Study On Transgenic Technology Through Male Germ Cells

Male germline cells could transmit genetic information to the next generation through sexual reproduction in which spermatogonial stem cells(SSCs) are the foundation for spermatogenesis to sustain male fertility. SSCs are the only adult stem cells that maintains germ cell pool by self-renewal and differentiation in male. Meanwhile, SSCs could transform to embryonic-like stem cells spontaneously or under induced conditions and have attracted much attention in biomedical, translational medicine and animal husbandry. Up to today, most studies about SSCs or male germline cells have been focused on these fields, such as elucidation of specific molecular markers of germ cells, development of long-term culture system in vitro, uncovering the classic or epigenetic signaling pathways and related protein functions, generation of transgenic animal via SSCs manipulation and testis tissue grafting.Immunodeficient animals were used in most of these studies which has severely restrained the key role of SSCs in scientific research and the application in animal husbandry industry. At the same time, generation of transgenic animal based SSCs transplantation is complicated and time-consuming as well. Therefore, a simple strategy to generate transgenic animals via SSCs is highly desirable. This research focuses on two key questions: immunocompetent recipients for male germline cell transplantation or grafting and an effective strategy of transgenesis with male germline cells. The main findings were presented as followed:1, In order to develop a novel strategy for preparation of recipient rat for male germ cell transplantation, different concentrations of adenine were administrated(200mg/kg body weight, 260mg/kg body weight, 320mg/kg body weight and 380mg/kg body weight).The observations obtained in the present study showed the adenine-treatment with the concentration of 320mg/kg is an alternative way of recipient preparation for transplantation of SSCs into rat testes. As a control, administration of busulfan was optimized as well in the present study. After enzyme digestion, SSCs were enriched via differentiation plating and magnetic activated cell sorting. The SSCs enriched germ cells(84% Lin28 positive cells testis cells) were infected with lentivirus and transplanted into seminiferous tubules of rat recipient.The mouse donor SSCs were survived and differentiated into spermatozoa in the recipient rat testis. In addition, male germ cell lines and primary testis cells were treated with adenine in vitro and cell apoptosis were observed in the adenine treated cells. The above observation indicate that administration of adenine is a novel strategy for recipient preparation in rat forSSC transplantation, in which a severe syndrome of side effects displayed when busulfan was administrated.2, Genomic manipulation of SSCs and transplantation can generate transgenic animals.However, the strategy needs expertise and expensive equipment. In the present study,lentivirus was directly injected into seminiferous tubules or parenchyma of 7-day-old and28-day-old mouse testes. The mice injected with lentivirus were mated with wild type female mice and yielded 67.8% of positive for transgene in F1 generation. And importantly the transgene was successfully transmitted to F2 generation. Meanwhile, the transgenic pups were generated in an injection site-and age-independent manner. Copy number and insertion of the transgene were also screened. To further explore the mechanisms by which the transgene was silenced, DNA methylation level in the EF-1 and CMV promoters were analyzed. DNA methylation in EF1 and CMV promoter were totally different and 87.8% and 45.5%,respectively, suggesting that a specific promoter of lentiviral vectors should be carefully considered for transgenic generation in vivo.3, At last, when rat testis tissue was heterogeneously xenografted under the skin of immunocompetent recipient Kuming mice, complete spermatogenesis was observed in the retrieved grafts. To our knowledge, it is the first time to observe the spermatozoa in xenografts from immuncompetent hosts. In the xenotransplanted testis tissue, seminiferous tubules were de-structured and re-formed, differentiated germ cells were initially lost and spermatogenesis were re-started afterward. To order to improve the survival rate and spermatogenesis, immunosuppressive agents were administrated and yielded higher survival rate and better spermatogenesis(from 2-5% to 8.3%).In conclusion, this research focused on two strategies of SSCs biology with immunocompetent recipients: SSCs transplantation and testis tissue grafting. The treatment of adenine could be an efficient and novel strategy for preparation of recipients before SSC transplantation in immunocompetent rats in which busulfan treatment results in systemic toxicity and even mortality. With induced germ cell apoptosis by adenine-treatment, the immunocompetent rats could offer an effective stem cell niche for SSC homing even complete spermatogenesis. Then lentiviral mediated transgenesis by in vivo manipulation of SSCs has been optimized and the silence of integration events was illuminated with different methylation of promoter. At last, an effective strategy for fertility preservation was explored under immunocompetent recipient mice with the treatment of immunosuppressive agents.This research provided a new way to study the SSC function, spermatogenesis and grafting.This research could expand the transgenic production by manipulation of male germline cells and SSCs. These strategies could be applied to other animal species, especially livestock andendangered animals, leading to significant advancement of animal transgenesis in agricultural and biomedical fields.