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. 2021 Nov 1;99(11):skab318. doi: 10.1093/jas/skab318

Effects of bone morphogenetic protein 4, gremlin, and connective tissue growth factor on estradiol and progesterone production by bovine granulosa cells

Leon J Spicer 1,✉, Luis F Schutz 1,✉, Pauline Y Aad 1,✉
PMCID: PMC8601128  PMID: 34724558

Abstract

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-β family of proteins that have been implicated in the paracrine regulation of granulosa cell (GC) function, but whether responses to BMPs change with follicular size or interact with connective tissue growth factor (CTGF) or BMP antagonists (e.g., gremlin [GREM]) to directly affect GC function of cattle is unknown. Therefore, to determine the effects of BMP4 on proliferation and steroidogenesis of GCs and its interaction with GREM or CTGF, experiments were conducted using bovine GC cultures. In vitro, BMP4 (30 ng/mL) inhibited (P < 0.05) follicle-stimulating hormone (FSH) plus insulin-like growth factor 1 (IGF1)-induced progesterone and estradiol production by large- and small-follicle GCs, but the inhibitory effect of BMP4 on estradiol production was much more pronounced in large-follicle GCs. In small-follicle GCs, BMP4 had no effect (P > 0.10) on IGF1-induced proliferation, but GREM inhibited (P < 0.05) cell proliferation and estradiol and progesterone production in IGF1 plus FSH-treated GCs. In large-follicle GCs, BMP4 (10 to 30 ng/mL) increased (P < 0.05) GC numbers and GREM (100 ng/mL) blocked this effect. In large-follicle GCs, CTGF inhibited (P < 0.05) FSH plus IGF1-induced progesterone and estradiol production, and CTGF blocked the stimulatory effect of BMP4 on GC proliferation. These results indicate that BMP4, GREM, and CTGF inhibit GC aromatase activity and progesterone production. Also, the stimulatory effect of BMP4 on GC proliferation and the inhibitory effects of BMP4 on GC steroidogenesis are more pronounced in large vs. small follicles.

Keywords: bone morphogenetic protein 4, connective tissue growth factor, estradiol, granulosa cell, gremlin, progesterone

Introduction

Throughout ovarian follicular development, proliferation of granulosa and theca cells and their differentiation are influenced by several endocrine and intraovarian factors secreted by both the oocyte and the surrounding somatic cells (Shimasaki et al., 2004; Juengel and McNatty, 2005; Sirard, 2016). Among the intraovarian factors are members of the transforming growth factor-ß (TGF-β) superfamily including TGFß1, bone morphogenetic proteins (BMPs), activins, and growth differentiation factors (GDFs; Mazerbourg and Hsueh, 2003; Shimasaki et al., 2004; Regan et al., 2018). Expression of these TGFß family members within the three main cell types of the follicle (i.e., oocyte, granulosa, and theca) varies dramatically among species. For example, in rodents, BMP4 mRNA is localized in theca but not oocytes or granulosa cells (GCs) (Erickson and Shimasaki, 2003), whereas BMP4 protein is localized in GCs (Tanwar and McFarlane, 2011). In cattle, BMP4 mRNA is localized in both GCs and theca cells (Fatehi et al., 2005; Kayani et al., 2009; Glister et al., 2011), whereas BMP4 protein is primarily localized in theca cells (Glister et al., 2004; Fatehi et al., 2005; Díaz et al., 2016). Importantly, cystic-follicle GCs from Holstein cows had dramatically less BMP4 mRNA abundance than control-follicle GCs (Díaz et al., 2016). Studies with mice indicate that BMP4 has an important role in suppressing GC apoptosis (Shimizu et al., 2012). However, little is known about the role of BMP4 and other BMPs in regulating GC function in cattle. Previously, BMP4 has been shown to stimulate estradiol production but inhibit progesterone production by rat GCs (Shimasaki et al., 1999; Hung et al., 2012), inhibit both estradiol and progesterone production by bovine GCs (Spicer et al., 2006; Kayani et al., 2009; Yamashita et al., 2011), inhibit progesterone production by ovine GCs (Mulsant et al., 2001; Fabre et al., 2003; Pierre et al., 2004), and either increase proliferation of ovine GCs (Fabre et al., 2003) or have no effect on bovine (Yamashita et al., 2011) or human (Miyoshi et al., 2006) GC proliferation. Thus, species differences may exist in terms of the GC response to BMP4.

Four subfamilies of antagonists for BMPs have also been identified: 1) differential screening-selected gene aberrative in neuroblastoma (DAN) subfamily, 2) twisted gastrulation subfamily, 3) chordin/noggin subfamily, and 4) follistatin-related subfamily. The DAN subfamily is of particular interest and includes several cysteine knot proteins including DAN, cerberus, and gremlin (GREM) 1 and 2 (Fenwick et al., 2011; Hung et al., 2012). In the ovary, GREM inhibits BMP4-induced prostaglandin E2 production by mouse GCs (Pangas et al., 2004) and stimulates androstenedione production by bovine theca cells (Glister et al., 2005). More recently, GREM1 gene expression in GCs was found to be downregulated by 3-fold in women with diminished ovarian reserve (Jindal et al., 2012) and decreased with follicle size in cattle (Glister et al., 2011). Also, loss of GREM delays primordial follicle assembly but does not affect fertility in mice (Myers et al., 2011).

Connective tissue growth factor (CTGF), a 349 amino acid protein, also called cellular communication network factor family member 2 (Mizutani et al., 2010), has been identified as an ovarian paracrine growth factor necessary for normal follicle development and ovulation in rodents (Harlow et al., 2002; Nagashima et al., 2011). Abundance of CTGF mRNA is 3-fold greater, whereas BMP4 mRNA is 1.7-fold less in 6 to 9 mm bovine follicles in the plateau vs. growing phase (Douville and Sirard, 2014) and CTGF mRNA increases in GCs and thecal cells of porcine ovaries during early antral follicle development (Wandji et al., 2000). Similarly in rats, CTGF mRNA expression increases 5-fold in GCs as follicles develop from preantral to large antral follicles and then decreases 30% in preovulatory follicles (Harlow and Hiller, 2002; Harlow et al., 2007). In addition, CTGF treatment of rat ovaries in culture induces the expression of several genes related to cell-cycle progression and cell differentiation and stimulates primordial follicle assembly (Schindler et al., 2010). Also in rats, follicle-stimulating hormone (FSH) inhibits, whereas estradiol, TGFβ1, GDF9, and activin stimulate CTGF mRNA expression in GCs both in vivo and in vitro (Harlow et al., 2002, 2007). Previously, CTGF has been shown to interact with BMP4 in cell-free systems (Abreu et al., 2002), but whether CTGF and BMP4 interact to regulate GC function is unknown.

Because insulin-like growth factor 1 (IGF1) is a major trophic hormone involved in reproduction and follicular development (Hunter et al., 2004; Velazquez et al., 2008; Shimizu, 2016), our first objective was to determine the effect of BMP4 on IGF1-induced cell proliferation and steroidogenesis in cultured bovine GCs from small (i.e., 1 to 5 mm) and large (i.e., ≥8 mm) follicles. These two sizes of follicles were evaluated based on previous observations indicating that: 1) follicles ≥ 8 mm in diameter have much greater androstenedione and estradiol concentrations than small follicles and have GCs that are more differentiated (i.e., greater amount of luteinizing hormone receptors; Stewart et al., 1996; Spicer et al., 2001, 2011), 2) selection of dominant follicles occurs at about 8 mm in diameter (Beg and Ginther, 2006), and 3) GCs from small and large bovine follicles show different responses to various hormones in vitro (Alpizar and Spicer, 1994; Spicer and Alpizar, 1994; Spicer et al., 2011). Our second objective was to determine if GREM influences the effects of BMP4 on bovine GC function. Our third objective was to determine if CTGF influences bovine GC function and alters the effects of BMP4. We hypothesized that BMP4, GREM, and CTGF act to attenuate the stimulatory effect of FSH and IGF1 on steroid production by GCs in cattle.

Materials and Methods

Tissues, hormones, and reagents

There were no live animals used in this study, so no ethical approval was required. Ovaries from beef heifers were collected at a slaughterhouse where humane slaughter practices were followed, according to USDA guidelines.

The hormones and reagents used in cell culture were: ovine FSH (F1913; FSH activity, 15 × NIH-FSH-S1 U/mg) from Scripps Laboratories (San Diego, CA, USA); recombinant human IGF1, CTGF, and BMP4, and recombinant mouse GREM (all carrier-free) from R&D Systems (Minneapolis, MN)—the homology of these proteins between bovine and human or mouse ranges from 96% to 100%; testosterone from Steraloids (Wilton NH); and fetal calf serum (FCS), Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F-12 (F12), collagenase, deoxyribonuclease (DNase), gentamycin, glutamine, and sodium bicarbonate from Sigma-Aldrich Inc. (St. Louis, MO).

Cell culture

Ovaries from nonpregnant beef cows were collected from a local slaughterhouse, and based on surface diameter, GCs were collected from small (1 to 5 mm) and large (8 to 22 mm) follicles as previously described (Langhout et al., 1991; Spicer and Chamberlain, 1998). Briefly, GCs were collected from small follicles via spiration of follicular fluid with needles and syringes and centrifuged at 200 × g for 5 min. Large follicles were bisected after aspiration of follicular fluid and GCs separated from the theca interna cells via blunt dissection, rinsed with basal serum-free medium (1:1 DMEM:F12; 0.12 mM gentamicin, 2.0 mM glutamine, and 38.5 mM sodium bicarbonate), as previously described (Spicer and Chamberlain, 1998; Dentis et al., 2016). Large-follicle GCs isolated from follicular fluid aspirates and after blunt dissection were combined. The GCs were re-suspended in serum-free medium containing collagenase (1.25 mg/mL) and DNase (0.5 mg/mL) to prevent cell clumping prior to plating. Viability of GCs from small and large follicles was determined by the trypan blue exclusion method and averaged 77 ± 4% and 69 ± 5%, respectively, and is within the range of viabilities previously reported for bovine GCs collected from abattoir tissues (Spicer et al., 2002, 2006; Schreiber and Spicer, 2012; Feng et al., 2018). For each experiment, GCs were collected into three separate pools, each containing cells from follicles of at least 10 individual animals for small-follicle GCs and at least 5 individual animals for large-follicle GCs.

Viable cells (2 × 105 cells in 22 to 88 μL of medium) were plated on 24-well Falcon multiwell plates (Becton Dickinson, Lincoln Park, NJ, USA) in basal medium (1 mL) containing 10% FCS. Cells were cultured at 38.5 °C in 10% FCS for the first 48 h with a medium change at 24 h. Cells were then washed twice with serum-free medium and the various treatments (see below) were applied in serum-free medium containing testosterone (500 ng/mL, as an estrogen precursor) for 48 h with a medium change at 24 h. This 24-h period of testosterone exposure has been shown to allow for direct measure of functional aromatase activity (Spicer et al., 1993). Medium was collected for steroid radioimmunoassay (RIA), and cells were collected for cell enumeration (see below). The concentrations of BMP4 used for the experiments were selected based on previously published studies, indicating that these concentrations of BMP4 had significant effects on steroid production by rat (Shimasaki et al., 1999), ovine (Mulsant et al., 2001), and bovine (Spicer et al., 2006) GCs. The concentrations of FSH and IGF1 were selected based on previous studies (Spicer and Stewart, 1996; Spicer and Chamberlain, 1998; Spicer et al., 2002). Because steroid production in this culture system is maximized with a combined FSH and IGF1 treatment and only weakly responsive to either FSH or IGF1 alone (Spicer and Chamberlain, 1998; Spicer et al., 2002), FSH was used in combination with IGF1 for all experiments.

Experimental design

Experiment 1 was designed to evaluate the dose–response effect of BMP4 and its interaction with GREM (a BMP antagonist) on hormone-induced proliferation and steroidogenesis of bovine GCs. The GCs from small or large follicles were collected, cultured for 48 h in 10% FCS, washed twice with serum-free medium as described earlier, and cells cultured for an additional 48 h in the presence of FSH (10 ng/mL), IGF1 (30 ng/mL), and testosterone (500 ng/mL, as an estrogen precursor) with various doses of BMP4 (0, 3, 10, or 30 ng/mL) and GREM (0 or 100 ng/mL). Medium was changed every 24 h. Cells were counted and medium was collected for RIA to measure estradiol and progesterone concentrations (see below). Dose of GREM was selected based on previous studies (Pangas et al., 2004; Sudo et al., 2004), and doses of BMP4 were selected based on previous studies (Shimasaki et al., 1999; Spicer et al., 2006; Yang et al., 2016). BMP4 rather than other BMPs was selected to be evaluated, because, in cattle, BMP4 increases during follicular development (Glister et al., 2010), is associated with cystic follicles (Díaz et al., 2016), and is produced by ovarian follicles (Glister et al., 2004; Fatehi et al., 2005). Also, BMP4 has the greatest amino acid sequence homology (i.e., 98%) between human BMP4 and bovine BMP4 compared with other BMPs. GREM was selected because it inhibits BMP4 action in GCs (Pangas et al., 2004) and theca cells (Glister et al., 2019), and GREM decreases with follicle development in cattle (Glister et al., 2011). The amino acid sequence homology between human and bovine GREM is 100% (http://www.ncbi.nlm.nih.gov).

Experiment 2 was designed to evaluate the dose–response effect of CTGF and its interaction with BMP4 on hormone-induced proliferation and steroidogenesis of large-follicle GCs. Large-follicle GCs were selected to be used in exp. 2 because they were more responsive to the effects of BMP4 in exp. 1 than small-follicle GCs. Cells were cultured for 48 h in 10% FCS, washed twice with serum-free medium as described earlier, and cells cultured for an additional 48 h in the presence of FSH (30 ng/mL), IGF1 (30 ng/mL), testosterone (500 ng/mL, as an estrogen precursor) with or without BMP4 (30 ng/mL), and various doses of CTGF (0, 3, 30, 300 ng/mL). Medium was changed every 24 h. Cells were counted and medium was collected for RIA to measure estradiol and progesterone concentrations (see below). Doses of CTGF were selected based on previous in vitro studies (Harlow et al., 2007; Duan et al., 2017; Li et al., 2019) and selected to include the range (i.e., 5 to 122 ng/mL) of plasma concentrations of CTGF reported in humans (Mizutani et al., 2010).

Determination of steroid concentrations and cell numbers

Medium was collected from individual wells and frozen at –20 °C for subsequent determination of concentrations of estradiol and progesterone via RIA as previously described (Langhout et al., 1991; Feng et al., 2018). A 24-h period of testosterone exposure to GCs allows for direct measure of functional aromatase activity (Spicer et al., 1993). The intra-assay coefficients of variation were 9.9% and 8.9% for the progesterone and estradiol RIA, respectively.

Numbers of cells in the same wells that medium was collected were determined via Coulter counting as previously described (Perego et al., 2017; Feng et al., 2018) and used to calculate steroid production on an ng or pg per 105 cell basis. Briefly, cells were gently washed twice with 0.9% saline (500 µL), exposed to 500 µL of trypsin solution (0.25%) for 30 min at 37 °C, and then scraped from each well. Cell aggregates were disrupted via pipetting the cell suspension back and forth through a 500-μL pipette tip three to five times, diluted in 9 mL of 0.9% saline, and counted using a Coulter counter (model Z2; Beckman Coulter, Inc., Miami, FL, USA). The intra-assay coefficients of variation averaged 2.9 ± 0.4%.

Statistical analysis

Data are presented as the least squares means (±SEM) of measurements from three or four pools of cells (i.e., biological replicates) with each pool of cells collected from at least 10 individual animals for small-follicle GCs and at least 5 individual animals for large-follicle GCs, and each replicated experiment (i.e., pool of cells) had three technical replicates of cells per treatment. Steroid production was expressed as ng or pg/105 cells per 24 h, and cell numbers at the termination of the experiment were used for this calculation. Treatment effects and interactions were determined via analysis of variance (ANOVA) using the general linear models procedure of SAS for Windows (ver. 9.3, SAS Institute Inc., Cary, NC). Exp. 1 was analyzed as 2 × 4 factorial ANOVA with GREM and BMP4 dose (0, 3, 10, and 30 ng/mL) as main effects and their interactions. Exp. 2 was analyzed as 2 × 4 factorial ANOVA with BMP4 and CTGF dose (0, 3, 30, and 300 ng/mL) as main effects and their interactions. Specific differences in cell numbers and steroid production were determined using Fisher’s protected least significant difference procedure (Ott, 1977). Significance was declared at P < 0.05.

Results

Experiment 1: BMP4 and GREM effects on GCs from small and large follicles

In small-follicle GCs, only the main effect of GREM was significant such that BMP4 (3, 10, or 30 ng/mL) did not affect (P > 0.10) cell numbers induced by IGF1, whereas GREM (100 ng/mL) inhibited GC numbers (Figure 1A). In large-follicle GCs, main effects of BMP4 and GREM were significant such that BMP4 at 10 and 30 ng/mL increased (P < 0.05) cell numbers and GREM blocked (P < 0.05) this increase (Figure 1B).

Figure 1.

Figure 1.

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Effects of BMP4 (3 to 30 ng/mL) and gremlin on proliferation of small- (A) and large-follicle (B) granulosa cells (exp. 1). a–dWithin a panel, means (±SEM of four separate experiments) without a common superscript differ (P < 0.05). All cells were treated with FSH (10 ng/mL) and IGF1 (30 ng/mL). Abbreviations: BMP4, bone morphogenetic protein 4; FSH, follicle-stimulating hormone; IGF1, insulin-like growth factor 1.

In small-follicle GCs, main effects of BMP4 and GREM were significant such that BMP4 caused a dose-dependent decrease (P < 0.05) in progesterone production, and GREM alone (100 ng/mL) or in combination with BMP4 further reduced (P < 0.05) progesterone production (Figure 2A). In large-follicle GCs, main effects of BMP4 and GREM were significant such that BMP4 caused a dose-dependent decrease (P < 0.05) in progesterone production, and GREM decreased (P < 0.05) progesterone production only in the presence of 10 ng/mL BMP4 (Figure 2B). At 30 ng/mL, BMP4 significantly decreased progesterone production by 39% and 29% in small- and large-follicle GCs, respectively.

Figure 2.

Figure 2.

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Inhibitory effect of BMP4 (3 to 30 ng/mL) and gremlin (100 ng/mL) on production of progesterone in small- (A) and large-follicle (B) granulosa cells (exp. 1). a–eWithin a panel, means (±SEM of four separate experiments) without a common superscript differ (P < 0.05). All cells were treated with FSH (10 ng/mL) and IGF1 (30 ng/mL). Abbreviations: BMP4, bone morphogenetic protein 4; FSH, follicle-stimulating hormone; IGF1, insulin-like growth factor 1.

In small-follicle GCs, main effects of BMP4 and GREM were significant such that only 30 ng/mL BMP4 decreased (P < 0.05) estradiol production (aromatase activity) and GREM (100 ng/mL) alone or in combination with BMP4 further reduced (P < 0.05) estradiol production (Figure 3A). In large-follicle GCs, main effects of BMP4, GREM, and their interaction were significant such that BMP4 caused a dose-dependent decrease (P < 0.05) in estradiol production (Figure 3B). GREM alone decreased estradiol production but did not affect BMP4-inhibited estradiol production (Figure 3B). In the absence of GREM, BMP4 at 30 ng/mL significantly decreased estradiol production by 40% and 76% in small- and large-follicle GCs, respectively. In large-follicle GCs, the estimated inhibitory concentration (IC50) of BMP4 inhibiting 50% of the maximal steroidogenic response (calculated from inhibition curves that were linearized using a semi-log plot) averaged 3 and 10 ng/mL for estradiol production in the absence and presence of GREM, respectively.

Figure 3.

Figure 3.

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Inhibitory effect of BMP4 (3 to 30 ng/mL) and gremlin (100 ng/mL) on production of estradiol (aromatase activity) in small- (A) and large-follicle (B) granulosa cells (exp. 1). a–dWithin a panel, means (±SEM of four separate experiments) without a common superscript differ (P < 0.05). All cells were treated with FSH (10 ng/mL) and IGF1 (30 ng/mL). Abbreviations: BMP4, bone morphogenetic protein 4; CTGF, connective tissue growth factor; FSH, follicle-stimulating hormone; IGF1, insulin-like growth factor 1.

Experiment 2: BMP4 and CTGF effects on large-follicle GCs

In large-follicle GCs, BMP4 (30 ng/mL) increased (P < 0.05) cell numbers in the absence of CTGF, whereas CTGF blocked (P < 0.05) this increase (Figure 4A). In the absence of BMP4, the various doses of CTGF had no effect (P > 0.10) on cell numbers (Figure 4A).

Figure 4.

Figure 4.

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Effect of CTGF (3 to 300 ng/mL) and BMP4 (30 ng/mL) on granulosa cell (GC) function (exp. 2). (A) Effect of CTGF and BMP4 on proliferation of large-follicle GCs. (B) Inhibitory effect of BMP4 and CTGF on production of progesterone in large-follicle GCs. (C) Inhibitory effect of BMP4 and CTGF on production of estradiol production (aromatase activity) in large-follicle GCs. a–dWithin a panel, means (± SEM of three separate experiments) without a common superscript differ (P < 0.05). All cells were treated with FSH and IGF1. Abbreviations: Abbreviations: BMP4, bone morphogenetic protein 4; CTGF, connective tissue growth factor; FSH, follicle-stimulating hormone; IGF1, insulin-like growth factor 1.

In large-follicle GCs, BMP4 decreased (P < 0.05) progesterone production induced by IGF1 plus FSH by 58% and CTGF had no effect on this inhibition (Figure 4B). However, CTGF at 300 ng/mL decreased (P < 0.05) progesterone production induced by IGF1 plus FSH (by 43%) to the levels seen with BMP4 alone (Figure 4B).

In large-follicle GCs, BMP4 decreased (P < 0.05) estradiol production (aromatase activity) induced by IGF1 plus FSH by 79% (Figure 4C) and CTGF had no effect on this inhibition. However, 30 and 300 ng/mL of CTGF (in the absence of BMP4) decreased (P < 0.05) estradiol production in the presence of IGF1 plus FSH by 47% and 71%, respectively (Figure 4C). The estimated IC50 of CTGF inhibiting 50% of the maximal steroidogenic response averaged 18.5 and 44 ng/mL for estradiol production in the absence and presence of BMP4, respectively.

Discussion

Results of the present study using cultured bovine GCs revealed that: 1) BMP4 inhibited progesterone and estradiol production induced by FSH plus IGF1 in small- and large-follicle GCs and that the inhibitory effect of BMP4 on estradiol production was much more pronounced in large-follicle GCs; 2) GREM decreased BMP4-induced proliferation of GCs from large follicles, and alone GREM decreased estradiol production by GCs from small and large follicles; and 3) CTGF inhibited estradiol and progesterone production in the absence of BMP4 and blocked the stimulatory effect of BMP4 on proliferation of large-follicle GCs.

For the first time, dose–response effects of BMP4 and CTGF on GC function have been compared. Previous studies with rodent GCs have indicated that BMP4 blocks progesterone production but increases estradiol production (Shimasaki et al., 1999; Hung et al., 2012). In agreement with the present study, previous studies in bovine GCs reported that BMP4 inhibited progesterone and estradiol production induced by FSH and IGF1 (Spicer et al., 2006; Yang et al., 2016; Sakaguchi et al., 2017). Also, BMP4 inhibited StAR mRNA but had no effect on CYP11A1 or 3BHSD mRNA in bovine GCs (Yamashita et al., 2011). In ovine GCs, BMP4 consistently decreased GC progesterone production (Mulsant et al., 2001; Fabre et al., 2003; Pierre et al., 2004) and both StAR and CYP11A1 mRNA induced by FSH (Pierre et al., 2004). Also, BMP4 inhibited CYP11A1 protein in ovine GCs (Fabre et al., 2003). In human GCs, BMP4 inhibited progesterone production but had no effect on estradiol production or on CYP11A1 or CYP19A1 mRNA (Miyoshi et al., 2006). Interestingly, the inhibitory effect of BMP4 on GC estradiol production was much greater in large than small follicles of the present study. Thus, species differences may exist with regard to the specific GC response to BMP4 and may be due in part to size or maturation differences of follicles utilized among studies. Results of the present study support the hypothesis that BMP4 acts to attenuate the stimulatory effect of FSH and IGF1 on steroid production by GCs in cattle.

A stimulatory effect of BMP4 was observed on IGF1-induced numbers of large-follicle GCs but BMP4 had no effect on numbers of small-follicle GCs of the present study. These results are in agreement with previous studies showing no effect of BMP4 on proliferation of GCs collected from small bovine follicles (Yamashita et al., 2011) and on proliferation of human GCs (Miyoshi et al., 2006). In sheep, BMP4 induces proliferation of small-follicle GCs collected from homozygous carriers of the prolificacy allele but not in GCs collected from homozygous noncarriers (Fabre et al., 2003). This suggests that the mitogenic response to BMP4 may be linked to the functionality of BMP receptors 1B and 2 (i.e., BMPR1B and BMPR2). In mouse chondrogenic precursor cells, BMPR1A is required for BMP4 to induce their differentiation into chondrocytes (Shukunami et al., 2000), further supporting the role of BMPRs in regulating BMP4 responses. The present finding that large-follicle GCs were more responsive to the mitogenic effects of BMP4 than small-follicle GCs suggests that BMP4 may preferentially stimulate GC proliferation during later phases of follicle development. Interestingly, abundance of BMPR2 mRNA is greater in GCs of large vs. small bovine (Glister et al., 2010) and ovine (Souza et al., 2002) follicles, and greater in bubaline GCs obtained from follicles with high estradiol concentrations (Rajesh et al., 2018). In contrast, GREM1 mRNA is less in large vs. small bovine GCs (Glister et al., 2011). Collectively, these studies support the idea that GCs from small (i.e., <5 mm) follicles have weaker responses to BMP4 than large (i.e., >8 mm) follicles (as observed in the present study) due to fewer BMPR2 receptors and greater GREM production in GCs, and this conclusion is supported by results of a microarray study, indicating that BMPR2 mRNA abundance is 3-fold greater in GCs from large (>12 mm) than small (≤5 mm) bovine follicles (Hatzirodos et al., 2014). Further research will be required to elucidate the mechanisms of the mitogenic effect of BMP4 on GCs and the role that GREM and BMPRs may play in the process.

The present study showed for the first time that CTGF inhibited both estradiol and progesterone production by bovine GCs and inhibited BMP4-induced cell proliferation. Previously, CTGF treatment of rat GCs in vitro had no effect on aromatase activity (Harlow et al., 2007), but in cultured rat ovaries, CTGF induced the expression of several genes related to cell-cycle progression and cell differentiation and stimulated primordial follicle assembly (Schindler et al., 2010). Previous studies in non-ovarian tissues have reported that CTGF can induce cell proliferation in cultured mouse adipose-derived stromal cells (Li et al., 2019) and human periodontal ligament fibroblasts (Duan et al., 2017) but not chicken embryo fibroblasts (Gygi et al., 2003). Although CTGF has been shown to interact with BMP4 in cell-free systems (Abreu et al., 2002), results of the present study indicate that CTGF does not block the inhibitory effects of BMP4 on steroidogenesis in cultured GCs. However, CTGF did block the stimulatory effect of BMP4 on GC proliferation. Furthermore, the IC50 for CTGF inhibition of estradiol production was calculated to be 18.5 ng/mL, and this was increased by 3-fold in the presence of BMP4, implying that BMP4 may alter the response of GCs to CTGF. Additional studies will be required to ascertain the mechanisms by which CTGF inhibits GC steroid production and interacts with BMP4.

Several high-affinity-binding proteins antagonize BMP signaling, including follistatin, noggin, chordin, and members of the DAN family, including DAN, cerberus, and GREM (Canalis et al., 2003; Fenwick et al., 2011). The primary mode of inhibition occurs by binding to BMPs, which prevents their association with the receptor complex (Zimmerman et al., 1996; Groppe et al., 2002). The extracellular antagonists are often part of negative feedback loops and thus are directly upregulated by the ligands that they antagonize (Canalis et al., 2003). In mice, ovarian GREM1 mRNA abundance increases between 12 and 21 d of age when follicle growth occurs (Fenwick et al., 2011), but in bovine GCs collected from 11 to 18 mm follicles, GREM1 mRNA does not differ between estrogen-active and estrogen-inactive follicles (Glister et al., 2011). In large-follicle GCs of the present study, GREM treatment blocked the stimulatory effect of BMP4 on cell proliferation, had little effect on progesterone production, and reduced estradiol production in control cultures. Interestingly, the IC50 for BMP4 inhibition of estradiol production by large-follicle GCs was calculated to be 3 ng/mL and this was increased by 3-fold in the presence of GREM. In small-follicle GCs, GREM reduced cell numbers and steroid production in control cultures, suggesting that GREM may also act independently of BMP4 to alter GC growth and steroidogenesis, but additional research is needed to verify this suggestion. Previous studies have shown that GREM directly regulates cell function independently of BMPs in bovine aortic endothelial cells (Mitola et al., 2010) and in murine renal tubular epithelial cells (Lavoz et al., 2015).

Conclusions

BMP4, GREM, and CTGF decreased hormone-induced steroidogenesis in bovine GCs and thus should be considered important regulators of follicular function in cattle. The inhibitory effects of BMP4 on GCs were more pronounced in large vs. small follicles, suggesting that BMP4 may be important in regulating dominant/ovulatory follicle development in cattle. In addition, GREM and CTGF altered the BMP4 responses of GCs. Further research will be required to identify comprehensive dose–response interactions among these and other ovarian factors as well as to ascertain if production of these factors changes with increased follicular size. A better understanding of the role of BMP4, GREM, and CTGF and their molecular mechanisms that govern GC function will allow for a greater perspective on how these signals impact follicular development and subsequent progression into ovarian cysts.

Acknowledgments

We thank Dustin Allen for laboratory assistance and Creekstone Farms (Arkansas City, KS) for generous donations of bovine ovaries. Approved for publication by the Director, Oklahoma Agricultural Experiment Station, Oklahoma State University.

Glossary

Abbreviations

BMP4

bone morphogenetic protein 4

BMPR1B

bone morphogenetic protein receptor 1B

BMPR2

bone morphogenetic protein receptor 2

CTGF

connective tissue growth factor

DAN

differential screening-selected gene aberrative in neuroblastoma

DNase

deoxyribonuclease

FSH

follicle-stimulating hormone

GC

granulosa cell(s)

GREM

gremlin

GDF9

growth differentiation factor 9

IC50

concentration inhibiting 50% of response

IGF1

insulin-like growth factor 1

RIA

radioimmunoassay

TGF-β

transforming growth factor-β

Conflict of interest statement

The authors declare no real or perceived conflicts of interest.

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