Follicle-stimulating hormone regulation of estradiol production: Possible involvement of WNT2 and β-catenin in bovine granulosa cells1,2

Abstract

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.

Non-canonical WNT5A is a potential regulator of granulosa cell function in cattle

“…We also failed to detect significant quantities of WNT2 mRNA, unlike previous studies in bovine and rodent granulosa cells (Castañon et al, 2012;Ricken et al, 2002). One possible explanation for the discrepancy with the previous bovine study is the differentiation status of the cultured cells, as Castañon et al (2012) cultured cells with serum under luteinizing conditions.…”

“…In humans, WNT2 but not WNT4 mRNA was detected in cumulus cells (Wang et al, 2009), whereas WNT4 and WNT5A transcripts have recently been reported in luteinized granulosa cells (Sanchez et al, 2014). In luteinized bovine granulosa cells, WNT2 mRNA was detected and upregulated by FSH (Castañon et al, 2012), and CTNNB1 has been shown to act downstream of LH to regulate STAR expression and progesterone synthesis (Roy et al, 2009). To our knowledge, there are no reports of WNT expression in non-luteinized granulosa cells of a monovulatory species.…”

“…Abundance of WNT5A mRNA was regulated by FSH, whereas WNT2B and WNT5B mRNA levels were not, which points to a specific regulation of WNT5A in the ovary. Other reports of WNT regulation in granulosa cells show that FSH stimulates abundance of mRNA encoding Wnt2 and Wnt4 (Castañon et al, 2012;Hsieh et al, 2002), therefore this is the first description of the negative regulation of a WNT by FSH. The reduction of WNT5A mRNA was reflected by a reduction of WNT5A protein levels, indicating that altered mRNA abundance has potential physiological relevance.…”

Regulation and Regulatory Role of WNT Signaling in Potentiating FSH Action during Bovine Dominant Follicle Selection

“…To our knowledge, expression of WNT pathway components at specific stages of a follicular wave has not been reported previously. Previous studies demonstrated greater abundance of CTNNB1 protein in large bovine follicles of abattoir origin with high versus low follicular fluid estradiol concentrations [18]. Although follicles were not grouped using known criteria indicative of follicle health status [19], [20], size range of follicles analyzed [21] suggest most had reached dominant follicle stage of follicular wave.…”

Expression and effect of fibroblast growth factor 9 in bovine theca cells

“…Cells were cultured as described earlier with treatments applied for 24 h as follows: control, TNFa (30 ng/ml), and WNT3A (100 ng/ml) with or without IGF1 (30 ng/ml) plus LH (30 ng/ml). Doses of IGF1, LH, WNT3A, and TNFa were selected based on previous studies showing that these doses significantly alter steroidogenesis (Spicer 1998, Spicer & Chamberlain 1998, Castañon et al 2012. After 24 h of treatment, cells were lysed for RNA extraction as described earlier.…”