A Preliminary Study On The Isolation And In Vitro Culture Of Buffalo Spermatogonial Stem-Like Cells

In order to optimize the culture system of buffalo spermatogonial stem-like cells, isolation methods and factors affecting the proliferation of buffalo type A spermatogonia in vitro were investigated in this study. Meanwhile, the biological characteristics of buffalo type A spermatogonia were also examined.(1) The cell type of3to7-month-old buffalo seminiferous epithelium was investigated in this study. The results showed that there are three types of cells: Sertoli cell, Leydig cell and type A spermatogonia in this stage. Type A spermatogonia located at the basilar membrane, had a big and stained dark cell nucleus and was expressed the markers Oct-4, c-kit, PGP9.5, THY-1and DBA. The expressions of Oct-4, c-kit, PGP9.5, THY-1were also detected in the testis by RT-PCR.(2) The approach of isolation and purify buffalo seminiferous epithelium cells was studied. These results showed that using a two-step enzymatic digest seminiferous tubule could obtain a lot of testis cells. Through different adhesion combined with Percoll discontinuous density gradient centrifugation could isolate and depurate the Sertoli cells and type A spermatogonia. The Sertoli cells expressed special protein vimentin, could be used as feed layer.(3) In vitro culture characterizations of buffalo type A spermatogonia and spermatogonial stem-like cells were examined. The results showed that on two days after implanted, type A spermatogonia began to proliferate, then formed the spermatogonial stem-like cells clone on day10to12. The clone showed AP stained positive, and expressed Oct-4, c-kit, PGP9.5, THY-1, the specific marker of the spermatogonial cells and DBA, the specific marker of bovine male germ cell.(4)The effects of serum and GDNF on proliferation of type A spermatogonia was investigated. These results showed that the rate of cells proliferation accelerated, the numbers of DBA positive clones increased accompany with the concentration of serum upgraded, and the2.5%FBS was the best. The rate of cells proliferation and the number of clones were obviously promoted by GDNF and in a dose-dependent manner with the concentration of GDNF, the best adding content was40ng/mL.In conclusions:(1) type A spermatogonia could be isolated from3to7-month-old buffalo testis;(2) buffalo type A spermatogonia could proliferate to form spermatogonial stem-like cells clone;(3) adding appropriate amount of serum and GDNF could promote the cells proliferation.