Long-term Culture And Identification Of Rat Sper-matogonial Stem Cells And Construction Of Its Specific Marker Gene Expression Vector

Continual spermatogenesis at a quantitatively normal level is required to sustain male fertility. Spermatogonial stem cells (SSCs) serve as the foundation of spermatogenesis by both committing to differentiation into functional sperm as well as replicating themselves in a process of self-renewal. The ability to isolate and experimentally manipulate SSCs has opened new doors for research on spermatozoan development, assisted reproduction, cellular therapy and genetics. In view of this potential, we have made whole protocols for isolating, purifying, propagating and differentiating rat SSCs in vitro. Chosen the rat as a species for these studies due to the rat models provide more physiological data for the study of human health and disease. In addition, various qualities of the rat, such as size, fecundity, ease of surgical techniques, tissue sampling, and general laboratory management make it a particularly attractive animal model. Constructing a plasmid vector which is specifically expressed fluorescent protein in SSCs will make it easy to identify SSCs, and will make it visible to show the dynamics of proliferation and differentiation of SSC research both in vivo and in vitro. In this study, we prepared a plasmid vector which will express red fluorescent protein in SSCs specificly. If transgenic rats can be prepared using this plasmid vector through SSC transplantation, it will be an important cell model and animal model in the following study of rat SSCs.1. Isolation and purification of rat SSCsIn this study,22-25-day-old Wistar-Iamichi rats were used. Testicular cell suspension was obtained by two-step enzymatic digestion. Then, according to the principle that testicular somatic cells bind tightly to plastic and collagen matrics when cultures in serum-containing medium, whereas spermatogonia and spermatocytes do not bind to plastic or collagen matrics when culture in serum-containing medium. Rat SSCs were then easily isolated from the purified spermatogenic population during a short incubation step on culture on laminin matrix. After purification,3×105SSCs were obtained from10testes. This method is simple and easy to operate, and the obtained SSCs have great viability and proliferation of high capacity. So, it is a highly efficient separation and purification methods.2. In vitro culture of rat SSCs In this study, we established rat SSCs long-term culture system. In this system, the SSCs were cultured on STO (SIM mouse embryo-derived thioguanine and ouabain resistant) mouse embryonic fibroblast feeder layers, using a defined serum-free medium. In the presence of GDNF, GFRα1, bFGF growth factors, rat SSCs formed tightly packed clumps of cells and continuously proliferated. Clump-forming germ cells kept on expanding for more than30passages and up to6months. The in vitro cultured SSCs can be cryopreserved at any time of the culture period, and resume growth after thawing rapidly. The stable establish of this system may lay the foundation for future studies and applied research of rat SSCs.3. Identification of in vitro cultured rat SSCsIn this study, frozen sections of testis immunofluorescence and whole-mount immunohistochemistry showed the topographic arrangement and cell morphology of SSCs in the seminiferous cutubles, and testified that the SSC specific marker genes CDH1, GFRα1, PLZF are reliable. Immunocytochemistry showed that the SSCs clones were positive for CDH1, GFRα1and PLZF. RT-PCR confirmed that except for CDH1, GFRal and PLZF, they also expressed other SSC specific genes, such as c-kit, POU5f1and C-Ret. This technique is simple, reliable and can be used to identify long-term cultured SSCs. So, it will be laid the foundation for the identification of SSCs of other large animals. 4. In vitro differentiation of rat SSCsIn this system, the long-tem cultured rat SSCs were co-cultured with testicular somatic cells using MEM-α+10%FBS culture medium, and adding the follicle-stimulating hormone (FSH) and testosterone, at34℃,5%CO2and saturated humidity. At the beginning24h of culture, the spermatogonia were dead abundantly, then gradually atabilized. After two weeks of culture, spermatocytes were observed, three weeks later, spermatid can be seen. The studies have been shown that long-term cultured rat SSCs have physiological functions. It has laid the foundation for further study of SSCs'in vitro spermatogenesis.5. Construction of SSCs specific marker gene expression vectorIn this study, a plasmid vector that is specificly expressed red fluorescent protein in spermatogonial stem cells was prepared. The vector carried CDH1promoter. CDH1has been shown to be expressed in undifferentiated spermatogonia of mouse and rats. Here, we used no promoter-based vector pDsRed2-1, in its multiple cloning site, we inserted CDH1-DsRed2gene expression frame and CMV-EGFP gene expression frame in the opposite direction. It will be expressed red fluorescent protein in SSCs specificly, and expressed green fluorescent protein in all cells. The human hepatoma cell line HepG2cell transfection experiments confirmed that the vector containing CDH1-DsRed2expression frame and CMV-EGFP expression frame has been constructed successfully.