Follicle-stimulating hormone regulation of estradiol production: Possible involvement of WNT2 and β-catenin in bovine granulosa cells

Follicle-stimulating hormone regulation of estrogen biosynthesis in the adult rodent ovary requires beta-catenin (CTNNB1), but whether CTNNB1 is involved in FSH-induced estrogen production in cattle is unknown. To elucidate the effect of FSH in regulating specific wingless-type mouse mammary tumor virus integration site (WNT)/CTNNB1 pathway components in bovine folliculogenesis and steroidogenesis, granulosa cells and follicular fluid were collected from large antral follicles (8 to 22 mm) from ovaries containing stage-III corpora lutea (d 11 to 17 of an estrous cycle). Follicles were categorized as high estradiol (n = 3; >= 25 ng/mL) or low estradiol (n = 3; <= 14 ng/mL) based on intra-follicular estradiol concentrations. Protein fractions were collected from granulosa cells and CTNNB1 abundance was analyzed by Western blot. Follicles with increased estradiol concentrations had 6-fold greater (P < 0.001) abundances of CTNNB1 compared with those classified as low-estradiol follicles, indicating that the hormonal milieu responsible for increased estradiol content could result in CTNNB1 accumulation. To ascertain specific contributions of FSH to increases in CTNNB1 protein abundances, granulosa cells were isolated from small ovarian follicles (1 to 5 mm) and cultured in the presence or absence of 100 ng/mL FSH for 24 or 48 h. Real-time PCR quantification of aromatase (CYP19A1) and select WNT family members were evaluated in response to FSH treatment. Successful stimulation of granulosa cells with FSH was confirmed by induction of CYP19A1 mRNA and parallel temporal increases of medium estradiol concentrations. Additionally, protein kinase b (AKT), a known FSH target, increased 1.7-fold (P = 0.07). Of the WNT family members analyzed, only WNT2 mRNA was induced after 24 h of FSH treatment compared with controls (0.12-fold and 3.7-fold for control and FSH-treated, respectively; P < 0.05), and WNT2 expression tended (P = 0.11) to remain increased at 48 h in FSH-treated cells compared with controls (1.0-and 3.14-fold, respectively). Furthermore, FSH-treated granulosa cells had greater abundances of total CTNNB1 (P = 0.04) protein. These data demonstrate for the first time that FSH regulates CTNNB1 protein and WNT2 mRNA expressions in bovine granulosa cells, suggesting a potential role of canonical WNT signaling in ovarian steroidogenesis and follicular growth of cattle. Future studies are necessary to determine if FSH directly regulates CTNNB1 through modulation of AKT or indirectly by up regulating WNT2, which subsequently activates the canonical WNT pathway.