Skip to main content
SpringerLink
Log in

OCT4 protein and gene expression analysis in the differentiation of spermatogonia stem cells into neurons by immunohistochemistry, immunocytochemistry, and bioinformatics analysis

  • Published:
Stem Cell Reviews and Reports Aims and scope Submit manuscript

Abstract

Background

Spermatogonia Stem Cells (SSCs) are potential candidates for reprogramming and regeneration. Recent studies have revealed that differentiated cells can be reverted to pluripotent by overexpressing a set of pluripotent transcription factors. OCT4 (encoded by pou5f1), a POU transcription factor family member, is essential to the potential that controls pluripotency, and it is widely expressed in pluripotent stem cells, although it decreased or suppressed after differentiation.

Methods

In this investigated research, we examined the OCT4 expression during the differentiation of SSCs into neurons (involving four stages in the following order: SSCs in vivo and in-vitro, embryonic Stem Cell-like (ES-like), Embryonic Bodies (EBs), and finally Neurons) by Immunocytochemistry (ICC), Immunohistochemistry (IMH), and Fluidigm Real-Time polymerase chain reaction. In addition, we use some databases like STRING to predict protein–protein interaction and enrichment analysis.

Results

We evaluated the expression of OCT4 in this process, and we observed that it is expressed in SSCs, ES-like, and EBs during the differentiation of spermatogonia stem cells into adult neurons. We show that by adding RA to EBs, the expression of OCT4 is reduced and is not expressed in the neuron cells. We observed that the expression of OCT4 is linked and interacts with the differentiation of spermatogonia stem cells into neuron cells, and it has been shown to be biologically functional, like stem cell maintenance and somatic cell reprogramming.

Conclusion

Our findings can help us better understand the process of differentiation of spermatogonia stem cells into neurons, and it can be effective in finding new and more efficient treatments for neurogenesis and repair of neurons.

Graphical Abstract

We examined the OCT4 expression during the differentiation of SSCs into neurons (involving four stages in the following order: SSCs in vivo and in-vitro, embryonic Stem Cell-like (ES-like), Embryonic Bodies (EBs), and finally Neurons) by Immunocytochemistry (ICC), Immunohistochemistry (IMH), and Fluidigm Real-Time polymerase chain reaction. In addition, we use some databases like STRING to predict protein–protein interaction and enrichment analysis. We evaluated the expression of OCT4 in this process, and we observed that it is expressed in SSCs, ES-like, and EBs during the differentiation of spermatogonia stem cells into adult neurons. We show that by adding RA to EBs, the expression of OCT4 is reduced and is not expressed in the neuron cells. We observed that the expression of OCT4 is linked and interacts with the differentiation of spermatogonia stem cells into neuron cells, and it has been shown to be biologically functional, like stem cell maintenance and somatic cell reprogramming.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are available from the corresponding author on reasonable request.

Abbreviations

SSCs:

Spermatogonia Stem Cells

ICC:

Immunocytochemistry

IHC:

Immunohistochemistry

ES-Like:

Embryonic Stem Cell-like

EBs:

Embryoid bodies

iPSCs:

Induced pluripotent stem cells

PPI:

Protein–protein interactions

RA:

Retinoic acid

References

  1. Kahroba, H., et al. (2021). The role of Nrf2 in neural stem/progenitors cells: From maintaining stemness and self-renewal to promoting differentiation capability and facilitating therapeutic application in neurodegenerative disease. Ageing research reviews, 65, 101211.

    CAS  PubMed  Google Scholar 

  2. Maher, P. (2019). The potential of flavonoids for the treatment of neurodegenerative diseases. International journal of molecular sciences, 20(12), 3056.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Bojnordi, M. N., et al. (2017). Differentiation of spermatogonia stem cells into functional mature neurons characterized with differential gene expression. Molecular neurobiology, 54(7), 5676–5682.

    CAS  PubMed  Google Scholar 

  4. Strnadel, J., et al. (2018). Survival of syngeneic and allogeneic iPSC-derived neural precursors after spinal grafting in minipigs. Science Translational Medicine, 10(440), eaam6651. https://doi.org/10.1126/scitranslmed.aam6651

  5. Little, D., et al. (2019). Using stem cell–derived neurons in drug screening for neurological diseases. Neurobiology of aging, 78, 130–141.

    CAS  PubMed  Google Scholar 

  6. Hall, B. K. (2018). Germ layers, the neural crest and emergent organization in development and evolution. Genesis, 56(6-7), e23103. https://doi.org/10.1002/dvg.23103

  7. Shafa, M., et al. (2018). Human-induced pluripotent stem cells manufactured using a current good manufacturing practice-compliant process differentiate into clinically relevant cells from three germ layers. Frontiers in medicine, 5, 69.

    PubMed  PubMed Central  Google Scholar 

  8. Katayama, S., et al. (2018). HDAC8 regulates neural differentiation through embryoid body formation in P19 cells. Biochemical and biophysical research communications, 498(1), 45–51.

    CAS  PubMed  Google Scholar 

  9. Yang, J., et al. (2017). Analysis of retinoic acid-induced neural differentiation of mouse embryonic stem cells in two and three-dimensional embryoid bodies. JoVE (Journal of Visualized Experiments), 122, e55621.

    Google Scholar 

  10. Sinha, N., et al. (2021). Roles of Stra8 and Tcerg1l in retinoic acid induced spermatogonial differentiation in mouse. Biology of Reproduction, 105(2), 503–518.

    PubMed  Google Scholar 

  11. Zhang, C., et al. (2017). Biological significance of the suppression of oxidative phosphorylation in induced pluripotent stem cells. Cell reports, 21(8), 2058–2065.

    CAS  PubMed  Google Scholar 

  12. Li, Y.-Q. (2017). Networks of transcription factors for OCT expression in mice. DNA and cell biology, 36(9), 725–736.

    PubMed  Google Scholar 

  13. Echigoya, K., et al. (2020). Nucleosome binding by the pioneer transcription factor OCT4. Scientific reports, 10(1), 1–11.

    Google Scholar 

  14. Zalc, A., et al. (2021). Reactivation of the pluripotency program precedes formation of the cranial neural crest. Science, 371(6529), eabb4776. https://doi.org/10.1126/science.abb4776

  15. Azizi, H., Asgari, B., & Skutella, T. (2019). Pluripotency potential of embryonic stem cell-like cells derived from mouse testis. Cell Journal (Yakhteh), 21(3), 281.

    Google Scholar 

  16. Conrad, S., et al. (2016). Expression of genes related to germ cell lineage and pluripotency in single cells and colonies of human adult germ stem cells. Stem Cells Int, 2016, 8582526. https://doi.org/10.1155/2016/8582526

  17. Azizi, H., et al. (2016). Derivation of pluripotent cells from mouse SSCs seems to be age dependent. Stem Cells Int, 2016, 8216312. https://doi.org/10.1155/2016/8216312

  18. Azizi, H., Hamidabadi, H. G., & Skutella, T. (2019). Differential proliferation effects after short-term cultivation of mouse spermatogonial stem cells on different feeder layers. Cell Journal (Yakhteh), 21(2), 186.

    Google Scholar 

  19. Mizrak, S. C., et al. (2010). Embryonic stem cell-like cells derived from adult human testis. Human Reproduction, 25(1), 158–167.

    CAS  PubMed  Google Scholar 

  20. Kaushik, A., & Bhartiya, D. (2018). Pluripotent very small embryonic-like stem cells in adult testes–an alternate premise to explain testicular germ cell tumors. Stem cell reviews and reports, 14(6), 793–800.

    CAS  PubMed  Google Scholar 

  21. Yamashiro, C., et al. (2018). Generation of human oogonia from induced pluripotent stem cells in vitro. Science, 362(6412), 356–360.

    CAS  PubMed  Google Scholar 

  22. Akamatsu, W., et al. (2009). Suppression of OCT4 by germ cell nuclear factor restricts pluripotency and promotes neural stem cell development in the early neural lineage. Journal of Neuroscience, 29(7), 2113–2124.

    CAS  PubMed  Google Scholar 

  23. Johnstone, V. P., et al. (2015). Experimental traumatic brain injury results in long-term recovery of functional responsiveness in sensory cortex but persisting structural changes and sensorimotor, cognitive, and emotional deficits. Journal of neurotrauma, 32(17), 1333–1346.

    PubMed  Google Scholar 

  24. Mattson, M. P. (2000). Apoptosis in neurodegenerative disorders. Nature reviews Molecular cell biology, 1(2), 120–130.

    CAS  PubMed  Google Scholar 

  25. Bhowmick, S., et al. (2018). Neurodegeneration and sensorimotor deficits in the mouse model of traumatic brain injury. Brain sciences, 8(1), 11.

    PubMed  PubMed Central  Google Scholar 

  26. Yang, H., et al. (2019). Sonic Hedgehog Effectively Improves OCT4-Mediated Reprogramming of Astrocytes into Neural Stem Cells. Molecular Therapy, 27(8), 1467–1482.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Carreon, L. Y., & Dimar, J. R. (2011). Early versus late stabilization of spine injuries: A systematic review. Spine, 36(11), E727–E733.

    PubMed  Google Scholar 

  28. Vismara, I., et al. (2017). Current options for cell therapy in spinal cord injury. Trends in molecular medicine, 23(9), 831–849.

    CAS  PubMed  Google Scholar 

  29. Baptiste, D. C., & Fehlings, M. G. (2007). Update on the treatment of spinal cord injury. Progress in brain research, 161, 217–233.

    CAS  PubMed  Google Scholar 

  30. Lepore, A., et al. (2006). Long-term fate of neural precursor cells following transplantation into developing and adult CNS. Neuroscience, 142(1), 287–304.

    CAS  PubMed  Google Scholar 

  31. Martínez-Serrano, A., & Björklund, A. (1997). Immortalized neural progenitor cells for CNS gene transfer and repair. Trends in neurosciences, 20(11), 530–538.

    PubMed  Google Scholar 

  32. Pessina, A., & Gribaldo, L. (2006). The key role of adult stem cells: Therapeutic perspectives. Current Medical Research and Opinion, 22(11), 2287–2300.

    CAS  PubMed  Google Scholar 

  33. Gruen, L., & Grabel, L. (2006). Concise review: Scientific and ethical roadblocks to human embryonic stem cell therapy. Stem cells, 24(10), 2162–2169.

    PubMed  Google Scholar 

  34. Swijnenburg, R.-J., et al. (2008). Immunosuppressive therapy mitigates immunological rejection of human embryonic stem cell xenografts. Proceedings of the National Academy of Sciences, 105(35), 12991–12996.

    CAS  Google Scholar 

  35. Gomes, K. M., Costa, I. C., Santos, J. F., Dourado, P. M., Forni, M. F., & Ferreira, J. C. (2017). Induced pluripotent stem cells reprogramming: Epigenetics and applications in the regenerative medicine. Rev Assoc Med Bras  (1992), 63(2), 180–189. https://doi.org/10.1590/1806-9282.63.02.180

  36. Mertens, J., et al. (2016). Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience. Nature Reviews Neuroscience, 17(7), 424–437.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Kim, S. M., et al. (2014). Direct conversion of mouse fibroblasts into induced neural stem cells. Nature protocols, 9(4), 871–881.

    CAS  PubMed  Google Scholar 

  38. Han, D. W., et al. (2012). Direct reprogramming of fibroblasts into neural stem cells by defined factors. Cell Stem Cell, 10(4), 465–472.

    CAS  PubMed  Google Scholar 

  39. Lujan, E., et al. (2012). Direct conversion of mouse fibroblasts to self-renewing, tripotent neural precursor cells. Proceedings of the National Academy of Sciences, 109(7), 2527–2532.

    CAS  Google Scholar 

  40. Zhang, M., et al. (2016). Pharmacological reprogramming of fibroblasts into neural stem cells by signaling-directed transcriptional activation. Cell Stem Cell, 18(5), 653–667.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Zhu, S., Wang, H., & Ding, S. (2015). Reprogramming fibroblasts toward cardiomyocytes, neural stem cells and hepatocytes by cell activation and signaling-directed lineage conversion. Nature protocols, 10(7), 959–973.

    CAS  PubMed  Google Scholar 

  42. Yang, H., et al. (2011). ErbB2 activation contributes to de-differentiation of astrocytes into radial glial cells following induction of scratch-insulted astrocyte conditioned medium. Neurochemistry international, 59(7), 1010–1018.

    CAS  PubMed  Google Scholar 

  43. Feng, G.-D., et al. (2014). Fibroblast growth factor 4 is required but not sufficient for the astrocyte dedifferentiation. Molecular neurobiology, 50, 997–1012.

    CAS  PubMed  Google Scholar 

  44. Takuma, K., Baba, A., & Matsuda, T. (2004). Astrocyte apoptosis: Implications for neuroprotection. Progress in neurobiology, 72(2), 111–127.

    CAS  PubMed  Google Scholar 

  45. Masoudi, M., Azizi, H., Sojoudi, K., Yazdani, M., & Gholami, D. (2022). Comparison of POU5F1 gene expression and protein localization in two differentiated and undifferentiated spermatogonial stem cells. Biol Futur, 73(4), 503–512. https://doi.org/10.1007/s42977-022-00149-w

    Article  CAS  PubMed  Google Scholar 

  46. Niazi Tabar, A., et al. (2022). Testicular Localization and Potential Function of Vimentin Positive Cells during Spermatogonial Differentiation Stages. Animals, 12(3), 268.

    PubMed  PubMed Central  Google Scholar 

  47. Amirian, M., et al. (2022). VASA protein and gene expression analysis of human non-obstructive azoospermia and normal by immunohistochemistry, immunocytochemistry, and bioinformatics analysis. Scientific Reports, 12(1), 17259.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Conrad, S., Azizi, H., Hatami, M., Kubista, M., Bonin, M., Hennenlotter, J., Renninger, M., & Skutella, T. (2014). Differential gene expression profiling of enriched human spermatogonia after short- and long-term culture. Biomed Res Int, 2014, 138350. https://doi.org/10.1155/2014/138350

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Hashemi Karoii, D., & Azizi, H. (2022). A review of protein-protein interaction and signaling pathway of Vimentin in cell regulation, morphology and cell differentiation in normal cells. J Recept Signal Transduct Res, 42(5), 512–520. https://doi.org/10.1080/10799893.2022.2047199

    Article  CAS  PubMed  Google Scholar 

  50. Karoii, D.H., Azizi H., & Amirian M. (2022) Signaling Pathways and Protein–Protein Interaction of Vimentin in Invasive and Migration Cells: A Review. Cellular Reprogramming.

  51. Azizi, H., Skutella, T., & Shahverdi, A. (2017). Generation of mouse spermatogonial stem-cell-colonies in a non-adherent culture. Cell Journal (Yakhteh), 19(2), 238.

    Google Scholar 

  52. Azizi, H., Hashemi Karoii, D., & Skutella, T. (2022). Whole Exome Sequencing and In Silico Analysis of Human Sertoli in Patients with Non-Obstructive Azoospermia. Int J Mol Sci., 23(20), 12570. https://doi.org/10.3390/ijms232012570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Hashemi Karoii, D., Azizi, H., & Skutella, T. (2022). Microarray and in silico analysis of DNA repair genes between human testis of patients with nonobstructive azoospermia and normal cells. Cell Biochem Funct., 40(8), 865–879. https://doi.org/10.1002/cbf.3747

    Article  CAS  PubMed  Google Scholar 

  54. Szklarczyk, D., et al. (2019). STRING v11: Protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic acids research, 47(D1), D607–D613.

    CAS  PubMed  Google Scholar 

  55. Doncheva, N. T., et al. (2018). Cytoscape StringApp: Network analysis and visualization of proteomics data. Journal of proteome research, 18(2), 623–632.

    PubMed  PubMed Central  Google Scholar 

  56. Zhang, S., et al. (2019). OCT4 and PAX6 determine the dual function of SOX2 in human ESCs as a key pluripotent or neural factor. Stem cell research & therapy, 10(1), 1–14.

    Google Scholar 

  57. Ding, S., et al. (2021). CHD8 safeguards early neuroectoderm differentiation in human ESCs and protects from apoptosis during neurogenesis. Cell death & disease, 12(11), 1–15.

    Google Scholar 

  58. Lei, I., et al. (2020). SWI/SNF component BAF250a coordinates OCT4 and WNT signaling pathway to control cardiac lineage differentiation. Frontiers in cell and developmental biology, 7, 358.

    PubMed  PubMed Central  Google Scholar 

  59. Masoudi, M., Azizi, H., Sojoudi, K., Yazdani, M., & Gholami, D. (2022). Comparison of POU5F1 gene expression and protein localization in two differentiated and undifferentiated spermatogonial stem cells. Biol Futur., 73(4), 503–512. https://doi.org/10.1007/s42977-022-00149-w

    Article  CAS  PubMed  Google Scholar 

  60. Niknejad, P., Azizi, H., & Sojoudi, K. (2021). POU5F1 Protein and Gene Expression Analysis in Neonate and Adult Mouse Testicular Germ Cells by Immunohistochemistry and Immunocytochemistry. Cell Reprogram., 23(6), 349–358. https://doi.org/10.1089/cell.2021.0108

    Article  CAS  PubMed  Google Scholar 

  61. Nogales, F. F., et al. (2018). Germ cell tumour growth patterns originating from clear cell carcinomas of the ovary and endometrium: A comparative immunohistochemical study favouring their origin from somatic stem cells. Histopathology, 72(4), 634–647.

    PubMed  Google Scholar 

  62. Tong, M.-H., et al. (2011). Expression of Mirlet7 family microRNAs in response to retinoic acid-induced spermatogonial differentiation in mice. Biology of Reproduction, 85(1), 189–197.

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Villodre, E. S., et al. (2019). Silencing of the transcription factors OCT4, Sox2, Klf4, c-Myc or Nanog has different effect on teratoma growth. Biochemical and biophysical research communications, 517(2), 324–329.

    CAS  PubMed  Google Scholar 

  64. Busada, J. T., et al. (2015). Retinoic acid regulates Kit translation during spermatogonial differentiation in the mouse. Developmental biology, 397(1), 140–149.

    CAS  PubMed  Google Scholar 

  65. Simmet, K., et al. (2018). OCT4/POU5F1 is required for NANOG expression in bovine blastocysts. Proceedings of the National Academy of Sciences, 115(11), 2770–2775.

    CAS  Google Scholar 

  66. Kirsanov, O., et al. (2022). Modeling mammalian spermatogonial differentiation and meiotic initiation in vitro. Development, 149(22), dev200713. https://doi.org/10.1242/dev.200713

  67. Wang, X., et al. (2015). Isolation and culture of pig spermatogonial stem cells and their in vitro differentiation into neuron-like cells and adipocytes. International Journal of Molecular Sciences, 16(11), 26333–26346.

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Gilmozzi, V., et al. (2021). Generation of hiPSC-derived functional dopaminergic neurons in alginate-based 3D culture. Frontiers in Cell and Developmental Biology, 9, 708389.

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This research was supported by Amol University of Special Modern Technologies (AUSMT) and the University of Heidelberg, Institute for Anatomy and Cell Biology III, Department of Neuroanatomy, Germany.

Funding

This research was funded by Centre for International Scientific Studies and Collaboration (CISSC), Ministry of Science, Research and Technology, and a BMBF grant (01DS13003). This research has been done with the financial support of the Centre for International Scientific Studies and Collaboration (CISSC) of the Ministry of Science, Research and Technology as well as the Iranian National Science Foundation (INSF) under project No.4006239, and a memorandum of understanding (MOU) agreement between the Institute for Anatomy and Cell Biology at the University of Heidelberg and Amol University of Special Modern Technology.

Author information

Author notes

  1. Danial Hashemi Karoii

    Present address: Department of Cell and Molecular Biology, School of Biology, College of Science, University of Tehran, Tehran, Iran

  2. Danial Hashemi Karoii and Hossein Azizi contributed equally to this work.

Authors and Affiliations

  1. Faculty of Biotechnology, Amol University of Special Modern Technologies, Amol, Iran

    Danial Hashemi Karoii & Hossein Azizi

Authors
  1. Danial Hashemi Karoii
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hossein Azizi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

H.A: Carried out and designed the experiment and edited the manuscript, and D.H.K: Assembly of data, do the experiment, data analysis, design PPI network, and enrichment analysis, wrote and edited the manuscript.

Corresponding author

Correspondence to Hossein Azizi.

Ethics declarations

Ethics Approval

All animal use was approved by University of Heidelberg and Amol Research Ethics Office and experiments were conducted in accordance with the Iranian Council of Animal Care Policies.

Consent to Participat

Not applicable.

Consent for Publication

We consent to publication.

Conflict of Interests

The authors declare that there is no conflict of interest regarding the publication of this study.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hashemi Karoii, D., Azizi, H. OCT4 protein and gene expression analysis in the differentiation of spermatogonia stem cells into neurons by immunohistochemistry, immunocytochemistry, and bioinformatics analysis. Stem Cell Rev and Rep 19, 1828–1844 (2023). https://doi.org/10.1007/s12015-023-10548-8

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12015-023-10548-8

Keywords

Search

Navigation