Establishment Of Cell Lines With Poricine Spermatogonial Stem Cell Properties

Spermatogonial stem cells(SSCs)are capable of both self-renewal and differentiation to mature functional spermatozoa,being the only adult stem cells in the males that can transmit genetic information to the next generation.Porcine SSCs hold great value in transgenic pig production and in establishment of porcine models for regenerative medicine.However,studies and applications of porcine SSCs have been greatly hampered by the low number of SSCs in the testis as well as the lack of an ideal stable long-term culture system to propagate porcine SSCs perpetually.In the present study,by lentiviral transduction of plasmids expressing the simian virus 40(SV40)large T antigen into porcine primary SSCs,we developed two immortalized cell lines with porcine SSC attributes.We found that the established cell lines:(1)Immoralized porcine spermatogonial stem cells underwent gradual morphological transformation in the culture process,they changed from round/oval to flat.By staining the microtubules and microfilaments,it was found that the morphology of the cells was normal,and no polyploid cells appeared.(2)After the immoralized cell line was treated with RA,it was found that the immoralized porcine sperm stem cell line responded to the treatment of RA,but the differentiation was limited,which was characterized by weak down-regulation of PLZF,up-regulation of STRA8,stable expression of c-kit,and no significant changes in cell morphology.(3)The pig spermatagonial stem cell marker UCHL1,PLZF and THY1 are expressed in the whole training process,germ cell markers VASA and DAZL have also expressed,homing related factors CXCR4 expression,the proliferation marker PCNA was expressed in all generations,and the immortalized SV40 t-antigen was also expressed.(4)Through allotransplantation of immoralized porcine spermatogonial stem cells,fluorescence observation of recipient mice,staining of convoluted spermatogonial tubes and frozen section,it was confirmed that immoralized porcine spermatogonial stem cells could colonize the basement membrane of convoluted spermatogonial tubes,and their stem cell characteristics were also confirmed.(5)Through the detection of cell karyotype and DNA content,the results showed that the number of chromosomes decreased and some chromosomes were distorted.The DNA content of immortalized cells was also changed compared with that of primary spermatogonial cells.The immortalized porcine spermatogonial stem cell line have now been cultured for more than 7 months and passaged for over 35 times without morphological abnormalities.Thus,we have for the first time established porcine SSC lines that could provide abundant cell sources for mechanistic studies on porcine SSC self-renewal and differentiation,thereby facilitating development of an optimal long-term culture system for porcine primary SSCs and their application to animal husbandry and medicine.