In-vitro Culture And Identification Of Buffalo Spermatogonial Stem Cells And Transgene Through Xenotransplantation

By studying the different conditions (including serum concentrations, feeder cells and several types of growth factors) on the proliferation of SSCs buffalo, we eventually establish in vitro culture system for proliferation of SSCs. By PB transposon vector, SSCs has been transfected with the SS gene fragment. After transplantation of genetically modified SSCs to the recipient’s testicles, we analysed the proliferation and differentiation of transgenic SSCs function on different time after transplatation. The study will provide new opportunities and pathways for clone and production of transgenic buffalo; provide a platform for research on other potential mechanisms of epigenetic biological characteristics of SSCs, directed differentiation and spermatogenesis; provide new tools for buffalo germplasm conservation by combination of cryopreserved SSCs; provide a theoretical reference for the study of other livestock and human SSCs.1. Buffalo spermatogonial stem cells in vitro culture and molecular markers explorationDevelopment of suitable selective marker for buffalo spermatogonial stem cells (SSCs), optimization of long term in-vitro culturing conditions and their pluripotent retention capacity in buffaloes can be of prime importance in selective genetic modifications of this species. In the present study, we identified CDH1as a specific marker for buffalo SSCs and revealed that it exists in two protein isoforms (large (135kDa) and small (90kDa) subunits) in the three-month-old cross-bred (Murrah×swamp,49chromosomes) buffalo testis. Furthermore, immunohistochemical analysis revealed that CDH1expression was present in spermatogonia but absent from somatic cells in testis. After7days of enrichment, expressions of CDH1were also detectable in in-vitro culture colonies (-53%enrichment efficiency by FACS). For long term culture of SSCs, proliferation studies with different factors showed that combination of20ng/mL GDNF,10ng/mL FGF2and1000U/mL LIF can significantly promote number of colonies (-2folds) and proliferation of buffalo SSCs (-3folds) compared to those of control or single treatment groups, furthermore, addition of these combination growth factors significantly up regulated the mRNA level of spermatogonial specific and pluripotency related markers (BCL6B, GFRA1, and POU5F1) while down regulated receptor tyrosine kinase (KIT). These findings indicate the identification of a new buffalo SSCs marker, furthermore, it may help in establishing culture that would help in genetic modifications of these buffaloes. 2. Transfection of BHK-21and SSCs colonies with somatostatin RNAi vectorSomatostatin (SS) play a major role in inhibiting the secretion of growth hormone (GH), so animal growth character can be promoted by inhibiting the secretion of SS. If we knock down SS gene out of SSCs, we can get long-term integration transgenic offspring through SSCs transplantion, and then furtherly get excellent individual in good growth condition. But previous studies in our laboratory have confirmed SSTR is not present in buffalo SSCs, so the moderating effects of need to be verified on other SS secreting cells. In this study, three PB delivered interference vector was designed and constructed for SS gene sequences, and lipo-mediated transfected into BHK-21cells (hamster kidney fibroblasts, identified by SS secreting cells). Result of radioimmunoassay (RIA) and fluorescence quantitative PCR (qPCR) showed that interference effects from one of the interfering carrier can reach more than50%at the cellular level. In addition, we found that the expression of somatostatin after RNAi, the early phase apoptotic of BHK-21cells significantly reduced, and showed more cells into S phase. The effects of Somatostatin in vitro interference on cell somatostatin receptor expression have not been reported, so we examined the changes in the expression of the five somatostatin receptors. The results showed that all somatostatin receptor expression was down regulated. Apoptosis and cell cycle-related genes were detected by RT-PCR and Western Blot, showing Caspase-3, p21and Bax expression levels were significantly reduced, while the expression of Bcl-2levels were significantly upregulated. These results suggest that somatostatin may regulate Caspase-3activity and Bcl-2/Bax genes through somatostatin receptor2and3to promote apoptosis in BHK-21cells. Through these results we have chosen the best interference pshRNA-2plasmid. After optimizing transfection methods coupled with SSCs in vitro conditions, we conducted successful SSCs transfection, which has laid a solid foundation for the production of transgenic animals.3. Transgenic SSCs colonies xenotranplantationFor confirmation of their stem cell potential, DBA-stained cells were identified in the basal membrane of seminiferous tubules of xenotransplanted mice testis. In order to examine the propagation of buffalo spermatogonial stem cells following transplantation into Kunming mice. For preparation of recipient animals, we also optimized the dosage of busulfan in Kunming mice for successful depletion of endogenous spermatogenesis. This was followed by microinjection of enriched donor buffalo germ cells (58%purified) into mouse seminiferous tubules. After treatment with different doses of busulfan (30,35,40 mg/kg b.w.), we found that a dose of40mg kg-1is optimal for the ablative treatment. This optimization was based on body weight, testicular histomorphology, and testosterone level of recipient mice. After transplantation, mouse testes were analyzed at day1d,4d,1wk,2wk,3wk,1mo and2mo after transplantation with Dolichos biflorus agglutinin (DBA) immunohistochemistry and PCR for the presence of donor germ cells. As fresh explants, buffalo testes cells were observed as small colonies of round cells within mouse seminiferous tubules, indicating successful proliferation and colonization of germ cells, however, at2months, the number of colonies decreased although, these cells appeared as fibrous tissues and devoid of spermatogenic capacity. In conclusion, busulfan-treated Kunming mouse testis are capable of colonizing the buffalo germ cells, thus making it suitable model for evaluation and further development of buffalo germ cells transplantation in mice. The buffalo spermatogonial stem cells are able to be alive and propagated for2months in mouse testis.