Effects Of Tauro Ursodeoxycholic Acid On Bovine Oocyte Maturation And Early Embryo Development In Vitro

The study was to investigate the effects of endoplasmic reticulum stress (ERS) inhibitors taurocholate ursodeoxycholic acid (TUDCA) on the efficiency of bovine granule cells (GCs) in vitro culture (IVC), oocytes in vitro maturation (IVM) and its subsequent embryonic development, so that to perfect the FVM systems for bovine oocytes, and further improve the efficiency of embryo production in vitro. This study was included two parts:Part I, the effects of TUDCA on the cell growth, apoptosis and ERS-related genes expression of bovine GCs was investigated, and then the apoptosis model of GCs was built by adding tunicamycin (TM) to study the inhibitory effect of TUDCA on apoptosis induced by TM. The results shown that:(1) There was no significant difference in the Cell growth and normal diploid karyotype rates among of bovine GCs treated with different concentrations of TUDCA (0,100,250,500,1000,1500μmol/L)(P>0.05), while the apoptosis rate of cells in the1500μmol/L TUDCA group was significantly lower than that of the Oμmol/L group (P<0.05). The QRT-PCR analysis found:with increasing concentration of TUDCA, the expression levels of the ERS-related genes Grp78, Irel, Chop and Bax genes shown a decreasing trend, and they were remarkedly lower than that of the Oμmol/L group (P<0.05), whereas, the expression levels of Bcl-2processed a upward trend.RT-PCR analysis showed the XBP-1mRNA splicing was no apparent change among of various concentration groups.(2) When the bovine GCs were respectively treated with TM (0,100,500,1000ng/ml) for12h, with increasing the treated concentration of TM, the expression levels of Grp78, Ire1,Chop,Bax genes and the apoptosis rates of cells had an enhancing trend, but the expression levels of Bcl-2gene shown a decreasing trend, and the apoptosis rate and the expression levels of Grp78, Ire1,Chop, Bax genes of GCs in the500and1000ng/ml TM groups were significantly higher than that of the0ng/ml group (P<0.05), and the expression levels of Bcl-2was remarkedly lower than that of the0ng/ml group (P<0.05), while the levels of spliced Xbpl gradually was upregulated with increasing treated concentration of TM.(3) The bovine GCs were cultured in the presence of different concentrations of TUDCA (0,500,1000,1500μmol/L) for24h, and then TM (500ng/ml) was added into the medium to co-culture for12h, and found that the apoptosis rate and the expression levels of Grp78, Irel, Chop and Bax genes of cells in the1500μmol/L TUDCA group were significantly improved compared with those of the positive-control group (TUDCA=Oμmol/L, TM=500ng/ml, P<0.05). However, the expression of Bcl-2shown a rising and then decreasing trend with increasing concentration of TUDCA, and it was remarkedly different in the groups of500,1000, and1500μmol/L TUDCA with the positive-control and the blank-control groups (TUDCA=Oμmol/L, TM=Ong/ml)(P<0.05). Moreover, with increasing concentration of TUDCA, the levels of spliced Xbp-1shown the decreasing trend compared with the positive-control group.Part Ⅱ, the effects of TUDCA on the bovine oocytes IVM and embryonic development were examined. Firstly, the splicing of XBP-1was observed during the bovine oocytes IVM and the development of IVF embryos, and found that the spliced Xbp-1form was especially obvious at the stages of oocytes IVM for12h,24h, as well as4-cell, morula, blastocyst of IVF embryos. Afterward, when the oocytes were treated with different concentrations of TUDCA (0、100、250、500、1000、1500μmol/L) during IVM, the first polar body exclusion rate of oocytes, and the subsequent embryonic cleavage rate and blastocyst rate of IVF embryos in500μmol/L TUDCA treatment group were significantly improved compared with the control group (P<0.05). The detection of genes expression shown:the relative expression of Grp78, Irel and Chop genes in oocytes in the500μmol/L TUDCA treatment group significantly decreased compared with the Oμmol/L group (P<0.05), and the expression ratio of Bcl-2and Bax genes was also increased, furthermore, the levels of spliced Xbp-1of oocytes also reduced. Additionally, when bovine IVF embryos were respectively treated with different concentrations (0,100,250,500,1000μmol/L) of TUDCA, and found that adding with500μmol/L TUDCA to culture the bovine IVF embryos could significantly improved the blastocyst rate and the cell number of blastocysts (P<0.05), and markedly decreased the apoptosis rate of blastocysts (P<0.05). Equally, the detection of genes expression also shown that treating embryos with500μmol/L TUDCA significantly reduced the expression of Ire1, Chop genes (P<0.05), and up-regulated the expression of Bcl-2gene (P<0.05), moreover, the levels of spliced Xbp-1of blastocysts also decreased.In conclusion:(1) The suitable concentration of TUDCA (≤1500μmol/L) have no significant effect on cell growth and karyotype of bovine GCs.(2) TM promote the expression of Grp78, Ire1, Chop, Bax gene and inhibit the expression of Bcl-2, as well as activate the XBP-1splicing to induce bovine GCs apoptosis.(3) Adding with1500μmol/L TUDCA can decrease the apoptosis rate of bovine GCs, down-regulate the expression of ERS related genes (Grp78, Ire1, Chop, Bax), and up-regulate the expression of Bcl-2gene, as well as reduce the levels of spliced Xbp-1to inhibit the ERS of bovine GCs.(4) ERS is present in the process of bovine oocytes IVM and embryos IVC, supplementing with500μmol/L TUDCA into the oocytes maturation medium or embryo culturing medium may inhibit ERS to facilitate the oocytes maturation and embryonic development.