A study of risk factors for hypospadias in the Rhône-Alpes region (France).

OBJECTIVE: Hypospadias is one of the most common congenital urogenital malformations in males with a significantly increasing incidence over the past 20 years. The causes of this insufficient virilization of the genital tubercle are essentially unknown. SUBJECTS AND METHODS: A hospital-based controlled study was realized with 225 hypospadias cases at Debrousse Hospital, Lyon, using a detailed questionnaire completed during a consultation with the patients' parents and those of controls of the same age. The chi(2), the P-value, the odds ratios and the 95% confidence interval were assessed. RESULTS: Hypospadias was found to be positively associated with genetic factors (as defined by the presence of other case(s) in the family in one case in four) and with neonatal low birth weight, fair-haired boys, maternal history such as viral infection during the first trimester, order of parity, toxaemia of pregnancy, delivery modality such as caesarean section, and environmental pollution. CONCLUSIONS: These results show that aetiological factors of hypospadias are likely to be related to three main fields which interact: genes, the placenta and environmental factors.

FLRG, an activin-binding protein, is a new target of TGFbeta transcription activation through Smad proteins.

The FLRG gene encodes a secreted glycoprotein that binds to activin and is highly homologous to follistatin, an activin ligand. We cloned the promoter region of the human FLRG gene, and defined the minimal region necessary for transcription activation in a reporter-system assay. We showed that the fragment between positions -130 and +6, which consists of multiple consensus Sp1-binding sites, is required for the constitutive expression of the FLRG gene. We demonstrate here that FLRG mRNA expression is rapidly induced by TGFbeta or by transfection with Smad protein expression vectors in human HepG2 cells. We investigated the transcription-regulation mechanism of FLRG expression in HepG2 cells following treatment with TGFbeta. By deletion and point-mutation analysis of the FLRG promoter, we identified a Smad-binding element involved in the TGFbeta-inducible expression of the FLRG gene. Moreover, transactivation of the FLRG promoter by TGFbeta was compromised by dominant-negative mutants of Smad3 and Smad4 proteins. In addition, gel electrophoresis mobility-shift assays demonstrated the specific interaction of Smad3 and Smad4 proteins with the Smad-binding element consensus motif found in the FLRG promoter. Taken together, our data imply that Smad proteins participate in the regulation of expression of FLRG, a new target of TGFbeta transcription activation.

MT1-MMP in rat testicular development and the control of Sertoli cell proMMP-2 activation.

Metalloproteases (MMPs) are likely to be involved in the restructuring events occurring in the testis throughout development. We here demonstrate that membrane-type 1 (MT1)-MMP, a physiological activator of proMMP-2 under TIMP-2 control, is present within the testis together with MMP-2 and TIMP-2. In the prepubertal testis MT1-MMP immunoreactivity was uniformly distributed, whereas in the adult it was confined to the apical compartment of the tubules, where meiosis and spermiogenesis occur. We further showed that the two cell lineages (somatic and germinal) expressed MT1-MMP and TIMP-2, whereas MMP-2 was of somatic origin. To get a better picture into proMMP-2 activation, use was made of a model of cultured Sertoli cells treated with FSH or co-cultured with germ cells to mimic an immature or a mature developmental period, respectively. We found that follicle-stimulating hormone enhanced the expression of MMP-2 and TIMP-2 but not of MT1-MMP, and promoted the activation of proMMP-2. In co-cultures, a tremendous elevation and activation of MMP-2 was observed, which might relate to the processed MT1-MMP form solely detected in germ cells. That MMP-2 synthesis and activation are under local (germ cells) and hormonal (follicle-stimulating hormone) regulation emphasizes the importance of MMPs in testicular physiology.

Tumor necrosis factor-alpha regulates plasminogen activator inhibitor-1 in rat testicular peritubular cells.

We examined the regulation by tumor necrosis factor-alpha (TNF alpha) of plasminogen activator inhibitor-1 (PAI-1) in cultured peritubular cells recovered from 20-day-old rat testes. We demonstrated that TNF alpha in a nanomolar dose range stimulated PAI-1 messenger RNA (mRNA; Northern blots) as well as immunoreactive (Western blots) and bioactive (Stachrom) PAI-1 protein. Induction of PAI-1 mRNA started 4 h after the addition of TNF alpha (2.5-fold increase) and peaked (7-fold increase) after 24 h of treatment. Actinomycin D and cycloheximide inhibited the effects of TNF alpha on PAI-1 mRNA, suggesting that ongoing RNA and protein syntheses were required. The combined actions of transforming growth factor-alpha (TGF alpha), a potent inducer of PAI-1, and TNF alpha on PAI-1 were less than additive, suggesting the activation of some common pathway. TNF alpha action on PAI-1, like that of TGF alpha demonstrated previously, was masked by a preexposure to phorbol myristate acetate (a stimulator of protein kinase C) and strongly reduced by staurosporine (an inhibitor of the protein kinase C). Furthermore, using genistein to inhibit tyrosine kinase activity, we not only blocked the action of TGF alpha on PAI-1 [initiated upon binding to the tyrosine kinase epidermal growth factor/TGF alpha receptor (EGFR)], but also markedly reduced that of TNF alpha. Finally, TNF alpha, at a dose range that stimulated PAI-1, enhanced EGFR mRNA levels and EGF binding. Together, the present findings suggest that some of the biological effects of TNF alpha on PAI-1 might be secondary to de novo synthesis of EGFR. Because TNF alpha probably originates from testicular macrophages, such a regulation of PAI-1 by TNF alpha may occur in the context of physiological interactions between the testis and the immune system.

Transforming growth factor beta receptor expression in cultured porcine granulosa cells.

By using primary cultures of porcine granulosa cells as a model, TGF beta receptors have been identified and characterized through three different approaches including cross-linking experiments, Western- and Northern-blotting analysis. In cross-linking experiments, labeled TGF beta was shown to bind to four different molecular species of 300, 168, 72 and 58 kDa. The 300-kDa species may correspond to beta-glycan, while the 72- and 58-kDa correspond to TGF beta type II and I receptors, respectively. The presence of these receptors was further demonstrated by Western-blotting analysis using specific polyclonal antibodies. Finally, both the expression of beta-glycan, type II and type I mRNA, was confirmed through Northern-blotting analysis as shown by the presence of 6.4, 4.6 and 5.8 kb mRNA, respectively. Additionally, we detected another TGF beta binding protein of 168 kDa which remains to be identified. Together, our present data indicate that the regulatory action of TGF beta on cultured granulosa cells previously reported in several laboratories may be mediated through the receptors identified here.

In vitro regulation of rat Sertoli cell inhibin messenger RNA levels by transforming growth factor-beta 1 and tumour necrosis factor alpha.

In the present study, we examined the in vitro regulation of 20-day-old rat Sertoli cell inhibin alpha- and beta B-subunits mRNA levels by transforming growth factor-beta 1 (TGF-beta 1) and tumour necrosis factor alpha (TNF alpha), two factors produced in the testis. Addition of TGF-beta 1 to highly purified cultured Sertoli cells resulted in a time- and dose-dependent enhancement in the alpha-subunit mRNA levels (ED50 = 2.4 pM; maximal increase of 2.6-fold after 48 h of treatment), without affecting the beta B-subunit mRNA levels. Similarly, activin A up-regulated the alpha- but did not modulate the beta B-subunit mRNA levels. By contrast, TNF alpha decreased in a time- and dose-dependent fashion the mRNA levels of the two inhibin subunits alpha and beta B (IC50 = 29 pM for both subunits; maximal decrease of 4.4- and of 4-fold after 72 and 24 h of treatment for respectively the alpha- and beta B-subunits). The effects of TGF-beta 1 and TNF alpha on inhibin mRNA levels occurred within a dose range that might be expected under physiological conditions. In addition, TGF-beta 1-treated Sertoli cells responded to FSH or dibutyryl cyclic AMP ((Bu)2cAMP) by a further and significant additive increase of the alpha-subunit mRNA levels. TNF alpha-treated Sertoli cells responded significantly to FSH and to (Bu)2cAMP, thus attenuating the inhibitory action of TNF alpha on the alpha-inhibin mRNA levels. Together, the present findings emphasize the ability of some local growth factors to modulate the effects of FSH on Sertoli cell function.

Expression of mRNAs for transforming growth factor-beta receptors in the rat testis.

In this study, we examined by means of northern blots, the mRNA expression of the three transforming growth factor-beta receptors (TGF-beta receptors) during rat testicular development and their cellular localization using fractionated testicular cells. Transcripts for receptors I and II were essentially detected in the immature testis, whereas receptor III mRNAs were present throughout testicular development. Somatic cells contained mRNAs for the three receptor types. In germ cells, transcripts for types I and II were in low abundance compared to type III mRNAs. In addition germ cells expressed three transcripts for the type III. Among the three transcripts, two were expressed exclusively in germ cells. TGF-beta being produced locally, our results are consistent with TGF-beta acting in testis via autocrine and/or paracrine mechanisms.

Fibroblast growth factor receptor type 1 expression during rat testicular development and its regulation in cultured Sertoli cells.

In the present study, we examined the ontogeny of the type 1 receptor for basic fibroblast growth factor (FGFR-1) in whole rat testis, its cellular localization, and its in vitro regulation in 20-day-old rat Sertoli cells. Gene expression of FGFR-1 was developmentally regulated; expression was higher in prepubertal testes and decreased with sexual maturity. The transcript was found to be expressed in Leydig-enriched fractions, peritubular cells, Sertoli cells, and, to a lesser extent, germ cells. FSH as well as (Bu)2cAMP enhanced FGFR-1 messenger RNA (mRNA) levels in cultured Sertoli cells, suggesting an involvement of the protein kinase-A pathway. Addition of basic FGF (bFGF), tumor necrosis factor-alpha (TNF alpha), or interleukin-1 alpha resulted in a dose- and time-related increase in FGFR-1 mRNA levels. The effect of bFGF was specific, because it was neutralized by cotreatment with an anti-bFGF. We tested medium conditioned by germ cells and found a stimulation of the Sertoli cell FGFR-1 mRNA levels, which was abolished by immunodepletion of the conditioned medium with anti-TNF alpha antibodies. It is suggested that in Sertoli cells, bFGF action, when mediated by FGFR-1, is under a complex hormonal (FSH) and paracrine and/or autocrine control exerted at least by bFGF, TNF alpha, and interleukin-1 alpha.

Effect of epidermal growth factor/transforming growth factor alpha on lactate production in porcine Sertoli cells: glucose transport and lactate dehydrogenase isozymes as potential sites of action.

Germ cell development is dependent upon the delivery of essential nutriments such as lactate originating from Sertoli cells. Lactate production is under the systemic control but probably also under a local control exerted via certain growth factors. By using a model of porcine cultured Sertoli cells, we have characterized the action of epidermal growth factor (EGF) on lactate production and further delineated the potential biochemical mechanisms involved in the EGF action. EGF stimulated lactate production in a time and dose dependent manner with a half-maximal (ED50) and maximal effects, respectively with 3.8 (0.6 x 10(-9) M) and 22 ng/ml of EGF. Lactate formation involves several biochemical steps among which the glucose substrate uptake and transport system as well as the lactate dehydrogenase (LDH) activity appear to play key roles. We report here that EGF increased the uptake of glucose evaluated through that of 2-deoxy-D-glucose (2-DOG), a non-metabolizable glucose analog. Such an increase in glucose substrate uptake occurs both after a long term (48 h) and a short term treatment (ED50 = 6.4 ng/ml, 1.1 x 10(-9) M EGF). Moreover, EGF was also able to enhance the activity of the Sertoli cell LDH. The maximal effect of the growth factor on LDH activity was observed after a long term (24 h) treatment with an ED50 of 7 ng/ml (1.2 x 10(-9) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Tri-iodothyronine effects on Sertoli cell protein turnover in the prepubertal pig.

The direct effect of Tri-iodothyronine (T3: 0.1-100 nM) on protein turnover was studied using primary cultures of Sertoli cells isolated from immature piglet testis. The results demonstrate that T3 significantly increases protein synthesis without altering the protein degradation rate. These data and previous ones, showing the presence of specific T3 receptors in Sertoli cell nuclei, indicate that T3 plays a fundamental role in the early regulation of porcine Sertoli cell growth and maturation.

Tri-iodothyronine effects on Sertoli cell protein turnover in the prepubertal pig.

The direct effect of Tri-iodothyronine (T(3): 0.1-100 nM) on protein turnover was studied using primary cultures of Sertoli cells isolated from immature piglet testis. The results demonstrate that T(3) significantly increases protein synthesis without altering the protein degradation rate. These data and previous ones, showing the presence of specific T(3) receptors in Sertoli cell nuclei, indicate that T(3) plays a fundamental role in the early regulation of porcine Sertoli cell growth and maturation.

Basic fibroblast growth factor enhances testosterone secretion in cultured porcine Leydig cells: site(s) of action.

The effect and mechanism of action of basic fibroblast growth factor (bFGF) on testicular steroidogenesis were investigated using as a model primary cultures of purified porcine Leydig cells from immature intact animals. Basic FGF increased basal and human chorionic gonadotrophin (hCG)-induced testosterone accumulation (with an ED50 of 0.64 ng/ml bFGF, 35 pM) in the medium following a long-term treatment. The effects of bFGF (10 ng/ml, 72 h) were found at all hCG concentrations tested (0.001-1 ng/ml), the growth factor affecting the maximal steroidogenic capacity of the Leydig cells but not their sensitivity to the gonadotrophin. In this context, we have therefore investigated whether the stimulatory effect of bFGF on testosterone formation was related to an increase of the steroidogenic enzyme activities. The data obtained indicate that the growth factor did not affect the gonadotrophin action on the formation of delta 5-steroid hormone, namely dehydroepiandrosterone (DHEA) (evaluated in the presence of 10(-5) M WIN 24540, an inhibitor of 3 beta-hydroxysteroid dehydrogenase/isomerase). By contrast, bFGF (10 ng/ml, 72 h) was found to increase in a comparable manner the conversion of pregnenolone, DHEA and delta 4-androstenedione into testosterone, suggesting a stimulatory effect on 17 beta-hydroxysteroid dehydrogenase activity. Indeed, bFGF enhanced in a dose-dependent manner (ED50 = 39 pM) this enzyme activity evaluated through the conversion of delta 4-androstenedione to testosterone. These effects of bFGF on Leydig cell steroidogenic activity are probably exerted through specific membrane bFGF receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of nuclear tri-iodothyronine receptors in Sertoli cells from immature piglet testes.

The existence of nuclear tri-iodothyronine (T3) receptors in both Sertoli and Leydig cells isolated from immature piglet testes was investigated. The results demonstrated the presence of high-affinity (Kd = 1.09 +/- 0.25 nM), low-capacity (185 +/- 24 pg T3/mg DNA) binding sites for T3 in nuclei from freshly isolated Sertoli cells. No specific binding for T3 was observed in nuclei isolated from Leydig cells. The localization of specific T3 receptors, which might mediate the onset of thyroid hormone action, at Sertoli cell level confirms that these cells are a target for thyroid hormone and strongly sustain the role of the thyroid in the regulation of testicular functions during postnatal development.

Interleukin-1 alpha as a potent inhibitor of gonadotropin action in porcine Leydig cells: site(s) of action.

The effects of interleukin on testicular steroidogenesis have been studied in several laboratories, most often by using cultured rat Leydig cells. Several reports have indicated that interleukin-1 beta (IL-1 beta), but not interleukin-1 alpha (IL-1 alpha), exert a potent effect on gonadotropin action in rat Leydig cells. By using cultured porcine Leydig cells as a model, we found that IL-1 alpha (and to a lesser extent IL-1 beta), contrary to previous reports, is a potent inhibitor of LH/hCG steroidogenic action; and we further localized the steroidogenic biochemical step(s) affected by IL-1 alpha. IL-1 alpha inhibited hCG-induced testosterone secretion (about 67%) in a dose- and time-dependent manner. Half maximal and maximal effects were obtained with 4 U/ml (approximately 0.4 ng/ml, 0.3 x 10(-10) M) and 20 U/ml (approximately 2 ng/ml, 1.4 x 10(-10) M) of IL-1 alpha, respectively. The inhibitory effect of IL-1 alpha on gonadotropin action was detected at 6 h and was maximal after 24 h of treatment with the cytokine. The IL-1 alpha inhibitory effect was more potent than that of IL-1 beta: the maximal inhibitory effect of IL-1 beta was obtained with 400 U/ml. Subsequent investigations indicated that IL-1 alpha inhibited different biochemical steps involved in gonadotropin-induced testicular steroidogenesis. In this context, although IL-1 alpha appears to inhibit Leydig cell membrane functions (through a decrease in LH/hCG binding and gonadotropin-induced cAMP production), the antigonadotropin action of the cytokine is probably exerted predominantly at a step(s) located beyond cAMP formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Sites of interaction between epidermal growth factor and transforming growth factor-beta 1 in the control of steroidogenesis in cultured porcine Leydig cells.

The present study examines how the hormonal action of gonadotropin is modulated by transforming growth factor-beta 1 (TGF beta 1) and epidermal growth factor (EGF) in primary cultures of purified porcine Leydig cells. Although TGF beta 1 (1 ng/ml; 48 h) and EGF (10 ng/ml; 72 h) individually enhanced hCG-stimulated testosterone formation, the effects of EGF were more pronounced than those of TGF beta 1. When studied in combination, the effects of maximal concentrations of TGF beta 1 and EGF were additive on gonadotropin hormonal action. In the present study we demonstrate that their additive effects resulted from a complex interaction occurring at the levels of cholesterol substrate availability in the mitochondria and of 3 beta-hydroxysteroid dehydrogenase/isomerase activity (3 beta HSDI). First, TGF beta 1 (1 ng/ml; 48 h) and EGF (10 ng/ml; 72 h) were, respectively, shown to reduce and enhance dehydroepiandrosterone (DHEA) formation (evaluated in the presence of 10(-5) M WIN 24540, an inhibitor of 3 beta HSDI) in Leydig cells when acutely (3 h) stimulated with hCG (0.01-1 ng/ml), but not when incubated with 22R-hydroxycholesterol (3 micrograms/ml). Such findings indicate that TGF beta 1 and EGF did not affect cholesterol side-chain cleavage cytochrome P450 activity, but, respectively, decreased and increased cholesterol substrate availability for this enzyme in the mitochondria. Furthermore, when Leydig cells were treated with the combined factors, the formation of delta 5-steroid intermediates (such as DHEA) in untreated (control) and EGF-plus TGF beta 1-treated cells was not significantly different whether the cells were acutely stimulated with the gonadotropin or incubated with 22R-hydroxycholesterol. Such findings indicate that the effects of EGF and TGF beta 1 on cholesterol substrate availability in the mitochondria are antagonistic. Second, EGF, TGF beta 1, and EGF plus TGF beta 1 significantly (P less than 0.001) increased delta 5-steroid intermediate (i.e. pregnenolone and DHEA), but not delta 4-steroid intermediate (i.e. progesterone and androstenedione), conversion into testosterone, indicating that the growth factors increased, individually or in combination in an additive manner, 3 beta HSDI activity (respectively, 90.7 +/- 0.6%, 80.6 +/- 2.6%, and 164 +/- 4.5% increase in the presence of EGF, TGF beta 1, and EGF plus TGF beta 1). Together, the reciprocal suppression of the effects of TGF beta 1 and EGF on the mitochondrial cholesterol substrate availability coupled to their stimulatory additive actions on 3 beta HSDI activity provide an explanation of the additive actions of the two growth factors on gonadotropin-induced testicular androgen formation.

Transforming growth factor beta 1 inhibits gonadotropin action in cultured porcine Sertoli cells.

In the present study, we have tested the effects of transforming growth factor beta 1 (TGF beta 1) on FSH action toward aromatase activity and lactate production in cultured Sertoli cells isolated from immature porcine testes. Whereas treatment of Sertoli cells with FSH resulted in a dose-dependent increase (about 7-fold) in aromatase activity (conversion of testosterone into estradiol) (ED50 = 80 ng/ml FSH), the addition of TGF beta 1 reduced this gonadotropin action. The inhibitory effect of TGF beta 1 on FSH aromatase activity was dose dependent (ED50 = 0.1 ng/ml, 4 pM TGF beta 1) with a maximal decrease (about 40%) observed after a long term (48-h) treatment. TGF beta 1 exerted its inhibitory effect on FSH action at the level(s) of cAMP accumulation, exerting no apparent effect on the gonadotropin receptor or at a site(s) related to cAMP action. TGF beta 1 (2 ng/ml) significantly (P less than 0.002) reduced (52% decrease) FSH-stimulated cAMP levels in cultured porcine Sertoli cells. However, such an inhibitory effect of the growth factor was no longer observed when stimulation of cAMP accumulation with FSH occurred in the presence of methyl isobutyl xanthine (0.5 mM), an inhibitor of cAMP-phosphodiesterase activity. This observation suggests that TGF beta 1 decreased cAMP levels by increasing catabolism of the cyclic nucleotide through an enhancement of cAMP-phosphodiesterase activity. The inhibitory effect of TGF beta 1 was not limited to the action of FSH on aromatase activity but also extended to the gonadotropin action (mediated by cAMP) on lactate production. As for the inhibitory effect of TGF beta 1 on FSH-induced aromatase activity, the inhibitory effect of the growth factor on FSH-stimulated lactate production was dose and time dependent with a maximal decrease (about 30%) observed in the picomolar range (1 ng/ml, 40 pM) after 48 h treatment with TGF beta 1. In conclusion, the present study demonstrates that TGF beta 1 attenuates FSH action on Sertoli cell activity and that such inhibitory action is potentially exerted through a decrease in cAMP levels. Because of the local production of TGF beta 1, it is suggested that the effects of the growth factor reported here might be exerted in the context of the testicular paracrine mechanisms.

Tumor necrosis factor alpha inhibits gonadotropin action in cultured porcine Leydig cells: site(s) of action.

In the present study, we have tested the direct effects of tumor necrosis factor-alpha (TNF-alpha) on basal and human (h)CG-stimulated testosterone secretion by cultured purified Leydig cells isolated from immature porcine testes. TNF-alpha reduced (as much as 90% decrease) hCG-stimulated, but not basal testosterone secretion in a dose- and time-dependent manner. The maximal and half-maximal effects were, respectively, 3.75 ng/ml (2.2 x 10(-10) M) and 0.66 ng/ml (3.9 x 10(-11) M) of TNF-alpha after 48 h treatment. TNF-alpha antagonizes the gonadotropin hormonal action by affecting at least two types of biochemical steps. First, TNF-alpha reduced LH/hCG binding to a maximal decrease of 45% obtained with 2 ng/ml of TNF-alpha after 48 h of treatment. TNF-alpha also inhibited (44% decrease) hCG-stimulated cAMP production in optimal conditions (20 ng/ml, 72 h). Second, TNF-alpha significantly (P less than 0.001) reduced testosterone secretion stimulated with 8-bromo-cAMP (3 x 10(-3) M) in a similar range (86% decrease) to that observed with the gonadotropin. Such an observation indicates that the antigonadotropic action of the cytokine is exerted in a predominant manner at a step(s) located beyond cAMP formation. Furthermore, incubation of Leydig cells with 22R-hydroxycholesterol (5 micrograms/ml, 2 h) reversed most of the inhibitory effect of TNF-alpha on androgen production. Indeed, the TNF-alpha (20 ng/ml, 72 h) inhibitory effect on testosterone production was limited to about 20% (P less than 0.03) in Leydig cells supplied with 22R-hydroxycholesterol. Such a moderate effect of the cytokine in the presence of 22R-hydroxycholesterol compared with that observed when androgen secretion was stimulated with the gonadotropin (up to 90% inhibition) indicate that TNF-alpha acts by dramatically reducing cholesterol substrate availability in the mitochondria. Such an effect of TNF-alpha is directly exerted on Leydig cells since TNF-alpha receptors (dissociation constant approximately 5.4 x 10(-10) M) are present in primary cultures of purified porcine Leydig cells. Together, the present findings show that in Leydig cells TNF-alpha antagonizes the gonadotropin action on testosterone formation predominantly through a decrease in the availability of cholesterol substrate in the mitochondria.

Effect of activin A on dehydroepiandrosterone and testosterone secretion by primary immature porcine Leydig cells.

In the present study, we evaluated the effect of the homodimer activin A on immature porcine Leydig cell functions in primary culture. Activin A (0.5-100 ng/ml) reduced hCG-stimulated dehydroepiandrosterone (DHEA) accumulation in a dose- and time-dependent manner, with a maximal inhibitory effect (58% decrease) at 20 ng/ml (8 x 10(-10) M). Activin A was found not to control steroidogenesis, either through a modulation of the gonadotropin LH/hCG binding or low-density lipoprotein cholesterol binding and internalization. However, activin A significantly decreased pregnenolone (p less than 0.002) and DHEA (p less than 0.001) formation (evaluated in the presence of 10(-5) M of WIN 24540, an inhibitor of 3 beta-hydroxysteroid dehydrogenase/isomerase [3 beta-HSDI]activity) in Leydig cells maximally stimulated with hCG (3 ng/ml, 3 h) or incubated in the presence of 22R-hydroxycholesterol (5 micrograms/ml, 2 h). These findings indicate that activin A probably exerts a partial inhibitory effect on cholesterol side-chain cleavage cytochrome P450 (P450scc) activity. On the other hand, activin A significantly (p less than 0.001) enhanced the conversion of exogenous pregnenolone and DHEA (500 ng/ml) but not of progesterone and androstenedione (500 ng/ml) into testosterone, suggesting that activin A potentially enhances 3 beta-HSDI activity in Leydig cells. Activin A action on 3 beta-HSDI activity was found to be closely related to that of transforming growth factor-beta 1 (TGF beta 1), since both activin A (20 ng/ml) and TGF beta 1 (2 ng/ml) induced a comparable and non-additive increase in 3 beta-HSDI activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor directly stimulates steroidogenesis in primary cultures of porcine Leydig cells: actions and sites of action.

The actions and the mechanisms of action of epidermal growth factor (EGF) in testicular steroidogenesis were investigated using a model of primary culture of purified porcine Leydig cells from immature intact animals. EGF decreased (1.7-fold) human CG (hCG)-induced dehydroepiandrosterone (DHEA) accumulation in the medium whereas it enhanced (2.5-fold) that of testosterone. The maximal and half-maximal effects on both DHEA and testosterone secretions were observed at similar concentrations which were, respectively, 3 (5 x 10(-10) M) and 0.7 (11 x 10(-11) M) ng/ml EGF, after 72-h treatment. EGF effect on DHEA and testosterone secretion was similarly observed whether the cells were acutely (3 h) stimulated with hCG (1 ng/ml) or with 8-bromo-cAMP (10(-3) M). To further localize the steroidogenic biochemical steps affected by EGF, the growth factor action on steroidogenic enzyme activities was investigated. EGF increased delta 5 steroid intermediate (i.e. pregnenolone and DHEA) formation [evaluated in the presence of 10(-5) M of WIN 24540, an inhibitor of 3 beta-hydroxysteroid dehydrogenase/iosomerase (3 beta-HSDI) activity]. However, this stimulation was observed in cells when acutely (3 h) stimulated with hCG (0.01-1 ng/ml) but not when incubated with 22R-hydroxycholesterol (0.01-10 micrograms/ml). Such findings indicate that EGF did not affect cholesterol side chain cleavage cytochrome P450 activity but probably increased cholesterol substrate availability for this enzyme in the inner mitochondria. Moreover, EGF significantly (P less than 0.001) increased delta 5 steroid intermediate (i.e. pregnenolone and DHEA) but not delta 4 steroid intermediate (i.e. progesterone and androstenedione) conversion into testosterone, indicating that EGF enhances 3 beta-HSDI activity. Such effects of EGF are directly exerted on Leydig cells since EGF receptors (Kd = 16 x 10(-11) M) are present in primary cultures of purified porcine Leydig cells. Together, the present findings show that in Leydig cells from intact animals, EGF enhances the gonadotropin action on testosterone formation through an increase in the availability of cholesterol substrate in the mitochondria as well as an increase in the activity of 3 beta-HSDI.