Expression of two insulin receptor subtypes, insra and insrb, in zebrafish (Danio rerio) ovary and involvement of insulin action in ovarian function

Present study reports differential expression of the two insulin receptor (IR) subtypes in zebrafish ovary at various stages of follicular growth and potential involvement of IR in insulin-induced oocyte maturation. The results showed that mRNA expression for IR subtypes, insra and insrb, exhibited higher levels in mid-vitellogenic (MV) and full-grown (FG) rather than pre-vitellogenic (PV) oocytes. Interestingly, compared to the levels in denuded oocytes, mRNAs for both insra and insrb were expressed at much higher level in the follicle layer harvested from FG oocytes. Immunoprecipitation using IRβ antibody could detect a protein band of desired size (∼95 kDa) in FG oocyte lysates. Further, IRβ immunoreactivity was detected in ovarian tissue sections, especially at the follicle layer and oocyte membrane of MV and FG, but not PV stage oocytes. While hCG (10 IU/ml) stimulation was without effect, priming with insulin (5 μM) could promote oocyte maturation of MV oocytes in a manner sensitive to de novo protein and steroid biosynthesis. Compared to hCG, in insulin pre-incubated MV oocytes, stimulation with maturation inducing steroid (MIS), 17α,20β-dihydroxy-4-pregnen-3-one (DHP) elicited higher maturational response. Potential involvement of insulin-mediated action on acquisition of maturational competence and regulation of oocyte maturation was further manifested through up regulation of 20β-hydroxysteroid dehydrogenase (20β-hsd), MIS receptor (mPRα), insulin-like growth factor 3 (igf3) and IGF1 receptor (igf1rb), but not cyp19a expression in MV oocytes. Moreover, priming with anti-IRβ attenuated insulin action on meiotic G2-M1 transition indicating the specificity of insulin action and physiological relevance of IR in zebrafish ovary.