Changes in expression and localization of connexin 43 mRNA and protein in porcine ovary granulosa cells during follicular atresia.

Gap junctions contain channels that connect neighboring cells by allowing the movement of molecules smaller than 1,200 Da. They are formed by connexins and may play a crucial role in the regulation of apoptotic cell death. To determine the role of connexin 43 (Cx43), which is dominantly expressed in granulosa cells, in the regulation of granulosa cell apoptosis during follicular atresia, we examined the changes in the expression and localization of Cx43 mRNA and protein in granulosa cells during atresia using the quantitative real-time revese transcription-polymerase chain reaction, in situ hybridization, Western blot, and immunohistochemistry. Stages of follicular atresia were assessed based on histochemical terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end-labeling (TUNEL) and/or the ratio of progesterone and 17beta-estradiol levels in follicular fluid measured by radioimmunoassay. Cx43 mRNA was detected in granulosa cells of secondary follicles and of healthy, early and progressed atretic tertiary follicles, but not in those of primordial or primary follicles. Both phosphorylated/activated and non-phosphorylated/native Cx43 proteins were detected in granulosa cells of secondary and tertiary follicles, but not in those of primordial or primary follicles. Moreover, in tertiary follicles, these Cx43 proteins were expressed most strongly in granulosa cells of healthy follicles, but only trace levels were noted in cells of early atretic and progressed atretic follicles, an indication that the expression levels of Cx43 protein decrease during follicular atresia. These findings indicate that Cx43 is involved in the apoptosis of granulosa cells during atresia in porcine ovaries.