| In order to study the differentiation system of Bama mini-pig spermatogonial stem cells, isolation, purification and identification methods of Bama mini-pig spermatogonial stem cells in vitro were investigated in this study. Meanwhile, the ways of differentiated into haploid cells or embryonic-like stem cells were also researched.(1) The approach of isolation and purification of3to4-week-old Bama mini-pig spermatogonial stem cells were investigated in this study. The results showed that using a three-step enzymolysis approach to digest seminiferous tubule which including collagenase IV, hyaluronidase and trypsnase digestion, could obtain a mass of testis cells. And a large number of spermatogonial stem cells would obtain after different adhesion by rat tail collagen and laminin. The STO cells treated for2hours by15μg.ml-1mitomycin-C could be used as feed layer, since its proliferation was restrained but still kept a good condition.(2) The characterizations and identification of Bama mini-pig spermatogonial stem cells from in vitro culture were examined. The results showed that, the spermatogonial stem cells begin to shape round or oval, and to proliferate and grow well one day after implanted, then formed the spermatogonial stem cells clone on day3to5. The clone expressed CDH1and UCHL1, the specific markers of the pig spermatogonial stem cells and DBA, the specific marker of porcine male germ cell.(3) The differentiation of Bama mini-pig spermatogonial stem cells in vitro both in cell and tissue levels were examined. It turned out that the spermatogonial stem cells could differentiate into haploid cells on the feed layer of testicular cells, under the condition of32℃,5%CO2, while differentiation culture medium was a-MEM+10%KOSR+RA+IGF+FSH. However, the tissues differentiation under the same cultural conditions did not support the spermatogenesis from Bama mini-pig spermatogonial stem cells.(4) The method inducing Bama mini-pig spermatogonial stem cells into embryonic-like stem cells was investigated. The results showed that under the inducing medium of a-MEM+10%KOSR+GDNF, on four days after implanted, colonies were formed, then the clone formed relatively obvious edge with surrounding cells on day14. The clone showed ALP stained positive, and expressed Oct-4, the specific marker of the pluripotent stem cells and UCHL1, the specific marker of pig male germ cell.In conclusions:(1) spermatogonial stem cells could be isolated from3to4-week-old Bama mini-pig testis by the three-step enzymolysis approach;(2) Bama mini-pig spermatogonial stem cells differentiate into haploid cells;(3) Bama mini-pig spermatogonial stem cells could be induced into embryonic-like stem cells. |
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