| Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins family, not only expresses widely in nervos system, but also in mammalian ovaries. It has been reported that BDNF plays a very important role in mammalian follicles development, oocytes maturation and early embryos development. The purpose of this study was to investigate the effect of BDNF on the in vitro maturation (IVM) efficiency of buffalo oocytes and its relevant regulation meschanism, so as to make further efforts to optimize the IVM culture system, and improve the efficiency of in vitro embryos production.1. The study of cloning and sequence analysis of buffalo BDNF gene and investigation of its expression pattern in different tissues. Sequence analysis showed that the whole length of buffalo BDNF gene was 800bp, and the CDS was 753bp, futhermore, the buffalo BDNF gene coded 250 amino acid, and it shared high homologies in nucleotide sequence and amino acid sequence with several other species. The protein structure prediction analysis showed that BDNF was a secretory protein which contained signal peptide sequence, and it was located in the cell, moreover, the tertiary structure of BDNF protein was composed of several a-helix and 3 pairs of antiparallel ?-sheet structures, which configuring an active center aslo called the NGF functional domain. The qRT-PCR result showed that BDNF mRNA completely expressed in 10 tissues including of heart, liver, spleen, lung, kidney, brain, skin, muscle, ovary and testies of fetal and adult buffalo, especially, it highly expressed in ovary. However, the TrkB and p75 mRNA expression pattern in tissues of fetal and adult buffalo was not completely similar with that of BDNF mRNA, but the TrkB mRNA highly expressed in ovary, while the p75 mRNA expressed quitely lowly in ovary.2. The study of the expression pattern of BDNF and its receptors in buffalo follicles, oocytes and early embryos. The experiment result by using immunochemistry, ELISA, Immunofluorescence and qRT-PCR technology showed that BDNF expressed completely during the development of buffalo follicles, and it mainly expressed in oocytes, granulosa cells and cumulus cells. Moreover, the BDNF concentration in follicular fluid and the expression level of BDNF mRNA and TrkB mRNA in follicular granulosa cells from the follicles with 4<??7mm diameter were the highest, and the follicles with ?>7mm diameter were the next, lastly, the follicles with 2???4mm diameter were the lowest, and there was significant differences among of them (P<0.05), however, the expression level of p75 in follicular fluid and granulosa cells was very low. During the buffalo COCs in vitro maturation (0h,6h,12h,24h), BDNF mRNA showed an increased gradually expression trend in oocytes and cumulus cells, TrkB mRNA showed an increased and then decreased expression trend in cumulus cells, but it was not present in oocytes, p75 mRNA showed a decreased and then increased rapidly expression trend in oocytes and cumulus cells. Furthermore, during the buffalo early embryos development (2cell,4cell,8cell, morula, blastocyst), BDNF mRNA showed an increased and then decreased expression trend, and it expressed highest in 4cell stage, p75 mRNA showed a decreased gradully expression trend, and it expressed highest in 2cell stage. Besides, the expression level of both BDNF and p75 mRNA was very weak in morulae and blastocysts stage. However, the TrkB mRNA was not present in early embryos.3. The study of the effect and mechanism of BDNF on buffalo oocytes in vitro maturation. The result showed that adding with 10ng/ml BDNF into the maturation culture medium could significantly improve the oocytes maturity rate (59.07% vs 48.96%, P<0.05) and the blastocysts rate of the subsequent IVF embryos (23.29% vs 15.58%, P<0.05), but it had no significant effect on denuded oocytes (50.82% vs 50.86%, P>0.05). When adding the receptor inhibitor of TrkB (K-252a) and p75 (Pep5) individually into in vitro maturation culture medium, the oocytes maturity rate of the BDNF+K-252a treatment group was significantly lower than the BDNF treatment group (49.50% vs 60.01%, P<0.05), but there's no significant difference between the BDNF+Pep-5 treatment group and the BDNF treatment group (59.04% vs 60.01%, P>0.05). The qRT-PCR result of the apoptotsis relevant genes (Bax, Bcl-2, Caspase-3, Caspase-9, Fas, p53), the receptor genes of BDNF (TrkB, p75) and the development relevant genes (CCNB1, COX2, Cx37, Cx43, CYP11A1, FSHR, HAS2, MAPK, PCNA, PGES, PTX3,TSG-6) in cumulus cells showed that BDNF could significantly down-regulate the expression level of Caspase-9 and Fas, and up-regulate the expression level of TrkB, CCNB1, PCNA, Cx37, Cx43, HAS2, PTX3, TSG-6.In conclusion:(1) The buffalo BDNF gene cloned is 800bp in whole length, and the CDS is 753bp while codes 250 amino acid, and it keeps high conservative among different species, moreover, BDNF and its receptor of TrkB express in high-level in buffalo ovary. (2) BDNF presents in different development stages of buffalo follicles, meanwhile BDNF and p75 mRNA completely express in the granulosa cells from different diameters follicles, and the oocytes and cumulus cells of COCs during in vitro maturation, as well as the early embryos in different development stages, but the TrkB mRNA only expresses in granulosa cells and cumulus cells, it can't be detected in oocytes and early embryos. (3) The suitable concentration of BDNF (10ng/ml) is good for the efficiency of buffalo oocytes in vitro maturation and the acquirement of embryos developmental potential, and to make this effect of BDNF to work, BDNF maybe need to combine with the receptor of TrkB in cumulus cells, and then it can down-regulate the expression level of apoptosis relevant genes Caspase-9 and Fas, and up-regulate the expression level of receptor Tr?B and proliferation relevant genes CCNB1 and PCNA, aslo it can up-regulate the gap junction genes Cx37 and Cx43, as well as the cumulus expansion relevant genes HAS2, PTX3, TSG-6. |
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