| Spermatogonial stem cells(SSCs) , the only stem cells that are capable of transmitting genetic information to subsequent generations in adult animals,have abilities to self-renew and differentiate into spermatozoa. Therefore, SSCs are not only the study object of stem cell biology, but also the valuable resource of in vitro spermatogenesis, gene analysis andfunctional genomics. Brister et al (1994) established the SSC transplantation technique, and Nagano et al (2001) and Hamra et al (2002) successfully in vitro manipulated SSCs by using different vectors and produced transgenic mice and rat after transplantation back into recipient testes respectively. These studies provided a new method for delineating spermatogenesis and making transgenic animals. However, unfortunately this technology has been applied to no birds. In this study, we use different doses of busulfan to induct adolescence and sexual rooster, then checked the change of convoluted seminiferous tubule in histology and testes in form.According to these,we choose the optimal dosage and induction time to prepare recipient and transplant SSCs(marked with EGFP) in the testis of recipient. Some sperm marked with EGFP is checked out in seven weeks after transplantation.We used 30 mg 40 mg 50 mg and 70 mg busulfan kg-1 body weight on 15 weeks old adolescence rooster and 50 mg and 70 mg busulfan kg-1 body weight on 24 weeks old sexual rooster.Injected busulfan in 37℃, solution water remains constant, prevent injection coagulation. Rooster are killed when it is 5 d,10 d,15 d,20 d,25 d,30 d,60 d after treated with busulfan,and weigh the weight of the testis and rooster.In the end all the testicle are embedded in paraffin.In this experiment, the development of the testes in adolescence rooster is effected in different degree after treated with busulfan. The weitht of the testis from the adolescence rooster treated with 30 mg busulfan kg-1 body weight rises synchronously with control group.This shows this dose of busulfan is small to eliminate endogenetic SSCs of the adolescence rooste. When it is 20 d and 25 d after treated with 40 mg busulfan kg-1 body weight, the weitht of the testis from the adolescence rooster is significant different with control group.This dose of busulfan is more severe than 30 mg busulfan kg-1 body weight to eliminate endogenetic SSCs.But there is no difference between the experimental group treatedwith this dose and control group 30 d and 60 d after being treated. In this experiment, there are significant effect to the development of testis after treated with 50 mg and 70 mg busulfan kg-1 body weight. The size of the testis can not return to normal levels until 60 d after it is treated with busulfan .But the time busulfan begins to take effect is 10 d after being treated with 70 mg busulfan kg-1 body weigh and the time busulfan begins to take effect is 15 d after being treated with 50 mg busulfan kg-1 body weigh. This shows there is a significant dose dependent effect.The development of the testes in sexual rooster is effected in different degree after treated with busulfan too. In 30 d after the experimental group is treated with busulfan, the weight of testis has been a steady decrease, it is significant different with control group. The size of the testis can not return to normal levels until 60 d after it is treated with busulfan.In this experiment, the weight of the testis treated with 70 mg busulfan kg-1 body weigh is smaller than the testis treated with 50 mg busulfan kg-1 body weigh, but it is not significant. This does not show there is a significant dose dependent effect.The effect to the body weigh is very large after the sexual rooster and the adolescence rooster are treated with busulfan. From 15 d to 60 d after the adolescence rooster is treated with 50 mg and 70 mg busulfan kg-1 body weigh, the weight of rooster is significant lower than the adolescence rooster treated with 30 mg , 40 mg busulfan kg-1 body weigh and control group. From 10 d to 60 d after the sexual rooster is treated with 70 mg busulfan kg-1 body weigh is significant lower than the sexual rooster treated with 50 mg busulfan kg-1 body weigh and control group.In order to verify the effects of busulfan on the development of testicular, we also conducted histology analysis in paraffin sections. We find the cells in the convoluted seminiferous tubules begin in the fall 15 d after the sexual rooster treated and the adolescence rooster are treated with 50 mg busulfan kg-1 body weigh and 10 d after they are treated with 70 mg busulfan kg-1 body weigh, the sperm in the convoluted seminiferous tubules begins to decrease. The cells constantly shed; the hollow convoluted seminiferous tubules continue to increase. There is only a layer of cells 30 d after it is treated with 50 mg busulfan kg-1 body weigh and 25 d after it is treated with 70 mg busulfan kg-1 body weight in the vast majority tubules, and provides a certain number of niches for the exogenous SSCs. The convoluted seminiferous tubules are gradually shrunk; there is only a layer of cells 30 d after it is treated with 70 mg busulfan kg-1 body weigh.In this experiment, the effect on the development of testicular is significantly faster and stronger than other doses. 50 mg/kg. The cells in the convoluted seminiferous tubules increase from 1-2 layers to 2-4 layers 60 d after the sexual rooster is treated with 50 mg busulfan kg-1 body weigh. This shows the ability of spermatogenesis comes back. This time may be the best for SSCs transplantation. There is only a layer of sertoli cells in the tubules 60 d after being treated with 50 mg busulfan kg-1 body weight. This shows it is not the optimal time and dose for SSCs transplantation. There is only a layer of sertoli cells in the tubules 60 d after the adolescence rooster being treated with 50 mg busulfan kg-1 body weigh. The ability of spermatogenesis does not come back .This shows it is not the optimal time and dose for SSCs transplantation too.In order to determine the above analysis, we transplant SSCs(marked with EGFP) from 16～19 d old PGCs into the testis of recipients treated with 50 mg busulfan kg-1 body weight when it is 30 d after these the sexual and the adolescence rooster are treated. Some sperm marked with EGFP is checked out in seven weeks after transplantation. Our research shows that the effect of busulfan on the development of testicular and the efficiency of SSCs transplantation. High dose of busulfancan can effectively eliminate endogenous SSCs, and provides a certain number of niches for the exogenous SSCs. But it is wrong that the more the endogenous SSCs eliminate, the higher the efficiency of SSCs transplant. We should turn our eyes to the testicles microenvironment after induction, and we should know does the ability of spermatogenesis come back. |
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