MicroRNA-30a-5p promotes differentiation in neonatal mouse spermatogonial stem cells (SSCs).

BACKGROUND: 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. 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. METHODS: SSCs 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. RESULTS: Based 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. CONCLUSIONS: It can be concluded that inhibition of miRNA-30a-5p by overexpression of differentiation markers promotes differentiation of Spermatogonial Stem Cells.

FSH regulates RA signaling to commit spermatogonia into differentiation pathway and meiosis.

BACKGROUND: Spermatogenesis is a complex process that is controlled by interactions between germ cells and somatic cells. The commitment of undifferentiated spermatogonia to differentiating spermatogonia and normal spermatogenesis requires the action of gonadotropins. Additionally, numerous studies revealed the role of retinoic acid signaling in induction of germ cell differentiation and meiosis entry. MAIN TEXT: Recent studies have shown that expression of several RA signaling molecules including Rdh10, Aldh1a2, Crabp1/2 are influenced by changes in gonadotropin levels. Components of signaling pathways that are regulated by FSH signaling such as GDNF, Sohlh1/2, c-Kit, DMRT, BMP4 and NRGs along with transcription factors that are important for proliferation and differentiation of spermatogonia are also affected by retinoic acid signaling. CONCLUSION: According to all studies that demonstrate the interface between FSH and RA signaling, we suggest that RA may trigger spermatogonia differentiation and initiation of meiosis through regulation by FSH signaling in testis. Therefore, to the best of our knowledge, this is the first time that the correlation between FSH and RA signaling in spermatogenesis is highlighted.

Determination of the Excitatory Effects of MicroRNA-30 in the Self-Renewal and Differentiation Process of Neonatal Mouse Spermatogonial Stem Cells.

BACKGROUND: Spermatogonial stem cells (SSCs) are considered as special stem cells since they have the ability of self-renewal, differentiation, and transferring genetic information to the next generation. Also, they considered as vital players in initiating and preserving spermatogenesis. The fate decisions of SSCs are mediated by intrinsic and extrinsic factors, among which microRNAs (miRNAs) are one of the most essential factors in spermatogenesis among endogenous regulators. However, the mechanisms by which individual miRNAs regulate self-renewal and differentiation of SSCs are unclear. The present study aimed to evaluate the impact of miRNA-30 mimic on fate determinations of SSCs. MATERIALS AND METHODS: The obtained SSCs from neonatal mice (3-6 days old) were purified by MACS and flow cytometry with a promyelocytic leukemia zinc-finger marker. Then, the cultured cells were transfected with miRNA- 30 mimic, and finally, the changes in expressing ID4 and c-kit proteins were assessed by western blot analysis. RESULTS: According to flow cytometry findings, the percentage of SSC purity was about 98.32. The expression of ID4 protein and colonization increased significantly through the transfection of miRNA-30 mimic (P<0.05). CONCLUSION: The miRNA-30 controls spermatogonial stem cell self-renewal and differentiation, which may have significant implications for treating male infertility.