The Effects Of Inducer Factors On Adult Mouse Spermatogonial Cells Colony Formation In Vitro

Document Type : Original Article




Introduction: The spermatogonial stem cells (SSCs) form the basis of spermatogenesis. In vitro culture of germ cells is useful for the purification and increasing of SSCs for transplantation. In the present study, we examined: in vitro the effects of co-culture with sertoli cells, GDNF, SCF and GM-CSF on efficiency of spermatogonial cells colony formation and spermatogenesis induction in the recipient testes after transplantation with SSCs in the colonies.
Materials and Methods: Both Sertoli and spermatogonial cells were isolated from adult mouse testes by enzymatic digestion. The identity of the cells was confirmed through analysis of alkaline phosphatase activity, immunocytochemistry against oct-4 and vimentin; and also transplantation of the cells. Isolated spermatogonial cells were treated either with various concentrations of GDNF (1, 40,100 ng/ml), SCF (1, 40,100 ng/ml) and GM-CSF (0.1, 0.1,1 ng/ml) or with co-culture with Sertoli cells during three weeks. Colony assay was done using a light microscopy. For transplantation, spermatogonial cells of the colonies were transplanted into the seminiferous tubules mouse which were irradiated with 14Gy at 10 weeks of age, via rete testis. The statistical significance between mean values was determined using repeated ANOVA test. p<0. 05 was considered to be significant.
Results: Results indicated that GDNF is the best factor for in vitro colonization of adult mice spermatogonial cells compared with the other cytokines and growth factors. Co-culture system with Sertoli cells was chosen as colonizer. Because co culture showed a significant increase in number (25.1±5.2) and diameter (205.8±50.1µm) of colonies compared with growth factors in both treated and control groups. Spermatogonial stem cells in the colonies may induce spermatogenesis in the recipient testes after transplantation.
Conclusion: co-culture system with Sertoli cells increases adult spermatogonial cell colony formation in vitro.