman, H. J. 1958. Estrogen inhibition of nutritional fatty liver. Metabohsm, 7, 154.

Pomer.'^ntz, L., and Mulinos, M. G. 1939. Pseudohypophysectomy produced by inanition (Abstr.). Am. J. Physiol., 126, P601.

Pomeranze, J., Piliero, S. J., Medeci, P. T., and Pl.\chta, a. 1959. Deficiency diets in young growing rats. Proc. Soc. Exper. Biol. & Med., 100, 207.

Poo, L. J., Lew, W., and Addis, T. 1939. Protein anabolism of organs and tissues during pregnancy and lactation. J. Biol. Chem., 128, 69.

PoPOFF, J. S., AND Okultilschew, G. Z. 1936. Der Einfluss der Ernahrung auf die Geschlechts-f unction. Ztschr. Zuchtung B., 1936, 221.

Pugsley, L. I. 1957. The biological assay of chorionic gonadotrophin and of the gonadotrophin of pregnant mare's scrum using dietary anoestrous adult rats. Canad. J. Biochcm. & Physiol., 35, 889.

QuiMBY, F. H. 1948. Organ weights of rats receiving hormone supplements during recovery from chronic starvation. Endocrinology, 42, 263.

Ralli, W. p., and Dumm, M. E. 1952. Nutritional fractions affecting survival in young adrenalectomized rats. Endocrinology, 5i, 135.

Ranney, R. E., and Drill, V. A. 1957. The ability of 17-ethyl-19-nortestosterone to block ethio

nine-induced fattv liver in rats. Endocrinology, 61, 476.

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

Reece, R. P. 1959. The role of nutrition in mammary gland physiology. In Reproductive Physiology and Protein Nutrition, p. 23. New Brunswick : Rutgers University Press.

Reece, R. P., Turner, C. W., Hathaway, I. L., AND D.wis, H. p. 1937. Lactogen content of pituitary glands from rats on vitamin deficient rations. Proc. Soc. Exper. Biol. & Med., 37, 293.

Reed, D. C., Browning, W. H., and O'Donnell, H. F. 1958. Male subf ertility : treatment with liothyronine (cytomel). J. Urol., 79, 868.

Reid, J. T. 1949. Relationship of nutrition to fertility in animals. J. Am. Vet. M. A., 114, 158.

Rice, E. G., and Bo, W. J. 1958. Effect of /3carotene and cortisone on vaginal epithelium of vitamin A deficient rats. Proc. Soc. Exper. Biol. & Med., 98, 877.

RiCHTER, K. M., AND WiNTER, C. A. 1947. A

ciuantative study of the effect of hyperthyroidism on genital structure and function. Am. J. Physiol., 150, 95. RiNALDiNi, L. M. 1949. Effect of chronic inanition on the gonadotrophic content of the pituitary gland. J. Endocrinol., 6, 54.

RiVERO, FONTAN, J., PaSCHKIS, K. E., WeST, E., AND

Cantarow, a. 1952. Influence of different dietary protein levels in starvation on the endocrine system of male and female rats. Endocrinology, 51, 100.

RoBSON, J. M., AND Shar.\f, A. A. 1951. Interruption of pregnancy with ACTH and cortisone. J. Physiol., 114,' lip.

Rosenthal, H. L., and Allison, J. B. 1956. Effects of caloric intake on nitrogen balance and organ composition of adult rats. Agric. & Food Chem., 4, 792.

Ross, M. L., AND Pike, R. L. 1956. The relationship of vitamin Bo to serum protein and nonprotein nitrogen in the rat during pregnancy. J. Nutrition, 60, 211.

Rowlands, I. W., and Singer, E. 1936. Gonadotrophic activity of the pituitaries of vitamin E-dcficient rats. J. Physiol., 86, 323.

Ryabinina, Z. a. 1952. Effect of protein deficieny on structure and functional state of white rat ovaries. Doklady Akad. Nauk S. S. R., 86, 877.

Salhanick, H. a., Hisaw, F. L., .^ND Zarrow, M. X. 1952. The action of estrogen and progesterone on the gonadotrophin content of the pituitary of the monkey {Macaca 7nulatta). J. Clin. Endocrinol.. 12,310.

Salvesen, H. a. 1957. Endocrine organs and undernutrition: paradoxical behavior of the pancreas. Acta endocrinol., 25, 173.

Samuels, L. T. 1948. Nutrition and Hormones. Springfield, 111.: Charles C Thomas.

Samuels, L. T. 1949. The metabolism of androgens by tissues. Recent Progr. Hormone Res., 4,65. '

Samuels, L. T. 1953. Effect of protein and amino acids of the diet on the endocrine system. In Protein Metabolism, Hormones and Growth, p. 11. New Brunswick: Rutgers Uniyersity Press.

Samuels, L. T., Reinecke, R. M., and Peterson, W. E. 1941. Relation of nutrition to mammary growth after estradiol administration to hypophysectomized rats. Proc. Soc. Exper. Biol. & Med., 46, 379.

Schultze, M. O. 1955. Effects of malnutrition in early life on subsequent growth and reproduction of rat. J. Nutrition, 56, 25.

Schultze, M. O. 1956. Reproduction of rats fed protein-free amino acid rations. J. Nutrition, 60, 35.

Scott, E. B. 1956. Histopathology of amino acid deficiencies. V. Isoleucine. Proc. Soc. Exper. Biol. & Med., 92, 134.

Segaloff, a., and Segaloff, a. 1944. The role of the yitamins of the B-complex in estrogen metabolism. Endocrinology, 34, 346.

Sherman, H. C, Pe.^rson, C. S., Bal, M. E. R., McCarthy, A., and L.\nford, C. S. 1956. Influence of protein and calcium additions to already adequate diet. Proc. Nat. Acad. Sc. U. S., 42, 682.

Sherman, W. C. 1941. The effect of certain fats and unsaturated fatty acids upon the utilization of carotene. J. Nutrition, 22, 153.

Shipley, E. G., .and Danley, K. S. 1947. Pituitary and ovarian dysfunction in experimental diabetes. Am. J. Physiol., 150, 84.

Shipley, R. A., Chudzik, E. B., Curtiss, C, and Price, J. W. 1953. Effect of lipotrophic deficient diets and steroid hormones on the pregnant rat. Metabolism, 2, 165.

Sims, F. H. 1951. Methionine and choline deficiency in the rat with special reference to the pregnant state. Brit. J. Exper. Path., 32, 481.

SiNDEN, J. A., and Longwell, B. B. 1949. Effect of alloxan diabetes on fertility' and gestation in the rat. Proc. Soc. Exper. Biol. & Med., 70, 607.

Skelton, F. R. 1950. Some specific and nonspecific effects of thiamine deficiency in the rat. Proc. Soc. Exper. Biol. & Med., 73, 516.

Smithberg, M., .^nd Runner, M. N. 1957. Pregnancy induced in genetically sterile mice. J. Hered., 48, 97.

Souders, H. J., .\ND Morgan, A. F. 1957. Weight and composition of organs during the reproductive cycle in the rat. Am. J. Physiol., 191, 1.

Srebnik, H. H.. and Nelson, M. M. 1957. Increased pituitary gonadotrophic content in protein deficient castrate rats. Anat. Rec, 127, 372.

Srebnik, H. H., Nelson, M. M., and Simpson, M. E. 1958. Response of exogenous gonadotrophins in absence of dietary protein. Proc. Soc. Exper. Biol. & Med., 99, 57.

Stenger-Haffen, K., and Wolff, E. 1957. La differenciation des organes sexues cultiyes en milieux synthetique. Role et mode d'action des hormones sexuelles. In Proceedings Fourth International Congress of Nutrition, p. 49.

Stephens, D. J. 1941. Anorexia nervosa; endocrine factors in undernutrition. J. Clin. Endocrinol., 1, 257.

Stephens, D. J., and Allen, W. M. 1941. The effect of refeeding and of the administration of a pituitarj^ extract on the ovaries of undernourished guinea pigs. Endocrinology, 28, 580.

Sutton, T. S., and Brief, B. J. 1939. Physiologic changes in the anterior hypophysis of vitamin A-deficient rats. Endocrinolog.y, 25, 302.

SzEGO, C. M., and Barnes, R. H. 1943. Dietary fatty livers in mice and sensitivity to exogenous estrogen. Endocrinology, 32, 367.

T.^lbert, G. B., and H.\milton, J. B. 1955. The effect of reduced rate of growth on maturation of the male reproductive system of the albino rat. Anat. Rec, 121, 763.

Thiersch, J. B. 1954. Effect of certain 2,4 diaminopyrimidine antagonists of folic acid on ]iregnancy and rat fetus. Proc. Soc. Exper. Biol. & Med., 87, 571.

Trentin, J. J., and Turner, C. W. 1941. Quantitative study of the effect of inanition on responsiveness of the mammaiy gland to estrogen. Endocrinology, 29, 984.

Tuchm.\nn-Duplessis, H., and Aschkenasy-Lelu, P. 1947. Modifications histologiques de I'ovaire et du tractus genital de la rate adulte soumise au regime hyperprotidique. Compt. rend. Soc. biol., 141, 689.

TUCHM.^NN-DUPLESSIS, H., AND AsCHKEN.A.SY-LeLU,

p. 1948. Basophilie et augmentation du pouvoir gonadotrope de I'hypophyse de la rate en hyperproteose. Compt. rend. Soc. biol., 142, 472.

Ullrey, D. E., Becker, D. E., Terrill, S. W., and NoTzoLD, R. A. 1955. Dietary levels of pantothenic acid and reproductive performance of female swine. J. Nutrition, 57, 401.

Umberger, E. J., AND Gass, G. H. 1958. The effect of dietary fat on the uterine weight response of immature mice to oral stilbestrol. Endocrinology, 63, 801.

Vanderlinde, R. E., and Westerfield, W. W. 1950. The inactivation of estrone by rats in relation to dietary effects on the liver. Endocrinology, 47, 265.

Van Pilsum, J. F., Speyer, J. F., and Samuels, L. T. 1957. Essential amino acid deficiency and enzyme activity. Arch. Biochem., 68, 42.

A^asington, F. D., Parker, A., Headley, W., and V.\nderlinde, R. E. 1958. Relationship of low protein diet and ascorbic acid in estrogen inactivation by liver. Endocrinology, 62, 557.

Velardo, J. T., AND HiSAW, F. L. 1953. Inhibitory effects of aminopterin on decidual development. Am. J. Physiol., 172, 535.

Visscher. M. B., King, J. T., and Lee, Y. C. P. 1952. Further studies on influence of age and diet upon reproductive senescence in strain A

female mice. Am. J. Physiol., 170, 72. VoLLMER, E. P. 1943. Relation of hypopituitarism

and starvation. Nature, London, 151, 698. W.AL.AAS, 0. 1952. Effect of estrogens on the

glycogen content of the rat uterus. Acta endo crinol., 10, 175.

\V.\LKER, D. G., AND WiRTSCHAFTER, Z. T. 1956.

Estrogenic inhibition of fetal reabsorption in lathyrism. J. Nutrition, 58, 161.

Ward, L. W., Slocumb, C. H., Polley, H. F., Lowman. E., AND Hexch, p. S. 1951. Clinical effects of cortisone administered orally to patients with rheumatoid arthritis. Proc. Staff Meet., Mayo Clin., 26, 361.

Warrant, J., and Schraffenberger, E. 1944. Congenital malformations of the eyes induced in rats by maternal vitamin A deficiency. Proc. Soc. Exper. Biol. & Med., 57, 49.

Weatherby, E. J., and Reece, R. P. 1941. Effect of a high- and low-protein ration on the gonadotrophic content of male rat pituitaries. J. Dairy Sc, 24, 508.

Weir, D. R., and Leuchtenberger, C. 1957. Low sperm desoxyribose nucleic acid as possible cause for otherwise unexplained human infertility. Fertil. & Steril.. 8, 373.

Werner, S. C. 1939. Failure of gonadotrophic function of the rat hypophysis during chronic inanition. Proc. Soc. Exper. Biol. & Med., 41, 101.

White, F. R., .and White, J. 1942. Effect of a low cystine diet on the growth of various strains of mice. J. Nat. Cancer Inst., 2, 449.

Whitnah, C. H., and Bogart, R. 1936. Reproductive capacity in female rats as affected by kinds of carbohydrate in the ration. J. Agric. Rec, 53, 527.

Wil.son, J. G., Roth, C. B., and Warkany, J. 1953. An analysis of the syndrome of malformations induced by maternal vitamin A deficiency. Effects of restoration of vitamin A at various times during gestation. Am. J. Anat., 92, 189.

Wolf, R. C, and Leathem, J. H. 1955. Hormonal and nutritional influences on the biochemical composition of the rat testis. Endocrinologv 57, 286.

WOOLLAM, D. H. M., AND MiLLEN, J. W. 1957. The effect of cortisone on the incidence of cleft palate in the offspring of female rats subjected to hypervitaminosis A. In Proceedings Fourth International Congress oj Nutrition, p. 52.

WooTEN, E., Nelson, M. M., Simpson, M. E., and Evans, H. M. 1955. Effect of pyridoxine deficiency on the gonadotrophic content of the anterior pituitary of the rat. Endocrinologv, 56, 59.

Wooten, E., Nelson, M. M.. Simpson, M. E., and Ev.ANs, H. M. 1958. Response of vitamin Bodeficient rats to hypophyseal follicle-stimulating and interstitial cell-stimulating hormones. Endocrinology, 63, 860.

Yamamoto, R. S., and Chow, B. F. 1950. Role of dietary proteins in development of sex organs of young rats under stimulus of a gonadotrophin. Fed. Proc, 9, 250.

Young, W. C, Rayner, B., Peterson, R. R., and Brown, M. M. 1952. The thyroid and reproductive performance in the adult male guinea pig. Endocrinology, 51, 12.

ZiMMER. R., Weill, J., and Dubois, H. 1944. Nutritional situation in camps of the occupied zone of France in 1941 and 1942 and its con.sequence. New England J. Med.. 230, 303.

Zondek, B. 1934. Fate of follicular hormone in the living body. Lancet, 227, 356.

ZuBiRAN, S., AND GoMEZ-MoNT, F. 1953. Endocrine di.sturbances in chronic human malnutrition. Vitamins & Hormones, 11, 97.

ZucKERMAN, S. 1953. The adrenogenital relationship. The suprarenal cortex. In Proceedings Fijth Symposium Colston Research Societij, p. 69. New York: Academic Press, Inc.