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)

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.

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.

[Interaction between gonadotropins and "Transforming Growth Factor Beta" (TGF Beta) in testicular function control]. / Interactions entre les gonadotrphines et le "Transforming Growth Factor beta " (TGF beta) dans le contröle des fonctions testiculaires.

Testicular function is regulated not only by circulating hormones, among which the gonadotrophins play the main role, but also by local factors originating in multiple and complex interactions among cells. In this review, the example of gonadotrophins (LH and FSH) and Transforming Growth Factor beta (TGF beta) was chosen to illustrate the role of interactions between circulating hormones and gonadal growth factors in testicular function control; TGF beta-like activity has been found in the male gonad and we have used a model of cultured purified testicular cells to show that the action of TGF beta on testicular function mainly involves antagonism of the effect of gonadotrophins. Conversely, TGF beta promotes differentiated Leydig and Sertoli cell function. The example of interactions between TGF beta and gonadotrophins reported here shows that locally produced growth factors can regulate the response of testicular cells to gonadotrophins, a finding that extends our concept of reproductive endocrinology to cell-cell interactions.

On the mechanisms involved in the inhibitory and stimulating actions of transforming growth factor-beta on porcine testicular steroidogenesis: an in vitro study.

By using immature porcine Leydig cells cultured in defined medium as a model, transforming growth factor-beta (TGF beta) was shown to exert a dramatic inhibitory effect on their basal and human chorionic gonadotropin (hCG) (or 8-bromo-cyclic AMP) stimulated dehydroepiandrosterone secretion, in the presence or absence of saturating concentrations of exogenous (low density lipoprotein) cholesterol substrate. In contrast, TGF beta exerted both a stimulating and inhibitory effect on testosterone secretion: while hCG-stimulated testosterone secretion was enhanced by low doses of TGF beta (0.06-0.4 ng/ml, 48 h), it was decreased with higher concentrations of TGF beta (2.5-10 ng/ml, 48 h). The data obtained show that the inhibitory action of TGF beta on testicular steroidogenesis was related to a decrease in pregnenolone formation by affecting a step(s) distal to cyclic AMP formation but before cholesterol association with cytochrome P-450 side-chain cleavage. As for the stimulatory effect of TGF beta on testosterone formation, this was mainly related to an increase (about 2-fold) in 3 beta-hydroxysteroid dehydrogenase/isomerase activity (ED50 0.05 ng/ml, 2 X 10(-13) M). The results indicate that the (short-term) steroidogenic stimulatory action of luteinizing hormone (LH)/hCG is antagonized by high concentrations of TGF beta by decreasing pregnenolone formation while it is enhanced by the stimulating action of low concentrations of TGF beta exerted on 3 beta-hydroxy steroid dehydrogenase/isomerase activity.

Fibroblast growth factor is a regulator of testosterone secretion in cultured immature Leydig cells.

The regulatory effect of fibroblast growth factor (FGF) on testosterone secretion was studied by using a model of immature porcine Leydig cells cultured in serum-free defined medium. FGF enhanced in a dose-dependent manner hCG-stimulated testosterone secretion (ED50 = 11 ng/ml FGF). The stimulatory effect of FGF on testosterone accumulation was time dependent; testosterone increased to a maximal value at 24 h treatment and then dramatically declined to near control value following 48 and 72 h treatment with FGF; such a decline was not related to FGF degradation in culture medium. Although FGF increased maximal secretion of testosterone, it did not affect the human chorionic gonadotrophin (hCG) concentrations required for maximal and half-maximal secretion of testosterone (1 and 0.2 ng/ml hCG, respectively). These effects of FGF are probably exerted in the context of the local control of testicular steroidogenesis.