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.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMedDate not supplied

The aging Leydig cell. VIII. Protein kinase activity.

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 had the lowest levels. Testosterone responses of 30-month-old rates 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.

Animals↗

PubMedDate not supplied

Mechanism of action of gonadotropin-releasing hormone-stimulated Leydig cell steroidogenesis. I. The stimulatory effect is calcium dependent and not mediated by cyclic nucleotides.

UNLABELLED: The present study was designed to elucidate mechanisms responsible for gonadotropin-releasing hormone (GnRH)-stimulated testosterone formation. Purified Leydig cells from adult Sprague-Dawley rats were incubated with varying concentrations of GnRH agonist (des-Gly10, (D-Ala6) GnRH N-ethylamide), hCG, 8-bromo cAMP or pregnenolone; testosterone, cAMP, cyclic GMP (cGMP) and cAMP-dependent protein kinase activity were measured after various time periods. Basal testosterone levels were 2.54 +/- 0.13 ng/10(5) cells, increasing to 3.18 +/- 0.14, 4.32 +/- 0.08, and 4.63 +/- 0.12 ng within 1 hour after the addition of 10(-9), 10(-8), and 10(-7) M GnRH agonist, respectively. After a 3-hour incubation a 10(-7) M dose of GnRH agonist increased testosterone production four-fold above control. GnRH agonist potentiated hCG-stimulated testosterone formation, but had no significant effects on cGMP levels and cAMP-dependent protein kinase activity. Cyclic AMP levels in the incubation medium increased slightly. GnRH agonist also enhanced 8-bromo-cAMP and pregnenolone-induced testosterone formation. Furthermore, GnRH agonist increased testosterone formation both in the absence and presence of phosphodiesterase inhibitor. These results suggest that the major effect of GnRH agonist is probably beyond the cAMP step. When purified Leydig cells were incubated in a calcium-free medium, the stimulatory effects of GnRH agonist on testosterone formation were completely abolished, but could be restored by the addition of calcium to the incubation medium. GnRH agonist-induced testosterone formation was also blocked by the addition of nifedipine (a calcium channel blocking agent, 0.1 to 10 micrograms/ml). Finally, GnRH antagonist in a concentration of 10 micrograms/ml completely inhibited GnRH agonist-stimulated testosterone formation. IN CONCLUSION: GnRH agonist stimulated Leydig cell testosterone formation in short-term incubations. The stimulatory effect is calcium dependent and not mediated by cyclic nucleotides.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMedDate not supplied

Mechanism of action of gonadotropin-releasing hormone-stimulated Leydig cell steroidogenesis. II. Gonadotropin-releasing hormone stimulates phospholipid labeling.

To investigate mechanisms responsible for gonadotropin-releasing hormone (GnRH)-stimulated Leydig cell steroidogenesis, the effects of GnRH agonist [des-Gly10, (D-Ala6) GnRH] on phospholipid turnover were studied. GnRH agonist in concentrations of 10(-9) to 10(-7)M increased phosphatidic acid labeling 292 +/- 16% (mean +/- SE), and phosphatidylinositol labeling 258 +/- 13.2%. GnRH agonist-stimulated phospholipid labeling was detectable as early as 2 minutes. GnRH antagonist completely blocked GnRH agonist-induced testosterone formation and phosphatidic acid and phosphatidylinosital labeling. Nifedipine in concentrations of 1 and 10 micrograms/ml inhibited GnRH agonist-stimulated testosterone formation but had no effect on 32P incorporation into phospholipids. Our results suggest that GnRH agonist-stimulated Leydig cell steroidogenesis is calcium dependent and correlated with increased phospholipid turnover.

Animals↗