Valério M. Portela

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.

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.

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.

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.

Regulation of Angiotensin Type 2 Receptor in Bovine Granulosa Cells

Angiotensin II (AngII) is best known for its role in blood pressure regulation, but it also has documented actions in the reproductive system. There are two AngII receptors, type 1 (AGTR1) and type 2 (AGTR2). AGTR2 mediates the noncardiovascular effects of AngII and is expressed in the granulosa cell layer in rodents and is associated with follicle atresia. In contrast, expression of AGTR2 is reported to occur only in theca cells in cattle. The objective of the present study was to determine whether AngII also plays a role in follicle atresia in cattle. RT-PCR demonstrated AGTR2 mRNA in both granulosa and theca cells of bovine follicles. The presence of AGTR2 protein was confirmed by immunofluorescence. Abundance of AGTR2 mRNA in granulosa cells was higher in healthy compared with atretic follicles, whereas in theca cells, it did not change. Granulosa cells were cultured in serum-free medium, and treatment with hormones that increase estradiol secretion (FSH, IGF-I, and bone morphogenetic protein-7) increased AGTR2 mRNA and protein levels, whereas fibroblast growth factors inhibited estradiol secretion and AGTR2 protein levels. The addition of AngII or an AGTR2-specific agonist to granulosa cells in culture did not affect estradiol secretion or cell proliferation but inhibited abundance of mRNA encoding serine protease inhibitor E2, a protein involved in tissue remodeling. Because estradiol secretion is a major marker of nonatretic granulosa cells, these data suggest that AngII is not associated with follicle atresia in cattle but may have other specific roles during follicle growth.

Role of Angiotensin II in the Periovulatory Epidermal Growth Factor-Like Cascade in Bovine Granulosa Cells In Vitro

ABSTRACT Angiotensin II (AGT-2) induces follicular prostaglandin release in a number of species and ovulation in rabbits. Conversely, AGT-2 antagonists block ovulation in cattle. To determine the mechanism of action of AGT-2, we used a bovine granulosa cell model in which luteinizing hormone (LH) increased the expression of genes essential for ovulation in a time- and dose-dependent manner. The addition of AGT-2 to LH-stimulated cells significantly increased abundance of prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA and protein, whereas AGT-2 alone had no effect. Upstream of PTGS2, AGT-2 increased abundance of mRNA encoding the epidermal growth factor-like ligands amphiregulin (AREG) and epiregulin (EREG) at 6 h posttreatment and abundance of a disintegrin and metalloprotease 17 (ADAM17), a sheddase, within 3 h of treatment. Inhibiting sheddase activity abolished the stimulatory effect of AGT-2 on AREG, EREG, and PTGS2 mRNA. The addition of selective AGT-2 antagonists to cells stimulated with LH plus AGT-2 demonstrated that AGT-2 did not act through the type 1 receptor and did not increase mitogen-activated protein kinase 3/1 phosphorylation. Combined with previous data from studies in vitro, we conclude that AGT-2 is an essential cofactor for LH in the early increase of ADAM expression/activity that induces the cascade of events leading to ovulation.

Regulation of inducible nitric oxide synthase expression in bovine ovarian granulosa cells.

Nitric oxide (NO) is a potential regulator of ovarian follicle growth, and ovarian granulosa cells reportedly generate NO in response to gonadotrophins, suggesting that the regulated form of nitric oxide synthase (iNOS) is present. The objectives of the present study were to gain insight into the expression and role of iNOS in the follicle. Messenger RNA encoding iNOS was detected in granulosa cells, and abundance was higher in growing dominant follicles compared to subordinate follicles (P<0.01). FSH (P<0.05) and IGF1 (P<0.01) stimulated oestradiol secretion and iNOS mRNA abundance in granulosa cells in vitro, whereas FGF2 (P<0.05) and EGF (P<0.01) decreased oestradiol secretion and iNOS expression. The addition of an anti-oestrogen prevented FSH-induced iNOS mRNA accumulation. Inhibition of endogenous NO production did not affect steroidogenesis in granulosa cells, but increased FasL mRNA abundance, caspase-3 activation and the incidence of apoptotic cell death (P<0.05). These results demonstrate that iNOS is expressed in ruminant granulosa cells and is regulated by gonadotrophins and oestradiol. Physiological levels of NO may contribute to the survival of granulosa cells.

Fibroblast Growth Factor-2 Regulation of Sprouty and NR4A Genes in Bovine Ovarian Granulosa Cells

Fibroblast growth factors (FGFs) alter ovarian function, at least in part by inhibiting steroid hormone secretion and affecting survival of granulosa cells. The mechanism of action of FGFs in ovarian follicle cells is largely unknown; in the present study we identified the major pathways used by FGF2 in non‐luteinizing granulosa cells cultured under serum‐free conditions. FGF2 increased abundance of mRNA encoding SPRY1, 2, and 4, but not SPRY3. Common pathways employed by FGF2 in the regulation of SPRY1, 2, and 4, as demonstrated by immunoblot and inhibitor studies, included ERK1/2 and Akt signaling. In contrast, PKC activation was necessary for FGF2‐stimulated expression of SPRY1 and 4, but not for SPRY2. Intracellular calcium flux is critical and sufficient for SPRY2 expression, but not for SPRY1 and 4. We also identified the orphan nuclear receptor NR4A1 as a potential early response gene in FGF2 signaling, whose expression, like that of SPRY2, is critically dependent on calcium signaling. Together, these data identify FGF2‐target genes in follicular granulosa cells, and demonstrate alternative pathway use for the differential control of SPRY genes. J. Cell. Physiol. 226: 1820–1827, 2011.

Regulation of MMP2 and MMP9 metalloproteinases by FSH and growth factors in bovine granulosa cells

Matrix metalloproteinases (MMP) are key enzymes involved in tissue remodeling. Within the ovary, they are believed to play a major role in ovulation, and have been linked to follicle atresia. To gain insight into the regulation of MMPs, we measured the effect of hormones and growth factors on MMP2 and MMP9 mRNA levels in non-luteinizing granulosa cells in serum-free culture. FSH and IGF1 both stimulated estradiol secretion and inhibited MMP2 and MMP9 mRNA abundance. In contrast, EGF and FGF2 both inhibited estradiol secretion but had no effect on MMP expression. At physiological doses, none of these hormones altered the proportion of dead cells. Although we cannot link MMP expression with apoptosis, the specific down regulation by the gonadotropic hormones FSH and IGF1 in vitro suggests that excess MMP2 and MMP9 expression is neither required nor desired for follicle development.

The Role of Fibroblast Growth Factor-18 in Follicular Atresia in Cattle

ABSTRACT Although the various members of the fibroblast growth factor (FGF) family are generally mitotic, one member, FGF18, has been shown to increase the rate of apoptosis of ovarian granulosa cells. In the present study, we first determined whether granulosa cells express FGF18 and we then explored the mechanism through which FGF18 increases apoptosis in vitro. Under culture conditions that favored estradiol secretion and CYP19A1 expression, granulosa FGF18 mRNA levels were barely detectable; however, withdrawing gonadotropic support (follicle-stimulating hormone or insulin-like growth factor 1) reduced levels of CYP19A1 mRNA and increased abundance of mRNA encoding the death ligand FASLG and FGF18. Addition of FGF18, but not FGF2, FGF10, or EGF, increased the proportion of apoptotic cells and frequency of caspase 3 activation, and these effects were abrogated by coculture with estradiol. Addition of FGF18 decreased abundance of mRNA encoding the antiapoptotic proteins GADD45B and MDM2, and increased that encoding the proapoptotic protein BBC3; these effects were reversed by coculture with estradiol. The physiological relevance of FGF18 was determined using an in vivo model: injection of FGF18 directly into growing bovine dominant follicles caused cessation of follicle growth by 24 h after injection. Collectively, these data demonstrate that FGF18 is proapoptotic in vivo and may act through a mechanism involving the BBC3-MDM2 pathway.