The Isolation And Characterization Of Mouse Oocyte Stem Cells From Ovaries

The recent publications of purification of oocyte stem cells from adult mouse and human ovaries have challenged the central dogma of female reproductive biology that female mammals are born with fixed number of nonrenewing pool of germ cells. Naturally, those innovative study evoked a huge wave of controversy in the filed of reproductive and stem cell biology. The facts of exist of oocyte stem cells in adult ovaries will force us to reconsider of the oogenesis in adult ovaries and the ovarian biology. However, there was no further relevant reports of oocyte stem cells since the the first one. And the follow-ups were not entirely consistent with the previous. Therefore, in this study, we firstly isolated mouse oocytes stem cells according to the published methods. We optimized the separation method of mouse oocyte stem cell and identified, analysed the stem cells.We isolated and purified the DDX4 positive cells from 2 week mouse ovaries with the method of MACS, and we obtained 4 cell lines with 2 cell lines cultured more than 30 passages in vitro. The chromosome karyotype analysis showed that the cell line had no chromosome loss during culture in vitro and the number of chromosome was 40 (38+XX). In this study, the alkaline phosphatase staining of the 4 oocyte stem cell lines were positive, but the expression of alkaline phosphatase decreased with the passages increased. From passage 9, alkaline phosphatase staining was stable. Cell immunofluorescence staining showed that the oocyte stem cells expressed germline stem cell marker proteins as Oct4, DDX4, C-kit and Nanog. We detected germ stem cell marker gene expression with RT-PCR, and the results showed that the cells expressed Oct4, DDX4, C-kit, Blimp1, Ifitm3 and other germline stem cell marker genes. In this study we simplified the separation method of oocytes stem cells by continuous adherent methods. Oocyte stem cells enriched in this repeatable method with experiment process simplified. By a series of detection of the oocyte stem cell’s developmental potential in vitro and in vivo, we had successfully established oocyte stem cell lines of mouse in vitro.