Canonical WNT Signaling Inhibits Follicle Stimulating Hormone Mediated Steroidogenesis in Primary Cultures of Rat Granulosa Cells

Abstract

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.

Fibroblast growth factor 9 (FGF9) regulation of cyclin D1 and cyclin-dependent kinase-4 in ovarian granulosa and theca cells of cattle

“…Membranes were then washed and the secondary goat anti-rabbit HRP-conjugated antibody (ImmunoPure® HRP-conjugated Goat Anti-Rabbit IgG; Pierce Biotechnology, Rockford, IL; diluted in 5% milk at 1:15,000 for CDK4 detection or at 1:20,000 to 1:25,000 for CCND1 detection) was incubated with membranes for 1 h at 25°C on a rocking platform shaker after which membranes were washed. CDK4 membranes were then dried and incubated with Immobilon® Chemiluminescent HRP Substrate for 3 min, wrapped in plastic, exposed to CL-XPosure Film for 1 sec up to 10 min, and developed with a Mini-Medical X-Ray film processor (AFP Imaging, Elmsford, NY) as previously described (Castanon et al, 2012; Stapp et al, 2014). CCND1 membranes were dried incubated with SuperSignal® West Femto Maximum Sensitivity Substrate for 5 min, placed in a plastic page protector, and developed with the FluorChem™ E FE0622 Imager from ProteinSimple (San Jose, CA) on movie mode for 180 min chemiluminescence detection and automatic marker detection.…”

“…CCND1 membranes were dried incubated with SuperSignal® West Femto Maximum Sensitivity Substrate for 5 min, placed in a plastic page protector, and developed with the FluorChem™ E FE0622 Imager from ProteinSimple (San Jose, CA) on movie mode for 180 min chemiluminescence detection and automatic marker detection. After developing, membranes were washed and re-probed for β-actin (that served as a loading control; Castanon et al, 2012; Stapp et al, 2014) using rabbit monoclonal antibody against human β-actin from Cell Signaling Technology, Inc. (Danvers, MA) at concentrations of 1:10,000 to 1:15,000 for CDK4 and CCND1 target membranes overnight at 4°C on a rocking platform shaker. The membranes were then washed and incubated with secondary antibody at 1:5,000 for CDK4 membranes or 1:25,000 to 1:40,000 for CCND1 membranes on a rocking platform shaker for 1 h at 25°C.…”

The role of WNT signaling in adult ovarian folliculogenesis

“…A series of studies have identified the expression and regulation of WNT ligands and downstream WNT signaling components in the developing follicle and corpus luteum of rats, mice, humans, and cattle (Hsieh et al 2002, Ricken et al 2002, Harwood et al 2008, Wang et al 2009, Castanon et al 2012, Gupta et al 2014) (Table 1). However, characterization of specific WNT molecules during folliculogenesis has been focused primarily on Wnt2 /WNT2 and Wnt4 /WNT4 in mice, rats and humans, although recent studies have unveiled contributions of frizzled receptor agonist, WNT3A in follicular development and steroid production of mice and rats (Li et al 2014, Stapp et al 2014). …”

“…Consistent with β-catenin's role in regulation of steroidogenesis is the demonstrated ability of FSH to directly increase β-catenin protein accumulation (Castanon et al . 2012, Stapp et al . 2014) and β-catenin/TCF dependent transcriptional activity in granulosa cells (Fan et al .…”

“…2014) and β-catenin/TCF dependent transcriptional activity in granulosa cells (Fan et al . 2010, Stapp et al . 2014).…”