The Effect Of HELQ On Germ Cells Differentiation

According to the World Health Organization(WHO) assessment,the proportion of global infertility has accounted for 15 % in the couples of child-bearing age. And the patients with male infertility have occupied about half of the total of human infertility patients. Therefore,establishing in vitro reproductive disease differentiation model is necessary to study the male infertility mechanism by using of stem cells in combination with gene editing techniques. It also will be have very good application prospect.HELQ, named POLQ-like helicase, a DNA-dependent ATPase and DNA helicase, plays an important role in the process of repair DNA double-strand breaks(DSB) and inter-strand crosslink(ICL). HELQ directly intact with RAD51 homologue RAD51B/C/D, XRCC2 and the kinase ATR corresponding effect on DNA damage. Some researchers suggests the defect of HELQ result in testicular atrophy in male mice of seminiferous tubules structure, and the number of spermatogonial stem cells reduced obviously, it appeared like the setroli cell only syndronie(SCOS) phenotype. So we supposed that HELQ may work in gonocyte cells stage or primordial germ cells stage.In our experiment, we have obtained the knockout of HELQ gene in mice embryonic stem cell which can stably growth and passage by using of CRISPR-cas9 gene editing techniques.Immunofluorescence staining showed that after knock out HELQ, the expression of OCT4 and Nanoghave no significant difference as compared with the control group.Meanwhile, tetraploid compensation results also showed that the pluripotency of embryonic stem cells did not affected, and we have get the health mouse tetraploid offspring. At the same time, we use the HELQ knockout embryos stem cells in specific culture conditions to differentiate in vitro and eventually acquired the Day2 epiblast like cells.By immunoflurescence staining and the real-time PCR analysis, the results illustrated that the differentiation of HELQ knockout embryonic stem cell is unaffected. The results indicated that knockout HELQ did not affect embryo stem cells pluripotency and directional differentiation.Therefore, according to the germ cell differentiation in vitro system, we continued to differentiate epiblast like cells into germ cell like cell. Flow cytometry analysis data showed that knockout HELQ formed lower Blimp1 and Stella positive cells compared with the control group, and between them have two to three times difference. This results demonstrated that the gene of HELQ have certain effect on germ cell differentiation. Although we do not have further research, but it provide theoretical and technical support for male infertility treatment, and also provide new research ideas and important basis for clinical application of stem cells.