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Exposure to the pesticide methoxychlor in rodents is linked to impaired steroid production, ovarian atrophy and reduced fertility. Following in vivo administration, it is rapidly converted by the liver to 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), the reported active metabolite. Both methoxychlor and HPTE have weak estrogenic and antiandrogenic activities, and these effects are thought to be mediated through the estrogen and androgen receptors, respectively. Previous in vivo studies on methoxychlor exposure to female animals have demonstrated decreased progesterone production but no change in serum estrogen levels. We recently showed that HPTE specifically inhibits the P450 cholesterol side-chain cleavage (P450scc, CYP11A1) step resulting in decreased androgen production by cultured rat testicular Leydig cells. The current studies examined the mechanism of action of HPTE on progesterone production by cultured ovarian cells (granulosa and theca-interstitial) from pregnant mare serum gonadotropin-primed immature rats. In addition, we evaluated whether the effects of HPTE on rat ovarian cell progesterone biosynthesis were mediated through the estrogen or androgen receptors. Exposure to HPTE (0, 10, 50 or 100 nM) alone progressively inhibited progesterone formation in cultured theca-interstitial and granulosa cells and the P450scc catalytic activity in theca-interstitial cells in a dose-dependent manner with significant declines starting at 50 nM. However, HPTE did not change mRNA levels of the P450scc system (P450scc, adrenodoxin reductase and adrenodoxin) as well as P450scc protein levels. Of interest, estradiol, xenoestrogens (bisphenol-A or 4-tert-octylphenol), a pure antiestrogen (ICI 182,780), or antiandrogens (4-hydroxyflutamide or the vinclozolin metabolite M-2), had no effect on progesterone production even at 1000 nM. Co-treatment of HPTE with ICI 182,780 did not block the effect of HPTE on progesterone formation. These studies suggest that the decline in progesterone formation following exposure to HPTE in cultured ovarian cells is associated with the inhibition of catalytic activity of P450scc at least in theca-interstitial cells. This action does not appear to be mediated through the estrogen or androgen receptor signaling pathways, and other chemicals exhibiting estrogenic, antiestrogenic or antiandrogenic properties do not mimic its effect on ovarian steroid production.

The initial goal of occupational reproductive health research is to effectively study the many toxicants, physical agents, and biomechanical and psychosocial stressors that may constitute reproductive hazards in the workplace. Although the main objective of occupational reproductive researchers and clinicians is to prevent recognized adverse reproductive outcomes, research has expanded to include a broader spectrum of chronic health outcomes potentially affected by reproductive toxicants. To aid in achieving these goals, the National Institute for Occupational Safety and Health, along with its university, federal, industry, and labor colleagues, formed the National Occupational Research Agenda (NORA) in 1996. NORA resulted in 21 research teams, including the Reproductive Health Research Team (RHRT). In this report, we describe progress made in the last decade by the RHRT and by others in this field, including prioritizing reproductive toxicants for further study; facilitating collaboration among epidemiologists, biologists, and toxicologists; promoting quality exposure assessment in field studies and surveillance; and encouraging the design and conduct of priority occupational reproductive studies. We also describe new tools for screening reproductive toxicants and for analyzing mode of action. We recommend considering outcomes such as menopause and latent adverse effects for further study, as well as including exposures such as shift work and nanomaterials. We describe a broad domain of scholarship activities where a cohesive system of organized and aligned work activities integrates 10 years of team efforts and provides guidance for future research.

Methoxychlor (MC) was developed as a replacement for the banned pesticide DDT. After in vivo administration, it is metabolized in the liver to 2,2-bis( p -hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which is proposed to be the active agent. Both MC and HPTE have been shown to exhibit weak estrogenic and antiandrogenic activities, and they are thought to exert their effects through estrogen and androgen receptors, respectively. Although in vitro studies using cultured rat Leydig cells have reported that HPTE inhibits both basal and hCG-stimulated testosterone formation, the response of circulating testosterone levels to in vivo MC has been more variable. Therefore, the current studies evaluated whether the daily in vivo administration of MC (0, 5, 40 and 200 mg/kg body weight) for a short duration (days 54–60 of age) by gavage altered serum testosterone levels and ex vivo Leydig cell testosterone formation in young adult male rats. These results demonstrate that both fluid-retained and fluid-expressed seminal vesicle weights declined to 44 and 60% of control, respectively, in the 200 mg/kg MC-exposed animals. Similarly, serum testosterone and dehydroepiandrosterone levels declined to 41 and 45% of control, respectively, in the 200 mg/kg MC-exposed animals; however, serum LH and FSH levels were unaffected. Ex vivo Leydig cell basal testosterone formation over 4 h declined to 49% of control in animals exposed to 200 mg/kg MC, and ex vivo Leydig cell P450 cholesterol side-chain cleavage activity declined to 79 and 50% of control in animals exposed to 40 and 200 mg/kg of MC, respectively, supporting previous in vitro studies which demonstrated the sensitivity of this step to MC.

Methoxychlor (MC) is an insecticide that is presently used on agricultural crops, especially after the ban on the use of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT) in the United States. Following administration in vivo, MC is converted to 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which is thought to be the active agent. However, both MC and HPTE have been reported to have weak estrogenic and antiandrogenic activities, and they are thought to exert their potential adverse (endocrine disruptive) effects through the estrogen and androgen receptors, respectively. In a recent study, HPTE was shown to inhibit both basal and hCG-stimulated testosterone production by cultured Leydig cells from immature and adult rats, and these effects were reported to be mediated through the estrogen receptor. Because fetal Leydig cells represent a separate population from adult Leydig cells and many of the reported adverse actions of endocrine disruptors are thought to have their effects during gestational exposure, the present studies examined the effects of HPTE on testosterone formation by cultured fetal Leydig cells from neonatal rats to determine whether these cells are sensitive to HPTE. Our studies demonstrated that HPTE inhibited both basal and hCG-stimulated testosterone formation in a dose-dependent manner. Significant declines in testosterone were observed at about 100 nM HPTE, and this effect was detected as early as 1 h after exposure. The main effects of HPTE appeared to be localized to the cholesterol side-chain cleavage step which converts cholesterol to pregnenolone. In addition, this effect did not appear to be mediated through the estrogen receptor as a weak estrogen or the androgen receptor as an antiandrogen, which are the currently proposed modes of action of MC and HPTE.

Methoxychlor (MC) is an insecticide that is currently used on a variety of agricultural crops, especially following the ban of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT) use in the United States. Following in vivo administration, MC is converted to 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), which is proposed to be the active agent. Both MC and HPTE have been demonstrated to exhibit weak estrogenic and antiandrogenic activities, and they are thought to exert their effects through estrogen or androgen receptors, respectively. A recent study reported that HPTE inhibited both basal and hCG-stimulated testosterone formation by immature and adult cultured rat Leydig cells and that this effect was mediated through the estrogen receptor. In the current studies, we examined the effects of HPTE on basal and hCG-stimulated testosterone formation by cultured Leydig cells from young adult rats. In addition, we evaluated whether the effects of HPTE on rat Leydig cell testosterone biosynthesis were mediated through the estrogen receptor as an estrogen agonist or the androgen receptor as an antiandrogen. The current studies demonstrated that HPTE inhibited both basal and hCG-stimulated testosterone formation in a dose-dependent manner with significant declines in testosterone being observed at approximately 100 nM. The effects of HPTE were localized to the cholesterol side-chain cleavage step; however, these effects were not mediated through the classic estrogen receptor or by its acting as an antiandrogen, the currently recognized modes of action of MC and HPTE.

4-Tert-octylphenol (OP) is a breakdown product of 4-tert-octylphenol ethoxylate, which is a surfactant additive widely used in the manufacture of a variety of detergents and plastic products. OP has been reported to exhibit weak estrogenic activity in many assay systems. The studies described herein examined an unusual effect of OP in increasing constitutive testosterone levels of cultured Leydig cells from young adult rats. The increase in testosterone was both dose and time sensitive, and this response was observed in medium lacking both calcium and magnesium and containing a membrane-permeable calcium chelator, suggesting that the increase in testosterone was not mediated by an increase in the permeability of extracellular calcium into cells or the redistribution/release of calcium from intracellular stores, respectively. Cellular cAMP levels also were unaffected by OP alone in cultured Leydig cells. Furthermore, initial exposure to 2000nM OP alone for 4h did not alter the subsequent conversion of endogenous cholesterol or exogenously added 22 (R)hydroxycholesterol to testosterone, suggesting that the increase in testosterone was not due to the enhanced availability of endogenous cholesterol or an increase in cholesterol side-chain cleavage activity, respectively. The increase in testosterone also was observed in the presence of the pure estrogen antagonist, ICI 182,780, or a 5alpha-reductase inhibitor, suggesting that this effect of OP was not mediated through the estrogen receptor alpha or beta pathway or by inhibition of Leydig cell testosterone metabolism, respectively. In addition, exposure of cells to comparable concentrations of two different detergents, Triton X-100 or sodium cholate, did not increase testosterone levels, suggesting that this effect of OP was not due to its potential detergent qualities. Although these studies did not identify specific mechanism(s) that increase constitutive testosterone levels by OP, they identify specific pathways that appear not to be involved. The physiological relevance of this observation is not known; nevertheless, they illustrate potential diverse actions of OP in modulating the level of androgen secreted by Leydig cells, and they emphasize that some actions of OP do not appear to be mediated through the estrogen receptor alpha or beta pathway.

The present studies evaluated the suitability of using cultured dispersed testicular cells from neonatal rats as a source for fetal Leydig cells and the use of these cells to examine direct toxic effects of environmental/occupational chemicals on androgen biosynthesis. For the current studies, the direct actions of octylphenol (OP), a surfactant additive widely used in the manufacture of various detergents, on testosterone biosynthesis by cultured rat neonatal Leydig cells were examined. Octylphenol is considered a xenoestrogen and has been reported to mimic the actions of estrogen in many cellular systems. Following exposure of cultured cells for 24 h to varying concentrations of OP (1 to 2000 nM) together with 10 mlU/mL human chorionic gonadotropin (hCG), the lower concentrations of OP (1 and 10 nM) consistently enhanced testosterone levels (approximately 10 to 70% above control), whereas higher OP concentrations (100 to 2000 nM) progressively decreased testosterone from peak levels to approximately 40 to 80% below control at the highest OP concentration. Interestingly, increasing concentrations of 17beta-estradiol (1 to 1000 nM) were without effect on testosterone biosynthesis under the same conditions, and the biphasic pattern of testosterone biosynthesis elicited by increasing OP concentrations was unaffected by concomitant treatment with 10 or 100 nM ICI 182,780, which is considered a pure estrogen antagonist. Therefore, the actions of OP on testosterone biosynthesis by cultured neonatal Leydig cells do not appear to be mediated through the classic estrogen receptor alpha or beta pathway. Although the increase in testosterone levels after exposure to lower OP concentrations and to 0.1 and 1.0 mM 8-Br-cAMP was attenuated, suggesting that lower OP concentrations may alter cellular cAMP levels, because hCG-stimulated cAMP levels were unaffected by any of the OP concentrations evaluated, it appears that its main site(s) of action occurs after the generation of cAMP. In addition, because pretreatment of cells with increasing OP concentrations and hCG had no effect on the conversion of steroid precursors (22(R)-hydroxycholesterol, pregnenolone, progesterone, or androstenedione) to testosterone, it seems that the main actions of OP under the present conditions occur before the mitochondrial cholesterol side-chain cleavage step. Furthermore, because concomitant treatment of cells with various antioxidants (alpha-tocopherol, butylated hydroxyanisole, or ascorbic acid) did not alter the biphasic pattern of testosterone response to increasing concentrations of OP and hCG, it seems that OP is not acting as an anti- or pro-oxidant in producing these effects. It will be important to determine whether this dose-sensitive response to OP is observed in vivo, and whether the maturational status of Leydig cells influences their pattern of response to OP and similar chemicals.

The present studies examined the effects of heparin, heparinase, insulin or insulin-like growth factor-I on basic fibroblast growth factor actions on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity of cultured immature rat Leydig cells. Treatment with basic fibroblast growth factor alone (0.025-20 ng/ml) for 2 days had a biphasic effect on enzyme activity, with lower concentrations (0.025-1 ng/ml) progressively inhibiting activity to approximately 20% of control, while higher concentrations (2.5-20 ng/ml) partially reversed the inhibitive effects. The inclusion of 10 micrograms/ml heparin, a concentration reported to inhibit growth factor binding to heparan sulfate proteoglycans, blocked the increase in enzyme activity elicited by higher growth factor concentrations, but had no effect on the progressive decline in activity due to lower concentrations. Concomitant treatment with heparinase I and III, which specifically hydrolyze heparan sulfate proteoglycans, had a similar effect. In addition, both insulin and insulin-like growth factor-I partially reversed the inhibition of enzyme activity due to treatment with 1 ng/ml basic fibroblast growth factor. These studies suggest that some basic fibroblast growth factor actions on cultured immature Leydig cells are mediated by binding to heparan sulfate proteoglycans, and that both insulin and insulin-like growth factor-I can reverse the inhibitive effects on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase activity.

The present studies examined the effects of acidic fibroblast growth factor (aFGF) on 5-ene-3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD) and 5 alpha-reductase activities and [125I]human chorionic gonadotrophin ([125I]hCG) binding in cultured immature rat Leydig cells. Increasing concentrations of aFGF (0.1-20 ng/ml) progressively decreased basal 3 beta-HSD activity from 0.474 +/- 0.0335 to 0.093 +/- 0.0004 nmol progesterone/30 min/10(5) cells. This inhibition by aFGF (10 ng/ml) was partially reversed by 1 micrograms/ml insulin or 100 ng/ml insulin-like growth factor-I. Increasing aFGF concentrations (0.1-10 ng/ml) also inhibited hCG-stimulated 5 alpha-reductase activity in a dose-dependent manner, but had only a modest effect on basal enzyme activity. Increasing aFGF (0.1-200 ng/ml) also progressively inhibited [125I]hCG binding in cultured immature Leydig cells. These studies demonstrate a similarity in the inhibitive effects of aFGF with bFGF effects on 3 beta-HSD and 5 alpha-reductase activities and [125I]hCG binding to LH receptors, although, generally, higher aFGF concentrations were required to elicit maximal inhibitive effects. However, a FGF differed from the actions of bFGF on 3 beta-HSD activity and LH receptor levels in that a secondary increase with higher growth factor concentrations was not observed.

The methoxychlor metabolite, HPTE, was shown to inhibit P450-cholesterol side-chain cleavage (P450scc) activity resulting in decreased progesterone production by cultured ovarian follicular cells in previous studies. It is not known whether HPTE has any effect on progesterone formation by the corpus luteum.Exposure to 100 nM HPTE reduced progesterone production by luteal cells with progressive declines to22% of control at 500 nM HPTE. Similarly, HPTE progressively inhibited progesterone formation and P450scc catalytic activity of hCG- or 8 Br-cAMP-stimulated luteal cells. However, HPTE did not alter mRNA and protein levels of P450scc. Compounds acting as estrogen (17 β-estradiol, bisphenol-A or octylphenol), antiestrogen (ICI) or antiandrogen (monobutyl phthalate, flutamide or M-2) added alone to luteal cells did not mimic the action of HPTE on progesterone and P450scc activity. These results suggest that HPTE directly inhibits P450scc catalytic activity resulting in reduced progesterone formation, and this action was not mediated through estrogen or androgen receptors.

Previous studies have shown that basic fibroblast growth factor (bFGF) can modulate basal and luteinizing hormone/human chorionic gonadotropin (LH/hCG)-stimulated Leydig cell functions. It has not been ascertained whether these actions are due to direct or indirect effects on Leydig cells. To resolve this question, a multi-step procedure was used to isolate highly-purified Leydig cells from immature rats. 125I-bFGF binding studies were performed on cultured cells. Scatchard analysis of the data indicated a single binding site with an apparent Kd of 82 pM and a binding capacity of approximately 2800 sites per cell. Both bFGF and acidic FGF similarly were effective in displacing 125I-bFGF, suggesting that the receptor binds both bFGF and aFGF. However, neither hCG, follicle-stimulating hormone (FSH), insulin, insulin-like growth factor-1 (IGF-1), prolactin, platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) were effective competitors. When binding studies were conducted on cultured testicular interstitial cellular fractions that are normally discarded during Leydig cell purification, bFGF receptors were identified in these fractions. These results demonstrate that bFGF can have direct effects on Leydig cells through specific receptors; however, because other interstitial cell type(s) also have bFGF receptors, they stress the importance of using highly purified cells when evaluating bFGF actions on Leydig cells.

Basic fibroblast growth factor inhibited basal delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in cultured Leydig cells from immature rats in a concentration- and time-dependent manner. Maximal inhibition was achieved with 5-10 ng/ml basic fibroblast growth factor following approximately 48 h of exposure. The inhibition of basal delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity was not altered by human chorionic gonadotropin; however, cycloheximide (0.5-2.0 micrograms/ml) partially reversed the effects of basic fibroblast growth factor in a dose-dependent manner. These studies suggest that locally-produced basic fibroblast growth factor may modulate Leydig cell testosterone formation by regulating delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity.

The present studies examined the effects of hCG on steroidogenic enzyme activities involved in the metabolism of testosterone or dihydrostestosterone in cultured rat neonatal interstitial cells. 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities, which are involved in the conversion of testosterone to dihydrotestosterone and androstenedione, respectively, were low in cultured neonatal interstitial cells, were unresponsive to hCG and declined to undetectable levels during 14 days of culture. However, delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity, which is involved in the biosynthesis of testosterone, and 5 alpha-androstane-3 alpha-hydroxysteroid dehydrogenase and 5 alpha-androstane-3 beta-hydroxysteroid dehydrogenase activities, which are involved in the conversion of dihydrotestosterone to 5 alpha-androstan-3 alpha, 17 beta-diol and 5 alpha-androstan-3 beta, 17 beta-diol, respectively, were maintained or increased by hCG during the same culture period. These results demonstrate that 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities do not play a significant role in regulating testosterone accumulation in fetal/neonatal Leydig cells, and they suggest that these cells are adapted to maintain high testosterone but low dihydrotestosterone levels. The present results demonstrate also that fetal/neonatal Leydig cells differ from immature or adult Leydig cells with respect to the sensitivity of 5 alpha-reductase activity to LH/hCG.

In the current studies, we evaluated the effects of 4-tert-octylphenol (OP), endosulfan, bisphenol A (BPA), and 17beta-estradiol on basal or hCG-stimulated testosterone formation by cultured Leydig cells from young adult male rats. Exposure of Leydig cells to increasing concentrations of OP (1 to 2000 nM), 17beta-estradiol (1 to 1000 nM), endosulfan (1 to 1000 nM) or BPA (1 to 1000 nM), alone or with 10 mIU/mL hCG for 4 or 24 h, did not lower ambient testosterone levels, although cells exposed to higher OP concentrations + hCG for 24 h often had modest declines in testosterone (10 to 20%). Of interest, exposure to the highest concentration OP (2000 nM) alone for 4 or 24 h increased testosterone levels (approximately 2-fold in 4-h exposed cells). Whether prior exposure to OP + hCG for 24 h affects the subsequent conversion of steroid substrates to testosterone over 4 h was evaluated. Progressive declines in 1 microM 22(R) hydroxycholesterol, 1 microM pregnenolone, or 1 microM progesterone conversion to testosterone was observed beginning at 100 to 500 nM OP exposure (maximal declines of 40 to 12% of controls were observed); however, the conversion of 1 microM androstenedione to testosterone was not affected by OP. These results suggested that 24-h exposure to OP + hCG has no effect on 17beta-hydroxysteroid dehydrogenase, which converts androstenedione to testosterone, but that it inhibits the 17alpha-hydroxylase/C17-20 lyase step, which converts progesterone to androstenedione. In addition, potentially, OP could inhibit cholesterol side/chain cleavage activity, which converts cholesterol to pregnenolone, and/or 3beta-hydroxysteroid dehydrogenase, which converts pregnenolone to progesterone. Of interest, exposure to increasing concentrations of 17beta-estradiol (1 to 1000 nM), endosulfan (1 to 1000 nM), or BPA (1 to 1000 nM) + hCG for 24 h had no effect on subsequent conversion of 22(R)hydroxycholesterol to testosterone. Furthermore, the inhibiting effects of OP + hCG exposure on subsequent conversion of progesterone to testosterone was unaffected by concomitant exposure to the pure estrogen antagonist, ICI 182,780, or the antioxidants, ascorbate or dimethyl sulfoxide, suggesting that the actions of OP are not mediated through binding to estrogen receptor alpha or beta or by free radical induced damage to steroidogenic enzymes, respectively. These results demonstrate that direct exposure of adult Leydig cells to OP may have subtle effects on their ability to produce testosterone, which may not be detected by measuring ambient androgen levels. In addition, the effects of OP on Leydig cell testosterone formation appear to be different from those of the native estrogen, 17beta-estradiol, and from other reported weak xenoestrogens such as endosulfan and BPA.

4-tert-octyphenol (OP) is a surfactant additive widely used in the manufacture of a variety of detergents and plastic products. OP has been reported to mimic the actions of estrogen in many cellular systems. The present studies evaluated the direct effects of OP on human chorionic gonadotropin (hCG)-stimulated testosterone biosynthesis by cultured precursor and immature Leydig cells from 23-day old (prepubertal) rats. Exposure to increasing OP concentrations (1 to 2000 nM) progressively decreased hCG-stimulated testosterone formation in both precursor and immature Leydig cells at higher OP concentrations (100 or 500 to 2000 nM). Testosterone levels were reduced approximately 30 to 70% below control at the highest concentration in both cell types. Similar reductions in testosterone associated with OP exposure were observed in cells stimulated with 1 mM 8-Br-cAMP, suggesting that the main actions of OP occur after the generation of cAMP. Increasing concentrations of 17beta-estradiol (1 to 1000 nM) had no effect on hCG-stimulated testosterone formation in both precursor and immature Leydig cells and the inclusion of 100 nM ICI 182,780, a pure estrogen antagonist, in precursor and immature Leydig cells exposed to OP and hCG, did not alter the inhibition by higher OP concentrations of testosterone formation in both cell types. These results suggest that OP is a hormonally active agent, but that some of its actions are distinct from those of 17beta-estradiol and are not mediated through the estrogen receptor alpha or beta pathway. To further localize the potential site(s) of action of OP, cultured precursor and immature Leydig cells were exposed to increasing concentrations of OP and hCG for 24 h. Next, fresh media containing 1 microM 22(R)-hydroxycholesterol, 1 microM pregnenolone, 1 microM progesterone, or 1 microM androstenedione was added, and the conversion of each substrate to testosterone was determined after incubation for 4 h. The conversion of androstenedione to testosterone was unaffected by exposure to OP, suggesting that the 17beta-hydroxysteroid dehydrogenase step is not inhibited. However, the conversion of 22(R)-hydroxycholesterol, pregnenolone and progesterone all were inhibited by prior exposure to OP and hCG. This finding suggests that the 17alpha-hydroxylase/c17-20-lyase step, which converts progesterone to androstenedione, is inhibited by OP, and that the cholesterol side-chain cleavage and 3beta-hydroxysteroid dehydrogenase -isomerase steps, which convert cholesterol to pregnenolone and pregnenolone to progesterone, respectively, are other potential sites of OP action. Because concomitant exposure to the antioxidants alpha-tocopherol or ascorbate did not alter the inhibition of testosterone formation by higher OP concentrations, it does not appear that OP is acting as a pseudosubstrate for the generation of free radicals, which can damage P450 enzymes.

We reported previously that coculture of immature rat Sertoli cells with Leydig cells or the addition of a concentrate from Sertoli cell-conditioned medium (SCCM) stimulated Leydig cell [(3)H]-thymidine incorporation, increased cell number, and altered Leydig cell morphology (Wu and Murono, 1994). In the present studies, the effect of various extracellular matrix proteins on immature Leydig cell binding, proliferation and response to SCCM concentrate was investigated. Pretreatment of culture wells with 50 μg/mL collagen I or 10 μg/mL laminin inhibited Leydig cell binding to culture wells about 95 and 89%, respectively; however, 5 μg/mL fibronectin did not change the level of attachment. The binding of Leydig cells to fibronectin was reduced by antifibronectin or-β1 integrin antibodies (66 and 91%, respectively). Treatment of culture wells with five or 50 μg/mL fibronectin alone increased [(3)H]thymidine incorporation about twofold. When Leydig cells were cultured in wells precoated with increasing concentrations of fibronectin and then treated with SCCM concentrate for 2 d, [(3)H]-thymidine incorporation increased progressively with the concentration of fibronectin, beyond the levels observed with SCCM concentrate alone. This response was associated with increases in both Leydig cell number and labeling indices. When Leydig cells were cultured on fibronectin, their numbers increased by 3.7-and 5.1-fold following treatment with SCCM concentrates or coculture for 6 d, respectively; whereas, they increased 2.6- and 3.9-fold, respectively, when cultured on plastic. Labeling indices of Leydig cells cultured on plastic for 2 d and treated with SCCM or cocultured were 6.9 and 11.9%, respectively, while labeling indices of Leydig cells grown on fibronectin increased further to 17.6 and 26.3%, respectively. α5β1 integrin complexes and α5 integrin mRNA were expressed in Leydig cells, suggesting that binding to fibronectin may be mediated by α5β1 integrins, a fibronectin receptor. These results suggest that Leydig cell proliferation stimulated by a Sertoli cell-secreted mitogenic factor(s) is enhanced by Leydig cell binding fibronectin, and that this binding may be mediated by α5β1 integrins.

Previous studies have shown that disruption or damage to the seminiferous tubules by radiation, antiandrogen, vitamin A deficiency or experimental cryptorchidism causes Leydig cell hypertrophy and hyperplasia, suggesting that Sertoli cells secrete a mitogenic factor(s) that stimulates Leydig cell proliferation. To study the possible paracrine regulation of Leydig cell proliferation by Sertoli cells, highly purified Leydig cells and Sertoli cells were co-cultured in a two-chambered co-culture system. Our results revealed that co-culture of immature rat Sertoli cells with Leydig cells stimulated Leydig cell DNA synthesis by 19-fold, increased cell number by about 3.9-fold and increased the labeling index from 0.5% to 15.8%. In addition to these changes, co-culture reduced Leydig cell testosterone formation and luteinizing hormone (LH) receptor levels, and dramatically altered the morphology of Leydig cells. The addition of concentrates from Sertoli cell conditioned medium (SCCM) mimicked these biological effects. The Leydig cell mitogenic activity in SCCM was trypsin sensitive and inactivated by boiling for 2 h, suggesting that it is a protein. However, it was resistant to acid and dithiothreitol. The molecular weight of this putative factor(s) is above 10 kDa. The responsiveness of Leydig cells to this mitogenic protein(s) decreased with age, whereas the secretion of this protein(s) by Sertoli cells in culture did not change with age. The addition of 10 ng/ml of follicle stimulating hormone (FSH) dramatically decreased the mitogenic activity in SCCM, indicating that the secretion of this mitogenic factor(s) is inhibited by FSH. This paracrine factor(s) may be as yet an unidentified testicular growth factor(s) because it differs in molecular weight, stability and other characteristics from all previously reported Sertoli cell-produced or expressed growth factors.

The present studies examined how 125I-labeled basic fibroblast growth factor (bFGF) bound to high affinity receptors and with lower affinity to heparan sulfate proteoglycans (HSPG) of cultured immature rat Leydig cells was processed. Following incubation for 2 h at 4°C with 125I-bFGF, cells were washed to remove unbound radioactivity. Fresh medium was added, and cells were incubated at 4° and/or 37°C. At time zero and at specific intervals over the next 6 h, the incubation medium was saved and cells washed to quantitate 125I-bFGF released into the medium, associated with HSPG of the cell surface or extracellular matrix (radioactivity released by washing cells with 2 M NaCl, pH 7.4), associated with cell surface receptors (radioactivity released by washing cells with 2 M NaCl, pH 4.0) or internalized (radioactivity resistant to high salt and acid washes, and solubilized with 0.5 M NaOH). Radioactivity released into the initial medium and the pooled washes was further divided into a trichloroacetic acid (TCA)-precipitated form (radioactivity precipitated by 10% TCA) and a TCA-soluble form (radioactivity remaining in the TCA supernatant). 125I-bFGF associated with both HSPG and surface receptors declined progressively during the first 4 h of incubation before stabilizing when cells were transferred to 37°C. These declines were associated with a corresponding increase in intracellular 125I-bFGF. These changes were blocked by maintaining cells at 4°C. The majority of internalized 125I-bFGF appeared to originate from the HSPG-bound fraction as there was a greater decline in HSPG-associated radioactivity and most of the increase in internalized radioactivity could be blocked by the inclusion of 10 μg/ml heparin (which mainly blocks 125I-bFGF binding to HSPG but not to high affinity receptors) during the initial incubation with 125I-bFGF for 2 h at 4°C. Furthermore, HSPG-mediated internalization appeared to have two components: the major fraction was blocked by the inclusion of 10 μg/ml heparin, while a heparin-resistant fraction, appeared to be closely linked both quantitatively and temporarily to receptor-mediated internalization. A minor fraction of internalized 125I-bFGF was metabolized in lysosomes, as the inclusion of 50 μM chloroquine during the 6 h incubation at 37°C inhibited most of the increase in TCA-soluble radioactivity appearing in the incubation medium. Exposure of cultured cells to increasing concentrations of bFGF (0.1–10 ng/ml) for ~ 16 h decreased bFGF receptor levels in a dose-dependent manner, suggesting that internalization, in part, is associated with down-regulation of bFGF receptors.

Previous studies have shown that basic fibroblast growth (bFGF) has a biphasic effect on 125I-hCG binding to LH receptors in cultured Leydig cells from immature rats. Low concentrations of bFGF (0.1-1.0 ng/ml) progressively decreased binding, while higher concentrations (10-100 ng/ml) progressively increased binding above nadir levels. In the present studies, treatment of cultured immature Leydig cells with heparinase I and/or heparinase III, which enzymatically remove heparan sulfate proteoglycans, had no effect on basal binding of 125I-hCG to LH receptors or the decrease in binding due to treatment with low bFGF concentrations; however, this treatment dramatically reduced the secondary increase in binding following the addition of higher bFGF concentrations. These results strongly support the idea that the secondary increase in 125I-hCG binding to LH receptors elicited by treatment with higher bFGF concentrations is mediated by bFGF binding to heparan sulfate proteoglycans associated with the plasma membrane and/or extracellular matrix.

The present studies examined the effects of basic fibroblast growth factor (bFGF or FGF-2) on 125I-human chorionic gonadotropin (hCG) binding to cultured immature rat Leydig cells. We found that low concentrations of bFGF (0.1-1.0 ng/ml) inhibited 125I-hCG binding to cultured immature Leydig cells in a dose- and time-dependent manner; however, this inhibition was reversed partially at higher bFGF concentrations (10-200 ng/ml). The decline in 125I-hCG binding by bFGF was due to a reduction in the number of binding sites per cell and not to a change in receptor affinity for the ligand. The inclusion of 10 micrograms/ml heparin (a concentration that is reported to block bFGF binding to heparan sulfate proteoglycans) with increasing bFGF concentrations had no effect on the inhibition of 125I-hCG binding by low bFGF concentrations, but completely blocked the secondary increase in binding by higher bFGF concentrations. In addition, neither varying heparin concentrations (0.1-25 micrograms/ml) nor insulin or insulin-like growth factor-I had any effect on the inhibition of 125I-hCG binding by 1 ng/ml bFGF. These studies suggest that receptor-mediated actions of bFGF (inhibition of hCG binding by low bFGF concentrations) on cultured immature Leydig cells are unaffected by heparin; however, the secondary increase in 125I-hCG binding observed with higher bFGF concentrations (mediated by bFGF binding to heparan sulfate proteoglycans) is blocked by heparin.

The present studies examined the responsiveness to human chorionic gonadotropin (hCG) or 8-bromoadenosine 3',5'-monophosphate (8-Br-cAMP) of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity of cultured immature Band 2 (low density) or Band 3 (high density) Leydig cells isolated on Percoll gradients. Enzyme activity increased in relation to the dose of hCG or 8-Br-cAMP in both bands; however, activity in Band 2 cells increased about 200% above control, while activity in Band 3 cells increased only about 30-60% above control following 6 days of treatment. Maximal responses were observed 4-6 days following exposure to hCG or 8-Br-cAMP in both bands. Because elevated 5 alpha-reductase activity prevents testosterone accumulation in immature Leydig cells, Band 2 or Band 3 cells were cultured in the presence of 4-methyl-4-aza-3-oxo-pregnan-(20S)-carboxylate, a 5 alpha-reductase inhibitor, to assess the relationship between changes in delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity and testosterone formation. Although hCG or 8-Br-cAMP-stimulated delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity began to decline by day 8 or 10 of treatment, testosterone levels progressively increased for 10 days before declining in both bands. Thus, changes in enzyme activity did not strictly correlate with testosterone synthesizing capacity of cultured Leydig cells. Enzyme activity also was measured in cells cultured with the 5 alpha-reductase inhibitor to determine whether the responsiveness of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity of Band 2 or Band 3 cells was due, in part, to low testosterone levels. delta 5-3 beta-Hydroxysteroid dehydrogenase-isomerase activity in Band 2 or Band 3 cells cultured without or with the inhibitor was similar, suggesting that testosterone does not inhibit the enzyme in immature Leydig cells. The greater responsiveness of delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in Band 2 cells was not due to Leydig cell replication as neither total DNA or [3H]thymidine incorporation into DNA was altered by hCG or cAMP.

The present studies examined responses to hCG and/or insulin of 3 beta-hydroxy-5-ene-steroid dehydrogenase and steroid 5----4-ene-isomerase activity (3 beta-HSD) in cultured Band 2 and Band 3 cells from 25- to 40-day-old rats isolated on Percoll gradients. In Band 2 cells, from 25-day-old rats enzyme activity increased about 3- and 2.5-fold, after 6 days of exposure to hCG or insulin, respectively. However, hCG did not stimulate enzyme activity in Band 2 cells from 30-, 35- and 40-day-old animals, and responses to insulin alone or insulin plus hCG declined with age. In Band 3 cells only insulin increased enzyme activity at each age. Neither hCG or insulin altered DNA levels in Band 2 or Band 3 cells, suggesting that increased activity in Band 2 cells from 25-day-old rats was not due to cellular replication. However, hCG increased the number of cells staining positive for 3 beta-HSD about 4-fold in Band 2 cells from 25-day-old rats. Insulin did not increase the number of positive staining cells in Band 2 and Band 3 cells from 25-day-old rats, suggesting that its major effect was to increase enzyme activity in existing cells. These results suggest that during a limited period of maturation precursor cells in Band 2, which are undetected by histochemical staining for 3 beta-HSD, can be converted to Leydig cells in culture by hCG.

Platelet derived growth factor inhibited both hCG- and 8-Br-cAMP-stimulated 5 alpha-reductase activity in cultured immature Leydig cells in a dose-dependent manner, while not significantly inhibiting basal enzyme activity. Platelet derived growth factor also inhibited basal delta 5-3 beta-hydroxysteroid dehydrogenase activity and hCG-stimulated testosterone formation. Maximal inhibitions were achieved with 10 ng/ml of platelet derived growth factor. These studies suggest that platelet derived growth factor should be included among the variety of locally produced regulatory factors which modulate Leydig cell function.

The present studies examined the effects of basic fibroblast growth factor (bFGF) on 5 alpha-reductase activity of cultured Leydig cells from immature rats. Basic FGF inhibited both hCG- and 8-bromo-cyclic AMP-stimulated 5 alpha-reductase activity in a dose-dependent manner; however, it had little or no effect on basal enzyme activity. Inhibition was achieved with as little as 0.1 ng/ml bFGF, and maximal inhibition was observed with 10 ng/ml bFGF. These studies suggest that locally produced bFGF may play a role in modulating the age-dependent decline in 5 alpha-reductase activity in Leydig cells.

The present results demonstrate for the first time in rat liver, that low ethanol concentrations (2.2 and 22 mM) directly stimulate dihydrotestosterone conversion to 5α-androstan-3α, 17β-diol and 5α-androstan-3β, 17β-diol. Because this effect was blocked by 4-methylpyrazole, an alcohol dehydrogenase inhibitor, or by the addition of a saturating NADH concentration, this action probably is mediated by hepatic alcohol dehydrogenase activity through elevation of the NADH/NAD + ratio. It remains to be determined whether this effect of ethanol actually reduces circulating and/or target tissue dihydrotestosterone levels; nevertheless, it is tempting to speculate that this action, in part, is responsible for the reported adverse effects of alcohol on male reproductive functions.

Gossypol, a compound isolated from cottonseed, has been used in the People’s Republic of China as an antifertility agent in men. It was reported that gossypol is highly effective in reducing sperm count. Our study suggests that gossypol inhibits luteinizing hormone, 8-bromo-adenosine 3′,5′-monophosphate-induced testosterone formation, and the conversion of pregnenolone to testosterone in isolated rat interstitial cells. Serum testosterone levels were also significantly reduced in rats after 1 week of treatment with gossypol. Oligospermia and azoospermia in men caused by gossypol may be secondary to decreased testosterone biosynthesis.

The present study examined the effects of cytochalasin B on various steps in the luteinizing hormone (LH)-stimulated increase in testosterone synthesis by collagenase-dispersed interstitial cells of adult rat testis. Cytochalasin B at a concentration range of 0.1–50 μM inhibited the LH-stimulated increase in testosterone synthesis in a dose-dependent manner. Both intracellular and medium (released) testosterone levels were reduced, thus indicating that the decrease was not due to the accumulation of testosterone inside the cell as a result of cytochalasin B treatment. Cytochalasin B also inhibited the 8-bromocyclic AMP and pregnenolone-stimulated testosterone synthesis in a similar dose-dependent manner. Cytochalasin B at the two higher doses (10 and 50 μM) also inhibited the LH-stimulated generation of cyclic AMP by interstitial cells. However, this drug had no effect on basal testosterone synthesis except at the highest concentration added. Previous studies on adrenocorticotropic hormone (ACTH)- and LH-stimulated increase in glucocorticoid and testosterone synthesis in adrenal and Leydig cells, respectively, demonstrated that cytochalasin B or anti-actin inhibited the transport of cholesterol into mitochondria. The present studies suggest that cytochalasin B inhibits at least two additional steps in the LH-stimulated increase in testosterone synthesis: (1) the generation of cyclic AMP at the level of the plasma membrane, and (2) the conversion of pregnenolone to the testosterone at the level of the smooth endoplasmic reticulum. It remains to be established whether these are direct effects of cytochalasin B, or whether they are mediated by disruption of microfilaments by cytochalasin B.

Plasma estradiol and cytosolic estradiol receptor levels of testes were determined in a group of young (2-3 months) and old (24 months) Sprague-Dawley rats. Estradiol binding sites for the young rats averaged 5.6 +/- 0.3 fmol/mg protein (x +/- SE, n=12), which was comparable to that of the old rats, 5.7 +/- 0.3 fmol/mg protein (n=12). Using Scatchard analyses, the association constants at equilibrium of estradiol receptor binding of the old and young rats were the same, 6.1 x 10 10 M-1. Plasma estradiol levels were also similar in both groups-19.6 +/- 2.8 pg/ml (n=14) for the young and 19.2 +/- 2.6 pg/ml (n=10) for the old rats. Our results suggest that impaired testosterone biosynthesis in old rats was not due to elevated plasma estradiol levels or to differences in testicular estradiol receptor content.

The optimal concentrations of molybdate (Mo) to prevent thermal inactivation of estrogen receptor (ER) were found to be 5-20 mM. Using metrizamide density centrifugation, interstitial cells of adult Sprague-Dawley rat testes (2-3 months of age) can be separated into 5 distinct bands. Band 2 (B2) and 3 (B3) cells represented functionally different Leydig cells. For measurement of cytosolic ER, purified Leydig cells (B2 and B3 cells) were homogenized in 10 mM Tris-EDTA buffer with 5 mM of Mo (pH 7.4) and centrifuged at 105,000xg for 60 min. Cytosols were used immediately for ER determination. ER of B2 cells was 24.0 +/- 3.1 fmol/mg protein (mean +/- SE, n = 7), which was significantly higher than that of B3 cells, 18.6 +/- 3.2 fmol/mg protein (n = 7), p less than 0.005. The association constants of these two populations of Leydig cells for E2 receptor were comparable, 5.1 X 10(10)M-1 for B2 cells and 4.2 x 10(10)M-1 for B3 cells. In response to hCG 100 IU s.c., ER of B2 cells was reduced to 2.0 +/- 0.38 fmol/mg protein (n = 5), which was 8.4% of the control B2 cells. ER of B3 cells was almost completely depleted by the same dose of hCG, to 0.70 +/- 0.38 fmol/mg protein, 3.7% of the control B3 cells.B2 cells contained higher amounts of ER under basal conditions than B3 cells, and hCG-induced desensitization caused depletion of ER of both B2 and B3 cells.

The effect of verapamil on interstitial cell steroidogenesis was studied. Collagenase-dispersed interstitial cells were incubated with varying amounts of LH, 8-bromo-cyclic AMP, or verapamil. Cyclic AMP and testosterone were determined in media after 4-hour incubations. Verapamil in the doses of 10-4 and 10-5M inhibited LH-induced testosterone biosynthesis, while 10-6M verapamil had no effect. The inhibition persisted even though LH was increased 100 fold. Verapamil (10-5M) also significantly reduced 8-bromo-cyclic AMP and 1-methyl-3-isobutylxanthine-stimulated testosterone formation. Cyclic AMP responses to LH stimulation were enhanced by low doses (10-6 and 10-5M), but were inhibited by a higher dose (10-4M) of verapamil. This study suggests that verapamil blocks LH- and 8-bromo-cyclic AMP-induced testosterone formation. In high doses, verapamil also decreases cyclic AMP response to LH stimulation. The main effect of verapamil appears to be beyond cyclic AMP formation.

The effects of aging on cyclic AMP (cAMP) dependent protein kinase activity of purified Leydig cells were evaluated. Purified Leydig cells from 20-day-, 40-day-, 60-day- and 30-month-old rats were incubated with or without hCG for 1 hour. At the end of incubation, tubes were centrifuged and supernatants were saved for cAMP and testosterone determinations, and the pellets were prepared for protein kinase assay. Basal cAMP levels of Leydig cells from 20-day- and 40-day-old rats were lower than those of 60-day- and 30-month-old rats. In response to hCG stimulation, Leydig cells of 40-day-old rats produced the highest amounts of cAMP, while 30-month-old rats had the lowest levels. Testosterone responses of 30-month-old rats were also significantly lower than those of 60-day-old mature rats. When cAMP-dependent protein kinase activities were measured, 30-month-old rats had the lowest total kinase activity. These results suggest that reduced protein kinase activity might contribute to the decreased testosterone synthesis of aged rats.

The present studies were performed to evaluate the role of luteinizing hormone (LH) and calcium on cyclic guanosine 3',5'-monophosphate (cyclic GMP) formation in rat testicular interstitial cells. Dose related increases were observed in both intra-and extra-cellular cyclic GMP when various concentrations of LH (1, 10, 100 mlU/ml) were added to the purified Leydig cells. Cyclic GMP was also increased significantly with calcium ionophore A23187 at concentrations of 0.1, 1 and 10 microgram/ml. Adding LH concomitantly with A23187 resulted in a synergistic response and greatly enhanced cyclic GMP formation. When interstitial cells were incubated in calcium-free medium with 1 mM EGTA, the effects of LH and 123187 on cyclic GMP formation were abolished. These data suggest that LH and A23187 can increase cyclic GMP formation in interstitial cells and this response requires extracellular calcium.

The present in vitro studies using interstitial cells of adult rat testes demonstrated that ethanol inhibits LH- and 8-bromo-cyclic AMP-stimulated testosterone synthesis, pregnenolone- and progesterone-stimulated testosterone synthesis, and basal testosterone synthesis. However, the patterns of inhibition following exposure to 0.22 to 880 or 1100 mM ethanol were different. In general, the inhibition curves for LH-, 8-bromo-cycllc AMP-, pregnenolone- and progesterone-stimulated testosterone synthesis were biphasic, with a gradual slope from 0.22 to 220 mM ethanol, and a sharper slope with concentrations of ethanol greater than 220 mM. Basal testosterone synthesis was reduced only to 74% of control with ethanol concentrations up to 44 mM, and higher concentrations of ethanol reduced testosterone synthesis no further. The effect of ethanol on LH-stimulated cyclic AMP accumulation showed an even different pattern: some of the lower concentrations of ethanol inhibited cyclic AMP accumulation, while higher levels of ethanol progressively increased cyclic AMP accumulation. These studies demonstrate that isolated interstitial cells are highly sensitive to the direct effects of ethanol; they also suggest that the principle site of ethanol inhibition may be at the level of the smooth endoplasmic reticulum where progesterone is converted to testosterone.

Basal circulating levels of progesterone, 17α-OH-progesterone, dehydroepiandrosterone, androstenedione, testosterone and oestradiol-17β were measured in 'young' (median age 34 years) and 'old' (median age 73 years) normal males between 08.30 and 17.00 h. Diurnal rhythms for 17α-OH-progesterone, dehydroepiandrosterone, androstenedione and testosterone were observed in both young and old men. No rhythms were discernable for progesterone or oestradiol-17β in both age groups.

The present study was performed to evaluate the effects of calcium ionophore A23187 on adenosine 3',5'-monophosphate (cyclic AMP) and testosterone production in rat interstitial cells. Interstitial cells were incubated in Krebs-Ringer solution with varying amounts of luteinizing hormone, pregnenolone, or A23187. Cyclic AMP and testosterone were measured in the incubation medium after 4 h incubation. A23187 (0.01--10 microgram/ml) caused progressive increases of cyclic AMP formation (from 0.18 +/- 0.02 (S.E.) pmol/10(6) cells for the control of 0.42 +/- 0.02 pmol/10(6) cells, P less than 0.025), while testosterone production remained unaltered. When varying amounts of A23187 were added concomitantly with luteinizing hormone (5 IU/l), A23187 inhibited luteinizing hormone-induced steroidogenesis in a dose-dependent manner, but it had no effect on luteinizing hormone-induced cyclic AMP formation. When pregnenolone (10(-6) M) was added to the cells, testosterone formation increased from 1.50 +/- 0.22 to 8.46 +/- 1.65 ng/10(6) cells. A23187 (1 microgram/ml) had no discernable effect on the conversion of pregnenolone to testosterone. The main effect of increased cytosol calcium on steroidogenesis seems to be at the steps beyond adenylate cyclase-cyclic AMP. These results suggest that calcium is important for the conversion of cholesterol to pregnenolone, while the steps beyond pregnenolone are relatively independent of Ca2+.

The present study examined changes in steroidogenic enzyme activities which metabolize testosterone or dihydrotestosterone between days 21-73 of maturation in Band 2 and Band 3 cells isolated by centrifugation of rat testicular interstitial cells on metrizamide density gradients. 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities increased progressively in Band 2 and Band 3 cells between days 21-35 of maturation, then both enzyme activities declined to reach low levels in adult Band 2 and Band 3 cells. The significantly higher activities of both enzymes in Band 3, which contains a greater concentration of Leydig cells at each age, suggest their localization in Leydig cells. 5 alpha-androstane-3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities increased in both Band 2 and Band 3 cells between days 21-50 of maturation and remained elevated; however, dihydrotestosterone was metabolized primarily to 5 alpha-androstane-3 beta,17 beta-diol in Band 2 cells, while 5 alpha-androstane-3 beta,17 beta-diol was the major metabolite of dihydrotestosterone in Band 3 cells. These studies suggest that testosterone accumulation during sexual maturation can be influenced by changing patterns of 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase activities which metabolize testosterone, and of 5 alpha-androstane-3 alpha- and 3 beta-hydroxysteroid dehydrogenase activities which metabolize dihydrotestosterone in both Band 2 and Band 3 cells.

The direct effect of ethanol on dihydrotestosterone (DHT) conversion to 5 alpha-androstan-3 beta,17 beta-diol (3 beta-diol) and 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol) by adult rat Leydig cells was examined. Concentrations of ethanol comparable to blood levels of alcoholic men (2.2 - 65 mM) increased DHT conversion to 3 beta - and 3 alpha-diol, in direct relation to the dose of ethanol added; a 2-fold or greater stimulation was observed. Because this effect was blocked by 4-methylpyrazole or a saturating NADH concentration, these results suggest that this action is mediated by Leydig cell alcohol dehydrogenase activity. These results may have significant impact in the testis and/or other DHT sensitive tissues because ethanol may decrease the availability of the proposed active androgen.

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 17β-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 (5×10−7M), testosterone levels in the incubation medium were 27.0 ± 1.9 ng 10 6 cells . Tamoxifen (10−5M) 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 indicate that several steps of steroidogenesis are inhibited by tamoxifen.

Hypophysectomized immature rats were treated with LH or FSH to assess the role of these hormones on the activity of testicular steroidogenic enzymes during sexual maturation. Rats were hypophysectomized on Day 21 and hormone treatment was initiated on Day 27 and continued for 5 or 10 days. A 10,000 X g supernatant of testicular homogenates was assayed for 17j3-hydroxysteroid dehydrogenase, � -3a-hydroxysteroid dehydrogenase-isomerase, 5�s-reductase and 5 n-androstane-3a+3�-hydroxysteroid dehydrogenase activities. All enzyme activities decreased after hypophysectomy. Treatment with LH for 10 days stimulated 5n-reductase by 6-fold, �‘-3�-hydroxysteroid dehydrogenase by 4-fold, 5�s-androstane-3o+3j3-hydroxysteroid dehydrogenase by 2-fold and 17�1-hydroxysteroid dehydrogenase by only 50%. In constrast, treatment with FSH for 10 days stimulated 17P-hydroxysteroid dehydrogenase more than 2-fold. Although FSH treatment for 5 days significantly increased �-3#{216}-hydroxysteroid dehydrogenase-isomerase activity (56%) and 5n-androstane-3n+3f3-hydroxysteroid dehydrogenase activity (30%), these increases were not maintained for the 10 day treatment period. FSH treatment had neither a maintenance nor a stimulatory effect on Scs-reductase activity. The demonstration that FSH treatment increased 1 713-hydroxysteroid dehydrogenase activity is consistent with our previous hypothesis (Payne et al., 1977) that the increase in 17f3-hydroxysteroid dehydrogenase activity during sexual maturation may be regulated by FSH and that this may be one mechanism by which FSH increases testicular responsiveness to LI-I.

Acute ethanol exposure has been demonstrated to inhibit testosterone synthesis both in vivo and in vitro ; however, the precise step(s) affected is controversial. Using intact collagenase-dispersed interstitial cells or 10,000xg supernatants of interstitial cell homogenates, studies were undertaken to determine whether ethanol specifically inhibited Δ5-3β-hydroxysteroid dehydrogenase-isomerase activity. In both cellular preparations, varing concentrations of ethanol (2.2 – 652 mM) inhibited this enzyme activity. Because alcohol dehydrogenase activity was identified specifically in Leydig cells and because the inhibition of Δ5-3β-hydroxysteroid dehydrogenase-isomerase activity by concentrations of ethanol normally observed in circulation of alcoholic men (2.2 – 65 mM) could be reversed by saturating concentrations of NAD+ (0.2 mM) or by 4-methylpyrazole (2 mM), these results suggest that the mechanism of this inhibition is by limitation of available cofactor.

LH is recognized as the Leydig cell stimulus for testosterone production. Yet earlier studies of bolus administered human pituitary LH did not result in testosterone elevations and spontaneous LH elevations infrequently cause recognizable parallel rises of testosterone in adult males. Therefore, 10 normal adult volunteers were given 3 h infusions of human pituitary LH A-3 (2 IU/kg) and sampling was continued for 3 h after the gonadotrophin was stopped. Testosterone, the precursor 17 OH-progesterone and LH were determined in blood samples. There was a mean 5.2-fold LH elevation. From a group baseline mean of 698 ng/100 ml ± 72 (± se) testosterone gradually rose and was significantly elevated at 90, 120 and 150 min (787 ± 67 P P < 0.04, and 804 ± 69 P < 0.001, respectively). 17 OH-progesterone was determined on 9 subjects and averaged 142 ng/100 ml ± 16 before LH and gradually rose to 178 ± 18 at 120 min (P < 0.05) and then declined slightly at 150 and 180 min. Both steroids significantly declined between 180 min (end of LH infusion) and 240 min with no further significant concentration changes. The two men with the highest basal testosterone titers showed the smallest testosterone increases, but in both 17 OH-progesterone rose. In conclusion tonic LH stimulation caused mean testosterone and 17 OH-progesterone to peak at 150 min and at 120 min, respectively. Factors within the Leydig cell appear to limit increases of testosterone and 17 OH-progesterone during the first few hours of enchanced gonadotrophin stimulation.

Using metrizamide gradient centrifugation two populations of Leydig cells were found in both 60–90 day-old and 24 month-old rats. Cells from both Band 2 (B2) and Band 3 (B3) responded to LH stimulation with increased cyclic AMP formation; however, only B3 cells produced significant amounts of testosterone. Cells from both B2 and B3 of the old rats synthesized less cyclic AMP and testosterone than cells from their younger counterparts. In response to LH stimulation, 0.01–1.0 mIU/ml, no appreciable difference of cyclic AMP formation could be detected between young and old Leydig cells. Maximal testosterone production occurred when 1 mIU/ml LH was used. Only when LH concentration was increased to 10 and 100 mIU/ml, did young Leydig cells produce significantly more cyclic AMP than old Leydig cells. After addition of 5 × 10−7M of pregnenolone or progesterone to the incubation medium, both young and old Leydig cells produced comparable amounts of testosterone. These results demonstrate no impairment of old rat Leydig cells to synthesize testosterone from pregnenolone and progesterone.

The effects of tamoxifen on rat testicular steroidogenesis were studied using dispersed interstitial cells. Tamoxifen significantly inhibited LH-, and 8-bromo-adenosine 3′,5′-monophosphate (8-bromo-cyclic AMP)-stimulated testosterone synthesis in a dose-dependent manner. Tamoxifen (10 −5 M ) also reduced LH-stimulated cyclic AMP formation. The addition of equimolar concentrations of 17β-estradiol or tamoxifen separately to interstitial cells resulted in similar inhibition of LH-stimulated testosterone synthesis. When equimolar concentrations of 17β-estradiol and tamoxifen were added concomitantly to interstitial cells, the inhibition was additive. Present studies demonstrate that tamoxifen has direct inhibitory effects on testicular steroidogenesis: both at the plasma membrane resulting in decreased cyclic AMP formation and also at steps subsequent to cyclic AMP.

Plasma testosterone levels before and after a single injection of hCG were significantly lower in 24-month old rats than 60--90 day old animals (p less than 0.001). Even with repeated hCG administration for three weeks, plasma testosterone levels of old rats could not be restored to levels present in unstimulated young rats. In response to in vitro LH and 8-bromo-cyclic AMP stimulation, purified young Leydig cells produced significantly higher amounts of testosterone than Leydig cells from old rats. Maximal testosterone formation of the young Leydig cells in response to LH was 42.0 +/- 6.88 ng/10(6) cells, while cells from old rats produced only 16.8 +/- 3.69 ng/10(6) cells (p less than 0.01). However, the dose of LH at which one half maximal response (ED50) occurred was 0.1 mIU/ml for young Leydig cells and 0.05 mIU/ml for old Leydig cells. Basal and 1.0 mIU LH-stimulated cyclic AMP formation were comparable in both groups, but cyclic AMP formation in response to 10 mIU of LH was significantly less in the old rats (p less than 0.05). Present results demonstrate impaired steroidogenic capacity of old rats both in vivo and in vitro. Decreased testosterone response in old rats most likely is the consequence of understimulation of Leydig cells by gonadotropin; however, there appear to be additional intrinsic defects in old Leydig cells.

We have examined hormonal regulation of ornithine decarboxylase (ODC) activity in decapsulated rat testis, isolated testicular interstitial cells, and purified Leydig cells under defined conditions in vitro. Both immature (15 to 26 days old) and adult (60 to 90 days old) rat testes were employed. Basal (fresh tissue) ODC activity varied widely among rats of the same age but was similar (less than 5% difference) in pairs of testes from the same animal. For this reason, pairs of testes were compared in subsequent in vitro studies. ODC activity of decapsulated testes of adult rats declined (to 25 to 30% of basal) during 4 hours of incubation in Medium 199 + 0.1% bovine serum albumin + 0.1 mM 3-isobutyl-1-methylxanthine at 34 C. The addition of FSH, LH, prolactin, prostaglandin E2, epidermal growth factor, insulin, or 10% fetal calf serum singly or in combination failed to prevent this decline in ODC activity. In contrast, ODC activity of decapsulated testes of immature rats remained stable (versus fresh tissue) during 4 hours of incubation. The addition of FSH (100 ng/ml) caused a small but statistically significant (P less than 0.005) stimulation of the enzyme activity, and 8-bromo cyclic AMP (0.5 mM) mimicked the effect of FSH. In isolated interstitial cells from adult rats, LH stimulated ODC activity in a dose- (10 pg-200 ng/ml) and time-dependent fashion. 8-Bromo cyclic AMP mimicked the effect of LH. Prolactin, FSH, estradiol, insulin, prostaglandin E2, and epidermal growth factor did not alter the enzyme activity. LH also stimulated ODC activity of purified Leydig cells. This study demonstrates for the first time direct in vitro stimulation of rodent testicular ODC activity by gonadotropins and reveals marked age-dependent differences in regulation of this enzyme in vitro.

We examined the possibility that cytochalasin B reduced LH-stimulated testosterone synthesis of Leydig cells in part by inhibiting glucose uptake. In the presence of 5.6mM glucose cytochalasin B (0.1–50μM) inhibited LH-stimulated testosterone synthesis in a dose-dependent manner, while effects with cytochalasin D or cytochalasin E were not evident until inhibitor concentrations reached 1 or 10μM. Increasing levels of glucose (0–5mM) progressively increased LH-stimulated testosterone synthesis by interstitial cells, while cytochalasin B at 1 or 10μM inhibited testosterone synthesis in a dose-dependent manner at each glucose concentration examined. Higher concentrations of glucose (10– 50mM) partially reversed this inhibition. In the absence of glucose LH-stimulated testosterone synthesis was inhibited only when the cytochalasin B concentration reached 10μM. Cytochalasin B (0.1–5μM) inhibited [14C]-2-deoxyglucose uptake by purified Leydig cells in a dose-dependent manner, while 1 or 10μM cytochalasin D or cytochalasin E had only limited effects. These results suggest that cytochalasin B inhibits Leydig cell LH-stimulated testosterone synthesis by at least two mechanisms: by blocking glucose uptake at lower concentrations (0.1–10μM), while at higher concentrations (10–50μM) it probably blocks microfilament function.

Our studies demonstrate that direct stimulation of dihydrotestosterone metabolism by ethanol (2.2 - 65 mM) in rat Leydig cells primarily involves an increase in 5 alpha-androstan-3 beta, 17 beta-diol. Although the enzyme catalyzing this conversion, 5 alpha-androstane-3 beta-hydroxysteroid dehydrogenase, is localized in the microsomal fraction of Leydig cells, ethanol does not increase 5 alpha-androstan-3 beta, 17 beta-diol formation in isolated microsomes, presumably because of the removal of soluble alcohol dehydrogenase activity, which we propose mediates this action. Because 5 alpha-androstan-3 beta, 17 beta-diol is generally considered a weak or inactive androgen, this effect may function to decrease dihydrotestosterone secretion by Leydig cells and/or to reduce the availability of this androgen in responsive tissues.

Possible functional relationship between luteinizing hormone-stimulated ornithine decarboxylase and testosterone production was examined in rat testicular interstitial cells in vitro . Although luteinizing hormone enhanced both ornithine decarboxylase activity and testosterone production at a similar physiological dose range, we found dissociation in the two responses in terms of their temporal aspect and the way they were affected by an irreversible inhibitor of ornithine decarboxylase, alpha-difluoromethylornithine, and protein synthesis inhibitor cycloheximide. The results suggest that there appears to be no causal coupling between luteinizing hormone-stimulated enzyme activity and testicular steroidogenesis.