Study Of Separating Immature Spermatogonia Cells Of Sheep By Percoll Gradients

In vitro culture of spermatogonia, induced cell differentiation，in vitro fertilizationand spermatogonia transplantation among same species offers a new way of spreadinggenetic resource, protecting rare species, and healing male infertility caused by impairedspermatogenesis. But the present techniques for isolation, purification and identificationof spermatogonia yet couldn’t meet the requirements of related study. The imperfectionof invitro culture, immaturation of induced differentiation and difficulties in detectingthe donor cells and their daughter cells are all hinders that limiting the use of these newtechnologies.Fresh sheep testes were collected as material, different enzymolysis methods anddifferent density gradients centrifugation (DGC) parameters were studied to separate thesheep spermatogonia. Flow cytometry (FCM) was used to evaluate the separationresults through assessing cell ploidy and antibody staining patterns.The study showed:(1) Cells acquired by digesting immature sheep testes with0.3%trpsin have good integrity and activity, and have potential to be used for further study.(2) Three clear cell layers were obtained from the bottom to the top of the tube bycentrifuging30min at850g in a swing-out roter. Cell ploidy and antibody stainingpattern were studied with flow cytometer(FCM). The first cell layer contained the mostdiploidic cells up to94.66%, in which the PGP9.5positive cells was76.76%, higherthan the datum reported by previous study.The results showed: The simple trypsin digestion method combined withdiscontinuous density gradients we explored in this study has a good isolation effect ofsheep spermatogonia, which lays a foundation for the further study of sheepspermatogonia.