THE REGULATION AND FUNCTION OF THE OVARIAN-DERIVED INSULIN-LIKE GROWTH FACTOR SYSTEM IN ZEBRAFISH (Danio rerio)

Insulin-like growth factors (IGF) are known paracrine/autocrine regulators of ovarian development in teleosts. Initial studies investigated the hormonal and intracellular signal cascades involved in regulating the expression of ovarian-derived IGFs in zebrafish (Danio rerio). Quantitative real-time PCR was used to quantify the expression of igf3, igf2a, and igf2b in full grown immature (FG; 0.57-0.65 mm) and mid-vitellogenic (MV; 0.45-0.56 mm) follicles. Addition of the gonadotropin analogue human chorionic gonadotropin (hCG) and the adenylate cyclase activator forskolin increased igf3 expression in FG and MV follicles, but had no effect on igf2a or igf2b expression. The effects of hCG were blocked by the addition of the protein kinase A inhibitor H-89. Pituitary adenylate cyclase activating peptide stimulated a small increase in igf3 expression in FG follicles, while growth hormone and salmon gonadotropin releasing hormone had no effect on igf3, igf2a, or igf2b expression. Treatment with melittin, prostaglandin F2α, and prostaglandin E2 inhibited igf3 and igf2b expression in FG follicles whereas the protein kinase C activators, PMA and A23187, significantly inhibited igf3, igf2a, igf2b expression in FG and MV follicles. Secondary studies investigated the involvement of ovarian-derived IGFs in mediating the ovarian actions of gonadotropins on cell survival and steroidogenesis. Treatment of FG follicles with recombinant human IGF-I, hCG, or forskolin inhibited the induction of caspase-3/7 activity, which was used as a measure of apoptosis. The effects of hCG and forskolin on caspase-3/7 were attenuated by co-treatment with NVP-AEW54, an IGF-I receptor antagonist. hCG increased production of the maturation-inducing steroid 17α, 20β-dihydroxy-4pregnen-3-one and co-treatment with NVP-AEW541 had no effect. These results suggest there is a high degree of hormonal specificity in regulating IGFs in the zebrafish ovary and the ovarianderived IGFs, presumably IGF-III, are downstream mediators of gonadotropin-dependent cell survival, but are not involved in gonadotropin-induced steroidogenesis.