Screening Of Developmental Markers Inspermatagonial Stem Cells

Spermatogonial stem cells transplantation has a potential clinical value for the treatment of male infertility. Especially for those nonobstructive azoospermia patients with severely impaired spermatogenesis, which is caused by receiving high-dose of chemotherapy before puberty, this technique is associated with a unique clinical value. It is possible to induce the proliferation and differentiation of spermatogonial stem cells to produce sperm if they could be found in the testes of patients with nonobstructive azoospermia; or it can be achieved through obtaining highpurity spermatogonial stem cells in vitro for the proliferation and culture or transplantation through sorting specific markers. However, how to determine the obtained cells are spermatogonial stem cells becomes the first and foremost condition of this work. In the proliferation and differentiation processes of spermatogonial stem cells, many proteins would be expressed both on the surface and inside the cells. If they are highly specific and sensitive, as the markers, they will play an important role in the isolation and identification of spermatogonial stem cells. After the isolation and purification of spermatogonial stem cells in mice of different ages using composite enzyme digestion and magnetic activated cell sorting technology, the differences in the protein expression of spermatogonial stem cells in mice of different ages were observed dynamically using two-dimensional gel electrophoresis technology and protein mass spectrometry analysis technology. Then, differential proteins or new proteins were screened and identified with the help of bioinformatics proteome database.Objective : To study the dynamic changes in the protein marker expression of spermatogonial stem cells(SSCs) at different age using i TRAQ protein mass spectrometry, as well as to screen new markers based on the bioinformatic proteome database.Methods : Normal male C57BL/6 mice were taken as the research objects and divided into eight groups according to age. The mice testicular tissues of each group were aseptically extracted and digested using compound enzyme, then the SSCs were purified using magnetic activated cell sorting(MACS) method. The proteins of each group were analyzed and identified using twodimensional electrophoresis and protein mass spectrometry in combination with the protein database information.Results : 248,510 spectra were obtained from this MS experiment, and 1132 proteins were identified according to the analysis of Mascot software. The proteins with over 1.2-fold difference in protein abundance and p value < 0.05 in statistical test were regarded as differential proteins. 298 differentially expressed proteins were identified in 8 groups. 9 makers of SSCs(PCNA, GFRA1, CDH1, Annexin A7, UCHL1, VASA, CD49 f, CD29, PLZf) that are currently known were identified. Through comparison between groups, their expression trends at different ages were obtained, and of them, the trends of GFRA1, CD49 f and CD29 in SSCs were consistent with previous literature data. Subsequently, 10 proteins(P63, CD71, CD98, K19, ACE, K18, K15, K17, SH2, SH3) were selected as SSCs markers to be further studied.Conclusion : The proteins in SSCs demonstrate differential expression with the changes in the age of the body. With the assistance of i TRAQ protein mass spectrometry, the proteome information of mouse SSCs can be analyzed and compared, in order to obtain the proteins with different expressions at each age, which provides a new approach to further analyze and study the function and role of these differential proteins.