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. 2019 Dec 13;98(1):skz376. doi: 10.1093/jas/skz376

Regulation of the transcription factor E2F1 mRNA in ovarian granulosa cells of cattle

Breanne C Morrell 1, M Chiara Perego 1,b, Excel Rio S Maylem 1, Lingna Zhang 1,c, Luis F Schütz 1,d, Leon J Spicer 1,✉
PMCID: PMC6986437  PMID: 31832639

Abstract

The E2F family of transcription factors plays an important role in the control of the cell cycle, cell proliferation, and differentiation, and their role in ovarian function is just emerging. Although some evidence suggests a possible role of E2F1 in ovarian follicular development, what regulates its production in ovarian cells is unknown. Objectives of this study were to determine whether: (i) E2F1 gene expression in granulosa cells (GCs) and theca cells (TCs) change with follicular development and (ii) E2F1 mRNA abundance in TC and GC is hormonally regulated. Using real-time PCR, E2F1 mRNA abundance in GC was 5.5-fold greater (P < 0.05) in small (SM; 1 to 5 mm) than large (LG; >8 mm) follicles, but in TC, E2F1 expression did not differ among follicle sizes. SM-follicle GC had 2.1-fold greater (P < 0.05) E2F1 mRNA than TC. In SM-follicle GC, FGF9 induced a 7.6-fold increase in E2F1 mRNA abundance; however, FGF9 did not affect (P > 0.10) abundance of E2F1 mRNA in LG-follicle TC or GC. Follicle-stimulating hormone (FSH) had no effect (P > 0.10) on E2F1 gene expression in SM- or LG-follicle GC. SM-follicle GC were concomitantly treated with insulin-like growth factor 1 (30 ng/mL), FSH (30 ng/mL), and either 0 or 30 ng/mL of FGF9 with or without 50 µM of an E2F inhibitor (E2Fi; HLM0064741); FGF9 alone increased (P < 0.05) GC numbers, whereas E2Fi alone decreased (P < 0.05) GC numbers, and concomitant treatment of E2Fi with FGF9 blocked (P < 0.05) this stimulatory effect of FGF9. Estradiol production was inhibited (P < 0.05) by FGF9 alone and concomitant treatment of E2Fi with FGF9 attenuated (P < 0.05) this inhibitory effect of FGF9. SM-follicle GC treated with E2Fi decreased (P < 0.05) E2F1 mRNA abundance by 70%. Collectively, our studies show that GC E2F1 mRNA is developmentally and hormonally regulated in cattle. Inhibition of E2F1 reduced FGF9-induced GC proliferation and attenuated FGF9-inhibited estradiol production, indicating that E2F1 may be involved in follicular development in cattle.

Keywords: cattle, E2F1 transcription factor, granulosa cell, ovarian follicle, theca cell

Introduction

Endocrine and paracrine regulators of ovarian follicular growth include gonadotropins: LH and follicle-stimulating hormone (FSH) (Kaipia and Hsueh, 1997; Howles, 2000); steroids: estrogens, progestins, and androgens (Hillier and Tetsuka, 1997; Schams and Berisha, 2002); and growth factors: insulin-like growth factors (IGFs) (Spicer and Echternkamp, 1995; Spicer, 2004), transforming growth factor β family (Mazerbourg and Hsueh, 2006; Regan et al., 2018), and fibroblast growth factors (FGF) (Berisha et al., 2004; Schütz, et al., 2016). One of the FGF family members, FGF9, has been described as an anti-differentiation factor because it stimulates proliferation while inhibiting steroidogenesis in both granulosa cells (GCs) and theca cells (TCs) of cattle (Schreiber and Spicer, 2012; Schreiber et al., 2012; Totty et al., 2017). In particular, FGF9 inhibits FSH plus IGF1-induced estradiol production and FSH receptor (FSHR) mRNA levels in cultured bovine GC (Schreiber and Spicer, 2012). Moreover, abundance of FGF9 mRNA is less in GC of cystic vs. normal follicles (Grado-Ahuir et al., 2011), is greater in GC of small (SM) vs. large (LG) follicles (Schreiber et al., 2012), is less in GC of estrogen-active vs. estrogen-inactive follicles (Schütz, et al., 2016), and is greater in GC than TC of cattle (Schreiber et al., 2012; Schütz, et al., 2016). In bovine GC and TC, FGF9 stimulates cell proliferation, in part, via induction of a mitogen-activated protein kinase pathway (Totty et al., 2017), but what additional intracellular mediators and transcription factors are induced by FGF9 remain unknown.

E2F transcription factors include a family of 10 proteins (i.e., E2F1 to 8) encoded by 8 genes (Ertoson et al., 2016; Johnson et al., 2016), and are involved in cell cycle progression (Lavia and Jansen-Dürr, 1999; Ertosun et al., 2016), playing roles in cell proliferation, apoptosis, and differentiation (DeGregori and Johnson, 2006; Zhang et al., 2017b). The E2F are classified as activators (E2F1 to 3), repressors (E2F4 to 6), and atypical repressors (E2F7 and 8) activating or repressing transcription of regulators involved in cell cycle progression (Zhan et al., 2014; Thurlings and de Bruin, 2016). We decided to investigate E2F1 and its possible role in bovine follicular development, its control by FGF9 and its regulation of FSH action in GC because: (i) E2F1 mRNA abundance is 2-fold greater in GC of presumed growing follicles than those in the plateau phase of growth (Douville and Sirard, 2014); (ii) in human and rat GC, overexpression of E2F1 decreased FSHR transcription (Putowski et al., 2001); (iii) E2F1 mRNA is overexpressed in some ovarian cancers (Reimer et al., 2006; Suh et al., 2008; Zhan et al., 2016) and is reported to be a marker and predictor of poor prognosis in ovarian cancer (Zhang et al., 2017b; Farra et al., 2019); and (iv) expression of FGFRs appear to be regulated by several E2F (Tashiro et al., 2003; Kanai et al., 2009). In addition, we evaluated the effect of inhibition of E2F, using an E2Fi, on FGF9-induced proliferation, steroidogenesis and gene expression in bovine GC. We hypothesized that increased FGF9 production in SM follicles (Schutz et al., 2016) stimulates E2F1 gene expression in bovine GC which directly stimulates cell proliferation. Thus, the objectives for the present study were to determine whether E2F1 mRNA abundance changes during bovine follicular development and determine the hormonal regulation of E2F1 mRNA in GC.

Materials and Methods

Tissue, Reagents, and Hormones

Ovaries from beef heifers were collected at a slaughterhouse where humane slaughter practices were followed, according to USDA guidelines.

Reagents used were: Dulbecco’s modified Eagle medium (DMEM) and Ham’s F-12 (F12), gentamicin, streptomycin/penicillin, TRI reagent, sodium bicarbonate, collagenase, and deoxyribonuclease (DNase) from Sigma-Aldrich Chemical Co. (St. Louis, MO); ovine follicle stimulating hormone (FSH) from National Hormone and Pituitary Program (Torrance, CA); carrier-free recombinant human IGF1 and FGF9 from R&D Systems (Minneapolis, MN); E2F inhibitor (E2Fi; HLM006474l) from EMD Millipore/CalBiochem (Billerica, MA) shown to inhibit E2F1, E2F2, E2F3, and E2F4 (Ma et al., 2008; Kurtyka et al., 2014); fetal calf serum (FCS) from Atlanta Biologicals Inc. (Flowery Branch, GA); testosterone Steraloids (Wilton, NH); and I125 labeled estradiol (E2) and progesterone (P4) from MP Biomedicals (Santa Ana, CA).

Cell Culture

Ovaries from nonpregnant beef heifers were collected at a local slaughterhouse and processed for isolation of GC and TC as previously described (Langhout et al., 1991; Stewart et al., 1995; Spicer and Chamberlain, 1998). Both GC and TC from SM (1 to 5 mm) and LG (8 to 20 mm) follicles were collected for RNA isolation as previously described (Zhang et al., 2017a). For cell culture, SM follicles were aspirated and GC isolated, whereas LG follicles were bisected after aspiration of follicular fluid, and GC and TC were isolated via blunt dissection as previously described (Spicer and Chamberlain, 1998; Schreiber et al., 2012). The TC were then enzymatically digested for 1 h at 37 °C, filtered and washed in a serum-free medium as previously described (Spicer and Chamberlain, 1998; Schreiber et al., 2012; Zhang et al., 2017a). Isolated GC and TC were then resuspended in serum-free medium (38 mM sodium bicarbonate, 0.12 mM of gentamicin, and 2 mM glutamine in 1:1 DMEM and F-12) containing enzymes (1.25 mg/mL collagenase and 0.5 mg/mL of DNase) to help prevent the cells from clumping as previously described (Pizzo et al., 2015).

Viability of GC and TC was determined by trypan blue exclusion method as previously described (Robinson et al., 2018; Nichols et al., 2019). Then, GC and TC were plated on 24-well Falcon multi-well plates (No. 3047, Becton Dickinson, Lincoln Park, NJ) with 1 mL of medium/well and cultured in 10% FCS for 48 to 72 h with medium changed every 24 h. Once cells reached 80% confluency, they were washed twice with 0.5 mL of serum-free medium, and treatments were applied in serum-free medium for 12, 24, or 48 h depending on the experiment. This culture system has been developed so that GC and TC retain hormonally responsive aromatase activity and do not luteinize with time in culture because: (i) P4 production remains constant or does not increase with time using this culture paradigm (Langhout et al., 1991; Spicer and Chamberlain, 1998), (ii) the morphology of the GC retain a fibroblastic appearance (Chamberlain and Spicer, 2001), (iii) aromatase activity of GC remains responsive to FSH and IGF1, and their responses increase between days 3 and 4 of culture (Spicer and Chamberlain, 1998; Spicer et al., 2002), and (iv) GC from SM and LG follicles have little or no P4 response to LH (Spicer et al., 2002).

Radioimmunoassays (RIA) and Cell Counting

To determine concentrations of E2 and P4 in medium, specific RIA for E2 and P4 were conducted as previously described (Schreiber and Spicer, 2012; Pizzo et al., 2015). Intra-assay coefficient of variation averaged 8.3% and 9.9% for E2 and P4 RIA, respectively.

Cell numbers were determined using a Z2 Coulter Particle Count and Size Analyzer (Beckman Coulter, Hialeah, FL) as previously described (Schreiber et al., 2012; Albonico et al., 2017). Briefly, after GC were incubated at 37 °C for 20 min with 0.5 mL of 2.5 mg/mL trypsin-saline solution, GC were scraped from the plates and cell clumps broken up by pipetting repeatedly through a 500-µL pipette tip three to five times, wells were rinsed with 0.5 mL saline, and both added to 9 mL of saline.

RNA Extraction and Real-Time PCR Analysis

For fresh GC, 0.5 mL TRI reagent was added to each sample, and samples were transferred to 1.5 mL Eppendorf tubes and stored at −80 °C. For fresh TC, samples were placed in 0.5 mL RNAlater stabilization solution and kept overnight at 4 °C before being homogenized with PCR Tissue Homogenizing Kits (Omni International, Inc. Warrenton, VA) as previously described (Aad et al., 2012), and then stored at −80 °C in 0.5 mL TRI reagent for RNA extraction. For in vitro TC and GC gene expression experiments, medium was aspirated, discarded, and 0.25 mL TRI reagent was added to each well. Treatments were applied to 4 wells and cells from 2 wells were combined into 1 sample for 2 samples of RNA for each treatment. Samples were stored at −80 °C, thawed, RNA extracted, dissolved in 16.5 µL of diethylpyrocarbonate (DEPC)-treated water (Life Technologies, Carlsbad, CA), and stored at −80 °C as previously described (Voge et al., 2004; Schreiber et al., 2012; Zhang et al., 2017a).

Total RNA from samples was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE) at 260 nm, and diluted to a final concentration of 10 ng/µL with DEPC-treated water for subsequent quantitative PCR as previously described (Schutz et al., 2016; Dentis et al., 2017). The E2F1 (2207 bp mRNA; Accession NM_001206079.1) forward primer was constructed between bp 1,060 and 1,080 with a Tm of 56.0 °C and a sequence of 5′-GAGCAAGAACCTCTGCTTTCC-3′. The reverse E2F1 primer was constructed between bp 1,190 and 1,209 with a Tm of 56.4 °C and a sequence of 5′-GGACAGGGTGATGAACTCCT-3′. The E2F1 probe was constructed between bp 1,148 and 1,171 with a Tm of 63.8 °C and a sequence of 5′-TCCTGGAGCACGTGAAGGAGGACT-3′ possessing 5′FAM/ZEN/3′IBFQ as the reporter and double quencher dyes. Primers and probe were purchased from Integrated DNA Technologies Inc. (San Diego, CA). The E2F1 primers spanned introns (forward primer spanned exons 6 and 7, and reverse primer and probe are located in exon 7). The bovine FSHR primers and probe (i.e., sequences and information) for quantitative PCR were described previously by Spicer and Aad (2007).

Quantification of E2F1 mRNA abundance was measured using one-step PCR with iTaq Universal Probe One-Step Kit (Bio-Rad, Hercules, CA), performed on a CFX96 Real-Time System in 96-well plates (Bio-Rad). Total reaction volume of 20 µL consisting of 400 nM of forward and reverse E2F1 primers and 200 nM of E2F1 probe. The protocol for setting the reverse transcription and amplification processes was: 10 min at 50 °C for reverse transcription, 95°C for 5 min for denaturing, 50 cycles of 95 °C for 10 s for annealing, and 60.4 °C for 30 s for extension. On the 96-well plates, samples were placed in duplicate wells to determine an average threshold cycle (Ct) value. Target gene expression was normalized to the housekeeping gene 18S rRNA as previously described (Zhang et al., 2017a; Nichols et al., 2019; Morrell et al., 2019). Quantity of target gene mRNAs was expressed as 2− ΔΔCt using the relative comparative threshold cycle method as previously described (Livak and Schmittgen, 2001; Voge et al., 2004). Briefly, ΔCt was calculated by subtracting the 18S Ct from the target gene Ct value. The ΔΔCt value was calculated by subtracting the highest ΔCt from all other ΔCt values. The fold change of mRNA abundance for target genes was calculated as 2−ΔΔCt, and for experiments 2, 3, and 4 expressed as fold of control values.

Experimental Design

Experiment 1 was designed to determine whether E2F1 mRNA abundance in GC and TC changes with follicle development. Cells were collected from SM (<6 mm) and LG (8–20 mm) follicles obtained from slaughterhouse ovaries as previously described (Zhang et al., 2017a). For each LG-follicle GC and LG-follicle TC sample, cells were collected from seven individual follicles from at least three animals. For each SM-follicle GC and SM-follicle TC sample, GC and TC were pooled from 3 to 5 SM follicles from individual ovaries from at least five animals. The fresh cells were lysed with TRI reagent and extracted for RNA as described earlier.

To determine the hormonal regulation of E2F1 in TC and GC, three experiments were conducted. Experiment 2 evaluated if FGF9 could induce E2F1 mRNA abundance in SM-follicle GC. Cells were cultured for 72 h in 10% FCS, washed, serum-starved for 24 h, and then treated with either 0 or 30 ng/mL of FGF9 for 12 h. Cells were lysed in TRI reagent and RNA extracted as described earlier. Dose of FGF9 was based on previous studies (Schreiber and Spicer, 2012; Totty et al., 2017). Cells were serum-starved to synchronize the cell cycle (Totty et al., 2017).

To determine if FGF9 also induced E2F1 in LG-follicle GC and compare the effect to that in LG-follicle TC, Experiment 3 evaluated the FGF9-induced E2F1 mRNA abundance in LG-follicle GC and TC. Cells were cultured as in Exp. 2, washed, serum-starved for 24 h, and then treated in serum-free medium with either 0 or 30 ng/mL of FGF9 for 12 h. Cells were lysed in TRI reagent and RNA extracted as described earlier.

Experiment 4 was designed to determine if FSH affected E2F1 mRNA in LG- and SM-follicle GC. Cells were cultured for 48 h in 10% FCS, washed, and treated with either 0 or 30 ng/mL of FSH for 24 h. Cells were lysed in TRI reagent and RNA extracted as described earlier. Dose of FSH was based on previous studies (Spicer et al., 2002; Schreiber and Spicer, 2012).

Experiment 5 was designed to evaluate the effects of E2Fi and FGF9 on GC proliferation and E2 and P4 production. Cells were cultured for 48 h in 10% FCS, washed, and then treated in serum-free medium for an additional 48 h as follows: control, E2Fi (50 µM), FGF9 (30 ng/mL), and E2Fi plus FGF9; medium was changed after 24 h of treatment. All cells had an additional treatment of testosterone (500 ng/mL as an estrogen precursor), IGF1 (30 ng/mL), and FSH (30 ng/mL) to induce cell proliferation and steroidogenesis (Spicer and Chamberlain, 1998; Spicer et al., 2002). Cell numbers and E2 and P4 production were measured as described earlier. Doses of testosterone, IGF1, FSH, E2Fi, and FGF9 were based on previous studies (Spicer et al., 2002; Ma et al., 2008; Schreiber and Spicer, 2012; Kurtyka et al., 2014; Nichols et al., 2019).

Experiment 6 was designed to evaluate the effects of an E2Fi on E2F1 and FSHR mRNA abundance in SM-follicle GC. Cells were cultured for 48 h in 10% FCS, washed, and treated for 24 h as follows: control or E2Fi (50 µM). The GC were lysed and RNA extracted as described earlier. Dose of the E2Fi was based on experiment 5 and previous studies (Ma et al., 2008; Nichols et al., 2019).

Statistical Analyses

Treatment effects on dependent variables (e.g., E2F1 mRNA abundance) were determined using ANOVA and the general linear models procedure of SAS (version 9.4, SAS Institute Inc., Cary, NY). Data of experiments 1 and 5 were analyzed using a two-way ANOVA. Data of experiments 2, 3, 4, and 6 were analyzed using one-way ANOVA. Significance was determined at P < 0.05. If significant main effects and interactions were observed in ANOVA, mean differences were determined using Fisher’s protected least significant differences test. Data were presented as means ± SEM.

Results

In experiment 1, follicle size significantly affected E2F1 mRNA such that abundance of E2F1 mRNA was 5.5-fold greater (P < 0.01) in SM-follicle GC when compared with LG-follicle GC (Figure 1). Also, abundance of E2F1 mRNA was 2.1-fold greater (P < 0.05) in SM-follicle GC vs. SM-follicle TC (Figure 1). Abundance of E2F1 mRNA in TC did not differ (P > 0.10) between LG and SM follicles (Figure 1).

Figure 1.

Figure 1.

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Expression of E2F1 in freshly collected granulosa and TCs from SM and LG follicles of experiment 1. Results are normalized to constitutively expressed 18S ribosomal RNA. a,bMeans ± SE (n = 7) without a common letter differ (P < 0.05).

To determine the hormonal regulation of E2F1 gene expression in TC and GC, three experiments were conducted. In experiment 2, 12-h treatment with 30 ng/mL of FGF9 tended to increase (P < 0.10) abundance of E2F1 mRNA by 7.6-fold compared with controls (Figure 2).

Figure 2.

Figure 2.

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Effect of FGF9 (30 ng/mL) on E2F1 mRNA expression GCs from SM follicles (experiment 2). Granulosa cells were serum starved for 24 h and then treated for 12 h with either 0 or 30 ng/mL of FGF9. Results are normalized to constitutively expressed 18S ribosomal RNA and expressed as fold (mean ± SE, n = 6) of control values with no additions. Asterisk indicates mean differs from its respective control mean (P < 0.10).

To determine if FGF9 also induced E2F1 in LG-follicle GC and compare the response with LG-follicle TC, experiment 3 evaluated the FGF9-induced E2F1 mRNA abundance in LG-follicle GC and TC. Treatment with 30 ng/mL of FGF9 for 12 h had no effect (P > 0.50) on E2F1 mRNA abundance in LG-follicle GC or TC (Figure 3).

Figure 3.

Figure 3.

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Comparison of the effect of FGF9 (30 ng/mL) on E2F1 mRNA expression in TCs and GCs from LG follicles (experiment 3). Both TC and GC were serum starved for 24 h and then treated for 12 h with either 0 or 30 ng/mL of FGF9. Results are normalized to constitutively expressed 18S ribosomal RNA and expressed as fold (mean ± SE, n = 6) of control values for each cell type with no additions.

To determine if FSH affected E2F1 in SM- and LG-follicle GC, experiment 4 was conducted. Treatment with 30 ng/mL of FSH for 24 h had no effect (P > 0.10) on E2F1 mRNA abundance in SM- or LG-follicle GC (Figure 4).

Figure 4.

Figure 4.

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Comparison of the effect of FSH (30 ng/mL) on E2F1 mRNA expression in GCs from SM and LG follicles ( experiment 4). Cells were treated for 24 h with either 0 or 30 ng/mL of FSH. Results are normalized to constitutively expressed 18S ribosomal RNA and expressed as fold (mean ± SE, n = 6) of control values for each cell type with no additions. Main effect of FSH was not significant (P > 0.10). Control values for SM-follicle GC were 28-fold greater than for control values for LG-follicle GC (data not shown).

To determine if E2F transcription factors are involved in steroidogenesis or proliferation of GC, experiment 5 was conducted. Treatment with FGF9 (30 ng/mL) increased (P < 0.05) cell numbers by 1.4-fold (Figure 5A). Alone, E2Fi (50 µM) decreased (P < 0.05) cell numbers by 36.8% and when E2Fi and FGF9 were combined, the stimulatory effect of FGF9 on cell numbers was blocked (P < 0.05). Treatment with FGF9 decreased (P < 0.05) E2 production by 88.6% (Figure 5B). Production of E2 was unaffected (P > 0.10) by E2Fi alone, but concomitant treatment of E2Fi and FGF9 attenuated (P < 0.05) the FGF9-induced decrease in E2 production (Figure 5B). Both E2Fi and FGF9 alone decreased (P < 0.05) P4 production by 44.3% and 63.3%, respectively, and P4 production was further decreased (P < 0.05) by 81.6% with concomitant treatment of FGF9 and E2Fi (Figure 5C).

Figure 5.

Figure 5.

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Effects of FGF9 (30 ng/mL), E2Fi (50 µM), and FGF9 plus E2Fi and on cell proliferation (Panel A), and E2 (Panel B) and P4 (Panel C) production in SM-follicle GCs (experiment 5). Treatments were applied for 48 h in serum-free medium containing testosterone, FSH, and IGF1. a–c Within a panel, means ±SE (n = 9)without a common letter differ (P < 0.05).

In experiment 6, the effect of an E2Fi on E2F1 mRNA abundance in GC was evaluated. The E2Fi treatment alone decreased (P < 0.01) abundance of E2F1 mRNA by 70% (Figure 6). The E2Fi treatment alone also decreased (P < 0.01) abundance of FSHR mRNA by 86% (Figure 6).

Figure 6.

Figure 6.

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Effect of an E2Fi (50 µM) on E2F1 and FSHR mRNA expression in SM-follicle GCs (experiment 6). Treatments were applied in serum-free medium for 24 h. Results are normalized to constitutively expressed 18S ribosomal RNA and expressed as fold (mean ± SE, n = 6) of control values with no additions. Asterisk indicates mean differs (P < 0.05) from controls.

Discussion

The present studies show that GC E2F1 gene expression is under hormonal and developmental control. In particular, FGF9-induced SM-follicle GC E2F1 gene expression and suppression of E2F1 mRNA caused changes in GC proliferation and E2 production. Also, GC (but not TC) E2F1 mRNA abundance was greater in SM than LG follicles. Thus, expression of E2F1 in GC may be involved in regulating follicular growth in cattle. Although no previous study has documented an FGF9 regulation of ovarian E2F1 expression, studies in other species have shown a link between E2F and ovarian function (Putowski et al., 2001; Yin et al., 2014; Nichols et al., 2019; Zhang et al., 2019). In addition, E2F1 is thought to be involved in ovarian cancer and survival outcome (Reimer et al., 2006; Suh et al., 2008; Zhang et al., 2017b).

Based on our TC microarray study (Schutz et al., 2018), E2F1, 4, 5, 6, and 8 are expressed in bovine TC but only E2F8 expression was significantly increased by FGF9 in TC (Morrell et al., 2019). In the present study, we confirmed that E2F1 gene expression is not induced by FGF9 in TC but discovered it was induced by FGF9 in SM-follicle GC. Therefore, we think that E2F1 is a prime candidate for the major E2F involved in FGF9 regulated GC proliferation and E2 production. In the present study, E2F1 mRNA abundance in GC (but not TC) was severalfold greater in SM vs. LG follicles suggesting that GC E2F1 gene expression decreases as follicles develop, similar to what has been observed for GC FGF9 mRNA (Schreiber et al., 2012; Schutz et al., 2016). Similarly, Douville and Sirard (2014) reported that E2F1 gene expression was 2-fold greater in GC from presumed growing follicles than in GC from follicles in their plateau phase of growth. In the present study, when SM-follicle GC were treated with an E2Fi, E2F1 mRNA expression was decreased and the stimulatory effect of FGF9 on cell proliferation was attenuated as was the FGF9-induced inhibition of E2 production, suggesting that E2F1 may be mediating the effect of FGF9 on these GC functions. However, the inhibitory effect of FGF9 on GC P4 production was further enhanced with E2Fi treatment in the present study. The latter observation suggests that FGF9 may be regulating P4 production through a mechanism other than via E2F transcription factors, but further work is needed to verify this suggestion.

In support of the present study, other studies indicate that overexpression of E2F1 in Chinese hamster ovarian (CHO) cells increases proliferation (Lee et al., 1996) and viable cell densities (Majors et al., 2008) and that decreased E2F1 expression inhibits CHO cell growth (Valle et al., 2013). In contrast to the present study, porcine GC transfected with E2F1 siRNA showed an increase in cell proliferation (Zhang et al., 2019), suggesting species differences may exist in terms of the role E2F1 may play in regulating GC proliferation. Interestingly, Kanai et al. (2009) showed that overexpression of E2F1 in human embryonic kidney cells upregulates FGFR1 expression, suggesting that there may be a reciprocal regulative feedback loop between E2F and FGF. Thus, in addition to directly stimulating genes involved in mitosis, E2F may also be feeding back to enhance the response to the FGFs inducing the E2F. Similarly, overexpression of E2F1, E2F2, and E2F3 in mouse NIH3T3 cells upregulates FGFR2 gene expression (Tashiro et al., 2003). Whether a feedback loop exists for E2F1 and FGF9 in bovine GC will require further study.

As mentioned earlier, possible roles of some E2F members in ovarian function have been identified previously. A recent study by Zhang et al. (2019) using porcine GC reported that transfection of GC with E2F1 siRNA caused an increase in CYP19A1 mRNA expression, which is consistent with findings of the present study. In human and rat GC, overexpression of E2F1 (an activator E2F) decreases FSHR transcription, whereas overexpression of E2F5 (a suppressor E2F) increases FSHR transcription (Putowski et al., 2001), further supporting the idea that E2F proteins may be involved in follicular growth in species other than cattle. Putowski et al. (2001) also reported that overexpression of E2F4 (a repressor E2F) had no effect on FSHR expression in human and rat GC, indicating that not all E2F affect FSHR gene expression. Moreover, because inhibition of E2F1 caused a decrease in FSHR mRNA as well as a decrease in E2F1 mRNA in bovine GC in the present study, and overexpression of E2F1 caused a decrease in FSHR mRNA in human and rat GC, species differences may exist in terms of the role E2F1 may play in follicular development. In mice, PMSG treatment in vivo and FSH treatment in vitro, both of which induce follicular growth, increase ovarian E2F1 mRNA levels, whereas hCG treatment in vivo decreases ovarian E2F1 mRNA levels (Yin et al., 2014), further supporting the idea that E2F1 may be involved in follicular growth. However, FSH treatment alone has no effect on bovine GC proliferation in vitro (Langhout et al., 1991). Consistent with this latter finding, the present studies show that FSH has no significant effect on E2F1 mRNA in bovine GC, further supporting the idea that the mechanism of action of FSH and the role of E2F1 differs among species. Previously, we reported that FGF9 inhibited steroid production in GC (Schreiber and Spicer, 2012), and thus, it is possible that the decrease in steroid production may be due, in part, to FGF9-induced increases in E2F1. Why FSHR mRNA decreased with E2F1 inhibition as well as with FGF9 treatment will require further investigation, but this decrease may be why E2 production was not fully restored to control levels. In particular, studies using knockdown of E2F1 expression in GC such as those of Zhang et al. (2019) may help identify specific targets of E2F1. More studies are also needed to determine what other factors/hormones regulate E2F1 (and other E2F) in bovine GC, and if these regulators can overcome pathways to upregulate or inhibit GC proliferation.

Funding

Supported in part by: The Endowment of Howard M. & Adene R. Harrington Chair in Animal Science (Project 21-58500; to LJS), and the Oklahoma State University Agricultural (Agric.) Experiment (Exp.) Station (Sta.) Project OKL02970 (to LJS).

Conflict of interest statement

None declared.

Acknowledgments

The authors thank A. Hemple and J. Nichols for technical assistance; Dr. A. F. Parlow, National Hormone & Pituitary Program (Torrance, CA) for purified FSH; and Creekstone Farms (Arkansas City, KS) for their generous donation of bovine ovaries. Approved for publication by the Director, Oklahoma Agric. Sta.

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