ABSTRACT
In the ovary, angiotensin II (ANGII) acts through the type 2 receptor (AGTR2) to induce ovulation and may play a role in follicle atresia. In this study, we determined the expression of AGTR2 mRNA and protein during follicle formation in the bovine ovary. Female fetuses at different gestational ages (60, 75, 90, 120, 150 and 210 days) were used for immunolocalization of AGTR2. At day 60, AGTR2 was localized to the cytoplasm of oogonia; from days 75 to 150, during follicle formation and development to secondary stage, AGTR2 immunostaining was weak and irregular, but from day 210 staining became evident in granulosa cells of preantral follicles and in both granulosa and theca cells of small antral follicles. These data differ from those in pigs, in which AGTR2 protein is detected in preantral follicles throughout gestation. Abundance of AGTR2 mRNA in whole ovaries did not change with fetal age. In conclusion, AGTR2 protein is expressed in ovigerous cords in fetal bovine ovaries but not in preantal follicles until the formation of antral follicles. These data suggest important species-specific differences in the expression of AGTR2 in fetal ovaries from polyovulatory and monovulatory animals.
Subject(s)
Ovary/embryology , Receptor, Angiotensin, Type 2/analysis , Animals , Cattle/embryology , Female , Fetus/chemistry , Fluorescent Antibody Technique/veterinary , Microscopy, Confocal/veterinary , Ovary/anatomy & histology , Ovary/chemistry , Receptor, Angiotensin, Type 2/physiology , Reverse Transcriptase Polymerase Chain Reaction/veterinaryABSTRACT
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.
Subject(s)
Fibroblast Growth Factor 2/metabolism , Granulosa Cells/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , Animals , Calcium Signaling , Cattle , Cells, Cultured , Female , Gene Expression Regulation , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Protein Kinase C/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA, Messenger/metabolism , Recombinant Proteins/metabolism , Time FactorsABSTRACT
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.
Subject(s)
Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/metabolism , Oogenesis/physiology , Ovarian Follicle/cytology , Ovarian Follicle/physiology , Animals , Cattle , Cell Differentiation/drug effects , Cell Differentiation/physiology , Cells, Cultured , Dose-Response Relationship, Drug , Estradiol/metabolism , Female , Fibroblast Growth Factors/pharmacology , Gene Expression Regulation/physiology , Granulosa Cells/cytology , Granulosa Cells/drug effects , Granulosa Cells/physiology , Insulin-Like Growth Factor I/pharmacology , Oocytes/cytology , Oocytes/drug effects , Oocytes/physiology , Progesterone/metabolism , RNA, Messenger/metabolism , Theca Cells/cytology , Theca Cells/drug effects , Theca Cells/physiologyABSTRACT
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.
