The isolation and identification of connexin in the porcine ovary



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Texas Tech University


Gap Junctions (GJ) are aggregates of intercellular channels responsible for the exchange of cytosolic materials (¡Ü.l kD) between adjoining cells. These channels are comprised of connexin (Cx) proteins. Several studies have suggested that the presence of channels between granulosa cells and the oocyte is needed for maintenance of meiotic arrest in growing follicles and for release of this arrest after the gonadotropin surge. Gap junction function in rat and Xenopus ovaries has drawn the most attention. Other studies have also dealt with mouse, bovine, and Atlantic Croaker (fish) ovaries. The purpose of this study was to isolate and characterize Cx present in the cycling porcine ovary through RT-PCR, sequencing, and Northern blot hybridization.

Polymerase Chain Reaction (PCR) based on mixed primers was used to amplify Cx cDNA fragments isolated from ovaries of cyclic gilts containing corpora lutea. One major fragment and one minor fragment were amplified, cloned, and sequenced. Sequence analysis showed that the major fragment had 94.2% amino acid identity with mouse Cx43, while the minor fragment showed 72% amino acid identity with human Cx26. The latter may be Cx26 or a novel protein. (Full cDNA sequencing is needed to make this determination.) In either case, this is the first report of a Cx26-like mRNA in the mammalian ovary. Ovaries and corpora lutea were removed from prepubertal and cycling gilts and used for mRNA isolation. Northern blotting for Cx43 showed detectable levels in ovaries containing small (<3mm), small-medium (<3 to 5mm), and small-large (<3 to >5mm) follicles and also in the corpora lutea. However, Northern blotting for the Cx26- like mRNA failed to show a positive signal, indicating the presence of low amounts of this Cx in the ovaries.



Connexins, Sows, Swine -- Reproduction