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 aromatase 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.


METHODS
PGCs, namely S-PGC and M-PGC, were incubated with the NO donor, NOC18, and a competitive blocker of NOS, N(3)-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 (unstimulated) 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 aromatase 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 culture. 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 this stimulatory effect was weaker in S-PGC than in M-PGC. These results demonstrate 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 suppresses the differentiation of S-PGC; however, the extent of suppression decreased with the progression of follicular growth. In addition, the activity of NOS in S-PGC was weaker than that in M-PGC.


CONCLUSIONS
We strongly suggest that the NO/NOS system in PGC regulates steroidogenesis differently during different phase of follicular development.
Department of Obstetrics and Gynecology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-0034, Japan.
M
M
Masuda
T
T
Kubota
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T
Aso
D000818:Animals; D001141:Aromatase / Q000378:metabolism; D002478:Cells, Cultured; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D004958:Estradiol / Q000096:biosynthesis / Q000378:metabolism; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D006107:Granulosa Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009603:Nitroso Compounds / Q000494:pharmacology; D011374:Progesterone / Q000096:biosynthesis / Q000378:metabolism; D013256:Steroids / Q000096:biosynthesis* / Q000378:metabolism; D013552:Swine / Q000378:metabolism*; D019323:omega-N-Methylarginine / Q000494:pharmacology
D006063:Chorionic Gonadotropin; C093285:NOC 18; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D013256:Steroids; D019323:omega-N-Methylarginine; D009569:Nitric Oxide; D011374:Progesterone; D004958:Estradiol; D005640:Follicle Stimulating Hormone; D019001:Nitric Oxide Synthase; D001141:Aromatase