Progestins inhibit FSH-stimulated granulosa estrogen production at a post-cAMP site

The purpose of this investigation is to examine the mechanism by which progestins inhibit FSH-induced estrogen (E) production by cultured rat ovary granulosa cells. We have demonstrated that the highly potent synthetic progestin, R5020, is able to inhibit the induction of granulosa cell aromatase activity by cholera toxin, prostaglandin E2, dibutyryl cAMP or oFSH. Since the induction of E synthesis by these compounds is mediated through activation of adenylate cyclase and increased cellular cAMP production, these observations indicate that the progestin inhibitory effect is a post-cAMP event. In addition, we have demonstrated that R5020 does not inhibit FSH-stimulated granulosa cell cAMP production. The involvement of the granulosa cell progesterone (P) receptor as a mediator of this post-cAMP progestin effect is suggested by the relative abilities of various progestins to both bind the P receptor and to block the induction of granulosa cell aromatase activity by dibutyryl cAMP. While the precise mechanism of progestin action remains unclear, the kinetic analysis of aromatase enzyme activity demonstrates that progestins are not acting as competitive inhibitors of granulosa cell aromatase. Since E is necessary for follicular development, our in vitro data are consistent with the hypothesis that P is a factor which can inhibit FSH-induced follicular growth and development in the rat ovary.     

Progestin receptors in human uterine cytosol

Progestin(norethindrone and norethindrone acetate)-binding protein, exhibiting characteristics similar to uterine progesterone receptor, has been identified in human uterine cytosol. The progestin receptor was characterized by sedimentation coefficient 4.2 S; Stokes radius, 39 Å; frictional ratio 1.29; isoelectric pH 4.6; molecular radius 2.7 nm; and molecualr weight in the range 67 000–74 000. The ammonium-sulfate-precipitated progestin-receptor complex was eluted from a DEAE-cellulose column at 0.18 M KC1. The progestin binding was saturable and stereospecific. The sequential variation in receptor concentration (early proliferative, 3800–4300 sites/cell; late proliferative, 9500–11200 sites/cell; early secretory, 4900–6200 sites/cell; late secretory, 1800–2300 sites/cell) was in conformity for progesterone and the progestins, when concurrently measured. Oral administration of norethindrone significantly reduced the cytoplasmic and nuclear receptor concentration for estradiol and progesterone. A significant observation was that the progestins stabilized the progestin receptor by forming a slowly dissociating complex with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～ 110?130}\text{min}$ as compared with the progesteronereceptor complex dissociating with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～41}\text{min}$. Thus, the uterine progestin receptor recognizes progestins in general, although with a varying degree of affinity, and the altered rate constants could be of putative importance in determining the biological potency of the progestins.     

Studies on the synergistic effect of androgen on the stimulation of progestin secretion by fsh in cultured rat granulosa cells: Progesterone metabolism and the effect of androgens

Metabolic transformations of progesterone in cultures of granulosa cells from immature hypophysectomized rats treated with diethylstilbestrol were studied in relation to the synergistic action of exogenous androgen and FSH on progestin (progesterone and 20α-dihydroprogesterone) accumulation. Androstenedione (Ad; 10 ng/ml) enhanced the sensitivity of rat granulosa cells to this steroidogenic action of FSH, lowering the threshold of the response from >4 ng/ml (FSH alone) to 0.8 ng/ml in the presence of Ad. A synergistic effect with FSH was also shown by various 5α-androstane derivatives. They were, however, less effective than the parent Δ⁴-3 keto androstenes. Progesterone underwent extensive 5α-reduction during culture, leading to accumulation of endogenous 5α-pregnane compounds, and to transformation of labelled progesterone into 5α-reduced radiometabolites. These compounds corresponded in gas-liquid and thin-layer Chromatographic behaviour to 3α-hydroxy-5α-pregnan-20-one, 20α-hydroxy-5α-pregnan-3-one and 5α-pregnane-3α,20α-diol. The rate of 5α-reduction of progestins was not affected by the presence of exogenous Ad (1 μg/ml), ruling out the possibility that the effect of androgen on progestin accumulation depends on competitive inhibition of 5α-reductase. An involvement of androgen of thecal origin in the enhancement of the sensitivity of the FSH-responsive mechanism in granulosa cells is suggested.     

Effect of 17 beta-estradiol on thyroliberin responsiveness in GH3/B6 rat prolactin cells

GH3/B6 rat prolactin cells were used to analyse at the cellular level the mechanisms by which 17 beta-estradiol (E2) regulates TRH responsiveness of prolactin cells. Before experiments, cells were grown for up to 7 days in 3 different media: normal medium (N) containing 15% horse serum and 2.5% fetal calf serum, CD medium prepared with charcoal-dextran extracted serum and CDE medium supplemented with 4 x 10(-8) M E2. The binding of 3H-TRH (30 min at 37 degrees C) and the TRH-induced percent increase of prolactin release as a function of TRH doses were compared in the 3 conditions. Preculture in E2 enriched medium increased by 50% the number of TRH high-affinity binding sites without modifying their affinity, increased by up to 3 times the percent of the TRH-induced stimulation of prolactin release and improved by one order of magnitude the ED50 of the TRH effect on prolactin release. The presence of HEPES (10 mM) during TRH challenge masked the effect of E2 on the increase in number of binding sites but respected its potentiating effect on prolactin release.     

Cortisol metabolism in lymphocytes from cancer-bearing patients

A study was conducted to determine the pattern of cortisol metabolism by lymphocytes obtained from four groups of subjects: 27 male and female patients suffering from various types of malignancy other than malignancy of lymphatic tissues; and 26 healthy male and female controls. Known concentrations of cells were incubated with 1,2-³H-cortisol and the products were isolated by thin-layer and paper chromatography. Three metabolites were found to be produced by lymphocytes from both normal and cancer-bearing patients: 20α-hydroxycortisol, 20β-hydroxycortisol, and tetrahydrocortisol. Cells from the female control group were found to be more active than those from the male controls, while cells from cancer-bearing patients were markedly more active than the normal cells, regardless of sex. It is suggested that this finding of increased metabolism of cortisol by lymphocytes from patients with different types of malignancy other than lymphoma may provide the basis for a new diagnostic aid.     

In vitro inhibition of aldosterone biosynthesis by 17-hydroxylated steroids in rat adrenal

The in vitro effect of the addition of non-17-hydroxylated and 17-hydroxylated steroids on the production of aldosterone has been studied in the rat. The biosynthesis of deoxycorticosterone, corticosterone, and aldosterone was increased when progesterone or pregnenolone was added to the incubation media, but neither cortisol nor 11-deoxycortisol was produced. The addition of 17α-hydroxyprogesterone or 17α-hydroxypregnenolone to the incubation media resulted in the biosynthesis of cortisol and 11-deoxycortisol by the rat adrenal; concomitantly, a decrease in aldosterone production occurred. However, no decrease in aldosterone production resulted when cortisol and 11-deoxycortisol were added directly to the incubation media. These studies show that neither cortisol nor 11-deoxycortisol inhibits the in vitro biosynthesis of aldosterone and suggest that other mechanisms are involved.     

Evidence suggesting 11 beta-hydroxylase inhibition during aminoglutethimide administration

Steroid hormone excretion during amino-glutethimide administration was studied in a patient with Cushing's syndrome due to bilateral adrenocortical hyperplasia. Plasma and urinary 17-OHCS showed a persistent decrease, as did urinary total 17-KS. Chromatographic fractionation of urinary 17-KS demonstrated a dramatic reduction of 11-oxy-17-KS, while 11-deoxy-17-KS, after a transient fall, tended to recover. Urinary THS showed an absolute increase, while pregnanetriolone diminished. Δ⁵-pregnenetriol and pregnanetriol, after initial decreases, showed a gradual trend upward. The sum of these data, and particularly the increase in THS excretion, suggests that, in this case, aminoglutethimide exerted an inhibitory effect not only on the early steps of adrenocortical steroidogenesis but also on 11 β-hydroxylation.     

Dissociation between cortisol-induced pycnosis and inhibition of [3H]uridine incorporation in rat thymocytes.

Treatment of rat thymocytes with cortisol induced an inhibition of [3H]uridine incorporation after 30-90 min, an accumulation of pycnotic cells after 90 min, and a decrease in cell viability after several hours. No cortisol-resistant cells could be distinguished, and dose-response curves for a number of glucocorticoids showed a correlation to the saturation of the glucocorticoid receptors. The pycnotic effect of cortisol increased between pH 5.2--7.0 in parallel with a stimulation of the spontaneous development of pycnotic cells. The cortisol-induced accumulation of pycnotic cells and inhibition of [3H]uridine incorporation varied independently as a function of the cell density, and in a glucose-salt medium only the pycnotic effect of cortisol became inhibited. The inhibition of [3H] uridine incorporation is therefore not an integral part of the pycnotic change of the cells. The glucocorticoid sensitivity was found to increase with the age of the animals, before the onset of thymus involution.     

Estrogen augmentation of gonadotropin-stimulated progestin biosynthesis in cultured rat granulosa cells

The influence of estrogens on gonadotropin-stimulated production of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) was examined in primary cultures of rat granulosa cells. Granulosa cells were cultured for 3 days with increasing concentrations of FSH in the presence or absence of either diethylstilbestrol (DES) or estradiol. FSH treatment increased progestin production in a dose-dependent manner, whereas treatment with estrogens alone were ineffective. In contrast, concomitant addition of either DES or estradiol augmented FSH-stimulated production of progesterone and 20 alpha-OH-P. Increasing concentrations of estradiol (10(-10) - 10(-7) M) augmented the stimulatory effect of FSH (30 ng/ml) on progesterone production in a dose-dependent manner with ED50 values of approximately 3 X 10(-9) M. The facilitatory action of estradiol was time-related, becoming significant after 36 h of treatment. Granulosa cells were also cultured for 2 days with FSH to induce functional LH receptors. The FSH-primed cells were treated for an additional 3 days with increasing concentrations of LH (0.3-30 ng/ml) in the absence or presence of DES (10(-7) M). LH stimulated progesterone and 20 alpha-OH-P production in a dose-dependent manner, whereas concomitant addition of DES further enhanced LH-induced progestin biosynthesis. (Bu)2cAMP also increased progesterone and 20 alpha-OH-P production by the granulosa cells; however, concurrent addition of DES did not augment the actions of (Bu)2cAMP. The effect of estrogens on gonadotropin-stimulated cAMP accumulation was also examined. FSH treatment dose-dependently increased cAMP accumulation, whereas concomitant treatment with estradiol further increased the FSH action. Similarly, LH treatment also stimulated cAMP accumulation in FSH-primed cells, whereas concurrent addition of DES further augmented LH action. Thus, the stimulatory effect of estrogens upon gonadotropin-stimulated progestin production may be related to the augmentation of cAMP biosynthesis. The present observations suggest that intraovarian estrogens may act locally to enhance the sensitivity of granulosa cells to FSH and LH, thereby increasing the biosynthesis of progestins and cAMP by the granulosa cells.     

Pregnancy without progesterone in horses defines a second endogenous biopotent progesterone receptor agonist, 5α-dihydroprogesterone

One of the most widely accepted axioms of mammalian reproductive biology is that pregnancy requires the (sole) support of progesterone, acting in large measure through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained. However, mares lack detectable progesterone in the latter half of pregnancy. Instead of progesterone, several (mainly 5α-reduced) pregnanes are elevated and have long been speculated to provide progestational support in lieu of progesterone itself. To the authors'' knowledge, evidence for the bioactivity of a second potent endogenously synthesized pregnane able to support pregnancy in the absence of progesterone has never before been reported. The 5α-reduced progesterone metabolite dihydroprogesterone (DHP) was shown in vivo to stimulate endometrial growth and progesterone-dependent gene expression in the horse at subphysiological concentrations and to maintain equine pregnancy in the absence of luteal progesterone in the third and fourth weeks postbreeding. Results of in vitro studies indicate that DHP is an equally potent and efficacious endogenous progestin in the horse but that the PR evolved with increased agonistic potency for DHP at the expense of potency toward progesterone based on comparisons with human PR responses. Sequence analysis and available literature indicate that the enzyme responsible for DHP synthesis, 5α-reductase type 1, also adapted primarily to metabolize progesterone and thereby to serve diverse roles in the physiology of pregnancy in mammals. Our confirmation that endogenously synthesized DHP is a biopotent progestin in the horse ends decades of speculation, explaining how equine pregnancies survive without measurable circulating progesterone in the last 4 to 5 mo of gestation.Since first crystallized almost eight decades ago, progesterone has remained the only endogenous member of the progestin class of steroids defined by its singular ability to maintain pregnancy (1), acting, in large measure, through nuclear progesterone receptors (PRs) in uterine and cervical tissues, without which pregnancy cannot be established or maintained (2). Birth is thought to be triggered by a decrease in systemic progesterone concentrations (withdrawal) (3), even though this is not evident in mares (4), women, or guinea pigs (5), a disparity that limits the utility of other animal models for preterm labor (6). The vast majority of studies have focused on measuring progesterone, with most using immunoassays that necessarily cross-react with multiple pregnanes (7), the bioactivity of which remain uncharacterized. This is reasonable because, in contrast to androgens, estrogens, and corticoids, for which multiple natural biopotent analogs are known, no other endogenous pregnane has ever been shown to substitute for progesterone in pregnancy in any mammal.However, over five decades ago, Short (8) reported that circulating progesterone concentrations in pregnant mares were surprisingly low at <4 ng/mL, as did Holtan et al. (4) subsequently. Indeed, Holtan et al. (9) further confirmed by GC-MS that maternal progesterone concentrations in middle to late equine gestation were <0.5 ng/mL, and were low even in fetal circulation (10). Conversely, 5α-reduced metabolites like 5α-dihydroprogesterone (DHP) were very high in both pregnant mares and their fetuses (9, 10). Some human pregnancies that have extremely low concentrations of progesterone can survive to term also, as in patients who have congenital hypobetalipoproteinemia (11), and progesterone is low or undetectable in plasma of pregnant zebras (12), elephants (13), and the rock hyrax (14). Thus, alternative endogenous progestins have been postulated to exist in species other than horses, but definitive evidence of bioactivity is lacking. For instance, the results of studies investigating the activity of identified circulating pregnanes on equine (15) or human (16) myometrial contractility have not been consistent. Consequently, speculation about alternative progestins has been based mostly on binding assays in tissue extracts (17). However, binding assays alone do not reliably predict bioactivity (18, 19), and, to date, an alternative endogenous progestin capable of sustaining pregnancy in the absence of progesterone has yet to be identified in any mammal.Here, we verify by a unique combination of in vivo and in vitro studies that endogenously synthesized pregnane DHP sustains pregnancy in the absence of detectable progesterone in mares by (i) inducing equine endometrial growth and stimulating expression of progesterone-responsive endometrial genes in vivo; (ii) maintaining equine pregnancy after progesterone withdrawal induced by luteal regression; (iii) activating the equine PR (ePR) in vitro with equal efficacy and potency to progesterone itself; and (iv) doing so at concentrations seen during the luteal phase and early pregnancy, as well as in the second half of equine gestation when progesterone itself is undetectable. Collectively, these data establish DHP as a biopotent progestin in the horse at concentrations seen during gestation. Evidence both in vivo and in vitro demonstrating that an endogenously synthesized pregnane is able to sustain pregnancy by activating the nuclear PR at physiological concentrations has not previously been reported for any species to our knowledge. Evolutionary implications relating to the synthetic enzymes involved, and the classical PR itself, were also explored and are discussed.     

Differentiation between steroid hormone receptors CBG and shbg in human target organ extracts by a single-step assay

The steroid complexes of (plasma) corticosteroid-binding globulin can be distinguished from intracellular steroid-receptor complexes by agar electrophoresis at low temperature in neuraminidase-treated tissue extracts. With this method, the presence of progesterone receptor has been demonstrated in heavily plasma-protein-contaminated human uterus “cytosol”, but not in human mammary carcinoma extracts. SHBG and “basic” receptors for estradiol and dihydrotestosterone in human uterus cytosol could also be assayed simultaneously.     

Glucocorticoid-induced cell-size changes and nuclear fragility in rat thymocytes

The events preceding glucocorticoid-induced lymphocytolysis have been studied in isolated rat thymocytes. Incubation of thymocytes at 37°C in the presence of 1 μM dexamethasone resulted in the progressive appearance of pyknotic cells of modal diameter 4.6 μm, distinct from normal cells of diameter 5.2 μm. The rate of appearance of the pyknotic cells was determined by selective electronic cell counting, and was shown to be accompanied by increased nuclear fragility. The production of pyknotic cells was glucocorticoid-specific, dose-dependent, blocked by cycloheximide, and preceded the loss of cell viability as determined by dye exclusion. The pyknotic cells were separated from the non-pyknotic cells by density-gradient centrifugation and shown to be solely responsible for the observed nuclear fragility.     

Effect of a teleost GnRH analog on steroidogenesis by the follicle-enclosed goldfish oocytes, in vitro

The influence of an agonist analog of teleost GnRH [(D-Arg6, Trp7, Leu8, Pro9-NEt)-GnRH; tGnRH-A] on steroidogenesis was studied in prophase-I arrested, follicle-enclosed, goldfish oocytes in vitro. Incubation of the follicles with carp gonadotropin (GtH) significantly increased production of 17 alpha-hydroxyprogesterone (HP) and testosterone following 24 hr of incubation in vitro. Concomitant incubation with tGnRH-A (10(-7) M) significantly attenuated the dose-related increase in GtH-induced testosterone production, but was without effect on the GtH-induced HP level. Time course studies indicated that tGnRH-A exerted its maximum inhibitory action on the GtH-induced testosterone production during the initial 8 hr of incubation in vitro. The inhibition of GtH-induced testosterone production by tGnRH-A was dose dependent with an ED50 of 1.39 +/- 2.88 nM. A significantly higher testosterone level was obtained in the incubation media containing HP as substrate; concomitant treatment with tGnRH-A reduced the conversion of HP to testosterone. The incubation media also contained low, but measurable levels of 17 alpha-hydroxy-20 beta-dihydroprogesterone (DHP), which increased in the presence of 3-isobutyl-methyl-xanthine; lower levels of DHP were obtained in the groups incubated with tGnRH-A. In view of our present findings and previous observations concerning inhibitory effects of tGnRH-A on the progestogen and GtH-induced reinitiation of meiosis in the follicle-enclosed goldfish oocytes (H. R. Habibi, G. Van Der Kraak, E. Bulanski, and R. E. Peter, Amer. J. Physiol. 255, R268-R273 (1988] the influence of testosterone on the GtH- and DHP-induced meiosis in vitro was also studied. Testosterone (1 micrograms/ml) enhanced both GtH- and DHP-induced oocyte meiosis in the goldfish oocytes. Testosterone alone was also found to significantly increase oocyte meiosis in the goldfish oocytes in a dose-related fashion. The present findings demonstrate an inhibitory effect of a GnRH agonist on GtH-induced testosterone production in goldfish oocytes and suggest that tGnRH-A might influence oocyte meiosis in part by influencing steroidogenesis.     

The role of cyclic AMP in the induction of estrogen and progestin synthesis in cultured granulosa cells

The role of cyclic AMP in the induction of enzymes involved in estrogen and progestin biosynthesis in undifferentiated granulosa cells was investigated. When granulosa cells from immature hypophysectomized, DES-treated rats were cultured for 2 days in serum-free medium with aromatase substrate (10^?7M androstenedione) together with graded doses of FSH, prostaglandin E₂ (PGE₂), cholera toxin (CT), or dibutyryl cyclic AMP (Bu₂cAMP), there was a dose-related increase in estrogen (E) production. The induction of E production by saturating doses of FSH, PGE₂, CT and Bu₂cAMP required a lag phase of ～24 h, after which the E response increased sharply to maximum levels at day 3 and then declined gradually to day 5. Treatment for 24 h (day 0–1) with FSH, together with 1 μg/ml of either actinomycin D or cycloheximide, completely abolished the stimulatory action of FSH on E production. When the inhibitors were removed, the FSH-induced increases in E returned to near normal levels after a 24-h lag period. Similar effects of the inhibitors upon E production by CT, PGE₂ and Bu₂cAMP were observed. As with E, the production of progesterone and 20 α-dihydroprogesterone was markedly stimulated by FSH, PGE₂, CT and Bu₂cAMP and the results of the time course, dose response and inhibitor experiments were similar to those for E production.These results indicate that FSH induces the de novo synthesis of enzymes required for both estrogen and progestin biosynthesis by undifferentiated granulosa cells and suggest that this action is mediated by cyclic AMP.     

LH stimulation of estrogen secretion by cultured rat granulosa cells

The direct effect of LH on estrogen secretion by rat granulosa cells was investigated. Ovarian granulosa cells from immature hypophysectomized diethylstilbestrol-treated rats were primed with FSH for 2 days in vitro to induce LH receptors. After the FSH priming, the granulosa cells were washed, and recultured for 4 additional days in media containing aromatase substrate (10(-7) M androstenedione) and purified FSH or LH. After the incubations, estrogen (E), progesterone (P) and 20 alpha-dihydroprogesterone (20 alpha-OH-P) in the media were measured by RIA. When granulosa cells from hypophysectomized DES-treated rats were cultured for 6 days with FSH and androstenedione, the production of E, P and 20 alpha-OH-P was stimulated to a maximum of 100-, 200- and 270-fold, respectively, above that of control levels. In contrast, LH did not increase steroidogenesis in these cells. Following 2 days of FSH priming in vitro, however, the cultured granulosa cells exhibited marked increases (400-600%) in E, P and 20 alpha-OH-P production in response to LH treatment over a 4-day incubation period. This stimulatory effect of LH on estrogen and progestin production was dose-related; the minimum and maximum effective doses of LH for steroid production were 3 and 30 ng/ml, respectively, and the ED50 was calculated to be 6 ng/ml of LH. As with LH, FSH also stimulated steroidogenesis in a dose-related manner and the apparent ED50 of FSH on steroidogenesis was 45 ng/ml. To investigate whether LH can also stimulate aromatase activity in granulosa cells primed with FSH in vivo, immature hypophysectomized DES-treated rats were injected for 2 days with FSH after which the granulosa cells were isolated and cultured for 4 days in medium containing 10(-7) M androstenedione and LH or FSH. Both LH and FSH stimulated E, P and 20 alpha-OH-P production, and the maximum steroidogenic responses of LH and FSH were similar to those observed in cultured granulosa cells primed with FSH in vitro. THese results have demonstrated that LH is effective in stimulating both estrogen and progestin secretion in rat granulosa cells pretreated with FSH. This suggests an important role of LH in the direct control of both aromatization and luteinization in the granulosa cell.     

Effect of aminoglutethimide on extraglandular metabolism of exogenous testosterone

Aminoglutethimide (AG), an inhibitor of steroid biosynthesis, seems to have an extraglandular site of action on steroid catabolism. To study this effect, five males with peripheral hypogonadism were first given testosterone propionate and then the same dose was repeated combined with AG and urinary testosterone, and its metabolites were measured. AG was shown to have a very evident effect on the peripheral degradation of exogenous testosterone. This may be responsible for a few signs of virilization and fetal masculinization in women taking AG.     

Hormonal regulation of androgen biosynthesis by primary cultures of testis cells from neonatal rats

Enzymatically dispersed testis cells derived from 7-day-old male rats maintained their gonadotropin-stimulated testosterone production for 18 days in culture. Treatment with hCG or LH stimulated androgen production in a dose-dependent manner, with ED50 values of 0.030 +/- 0.007 and 1.0 +/- 0.4 ng/ml for hCG and LH, respectively. Concomitant treatment with a phosphodiesterase inhibitor further enhanced LH action. In contrast, treatment with FSH, GH, or PRL was without effect. Treatment with forskolin, cholera toxin, or 8-bromo-cAMP induced dose-dependent increases in testosterone biosynthesis; this was accompanied by stimulation of 3 beta-hydroxysteroid dehydrogenase activity after treatment with hCG, forskolin, or 8-bromo-cAMP. RIA measurement of different androgens in HPLC-fractionated medium revealed that the main androgen secreted by the neonatal testis cells was testosterone, with lower production of 5 alpha-androstane-3 alpha,17 beta-diol and negligible 5 alpha-dihydrotestosterone, androstenedione, and androsterone. Treatment with epidermal growth factor, GnRH, and arginine vasopressin (AVP) decreased hCG-induced testosterone biosynthesis. Since the inhibitory actions of GnRH and AVP were blocked by concomitant addition of specific hormone antagonists, their inhibitory actions were probably mediated by specific testis receptors. In contrast, treatment with several potent synthetic steroid hormone analogs [diethylstilbestrol (an estrogen), dexamethasone (a glucocorticoid), R5020 (a progestin; 17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione), R1881 (an androgen; 17 beta-hydroxy-17 alpha-methyl-4,9,11-estratrien-3-one), or cyproterone acetate (an antiandrogen; 17 alpha-acetyloxy-6-chloro-1,2-dihydro-(1 beta,2 beta)3'-H-cyclopropa-(1,2) pregna-1,4,6-trien-3,20-dione)] did not affect testosterone biosynthesis in hCG-treated cells. These results demonstrate that testosterone production by neonatal testis cells is maintained by gonadotropins during prolonged culture; the ability of cAMP-generating drugs and a cAMP analog to mimic gonadotropin actions on testosterone biosynthesis and 3 beta-hydroxysteroid dehydrogenase activity suggests a mediatory role of cAMP in gonadotropin action; and AVP, epidermal growth factor, and GnRH, through their putative testis receptors, directly inhibit gonadotropin-stimulated testosterone synthesis, while various steroids (androgens, estrogens, progestins, and glucocorticoids) do not affect Leydig cell function in the neonatal testis. The present culture system offers a unique model for elucidating the hormonal control of Leydig cell androgen biosynthesis during neonatal development.     

Androgen-binding proteins in rat epididymis: properties of a cytoplasmic receptor for androgen similar to the androgen receptor in ventral prostate and different from androgen-binding protein (ABP)

The cytoplasmic receptor (CR) in rat epididymal 105,000 $\text{g}$ supernatant was separated from the androgen-binding protein (ABP) by gel electrophoresis following labeling with [1,2,6,7-³H]-testosterone in vivo. ABP disappeared from epididymal supernatants after castration 01 hypophysectomy, while CR remained unchanged. CR was evenly distributed between caput and cauda, while much more ABP was present in caput. Properties of CR in epididymis and prostate were similar and distinctly different from ABP. Binding to CR was destroyed by charcoal treatment (1 mg/mg protein) of supernatant at 0 °C for 6 h, heating at 50 °C for 30 min, or exposure to the sulfhydryl blocking reagent, $\text{p}$-chloromercuriphenylsulfonate (1 mM) at 25 °C for 30 min, while binding to ABP was unaffected. The isoelectric pH of CR (5.8) was higher than that of ABP (4.6). Dissociation of radioactive 5α-dihydrotestosterone (DHT) from CR and nuclear receptors was extremely slow (half-time at 0 °C >2 days), while dissociation from ABP was rapid (half-time at 0 °C ~ 6 min). Cyproterone acetate (250 mg/100g body weight) inhibited binding to CR both in epididymis and ventral prostate but did not affect binding to ABP. Nuclear uptake was inhibited by cyproterone to the same extent as binding to CR, indicating that nuclear uptake and binding are dependent on CR and independent of ABP. The time-course of uptake and binding in epididymal supernatant and nuclear fractions was essentially the same 1 day after bilateral castration when both CR and ABP were present or 8 days after castration when CR alone was present. It is concluded that the cytoplasmic receptor for androgen in rat epididymis has properties very similar to the androgen receptor in ventral prostate but different from ABP.     

Interaction of glucocorticoid hormones with rat skeletal muscle: catabolic effects and hormone binding.

The mechanism of action of glucocorticoid hormones on rat skeletal muscle was studied by following their effect on muscle weight, free amino acid content, activity of amino acid-metabolizing enzymes, and binding to cytoplasmic receptor proteins. A significant reduction of gastrocnemius muscle and body weight occurred following administration of cortisol, triamcinolone diacetate, and triamcinolone acetonide to adrenalectomized rats. Treatment with triamcinolone diacetate also reduced the level of several free amino acids and enhanced the activity of a myofibrillar protease in skeletal muscle. The hormone had, however, no effect on the activity of various enzymes involved in amino acid catabolism in muscle. In nephrosis, another condition of muscle wasting, the level of several muscle amino acids were also reduced to a lesser extent. Cortisol and triamcinolone acetonide, both of which induce muscle wasting, were found to bind to two distinct cytoplasmic proteins in muscle. Binding of the labeled hormones was followed at 0 C and could be observed in presence of a 1000-fold excess of the catabolically inactive steroid epicortisol. Binding of 3H-triamcinolone acetonide. In vitro competition experiments further suggest a correlation between steroid binding to the 3H-dexamethasone or 3H-triamcinolone acetonide site and their potency to induce muscle catabolism. It is concluded that skeletal muscle is a direct target organ for glucocorticoids, and that muscle responsiveness involves binding of the active hormones to cytoplasmic receptor sites.