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The aim of this study was to enrich and in vitro culture of the ovine spermatogonial stem cells(SSCs).Theappropriate age of the donor ram for SSCs isolation was investigated firstly.Spermatogonial stem cell specificmarker protein promyelocytic leukaemia zinc-finger (PLZF) and male germ cell marker VASA double staining werechecked on the paraffin sections of the neonatal and the peripubertal ram testis.Type A spermatogonial markerPGP9.5 and sertoli cell marker Vimentin double staining were also used to confirm testes development.Resultsindicated that testes from neonatal rams had only one type of undifferentiated germ cell gonocyte and should besuitable for ovine SSCs enrichment.Therefore neonatal rams were used to isolate germ cells using a two-stepenzymatic digestion followed by differential plating for SSCs enrichment.Isolated and enriched cells werecharacterized by using PLZF and VASA antibody.The effects with either sertoli cells or stromal cells as feeder onprimary development of SSCs were investigated secondly.During the first week of culture,gonocyte formed pairsand chains of type A spermatogonia.After one week.colonies started to increase in size.Sertoli cells were moreeffective as feeder cell regarding proliferation,self-renewal,survival rates and colony formation of SSCs comparedwith stromal cells in the ovine spermatogonial stem cell culture system.