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Androgen regulates the expression and function of connexin 43 in granulosa cells via the PKC signalling pathway


 Background

Poor ovarian response (POR) is one of the most challenging issues in assisted reproductive technology. Given the importance of androgens in follicle recruitment, growth and development, domestic and foreign reproductive centers have added androgen as an adjuvant to in vitro fertilization protocols for patients with POR to improve the ovarian response. The mechanism underlying the effects of androgens is unclear. The aim of this research was to study the effects of testosterone on granulosa cells and the underlying mechanisms.
Methods

The human ovarian granulosa cell line KGN was treated with testosterone at various concentrations, an androgen receptor antagonist (flutamide) and a protein kinase C (PKC) inhibitor (GF109203X). The protein levels of PKC and phosphorylated connexin 43 (p-Cx43) were measured using Western blot analysis. The location and distribution of Cx43 and p-Cx43 were determined by immunofluorescence. Gap junction intercellular communication (GJIC) was monitored using the scrape loading/dye transfer method.
Results

The results showed that treatment with testosterone at a physiological level significantly increased the PKC and p-Cx43 levels and GJIC activity in a concentration-dependent manner. However, the expression of PKC, p-Cx43 levels and GJIC activity in the cells treated with testosterone above physiological concentrations (10− 5 M) decreased compared with those in the cells treated with 10− 7 M testosterone. Compared to the 10− 7 M testosterone-treated cells, the KGN cells treated with flutamide had much lower levels of PKC and p-Cx43. The levels of p-Cx43 and GJIC activity in the GF109203X-treated group were decreased compared to those in the 10− 7 M testosterone group. Immunofluorescence showed that testosterone treatment had no effect on the localization and distribution of gap junction proteins.
Conclusions

This study suggests that androgen can increase GJIC activity in granulosa cells by phosphorylating Cx43 via the PKC signalling pathway to promote oocyte development. The results of this study might provide a theoretical basis for androgen pretreatment of patients with POR.