Follicle-stimulating hormone regulation of estrogen biosynthesis in the adult rodent ovary requires β-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.

Beta-catenin (CTNNB1), a key component of wingless-type mouse mammary tumor virus integration site family (WNT) signaling, participates in follicle stimulated hormone-mediated regulation of estrogen (E2) production. The purpose of these studies was to determine if CTNNB1’s contribution to FSH-mediated steroidogenesis in primary rat granulosa cells was due in part to extracellular stimulation of the canonical WNT signaling pathway. To achieve this purpose, primary cultures of rat granulosa cells were exposed to vehicle or a canonical member of the WNT signaling pathway, WNT3A, before co-culture and in the presence or absence of FSH for 24 h. Activation of the canonical WNT signaling pathway was determined by dose-dependent induction of Axin2 mRNA expression and stimulation of the CTNNB1/T cell factor promoter-reporter TOPflash. WNT pathway induction was demonstrated at doses of 50 and 500 ng/mL of WNT3A. Granulosa cells treated with WNT3A in combination with FSH had enhanced CTNNB1/T cell factor transcriptional activity above cells treated with WNT3A alone. Steroidogenic enzymes and ovarian differentiation factor mRNAs were quantified via quantitative PCR. Expression of steroidogenic enzyme mRNAs aromatase (Cyp19a1), P450 side chain cleavage (Cyp11a1), and steroidogenic acute regulatory protein (Star) were increased following FSH treatment. Co-incubation of WNT3A and FSH reduced the ability of FSH to stimulate steroidogenic enzymes and subsequent E2 and progesterone (P4) production. Concomitant activation of FSH and WNT pathways results in marked reduction of ovarian differentiation factors, LH receptor (Lhcgr) and inhibin-alpha (Inha). Therefore, WNT inhibits FSH target genes and steroid production associated with maturation and differentiation of the ovarian follicle.

BackgroundHeifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments: non-implanted (n = 5), Revalor 200 for 28 d (n = 5), or Revalor 200 for 84 d (n = 6). Small follicle (1 to 5 mm) granulosa cells were isolated from each pair and incubated with phosphate buffered saline (n = 16) or 100 ng/mL follicle stimulating hormone (n = 16) for 24 h.ResultsGranulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar (P = 0.22) to non-implanted heifers, while medium estradiol concentrations were increased (P < 0.10) at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein (P = 0.05) mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 (P < 0.10) or 84 d (P < 0.05) compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase (P = 0.57) and aromatase (P = 0.23) were demonstrated in implanted or non-implanted heifers.ConclusionsThese results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle stimulating hormone stimulation. Thus, efforts should be made to avoid the use of implanted heifers to study steroidogenesis in small follicle granulosa cell culture systems.

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