Effects Of Vascular Endothelial Growth Factor On The In Vitro Maturation Of Buffalo Oocytes

At present, the technology of oocytes in vitro maturation (IVM) had made great progress, but there were still problems of poor developmental ability for oocytes obtained by IVM. The purpose of this study was to investigate the effects of vascular endothelial growth factor (VEGF) on buffalo oocytes maturation efficiency and its subsequent embryonic development, and also study the relevant meschansm, so as to further improve the IVM system for buffalo oocytes, and then improve the efficiency of in vitro embryo production.Firstly, the expression of VEGF and its receptor VEGFR1and VEGFR2in the ovarian tissue and cumulus oocyte complexes (COCs) duing IVM were detected. The result of immunohistochemistry showed that VEGF present all developmental stages of follicle and the granulosa cells. Immunofluorescence staining observed that it was a strong fluorescence signal for VEGF in cumulus cells of buffalo COCs, but no in the oocyte. Real-time fluorescence quantitative PCR (Q-PCR) was used to detect the mRNA level of VEGF and its receptor (VEGFR1and VEGFR2) in buffalo COCs during IVM (0,6,12and24h), and found that the expression of VEGF processed a downward trend, in which the expression of VEGF in the group of Oh was significantly higher than the group of culture for6h (p<0.05), while it was no significant difference among of the groups of culture for6h,12h and24h (p>0.05). By contrast, the expression of VEGFR1and VEGFR2during COCs IVM showed an increased and then decreased trend, wherein, the amount of expression of VEGFR1reached to the maximum value when the COCs culture for6h, and VEGFR2expression level reached to the maximum in12h.Secondly, the effect of different concentrations of VEGF on the buffalo oocytes maturation and subsequent embryonic development were examined, as well as its active mechanism was analyzed. The results showed that adding with5ng/ml of VEGF to culture the buffalo COCs could significantly improve the mature rate of buffalo oocyte (34.81%vs43.73%, P<0.05). Moreover, the rate of subsequent cleavage (35.11%vs44.81%;48.65%vs86.27%; P<0.05) and blastocyst development (11.45%vs20.18%;18.92%vs31.69%; P<0.05) in the embryos derived from IVF and SCNT were also increased. However, adding with5ng/ml VEGF had no significant effect on the denuded oocytes during IVM (22.67%vs18.18%, P>0.05). TUNEL analysis showed that adding with 5ng/ml VEGF could significantly reduce the apoptosis rate of cumulus cells during COCs IVM. Furthermore, the expression of genes related to apoptosis (bax, bc12, birc4, caspase-3and fas) and development (CX37, PTGS2, CX43, CYP11A1, CYP19A1, PCNA, CCNB, FSHR, MAPK, VEGFR1and VEGFR2) in the cumulus cells were detected by Q-PCR technology after IVM, and found that the expression levels of birc4, CX37, CYP11A1and PGES increased significantly when the COCs were treated with5ng/ml VEGF (P<0.05), and the expression levels of caspase-3and fas decreased significantly (P<0.05), but it had no significant effect on bc12, bax, CX43, CY19A1, PCNA, FSHR, MAPK, CCNB, PTGS2, VEGFR1and VEGFR2.In conclusions, the present study demonstrated that VEGF was present in all development stages of the buffalo follicle, and VEGF was mainly expressed in the granulosa cells but not in oocytes. Endogenous VEGF in buffalo COCs during IVM showed a downward trend, while VEGFR1and VEGFR2expression had a increasing and then decreasing trend. Adding with5ng/ml of the VEGF during COCs IVM was beneficial to improve buffalo oocytes maturation and its subsequent developmental competence, but had no effect on denuded oocyte. Moreover, supplementing with5ng/ml VEGF could inhibit the apoptosis of cumulus cells, and up-regulate the mRNA level of birc4, CX37, CYP11A1and PGES in the cumulus cells, as well as down-regulate the expression of caspase-3and fas, but it had no effect on bcl2, bax, CX43, CY19A1, PCNA, FSHR, MAPK, CCNB, PTGS2, VEGFR1and VEGFR2.