Development Of Bovine Androgenetic Embryos Derived From The Oocytes Treated With Tsa And/or 5-aza-dc, And The Sperm Treated With 5-aza-dc

Oocyte cytoplasm plays a key role in in vitro production (IVP) of embryos, and it has capability to decide the fate of the embryo development. In the recent view, epigenetics reprogramming of the somatic cell nuclear in the oocyte cytoplasm is a major factor that can influence the efficiency of the somatic cell nuclear transplantation. In 2004, Kono, a Japanese researcher, got an adult parthenogenetic mouse derived from the knock-out H19 mouse through the regulation of the imprint genes Igf2 and H19 which suggests that imprint genes are important for the development of parthenogeneticembryos. Main parent imprint is established through DNA methylation, and gene expression pattern expressed by the embryos can be regulated by DNA methylation and histone acetylation. Sperm-clone is a technology that can produce androgenetic embryo in vitro and based on both the technology of somatic cell nuclear transplantation (SCNT) and Intracytoplasmic Sperm Injection (ICSI), and the correct expression of imprint genes is also the important question for the androgenetic embryo development. sperm decondensation is a main issue in sperm-clone-embryo development. Male pronucleus undergoes a quick demethylation active in the normal embryo development, some scientists foundthat the process of sperm decondensation is related with the process of demethylation in the Male pronucleus. Therefore, in this study, TSA and 5-aza-dc which are common drugs to change the epigeneticmodification, maturing oocytes and sperm were treated with these chemicalsand found that 5-aza-dc can change DNA methylation level of both the oocytes and sperms, TSA/5-aza-dc-treated oocytes and 5-aza-dc-treated sperm can improve the development ability of the androgenetic embryos derived from these treatments. Main findings of present study are described below:1 Development of the androgenetic embryos derived from the oocytes treated with TSA and 5-aza-dcTSA and/or 5-aza-dc in appropriate concentration can raise the rate of matured oocytes. 10nmol/L TSA have the highest maturation rate ofoocytes (55.5%) in the TSA-treated groups, and differ significantly (p<0.05) compared with the control (37.8%). Moreover,20nmol/L TSA can inhibit the oocyte mature. In 5-aza-dc-treated groups the concentration with the highest rate of matured oocytes is 20nmol/L (53.8%). All the oocytes treated with TSA and 5-aza-dc have high maturation rate. And 10 nmol/L 5-aza-dc+10nmol/L TSA-treated oocytes have the highest rate (71.9%) among all the groups.Oocytes treated with TSA and/or 5-aza-dc in appropriate concentration can improve the development ability of the androgenetic embryos. Androgenetic embryos derived from the oocytes treated with TSA have high cleavage rates and morula rates, but 10nmol/L 5-aza-dc+10nmol/L TSA-treated oocytes had highest blastocyst rate.From DNA methylation immunofluorescence of the matured oocytes treated with TSA and/or 5-aza-dc, we can find that 5-aza-dc decreases DNA methylation level of oocytes, but TSA have little effect to the DNA methylation level of oocytes.2 Developmet of androgenetic embryos derived from 5-aza-dc-treated spermsSperms were treated with 5-aza-dc for 2h, and then DNA methylation immunofluorescence showed that 5-aza-dc can decrease DNA methylation level of sperms. And 50nmol/L 5-aza-dc have the best effect to the DNA methylation level of sperms, interestingly, the higher concentration (100nmol/L) 5-aza-dc could not decrease the DNA methylation level of sperm, because of sperm death due to the cytotoxiceffects of 5-aza-dc.Androgenetic embryos derived from 50nmol/L 5-aza-dc-treated sperms have the highest morula rate (71.4%) in all the teams of this study, and in the same time, have the highest blastocyst rate in the sperm-treated groups.The results of DNA metheylation immunofluorescence from androgenetic embryos that have a good development ability derived from 20nmol/L and 50nmol/L 5-aza-dc-treated sperms showed that,16-cell and morula stage from 20nmol/L have higher DNA methylation level than 2-cell,4-cell, and 8-cell stage, but not significantly. Moreover, the embryos from 50nmol/L have significantly higher DNA methylation level than 20nmol/L.