Catecholamine stimulation of testosterone production via cyclic AMP in mouse Leydig cells in monolayer culture

The effects of the beta-adrenergic hormone agonist, isoproterenol, on testosterone and cyclic AMP production in mouse Leydig cells in culture have been investigated. It was found that isoproterenol increased testosterone production on days 1, 2 and 3 of culture but not in freshly cultured cells. Cyclic AMP production was however increased on all days of culture. In subsequent studies carried out on day 2 of culture the amounts of testosterone formed during incubation with isoproterenol were 20-90% of those obtained with maximum stimulating levels of luteinizing hormone. The amounts of cyclic AMP formed were extremely low compared with those obtained with luteinizing hormone (22 +/- 5.3 and 2320 +/- 100 pmoles/10(6) cells/2 h respectively). Isoproterenol (10(-8) -10(-7) M) gave a significant increase in testosterone production and reached a maximum with 10(-6) M. Similar dose-response curves for cyclic AMP production were obtained. The stimulation of cyclic AMP and testosterone by isoproterenol was highly dependent on the presence of the phosphodiesterase inhibitor, methylisobutylxanthine. Propranolol blocked, in a dose-dependent manner, both isoproterenol-stimulated cyclic AMP and testosterone production. In the presence of excess luteinizing hormone no additional effects of isoproterenol were detected. Epinephrine also stimulated testosterone production. It is concluded that catecholamines stimulate testosterone production in mouse Leydig cells in monolayer culture and that this effect if mediated by cyclic AMP.     

The functional activity of adult mouse Leydig cells in monolayer culture: effect of lipoproteins, pregnenolone and cholera-toxin

Further studies were carried out on purified mouse Leydig cells to determine why they lose their hormone responsiveness after several days in monolayer culture. The effects of cholera-toxin on cyclic AMP and testosterone production were examined. It was found that cyclic AMP production could still be maximally stimulated by cholera-toxin after 4 days in culture when response to luteinizing hormone (LH) has declined. Testosterone production was, however, not maintained. Because this decline in testosterone production may have been due to the lack of a suitable substrate after several days in culture, cells were cultured initially in the presence of exogenous pregnenolone and low-density lipoproteins (LDL). Both substances were found to enhance basal and LH-stimulated testosterone production and to extend responsiveness of the cells until at least day 4, but by day 7 response was lost. Cells were then cultured in the presence of rat and human LDL and HDL and in both cases LDL was found to enhance consistently testosterone production, but HDL was much less effective. Scanning and transmission electron micrographs showed that changes in cell shape occurred during culture, but indicated that the cells were not depleted of lipid droplets by the end of culture or after LH stimulation. It is concluded that the eventual decline in testosterone synthesis is not due to lack of substrate, although the addition of exogenous substrate does extend the period of responsiveness. Nor is it due to a decrease in adenylate cyclase activity. At least part of the lesion is caused by a decrease in the enzymes required for the conversion of pregnenolone to testosterone.     

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.     

Evaluation of steroid biosynthetic lesions in isolated Leydig cells from the testes of streptozotocin-diabetic rats

Summary In streptozotocin-treated adult male rats, the mechanisms of reduced response of testicular Leydig cells in vitro to stimulation with human chorionic gonadotropin was investigated. A decrease in testosterone formation by 70% after 6 weeks of diabetes mellitus is combined with diminished intracellular cyclic AMP accumulation as well as with a disturbance in progesterone conversion to androgens. The latter is also reflected by reduced progesterone binding to the cytochrome P-450 moiety of the steroid-17-hydroxylase system.     

LH/hCG-receptor is coupled to both adenylate cyclase and protein kinase C signaling pathways in isolated mouse Leydig cells

The aim of this study was to examine whether or not a protein kinase C-dependent pathway is involved in the desensitization process of the LH/hCG-receptor-linked adenylate cyclase system in isolated mouse Leydig cells. Treatment of these cells with the phorbol ester, 4-β-phorbol 12-myristate 13-acetate (PMA) leads to a translocation (and a putative activation) of protein kinase C from the cytosol to the plasma membrane, as evidenced by the Western blotting procedure using particulate and cytosolic fractions of Percoll-purified mouse Leydig cells. A similar translocation is also observed following the treatment of mouse Leydig cells with hCG. Data obtained show that this effect is time-dependent and is mediated specifically through the LH/hCG-receptor. Furthermore, we show that the treatment of Leydig cells with either PMA or hCG leads to a desensitization of the adenylate cyclase stimulated with hCG, hCG plus GppNHp or AIF ₄ ⁻ . This desensitization was not accompanied by a change in the [¹²⁵I]-hCG binding to membrane receptors. Thus we provide here direct evidence that hCG is capable of activating protein kinase C. In addition, we postulate that PMA as well as hCG-treatment leads to a lesion located at a site distal to the receptor/G-protein interaction but proximal to the adenylate cyclase activation and that the translocation (and activation) of protein kinase C may be a common mechanism involved in this desensitizing effect caused by both PMA and hCG on Leydig cells     

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.     

Androgen production in primary culture of immature porcine Leydig cells

The present paper examines the steroidogenic responsiveness of immature porcine Leydig cells in primary culture. Both testosterone (T) and dehydroepiandrosterone sulfate (DHAS) secretion were measured under basal conditions and after stimulation with human chorionic gonadotropin (hCG) (25 ng/ml). In medium supplemented with insulin, transferrin, epidermal growth factor (3H) and 0.1% calf serum, cells survived 3-5 days in culture. The production of steroids (under hCG stimulation) is poor on day 0-1 of the culture. On day 2-4 basal T and DHAS levels are 1.9 and 17.0 ng/10(6) cells/24 h. The addition of hCG stimulated T and DHAS production 19- and 6-fold respectively and the average productions were 37 and 109 ng/10(6) cells/24 h. Increasing the serum to 0.5% did not change the viability of the cultures, but increased hCG stimulated T and DHAS production (183 and 188 ng/10(6) cells/24 h). The addition of alpha-tocopherol (vitamin E) to 0.1% calf serum led to a 4-fold increase in stimulated T production (142 ng/10(6) cells/24 h) and maintained full cell viability for more than 5 days. Measurement of 3 beta-ol steroid dehydrogenase activity indicates that the amount of enzyme is 4 times higher at day 2 than at day 0 and 1 (with or without hCG), suggesting a spontaneous maturation of the cells in culture. This might explain the increased T production with time in culture. In cumulative experiments (24 h) the cells do not seem to be desensitized to hCG stimulation following prolonged exposure to 25 ng hCG since the daily steroid production is increasing with time in culture. However, kinetic studies show that steroidogenesis is not linear over a 24 h period. In cumulative experiments the steroid production stops between 12 and 16 h following hCG exposure (5 and 100 ng/ml) and resumes following a medium change. These results suggest that some inhibitory compounds are accumulated in the medium and are controlling the Leydig cell function. Moreover high doses of hCG (100 ng/ml) result in a lower production of steroids and an earlier plateau in the case of DHAS. These results demonstrate that porcine Leydig cells can live and differentiate in hormone- and vitamin-supplemented medium and that auto-feedback mechanisms inhibiting steroid accumulation take place under in vitro conditions.     

Effect of cholera toxin on insulin release in monolayer cultures of the endocrine pancreas

Summary The effect of cholera toxin on insulin release by monolayer cultures of endocrine pancreas has been studied. The toxin has a marked stimulatory effect upon insulin release at concentrations as low as 10^–10M. The toxin had a small effect at low glucose concentrations, but was strongly stimulatory at high glucose concentrations and in the presence of arginine. Its effect could be detected within 30 min of application and only two minutes' exposure to the toxin was required for it to subsequently stimulate release. In comparative studies on insulin release the toxin was equal to, or slightly more potent than, 1.5 M glucagon and significantly more potent than cyclic AMP (10 mM) or theophylline (5 mM).     

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.     

Stimulatory effect of Sertoli cell secretory products on testosterone secretion by purified Leydig cells in primary culture

We investigated the influence of media from nonstimulated (SCCM) and FSH-stimulated (F-SCCM) cultured rat Sertoli cells on testosterone secretion by purified rat Leydig cells maintained in culture for 4 days. Both SCCM and F-SCCM stimulated Leydig cell secretory activity to a level 2-6 times that of the control, the effect being always maximal on day 3 of culture. On day 3, concentrated SCCM had a greater stimulatory effect on testosterone secretion than the original (i.e. non-concentrated) one, the effect being dose-related and similar to that exerted by concentrated F-SCCM. On the other hand, original as well as concentrated F-SCCM stimulated the basal testosterone secretion in a dose-dependent manner on day 1 to about 200% and 400% of the control level, respectively, whereas SCCM exerted the 'early' effect only as a concentrated preparation. Preincubation of Leydig cells with F-SCCM enhanced both basal (190% control) and LH-stimulated (274% control) testosterone secretion when the LH (10 ng/ml) was added for 3 h on day 1. The enhanced influence of SCCM was noted only with the LH-stimulated cells (140% control). It is concluded that, in culture, Sertoli cells release at least 2 factors which enhance testosterone secretion by Leydig cells in vitro. One of them seems to be FSH-dependent and increases both basal and LH-stimulated testosterone secretion. This factor (MW greater than 1 kDa) is heat-labile and exerts its maximal effect between 12 and 18 h of culture. The second factor(s) acts predominantly on day 3 of culture, is apparently FSH-independent, and its influence on Leydig cell testosterone may be, at least in part, nonspecific.     

Regulation of seminiferous tubule-associated stem Leydig cells in adult rat testes

Testicular Leydig cells are the primary source of testosterone in males. Adult Leydig cells have been shown to arise from stem cells present in the neonatal testis. Once established, adult Leydig cells turn over only slowly during adult life, but when these cells are eliminated experimentally from the adult testis, new Leydig cells rapidly reappear. As in the neonatal testis, stem cells in the adult testis are presumed to be the source of the new Leydig cells. As yet, the mechanisms involved in regulating the proliferation and differentiation of these stem cells remain unknown. We developed a unique in vitro system of cultured seminiferous tubules to assess the ability of factors from the seminiferous tubules to regulate the proliferation of the tubule-associated stem cells, and their subsequent entry into the Leydig cell lineage. The proliferation of the stem Leydig cells was stimulated by paracrine factors including Desert hedgehog (DHH), basic fibroblast growth factor (FGF2), platelet-derived growth factor (PDGF), and activin. Suppression of proliferation occurred with transforming growth factor β (TGF-β). The differentiation of the stem cells was regulated positively by DHH, lithium- induced signaling, and activin, and negatively by TGF-β, PDGFBB, and FGF2. DHH functioned as a commitment factor, inducing the transition of stem cells to the progenitor stage and thus into the Leydig cell lineage. Additionally, CD90 (Thy1) was found to be a unique stem cell surface marker that was used to obtain purified stem cells by flow cytometry.Testicular Leydig cells are the primary source of testosterone (T) in males. T is essential for the development of the male reproductive system and the maintenance of male reproductive functions (1, 2). In addition to defects in reproductive system, its deficiency in the adult contributes to other symptoms that include increased body fat, decreased muscle mass, increased fatigue, depressed mood, decreased cognitive function (3, 4), and reduced immune response (5, 6). In aged men, low T has been reported to contribute to mortality (7). Thus, the formation and maintenance of a functional Leydig cell population throughout adult life is of fundamental importance.In rodents and humans, T production gradually increases from the peripubertal period through the adult, coincident with the development of adult Leydig cells. There now is compelling evidence that most, if not all, of the adult Leydig cells arise from stem cells, not from the transdifferentiation of fetal Leydig cells (8, 9). In previous studies, we and others isolated cells from neonatal testes that expressed platelet-derived growth factor receptor-α (PDGFα) or nestin (9–11). Depending on culture conditions, these cells were shown to be capable of proliferating indefinitely or of differentiating into T-producing Leydig cells and, thus, were identified as stem Leydig cells (9–11). During their differentiation, these cells proceeded through two intermediate stages, progenitor Leydig cells and immature Leydig cells, before becoming adult Leydig cells (12). The gene expression pattern by the stem Leydig cells was similar to that of bone marrow stem cells and quite different from the patterns of the cells in the Leydig cell lineage (13).The adult Leydig cells, once established, turn over slowly during adult life. However, when these cells are eliminated from adult testes by treating the rats with ethane dimethanesolfonate (EDS), new, fully functional adult Leydig cells reappear (14, 15). In initial efforts to localize the precursor cells, seminiferous tubules were isolated from EDS-treated testes and cultured with luteinizing hormone (LH) (16). Cells on the tubule surfaces first underwent division, and then differentiated and produced T (17). These results suggested that in addition to reported perivascular locations in the interstitial compartment (18), stem cells also were located on the surfaces of the seminiferous tubules (16, 19, 20).Studies of a number of tissues have shown that stem cell self-renewal and differentiation are regulated by the interactions between cues that are intrinsic to the cells and extracellular signaling from the local environment, the latter referred to as the niche. In many tissues, including the testis, anatomic complexity combined with the inability to specifically mark stem cells make it difficult to identify these cells, characterize the niche, or determine the extrinsic factors involved in stem cell functions. Thus, as yet the extent to which the testicular environment influences the ability of the stem cells to proliferate and/or differentiate remains unknown. In the present study, we used a unique in vitro system of cultured seminiferous tubule to identify stem Leydig cells on the surface of seminiferous tubules and to assess the ability of factors associated with the tubules to regulate their proliferation and entry into the Leydig cell lineage (i.e., their differentiation). We provide evidence that the proliferation and subsequent differentiation of the stem Leydig cells are regulated by multiple niche factors from the seminiferous tubules, including PDGF, basic fibroblast growth factor (FGF2), transforming growth factor β (TGF-β), activin, Notch, Wnt, and most importantly, Desert hedgehog (DHH). Additionally, we report on the isolation of the stem cells by flow cytometric sorting through a specific cell surface marker protein, CD90.     

Differences between dispersed Leydig cells and intact testes in their sensitivity to gonadotrophin-stimulation in vitro after alteration of LH-receptor numbers

The sensitivity to hCG-stimulation in vitro of intact hemi-testes and collagenase-dispersed Leydig cells has been compared directly following either an hCG-induced loss of LH-receptors or after a hyperprolactinaemia-induced increase in LH-receptors. Injection of hCG 65 h previously, reduced hCG-binding to dispersed Leydig cells by over 84%. The sensitivity of the steroidogenic response of these cells to hCG-stimulation in vitro was reduced 22-fold whereas intact testes from the same animals showed only a 3-fold reduction in sensitivity to hCG. Dispersed Leydig cells from control rats were 8 times more sensitive to hCG-stimulation than intact testes from the same rats, a difference not evident with hCG-injected rats. In contrast, there was no difference between intact testes and dispersed Leydig cells from control and hCG-injected rats in their sensitivity to stimulation with dibutyryl cyclic AMP in vitro. Induction of hyperprolactinaemia increased hCG-binding to dispersed Leydig cells by 55%. The sensitivity of these cells to hCG-stimulation in vitro was increased by a factor of 4.5, a difference not found with intact testes from the same animals. These results show that experimental manipulation of LH-receptor numbers alters the sensitivity of dispersed Leydig cells, but not of the intact testis, to hCG-stimulation in vitro, a difference which appears to reside at the receptor level. Possible explanations for these findings are discussed together with their implications with respect to the distribution of LH-receptors over the Leydig cell surface.     

Morphological and functional characteristics of rat leydig cells isolated on Percoll gradients: is Leydig cell heterogeneity in vitro an artifact?

Rat testicular intertubular cells have been isolated on Percoll density gradients. Detailed light and electron microscopic studies have determined the sedimentation positions for Leydig cells, macrophages, fibroblasts, endothelial cells, germ cells and residual bodies. Stereological techniques have been utilized to determine the number of cells in the region of the gradient where Leydig cells sediment. Morphologically intact Leydig cells were present in the more dense region of the gradient (1.0590-1.0900 g/ml), and they responded to hCG stimulation with an 11-fold increase in testosterone production and contained LH/hCG receptors. Leydig cells in the less dense region of the gradient (1.0440-1.0589 g/ml) secreted less testosterone and contained less hCG receptors than those obtained from denser regions. However, the morphological studies described herein provide evidence for the first time that the majority of these less functional 'Leydig cells' from the lighter region of the gradients do not contain a nucleus and represent pieces of Leydig cell cytoplasm with variable size, shape and complement of organelles.     

Acquisition of sensitivity to LH in relation to foetal development. Stimulation of cyclic AMP and testosterone production in the rat testis

The ability of LH to stimulate, in vitro, adenylate cylcase activity and testosterone secretion was studied in foetal rat testes after prelabelling with [14C]adenine. As little as 0.1 ng/ml LH produced significant synthesis of cyclic AMP and testosterone secretion. Increase of cyclic AMP production was observed as early as 1 min after addition of LH (100 ng/ml), preceding the rise in testosterone synthesis and secretion. FSH and prolactin were not effective. LH-stimulation of cyclic AMP and testosterone production appeared concomitantly in rat foetal testes on day 15 of gestation and reached a maximum on day 18. Immature testes (14 days) developed functional receptors when cultured in a medium devoid of hormone. The results of the present study suggest that cyclic AMP mediates the effect of LH on steroidogenesis in foetal testes and that the differentiation of functional receptors occurs at the same time as the capacity for testosterone synthesis.     

Steroidogenesis of cultured purified pig Leydig cells: secretion and effects of estrogens

Using a primary culture of purified immature pig Leydig cells we have demonstrated: (1) that during the first 3 days of culture there is a 'spontaneous' maturation of the steroidogenic response to hCG, as expressed by a 50-fold increase of the steroidogenic capacity, an increased secretion of both dehydroepiandrosterone sulfate (DHAS) and testosterone (T), a shift of the DHAS/T ratio (5 on day 0 vs. 0.5 on day 3) without significant changes in the number of hCG-binding sites; (2) that purified cells, on day 3 following hCG stimulation, secrete large amounts of T and small amounts of E2 (T/E2 congruent to 150) and detectable amounts of estrone, while crude pig interstitial cells under the same conditions secrete less T but 40-50 times more estrogens (T/estrogens congruent to 1.5); (3) that the steroidogenic responsiveness of purified Leydig cells is not impaired by E2 treatment, in spite of the fact that Leydig cells contain specific estradiol receptors (approximately 10000 sites/cell). These data suggest that in this model the main source of testicular estrogens are not Leydig cell but some other testicular cell types, and that the lack of effect of estrogens on pig Leydig cell steroidogenesis is not due to absence of estrogen receptors.     

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.     

Increase in catecholamine-stimulated cyclic AMP and progesterone synthesis in rat granulosa cells during culture

Catecholamines stimulated cyclic AMP and progesterone synthesis in rat corpora lutea but not in the pre-ovulatory follicles. We collected granulosa cells from follicles of immature rats treated with pregnant mare's serum gonadotropin (PMSG) and cultured them either in monolayer or in suspension. Freshly collected granulosa cells responded to isoproterenol and epinephrine with 2-fold increases in cyclic AMP accumulation and progesterone synthesis. However, granulosa cells cultured in monolayer for 2 days responded to isoproterenol and epinepherine with a 90-fold and a 6-fold increase in cyclic AMP accumulation and progesterone synthesis, respectively. The accumulation of cyclic AMP in response to Catecholamines gradually increased in cells cultured in suspension, from 2-fold over control after 5 h, to 8-fold after 24 h. Granulosa cells isolated from hypophysectomized and diethylstilbestrol (Hx-DES) treated immature rats (containing pre-antral follicles) also showed an increase in cyclic AMP accumulation in response to Catecholamines during culture. Because these cells are devoid of an LH-responsive adenylate cyclase system, we conclude that luteinization of granulosa cells in culture is not necessarily the process responsible for the increased response to Catecholamines.During culture, the number of β-adrenergic receptors in granulosa cells rose from 6020 ± 400 per 10⁶ cells shortly after isolation to 26400 ± 1800 after 2 days in culture. This increase in receptor density during culture may be responsible for the change in the responsiveness to Catecholamines, although other factors, such as changes in coupling efficiency between the hormone-receptor complex and the adenylate cyclase moiety and/or supersensitivity to Catecholamines, should also be considered.     

Regulation of aromatase activity of rat granulosa cells: induction of synthesis of NADPH-cytochrome P-450 reductase by FSH and dibutyryl cyclic AMP

Aromatase is an enzyme complex that is composed of a specific form of cytochrome P-450 and a flavoprotein, NADPH-cytochrome P-450 reductase. Aromatase activity of granulosa cells is increased markedly by follicle-stimulating hormone (FSH) and by analogs of cyclic AMP. It was the objective of the present study to investigate the effects of FSH and dibutyryl cyclic AMP (Bt₂cAMP) on the synthesis of NADPH-cytochrome P-450 reductase in rat granulosa cells maintained in vitro. Granulosa cells were obtained from the ovaries of diethylstilbestrol (DES)-treated immature rats and were incubated in the presence of DES (10⁻⁷ M), DES + FSH (250 ng/ml), or DES + Bt₂cAMP (1 mM) for up to 72 h. After 72 h of incubation, aromatase activity of cells incubated with DES alone was 5 pmoles estrogen formed 2 h⁻¹ mg⁻¹ protein and was increased > 60-fold in cells incubated with FSH or Bt₂cAMP. NADPH-cytochrome P-450 reductase was immunoisolated from [³⁵S]methionine-labeled lysates of granulosa cells incubated for 72 h in the absence or presence of stimulatory factors. The rate of synthesis of reductase was found to be increased about 3-fold in cells incubated with DES + FSH or DES + Bt₂cAMP as compared to cells incubated with DES alone. By immunoblot analysis we found that the cellular content of reductase was increased about 2-fold by FSH and Bt₂cAMP treatment. Reductase specific activity was 10 nmoles min⁻¹ mg⁻¹ protein in membrane fractions of DES-treated cells and was increased 1.6-fold by FSH treatment. These findings are indicative that FSH increases the rate of synthesis, cellular content and specific activity of NADPH-cytochrome P-450 reductase in rat granulosa cells in vitro. The finding that Bt₂cAMP causes a similar induction of reductase synthesis is suggestive that the stimulatory effect of FSH on this component of the aromatase enzyme complex is mediated by an increase in cyclic AMP formation.     

Inhibitory effects of TNFα on mouse tumor Leydig cells: possible role of ceramide in the mechanism of action

TNFα is reported to inhibit steroidogenesis in mouse Leydig cells. In primary cells this inhibition resulted mainly from a reduced expression of Cyp-17 gene. Mouse tumor Leydig cells, MA-10, being free of macrophages and lacking Cyp-17, appear to be an excellent model to investigate those effects of TNFα which are independent of either macrophages or Cyp-17. We report here that TNFα receptors are expressed in this cell line. Treatment of the cells with TNFα had no effect on basal progesterone production. In contrast, LH-, 8Br-cAMP and forskolin-stimulated progesterone production was inhibited by TNFα. Neither enzymes involved in the conversion of cholesterol to pregnenolone nor hormone-induced hydrolysis of [¹⁴C] cholesterol-ester were affected by TNFα. The hormone- induced expression of StAR protein was diminished in mitochondrial fractions from TNFα-treated cells. Also cell permeable ceramides markedly inhibited StAR protein levels. We show further that TNFα was able to induce [¹⁴C]-ceramide accumulation in MA-10 cells and suggest that this sphingolipid may be considered as a transmitter of TNFα signals to the StAR protein.     

The expression of the RLF/INSL3 gene is reduced in Leydig cells of the aging rat testis

The relaxin-like factor (RLF), which is the product of the INSL3 gene, is highly expressed in the fetal and adult-type Leydig cells of all species so far examined. In adult testes it is upregulated at puberty but appears subsequently to be expressed in a constitutive manner, independently of acute changes in the hypothalamic-pituitary-gonadal (HPG) axis. Functional hypogonadism with decreased testosterone is prevalent in the aging male. In order to test whether this is a property of the HPG axis, or of the Leydig cells themselves, RLF/INSL3 was used as an independent marker to assess rat Leydig cell differentiation status. Hybridization analysis showed that in the testes of old (2 years) rats, RLF/INSL3 mRNA expression was dramatically reduced, compared to young (3 months) animals. This was also evident at the protein level using immunohistochemistry. The results suggest that increasing functional hypogonadism in older male mammals is likely caused by a dedifferentiation of the Leydig cells themselves.