Abstract
BackgroundThe importance of spermatogonial stem cells (SSCs) in spermatogenesis is crucial and intrinsic factors and extrinsic signals mediate fate decisions of SSCs. Among endogenous regulators, microRNAs (miRNAs) play critical role in spermatogenesis. However, the mechanisms which individual miRNAs regulate self- renewal and differentiation of SSCs are unknown. The aim of this study was to investigate effects of miRNA-30a-5p inhibitor on fate determinations of SSCs.MethodsSSCs were isolated from testes of neonate mice (3–6 days old) and their purities were performed by flow cytometry with ID4 and Thy1 markers. Cultured cells were transfected with miRNA- 30a-5p inhibitor. Evaluation of the proliferation (GFRA1, PLZF and ID4) and differentiation (C-Kit & STRA8) markers of SSCs were accomplished by immunocytochemistry and western blot 48 h after transfection.ResultsBased on the results of flow cytometry with ID4 and Thy1 markers, percentage of purity of SSCs was about 84.3 and 97.4 % respectively. It was found that expression of differentiation markers after transfection was significantly higher in miRNA-30a- 5p inhibitor group compared to other groups. The results of proliferation markers evaluation also showed decrease of GFRA1, PLZF and ID4 protein in SSCs transfected with miRNA-30a-5p inhibitor compared to the other groups.ConclusionsIt can be concluded that inhibition of miRNA-30a-5p by overexpression of differentiation markers promotes differentiation of Spermatogonial Stem Cells.
Highlights
The importance of spermatogonial stem cells (SSCs) in spermatogenesis is crucial and intrinsic factors and extrinsic signals mediate fate decisions of SSCs
Recent studies have shown that microRNAs are a class of endogenous factors involved in different cellular processes, including self-renewal, proliferation, differentiation, and apoptosis [10]. miRNAs are small single-stranded RNA molecules (18–25 nucleotides) that act as vital factors for post-transcriptional gene silencing
The results indicated that the expression level of miR-30a-5p significantly decreased in the miR30a-5p inhibitor group (22.51 ± 3.51) compared to the untransfected group (p ≤ 0.001)
Summary
The importance of spermatogonial stem cells (SSCs) in spermatogenesis is crucial and intrinsic factors and extrinsic signals mediate fate decisions of SSCs. Among endogenous regulators, microRNAs (miRNAs) play critical role in spermatogenesis. The aim of this study was to investigate effects of miRNA-30a-5p inhibitor on fate determinations of SSCs. Spermatogenesis is a complex developmental process in the mammalian reproductive system that results in generation of highly specialized sperm from spermatogonial stem cells (SSCs) [1, 2]. MiRNAs are small single-stranded RNA molecules (18–25 nucleotides) that act as vital factors for post-transcriptional gene silencing. They bind to three untranslated regions of target mRNAs and result in either endonucleolytic cleavage of the target mRNA or translation inhibition [8, 11]. The function and molecular mechanisms of individual miRNAs in regulating the SSCs fate determination are not clear warranting further research
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