A Study On Proliferation And Undifferentiation Of Spermatogonial Stem Cells

Objiect:To study the effecting factors on spermatogonial stem cells proliferation and undifferen- tiation in vitro, which could provide a valuable approache to study the survival conditions for spermatogonial stem cell and establish a long-time system in vitro.Methods:Spermatogonial stem cells were separated and purified from 2-5 mouth postnatal sheep by enzymatic digestion, Discontinuous density gradient centrifugation and centrifugal elutria- tion, and then cultured in vitro. Cells were cultured in DMEM/F12 or DMEM with different concentrations of fetal calf serum (FCS)and different feeder layer at 33℃,35℃or 37℃in a humidified atmosphere with 5% CO2. Medium was changed twice a week. spermatogonia was identified by inverted microscope or Alkaline Phosphatase Assay or Antibody Staining.Results:1. Sheep SSCs could be efectively seperated and purifiedby Percoll discontinuous density gradient centrifugation. Spermatogonia mainly distribute between the gradient 27 % to 35% (density between 1 .0410g/ml to 1 .0508g/m1).The average purity rate of spermatogonia was 56.7% in this study (P<0.01).2. The temprature at 37℃could reduce the cell differentiation and promote stem cell proliferation. The cells proliferation rate at 35℃was more quickly and the survival period was longer than those of other temptature. The temperature of 35℃and 37℃are suitable temperature to cultured sheep spermatogonial stem cells.3. Cultured in the mediumwithout serum 1 wk, only 20% of the cells were alive. However, in the presence of 2.5% FCS, approximately 80% of cells were alive and proliferating. Higher concentrations of FCS only enhanced numbers of somatic cells.Although more serum resulted in more cells, the proportion of Spermatogonia was dropped. These results in dicated that culture medium with 2.5% FCS clearly enhanced proliferation of somatic cells.4. The viability and proliferation of cell cultured in DMEM/F12 or DMEM medium were no significant difference.5.In the beginning the biological behavior of Spermatogonia was similar on two types feeder layer, after a week, spermatogonial stem cells on the Sertoli feeder layer much more than SFFCs feeder layer. About 30% of the SSCs cultured on Sertoli cells can survive more than 60 days . So we concluded that Sertoli cells can not only promote the survival and proliferation, but also simple and quickly.Conclusion:1. The methods such as Percoll diacontinuous density gradient centrifugation according to the different adhesiveness can be used to isolate and purify sheep SSC effectively. 2.The temperature at 35℃and 37℃are ideal temperature to cultured sheep spermatogonial stem cells.3. DMEM/F12 and DMEM medium are suitable culture medium for sheep spermatogonial stem cells.The mudium with 2.5% FCS can promote sheep spermatogonial stem cell proliferation, it is optimum serum concentration for sheep spermatogonial stem cells cultured in vitro.4.Without growth factor, feeder layer is essential to suppot sheep spermatogonial stem cell survival and proliferation. Sertoli cells can not only promote the survival and proliferation, but also simple and quickly.