Effects of nitric oxide on steroidogenesis in porcine granulosa cells during different stages of follicular development

Background: We have previously demonstrated that nitric oxide (NO) inhibits steroidogenesis via a cGMP-independent process, by inhibiting P450 aromata se activity in porcine granulosa cells (PGCs) derived from medium-sized (3- 5 mm) ovarian follicles (M-PGC). Objective: To determine whether the NO/NO synthase (NOS) system exerts any significant effects on steroidogenesis in PGCs derived from small follicles (<3 mm) (S-PGC) in comparison with those derived from medium follicles, Design and methods: PGCs, namely S-PGC and M-PGC, were incubated with the N O donor, NOC18, and a competitive blocker of NOS, NG-monomethyl-L-arginine (LNMMA), either alone or in the presence of FSH (200 ng/ml) or hCG (5 IU/ml ). Results: NOC18 significantly (P < 0.01-0.001) suppressed basal (unstimulate d) and gonadotropin-stimulated estradiol (E2) release from both S-PGC and M -PGC in a 2-h culture. NOC18 significantly (P ( 0.01-0.001) decreased basal and gonadotropin-stimulated progesterone release from S-PGC, but not from M-PGC, In addition, NOC18 significantly (P < 0.05-0.001) inhibited aromatas e activity in S-PGC, LNMMA had a significantly (P < 0.01-0.001) stimulatory effect on the basal release of E2 and progesterone from M-PGC; however, it had no significant effect on basal steroidogenesis in S-PGC in a 24-h cult ure. In the presence of gonadotropin, LNMMA significantly (P < 0.01-0.001) stimulated the release of E2 and progesterone from both S- and M-PGC, and t his stimulatory effect was weaker in S-PGC than in M-PGC. These results dem onstrate that NO inhibits E2 secretion by directly inhibiting the aromatase activity in S-PGC, as in M-PGC. It has been shown that the NO system suppr esses the differentiation of S-PGC: however, the extent of suppression decr eased with the progression of follicular growth. In addition, the activity of NOS in S-PGC was weaker than that in M-PGC. Conclusion: We strongly suggest that the NO/NOS system in PGC regulates ste roidogenesis differently during different phase of follicular development.