Treatment Nuclear Donor Cells With Taurousodeoxycholic Acid Enhances The Development Of Somatic Cell Cloned Embryos In Bovine

Treating donor cells with serum starvation is an important step in somatic cell nuclear transfer technology. Serum starvation can make the most of the donor cells’ cycle arrested in G0/G1 phase, increasing the capacity of egg cytoplasm programming and development after nuclear transfer. However, sudden removing of the serum in the culture medium is able to make cultured cells in difficult environment which can cause cell stress response, thus increasing the risk of cell damage. Tauroursodeoxycholic Acid(TUDCA) is an effective inhibitor of small molecules for endoplasmic reticulum stress. In the present study, we added TUDCA into serum starvation of bovine fetal fibroblasts to reduce the stress response caused by serum starvation, thus studying its influence on cloning embryos. This study achieved results as follows:1. In order to identify optimal TUDCA treatment concentration through the detection of the expression of ER stress related genes., bovine fetal fibroblasts were treated with different concentrations of TUDCA(50、100、150 and 200 μM) The relative m RNA abundance of GRP78 and CHOP was found to be significantly(P < 0.05) lower in the cells treated with 100 μM than those in control group. Hence 100 μM TUDCA was chosen for the following experiment.2. Treating bovine fetal serum-starved fibroblasts to detect its effect on cell proliferation and genes of apoptosis and reprogramming. As revealed by the results, TUDCA could significantly decrease the percentages of apoptosis cells(1.1±0.2% vs 6.4±0.4%, P < 0.05) and not affect the cell cycle. The relative m RNA abundance of P53 and BAX were lower compared to the untreated control group(P < 0.05). The expression levels of HDAC1 and DNMT1 in donor cells after TUDCA treatment were decreased(P < 0.05). And the level of H3K9 acetylation was 2.3-fold higher in the TUDCA-treated group than in the control group(P < 0.05).3. Fetal fibroblasts treated by TUDC A were used as somatic cell nuclear of transfer donor cells. Results showed that the fusion rates, cleavage rates and blastocyst formation rates of reconstructed embryos were significantly higher in comparison to the untreated control group(78.46% vs 68.89%, 92.09% vs 86.56%, 37.87% vs 30.43%, respectively, P < 0.05). In the case of transcript abundance, the expression levels of HDAC1,DNMT1 and P53 were significantly lower(P < 0.05) in blastocysts produced from treated donor cells than the control group. Besides, the blastocyst cell apoptotic rate was also decreased significantly(P < 0.05).In summary, bovine fetal fibroblasts with 100 μM TUDCA treatment decreased stress response of donor cells with serum s tarvation and significantly enhanced developmental competence of bovine embryoscells..