Expression Of BMP15 And GDF9 In Bovine Oocytes Matured In Vitro And Their Effects On Apoptosis Of Cumulus Cells

In animal reproduction, in vitro maturation(IVM),in vitro fertilization(IVF), and embryo development were important techniques for elevating the domestic animal reproduction. The IVM technology was well studied in various animals and a number of studies were carried to research the high efficiency of IVM in embryo development.Also, oocyte quality and developmental competence during IVM were very important factors to decide high efficiency in embryo in vitro production(IVP) systems. The importance of CCs in oocyte developmental competence was already elucidated in various livestock such as sheep, pig and cattle. Most of oocyte-secreted factors are members of the transforming growth factor-beta superfamily(TGF-beta), in which growth differentiation factor 9(GDF9) and bone morphogenetic protein 15(BMP15)are best understood in IVM. GDF9 and BMP15 which could act to CCs apoptosis are expressed in animal oocytes during IVM and embryo development. GDF9 and BMP15 may be important factors for preventing the apoptosis of CCs during COCs IVM, but the expression levels and time of GDF9 and BMP15 may be different among animals species and theirs signaling pathway is also special-species. Here, this paper was carried out to investigate the expression of BMP15 and GDF9 mRNA during IVM of bovine cumulus cell-oocyte complexes(COCs). There were three experiments in this thesis,(1) Experiment One: BMP15 and GDF9 mRNA expression in bovine oocytes during IVM. The expression of BMP15 and GDF9 mRNA in bovine oocytes at 0 h(GV), 16 h(MII) and 24 h(MII) after IVM were evaluated by qRT-PCR;(2) Experiment Two: Effects of BMP15 and GDF9 on apoptosis of bovine CCs.BMP15 or(and)GDF9 were added to IVM medium at 0 ng/ml(control), or 100 ng/ml GDF9 D, or 100 ng/ml BMP15 or 100 ng/ml BMP15 + 100 ng/ml GDF9 during IVM.At 24 h post-IVM, mRNA expression of pro-apoptotic(Bim, Bax, p53) and anti-apoptotic(Bcl2) genes in bovine CCs were evaluated by q RT-PCR, and Bim and p53 protein expression in bovine CCs were evaluated by Western Blot.(3) Experiment Three: the signaling pathway of GDF9 on apoptosis of bovine CCs during IVM. Bovine CCs were pretreated with various inhibitors(â… )SB203580,(â…¡) PDTC,(â…¢)SB431542or( â…£)LY294002),respectively, for 1 h after 24 h COCs cultured with GDF9(100ng/ml), then the expression of Bim and p53 protein were evaluated by Western Blot.The results showed that:(1) BMP15 and GDF9 were both expressed in bovine oocytes,and the expression levels were different among IVM stage;(2) GDF9 significantly decreased the mRNA expression levels of Bax, Bim and p53 and increased the mRNA expression of Bcl-2. But GDF9 did not significantly decrease the mRNA expression levels of Survivin. GDF9 significantly decreased the apoptosis of CCs by inhibiting the expression of Bim protein;(3) GDF9 might inhibit apoptosis of bovine CCs by inhibiting BIM expression via the SMAD and PI3K pathways.