Isolation, Purification And Identification Of Mouse Male Germ Cells

Spermatozoa are the vehicle by which male genetic information or germplasm is transmitted to successive generations. Thus, normal spermatogenesis is essential for species preservation and genetic diversity. The normal spermatogenesis depends on the proper regulation of gene expression. piRNA is a class of small RNA found in germ line cells, previous studies show that the piRNA may be involved in epigenetic regulation, gene translocation inhibition, transcriptional regulation and so on. PIWI is a piRNA binding protein and functioning in the form of piRNA/PIWI complex. So, The purpose of this project is to study the relationship between piRNA and PIWI and metabolism of piRNA/PIWI complex both in vitro and in vivo in the way of separation and purification of mouse male germ cells in different stages of differentiation and development.Mouse male germ cells includ spermatogonia, primary spermatocytes, round spermatids and elongating spermatids. First, we established the specific identification method of spermatogonia. Immunofluorescence technique was used to detect and analysis the expression of mvh and vimentin in testis, finding that mvh is mainly expressed in the germ cells and no expression of somatic cells, while vimentin is mainly expressed in somatic cells and no expression of germ cells, there is no overlap between the two signals. Since we use 7-8 day old mice to isolate spermatogonia, in which there is no other period of germ cell, we have identified mvh as the molecular MARKER of spermatogonia, vimentin as the molecular MARKER of sertoli cells.After determining the molecular MARKER of spermatogonia, we first investigated on several kinds of cell isolation methods to obtain spermatogenic cells from mouse testicular tissues. The combination of enzymatic digestion obtained the largest number of cells,20 mice were obtained 4×107 cells. Since spermatogonia and Sertoli cells have different adherent speed, we use differential adhesion to enrich spermatogonia. Then, spermatogonias were identified by IF and flow cytometry, the purity was 70%-80%.We obtained testes from 5 week-old mouse. A combination of enzymatic digestion was used to prepare seminiferous tubule suspension and STAPUT was used to purify primary spermatocytes, round spermatids and elongating spermatids. Identified by IF and flow cytometry, each purity were 66%,54% and 69%.In summary, the project successfully established a purification and identification methods of spermatogonia, primary spermatocytes, round spermatids and elongating spermatids.