Purification,Culture And Gene Modification Of Monkey Testicular Germ Cells

The transmission of human genetic information is closely related to the process of gametogenesis,and spermatogenesis represents half of the process.At present,animal models from different rodent species including mouse have been wildly used to study the mechanism of gene regulation in the process of spermatogenesis.However,there is phylogenetic divergence between rodents and human,and the clues on primate genesis are very limited.In contrast to rodents,the physiological and biochemical aspects are identical between primates and humans,and thus primates have more important research value.The isolation of spermatogenic cell and culture of spermatogonia in rodents have been established over the past years.However,it is difficult to make effective progress in the study of primate spermatogenic cells and the exploration of spermatogonial cells,due to the difficulty in obtaining the material and the lack of selection cell surface markers.Here,we successfully isolated monkey gonocytes,pachytene spermatocytes and round spermatids,elongated spermatids,and mature sperms from 0.5-and 5-year old monkey testis with the STA-PUT method.We verified the purity by fluorescence staining with specific molecule markers.The harvested cells were further subjected to disclose the dynamic of genetic or epigenetic changes of genes in primate spermatogenesis.In the meanwhile,we established monkey recepient testis by busulfan treatment,we also established the 14-day culture for spermatogonial stem cells,and conducted initial gene transfection on these cells,which are ready for cell transplantation to generate gene-modified monkeys from spermatogonial stem cells.For monkey spermatogonial stem cell culture,we applied the rat spermatogonial stem cell culture medium and a combination of multiple growth factors to maintain these cells in vitro.The cultured cells were verified as spermatogonial stem cells by their gene expression of molecular markers.Collectively,we have applied the STA-PUT method to the monkey testicular cells to successfully isolate five different stages of spermatogenic cells,and complete the short-term culture of spermatogonial stem cells.