Book - Sex and internal secretions (1961) 11

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

Section A Biologic Basis of Sex Cytologic and Genetic Basis of Sex | Role of Hormones in the Differentiation of Sex

Section B The Hypophysis and the Gonadotrophic Hormones in Relation to Reproduction Morphology of the Hypophysis Related to Its Function | Physiology of the Anterior Hypophysis in Relation to Reproduction

The Mammalian Testis | The Accessory Reproductive Glands of Mammals | The Mammalian Ovary | The Mammalian Female Reproductive Cycle and Its Controlling Mechanisms | Action of Estrogen and Progesterone on the Reproductive Tract of Lower Primates | The Mammary Gland and Lactation | Some Problems of the Metabolism and Mechanism of Action of Steroid Sex Hormones | Nutritional Effects on Endocrine Secretions

Section D Biology of Sperm and Ova, Fertilization, Implantation, the Placenta, and Pregnancy Biology of Spermatozoa | Biology of Eggs and Implantation | Histochemistry and Electron Microscopy of the Placenta | Gestation

Section E Physiology of Reproduction in Submammalian Vertebrates Endocrinology of Reproduction in Cold-blooded Vertebrates | Endocrinology of Reproduction in Birds

Section F Hormonal Regulation of Reproductive Behavior The Hormones and Mating Behavior | Gonadal Hormones and Social Behavior in Infrahuman Vertebrates | Gonadal Hormones and Parental Behavior in Birds and Infrahuman Mammals | Sex Hormones and Other Variables in Human Eroticism | The Ontogenesis of Sexual Behavior in Man | Cultural Determinants of Sexual Behavior

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Section C Physiology of the Gonads and Accessory Organs

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

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. Progesterone

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

Fig. 11.1. Biogenesis of cholesterol.

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.

Fig. 11.2. Biosynthetic paths from cholesterol.

Estrone

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 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-hydroxy 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 ) .

Fk;. 11.3. Excretory products of progesterone and androgen

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 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 conditions 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).

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 experiments 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 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 components 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 addition 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 from TPNH to DPN: 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 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 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 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 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 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 membrane 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.

Most 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, 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.

BaRR0N, E. S. G., aND HUGGIN.S, C. 1944. The metabolism of isolated prostatic tissue. J. Urol., 51, 630-634.

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