Effects of protein kinase C activation on cyclic AMP and testosterone production of rat Leydig cells in vitro

The role of protein kinase C in modulation of the endocrine function of rat Leydig cells was studied. Percoll-purified rat Leydig cells were stimulated with hCG, forskolin, cholera toxin, pertussis toxin and 8-bromo-cAMP in the presence and absence of two activators of protein kinase C, 12-0-tetradecanoylphorbol 13-acetate (TPA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG). The two activators had no effect on basal cAMP, but decreased hCG-stimulated, and increased cholera toxin- and forskolin-stimulated cAMP production. Cells pre-incubated with pertussis toxin showed enhanced rate of cAMP production in response to forskolin, but were no more responsive to TPA and OAG stimulation. These findings suggest that protein kinase C activation may on one hand inhibit the LH-receptor and Gs-protein coupling and on the other hand inhibit the Gi-protein mediated suppression of adenylyl cyclase activity. TPA and OAG effects on testosterone production were measured in the absence and presence of 8-bromo-cAMP stimulation. TPA enhanced basal testosterone production, but this effect was shifted to inhibition when steroidogenesis was stimulated by 8-bromo-cAMP. The OAG effect on testosterone production was inhibitory throughout the dose-response curve of 8-bromo-cAMP. The basal stimulation of testosterone production by TPA was probably due to a marginal increase of cAMP caused by inhibition of the Gi-protein, since a similar effect was observed by pertussis toxin, and thereafter TPA was without effect on testosterone. The inhibition of stimulated testosterone production by TPA and OAG indicates that protein kinase C activity also affects steroidogenesis at a step(s) beyound cAMP formation.     

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.     

Prolactin and antiprolactin receptor antibody inhibit steroidogenesis by purified rat Leydig cells in culture

The in vitro effects of ovine PRL (oPRL) on testicular testosterone synthesis were determined using isolated, collagenase-dispersed, adult rat Leydig cells in culture. oPRL (50-1000 ng/ml) had no effect either on basal or on LH (50, 100 or 2000 pg/ml)-stimulated testosterone secretion by Leydig cells in short-term culture (4 h). 125I-oPRL binding studies revealed a single class of high affinity sites (Ka 8.7 nM) with a low capacity (Bmax 6.7 fmol/mg protein identical to approximately 980 sites/Leydig cell). Isolated Leydig cells were further purified on a continuous Percoll gradient and cultured in serum-free medium, at 34 degrees C, in 5% CO2 and 95% air. After 3 days of culture, the media were collected, the cells washed and then stimulated with hCG (3 ng/ml) for 3 h. oPRL (1-1000 ng/ml) added at plating, caused a log dose-dependent inhibition of testosterone accumulation during the 3-day culture period; the highest and most consistent inhibition (31%) was with 500 ng/ml oPRL. hCG increased the sensitivity to the inhibitory effect of PRL, 10 ng/ml oPRL causing 40% inhibition and 100 ng/ml causing a maximal inhibition of 50%. PRL in fact caused a reduction in the maximal effect (efficacy) of hCG on steroidogenesis, without significantly affecting the ED50 (sensitivity). The effects of an antiPRL receptor antibody raised by the antiidiotypic route and previously shown to bind to rat testis PRL receptors were tested. The antiPRL receptor IgG (13 micrograms/ml) mimicked the PRL inhibitory effect and acted synergistically with PRL (100 ng/ml) in inhibiting both testosterone accumulation in 3-day cultured Leydig cells and their subsequent response to hCG. In summary, a clear inhibitory effect of PRL and a synergistic effect of antiPRL receptor antibody were demonstrated on testosterone synthesis by rat Leydig cells in 3-day culture.     

Effect of a tumour-promoting phorbol ester on atrial peptide-induced testosterone production and cyclic GMP accumulation by isolated mouse Leydig cells

The objective of this study was to investigate the effects of 4 beta-phorbol 12-myristate 13-acetate (4 beta-PMA)--a potent activator of protein kinase C--on the responsiveness of mouse Leydig cells to stimulation with rat atriopeptin II (rAP-II). We report that, in these cells, the stimulation of testosterone production by rAP-II could be inhibited in a dose-dependent manner by 4 beta-PMA (1-200 nM). In contrast, the basal steroidogenesis was stimulated 2-fold by 4 beta-PMA. There was no inhibition of testosterone production when the cells were stimulated with 8-bromo cyclic GMP (8Br-cGMP) in the presence of 4 beta-PMA. Furthermore, addition of 4 beta-PMA resulted in a marked reduction in the amount of cGMP accumulated in response to rAP-II stimulation. 4 alpha-Phorbol 12-myristate 13-acetate (4 alpha-PMA) was found to have no effect at all. The inhibitory effect of 4 beta-PMA on steroidogenesis could be completely reversed by the addition of 0.25 mM 3-isobutyl 1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Also, the 4 beta-PMA-induced lowering of cGMP content could be partially reversed by IBMX. Membrane fractions from cells treated with 4 beta-PMA or 4 alpha-PMA did not differ in their contents of either basal or rAP-II-stimulated guanylate cyclase activities. We conclude that the 4 beta-PMA-mediated inhibition of testosterone production by Leydig cells stimulated with rAP-II results from an activation of a phosphodiesterase enzyme, hypothetically through an activated protein kinase C. This leads to a reduction in the cellular cGMP content through an increased metabolic removal of cGMP formed in response to rAP-II stimulation.     

Measurement of steroidogenesis in rodent Leydig cells: a comparison between pregnenolone and testosterone production

The efficiency and specificity of inhibition of pregnenolone metabolism in mature, immature rat Leydig cells, mouse and tumour Leydig cells by SU-10603, a 17 alpha-hydroxylase inhibitor and epostane (WIN-32729), a 3 beta-hydroxysteroid dehydrogenase inhibitor, were studied. Metabolism of [14C]pregnenolone by mature rat Leydig cells was inhibited for more than 95% in the presence of 20 microM SU-10603 and 5 microM epostane. The sum of the different steroids produced by Leydig cells from immature rats incubated in the presence of a 5 alpha-reductase inhibitor was only 80% of pregnenolone production in the presence of SU-10603 and epostane. Pregnenolone metabolism could also be inhibited in tumour Leydig cells but not in mouse Leydig cells. Pregnenolone and testosterone production by Leydig cells from mature rats were similar when steroidogenesis is maximally stimulated by luteinizing hormone (LH). However, in the presence of LH and bovine serum albumin (bSA), or 22 R-hydroxycholesterol and bSA, pregnenolone production was 1.7- and 6-fold higher respectively, than testosterone production. The data show that for measuring the steroidogenic activity of Leydig cells estimation of pregnenolone production is more reliable than measuring testosterone production. At high activities of the cholesterol side-chain cleavage (CSCC) the conversion of pregnenolone into testosterone may become the rate-limiting step for testosterone production. Under all conditions the conversion of cholesterol into pregnenolone is the (hormonal regulated) rate-determining step for steroidogenesis.     

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.     

Biphasic effect of gonadotropin-releasing hormone and its agonist analog (HOE766) on in vitro testosterone production by purified rat Leydig cells

GnRH and GnRH agonists have stimulatory and inhibitory effects on testicular testosterone secretion both in vivo and in vitro. To determine whether they are exerted directly on the Leydig cells and to explore the temporal relationships, we examined the effects of acute (3 h) and chronic (24-72 h) exposure of purified (greater than or equal to 80%) rat Leydig cells to GnRH and its agonist analog HOE766 (D-Ser-t-BU6,des-Gly-NH2 10LHRH ethylamide; Hoechst, Frankfurt, Germany) on testosterone production. GnRH and HOE766 enhanced basal testosterone secretion by freshly isolated or cultured Leydig cells. HOE766 was at least 100 times more potent than GnRH. However, exposure of Leydig cells to HOE766 for 24 h or longer lead to a significant reduction in hCG responsiveness without altering basal testosterone secretion. Both the stimulatory and inhibitory effects were dose related, with a maximal response elicited by 10(-9) M HOE766. HOE766 reduced Leydig cell sensitivity to hCG (ED50) stimulation, but did not alter the slope of the dose-response curves. Thus, GnRH and its agonist appear to have a dual and biphasic effect on the Leydig cells. Acute exposure stimulates basal testosterone secretion (and occasionally the hCG response), whereas chronic exposure decreases the response to hCG stimulation. These data provide additional evidence that GnRH has a direct effect on Leydig cell steroidogenesis.     

Direct effect of arachidonic acid on protein kinase C and LH-stimulated steroidogenesis in rat Leydig cells; evidence for tonic inhibitory control of steroidogenesis by protein kinase C.

The role of arachidonic acid in the regulation of steroidogenesis in rat Leydig cells was studied. A dose- and time-dependent biphasic effect on maximal and submaximal LH- and dibutyryl-cAMP-stimulated testosterone production was found. The locus of the inhibition, which occurred during 3 h incubation, was prior to the side chain cleavage of cholesterol and after cAMP production. The same inhibitory effect was found with the protein kinase C (PKC) activators, phorbol-12-myristate, 13-acetate (PMA) and oleic acid, also with no change in LH-stimulated cAMP production. Arachidonic acid, PMA, and diolein, all stimulated PKC activity in a dose-dependent fashion in partially purified Leydig cell homogenates. When the cells were incubated for 5 h, arachidonic acid potentiated LH- and dibutyryl-cAMP-stimulated testosterone production. Similarly, incubation with PMA for 5 h, potentiated subsequent basal and dibutyryl-cAMP-stimulated testosterone production. PKC was down-regulated over 5 h (but not during 3 h) by pretreating Leydig cells with PMA or arachidonic acid in the presence of LH. Lipoxygenase and cyclooxygenase inhibitors did not alter the stimulatory effects of arachidonic acid. We conclude that the short-term inhibitory effect of arachidonic acid (and PMA) is via activation of PKC, but when protein kinase C (PKC) is down-regulated by these ligands, steroidogenesis is enhanced. These results suggest that steroidogenesis is normally under tonic inhibitory control by PKC.     

Evidence for the involvement of lutropin-independent RNA synthesis in Leydig cell steroidogenesis.

The effect of incubating purified Leydig cells in Eagle's medium and the subsequent effect of the RNA synthesis inhibitors, actinomycin D and cordycepin, on lutropin-stimulated testosterone synthesis have been investigated. The inhibiting effect was found to be inversely related to the time of preincubation; with cells preincubated for 0, 1, 2 and 3 h with Eagle's medium only, followed by 2-h incubation with lutropin with and without actinomycin D, testosterone synthesis was inhibited by 37 +/- 4, 31 +/- 3, 18 +/- 4 and 14 +/- 3% respectively (means +/- s.e.m., n = 5). In cells that had been preincubated for 3 h there was no significant effect of actinomycin D on testosterone synthesis during the first hour of incubation with lutropin. Thereafter the inhibition increased with time reaching a maximum of 30% after 5 h. The effects of preincubation were not due to endogenous lutropin in the Leydig cells because cells isolated from hypophysectomized rats gave similar results. The inhibition of [3H]uridine incorporation into the Leydig cell RNA was 80 +/- 1% with 8 microgram/ml actinomycin D. Increasing the concentration of this inhibitor to 80 microgram/ml did not significantly increase the inhibition of [3H]uridine incorporation or lutropin-stimulated steroidogenesis in preincubated and non-preincubated cells. With cordycepin the inhibition of both RNA synthesis and lutropin-stimulated testosterone synthesis in non-preincubated cells were the same; with 25.1--251 microgram/ml approx. 30--70% resp. With preincubated cells (3 h), 0--50% inhibition of testosterone synthesis was obtained respectively. The inhibitory effect of actinomycin D oimilar to that obtained with lutropin. These observations suggest that during preincubation and independently of lutropin, synthesis of intermediates, including RNAs required for stimulation of steroidogenesis, takes place and that subsequent stimulation of steroidogenesis by lutropin occurs without further de novo RNA synthesis. These results provide evidence for a permissive role of specific RNA and protein synthesis in the action of lutropin on testosterone synthesis in the Leydig cell.     

Cyclooxygenase-2 regulation of the age-related decline in testosterone biosynthesis

The age-related decline in testosterone biosynthesis in testicular Leydig cells has been well documented, but the mechanisms involved in the decline are not clear. Recent studies have described a cyclooxygenase-2 (COX2)-dependent tonic inhibition of Leydig cell steroidogenesis and expression of the steroidogenic acute regulatory protein (StAR). The present study was conducted to determine whether COX2 protein increases with age in rat Leydig cells and whether COX2 plays a role in the age-related decline in testosterone biosynthesis. Our results indicate that from 3 months of age to 30 months, COX2 protein in aged rat Leydig cells increased by 346% over that of young Leydig cells, StAR protein decreased to 33%, and blood testosterone concentration and testosterone biosynthesis in Leydig cells decreased to 41 and 33%, respectively. Further experiments demonstrated that overexpressing COX2 in MA-10 mouse Leydig cells inhibited StAR gene expression and steroidogenesis and that the inhibitory effects of COX2 could be reversed by blocking COX2 activity. Notably, incubation of aged Leydig cells with the COX2 inhibitor NS398 enhanced their testosterone biosynthesis. Blood testosterone concentrations in aged rats fed the COX2 inhibitor DFU, at doses of 5, 10, 15, and 20 mg/kg body weight per day were increased by 15, 23, 56, and 120%, respectively, over the levels in the rats receiving no DFU. The present study suggests a novel mechanism in male aging involving COX2 and a potential application of the mechanism to delay the age-related decline in testosterone biosynthesis.     

Isolation of a protein from human ovarian follicular fluid which exerts major stimulatory effects on in vitro steroid production of testicular, ovarian, and adrenal cells

We describe the isolation of a steroidogenesis-inducing protein (SIP) from human ovarian follicular fluid and its effects on in vitro steroid synthesis of testicular, ovarian, and adrenal cells. After heating at 60 C, precipitation with 80% ammonium sulfate and dialysis, the human ovarian follicular fluid proteins were fractionated by gel chromatography on Sephacryl S-200. The bioactivity was eluted in the mol wt region between 40 and 60 K. SIP was further purified by affinity chromatography on blue Sepharose (CL-6B), preparative isoelectrofocusing on sucrose density gradients, anion exchange chromatography on Mono Q by using fast protein liquid chromatography system and gel chromatography on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified SIP under both reducing and nonreducing conditions revealed a single band with an approximate mol wt of 60 K. SIP exhibited a pI value of 4.8, was heat sensitive, and lost activity upon lyophilization. SIP copurified with albumin in various isolation procedures and was distinct from human serum albumin but may represent a modified form of human albumin. SIP stimulated testosterone production by testicular pieces, interstitial cells, or purified Leydig cells from rats under different in vitro conditions. The SIP also stimulated basal and human CG stimulated in vitro progesterone production of human ovarian granulosa-lutein cells and corticosterone production of rat adrenal cells. The effects of SIP on testicular, ovarian, and adrenal cells were evident in the presence of maximal concentrations of tropic hormones. The steroid-free spent media from human granulosa-lutein cell cultures also stimulated testosterone production by rat interstitial cells, suggesting that granulosa cells may be the cellular source of SIP. In conclusion, human ovarian cells secrete a hitherto unknown albumin-like protein which enhances both basal and tropic hormone stimulated steroidogenesis in gonadal and adrenal cells. This protein may play a significant role in ovarian function by modifying steroidogenesis in the preovulatory follicle.     

Characterization of insulin and insulin-like growth factor I receptors of purified Leydig cells and their role in steroidogenesis in primary culture: a comparative study

Characterization of insulin and type I insulin-like growth factor (IGF-I) receptors and the effects of insulin and IGF-I on steroidogenesis were evaluated by using purified adult Leydig cells from Sprague-Dawley rats. Purified Leydig cells were found to contain both high and low affinity binding sites for insulin, with Ka values of 1.08 X 10(9) and 1.1 X 10(7) M-1, respectively. Using affinity cross-linking of [125I]iodoinsulin to plasma membrane insulin receptor, several bands were identified by autoradiography under nonreduced conditions with mol wt of 230,000, 280,000, and 300,000. After reduction with 50 mM dithiothreitol, only one band was identified with a mol wt of 130,000, consistent with the alpha-subunit of insulin receptor. Purified Leydig cells also contain specific type I IGF receptors with estimated binding affinity of 0.6 X 10(9) M-1. Multiple high mol wt bands (greater than 250,000) were identified under nonreduced conditions by affinity cross-linking. Under reduced conditions, one band with an approximate mol wt of 135,000 was identified. Purified Leydig cells (10(5) cells/ml) were cultured in Dulbecco's Modified Eagle's Medium-Ham's F-12 Nutrient Mixture (1:1) containing 0.1% fetal calf serum at 37 C in a humidified atmosphere of 5% CO2-95% air. Insulin and IGF-I stimulated testosterone formation as early as 3 h after administration, and their effects were completely blocked by the addition of a protein synthesis inhibitor, cycloheximide (1 microgram/ml). Insulin and IGF-I also significantly potentiated hCG-and 8-bromo-cAMP-induced testosterone formation. Furthermore, insulin and IGF-I potentiated hCG-stimulated cAMP formation. This suggests that insulin and IGF-I have effects at both the LH receptor sites and the steps beyond adenylate cyclase. The ED50 values of insulin and IGF-I-stimulated testosterone formation were comparable (25 ng/ml). In conclusion, we found that Leydig cells contain specific insulin and type I IGF receptors, and both insulin and IGF-I are capable of modulating Leydig cell steroidogenesis.     

Epidermal growth factor stimulates testosterone production of human Leydig cells in vitro.

Epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) have been shown to regulate Leydig cell steroidogenesis in several species. We have investigated the effects, if any, of EGF and IGF-I on in vitro testosterone production of human Leydig cells. Interstitial cells or Percoll purified Leydig cells were isolated from the testes obtained from patients (n = 9) undergoing orchidectomies for treatment of prostate cancer and were cultured for different time periods with hCG, dibutyryl cAMP, EGF and IGF-I. Testosterone in the culture media was measured by radioimmunoassay. While EGF had a stimulatory effect on basal testosterone production of isolated interstitial cells or purified Leydig cells, IGF-I was ineffective. When the interstitial cells were cultured in the presence of hCG or EGF for 3, 6 or 24 h, the stimulatory effects of EGF on testosterone production were only evident after 24 h. On the other hand, hCG stimulated testosterone production at all time points (i.e after 3, 6, 24 h of incubation). When added in the presence of maximal concentrations of hCG and dibutyryl cAMP, EGF did not further enhance steroidogenesis. On the other hand, IGF-I potentiated the effects of hCG on testosterone production. These studies suggest that EGF and IGF-I may play a regulatory role in steroidogenic function of the human testes.     

Ethane dimethane sulphonate (EDS) specifically inhibits LH stimulated steroidogenesis in Leydig cells isolated from mature rats but not in cells from immature rats

Effects of ethane dimethane sulphonate (EDS) on the pattern of protein synthesis, steroid production and ATP levels in isolated Leydig cells have been investigated. After incubation of Leydig cells isolated from mature rats with EDS (75 μg/ml) for 3–5 h, the synthesis of a 33 kDA and 50 dKa protein and LH stimulated steroid production was inhibited, but the LH stimulated cAMP production and conversion of 22 R-hydroxycholesterol to testosterone were not affected. Busulphan or ethyl methyl sulphonate (EMS) at similar molar concentrations had no effect on steroid production. After 24 h incubation with EDS Leydig cells were detached from the plastic surface and had rounded up, but the cellular ATP levels were the same as in control cells. Leydig cells were destroyed after incubation with EMS 2000 μg/ml for 24 h. EDS had no detectable effects on steroid production by isolated Leydig cells from mice, from Leydig cell tumour tissue or from immature rats, nor on rat adrenal cells or on LH and FSH secreting pituitary cells. The data indicate that EDS specifically inhibits LH regulated functional properties of mature Leydig cells possibly via alkylation of proteins. EDS could be a valuable tool to study possible regulator proteins for control of steroidogenesis in Leydig cells from adult rats.     

The role of calcium in luteinizing hormone/human chorionic gonadotrophin stimulation of Leydig cell immunoactive inhibin secretion in vitro.

The mechanism by which luteinizing hormone (LH) stimulates Leydig cell immunoactive inhibin (I-inhibin) secretion was investigated using Percoll-purified adult rat Leydig cells. Using a maximally stimulating dose of LH (16 ng/ml). Leydig cell I-inhibin secretion was non-detectable at 1-2 h of incubation, but subsequently increased at all time points during a 25 h incubation period. LH stimulated both Leydig cell content and release of I-inhibin. Increasing concentrations of LH stimulated both inhibin and testosterone immunoactivity in the incubation media over a similar dose-response range, with a 2- to 4-fold rise in I-inhibin secretion at maximal doses of LH. Dibutyryl cAMP stimulated testosterone secretion in a manner similar to that of LH, but I-inhibin secretion was less sensitive than testosterone and a significant stimulation was observed only at the highest doses (200-1000 micrograms/ml). LH-stimulated I-inhibin secretion was significantly decreased when Leydig cells were incubated in calcium-depleted (0.15 mM Ca2+ + 1 mM EGTA) or low [Ca2+] media (0.15 mM) as compared to normal (1.15 mM) or high [Ca2+] (2-5 mM) media. In contrast, LH-stimulated testosterone secretion remained unchanged by altering extracellular [Ca2+], and although decreased in the presence of EGTA, testosterone secretion remained significantly greater than basal levels. Furthermore both diltiazem and verapamil completely blocked the LH and dibutyryl cAMP-stimulated increase in Leydig cell I-inhibin, but did not reduce either LH or dibutyryl cAMP-stimulated testosterone production to basal levels. We conclude that LH stimulates both I-inhibin synthesis and release by adult rat Leydig cells in culture, by mechanisms involving calcium.     

Regulation of corticotropin-releasing factor secretion from Leydig cells by serotonin.

CRF is produced in the Leydig cells and acts as a negative autocrine regulator of Leydig cell function. To clarify the hormonal control of CRF secretion by Leydig cells, we evaluated the participation of serotonin (5HT) and serotonin agonists in the release of CRF from Leydig cells and their effects on hCG-induced cAMP generation and steroidogenesis. Serotonin stimulated CRF secretion up to 4-fold above basal levels and inhibited basal and hCG-stimulated cAMP generation and testosterone production (ID50, 1 nM). The inhibitory action of 5HT was prevented by a CRF antibody and the alpha-helical CRF-(9-41) antagonist. The selective 5HT2 receptor agonist (+-)1-[2,5-dimethoxy-4-iodophyryl]2-amino propane hydrochloride (DOI) also stimulated CRF secretion and inhibited hCG-stimulated cAMP generation and testosterone production to control levels (ID50, 7 microM). Serotonergic 5HT1A, 5HT1B/1C, 5HT1D, and 5HT3/5HT2 agonists were less effective inhibitors of hCG-stimulated cAMP and testosterone production, while agonists for the 5HT3 receptor had no effect. [125I]DOI binding studies in Leydig cells demonstrated two sets of receptors with Kd values in the nanomolar and micromolar range, with low and high capacities, respectively. The low affinity site differed from that of brain receptors (Kd, 4.2 nM) and displayed higher binding capacity (50-fold). The selective 5HT2 receptor antagonist ketanserin prevented CRF stimulation and blocked the inhibitory actions of 5HT and DOI, while the alpha 1-adrenergic antagonist prazosin had no effect. Also, treatment of cells with ketanserin increased sensitivity to hCG and raised maximal cAMP and testosterone production. 5HT was a more effective stimulus than hCG in stimulating CRF secretion, and gonadotropin-induced CRF release was inhibited by ketanserin. Inhibitory effects of exogenous CRF were demonstrable after blockade of 5HT action by ketanserin. The inhibitory actions of 5HT were unaffected by pertussis and cholera toxins and were reversed by the addition of 8-bromo-cAMP. These results demonstrate that 5HT acts on 5HT2 receptors in Leydig cells that are distinct from those in the brain to stimulate CRF secretion through a pertussis toxin-insensitive G-protein. This action of 5HT is predominantly mediated by the low affinity 5HT2-binding site and requires full occupancy for maximal CRF stimulation, indicating the absence of spare receptors. 5HT-stimulated CRF inhibits basal and hCG-induced cAMP generation and steroidogenesis. Furthermore, 5HT mediates the stimulatory action of LH/hCG on CRF secretion from Leydig cells and, thus, participates in a negative autoregulatory loop to limit the testosterone response to the gonadotropic stimulus.     

Stimulatory effect of LHRH and its agonists on Leydig cell steroidogenesis in vitro

Short-term (4 h) incubation of collagenase-dispersed Leydig cells from adult rats in the presence of an LHRH agonist caused a 2-3-fold stimulation (P less than 0.001) of testosterone production. This effect was dose-dependent and as little as 5 x 10(-11) M LHRH agonist caused significant stimulation whilst maximal effects were achieved with 10(-9) M concentrations. Stimulation of steroidogenesis by LHRH agonist was prevented by addition of an antiserum specific for the peptide, but was exaggerated in the presence of the phosphodiesterase inhibitor MIX, suggesting the involvement of cyclic AMP in the response of the Leydig cells to the agonist. Native LHRH caused a similar degree of stimulation of testosterone secretion to LHRH agonist but concentrations 1000 times greater than those of the agonist were required to achieve this, a finding consistent with the known affinities of these 2 peptides for the Leydig cell LHRH-receptor. The addition of LHRH agonist also enhanced (P less than 0.001) testosterone secretion by adult rat Leydig cells in response to hCG or dibutyryl cyclic AMP, and this effect was still evident in the presence of maximally-stimulating concentrations of these factors. LHRH agonist also stimulated testosterone secretion by Leydig cells from immature rats, but this effect differed from that in the adult in being of smaller magnitude and being restricted to effects on basal secretion or secretion elicited by low concentrations of hCG. These results show for the first time (a) that LHRH and its agonists can exert effects on Leydig cell steroidogenesis during short-term incubation, and (b) that these effects are stimulatory, which contrasts with the inhibitory effects reported after long-term (2-3 days) exposure of Leydig cells to LHRH agonists in vivo and in vitro. The availability of this simple and rapid measure of a biological action of LHRH on the Leydig cell should enable its precise mode of action to be determined, and should throw light on the physiological role of endogenously produced testicular 'LHRH'.