Primary Studys On Buffalo Seminiferous Epithelium Cell In Vitro Culture

The objective of this study was taken to establish a culture system supporting proliferation and differentiation of spermatogonium for long-term. The local buffalo testicles were used for material.Contents had been studied as follows:(1) Studying the changes of germ cells with the development of seminiferous tubular.The results found that: the seminiferous tubulars diameter were growing from 37.5～75.0μm at the postnatal to 125.0～150.0μm at the 10～12months age, meanwhile germ cells can developed from gonocytes to spermatids and sperms. The tubulars diameter were about 75.0～87.5μm at the age of 3～5months, and the most germ cells were spermatogonium, so these phase tubulars were the best material for isolation and culture of spermatogonium.(2) Studying the isolation, purification and culture of sertoli cells. The results found that: 2.61×10~6 cells were obtained from 1 gram testis parenchyma and the average percentages of living cells was 89.37%; the method of the different Adhesive and high temperature culture (culture for 24h at 38.5℃) can be used to isolate and purify buffalo sertoli cells, and the purity can reach to 90%; the espression of GATA4 and GDNF gene for sertoli cells and GFRAL gene for spermatogonium were confirmed by RT-PCR.(3) Studying the isolation, purification and culture of spermatogonium. The results found that: seminiferous tubular fragments floated easily in culture, but cells suspension cultured after Collagenase and trypsinase sequential two-step digestion can obtain more spermatogonium; the purity of spermatogonium can reach to 62.95% using Percoll to purify; AKP staining found that sertoli cells were negative, peritubular cells and undifferentiation spermatogonium were positive; differentiation spermatogonium expressed C-Kit ;Spermatids were observed after 4wk cultured and confirmed for the expression PRM-2 gene by RT-PCR.In conclusions: (1) With the development of buffalo seminiferous tubular , the kinds of compound germ cell can changes. The most germ cells in tubular were spermatogonium at the age of 3～5 months,wich phase tubulars can be used to isolate spermatogonium; (2) The method of different adhesbeness and high temperature culture can use to purify sertoli cells; (3) For culture spermatogonium, culture cells suspension after collagenase and trypsinase sequential two-step digestion were better than culture seminiferous tubular fragments; (4) The staining for AKP and C-Kit can be use to identify buffalo testicle somatic cells, differentiation and undifferentiation spermatogonium; (5) Buffalo spermatogonium can be purified using Percoll and can develop to spermatids in vitro culture.