To determine the specificity of activin A and inhibin A action, the interaction between activin A and FS or inhibin A and FS was examined. Furthermore, the combined effect of activin A and inhibin A on oocyte maturation was also tested. As shown in Figure 4, activin A (25 ng/ml) or inhibin A (25 ng/ml) alone significantly increased the maturation rate after 24-h incubation. The stimulatory effects of activin A and inhibin A were completely neutralized by FS; in the presence of FS-288 (100 ng/ml), neither activin A nor inhibin A induced oocyte maturation. FS-288 alone did not significantly alter basal level of maturation. When activin A and inhibin A were added together, there was no further increase in maturation rate compared to that of either activin A- or inhibin A-treated groups, indicating that activin A and inhibin A do not have additive effects. These results were consistently seen in follicles incubated with these agents for either 18 h or 24 h.
FS-288 Blocked Gonadotropin- and MIH-Induced Maturation
Gonadotropin and MIH are known to play major roles in oocyte maturation. To test the possibility that activins and/or inhibins are mediators of gonadotropin and MIH action, the interaction between FS-288 and hCG or MIH was examined. Human CG, previously shown to function as a gonadotropin in fish, stimulated oocyte maturation. Combined treatment of hCG and FS-288 resulted in a significant reduction in the rate of maturation compared to that of the hCG-treated group. Furthermore, in the presence of FS-288, hCG no longer had a significant stimulation effect on oocyte maturation (Fig. 5A). MIH alone also increased the rate of maturation significantly. When FS-288 was added together with MIH, the rate of maturation was significantly lower than that found in the MIH-treated group but was still significantly higher than the control value (Fig. 5B). These results indicate that FS completely blocks gonadotropin-induced maturation but only partially blocks MIH-stimulated oocyte maturation.
Gonadotropin Up-Regulated the Expression of Activin A-and Inhibin A-Like Molecules
The finding that FS-288 neutralized the effect of hCG on oocyte maturation suggests that activins and/or inhibins are local regulators mediating gonadotropin-induced oocyte maturation. To further test this hypothesis, fish were given injections of hCG, and ovaries were removed at 12, 24, and 30 h after injection. Proteins were then extracted from the ovaries and subjected to Western blot analysis using the antibody against the (3A subunit. At 12 and 24 h after hCG injection, there were significantly higher levels of both ac-tivin A- and inhibin A-like molecules in the ovaries of hCG-treated fish than in those of the control fish (Fig. 6). However, the expression levels of activin A and inhibin Alike molecules were similar between the control group and the hCG-treated group at 30 h after injection (Fig. 5B).
FIG. 4. Interaction among activin A, inhibin A, and FS on final oocyte maturation. Follicles were treated for 24 h with activin A (25 ng/ml), inhibin A (25 ng/ml), FS-288 (100 ng/ml) alone, activin A plus FS-288 or inhibin A, or inhibin A plus FS-288. Data are mean ± SEM of three experiments with three replicates in each experiment. Different letters denote statistical significance (P < 0.05). Act, activin A; Inh, inhibin.
FIG. 5. Interaction between hCG and FS-288 (A) or MIH and FS-288 (B) on the induction of final oocyte maturation. A) Follicles were incubated with hCG (10 IU/ml) and FS-288 (100 ng/ml), either alone or in combination, for 24 h. B) Follicles were treated with MIH (1 ng/ml) and FS-288 (100 ng/ml), either alone or in combination, for 24 h. Different letters denote statistical significance (P < 0.05). Data are mean ± SEM of three experiments with three replicates in each experiment.
FIG. 6. Effects of hCG on the expression of activin A- and inhibin A-like molecules. Fish received injections of saline (control) or 1 IU hCG; and 12, 24, and 30 h after injection, ovaries were removed and protein was extracted. Western blot analyses were performed, and the intensities of 26- and 32-kDa bands were analyzed by a densitometer. Data are expressed as the percentage of control at the respective time point. * Significantly higher than the control (P < 0.05).