Identification And Characterization Of Key Maternal Factors To Porcine Nuclear Transfer Reprogramming

Somatic cell nuclear transfer(SCNT) holds great potential for many aspects, but the practical use is limited by the extremely low efficiency of SCNT. To improve SCNT efficiency and deepen our undertanding of nulear reprogramming, the key maternal factors to nulear reprogramming were needed to be explored. In our previous studies, we found that most of the oocytes cultured in vitro for 33 h had completed nuclear maturation. But the cloned embryonic developmental rate of 33 O was significantly lower than that of 42 O which were generally used in porcine SCNT. This suggests that 42 O may contain more maternal factors beneficial to SCNT reprogramming than 33 O, and that provides us an opportunity to find out key maternal factors to nuclear reprogramming. In this study, the proteomes were compared between 33 O and 42 O to uncover the differentially expressed proteins, and it was attempted to identify key maternal factors to SCNT reprogramming, which could expand our knowledge of nulear reprogramming. The main results were as following:(1) The method of 2D-PAGE+MALDI-TOF/TOF was used to identify the differentially expressed proteins between 33 O and 42 O. It was found that 994 pairs of protein between 33 O and 42 O. And 18 differentially expressed proteins were identified, which 7 proteins expressed higher in 33 O while the other 11 proteins expressed higher in 42 O. Bioinformatics analysis found that the functions of the differentially expressed proteins mainly concentrated in oocyte maturation, embryo development and chromotine remolding et al. The results provide valuable information to understand the molecular mechanism of porcine oocyte maturation, and give us a candidate protein list to identify new key maternal factors to SCNT reprogramming.(2) Vimentin(VIM) is a maternal protein expressed higher in 42 O. First we tested the expression pattern of VIM in early embryos, and the results showed that VIM expressed highly in oocytes and decreased rapidly after fertilization and activation, and enriched in the donor nuclei after NT, which suggest VIM may function in early reprogramming process. After inhibition of VIM function by injection of anti-VIM antibody in MII oocytes, the cloned embryonic development was significantly lower than that of Ig G-NT and Con-NT embryos(12.24 v.s. 22.57 and 21.10%, respectively; P < 0.05), and more anti-VIM-NT embryos arrested at the two- or fourcell stage. But inhibition of VIM did not disturb in vitro development of IVF and PA embryos. This demonstrated that maternal VIM is indispensible for normal pig cloned embryonic development. To explore the function mechanism of maternal VIM, γ-H2 AX, as a symbol of DNA double strand breaks(DSBs), were tested on cloned embryos after injection of anti-VIM antibody. Inhibition of maternal VIM function resulted in increasedγ-H2 AX level in cloned embryos, and the expression of P53 in 4-cell was significantly higher in anti-VIM group than that of control group. These results suggest that maternal VIM acts as a genomic protector by inhibiting DSBs and leads to down-regulation of p53 during nuclear reprogramming, which is essential for successful porcine nuclear reprogramming.(3) EZH2, as H3K27me3 methylase, is also a maternal factor. The results showed that EZH2 was highly expressed in oocytes and enriched in nuclei after NT. The H3K27me3 level was decreased in cloned embryos after inhibition of EZH2 function by injecting anti-EZH2 antibody into MII oocytes. Meanwhile, the cloned embryonic development was significantly higher in antiEZH2 group than that of Ig G-NT embryos(Ig G v.s. anti-EZH2, 20.60% v.s. 30.63%, P < 0.05). This suggests that H3K27me3 level may be abnormal high level in cloned embryos, and the improvement of cloned embryonic development may be achieved by decreasing H3K27me3 level in cloned embryos after inhibition of EZH2. Thus the global H3K27me3 levels were determined at various stages of IVF and cloned embryos. The results demonstrated that the H3K27me3 level of early cloned embryos was higher than that of IVF embryos, which may related to higher H3K27me3 level in PEF cells than that of oocytes and sperms. Then, the two specifical inhibitors for H3K27me3 methylase(GSK126 for EZH2) and demethylase(GSK-J4 for UTX/JMJD3) were used for regulating H3K27me3 level in cells and embryos. The results showed that H3K27me3 level was reduced in cloned embryos by treatment of PEF with 0.75 μM GSK126 for 48 h or incubation of 1-cell embryos with 0.1 μM GSK126. Meanwhile, it was observed that the development of cloned embryos were significantly improved, which was similar with injection of anti-EZH2 experiment. On the contrary, GSK-J4 treatment increased the H3K27me3 level in cloned embryos and decreased the cloned embryonic development. Furthermore, i PS efficiency were increased by reducing the H3K27me3 level in donor cells or during the early reprogramming phase. In summary, we found that maternal EZH2 is adverse to porcine SCNT reprogramming, and reducing H3K27me3 level by inhibiton of EZH2 could improve SCNT and i PS efficiency.