|Budget Amount *help
¥1,900,000 (Direct Cost : ¥1,900,000)
Fiscal Year 1995 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1994 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Nitric oxide (NO) has been recently implicated in a variety of physiological precesses, including reproductive functions. The present study was undertaken to investigate whether NO might significantly induce any change in steroidogenesis in cultured porcine granulosa cells (PGC).
Cells were obtained from medium follicles by the needle aspiration method. After pre-incubation, cells were incubated with N^G-monomethyl-L-arginine (LNMMA), a competitive blocker of NO synthase (NOS), and the NO donor, NOC 18, either alone or in the presence of FSH (200ng/ml) / hCG (5IU/ml). Estradiol (E_2) and progesterone (P) concentrations in media were measured by EIA.LNMMA (0.01- 1 mM) significantly (P<0.05) stimulated the basal E_2 release and dosedependently enhanced the E_2 and P release from FSH-stimulated PGC for 24-h culture. LNMMA also significantly (P<0.001) suppressed the NO^- concentrations in media, measured by chemiluminescence. NOC 10 (0.1 - 1 mM) significantly (P<0.01) suppressed the E_2 release from basal and gonadotropin stimulated PGC for 2-h culture. Cells were immunostained with an avidin-biotin-peroxidase (ABC) kit, and the localization of constitutive (c) NOS and inducible (i) NOS was immunohistochemically comfirmed. Additionnally, we evaluated the possible involvement of cGMP and aromatase activity on NO-induced inhibition of steroidogenesis. The cGMP analog, 8-bromo-cGMP,had no significant effect on the accumulation of E_2 and P in basal and gonadotropin simulated PGC during 24-h culture. However, NOC 18 (0.1 - 1 mM) significantly (P<0.01) inhibited the aromatase activity of basal and gonadotropin stimulated PGC,measured by a modified tritiated water method.
These results show that no inhibits E_2 secretion independent of cGMP by directly inhibiting aromatase activity in moderately mature PGC,and suggest that NO is an important local modulator of the follicular development.