The Culture And Identification Of The Chicken Spermatogonial Stem Cells And Sertoli Cells

Spermatogonial stem cells were located at seminiferous tubule which not only can maintain the number of themselves but also can differentiate to spermatocyte because of its potential at transgenc animals production. And sertoli cells as only one kind of seminiferous epithelium cells play an important role in the support and nutrition. Poultry spermatogonial stem cells research is still immature, and the culture of poultry sertoli cells has not been reports.Spermatogonial stem cells and sertoli cells have become increasingly significant with the deepening of stem cells research. In this study, the rooster testicle of 20 to 30 days chick and the chicken embryo of hatching 18 days was as experimental material. The chicken testis cells was obtained using two-step enzymatic digestion. And we culture the spermatogonial stem cells using the methods of different temperatures and different density and different feed levels. To expect that establish the feeding system which is suitable for chicken's spermatogonial stem cells that keep alive in vitro for a long time. At the same time, we identify the biological speciality of spermatogonial stem cells. then do the experiment of transplantation in vivo using the cultured spermatogonial stem cells. And the sertoli cells of primary culture and subculture are identified. The conclusion of this research is:1. The motility rate of testis cells reach 95 percents high above. It is a suitable digestion method for testis.2. The chicken SSCs are able to grow up well and form multiplication under 37 degree and 38.5 degree. and the difference of culture results is unobvious(p>0.05). But the sertoli cells and other cells including the chicken SSCs could grow up more quickly and restrain the growth of chicken SSCs under 38.5 degree. So 37 degree is more suitable for chicken SSCs as culture degree.3. The culture effect of inoculating density in 5×10~5cell/mL is better than that in 1×10~5cell/mL obviously(p<0.05). And has no clear difference compared as in 1×10~6cell/mL(p>0.05). But the inoculating density in 5×10~5cell/mL is more suitable for chicken SSCs culture in vitro because the growth rate in 5×10~5cell/mL is higher than that in 1×10~6cell/mL.4. The culture result have no obvious differences between CEF and SCs(p>0.05). but the number of cultured cells after 24 hours and 96 hours is a little higher than that cultured in CEF, and the growth rate in SCs is also higher than in CEF. So SCs is more suitable for chicken SSCs as the feeder layer cells which are cultured in vitro.5. The chicken SSCs cultured in vitro still keeps the undifferentiated characteristics after the identification of AKP, PAS and the immunochemistry. So the mentioned identification methods are suitable for chicken SSCs, and they are easy and convenient.6. The successful rate of subcutaneously transplantation is higher than muscle transplantation and heart transplantation obviously(p<0.05). So subcutaneously is the ideal position of transplantation and differentiation in vivo. And the test of transplantation and differentiation in vivo also proved that the poultry SSCs have multi-directional differentiation potentiality undifferentiated characteristic of chicken SSCs cultured in vitro.7. Majority gonocyte can be removed after difference adherence and hypotonic treatment in the primary culture, and the monolayer sertoli cells can be obtained that the purity is about 90%.The methods of AKP, red O, acridine orange staining and rhodamine 123 are easy, and they are the ideal methods to identify testis sertoli cells.