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Divergence of intracellular signaling pathways and early response genes of two closely related fibroblast growth factors, FGF8 and FGF18, in bovine ovarian granulosa cells. Jiang Z et al. Fibroblast growth factors (FGFs) modulate ovarian function, including FGF8 and FGF18. These FGFs activate the same receptors, although FGF18 is unusual in that it increases apoptosis in ovarian granulosa cells whereas the 'typical' response to FGF is increased proliferation. The objective of the present study was to determine which early response genes and pathways are activated by FGF8 and FGF18 in bovine granulosa cells. FGF8 increased abundance of mRNA encoding the FGF-responsive genes SPRY1, SPRY2, SPRY4, NR4A1 and NR4A3 whereas FGF18 did not. FGF8 increased but FGF18 decreased levels of mRNA encoding the growth arrest associated protein, GADD45B. FGF8 increased ERK1/2 phosphorylation but FGF18 did not. Microarray analysis identified EGR1, FOS, FOSL1, BAMBI, XIRP1 and PLK2 as other FGF8 immediate-early response genes, and FGF18 stimulated EGR1, FOSL1, BAMBI and PLK2, but not FOS or XIRP1. This study demonstrates that FGF8 and FGF18 signal through divergent pathways in ovarian granulosa cells, despite reportedly similar receptor activation patterns.
//////////257 effects of fibroblast growth factor 8 on cumulus expansion and nuclear maturation in bovine cumulus-oocyte complexes. Ormond CM et al. Recent data indicate that fibroblast growth factor (FGF) signalling regulates oocyte developmental competence. Fibroblast growth factor 10 enhanced nuclear maturation, cumulus expansion, and embryo development in cattle (Zhang et al. 2010 Reproduction 140, 815-826). Like FGF10, FGF8 is expressed in the bovine oocyte, but whereas FGF10 activates FGF receptors (FGFR) 1B and 2B with higher affinity, FGF8 preferentially activates FGF receptor (FGFR) 2C, FGFR3C, and FGFR4. The involvement of FGF8 in the regulation of bovine cumulus-oocyte complex (COC) maturation has remained unknown. This study aimed to assess the effects of FGF8 supplementation in the in vitro maturation medium on nuclear maturation, degree of cumulus expansion, and expression of the genes necessary for expansion in bovine COC. Groups of 20 immature COC (grades 1 and 2) aspirated from 3- to 8-mm follicles were cultured in 200-?L drops of TCM-199 supplemented with FSH (1?gmL(-1)), LH (10IUmL(-1)), pyruvate (22?gmL(-1)), amikacin (75?gmL(-1)), and graded doses of recombinant human FGF8 (Peprotech, Rocky Hill, NJ, USA; 0, 1, 10, and 100ngmL(-1)) for 22h at 38.5?C and 5% CO(2). After culture, COC were visually classified according to the degree of cumulus expansion (grades 1 to 3, indicating absent, moderate, and full expansion, respectively). Oocytes were mechanically separated from cumulus cells and stained with Hoechst 33342 to assess meiosis progression. Total RNA was extracted from cumulus cells using RNeasy (Qiagen, Venlo, the Netherlands), and 100ng of RNA was reverse-transcribed using Omniscript (Qiagen). Expression levels of messenger RNA encoding genes necessary for cumulus expansion [prostaglandin endoperoxide synthase 2 (PTGS2), hyaluronan synthase 2 (HAS2), pentraxin 3 (PTX3) and tumour necrosis factor-stimulated gene-6 protein (TSG6)] were assessed by real-time PCR, with cyclophilin (CYCA) as the housekeeping gene. Data were derived from 5 replicates. Maturation and expansion data were transformed to arcsine, and gene expression data were log transformed. Effects of treatments were tested by ANOVA, and means were compared with the Tukey-Kramer honestly significant difference test. The FGF8 at 10 and 100ngmL(-1) reduced the proportion of oocytes reaching metaphase II (70, 64.8, 52.8, and 36% for FGF8 at 0, 1, 10, and 100ngmL(-1), respectively; P=0.005) and increased the proportion of oocytes in metaphase I at 22h of culture (30, 35.2, 47.2, and 64% for FGF8 at 0, 1, 10, and 100ngmL(-1), respectively; P=0.004). Fibroblast growth factor 8 did not affect the degree of cumulus expansion as visually assessed. However, FGF8 at 10 and 100ngmL(-1) increased the messenger RNA abundance of PTGS2 [P=0.0002; relative values (?SEM) of 0.69?0.10, 0.63?0.11, 1.56?0.44, and 1.67?0.20 for FGF8 at 0, 1, 10, and 100ngmL(-1), respectively] and HAS2 [P=0.0002; relative values (?SEM) of 1.38?0.15, 1.37?0.24, 3.58?0.61, and 4.14?0.27 for FGF8 at 0, 1, 10, and 100ngmL(-1), respectively] in cumulus cells. In conclusion, the present data suggest the involvement of FGF8 in the mechanisms regulating transcription of expansion-inducing genes in cattle. In contrast with previous findings with FGF10, FGF8 inhibited nuclear maturation, suggesting different actions for different FGF in the regulation of COC maturation. Further research is needed to clarify the roles of FGF8 in the bovine COC.
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Comment |
Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth factor receptor-3c and -4, in bovine antral follicles Buratini J Jr, et al .
Paracrine cell signaling is believed to be important for ovarian follicle development, and a role for some members of the fibroblast growth factor (FGF) family has been suggested. In the present study, we tested the hypothesis that FGF-8 and its cognate receptors (FGFR3c and FGFR4) are expressed in bovine antral follicles. RT-PCR was used to analyze bovine Fgf8, Fgfr3c and Fgfr4 mRNA levels in oocytes, and granulosa and theca cells. Fgf8 expression was detected in oocytes and in granulosa and theca cells; this expression pattern differs from that reported in rodents. Granulosa and theca cells, but not oocytes, expressed Fgfr3c, and expression in granulosa cells increased significantly with follicle estradiol content, a major indicator of follicle health. Fgfr4 expression was restricted to theca cells in the follicle, and decreased significantly with increasing follicle size. To investigate the potential regulation of Fgfr3c expression in the bovine granulosa, cells were cultured in serum-free medium with FSH or IGF-I; gene expression was upregulated by FSH but not by IGF-I. The FSH-responsive and developmentally regulated patterns of Fgfr3c mRNA expression suggest that this receptor is a potential mediator of paracrine signaling to granulosa cells during antral follicle growth in cattle.
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Mutations |
1 mutations
Species: None
Mutation name: None
type: naturally occurring
fertility: fertile
Comment: Single nucleotide polymorphisms in the bovine genome are associated with the number of oocytes collected during ovum pick up. Santos-Biase WK et al. The number of follicles recruited in each estrous cycle has gained practical importance in artificial reproductive technology, as it determines the oocyte yield from ultrasound-guided ovum pickup for in vitro embryo production. We aimed to identify single nucleotide polymorphisms (SNPs) in bovine genes related to reproductive physiology and evaluate the association between the candidate SNPs and the number of oocytes collected from ultrasound-guided ovum pickup. We sequenced genomic segments of GDF9, FGF8, FGF10 and BMPR2 and identified seventeen SNPs in the Bos taurus and Bos indicus breeds. Two SNPs cause amino acid changes in the proteins GDF9 and FGF8. Three SNPs in GDF9, FGF8 and BMPR2 were genotyped in 217 Nelore cows (B. indicus), while two previously identified mutations in LHCGR and mitochondrial DNA (mtDNA) were genotyped in the same group. The polymorphisms in GDF9, FGF8, BMRP2 and LHCGR were significantly associated (P<0.01) with the number of oocytes collected by ovum pickup, whereas the SNP in the mtDNA was not. In addition, we estimated an allelic substitution effect of 1.13?0.01 (P<0.01) oocytes for the SNP in the FGF8 gene. The results we report herein provide further evidence to support the hypothesis that genetic variability is an important component of the number of antral follicles in the bovine ovary.
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