The Differential Study On The Development Of Primordial Germ Cells Between Male And Female Embryos In Mice

C57BL/6J mice are standard Inbred Strains Mice, the embryo reproductive development of which has much in common with humans. And they have so clear genetic background that be considered as a better model of biological materials. Tissue processing technique is an important means indispensable for studying embryo reproductive development. At present, there are still many problems remained about embryo development of genital ridge, and a lot of data are obtained from separation, culture and investigation in vitro. The gestational period of C57BL/B6 mice is 19 to 21 days. During the lldpc (days post-conception) to 13.5dpc, the development of genital ridge and primordial germ cells have undergone tremendous changes. From 8.5 to 10.5 dpc, primordial germ cells migrate towards the genital ridge and increase their numbers through mitotic divisions. In the normal mouse, most of them enter the genital ridge at 11.5 dpc, lose their locomotive ability and expand. This expansion gradually ends after 13.5 dpc. After 13.5dpc, germ cells in female enter into the prophase of meiosis I and arrest as diplotene oocytes, and germ cells in male are sequestered. At present there is a little complete report about the morphology development of mouse embryo genital ridge at home and abroad, and the development difference of embryo genital ridge between male and female. In this study, we use the ways of hematoxylin-eosin staining, immunofluorescent histochemistry and western blot, try to explore the normal development morphology of genital ridge and the difference in number and growth rate of primordial germ cells between female and male from 11 to 13.5 days post-conception, providing reference for the further studies on molecular regulating mechanism of genital ridge development.1. Slice-making of mouse embryosAfter obtaining mouse embryos from 11 to 13.5 days post-conception, we fixed them with 4% paraformaldehyde. Then these embryos were dehydrated by gradient ethanol, transparent in xylene, immersed in paraffin, and then made into slices in 5um. With optical microscope HE staining would show us the morphology change of genital ridge from 11 to 13.5 days post-conception.The results are as follows. At the 11th days post-conception, genital ridge has been formed, in which primordial germ cells can be seen. Genital ridge are thicken and longer and primitive sex cord emerges at 11.5 days post-conception. Genital gland wouldn't differentiate until the 12.5th day. At the 13th day, we can see obvious character of sex differentiation.In male primitive sex cord develop into primitive seminiferous tubule. Then at 13.5 day, in female there are some primitive follicles in the cortex. Slice-making of mouse embryos provides valuable materials for immunofluorescent histochemistry and provides histological reference for the further studies.2. Double Immunoflurescence staining of slicesThe gene, Mouse vasa homology (Mvh) is specifically expressed in the germ cell lineage in both sexes. Proliferating cells are labeled by Proliferating Cell Nuclear Antigen (PCNA) antibody. After immunoflurescence staining, we choose 3 to 4 slides at an interval and intercepted the same area, then count the number of Mvh positive cells and Mvh-PCNA positive cells. We counted the number of Mvh positive cells and Mvh-PCNA positive cells, and then calculated the growth rate of primordial germ cells which equal to the ratio of Mvh-PCNA positive cells and Mvh positive cells. We did not detect any difference on the growth rate of primordial germ cells between female and male at the 11.5th,12th,12.5th and 13.5th day (P>0.01) except 13dpc. At the 13th day, growth rate of primordial germ cells in male was higher than it in female (P=0.000). And the number of Primordial germ cells in male was more than it in female at the 13th and 13.5th day (P=0.000)3. Western blotting detection of Mvh and PCNA protein expressionWe isolate the genital ridge of embryos at 13 and 13.5 day, and then extract protein for analyzing the expression of Mvh and PCNA. The result shows us that withβ-tubulin as the internal reference, expression of Mvh protein in male is higher than it in female at 13th and 13.5th day, and PCNA expression is higher in male at 13th day, which is the same as the result of double immunoflurescence staining.These results show that there are evident differences of the primordial germ cells development between male and female. After observing obvious character of sex differentiation and before expansion ends, especially at the 13th day, proliferation rate and the number of primordial germ cells in male are higher than them in female. The result provides experiment evidence for gene expression change in the development process of primordial germ cells and the reproductive study of gene knockout mice.