Molecular cloning and expression of connexin 32.3 cDNA in the ovary from the red seabream (Pagrus major).

This study examined connexin (Cx) gene activity in relation to oocyte maturation in the red seabream (Pagrus major) ovary. Mixed primers for the polymerase chain reaction (PCR) were designed based on the high sequence homology of selected regions of known Cx genes. PCR-amplified cDNA fragments generated by 3' and 5' rapid amplication of cDNA ends (RACE) were combined to generate full-length cDNA sequences. The 1212-bp cDNA has an open reading frame encoding 282 amino acids, with a molecular mass of 32.3 kDa (red seabream Cx32.3). Hydropathy plots of red seabream Cx32.3 show the four typical major hydrophobic and four major hydrophilic regions of Cx proteins. Typical Cx consensus sequences are observed in the first and second extracellular loops. The ovarian follicles of matured female seabream were incubated in the presence of 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP, 10 ng/ml), gonadotropin (GtH)-I (300 ng/ml) and GtH-II (300 ng/ml). Northern blot analysis of poly(A)(+) RNA extracted from the ovarian follicles were hybridized with red seabream Cx32.3 and beta-actin probes. The transcription level of PmCx32.3 in the presence of DHP, PmGtH-I and PmGtH-II was significantly higher than in the control.