Study On The Effect Of Zebularine On Cumulus Cells And Development Of Somatic Cell Nuclear Tansfer Embryos

Somatic cell nuclear transfer technology has a grow increasing importance in biology, medicine and many other research areas, but low efficiency of nuclear transfer limit its development. Now widely recognized that nuclear transfer is inefficient mainly due to donor nucleus reprogramming incompletely in oocytes.In this study, sheep cumulus cells as donor cells, treated with DNA methyltransferase inhibitor Zebularine. and its effects on the biological characteristics of sheep cumulus cells, the Developmental Competence of SCNT embryos and the embryonic methylation level were studied. Try to establish a new method that affects neither the biological characteristics of the cells, but also reduce the methylation status of methods for somatic cell nucleus, thereby providing an effective way to improve the efficiency of somatic cell nuclear transfer.sheep cumulus cells treated with different concentrations(0, 2.5, 5, 10 nmol/L) of Zebularine for 12 h,to evaluate different treatment on sheep cumulus cell survival, chromosome ploidy, cell cycle, DNA methylation level, Dnmt1 gene expression level, nuclear transfer embryo development capabilities. At the same time, we examined the effect of DNA methylation levels of different developmental stages of embryos constructed from cumulus cells treated with 5nmol/L Zebularine for 12 h with indirect immunofluorescence staining.The results showed that:(1) The cumulus cell survival rate of cumulus cells treating with Zebularine(2.5~10 nmol/L) was not significantly fifferent with control group.(2) Karyotype analysis found that it was no significant difference in the percentage of chromosome diploid(96.11%, 96.53%, 95.13%, 96.77%) when the donor cells treated with different concentration of Zebulrine(0, 2.5, 5, 10 nmol/L).(3) The cell cycle was detected by flow cytometry showed that: Sheep cumulus cells were treated with different concentration of Zebularine for 12 h, the proportion of cells G0/G1 showed a gradual upward trend and the proportion of cells in S phase showed a gradual downward trend, but there was no significant difference with the control group(P>0.05);(4) Using indirect immunofluorescence staining to detect DNA methylation level, DNA methylation level of cumulus cells was decreased as Zebularine concentration increased; 5 and 10 nmol/L Zebularine treatment groups DNA methylation levels were significant lower than the control group(P<0.05), but no significant difference between the two treatment groups(P>0.05);(5)By real-time quantitative PCR detected that Dnmt1 experssion level of cumulus cells was decreased as Zebularine concentration increased; 5 and 10 nmol/L Zebularine treatment groups Dnmt1 experssion levels were significant lower than the control group(P<0.05), and 10 nmol/L Zebularine treatment group was significantly lower than 5 nmol/L Zebularine treatment group(P<0.05);(6) the effects of Zebularine on the developmental ability of sheep SCNT embryos derived from the donor cumulus cells treated with different concentrations of Zebulrine were also examined. There was no significant difference in cleavage rate of reconstructed embryos between Zebularine treatment groups and control group(P>0.05); the morulae rate of 5 nmol/L Zebularine treatment group was signifiantly higher than control group(P<0.05), but there was no significant difference with other groups(P>0.05). The blastocyst rate of 5 nmol/L Zebularine treatment group was signifiantly higher than control group and other Zebularine treatment groups(P<0.05).(7) Using indirect immunofluorescence staining to detect the DNA methylation level of SCNT embryos derived from the donor cumulus cells treated with 5nmol/L Zebularine, showed that: DNA methylation level trend of SCNT-Zebularine group embryos was similar to the in vitro fertilization group embryos and the SCNT-Control group embryos. And DNA methylation level of different developmental stages in SCNT-Zebularine group was lower than the SCNT-Zebularine group, which the 2-cell and 8-cell stage were significantly lower(P<0.05); but the DNA methylation level of different developmental stages embryos of SCNT-Zebularine group is still higher than the corresponding period of IVF embryos(P>0.05).In summary, the donor cumulus cells treated with different concentrations(2.5、5、10 nmol/L)of Zebulrine,there was no different in cell survival, chromosome ploidy, cell cycle,but after treating with 5, 10 nmol/L Zebularine for 12 h, DNA methylation level and Dnmt1 gene expression levels was significantly reduced, and 5 nmol/L Zebularine group can significantly improve the efficiency of nuclear transfer embryo development, reducing nuclear transfer embryos DNA methylation levels.