Tamoxifen inhibits Leydig cell steroidogenesis: in vivo and in vitro studies

Using isolated interstitial cells from testes of Sprague-Dawley rats, we have shown previously that tamoxifen inhibits LH and 8-bromo-cyclic AMP stimulated testosterone synthesis in a dose-dependent manner. The inhibitory effect of tamoxifen could not be reversed with 17 beta-estradiol. The present studies indicate that tamoxifen directly inhibits testosterone response to gonadotropin stimulation both in immature and mature hypophysectomized rats. When interstitial cells were incubated with pregnenolone (5 x 10(-7) M), testosterone levels in the incubation medium were 27.0 +/- 1.9 ng/10(6) cells. Tamoxifen (10(-5) M) significantly inhibited pregnenolone-induced testosterone formation. Tamoxifen also significantly diminished adenylate cyclase activity whereas the binding of hCG to receptor was not affected. These results indicated that several steps of steroidogenesis are inhibited by tamoxifen.     

Stimulation and inhibition by LHRH analogues of cultured rat Leydig cell function and lack of effect on mouse Leydig cells

The effect of 2 luteinizing hormone-releasing hormone (LHRH) analogues (10^?8–10^?6 M) on the functional activity (testosterone and cyclic AMP production and [¹²⁵I]hCG binding) of purified mouse Leydig cells in culture was examined. The analogues were found to have no significant effect on the cells over a period of 3 days. No specific binding of a labelled analogue to impure or pure mouse Leydig cells could be detected. In contrast high levels of specific binding to impure rat interstitial cells occurred. Centrifugation of the rat interstitial cells on 0–90% Percoll gradients showed that the LHRH analogue bound specifically to the active lutropin-responsive Leydig cells.The purified rat Leydig cells were cultured in the presence of LHRH analogue (ICI 118630) (10^?7 M) and after an initial lag period (2 h) a marked stimulation of testosterone production occurred over a 32-h period (up to 400 ng/10⁶ cells). The response to LH alone increased with time in culture up to 10 h, and the LHRH analogue enhanced this LH-stimulated testosterone production. When the cells were cultured for longer time periods (24 h) the LHRH analogue was found to inhibit LH-stimulated testosterone production at all concentrations of LH used (P < 0.01). The LHRH analogue had no consistent effect on LH-stimulated cyclic AMP production, although when added alone, cyclic AMP production was increased.These results show that LHRH analogues do not bind to or have any detectable effect on mouse Leydig cells in vitro. However, LHRH analogue does bind specifically to purified rat Leydig cells. After a short lag period the analogue stimulates testosterone production which turns to inhibition after 20 h in culture.     

Adult rat Sertoli cells secrete a factor or factors which modulate Leydig cell function

Production of testosterone and oestradiol-17 beta by Leydig cells from adult rats was stimulated by LH or dibutyryl cyclic AMP (10 and 2.5-fold respectively). The addition of spent medium from normal, hemicastrated or gamma-irradiated rat seminiferous tubule cultures, as well as from Sertoli cell cultures, to purified Leydig cells further enhanced both basal (44 and 53% for testosterone and oestradiol-17 beta respectively) and LH-stimulated (56 and 18%) steroid output. Simultaneously, a decrease (20-30%) in intracellular cyclic AMP levels was observed. This stimulating factor (or factors) secreted by the Sertoli cells is different from LHRH, is of proteinic nature and has a molecular weight ranging between 10,000 and 50,000; its synthesis is not controlled by FSH nor by testosterone. This factor(s) involved in rat Leydig cell steroidogenesis, at a step beyond the adenylate cyclase, does not require protein synthesis for testosterone formation whereas it does for oestradiol-17 beta production. It should be noted that a germ cell-Sertoli cell interaction modulates the synthesis of this factor(s).     

Danazol inhibits steroidogenesis in the rat testis in vitro.

The effects of danazol on steroidogenesis in vitro in the rat testis were examined by studying: 1) androgen synthesis in rat Leydig cells cultured with danazol, 2) danazol binding to rat testis microsomal cytochrome P-450, and 3) enzyme kinetics of danazol inhibition of the microsomal enzymes of testicular steroidogenesis. Concentrations of danazol as low as 1 micrometer suppressed LH-stimulated testosterone and androstenedione production in cultured Leydig cells. The addition of danazol to a preparation of testicular microsomes elicited a type I cytochrome P-450 binding spectrum, with an apparent spectral dissociation constant (Ks) of 4.8 micrometer. Danazol inhibited progesterone and 17alpha-hydroxy-progesterone binding to microsomal P-450 with apparent spectral inhibition constants of 2.4 micrometer and 2.8 micrometer, respectively. Danazol competitively inhibited 3beta-hydroxy-delta5-steroid dehydrogenase-isomerase (apparent enzymatic inhibition constant, KI = 5.8 micrometer), 17alpha-hydroxylase (KI = 2.4 micrometer), 17,20 lyase (KI = 1.9 micrometer), and 17beta-hydroxysteroid dehydrogenase (KI = 4.4 micrometer). These findings indicate that low concentrations of danazol directly inhibit steroidogenesis in the rat testis in vitro.     

The acute effect of oestrogens on testosterone production appears not to be mediated by testicular oestrogen receptors

Scatchard binding analysis was performed to measure the cytoplasmic oestrogen receptor in the testis of rats. After treatment of rats with the antioestrogen tamoxifen no oestrogen receptor binding was found in testicular low speed supernatant between 12 and 96 h after treatment. Such tamoxifen-treated rats were used to study the acute effect of oestrogens on testosterone secretion, both in vivo and in vitro. Injection of oestradiol benzoate (50 μg, 24 h prior to experiment) resulted in a significant depression of basal and LH-stimulated plasma testosterone levels in control rats and this effect was unchanged in tamoxifen-pretreated rats. In vitro, oestradiol-17β also inhibited the LH-induced rise in testosterone secretion by isolated testicular interstitial cells. This inhibition was not affected if the rats had been pretreated with tamoxifen. These results indicate that the inhibitory effects of exogenous oestrogens on testicular testosterone production are probably not mediated by the oestrogen receptor.     

hCG-induced inhibition of testicular steroidogenesis: An oestradiol-mediated process?

Single i.v. injections of hCG (10 U) in adult male rats resulted, within 24 h, in a 2-fold decrease in the maximal LH-stimulated testosterone production in vitro, while pregnenolone production was not changed. In addition to the changes in steroidogenesis, a concomitant depletion of the oestradiol-cytosol receptor was observed. In contrast with the observations after 24 h, a 2-fold increase in both testosterone and pregnenolone production was observed at 72 h after a single i.v. injection of hCG (10 U). At 72 h after the hCG treatment, oestradiol-cytosol receptor levels were not different from values observed after injection of saline. Tamoxifen administration (50-500 microgram s.c. injections) resulted, within 24 h, in depletion of oestradiol-cytosol receptor levels. This decrease of oestradiol binding, however, was not paralleled by decreases in testosterone production. Simultaneous administration of tamoxifen and hCG did not prevent the hCG-induced inhibition of testosterone production observed 24 h after administration of hCG. It is concluded that the present results offer no proof for an obligatory role of the oestradiol receptor in gonadotropin-induced inhibition of testosterone production in testicular Leydig cells.     

Leptin effects on testis and epididymis in the lizard Podarcis sicula, during summer regression

In this study we assessed the effect of leptin treatment on testicular morphology, spermatogenesis, Peroxisome Proliferator Activated Receptor (PPAR) α, 17β-hydroxysteroide dehydrogenase, 17β-estradiol and testosterone levels in the testis and blood of the lizard Podarcis sicula at the beginning of summer regression before entering the refractory period, when lizards no longer respond to hormonal and environmental stimuli. Lizards treated with five injections of leptin showed seminiferous tubules with germinal cells at all stages and wider lumina with respect to the controls. After 10 injections, the diameter of the lumina increased compared to the controls and 5 injection-group. After 10 injections plus 20 days before the sacrifice, the seminiferous tubules with open lumina and germinal cells were less abundant than in the 5 and 10 injection-groups. In all groups, the epididymis epithelium was higher than in the controls, with mitosis and binucleated cells. In both the control and treated animals secondary spermatocytes and spermatids were immunoreactive to leptin receptor and PPARα. In treated animals the interstitial cells and peritubular fibrocytes were also leptin receptor immunoreactive, while PPARα immunoreactivity translocated from the cytoplasm to the nucleus. 17β-HSD immunoreactivity was present in the spermatids and interstitial cells of control lizards and in secondary spermatocytes and spermatids of treated lizards. Leptin treatment had no statistically significant effect on testicular and circulating 17β-estradiol and testosterone levels. These observations indicate that leptin brings about a delay in testis summer regression in Podarcis sicula, playing a regulatory role in reproduction in this species as already hypothesized for mammals.     

Negative regulation of glucose metabolism in human myotubes by supraphysiological doses of 17β-estradiol or testosterone

Objective

Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance, but evidence regarding the direct role of either sex hormone on metabolism is limited. Therefore, the aim of this study was to investigate the direct effect of acute sex hormone exposure on glucose metabolism in skeletal muscle.

Materials/Methods

Differentiated human skeletal myotubes were exposed to either 17β-estradiol or testosterone and metabolic characteristics were assessed. Glucose incorporation into glycogen, glucose oxidation, palmitate oxidation, and phosphorylation of key signaling proteins were determined.

Results

Treatment of myotubes with either 17β-estradiol or testosterone decreased glucose incorporation into glycogen. Exposure of myotubes to 17β-estradiol reduced glucose oxidation under basal and insulin-stimulated conditions. However, testosterone treatment enhanced basal palmitate oxidation and prevented insulin action on glucose and palmitate oxidation. Acute stimulation of myotubes with testosterone reduced phosphorylation of S6K1 and p38 MAPK. Exposure of myotubes to either 17β-estradiol or testosterone augmented phosphorylation GSK3β^Ser9 and PKCδ^Thr505, two negative regulators of glycogen synthesis. Treatment of myotubes with a PKC specific inhibitor (GFX) restored the effect of either sex hormone on glycogen synthesis. PKCδ silencing restored glucose incorporation into glycogen to baseline in response to 17β-estradiol, but not testosterone treatment.

Conclusion

An acute exposure to supraphysiological doses of either 17β-estradiol or testosterone regulates glucose metabolism, possibly via PKC signaling pathways. Furthermore, testosterone treatment elicits additional alterations in serine/threonine kinase signaling, including the ribosomal protein S6K1 and p38 MAPK.     

Biochemical, histochemical, and ultrastructural analyses of presumed steroid-producing tissues in the sexually mature sea lamprey, Petromyzon marinus L

In vitro incubations of testicular, ovarian, and presumed adrenocortical tissues (PAT) from the mature sea lamprey, Petromyzon marinus, with [1,2-³H]cholesterol failed to form cortisol, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, pregnenolone, and progesterone. “Isopolarity” and “isomorphicity” were establidhed for testosterone and androstenedione from the PAT incubation, but subsequent attempts at derivative formation indicated that no testosterone or androstenedione was formed. The interstitial cells of the testes and the cells within the ovarian follicles of P. marinus have a fine structure similar to those in other species of lamprey. The appearance of these tissues and of PAT remains unaltered after 4 hr of incubation. Histochemical procedures did not provide evidence for 3β-hydroxysteroid dehydrogenase (3β-HSD) activity in ovary and PAT, and only a weak 3β-HSD activity was observed over the interstitial tissue of the testes. Spectrophotometric evidence for 3β-HSD activity was obtained from PAT and testicular tissue homogenates. No 3β-HSD activity was observed in mature ovarian tissue homogenates.     

Role of calcium in luteinizing hormone-induced progesterone and cyclic AMP production in granulosa cells of the hen (Gallus domesticus)

The effects of calcium on steroidogenesis and cyclic AMP production in chicken granulosa cells were examined. For the expression of full steroidogenic effects by LH, forskolin, and 8-bromo cyclic AMP the presence of calcium in the incubation medium was essential, the optimal concentration being 1 mM. Moreover, calcium antagonists (verapamil, TMB-8) significantly suppressed steroidogenesis in response to all three agonists. The metabolism of 25-hydroxycholesterol and the conversion of pregnenolone to progesterone, however, were not affected by the lack of calcium or by coincubation with calcium antagonists. LH-stimulated cyclic AMP production was also suppressed in calcium-deficient medium and in the presence of the putative calcium channel blocker, verapamil. However, TMB-8 did not affect LH-induced cyclic AMP production. Moreover, neither forskolin- nor IBMX-induced cyclic AMP accumulation was significantly affected by lack of calcium or verapamil. These results are interpreted to indicate that the continuous presence of extracellular calcium is essential for hormonal regulation of steroidogenesis and is important for events both proximal and distal to cyclic AMP generation up to pregnenolone synthesis.     

The effect of calcium on the potentiation of LH-stimulated steroidogenesis and inhibition of LH-stimulated cyclic AMP production by LHRH agonist (ICI 118630) in rat Leydig cells

A luteinizing hormone-releasing hormone (LHRH) agonist (ICI 118630) potentiated the effects of luteinizing hormone (LH) and dibutyryl cyclic AMP on steroidogenesis during 4 h incubations with rat Leydig cells. LH-stimulated cyclic AMP levels were decreased by the addition of the LHRH agonist. The potentiation of the LH-increased steroidogenesis was dependent on Ca2+; maximum effects required at least 2.5 mM Ca2+ in the incubation medium. The calcium ionophore A23187 negated the potentiation in a dose-dependent manner (ED50 = 0.2-0.3 microM), but had no effect on LH-induced steroidogenesis, despite a 90% decrease in cyclic AMP production. The latter decrease was found to be dependent on the Ca2+ concentration. In the presence of the phosphodiesterase inhibitor methylisobutylxanthine (MIX), the ionophore A23187 induced a dose-dependent decrease in both LH and LH plus LHRH agonist-stimulated steroidogenesis and cyclic AMP production. The results obtained indicate that calcium, rather than cyclic AMP, is the mediator of the potentiating effects of LHRH agonist on LH-increased steroidogenesis in rat Leydig cells. The marked inhibition of the synergism in the presence of calcium ionophore A23187 suggests that Leydig cell calcium homeostasis must be intact for LHRH agonist action to occur. LHRH agonist causes a Ca2+-dependent decrease in LH-stimulated cyclic AMP production.     

Steroid hormone synthesis and secretion by testes,ovary, and adrenals of embryonic and postembryonic ducks

To elucidate the relationship between steroidogenesis and sex differentiation or sexual development in the birds, plasma LH, testosterone, 17β-estradiol, progesterone, corticosterone and cortisol, testicular and ovarian testosterone, 17β-estradiol and progesterone, and adrenal corticosterone, cortisol, progesterone, and testosterone concentrations from 15- to 26-day-old embryonic and 1- to 14-day-old postembryonic male and female ducks were determined by radioimmunoassays. Plasma LH level was high in embryos and decreased after haiching in both sexes. Both plasma testosterone and 17β-estradiol concentrations were statistically higher in female embryos than in male embryos, while sex differences were not observed in plasma progesterone, corticosterone, or cortisol concentrations. Both plasma corticosterone and cortisol reached a peak at hatching, and decreased thereafter. Testicular and ovarian testosterone, 17β-estradiol, or progesterone was much higher in female embryos than in male embryos. Adrenal corticosterone, cortisol, progesterone, and testosterone concentrations reached a peak 1 day after hatching, and decreased thereafter in both sexes. These results indicate that duck embryonic ovary is much more active in production and secretion of sex steroid hormones than the embryonic testes. It is suggested that the sex of the avian species is basically male having homozygoty of sex chromosomes (ZZ), and that estrogens secreted by the embryonic ovary have important roles in female sex differentiation. Very high levels of corticosterone and cortisol either in plasma or adrenals observed shortly after hatch suggest that corticosteroids have an important role in hatching of ducks to adapt themselves to a new environment.     

LHRH agonist decreases LH- but not forskolin-stimulated cyclic AMP levels in rat Leydig cells in vitro

The effects of an LHRH agonist on LH- and forskolin-stimulated cyclic AMP and testosterone production have been investigated in purified rat Leydig cells in vitro. In agreement with previous results it was found that preincubation with LHRH agonist inhibited subsequent LH-stimulated cyclic AMP production. At least 2 h preincubation was required and this effect of the LHRH agonist was negated by the protein synthesis inhibitor cycloheximide and by the phosphodiesterase inhibitor methylisobutylxanthine (MIX). Forskolin-stimulated cyclic AMP production was not inhibited by the LHRH agonist. Forskolin increased testosterone production to the same levels attained by LH and preincubation with LHRH agonist increased both forskolin- and LH-stimulated testosterone production. The data obtained suggest that LHRH agonist increases the synthesis of an inactive form of phosphodiesterase (or associated protein) which is activated by LH via a mechanism not involving cyclic AMP.     

Corticosteroid and ribonucleic acid synthesis in isolated adrenal cells: Inhibition by actinomycin D

Both adrenocorticotrophic hormone (ACTH) and cyclic AMP rapidly stimulate corticosterone synthesis in isolated adrenal cells prepared by collagenase disaggregation of decapsulated rat glands. This steroidogenic response is not accompanied by any acute increase in the incorporation of [³H]uridine into acid insoluble RNA; indeed a slight decrease is observed during the incubations. A wide variety of different effects of actinomycin D on adrenal steroidogenesis have previously been reported. The effects of a range of actinomycin D concentrations (1–50 μmol/l) on the steroidogenesis brought about by different concentrations of ACTH (0.1–100. mi.u./ml) and cyclic AMP (1–5 mmol/l) were therefore examined. Actinomycin D (1 μmol/l) inhibits overall RNA synthesis by over 91% but has little or no effect on the cellular response to low concentrations of ACTH, although both basal (non-stimulated) corticosterone output and cyclic AMP stimulated steroidogenesis are appreciably inhibited (by 29–54%). Even at very high doses of actinomycin D (50 μmol/l), which inhibit RNA synthesis by 96% a substantial steroidogenic stimulation is obvious at all concentrations of ACTH and cyclic AMP studied. There is a greater inhibition of stimulated steroidogenesis not only with increasing actinomycin D concentrations, but also with increasing time of cellular exposure to actinomycin D. It is concluded that the acute steroidogenic ACTH mechanism does not require newly synthesized RNA and that if the inhibition by actinomycin D is simply due to an effect on synthesis of various RNA species, then the shortest estimate of the half-life of any RNA involved is 70 min.     

Steroidogenic cellular sites in the cat ovary: a histochemical study.

The distribution of lipids, NADH, NADPH diaphorase, glucose 6-phospate, the isocitrate dehydrogenases, Δ⁵-3β- and 17β-hydroxysteroid dehydrogenases, and acid phosphatase in immature, cycling, and pregnant cat ovaries has been histochemically studied. Corpora lutea were found only in pregnant cats. Acid phosphatase was present in the degenerating follicles and only in trace amounts in the corpora lutea and was absent in the interstitial gland cells, germinal epithelium, and the normal developing follicles. Lipids and the various enzymes studied were localized in the theca interna of preantral and antral follicles, interstitial gland cells, and in the corpora lutea. Germinal epithelium lacked lipids, diaphorases, and all the enzymes investigated. It is suggested that theca interna and the interstitial gland cells form principal sites of steroid synthesis in the ovaries of immature as well as cycling cats while corpora lutea form additional sites of steroidogenesis during pregnancy.     

Fibroblast growth factor inhibits luteinizing hormone-stimulated androgen production by cultured rat testicular cells

The effect of fibroblast growth factor (FGF) on LH-stimulated testosterone production was investigated using primary cultures of rat testicular cells. Testicular cells obtained from neonatal rats (8-9 days of age) were maintained in culture for 3 days and then challenged with LH with or without basic FGF. After 3 additional days of culture, the media were collected for steroid RIA. LH treatment of cultured cells stimulated testosterone production in a dose-dependent fashion whereas FGF alone did not affect androgen biosynthesis. In contrast, cotreatment with FGF caused a dose-dependent decrease of LH-stimulated testosterone production, with an IC50 value of 1.1 X 10(-9) M (as calculated from three separate experiments). The inhibitory effect of FGF was evident 24 h after the initiation of treatment and this effect was reversible 1 day after the cessation of FGF treatment. The inhibition of LH-induced testosterone production by FGF (maximal inhibition greater than 90%) was accompanied by a 12-fold increase in progesterone levels, suggesting that the inhibitory effect of FGF was distal to the step of progesterone formation. FGF also inhibited forskolin (10(-5) M)- and (Bu)2cAMP (5 X 10(-4) M)-stimulated testosterone production. Furthermore, FGF inhibited the conversion of exogenously added androgen precursors (progesterone and 17 alpha-hydroxyprogesterone) to testosterone in LH-stimulated cultures indicating that FGF might inhibit 17 alpha-hydroxylase activity. The concept of a direct testicular action of FGF was further supported by the demonstration of high affinity (Kd: 3.9 X 10(-10) M; n = 3 experiments) and low capacity (46,900 sites per cell) FGF receptors in cultured testis cells. The binding of [125I]FGF was inhibited by basic and acidic FGF but not by several other growth factors. In conclusion, we suggest that FGF binds to testicular cells and inhibits LH-stimulated testosterone production by inhibiting, at least partially, 17 alpha-hydroxylase enzyme activities. Because FGF has been purified from testis extracts, this growth factor may have intratesticular paracrine or autocrine functions.     

Effect of myxothiazol on Leydig cell steroidogenesis: inhibition of luteinizing hormone-mediated testosterone synthesis but stimulation of basal steroidogenesis

Studies of MA-10 Leydig cells have shown that intact mitochondria with active respiration are essential for LH-induced Leydig cell steroidogenesis. To further elucidate the role played by mitochondria in steroidogenesis, we examined the effects of the perturbation of the mitochondrial electron transport chain with myxothiazol (MYX) on testosterone production by primary cultures of Brown Norway rat Leydig cells. Analysis of the steroidogenic pathway revealed that cAMP production and the activities of each of 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase/C17-20 lyase, and 17beta-hydroxysteroid dehydrogenase were inhibited by MYX and that LH-stimulated testosterone production was suppressed. In contrast to the inhibition of LH-stimulated testosterone production by MYX, the incubation of Leydig cells with MYX in the absence of LH stimulated testosterone production. Although testosterone production was increased, steroidogenic acute regulatory protein was decreased in response to MYX, not increased as could be expected. Additional electron transport chain inhibitors had stimulatory effects on testosterone production that were similar to those of MYX, strongly suggesting that the effect of MYX on basal testosterone production is related to its effect on the mitochondrial electron transport chain. Finally, incubation of the cells with a combination of MYX and the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid tetrakis acetoxymethyl ester suppressed MYX-mediated increased basal steroidogenesis but had no effect on hydroxycholesterol-mediated steroidogenesis. Taken together, these results indicate that inhibition of the mitochondrial electron transport chain can block LH-stimulated testosterone production through suppression of a number of steps of the steroidogenic pathway but also stimulates basal testosterone production through a calcium-mediated mechanism.     

Adenylate cyclase activity of avian granulosa: Effect of gonadotropin-releasing hormone

The activity of adenylate cyclase in crude and purified preparations of hen granulosa was investigated by measuring the production of cyclic AMP during a 20-min incubation at 30°. Both NaF and guanosine 5′-β,γ-imidotriphosphate (Gpp(NH)p), the nonhydrolyzable analog of GTP, stimulated enzyme activity in a dose-related manner. Ovine LH and, to a lesser extent, ovine FSH also activated adenylate cyclase in the presence of half-maximally stimulating concentrations of Gpp(NH)p (10^?7M). Gonadotropin-releasing hormone (10^?10–10^?4M) failed to significantly affect basal- or gonadotropin-promoted adenylate cyclase activity or the production of cyclic AMP by intact granulosa cells. Progesterone production, on the other hand, was enhanced by gonadotropin-releasing hormone (GnRH) (10^?8–10^?6M). It is suggested that in chicken granulosa cells, as in the mammalian pituitary cells, the adenylate cyclase/cyclic AMP system is not a mediator of GnRH action.     

Direct inhibitory effect of estrogen on LH-stimulated androgen synthesis by ovarian cells cultured in defined medium

The direct inhibitory effect of estrogen on ovarian androgen synthesis was investigated. When primary cultures of rat ovarian theca-interstitial cells were grown in defined medium with LH there was a marked increase in androgen synthesis of which 98% was androsterone (control = 11 ± 2 ng; LH = 1219±217 ng/ml/10⁶ cells). Diethylstilbestrol (DES), estrone (E₁), estradiol (E₂), and estriol (E₃) inhibited LH-stimulated androsterone synthesis by 81%, 81%, 81%, and 47%, respectively. The ED₅₀'s of the estrogens were: DES = 4.2±2.l × 10^?9M; E₁ = E₂ = 9.5±2.4 × 10^?8 M; and E₃ = 3.8±2.6 × 10^?7 M. The estrogen effect was very rapid ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ = 10 min) and long-lasting. Metabolic studies revealed that estrogen inhibited androsterone, androstenedione, 5α-androstane-3α, 17β-diol, and testosterone accumulation by 80%, dehydroepiandrosterone and 17α-hydroxypregnenolone by 40%, 17α-hydroxyprogesterone by 30%, while pregnenolone and progesterone were unchanged. These results prove, for the first time, that estrogen can directly inhibit LH-stimulated androgen production in ovarian theca-interstitial cells and suggest that mechanism involves, at least in part, a rapid selective inhibition of the 17α-hydroxylase/C_17–20 desmolase activities.     

Stimulation of prostaglandin E and testosterone production in rat interstitial cells by a gonadotropin-releasing hormone agonist

The early direct effects of a GnRH agonist analog [D-Ala6]des-Gly10-GnRH N ethylamide (GnRHa) on rat testicular interstitial cells include increased production of prostaglandin E (PGE) and testosterone (T) at 3 h (ED50 values of 0.5 and 0.75 nM, respectively). On the other hand, LH action on testicular function, which is mediated by increased cAMP, involves an increase in T production at 30 min followed by increased PGE formation at 3 h. GnRHa at concentrations of 10(-12)-10(-8) M had no effect on basal or LH-stimulated cAMP production during a 4-h incubation test. The stimulatory effect of GnRHa on PGE, but not on T production, was abolished by the prostaglandin synthesis inhibitor indomethacin (1.5 microns). We conclude that cAMP does not play a role in mediating the direct testicular effects of GnRH on PGE and T production; that PGE is not involved in mediating GnRH-induced T production; and, finally, that increased PGE and T production might be involved in mediating the direct inhibitory and stimulatory testicular effects of GnRH and its agonist analogs.