Preliminary Study On Gene Expression Related To Self-renewal And Differentiation Spermatagonial Stem Cells During Rat Prepuberty

Spermatogonial stem cells are multi-functional stem cells with the characteristics such as self-renewal and multi-directional differentiation potential; regulated by their own genes and/or external signals, they can finally differentiate into sperms. Recently, their studies have drawn much attention. The molecular mechanism of spermatogenesis has been a hot research on spermatogonial stem cells and also one of the most value topics in this area.At present, the study on the Molecular mechanism of the early differentiation of spermatogonial stem cells has focused largely on such animals as mice and fruit flies, but the report about rats is relatively less. Testis is the place of spermatogenesis which is a complex and regular cell differentiation process. The spermatogenic cells' development requires strict special condition and is regulated synergistically by many genes and hormones from the proliferation of spermatogonia, to meiosis of spermatocytes and to the differentiation of sperm cells and their maturation when moving to the epididymis. The study on the molecular mechanism of spermatogenesis needs to take into account not only the spermatogonia's gene expression, but also the effect of the surroundings on the differentiation of spermatogonia. Therefore, testis, the main object in this study, is used to research the expression pattern of the related key genes in the early stage of spermatogenesis from day2to day30after the rat was born, in order to provide the experimental basis for the further discovery of new key genes and reveal of the molecular mechanism of spermatogenesis.1. The transcriptional expression of the genes related with spermatogenesis in rat testis co-culture systemIn this study, vitro co-culture of the rat germ cells from its seminiferous tubules is used to culture testis germ cells of the15-day-old rat and in the process of long-term vitro culture, Morphological observation of germ cells at all levels was made; by RT-PCR, after1,1.5,2and2.5months'vitro culture of co-culture system of rat germ cells, the analysis of gene transcription expression of such genes as marker genes of rat germ cells at all levels (Type A spermatogonial stem cells, E-type cadherin gene (E-cadherin, CDH1), spermatocytes, SYCP3, round spermatids, transition protein-2(TP2) as internal control genes and glyceraldehyde-3-phosphate dehydrogenase (gapdh) was made. The result shows that the rat germ cells and Sertoli cells in the co-culture system can be symbiotic as long as possible. In the co-culture system, a large number of Sertoli cells can be observed, in addition, Spermatocytes and round spermatids are dominant. Two months later, it still can be seen that the symbiosis of a large number of spermatocytes and sperm cells in the Sertoli cells exists, which indicates that, during this period, spermatogonial stem cells perform self-renewal and differentiation in the co-culture system. In vitro co-culture system, the transcriptional expression of the CDH1, SYCP3and TP2genes have been detected in the1-2.5-month cell samples. This, at the molecular level, determines that there exist the germ cells at all levels in the process of spermatogenesis. RT-PCR of rat spermatogenic cells is expected to help initially establish the identification method of vitro culture of rat testicular germ cells at molecular level, provide a cell model for the study of the spermatogenesis and lay the foundation for the further study on molecular mechanism of the proliferation and differentiation of spermatogonial stem cells in the process of long-term vitro culture.2. The transcription expression of the genes related with the proliferation and differentiation of spermatogonial stem cells in rat's testis in its early pubertyIn the rat early puberty, a critical period of spermatogenesis, testicular tissue was taken on days2,4,6,8,10,12,15,20,25and30as the object of study, RT-PCR is used to detect the transcription expression of SYCP3and TP2; by semi-quantitative RT-PCR, the transcription expression of CDH11, GFRα-1, C-KIT, SCF and GDNF is detected separately. It can be seen that day8is the key time of SSCs'development. On day8, primordial germ cells and germ cells differentiate into spermatogonial stem cells, and further differentiate into type A spermatogonia. From day8to day15, they self-renew as they continue to differentiate. On day15, SSCs enter meiosis and produce primary spermatocytes. After day15, the self-renewal and differentiation of SSCs tend to be in dynamic equilibrium; to day30, SSCs differentiate and produce round spermatids. In the rat's early puberty, the unique spatial and temporal expression patterns of these critical genes are essential for the self-renewal and differentiation of spermatogonial stem cells.3. The analysis of different expression genes of testicular tissue in the rat's spermatogenesis early developmentIn this study, based on the transcriptional expression patterns of the key genes including CDH1, GDNF, GFRα-1, SCF and C-KIT responsible for testis-specific expression in the early spermatogenesis. It can be seen that day8is a critical period of SSCs'development because the mRNA levels of these genes showed significant changes from postnatal day6to day10. Therefore, the testicular tissues of the rats of postnatal day6,8,10were taken; the gene expression pattern of the rat was analyzed and compared on postnatal day6,8and10by high-throughput genome-wide expression microarray. The differentially expressed genes in the rat's testicular tissues on the three days were compared and the result shows that the number of the genes with more than2-times difference is between700and4519including up-regulated and down-regulated genes. The number of biological pathways involved is between46and154; and there are620to3237gene ontology annotations. Some significantly differentially expressed genes were further verified by quantitative PCR. From the results of gene chip technology, we can draw at least the following conclusions:1. The period of postnatal day6to day10is the very critical period for spermatogenesis in the process of testicular development in the rat's prepuberty.2. Early spermatogenesis is very complex and highly orderly process, which works through the proteins playing a very important role in the process of early development of germ cells through thousands of genes encoding.3. On each procedur,, the rat's early spermatogenesis is a process involving many genes.4. The expression of genes Trib3, Cxc16and C2cd4d in the testicular tissueBased on the results of gene chip technology, we have selected significantly differentially expressed3genes C2cd4d, Cxcl6and Trib3. By quantitative PCR and immunofluorescence staining, the transcription of genes C2cd4d, Cxcl6and Trib3was detected in the rat's testicular tissue in the prepuberty and the expression of CXCL6and TRIB3protein was detected in order to determine the correlation between these3genes, the early development of the rat's testicular tissue and the self-renewal and differentiation of spermatogonial stem cells. By immunofluorescence, the expression location of the proteins produced by CXCL6and TRIB3was analyzed and determined in order to reveal the fact that the expression products of the two genes may be related to the self-renewal and differentiation of spermatogonial stem cells in the early development of the rat testis and its early puberty. The results showed that:the transcription of genes C2cd4d, Cxcl6and Trib3in rat's testicular tissue in its early puberty is obviously dynamic. C2cd4d expression showed wavy curve, the first crest appearing on postnatal day4and then dropping; the second crest appeared on postnatal day8and then dropped; to postnatal day12, the peak came and then dropped slowly. The trough came on day20and then rose gradually from day25. The expression of Trib3reached highest point on postnatal day2and then decreased rapidly. Its expression was relatively stable between day4and8, increased to a crest from day8to day10and dropped slowly. Cxc16level decreased slowly from day2and reached the lowest value from day10to day20and then began to rapidly increase linearly on day20. The results showed that: the transcription of genes C2cd4d, Cxcl6and Trib3in rat's testicular tissue in its prepuberty is obviously dynamic. According to the results from Immunofluorescence staining, from the morphological point of view, TRIB3is expressed in spermatogonial stem cells. It suggests that the expression of this gene is related to the function of spermatogonial stem cells. The expression site of CXCL6(GCP-2) is very similar to the acrosome and CXCL6is also very likely to participate in the occurrence and reaction of the acrosome. Of course, this needs to be further verified by a lot of research. These results will provide new ideas and information for our further study on the molecular mechanism of spermatogenesis.