257 EFFECTS OF FIBROBLAST GROWTH FACTOR 8 ON CUMULUS EXPANSION AND NUCLEAR MATURATION IN BOVINE CUMULUS–OOCYTE COMPLEXES

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-µL drops of TCM-199 supplemented with FSH (1 µg mL–1), LH (10 IU mL–1), pyruvate (22 µg mL–1), amikacin (75 µg mL–1), and graded doses of recombinant human FGF8 (Peprotech, Rocky Hill, NJ, USA; 0, 1, 10, and 100 ng mL–1) for 22 h at 38.5°C and 5% CO2. 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 100 ng 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 100 ng mL–1 reduced the proportion of oocytes reaching metaphase II (70, 64.8, 52.8, and 36% for FGF8 at 0, 1, 10, and 100 ng mL–1, respectively; P = 0.005) and increased the proportion of oocytes in metaphase I at 22 h of culture (30, 35.2, 47.2, and 64% for FGF8 at 0, 1, 10, and 100 ng mL–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 100 ng mL–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 100 ng mL–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 100 ng mL–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. Supported by FAPESP.

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