Effects of hCG and PACAP on kitlga expression and Akt phosphorylation. (A) Primary culture of zebrafish follicle cells at the end of a 6-day incubation. The oocytes were removed during sub-culturing. GC, granulosa cells; TC, theca cells; O, oocyte. (B and C) Effects of hCG and PACAP on kitlga expression. The follicle cells were treated with IGF-I (50 ng/mL), hCG (5 or 20 IU/mL) or PACAP (100 nM or 200 nM) alone or in combination for 6 h followed by total RNA extraction for real-time qPCR analysis. The data were normalized to the housekeeping gene, ef1a, and expressed as a percentage of the control group (mean ± SEM, n = 4). **P < 0.01; ***P < 0.001 versus control. (D and E) Effects of hCG and PACAP on Akt phosphorylation. Cultured zebrafish follicle cells were treated with IGF-I (50 ng/mL), hCG (20 IU/mL) and PACAP (200 nM) alone or in combination for 15 min followed by protein extraction for Western blot analysis. The densitometric quantification of the Western blot signals is shown at the bottom of each graph. The level of phosphorylated Akt (p-Akt) was normalized to that of total Akt (t-Akt) and expressed as the fold change compared with the control group (mean ± SEM, n = 3). Different letters indicate statistical significance (P < 0.05).

response of the effects of db-cAMP (A) and forskolin (B) on the expression of kitlga in cultured zebrafish follicle cells after 6 h of treatment. The data were normalized to the housekeeping gene, ef1a, and expressed as a percentage of the control group (mean ± SEM, n = 4). **P < 0.01; ***P < 0.001 versus control.

Stage dependence of the effect of forskolin on the expression of kitlga during folliculogenesis. The follicles at the early vitellogenic (EV) and full-grown (FG) stages were treated with forskolin (10 µM) for 6 h. The data were normalized to the housekeeping gene, ef1a, and expressed as a percentage of the control group at the EV stage (mean ± SEM, n = 4). **P < 0.01; ***P < 0.001 versus control.

Effects of IGF-I on the expression of kitlga in the presence of db-cAMP (A), forskolin (B), and H89 (C). Cultured zebrafish follicle cells were pretreated with db-cAMP (1.5 mM), forskolin (10 µM), or H89 (10 µM) for 30 min before treatment with IGF-I (50 ng/mL) for 6 h. (D) Interactive effects of the PKA and PI3K-Akt pathways on kitlga expression. Cultured zebrafish follicle cells were treated with H89 (10 µM) and LY294002 (50 µM) alone or in combination for 6 h. The data were normalized to the housekeeping gene ef1a and expressed as a percentage of the control group (mean ± SEM, n = 4). Different letters indicate statistical significance (P < 0.05).