Connexin messenger ribonucleic acids in the ovary of Atlantic croaker: Molecular cloning and characterization, hormonal control, and correlation with appearance of oocyte maturational competence

Abstract

Intercellular gap junction channels are composed of connexin (Cx) protein. In the Atlantic croaker ovary, gonadotropin (GtH)-induced oocyte maturational competence is accompanied by an obligatory synthesis of RNA and protein and by the formation of granulosa cell-oocyte gap junctions. However, the GtH control of Cx gene expression in relation to follicular differentiation (including maturational competence) has not been examined in Atlantic croaker or in any other vertebrate species. Moreover, Cx genes and their products have not been characterized in fishes. Therefore, the objectives of this study were to characterize ovarian Cx mRNAs in Atlantic croaker and to determine their regulation by GtH during the acquisition of maturational competence. Polymerase chain reaction was used to amplify Cx cDNA prepared from maturationally incompetent or competent ovaries. Mixed primers for this reaction were based on the high sequence homology of selected regions of known connexins. One major fragment (Cx1) was amplified from incompetent ovary cDNA, and two major fragments (Cx1' and Cx2) from competent ovary cDNA. Sequence analysis showed that Cx1 and Cx1' are identical and that Cx1 and Cx2 have high sequence homology to other Cx cDNAs. A cDNA library made from competent ovaries was screened with Cx1 or Cx2 used as probes. Two cDNAs were isolated: one (Cx1) encoded 282 amino acids (32693 daltons; Cx32.7), and the other (Cx2) encoded 285 amino acids (32 169 daltons; Cx32.2). Both amino acid sequences showed all key features of Cx. Northern blot analysis showed a negligible level of Cx32.2 mRNA in incompetent ovaries, but a substantial rise in its levels accompanied the GtH induction of maturational competence. However, Cx32.7 mRNA levels in incompetent and competent ovaries were similar. In conclusion, selective activation of the ovarian Cx32.2 gene by GtH seems to occur during the acquisition of oocyte maturational competence.