Effects Of GDNF And LIF On The Proliferation Of Mouse Spermatogonial Stem Cells In Vitro

Spermatogonial stem cells (SSCs) are the only cells in postnatal mammals that theyundergo self-renewal and transmit genes to subsequent generations. Isolation, purification,identification, culture and cryopreservation of SSCs in vitro lay foundation for subsequentstudies. Six to eight day-old mouse testes were harvested and cell suspension was obtained bymechanical method and two-step enzyme digestion method. The SSCs and Sertoli cells wereseparated by differences of cells adherence. The two kinds of cells were identified by alkalinephosphatase (AP) staining and RT-PCR. Serum-free DMEM/F12media added with GDNF orLIF alone and in combination was used to culture SSCs. Single-factor effects anddouble-factor effects of GDNF and LIF on SSCs proliferation were separately tested bymethyl thiazolyl tetrazolium (MTT) assay. In this experiment, to get the best cryoprotectantformula, different concentrations of glycerol and DMSO were used to cryopreserve SSCs. Theresults are as follows:1. In order to obtain single cell suspension, a two-step enzymatic way of1mg/ml type IVcollagenase and0.25%trypsin was used. The survival rate was about90%. The improveddifferential adherence method can separate SSCs from sertoli cells, and the purity of SSCs canreach about80%. This method is a reliable and simple SSCs isolation and purificationmethods. AP staining to SSCs was positive. The SSCs cloning clusters showed brown. Therewas the β-actin gene in both Sertoli cells and SSCs. SSCs exist Ngn3gene and Oct4gene, butSertoli cells don’t express Ngn3and Oct4gene. Both methods show that the purified cellswere the purpose cell, SSCs.2. Compared with the control,20ng/mL and40ng/mL of GDNF could largely promotecell growth, regardless of the culture time(P＜0.05). However, there was no significantdifference to promote proliferation of SSCs between LIF treatment groups and the control (P＞0.05); Adding20ng/mL GDNF and1000U/mL LIF in combination could significantlyenhance mouse SSCs proliferation in vitro (P<0.05), and under this condition, the OD490value was0.696at5th day culture when the concentration of SSCs was6~10×10~4cells/mL. 3. The survival rate of10%and5%DMSO were higher than90%at1st daycryopreservation, while the survival rate of15%DMSO was only80%, the difference wassignificant (P<0.05). Glycerol as a protective agent, regardless of the concentration, the cellsurvival rate did not exceed30%. The cell survival rate of different concentrations of glycerolwere less than10%at7d cryopreservation, so subsequent AP staining and in vitroexperiments were terminated. A month later,10%DMSO cell survival rate was significantlyhigher than the5%DMSO and15%DMSO (P<0.05).10%DMSO+20%FBS+70%DMEM is the best cryoprotectants in this experiment. The survival rate of the cryoprotectantsbasically maintained at about78%. It can be used as cryoprotectants for futurecryopreseration of SSCs. Frozen for1month; SSCs were inoculated to feeder layer. The SSCswere basicly adherent to feeder layer and the SSCs showed a single round state culture for24h. SSCs proliferated and spreaded on the bottom of the flask, and round proliferating cellsformed clusters of cells after having been cultured for48h. At5d, most of SSCs formed cellmass, just like clusters of grapes. When the SSCs were co-cultured for7d, they formed largecolonies on feeders. AP staining showed that SSCs be dyed brown, the AP-positive.