Exploration Of The Culture Of Spermatogonial Stem Cells In Vitro And Transplantation

Spermatogonial stem cells (SSCs) which residented in the basal membrane inside the seminiferous tubules of the testes of male mammals are the colony of spermatogenic cells with a high degree capacity to self-renew and potential to differentiate in mammals, which generally refers to the undifferentiated type A spermatogonia. The culture of SSCs in vitro has been carried out to study the mechanism of spermatogenesis and served as the basis of the utilization and research of the. The aim of our research is to find a better way to culture the SSCs and its preliminary application under in vitro conditions. This study is mainly consists of three parts:the first part; mouse spermatogonial stem cell isolation and purification and identification of the second part is the role of Polysaccharides proliferation of spermatogonial stem cells in vitro; The third part is on the use of spermatogonia transplant explore.The first part of the study is the separation,purification and preliminary identification of of mouse testicular germ cells and spermatogonia in vitro. We obtained the testes from6-8day-old after birth mouse Kunming white mouse testis parenchyma. Combination of enzymatic digestion including type IV collagenase, Dnase I and trypsin was used to separate spermatogonia and prepare single cell suspension. Then differential adherent was repeatedly used to purify the spermatogonia. SSCs were identified by alkaline phosphatase (AP) staining and RT-PCR testing. The method of seperating and purifying spermatogonia could meet the needs of the culture invitro, and the spermatogonia we gained could be used as the material for application in some fields.The second part of the study was conducted by adding Rhodiola Sachalinensis polysaccharides (RSP)into the culture mdium in vitro with different concerntractions to explore the effect of RSP on the proliferation of spermatogonia. RSP was used both in co-culture system of spermatogonia and Sertoli cells and the system of Sertoli cells cultured alone. The effect was tested by methyl thiazolyl tetrazolium (MTT) assay. The result of MTT test showed that the amount of SSCs was apprently increased (P<0.05) by adding Rhodiola polysaccharides compared with control and the optimal concerntration was300ng/mL. However, hardly any effects can be discerned in the group of Sertoli cells cultured alone.Our findings indicate that the Rhodiola polysaccharides can singnificantly promote the proliferation SSCs in vitro.The third part of the study was to determine the feasibility of transfection by means of testicular effernt duct injection using sprematogonia carrying eexgenous gene. We obtained the testes from nearly3months old Rex rabbit testicular parenchyma, using the method described above. The human cytomegalovirus promoter-controlled enhanced green fluorescent protein (pCMV-EGFP) was selected as the marker protein and then construct the adenovirus vector. The vector was transfected into the spermatogonia cultured in vitro. Then transfected the cells which carried with exogenous gene into Rex rabbit testicular parenchyma by means of testicular effernt duct injection. About a week after the injection, frozen sections of the male rabbit testis were prepared for fluorescent microscopic observation; the protein was extracted simultaneously from testis tissue, followed by Western-blot analysis. Fluorescence microscopy detected different EGFP expression in the frozen section in comparison with normal rabbit; Western-blot results have determined the expression of the target protein in the testes of the recipient animal. These results suggest that testicular efferent duct injection can be operated on rabbit.