Elimination Of Ovine Spermatogonia In Vivo And Culture In Vitro

Spermatogonial stem cells are the only source of potentially totipotent diploid cells in the adult mammalian body resides within the basal layer of the seminiferous tubules of the testis.They are the ideal stem cells to manipulate in vitro because they are responsible for maintaining spermatogenesis throughout life in the male by continuous production of daughter cells that differentiate into spermatozoa. Many new developments, such as recipient eliminated germ cells in testis to make space for donor cell colonization, transgenic technology, syngeneic and xenogeneic transplants, purification, and culturing of spermatogonial stem cells, have been achieved and are still under investigation. The aim of this study was to derive the ram recipients for transplantation of SSCs, isolate, purify and culture the ovine spermatogonia.Three groups of rams were intervened respectively by chemotherapy (injection of Busulfan into scrotum once per three-week, 7.5mg/kg weight, four times), immunity 1 (injection of LHRH-vaccine s.c., once per three-week, 200μg of each, four times), and immunity 2 (injection of LH-vaccine s.c., once per three-week, 5mg of each, four times). The resule indicated that the treatment with busulfan could completely eliminate spermatogonia stem cells and other germ cells. Active immunization against LHRH and LH inhibited the process of the spermatogenesis in seminiferous tubules.Single enzyme digestion with collagenase I(1mg/mL,37℃, a water-saturated air,5%CO2 and 1 h), percoll uncontinuous density gradient entrifugation and isolation according to the different adhesiveness were used to purify the ram spermatogonia. Spermatogonia mainly distribute between the gradient 27% to 35% (density between 1.0410g/mL to 1.0508g/mL). A high purity of spermatogonia was obtained. The average purity of spermatogonia is 60.79% in this study.The spermatogonia can survive for a long period of time in the co-culture system with Sertoli cell. The purified spermatogonia was planted into two kinds of culture media of DMEM, DMEM/F12 in which the single cell suspension originated from the single enzyme digestion was used as the Sertoli cell-germ cell co-culture in two culture medium. The proportion of spermatogonia was decreased in two co-culture systems after 24 hours culture. The proportion was significantly decreased in two co-culture systems using DMEM medium after 3 days culture. After a week culture, bird-nest-like and mountain-like colonies were observed with positive alkaline phosphatase (AKP) staining.