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

Abstract

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.

Melatonin in cryopreservation media improves transplantation efficiency of frozen–thawed spermatogonial stem cells into testes of azoospermic mice

“…The high expression for these three markers (PLZF, Thy-1 and α-6 integrin) in SSCs indicated the primary phases of the evolvement of the spermatogenic lineage in isolated SSCs whose differentiation process had not yet initiated and confirmed that SSCs collected after digestion enzymatic were in the undifferentiated stage. The high expression of SSC identification markers in this study was consistent with other studies [ 35 – 37 ].…”

Identification of sex-biased miRNA markers informative of heat-past events

“…Similarly, in olive flounder ( Paralichthys olivaceus ), miR-143, miR-26a and let-7a were also identified as the most expressed miRNAs in both ovaries and testes (Gu et al, 2014). In mouse and human, miR-30a-5p was crucial for spermatogonial stem cell differentiation (Khanehzad et al, 2021) and was used as a marker for men suffering from non-obstructive azoospermia (Arefnia et al, 2022). In our data, however, we observed a conserved overexpression of miR-30a in adult fish ovaries compared to adult testes in the ten studied species.…”

MicroRNAs in spermatogenesis dysfunction and male infertility: clinical phenotypes, mechanisms and potential diagnostic biomarkers

“…Intrinsic factors and extrinsic signals play a crucial role in maintaining a delicate balance between SSC proliferation and differentiation, which ensures a continuous supply of differentiating spermatogonia while preserving an adequate population of stem cells ( 75 ) and ensuring the lifelong production of spermatozoa. miRNAs are important endogenous regulators of this process in spermatogenesis ( 76 ).…”

“…The inhibition of SSC differentiation by downregulating Plzf expression and upregulating Stra8, Dazl, and Sycp3 expression ( 80 ). Inhibition of miR-30a-5p triggers the differentiation of neonatal mouse SSCs ( 76 ), whereas its overexpression increases cell viability and promotes antiapoptotic and antioxidant activities during the freeze-thaw process of SSCs ( 81 ). SC-derived exosomal miR-30a-5p promotes the proliferation and differentiation of SSCs by regulating the MAPK signaling pathway through Zeb2/Fgf9 ( 82 ).…”