Regulation of NO by hCG in ovarian follicles of zebrafish.

(A) The oocyte maturation could be dynamically induced by injection of hCG (15 IU/fish). (B) Gross morphology of ovaries of FG stage intact follicles, which were manually separated into two comportments, follicular cells and denuded oocytes. (C) PI staining of intact follicles (upper panel) and denuded oocytes (lower panel). (D) After injection of hCG (15 IU/fish), the NO concentration was assessed in intact follicles (a), denuded oocytes (b) and follicular cells (c) at different times. (E) Morphology of primary cultured zebrafish ovarian follicular cells after 6 days of incubation. The two types of epithelial cells present in the culture with distinct morphologies: G, granulosa cells; T, theca cells. (F) The NO level was assessed in primary culture follicular cells by treatment with hCG (20 IU/mL) for 2 h. Each value represents the mean value ± SEM of quintuplicate assays from three independent experiments (*, P < 0.05 vs. control).

Regulation of cGMP by hCG in ovarian follicles of zebrafish.

(A) The cGMP concentration was assessed in intact follicles (a), denuded oocytes (b) and follicular cells (c) after injection of hCG (15 IU/fish) at different times. (B) The cGMP concentration was assessed in primary cultured follicular cells after treatment with hCG (20 IU/ml) for 0.5 h. (C) The cGMP production in preovulatory follicles after injection of hCG (15 IU/fish, 1.5 h) was determined by immunostaining. Scale bar: 200 μm. Each value represents the mean value ± SEM of quintuplicate assays from three independent experiments (*, P < 0.05; **, P < 0.01 vs. control). (D) A hypothetic model for the dual role of cGMP in regulation of oocyte maturation in zebrafish. After the LH surge, the increased NO and cGMP level in follicular cells could induce the oocyte maturation through some unknown factors, while a decreased cGMP level in oocyte could also initiate oocyte maturation in zebrafish.