Fibroblast growth factor 9 (FGF9) protein affects granulosa cell (GC) function but is mostly localized to theca cell (TC) and stromal cell of rat ovaries. The objectives of this study were to determine the 1) effects of FGF9 on TC steroidogenesis, gene expression, and cell proliferation; 2) mechanism of action of FGF9 on TCs; and 3) hormonal control of FGF9 mRNA expression in TCs. Bovine ovaries were collected from a local slaughterhouse and TCs were collected from large (8–22 mm) follicles and treated with various hormones in serum-free medium for 24 or 48 h. FGF9 caused a dose-dependent inhibition (P<0.05) of LH- and LH+IGF1-induced androstenedione and progesterone production. Also, FGF9 inhibited (P<0.05) LH+IGF1-induced expression of LHCGR, CYP11A1, and CYP17A1 mRNA (via real-time RT-PCR) in TCs. FGF9 had no effect (P>0.10) on STAR mRNA abundance. Furthermore, FGF9 inhibited dibutyryl cAMP-induced progesterone and androstenedione production in LH+IGF1-treated TCs. By contrast, FGF9 increased (P<0.05) the number of bovine TCs. Abundance of FGF9 mRNA in GCs and TCs was several-fold greater (P<0.05) in small (1–5 mm) vs large follicles. Tumor necrosis factor α and WNT5A increased (P<0.05) abundance of FGF9 mRNA in TCs. In summary, expression of FGF9 mRNA in TCs is developmentally and hormonally regulated. FGF9 may act as an autocrine regulator of ovarian function in cattle by slowing TC differentiation via inhibiting LH+IGF1 action via decreasing gonadotropin receptors and the cAMP signaling cascade while stimulating proliferation of TCs.

Abstract

Studies were conducted to determine the importance of de novo cholesterol synthesis and cholesterol side-chain cleavage enzyme in the action of IGF-I in bovine granulosa and thecal cells. Granulosa and thecal cells from bovine follicles were cultured for 2 days in 10% fetal calf serum and then treated with luteinizing hormone (100 ng/ml) and IGF-I (0 or 100 ng/ml) for an additional 2 days in serum-free medium. During the last 24 h of treatment, cells were concomitantly treated with simvastatin (0, 0·5 or 5 μg/ml) or 25-hydroxycholesterol (0 or 10 μg/ml). Simvastatin, a potent inhibitor of the key enzyme controlling de novo cholesterol synthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, completely inhibited (P<0·05) progesterone production by granulosa cells and progesterone and androstenedione production by thecal cells. Simvastatin also inhibited (P<0·05) granulosa cell and thecal cell proliferation. Concomitant treatment with mevalonate, an immediate product of HMG-CoA reductase, attenuated the inhibitory effect of simvastatin on progesterone and androstenedione production by thecal cells and blocked the inhibitory effect of simvastatin on cell proliferation. The addition of 25-hydroxycholesterol, a substrate for cholesterol side-chain cleavage enzyme, had no effect (P>0·10) on IGF-I-stimulated progesterone or androstenedione production by thecal cells and actually inhibited (P<0·05) IGF-I-stimulated progesterone production by granulosa cells. These results provide indirect evidence indicating that stimulation of HMG-CoA reductase is an important locus of IGF-I action in bovine granulosa and thecal cells, whereas IGF-I has little or no effect on side-chain cleavage enzyme activity in these same cell types under the culture conditions employed.

Journal of Endocrinology (1996) 151, 365–373

In addition to gonadotropins, steroidogenesis and proliferation of granulosa cells during follicular development are controlled by a number of intraovarian factors including growth differentiation factor-9 (GDF-9), bone morphogenetic protein-4 (BMP-4), and IGF-I. The objective of this study was to determine the effect of GDF-9 and BMP-4 and their interaction with IGF-I and FSH on ovarian granulosa cell function in cattle. Granulosa cells from small (1–5 mm) and large (8–22 mm) follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% fetal calf serum and then treated with various hormones in serum-free medium for an additional 48 h. We evaluated the effects of GDF-9 (150–600 ng/ml) and BMP-4 (30 ng/ml) during a 2-day exposure on hormone-induced steroidogenesis and cell proliferation. In FSH plus IGF-I-treated granulosa cells obtained from small follicles, 300 ng/ml GDF-9 reduced (P<0.05) progesterone production by 15% and 600 ng/ml GDF-9 completely blocked (P<0.01) the IGF-I-induced increase in progesterone production. In comparison, 300 and 600 ng/ml GDF-9 decreased (P<0.05) estradiol production by 27% and 71% respectively, whereas 150 ng/ml GDF-9 was without effect (P>0.10). Treatment with 600 ng/ml GDF-9 increased (P<0.05) numbers (by 28%) of granulosa cells from small follicles. In the same cells treated with FSH but not IGF-I, co-treatment with 600 ng/ml GDF-9 decreased (P<0.05) progesterone production (by 28%), increased (P<0.05) cell numbers (by 60%), and had no effect (P>0.10) on estradiol production. In FSH plus IGF-I-treated granulosa cells obtained from large follicles, GDF-9 caused a dose-dependent decrease (P<0.05) in IGF-I-induced progesterone (by 13–48%) and estradiol (by 20–51%) production. In contrast, GDF-9 increased basal and IGF-I-induced granulosa cell numbers by over 2-fold. Furthermore, treatment with BMP-4 also inhibited (P<0.05) steroidogenesis by 27–42% but had no effect on cell numbers. To elucidate downstream signaling pathways, granulosa cells from small follicles were transfected with similar to mothers against decapentaplegics (Smad) binding element (CAGA)- or BMP response element (BRE)-promoter reporter constructs. Treatment with GDF-9 (but not BMP-4) activated the Smad3-induced CAGA promoter activity, whereas BMP-4 (but not GDF-9) activated the Smad1/5/8-induced BRE promoter activity. We have concluded that bovine granulosa cells are targets of both GDF-9 and BMP-4, and that oocyte-derived GDF-9 may simultaneously promote granulosa cell proliferation and prevent premature differentiation of the granulosa cells during growth of follicles, whereas theca-derived BMP-4 may also prevent premature follicular differentiation.