Study On The Isolation, Culture And Histone H3 Lysine 9 Trimethylation Modification Of Bovine Spermatogonial Stem Cells

Spermatogonial stem cells(SSCs) are natural mammalian pluripotent adult stem cells, which can continuously self-renew and transfer genes to offspring in vivo, with potential application prospect in fundamental research and livestock husbandry. Great progress has been achieved in the research regarding the culture, identification and developmental mechanism of SSCs in rodents and human. Currently, there are still several essential issues regarding SSCs researches of large domestic animals especially cattle, such as difficult separation and identification, incompetent culture system as well as the insufficient maintenance of SSCs pluripotency.Faced with these problems, we firstly explored the cryopreservation of bovine testes tissue as well as the identification, separation and enrichment of SSCs. The results indicated that fetal bovine serum(FBS) can be replaced with knockout serum replacer(KSR) in the freezing medium for cryopreservation of bovine testes tissue. The morphology and structure of the testis tissues were intact and could be clearly observed after tissue recovery. Real-time PCR and immunofluorescence detection indicate that GFRα-1 are available for marking bovine SSCs. We prepared the seminiferous epithelia of bovine testis for SSCs enrichmet, and found that the enrichment efficiency can be improved through the conbination of differentiating plating and Percoll density gradient centrifugation.In this study, we further investigated the effects of 2i-based culture system on the stemness maintenance of bovine SSCs. Real-time PCR results showed that 2i condition remarkably up-regulated the expression of pluripotency-related genes(Oct4, Sox2, Nanog), SSCs marker genes(Gfrα-1, Plzf), anti-apoptosis gene(Bcl2) and leukemia inhibitory factor receptor(LIFR), while down-regulated the differentiating marker(c-kit) and pro-apoptosis gene(Bax). Meanwhile, the expression of PLZF protein was also up-modulated in 2i system. Results of cell proliferation activity detection showed that the 2i culture condition had a slight inhibitory effect on the proliferation activity of the cells(including SSCs) in the co-culture system. Additionally, apoptosis of bovine SSCs could be induced by hydrogen peroxide and 2i condition efficiently overcomed the hydrogen peroxide-mediated apoptosis stimulating, which further solidated the anti-apoptosis effect of 2i condition. These results suggest that 2i condition is beneficial to the maintenance of bovine SSCs.It’s reported that modifications of histone are closely connected with the state of pluripotent cells. We initially explored the effects of 2i condition on histone H3 lysine 9 trimethylation(H3K9me3) of bovine SSCs. Immunostaining results showed that 2i condition remarkably down-regulated H3K9me3 level in bovine SSCs. Furthermore, Real time-PCR detection showed that the m RNA level of Suv39h1/h2 were significantly down-regulated in 2i-treated SSCs. These results indicate that 2i condition may regulate the H3K9me3 modification of bovine SSCs through Suv39h1/h2.In summary, based on the cryopreservation of bovine testicular tissue, separation, enrichment and identification of bovine SSCs, 2i culture conditions were applied in the in vitro culture of bovine SSCs, revealing the role of small molecules inhibitors in the stemness maintenance as well as its influence on histone H3K9me3 of bovine SSCs. Our research laid a foundation for investigating the mechanism of maintainence of stemness at the point of histone methylation modifications.