Christopher A. Price

Expression and Function of Fibroblast Growth Factor 10 and Its Receptor, Fibroblast Growth Factor Receptor 2B, in Bovine Follicles

Abstract Some fibroblast growth factors (FGFs) affect ovarian follicle cell growth and/or differentiation. Whereas many FGFs activate several FGF receptors, FGF7 and FGF10 primarily activate only one, FGFR2B. As FGF7 is produced by bovine theca cells and acts on granulosa cells, we tested the hypothesis that FGF10 may also play a role in folliculogenesis in cattle. Reverse transcription-polymerase chain reaction demonstrated the presence of FGF10 mRNA in the oocytes and theca cells of the antral follicles, as well as in the preantral follicles. FGF10 protein was detected by immunohistochemistry in the oocytes of the preantral and antral follicles, and in the granulosa and theca cells of the antral follicles. FGF10 expression in theca cells changed during follicle development; mRNA abundance decreased with increasing follicular estradiol concentration in healthy follicles, and was lowest in highly atretic follicles. Culturing of granulosa cells in serum-free medium revealed FSH regulation of FGF10 receptor expression. The addition of FGF10 to cultured granulosa cells decreased the level of estradiol but did not alter cell proliferation. These data support a role for FGF10 in signaling to granulosa cells from theca cells and/or the oocyte.

Effect of Follicle-Stimulating Hormone on Steroid Secretion and Messenger Ribonucleic Acids Encoding Cytochromes P450 Aromatase and Cholesterol Side-Chain Cleavage in Bovine Granulosa Cells In Vitro

Abstract We determined 1) whether the previously observed induction of estradiol secretion in bovine granulosa cells cultured in serum-free conditions is associated with an increase in cytochrome P450 aromatase (P450arom) mRNA abundance and 2) whether P450arom mRNA levels are responsive to FSH in vitro. Granulosa cells from small (2–4-mm) follicles were cultured in serum-free medium. Estradiol secretion increased with time in culture and was correlated with increased P450arom mRNA abundance. Progesterone secretion also increased with time in culture, but P450 cholesterol side-chain cleavage (P450scc) mRNA abundance did not. FSH stimulated estradiol secretion and P450arom mRNA abundance; the effect was quadratic for both estradiol and P450arom mRNA. Estradiol secretion and P450arom mRNA levels were correlated. FSH stimulated progesterone secretion and P450scc mRNA abundance, although the minimum effective dose of FSH was lower for estradiol (0.1 ng/ml) than for progesterone (10 ng/ml) production. Insulin alone stimulated estradiol secretion and P450arom mRNA levels but not progesterone or P450scc mRNA abundance. We conclude that this cell culture system maintained both estradiol secretion and P450arom mRNA abundance responsiveness to FSH and insulin, whereas P450scc mRNA abundance and progesterone secretion were responsive to FSH but not insulin.

Regulation and action of fibroblast growth factor 17 in bovine follicles

Fibroblast growth factor 17 (FGF17) is a member of the FGF8 subfamily that appears to be relevant to folliculogenesis and oogenesis, as the prototype member FGF8 is an oocyte-derived protein that signals to cumulus cells. FGF8 has structural and receptor-binding similarities to FGF17, whose expression in the ovary has not been reported. In this study, we demonstrate localization of FGF17 protein to the oocyte of preantral follicles, and to the oocyte and granulosa cells of antral follicles. Real-time PCR demonstrated the presence of mRNA in oocytes and, to a lesser extent, in granulosa and theca cells. FGF17 mRNA abundance was low in granulosa and theca cells from healthy follicles and increased significantly in atretic follicles. Addition of FSH or IGF-I to granulosa cells in vitro decreased FGF17 mRNA abundance, and treatment with FGF17 inhibited estradiol and progesterone secretion from granulosa cells in relation to control cultures without these additives. We conclude that FGF17 is a potential mediator of granulosa cell differentiation.

Effects of dominant follicle aspiration and treatment with recombinant bovine somatotropin (BST) on ovarian follicular development in nelore (Bos indicus) heifers.

Follicle ablation has been recognized as an efficient method of follicular wave synchronization. Treatment with recombinant bovine somatotropin (BST) has been shown to enhance follicular development in Bos taurus. This experiment assessed the effects of these treatments in Nelore (B. indicus) heifers. Eight cycling Nelore heifers were randomly assigned to 3 different treatments. On Day 2 of a synchronized cycle (Day 0 = day of ovulation), heifers assigned to Treatments 1 and 2 received 2 mL of saline, whereas heifers assigned to Treatment 3 received 320 mg of BST. On Day 5, the first-wave dominant follicle was ablated by ultrasound-guided transvaginal aspiration in heifers in Treatments 2 and 3, and all heifers received an injection of prostaglandin on Day 11. Aspiration of the dominant follicle advanced and synchronized (P < 0.05) the day of second-wave emergence (6.9 +/- 0.1 vs. 8.4 +/- 0.4) and the day of the pre-wave FSH peak (6.0 +/- 0.0 vs. 6.9 +/- 0.4), and increased FSH peak concentrations (381 +/- 21 vs. 292 +/- 30; pg/mL; P < 0.01). Recombinant bovine somatotropin treatment caused a two-fold increase in plasma insulin-like growth factor-I (IGF-I) concentrations (P < 0.001) and resulted in a 36% increase in the number of small follicles (<5 mm; P < 0.001) compared with saline-treated heifers. In summary, in agreement with previous reports on B. taurus, dominant follicle aspiration synchronized ovarian follicular development, and BST treatment increased peripheral concentrations of IGF-I in Nelore heifers. Recombinant bovine somatotropin also increased the number of small follicles, but this response appeared to be inferior to that reported for B. taurus.

Expression and Function of Fibroblast Growth Factor 18 in the Ovarian Follicle in Cattle

Fibroblast growth factors (FGF) are involved in paracrine signaling between cell types in the ovarian follicle. FGF8, for example, is secreted by oocytes and controls cumulus cell metabolism. The closely related FGF18 is also expressed in oocytes in mice. The objective of this study was to assess the potential role of FGF18 in follicle growth in a monovulatory species, the cow. Messenger RNA encoding FGF18 was detected primarily in theca cells, and in contrast to the mouse, FGF18 was not detected in bovine oocytes. Addition of FGF18 protein to granulosa cell cultures inhibited estradiol and progesterone secretion as well as the abundance of mRNA encoding steroidogenic enzymes and the follicle-stimulating hormone receptor. In vivo, onset of atresia of the subordinate follicle was associated with increased thecal FGF18 mRNA levels and FGF18 protein in follicular fluid. In vitro, FGF18 altered cell cycle progression as measured by flow cytometry, resulting in increased numbers of dead cells (sub-G1 peak) and decreased cells in S phase. This was accompanied by decreased levels of mRNA encoding the cell cycle checkpoint regulator GADD45B. Collectively, these data point to a unique role for this FGF in signaling from theca cells to granulosa cells and suggest that FGF18 influences the process of atresia in ovarian follicles.

Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro.

The objective of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate cytochrome P450 aromatase (Cyp19) mRNA and oestradiol accumulation in oestrogenic bovine granulosa cells in vitro. Bovine granulosa cells from small follicles (2-4 mm diameter) were cultured for 6 days under non-luteinizing conditions in the presence of insulin at 100 ng/ml, or insulin (10 ng/ml) and FSH (1 ng/ml). On day 4 of culture, specific inhibitors of phosphatidylinositol 3-kinase (PI3K; LY-294002), protein kinase C (PKC; GF-109203X), protein kinase A (PKA; H-89) or mitogen-activated protein (MAP) kinase activation (PD-98059) were added. The addition of PI3K and PKC inhibitors, but not of PKA inhibitor, significantly decreased insulin-stimulated Cyp19 mRNA levels and oestradiol accumulation (P < 0.001). The PKA inhibitor significantly decreased FSH-stimulated Cyp19 mRNA abundance and oestradiol secretion, whereas PI3K and PKC inhibitors decreased oestradiol secretion without affecting Cyp19 mRNA accumulation. Inhibition of MAP kinase pathway significantly increased Cyp19 mRNA abundance in insulin- and FSH-stimulated cells. P450scc mRNA levels and progesterone secretion were not affected by any inhibitor in either experiment. Although FSH stimulates Cyp19 expression predominantly through PKA, oestradiol secretion is altered by PI3K and PKC pathways independently of Cyp19 mRNA levels. In addition, we suggest that Cyp19 is under tonic inhibition mediated through a MAP kinase pathway.

Expression of 17β- and 3β-hydroxysteroid dehydrogenases and steroidogenic acute regulatory protein in non-luteinizing bovine granulosa cells in vitro

Granulosa cells of small follicles differentiate in vitro in serum-free medium, resulting in increased estradiol secretion and abundance of mRNA encoding cytochrome P450aromatase (P450arom). We tested the hypothesis that differentiation in vitro also involves increased expression of 3beta- and 17beta-hydroxysteroid dehydrogenases (HSD) in the absence of steroidogenic acute regulatory protein (StAR) expression, as has been observed in vivo. Granulosa cells from small (<6 mm diameter) follicles were cultured for up to 6 days, and mRNA levels quantified by Northern hybridization or RT-PCR. Estradiol and progesterone concentrations in medium increased with time in culture, as did mRNA encoding P450arom, 3beta- and 17beta-HSD but not P450scc. Both P450arom and 17beta-HSD were significantly correlated with estradiol accumulation in culture medium. Progesterone secretion was correlated with 3beta-HSD but not P450scc mRNA levels. StAR mRNA was detectable by RT-PCR, did not change with duration of culture and was not correlated with progesterone secretion. FSH significantly stimulated P450arom and 17beta-HSD mRNA levels. Cell origin (from the antral or the basal layer of the membrana granulosa) did not affect steroidogenesis. We conclude that under the present cell culture system granulosa cells do not luteinize, and show expression of key steroidogenic enzymes in patterns similar to those occurring in differentiating follicles in vivo. Further, the data suggest that 17beta-HSD may be as important as P450arom in regulating estradiol secretion, and that 3beta-HSD is more important than P450scc as a regulator of progesterone secretion in non-luteinizing granulosa cells.

Cell plating density alters the ratio of estrogenic to progestagenic enzyme gene expression in cultured granulosa cells.

OBJECTIVE To determine if initial cell plating density alters steroidogenesis and the E(2):P ratio in granulosa cells in long-term serum-free culture. DESIGN Experimental study. SETTING Academic institution. ANIMAL(S) Cattle of slaughterhouse origin. INTERVENTION(S) Culture of granulosa cells in vitro at different cell plating density. MAIN OUTCOME MEASURE(S) Steroid secretion was measured by RIA, mRNA levels were measured by real-time polymerase chain reaction, and cell death was assessed by flow cytometry. RESULT(S) Low plating density favored E(2) secretion and mRNA encoding estrogenic enzymes, whereas higher density inhibited E(2) secretion and enhanced P secretion and levels of mRNA encoding progestagenic enzymes. Increasing plating density decreased the E(2):P ratio and cell health. CONCLUSION(S) Lower cell density favors an estrogenic granulosa cell phenotype, whereas higher density favors luteinization. Serum-free culture systems should be optimized with this in mind.

Role of transforming growth factor-β1 in gene expression and activity of estradiol and progesterone-generating enzymes in FSH-stimulated bovine granulosa cells

Survival and inhibitory factors regulate steroidogenesis and determine the fate of developing follicles. The objective of this study was to determine the role of transforming growth factor-beta1 (TGFB1) in the regulation of estradiol-17beta (E(2)) and progesterone (P(4)) secretion in FSH-stimulated bovine granulosa cells. Granulosa cells were obtained from 2 to 5 mm follicles and cultured in serum-free medium. FSH dose (1 and 10 ng/ml for 6 days) and time in culture (2, 4, and 6 days with 1 ng/ml FSH) increased E(2) secretion and mRNA expression of E(2)-related enzymes cytochrome P450 aromatase (CYP19A1) and 17beta-hydroxysteroid dehydrogenase type 1 (HSD17B1), but not HSD17B7. TGFB1 in the presence of FSH (1 ng/ml) inhibited E(2) secretion, and decreased mRNA expression of FSH receptor (FSHR), CYP19A1, and HSD17B1, but not HSD17B7. FSH dose did not affect P(4) secretion and mRNA expression of 3beta-hydroxysteroid dehydrogenase (HSD3B) and alpha-glutathione S-transferase (GSTA), but inhibited the amount of steroidogenic acute regulatory protein (STAR) mRNA. Conversely, P(4) and mRNA expression of STAR, cytochrome P450 side-chain cleavage (CYP11A1), HSD3B, and GSTA increased with time in culture. TGFB1 inhibited P(4) secretion and decreased mRNA expression of STAR, CYP11A1, HSD3B, and GSTA. TGFB1 modified the formation of granulosa cell clumps and reduced total cell protein. Finally, TGFB1 decreased conversion of androgens to E(2), but did not decrease the conversion of estrone (E(1)) to E(2) and pregnenolone to P(4). Overall, these results indicate that TGFB1 counteracts stimulation of E(2) and P(4) synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E(2) and cholesterol to P(4) without shutting down HSD17B reducing activity and HSD3B activity.

Effect of growth factors and co-culture with ovarian medulla on the activation of primordial follicles in explants of bovine ovarian cortex.

It has been proposed that the ovarian medulla exerts an intra-ovarian inhibitory effect on primordial follicle activation in cattle. We tested this hypothesis using cortical ovarian explants and determined whether growth factors could alter follicle activation or primary follicle health. Ovaries were obtained from bovine fetuses, and cortical slices were cultured on Millicell culture inserts for up to 8 days. Within 2 d of culture, the proportion of primordial follicles decreased from 70.1 +/- 3.5 to 6.4 +/- 3.4% (P<0.05), and the proportion of primary follicles increased from 23.8 +/- 3.3 to 79.7 +/- 5.5% (P<0.05). The proportion of secondary follicles was relatively stable (6 to 13%). Morphological examination indicated that 91.9 +/- 3.7, 76.7 +/- 8.8, and 71.8 +/- 10.4% of primordial, primary, and secondary follicles, respectively, were considered to be healthy in slices of fresh tissue; these proportions were not altered by up to 8 d of culture (P>0.05). The proportion of all classes of follicles and their morphological health were not affected by the addition of medullary slices to the culture well, nor by the culture of corticomedullary slices (P>0.05). The addition of FSH, insulin-like growth factor-I, epidermal growth factor, basic fibroblast growth factor, or transforming growth factor-beta did not alter primordial follicle activation or the morphological health of primary or secondary follicles. The addition of transforming growth factor-alpha (TGFalpha) decreased the proportion of primary follicles that were healthy from 67.6 +/- 5.1 to 36.8 +/- 4.7% (P<0.05). In conclusion, these data do not support the existence of a medullary inhibitor of primary follicle activation but suggest a role for TGFalpha in the regulation of primary follicle development.