Skip to main content

miR-106b-5p Intensifies the Proliferative Potential of Spermatogonial Stem Cells as a Prerequisite for Male Infertility Treatment


Although numerous studies have investigated the molecular basis of male infertility, various aspects of this area have remained uncovered. Over the past years, researchers have reported the significant potential of miRNAs in posttranscriptional regulatory roles. By targeting mRNAs, these notable molecules can modulate the processes related to male infertility. On the other side, the outstanding potential of male germline stem cells, SSCs, includes their application in infertility treatment. SSCs retain normal spermatogenesis and fertility by adjusting both SSC self-renewal and differentiation. Therefore, for the characterization and manipulation of SSCs, effective and efficient in vitro culture methods are essential in supporting their maintenance and development. In this regard, the present investigation was undertaken to evaluate the impact of one of the recently conspicuous miRNAs, miR-106b, in SSCs enrichment. As a result, we first found that the SSCs induced with miR-106b-5p highly express TGF-β1, which is known as a regulator of epigenetic modifiers and downstream genes. We next sought to show that self-renewal markers, including c-Myc, Oct-4, and Sox2, are increased in the induced SSC group. The intended miRNA also induced the inhibitor of differentiation 4 (ID4) and aided to remain unmethylated in SSCs. Additionally, for the tumorigenicity possibility of the manipulation, we indicated that PTEN, a tumor-suppressor gene, expressed remarkably in the induced SSCs. In conclusion, our findings showed that miR-106b-5p enhances the proliferative potential of SSCs, making it a substantial factor for therapeutic strategies of male infertility.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Code availability

Not applicable.


  1. Guerri G, Maniscalchi T, Barati S, Gerli S, Di Renzo GC, Della Morte C, et al. Non-syndromic monogenic female infertility. Acta Bio Medica: Atenei Parmensis. 2020;90(Suppl 10):68.

    Google Scholar 

  2. Bracke A, Peeters K, Punjabi U, Hoogewijs D, Dewilde S. A search for molecular mechanisms underlying male idiopathic infertility. Reprod Biomed Online. 2018;36(3):327–39.

    CAS  Article  Google Scholar 

  3. Pan MM, Hockenberry MS, Kirby EW, Lipshultz LI. Male infertility diagnosis and treatment in the era of in vitro fertilization and intracytoplasmic sperm injection. Med Clin North Am. 2017;102(2):337–47.

    Article  Google Scholar 

  4. Dong L, Kristensen SG, Hildorf S, Gul M, Clasen-Linde E, Fedder J, et al. Propagation of spermatogonial stem cell-like cells from infant boys. Front Physiol. 2019;10:1155.

    Article  Google Scholar 

  5. Mei Y, Bian C, Li J, Du Z, Zhou H, Yang Z, et al. miR-21 modulates the ERK–MAPK signaling pathway by regulating SPRY2 expression during human mesenchymal stem cell differentiation. J Cell Biochem. 2013;114(6):1374–84.

    CAS  Article  Google Scholar 

  6. Vlajković S, Čukuranović R, Daković Bjelaković M, Stefanović V. Possible therapeutic use of spermatogonial stem cells in the treatment of male infertility: a brief overview. The Scientific World Journal. 2012;2012.

  7. Gul M, Hildorf S, Dong L, Thorup J, Hoffmann ER, Jensen CFS, et al. Review of injection techniques for spermatogonial stem cell transplantation. Hum Reprod Update. 2020;26(3):368–91.

    CAS  Article  Google Scholar 

  8. Lian J, Tian H, Liu L, Zhang X, Li W, Deng Y, et al. Downregulation of microRNA-383 is associated with male infertility and promotes testicular embryonal carcinoma cell proliferation by targeting IRF1. Cell death & disease. 2010;1(11):e94-e.

  9. Vashisht A, Gahlay G. Using miRNAs as diagnostic biomarkers for male infertility: Opportunities and challenges. Mol Hum Reprod. 2020;26(4):199–214.

    CAS  Article  Google Scholar 

  10. Salas-Huetos A, James E, Aston K, Carrell D, Jenkins T, Yeste M. The role of miRNAs in male human reproduction: A systematic review. Andrology. 2020;8(1):7–26.

    CAS  Article  Google Scholar 

  11. Li MA, He L. microRNAs as novel regulators of stem cell pluripotency and somatic cell reprogramming. BioEssays. 2012;34(8):670–80.

    Article  Google Scholar 

  12. Khanehzad M, Nourashrafeddin SM, Abolhassani F, Kazemzadeh S, Madadi S, Shiri E, et al. MicroRNA-30a-5p promotes differentiation in neonatal mouse spermatogonial stem cells (SSCs). Reprod Biol Endocrinol. 2021;19(1):1–14.

    Article  Google Scholar 

  13. Tong M-H, Mitchell DA, McGowan SD, Evanoff R, Griswold MD. Two miRNA Clusters, Mir-17–92 (Mirc1) and Mir-106b-25 (Mirc3), Are Involved in the Regulation of Spermatogonial Differentiation in Mice1. Biology of Reproduction. 2012;86(3).

  14. Ogawa T, Arechaga J, Avarbock M, Brinster R. Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol. 2003;41(1):111–22.

    Google Scholar 

  15. Zhou W, Shao H, Zhang D, Dong J, Cheng W, Wang L, et al. PTEN signaling is required for the maintenance of spermatogonial stem cells in mouse, by regulating the expressions of PLZF and UTF1. Cell Biosci. 2015;5(1):1–10.

    Article  Google Scholar 

  16. Olariu V, Lövkvist C, Sneppen K. Nanog, Oct4 and Tet1 interplay in establishing pluripotency. Sci Rep. 2016;6(1):1–11.

    Article  Google Scholar 

  17. Hurtado A, Palomino R, Georg I, Lao M, Real FM, Carmona FD, et al. Deficiency of the onco-miRNA cluster, miR-106b∼ 25, causes oligozoospermia and the cooperative action of miR-106b∼ 25 and miR-17∼ 92 is required to maintain male fertility. Mol Hum Reprod. 2020;26(6):389–401.

    CAS  Article  Google Scholar 

  18. Khuu C, Utheim TP, Sehic A. The three paralogous microRNA clusters in development and disease, miR-17–92, miR-106a-363, and miR-106b-25. Scientifica. 2016;2016.

  19. He Z, Jiang J, Kokkinaki M, Tang L, Zeng W, Gallicano I, et al. MiRNA-20 and mirna-106a regulate spermatogonial stem cell renewal at the post-transcriptional level via targeting STAT3 and Ccnd1. Stem cells. 2013;31(10):2205–17.

    CAS  Article  Google Scholar 

  20. Huang YL, Huang GY, Lv J, Pan LN, Luo X, Shen J. miR-100 promotes the proliferation of spermatogonial stem cells via regulating Stat3. Mol Reprod Dev. 2017;84(8):693–701.

    CAS  Article  Google Scholar 

  21. Itman C, Mendis S, Barakat B, Loveland KL. All in the family: TGF-β family action in testis development. Reproduction. 2006;132(2):233–46.

    CAS  Article  Google Scholar 

  22. Rombaut C, Mertes H, Heindryckx B, Goossens E. Human in vitro spermatogenesis from pluripotent stem cells: in need of a stepwise differentiation protocol? Mol Hum Reprod. 2017;24(2):47–54.

    Article  Google Scholar 

  23. Spiller C, Burnet G, Bowles J. Regulation of fetal male germ cell development by members of the TGFβ superfamily. Stem cell research. 2017;24:174–80.

    CAS  Article  Google Scholar 

  24. Deng M, Hou SY, Tong BD, Yin JY, Xiong W. The Smad2/3/4 complex binds miR-139 promoter to modulate TGFβ-induced proliferation and activation of human Tenon’s capsule fibroblasts through the Wnt pathway. J Cell Physiol. 2019;234(8):13342–52.

    CAS  Article  Google Scholar 

  25. Hata A, Chen Y-G. TGF-β signaling from receptors to Smads. Cold Spring Harbor perspectives in biology. 2016;8(9):a022061.

  26. Miyazawa K, Miyazono K. Regulation of TGF-β family signaling by inhibitory Smads. Cold Spring Harbor Perspectives in Biology. 2017;9(3):a022095.

  27. Yang J, Jiang W. The role of SMAD2/3 in human embryonic stem cells. Frontiers in Cell and Developmental Biology. 2020;8:653.

    Article  Google Scholar 

  28. Smith AL, Iwanaga R, Drasin DJ, Micalizzi DS, Vartuli RL, Tan A-C, et al. The miR-106b-25 cluster targets Smad7, activates TGF-β signaling, and induces EMT and tumor initiating cell characteristics downstream of Six1 in human breast cancer. Oncogene. 2012;31(50):5162–71.

    CAS  Article  Google Scholar 

  29. Helsel AR, Yang Q-E, Oatley MJ, Lord T, Sablitzky F, Oatley JM. ID4 levels dictate the stem cell state in mouse spermatogonia. Development. 2017;144(4):624–34.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Sachs C, Robinson BD, Andres Martin L, Webster T, Gilbert M, Lo HY, et al. Evaluation of candidate spermatogonial markers ID 4 and GPR 125 in testes of adult human cadaveric organ donors. Andrology. 2014;2(4):607–14.

    CAS  Article  Google Scholar 

  31. Zhang J, Cao H, Xie J, Fan C, Xie Y, He X, et al. The oncogene Etv5 promotes MET in somatic reprogramming and orchestrates epiblast/primitive endoderm specification during mESCs differentiation. Cell Death Dis. 2018;9(2):1–16.

    Google Scholar 

  32. Chen Z, Hong F, Wang Z, Hao D, Yang H. Spermatogonial stem cells are a promising and pluripotent cell source for regenerative medicine. American Journal of Translational Research. 2020;12(11):7048.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Katz DJ, Kolon TF, Feldman DR, Mulhall JP. Fertility preservation strategies for male patients with cancer. Nat Rev Urol. 2013;10(8):463–72.

    Article  Google Scholar 

  34. Tang X, Chang C, Hao M, Chen M, Woodley DT, Schönthal AH, et al. Heat shock protein-90alpha (Hsp90α) stabilizes hypoxia-inducible factor-1α (HIF-1α) in support of spermatogenesis and tumorigenesis. Cancer gene therapy. 2021:1–13.

  35. Luo S-Q, Xiong D-H, Li J, Li G, Wang Y, Zhang J-M, et al. C1orf35 contributes to tumorigenesis by activating c-MYC transcription in multiple myeloma. Oncogene. 2020;39(16):3354–66.

    CAS  Article  Google Scholar 

  36. Melnik S, Werth N, Boeuf S, Hahn E-M, Gotterbarm T, Anton M, et al. Impact of c-MYC expression on proliferation, differentiation, and risk of neoplastic transformation of human mesenchymal stromal cells. Stem Cell Res Ther. 2019;10(1):1–18.

    Article  Google Scholar 

  37. Yang X, Shao F, Guo D, Wang W, Wang J, Zhu R, et al. WNT/β-catenin-suppressed FTO expression increases m 6 A of c-Myc mRNA to promote tumor cell glycolysis and tumorigenesis. Cell Death Dis. 2021;12(5):1–14.

    PubMed  PubMed Central  Google Scholar 

  38. Hasani Fard AH, Kamalipour F, Mazaheri Z, Hosseini SJ. Evaluation Of MiR-106b-5p Expression In The Production Of IPS-Like Cells From Mice SSCs During The Formation Of Teratoma And The Three Embryonic Layers. Cell Journal (Yakhteh). 2022;24(6): In press.

Download references


Acknowledgments and Funding Information

This investigation was financially supported by the Men’s Health & Reproductive Health Research Center, Shahid Beheshti University of Medical Sciences. We would like to thank the technical staff in Histogenotech company for their assistance with the collection of our data. This study was supported by a research grant from Men’s Health & Reproductive Health Research Center (MHRHRC), Shahid Beheshti University of Medical Sciences.


This study was supported by a research grant from Men’s Health & Reproductive Health Research Center (MHRHRC), Shahid Beheshti University of Medical Sciences.

Author information

Authors and Affiliations



AHF and ZM and SJH proposed the work. AHF performed experimental works and data collection. MV performed bioinformatics work. MV and AHF contributed to article writing and manuscript approving. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Seyed Jalil Hosseini.

Ethics declarations

Ethics approval

All experiments were approved by the Ethics Committee of Shahid Beheshti University of Medical Sciences and following the Declaration of Helsinki (Approval ID: IR.SBMU.REC1398.073).

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Conflicts of interest

Not applicable.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hasani Fard, A.H., Valizadeh, M., Mazaheri, Z. et al. miR-106b-5p Intensifies the Proliferative Potential of Spermatogonial Stem Cells as a Prerequisite for Male Infertility Treatment. Reprod. Sci. (2022).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Spermatogenesis
  • Stem Cells
  • MicroRNAs
  • Infertility
  • Male
  • Spermatogonia
  • Fertility