130 THE SYNERGIC EFFECT OF NERVE GROWTH FACTOR AND VASCULAR ENDOTHELIAL GROWTH FACTOR ON IN VITRO MATURATION AND DEVELOPMENTAL COMPETENCE IN BOVINE OOCYTES

Abstract

Nerve growth factor (NGF) has been reported to increase the mRNA expression of vascular endothelial growth factor (VEGF) in granulose cells of human, rat via TrkA signaling; VEGF has been shown to exert beneficial effects during bovine in vitro maturation (IVM) as well as early embryonic development. The aims of this study were 1) to investigate not only the direct effect of NGF but also the collaborative effect of NGF and VEGF during bovine in vitro maturation (IVM), in vitro culture (IVC), or both; and 2) to validate the correlation among transcript abundance of 7 genes (VEGF164, VEGF120, Flt-1, Flk-1, TrkA, PTGS2, and CYP11A1) in bovine cumulus cells and the results of IVM or IVC among the differently treated groups. In Experiment 1, concentrations of 0, 10, and 100 ng mL–1 NGF were added to our established IVM medium without serum, and in Experiment 2, control and treatment groups (concentration of 0, 10, and 100 ng mL–1 NGF with VEGF 100 ng mL–1) were added into chemically defined media. The oocytes of each group in Experiments 1 and 2 were determined by the proportion of MII oocytes after 24 h, and embryos were assessed after parthenogenetic activation. Cumulus cells from the differently treated matured cumulus cell–oocyte complexes (COC) were separated and synthesised into cDNA for RT-PCR and real-time PCR in order to measure relative abundance of 7 genes in a dose-dependent manner or a time-dependent manner. In Experiment 1, the concentration of 10 ng mL–1 (57.40%) and 100 ng mL–1 (62.75%) NGF treatment groups did not significantly increase the proportion of MII oocytes compared with the control group (55.06%). In Experiment 2, both the NGF 10 ng mL–1 with VEGF 100 ng mL–1 treated group (67.69%; P ≤ 0.01) and the NGF 100 ng mL–1 with VEGF 100 ng mL–1 treated group (72.24%; P ≤ 0.001) had a significantly higher percentage of polar body extrusion than control group (51.77%) and the group which was treated with VEGF 100 ng mL–1 (56.39%). The NGF treatment group with VEGF increased transcriptional level of VEGF164 and VEGF120 compared with the control group and only NGF- or VEGF-treated groups. In addition, either the NGF-treated group or NGF plus VEGF showed significantly increased mRNA abundance in VEGF164, VEGF120, Flt-1, Flk-1, and TrkA genes, whereas the NGF plus VEGF-treated group indicated the up-regulation of VEGF164, VEGF120, CYP11A1, and PTGS2 genes. In conclusion, NGF and exogenous VEGF have a synergic effect during bovine IVM and the early stage of embryo development; the elevated VEGF mRNA abundance in cumulus cells might contribute to the viability of bovine oocytes and early embryonic development. This study was supported by grants from IPET (#109023-05-1-CG000), NRF (#M10625030005-10N250300510), MKE (#2009-67-10033839, #2009-67-10033805), and BK21 program.