Aim: Gonocytes are primitive germ cells in neonatal male testes. Germ cells from the neonatal testes of mice have a self-renewal activity and have pluripotential characteristics in established stem-cell lines. Therefore, these germ cells are reliable sources for the preservation of genetic resources of domestic animals and endangered species. The aim of the present study was to examine the cryopreservation of porcine gonocytes in liquid nitrogen (LN2) from neonatal testes that were freshly collected or stored at 4°C for 24 h. Methods: Gonocytes were isolated as lectin Dolichos biflorus agglutinin (DBA) positive cells from porcine testes 2-5 days after birth. The effects of the cryoprotectants used in the cryopreservation of the gonocytes, which were isolated from testes stored at 4°C in various solutions for 24 h, were examined on the results of cell viability after cryopreservation and cell proliferation in culture. Testis tissues from stored testes were transplanted into immunodeficient mice to evaluate the ability of the gonocytes to differentiate 5 weeks after transplantation. Results: The portion of the gonocytes that was isolated from stored testes at 4°C was approximately 70%. The viability of the gonocytes from stored testes was significantly higher in HEPES-supplemented Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12) and HEPES-supplemented phosphate-buffered saline than from stock solutions without HEPES. The addition of 10% dimethylsulfoxide (DMSO) and 0.07 mmol/L sucrose to cryopreservation solutions supported high viability of gonocytes after freezing and thawing. The cryopreserved gonocytes formed colonies with DBA activity in DMEM/F12 supplemented with 10% fetal bovine serum 3 days after culture and continued to proliferate for at least 12 days in culture. The germ cells in the testis tissues that were xenografted into immunodefficient mice differentiated into primitive spermatogonia. Conclusion: Gonocytes in the testis stored at 4°C for at least 24 h, isolated and cryopreserved can survive. The cryopreserved gonocytes differentiated in immunodefficient mice and proliferated along with the formation of colonies in vitro. (Reprod Med Biol 2008; 7: 153-160).
Keywords: cryopreservation; gonocytes; in vitro culture; pig; testis.