| Background and purpose: Spermatogonial stem cells (SSCs) are the only stem cells in the body that self-renew and transmit genetic information to the next generation. Therefore, SSCs are not only the valuable resource of in vitro spermatogenesis, gene analysis and functional genomics, but also became a new assisted reproductive technology in the preservation of endangered wildlife. After the original description of mouse germ cell transplantation in 1994, SSCs had become the fouces of stem cell reasearch. Subsequently, SSCs technique had succeeded in goat, bull, pig, and other animal species. However, little is known pertaining to the biological characteristics in vitro of SSCs in canines. The dissertation described the isolation, culture and transfection techniques of canine spermatogonium in vitro, which provided a useful tool to establish the basal culture system of SSCs in vitro, prepared for transplantation techniques and facilitated in vitro manipulate studies on transgenesis for future.Methods: The spermatogonial stem cells were collected from 4-4.5 month-old Beagle testes by aseptic operation and isolated by four kind of enzymatic combinations (collagenase; collagenaseⅣ+ collagenaseⅣ; collagenaseⅣ+ trypin; collagenaseⅣ+ hyaluronidase + trypin). The cell viability was determined by Trypan blue dye exclusion test and the total number of seminiferous epithelial cells was calculated by counting plate. According to the differentiation of velocity of cells sticking to the wall in vitro, SSCs were purified without any chemical treatment in order to maintain the initial activity. Purified spermatogonia were cultivated with DMEM containing different concentrations of serum(0%, 1%, 5%, 10% and 20%) or EGF (0ng/ml, l0ng/ml, 10 ng/ml, 20 ng/ml, 40 ng/ml, 60 ng/ml and 100 ng/ml) respectively. By counting the clusters every day, the growth efficiency of SSCs can be readily detected within 1 week in a semi-quantitative manner. The SSCs were transfected with EGFP plasmids comparing cationic polymer method with calcium phosphate method and cultured in vitro for 72 h.Results: The suitable ages of beagle were 4-4.5 months. By four kind of enzymatic combinations, the total number of dissociated cells (×106) per 0.10 gram testis parenchyma was: 3.37±0.101×106, 3.96±0.062×106, 6.67±0.692×106 and 4.35±0.110×106 respectively; the homologus viability assessed by trypan blue exclusion was: 82.4±1.94%, 87.7±1.45%, 95.5±0.68% and 89.7±0.88% respectively. The best enzymatic digestion method was the group of collagenaseⅣ+ trypin. Purity of spermatogonia was 50.86%, but it had satisfied the need of tests in vitro. Among 0%, 1%, 5% , 10% and 20% groups, 10% FBS had promoted spermatogonial cells growing and forming more cell colonies. EGF enhanced more clusters of cell than control. Compared with EGF groups, the clusters of cell in the 20ng/ml group increased markedly(P<0.01). The transfection efficiency of cationic polymer method was extremely higher than that of calcium phosphate method (22.14% vs 8.12%).Conclusions: We are the first to develop an efficient and economical isolation method of canine spermatogonium, which is enough to obtain germ cells and has optimal cell viability. The technique of SSCs culture demonstrated that canine SSCs can be maintained in culture for approximately 1 week. The clusteres originating from a single cell indicated that SSCs in vitro were able to achieve proliferation in the culture systems we designed. However, the long-term culture systems is expected. The isolation, culture and transfection techniques provide a useful tool for transplantation of canine SSCs in vitro.
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