Methylation Patterns In 5' Terminal Regions Of Pluripotency-related Genes In Bovine Mature Gametes As Well As In Vitro Fertilized And Cloned Embryos

In vitro fertilization and somatic nuclear transfer was and is a focus both at home and abroad. Their efficiencies could be influenced by such factors as in vitro maturation of gametes and in vitro culture of embryos to a large extent. In particular, the stage between 8-cell and morula is very critical, during which so-called maternal to zygotic transition occurs. Influences from in vitro system transmit by means of epigenetic modifications on the molecular level, in which DNA methylation is not only one of the important epigenetic marks, but also one of the pathways of gene regulation. For embryogenesis, it is in the control of delicate and complicated gene regulation network, during which, particularly for the early embryo development, pluripotency-related genes play the vital roles. With the genes harboring CpG islands in their promoter regions, their expression is preferentially regulated in a DNA methylation-mediated manner. Hence, the methylation patterns of pluripotency-related genes are closely associated with embryo development competence. Their aberrant methylatin levels always lead to developmental block and abnormalities. In the present study, we investigated methylation profiles of 5 pluripotency-related genes(Oct4, Sox2, Nanog, Rex1 and Fgf4) which serve as the indicators in mature gametes, in vitro fertilized 8-cell embryos, in vitro fertilized morulae, 8-cell nuclear transfer embryos, nuclear transfer morulae as well as donor cells and made statistics on developmental rates of embryos, which could serve as the foundation for future study on the epigenetic reprogramming in bovine mature gametes and maternal to zygotic transition, provide a better insight into aberrant development of cloned embryos and act as the possible indicators for assessing embryo developmental competence.1. We investigated methylation profiles of 5 pluripotency-related genes (Oct4, Sox2, Nanog, Rex1 and Fgf4) in bovine capacitated sperms and mature oocytes. The results showed that the methylation levels of Sox2 and Nanog were 1.7% and 24.6%, respectively, in oocytes, 0% and 22.3%, respectively, in sperms. Both two genes exhibited the nongamete-specific pattern, namely hypomethylation in both sperms and oocytes. Reversely, the methylation levels of Oct4, Rex1 and Fgf4 were 49.6%, 75% and 13.6%, respectively, in oocytes, 79.1%, 98.6% and 99.1%, respectively, in sperms. These three genes displayed gamete-specific methylation profiles, namely hypomethylation in oocytes and hypermethylation in sperms.2. We investigate DNA methylation profiles of five pluripotency-related genes (Oct4, Sox2, Nanog, Rex1 and Fgf4) during maternal to zygotic transition in bovine in vitro fertilized embryos. The results showed that methylation levels of Oct4, Sox2, Nanog, Rex1 and Fgf4 were 56.52%(130/230), 0.83%(2/240), 31.54%(41/130), 91.36%(201/220) and 50.91%(56/110) in in vitro fertilized 8-cell embryos, and 31.3%(72/230), 2.08%(5/240), 27.70%(36/130), 59.54%(131/220) and 40.91%(45/110) in in vitro fertilized morulae. Taken together, it is apparent that local methylation level decreases for the four pluripotency-related genes and that Sox2 maintains unmethylated status during MZT, although the global methylation level gradually increases.3. We investigated DNA methylation profiles of five pluripotency-related genes (Oct4, Sox2, Nanog, Rex1 and Fgf4) during maternal to zygotic transition in bovine nuclear transfer embryos. The results showed that methylation levels of Oct4, Sox2, Nanog, Rex1 and Fgf4 were61.74%(142/230), 4.16%(10/240), 3.84%(5/130), 91.82%(202/220) and 7.27%(8/110) in 8-cell nuclear transfer embryos, and 38.70%(89/230), 5.0%(12/240), 9.23%(12/130), 85.50%(188/220) and 12.73%(14/110) in nuclear transfer morulae, together with 94.78%(218/230), 0.83%(2/240), 48.46%(63/130), 90%(198/220) and 6.36%(7/110) in donor cells, respectively. As shown above, Oct4 and Rex1 respectively underwent demthylation by 23.04% (P<0.01) and 6.02% (P>0.05), and, reversely, Sox2, Nanog and Fgf4, respectively experienced remethylation by 0.84% (P>0.05), 5.39% (P>0.05) and 5.46% (P>0.05) during maternal to zygotic transition in nuclear transfer embryos. Interestingly, the CpG 14 site of Sox2 was specifically methylated both in 8-cell and in morula nuclear transfer embryos, while other sites stayed normal. The comparison between in vitro fertilized and nuclear transfer embryos showed that only Oct4 and Sox2 underwent the correct methylation reprogramming process. In conclusion, the five pluripotency-related genes experienced incomplete DNA methylation reprogramming during maternal to zygotic transition in bovine nuclear transfer embryos.4. We made statistics on developmental rates of in vitro fertilized and cloned embryos in different stages. The results showed that embryonic developmental rates in 2-cell, 8-cell, morula and blastocyst stage were respectively 81.44%(215/264), 56.44%(149/264), 42.80%(113/264) and 29.55%(78/264) for in vitro fertilized embryos, together with 77.02%(191/248), 51.61%(128/248), 38.71%(96/248) and 24.60%(61/248) for cloned embryos. The comparison between in vitro fertilized and nuclear transfer embryos showed that no significant difference was observed in developmental rates. This can be explained by the correct DNA methylation reprogramming of Oct4 and Sox2, for these two genes play vital roles during 8-cell, morula and blastocyst stages. However, there existed low implantation rate, high abortion and abnormalities rates which were reported previously. Based on the study, it can be explained by aberrant methylation reprogramming of Nanog, Rex1 and Fgf4, for these genes participate the late embryonic development. Collectively, methylation profiles of Oct4, Sox2, Nanog, Rex1 and Fgf4 could be used as indicators for assessing the developmental competence of cloned embryos.