Hormonal Regulation and Cellular Distribution of Connexin 32.2 and Connexin 32.7 RNAs in the Ovary of Atlantic Croaker

Abstract

The in vitro effects of human chorionic gonadotropin (hCG) on ovarian connexin (Cx) 32.2 and 32.7 RNA levels and ovarian follicle maturation were assessed, and the cellular distribution of Cx transcripts in the ovary was determined. hCG caused a concentration-dependent induction of Cx32.2 RNA, which peaked coincidentally with the appearance of morphological indices of oocyte maturational competence (OMC). Cx32.2 RNA levels declined thereafter in all treatment groups, although this decline was not accompanied by the onset of germinal vesicle breakdown (GVBD) at the lowest hCG concentration used. The levels of Cx32.7 RNA initially declined and subsequently increased to preincubation values after hCG treatment, but these changes were not dependent on hCG concentration. In a separate experiment, the decline in Cx32.7 RNA occurred in the presence or absence of hCG and was prevented by low (physiological) concentrations of estradiol-17β (E2) or by protein kinase C (PKC) inhibitor, but was enhanced in the presence of high E2 concentrations or of PKC activator. These changes in Cx32.7 RNA abundance were not associated with any indices of oocyte maturation. In situ hybridization of tissue sections showed the presence of Cx32.2 and Cx32.7 RNA in somatic cells of the ovarian follicle but not in oocytes. Cx32.2 RNA seemed to be present in granulosa and thecal cells, but the assay resolution was insufficient to reliably determine the distribution of Cx32.7 transcript by somatic cell type. In view of earlier findings that Cx32.2-based (but not Cx32.7-based) connexons can form functional homotypic channels, these results indicate that Cx32.2 gene expression in granulosa cells is sufficient for the formation of homologous gap junctions (GJ). Northern blot of RNA extracts from ovulated eggs, which are free of follicle cells, showed the presence of relatively low levels of both Cx RNAs. Thus, it is possible that Cx32.2 is present in oocytes and that it participates in heterologous (homotypic) GJ formation between the oocyte and the granulosa cells. In conclusion, Cx32.2 RNA levels in somatic cells of the ovarian follicle correlated positively with morphological indices of OMC acquisition, but subsequently declined during GVBD. These changes in Cx32.2 RNA may function in the regulation of GJ contacts during follicular maturation.