Fundamental Research On Gene Transfer In Vitro And Transplantation Of Spermatogonial Stem Cells

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. Since the original establishment of SSCs transplantation technology by Brinster in 1994, it had succeeded in making transgenic goat, bull, pig, fowl and other animal species by SSCs transplantation technology. However, little is known about the biological characteristics in vitro of SSCs in canines. And no transgenic dog was reported. Based on the research of isolation, culture and transfection and transplantation of mice SSCs, we have made a primary similar research on canine, which provided a useful tool and fundamental reserch for us to establish the transgenic dog by SSCs transplantation technology in the future research.Materials and Methods:①Animals: The mouse SSCs were collected from 7-8d new-born ICR mice; While the canine SSCs were collected from the dogs of 4-4.5 months old;②The isolation of SSCs: Sseminiferous epithelium was dissociated to prepare a suspension of single cells by 0.2g/L collagenase-0.25% trypsin digestion.③The proliferation and cultivation of SSCs: The SSCs were purified by differential plate and cultured on sertoli cells(STO) feeder layer with DMEM media enriched with 10%FBS, 1%glutamine, 1%non-essential amino acid, 1%Pen/Strep, 1%sodium pyruvate;④The gene transfer in vitro of SSCs: The SSCs were transfected with EGFP plasmids both by cationic polymer method(including serum and no serum) and calcium phosphate method;⑤The establishment of animal models of azoospermia: Different concentration of busulfan were injected intraperitoneally (in mouse) and intramuscularly(in canine)in order to select an suitable concentration to establish the animal models of azoospermia.⑥The transplantation of transgenic SSCs: The SSCs transfected with EGFP were transplanted into testes of recipient mice and canine which had been treated with bulsafan to delete the endogenous spermatogenesis.The testes of recipient mice and canine were excised 1 week, 2 weeks, 3 weeks, 4 weeks and 5 weeks after transplantation, respectively. The frozen-cut sections of the testes were prepared, examined and photographed under a fluorescence microscope. The genomic DNA PCR amplification of recipient testes were also examined. Results:①The isolation, proliferation and cultivation of SSCs: The mouse SSCs were collected from 7-8d new-born ICR mice; While the canine SSCs were collected from the dogs of 4-4.5 months old. By using 0.2g/L collagenase-0.25% trypsin digestion to dissociation seminiferous epithelium, the percentage of viable cells were 97.73%(mouse)and 95.81%(canine). Purity of SSCs purified by differential plate were 60.78%(mouse) and 50.86%(canine), SSCs could adhere on the STO feeder layer after seeding 8-12h in DEME supplemented with 10%FBS, 1%glutamine, 1%non-essential amino acids, 1%Pen/Strep, and 1%sodium pyruvate. Under phase contrast microscope, they retained the typical morphological characteristics such as having a spherical or oval outline, homogeneous morphology and size, existing as scattered single cell or as aligned/clumped population.②The EGFP gene can be transfected into SSCs by all the three methods. The transfection efficiency of calcium phosphate method was 8.12%(mice) and 7.96%(canine); The transfection efficiency of cationic polymer method(including serum) was 22.14%(mice) and 23.69%(canine); The transfection efficiency of cationic polymer method(no serum) wae 21.58%(mice) and 24.01%(canine).③The establishment of animal models of azoospermia: In mouse, the results showed that 40mg/kg was a suitable dose to establish a stable model of azoospermia; while in canine, it showed that 12mg/kg was the suitable dose to establish a model of azoospermia.④The results of SSCs transplantationIn: Positive cells were observed in the testis frozen sections of mouse at 1 week, 2 weeks and 3 weeks after transplantation; while the genomic DNA PCR amplification of recipient testes showed that the mice carrying the target gene; No result was observed in canine.Conclusions: We have isolated mouse and canine SSCs successfully and efficiently. After transfecting EGFP gene into mouse and canine SSCs, then we transplanted transfected SSCs into recipient testes of models of azoospermia. Positive cells were observed in the testis frozen sections at 1 week, 2 weeks and 3 weeks after transplantation in mouse.All these techniques including isolation, cultivation, transfection and transplantation provide a useful tool for the establishment of transgenic canine in future.