Study On Regulation Of Natal Bovine Spermatogonial Stem Cells Proliferation In Vitro

Spermatoginial stem cells are a population of pluripotent cells that can self-renew and pass heredity information to the offspring in male animal's gonad. Spermatoginial stem cells have become a hit of many researchers due to their broad prospect in medicine, genetics and study on transgenic animal. However, spermatoginial stem cells are difficult to be maintained for a long term in vitro, which is a barrier for a further study. This study separated, purified and identified natal bovine spermatogonial stem cells; and optimized the culture system of spermatogonial stem cells in vitro ; transfected human telomerase reverse transcriptase (hTERT) gene into spermatogonial stem cells to prolong their life in vitro. The results are as follow:1. The average viability of natal bovine testicular cells obtained by two-step enzyme digestion was 89.00%, spermatogonial stem cells ratio was 21.61%, and cell attachment rate was 23.34%.2. Spermatogonial stem cells distributed mainly in 27 %～35 % Percoll gradient, spermatogonial stem cells purity was up to 69.27% after natal bovine testicular cells'purification by Percoll density gradients centrifugation .3. The colonies of natal bovine spermatogonial stem cells identified by immunohistochemical staining showed that they were weakly positive for Alkaline phosphatase (AKP) and positive for Integrinβ1 and C-kit. Spermatogonial-stem-cell- specific gene gfrα1 and c-kit were also found to express in natal bovine spermatogonial stem cell by RT-PCR.4. After optimizing natal bovine spermatogonial stem cells'culture system in vitro, the result showed that adding 2.5% fetal bovine serum(FBS),10μg/L leukemia inhibitory factor (LIF), 20μg/L stem cell factor (SCF), 80μg/L glial cell linederived neurotrophic factor(GDNF) into basal medium could promoted spermatogonial stem cells'proliferation significantly; Sertoli cells as feeder layer seeding in 5.0×105 cells/mL was best for spermatogonial stem cells'proliferation.5. Natal bovine spermatogonial stem cells were transfected with pCl-Neo-hTERT vector mediating by lipofectamine and screened by 600μg/mLG418. Telomerase activity test showed that OD450/630 value in cells positive for pCl-Neo-hTERT were 4 times as high as in untransfected cells. The growth curve showed that population double time was 1.9 times shorter than that in untransfected cells.The results above provided evidences that two-step enzyme digestion combining with Percoll density gradients centrifugation could satisfy the requirement for natal bovine spermatogonial stem cells'further study; natal bovine spermatogonial stem cells could be promoted to proliferate significantly in vitro by optimizing culture system and transfecting hTERT gene.