| RAD18is a RING structure protein binding with single-strand DNA, and has the role of E3ubiquitin ligase. RAD18gene has many aspects of function in DNA damage repair, also plays a role in protein degradation, gene transcriptional regulation. Currently, studies related to the DNA repair function of RAD18gene mainly focused on its role in somatic cells and tumor cells, whether it has the similar role on DNA repair during development of animal embryos is unclear. Therefore, in this study, DNA damage model of mouse embryos induced by H2O2was first established. Then, shRNA virus particles of the RAD18gene were injected into the mouse fertilized eggs using RNA interference technology, the expression of RAD18gene was analyzed by qRT-PCR method, and the expression of two specific markers of DNA double-strand breaks (DSB)(yH2AX and pATM) was checked by immunofluorescence technology in mouse early embryos. The aim was to explore the function of RAD18gene on DNA damage repair mechanism in mouse embryos. The results would help us to clarify the mechanisms of embryonic oxidative DNA damage repair, and to improve the embryo quality cultured in vitro by inhibiting the oxidative damage.1. Expression of RAD18gene in mouse early embryos and the establishment of mouse embryo DNA damage model In order to study the role of RAD18gene on DNA damage repair in mouse early embryos, in this study, the expression of RAD18gene in mouse early embryos was firstly analyzed by qRT-PCR and immunofluorescence methods. And then by using H2O2as the DNA damage agent, the effects of different concentrations of H2O2on mouse embryo development were observed, the expression of yH2AX and pATM, and the DNA damage induced by H2O2were detected by immunofluorescence technique, the purpose was to construct DNA damage model of mouse embryos. The results showed that RAD18gene expressed in mouse embryos at each developmental stages before implanted, among them, compared with the zygotes, the expression in embryos of2-cell and8-cell stages was higher (P<0.01), but significantly decreased in morula and blastocyst (P<0.01). The treatment of H2O2influenced the development of mouse early embryos, compared with the KOSM (-) control groups, the cleavage and blastocyst rates of mouse embryos in H2O2treated groups were graducally decreased. Among them, the cleavage rates of embryos in the KOSM (-) control group,1.5μM,1.8μM,2.1μ,2.4μgroups were87.96%,77.95%,53.69%,45.55%,36.98%,49.89%, respectively. The blastocyst rates were49.89%,48.82%,19.21%,2.29%,0%, respectively. The results of immunofluorescence test showed that the foci numbers of yH2AX of embryos at2-cell stage were more than1-cell stage embryos in the KOSM (-) control group, pATM was not detected in embryos of two stages. In1.5μM and1.8μM treated groups, yH2AX and pATM were not detected in the embryos of1-cell stage, yH2AX expressed in2-cell stage embryos, pATM was not detected. In2.1μM and2.4μM groups, yH2AX and pATM were detected in embryos of1-cell and2-cell stages, among them, the foci numbers of yH2AX of embryos of two periods in2.4μM group were more than that of in embryos at the same time of2.1μM group, indicated that treatment of2.1μM and2.4μM H2O2could induce obvious DSB phenomenon. The results of qRT-PCR analysis revealed that with the increase of H2O2added, the expression of RAD18gene in mouse early embryos was significantly up-regulated accordingly (P<0.01).2. Effects of RAD18gene down-regulation on mouse embryo development and DNA damage repair.To further explore the role of RAD18gene on mouse embryo development and DNA damage repair, based on the DNA damage model of mouse embryos constructed above, by micromanipulation method, virus particles of RAD18gene shRNA were injected into the perivitelline space of mouse zygotes, the effects of expression down-regulation of RAD18gene on development and DNA damage repair of mouse embryos were analyzed respectively. The results showed that, compared the groups of different H2O2injected with the non-injected groups, in injection groups of different H2O2concentration, after injection of shRNA virus particles, both of the mouse embryo cleavage and blastocyst rates in1.5μM group increased, but had no significant difference (P>0.05).The mouse embryo cleavage rate in1.8μM group was significantly higher (P<0.05), the blastocyst rate increased with no significant difference(P>0.05), the mouse embryo cleavage rate of2.1μM group was significantly higher (P<0.05). The blastocyst rate decreased, but the difference was not significant (P>0.05). The mouse embryo cleavage rate of2.4μM group increased, with no significant difference (P>0.05), the blastocyst rate did not change. The above results suggested that the inhibition of RAD18gene could increase cleavage rates of mouse embryos in all H2O2treated groups, but had different effects on blastocyst rates.The results of immunofluorescence test showed that, after injection of RAD18shRNA virus particles, either1-cell or2-cell stage embryos in2.1μM and2.4μM treatment groups, the foci numbers of yH2AX decreased,pATM expressed in embryos of before and after injection of RAD18shRNA virus particles, but had no significant change.The results indicated that RAD18gene was associated with the embryonic oxidative DNA damage. In summary, RAD18gene expressed in mouse preimplantation embryos. The down-regulation of RAD18could increase the cleavage rate of mouse embryos in H2O2treatment groups, and RAD18was associated with fetal DNA oxidative damage. |
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