ABSTRACT
Objective: The properties of self-renewal and division in spermatogonial stem cells [SSCs] support spermatogenesis. There is a number of reported methods for in vitro SSC culture systems. The development of a culture system that effectively supports isolation and self-renewal of germline stem cells [GSCs] is of tremendous benefit for clinical trials, experimental research, and as potential treatment for male infertility. The current study aims to consider the cultivation and behavior of GSCs in a non-adherent culture system
Materials and Methods: In this experimental study, we cultured testicular cells from neonatal mice in agarose coated plates in the presence of Dulbecco's modified Eagle's medium [DMEM] medium [CTRL group], 10% fetal bovine serum [FBS]+DMEM [10% group], and growth factor [G group] that contained 2% FBS, glial cell-derived neurotrophic factor [GDNF], epidermal growth factor [EGF], and fibroblast growth factor [FGF]. Mouse spermatogonial stem-like colonies were isolated approximately 3 weeks after digestion of the testis tissue. After passages 2-3, the identity of the mouse spermatogonial stem-like cells was confirmed by immunocytochemistry, reverse transcription-polymerase chain reaction [RT-PCR], and flow cytometry against the germ cell markers alpha 6, beta 1, c-Kit, Thy-1, c-Ret, Plzf, and Oct4. The statistical significance between mean values in different groups was determined by one-way analysis of variance [ANOVA]
Results: We observed spermatogonial stem-like colonies in the G and 10% groups, but not the CTRL group. Immunocytochemistry, flow cytometry, and RT-PCR confirmed expressions of germ cell markers in these cells. In the spermatogonial stem-like cells, we observed a significant expression [P<0.05] of germ cell markers in the G and 10% groups versus the testis cells [T]. Their proliferative and apoptotic activities were examined by Ki67 and PI/annexin V-FITC. Alkaline phosphatase assay showed that mouse spermatogonial stem-like colonies were partially positive
Conclusion: A non-adherent culture system could provide a favorable method for in vitro short-term culture of spermatogonial stem-like cell colonies