Epidermal growth factor regulation of connexin 43 in cultured granulosa cells from preantral rabbit follicles.

Connexin 43 (Cx43), a gap junction protein expressed in differentiated granulosa cells, is necessary for normal follicular development. Cx43 expression and regulation by epidermal growth factor (EGF) were characterized in immature rabbit granulosa cells. Cx43 mRNA was expressed in the granulosa cells of primary follicles, but was undetectable in primordial follicles. Abundant expression of Cx43 mRNA was maintained in the granulosa cells of growing follicles through maturity. Granulosa cells were isolated from early preantral follicles and maintained in monolayer cultures for 72 hr. After the first 24 hr of culture, they were maintained for 48 hr in serum-free medium supplemented with 0, 1, 5, or 10 ng/ml of mouse EGF. Granulosa cell proteins were isolated, solubilized, and evaluated for Cx43 by Western blot analysis using antibodies to rat Cx43. Relative amounts of Cx43 protein (both phosphorylated and nonphosphorylated) were increased (P < 0.05) by EGF in a dose-dependent manner. Northern blot analysis of RNA from cultured granulosa cells demonstrated increased amounts of Cx43 mRNA in the EGF treated cultures (10 ng EGF/ml) relative to controls (P < 0.03). In summary, Cx43 gap junctions are synthesized in granulosa cells following the onset of folliculogenesis in vivo and their expression is enhanced by EGF in vitro.

Epidermal growth factor enhances expression of connexin 43 protein in cultured porcine preantral follicles.

Connexin 43 (Cx43) and gap junctional coupling appear to play a critical role in early follicular development because absence of Cx43 disrupts progression of follicles beyond primary stages in transgenic mouse ovaries. Two experimental culture systems were used to determine whether epidermal growth factor (EGF) stimulates expression of Cx43 in early porcine follicular development. Ovarian explants were collected from 32- to 40-day-old gilts and cultured for 6 days on membrane inserts in Waymouth MB 752/1 medium supplemented with 0, 50, or 500 ng/ml mouse EGF. Western blot analysis demonstrated significant increases (P < 0.05) in relative amounts of Cx43 protein (both phosphorylated and nonphosphorylated) with 50 and 500 ng/ml of EGF as compared with control cultures. Preantral follicles were enzymatically isolated from 70- to 86-day-old gilts and cultured for 8 days in collagen matrices. Medium and EGF treatments were the same as previously described. Western blot analysis demonstrated a significant increase (P < 0.05) in relative amounts of Cx43 protein with 50 and 500 ng/ml of EGF as compared with control cultures. EGF increased expression of Cx43 protein in secondary preantral follicles in a dose-dependent manner, which suggests that EGF or similar growth factor molecules may modulate early folliculogenesis by stimulating expression of Cx43 gap junctions.