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Cryoprotective Effect of Pentoxifylline on Spermatogonial Stem Cell During Transplantation into Azoospermic Torsion Mouse Model

  • Male Reproduction: Original Article
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Abstract

Preserving the spermatogonial stem cells (SSCs) in long periods of time during the treatment of male infertility using stem cell banking systems and transplantation is an important issue. Therefore, this study was conducted to develop an optimal cryopreservation protocol for SSCs using 10 mM pentoxifylline (PTX) as an antioxidant in basal freezing medium. Testicular torsion—a mouse model for long-term infertility—was used to transplant fresh SSCs (n = 6), fresh SSCs treated with PTX (n = 6), cryopreserved SSCs with basal freezing medium (n = 6), and cryopreserved SSCs treated with PTX (n = 6). Eight weeks after germ cell transplantation, samples were assessed for proliferation, through evaluation of Ddx4 and Id4 markers, and differentiation via evaluation of C‐Kit and Sycp3, Tnp1, Tnp2, and Prm1 markers. According to morphological and flow cytometry results, SSCs are able to form colonies and express Gfra1, Id4, α6‐integrin, and β1‐integrin markers. We found positive influence from PTX on proliferative and differentiative markers in SSCs transplanted to azoospermic mice. In the recipient testis, donor SSCs formed spermatogenic colonies and sperm. Respecting these data, adding pentoxifylline is a practical way to precisely cryopreserve germ cells enriched for SSCs in cryopreservation, and this procedure could become an efficient method to restore fertility in a clinical setup. However, more studies are needed to ensure its safety in the long term.

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Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code Availability

In this study, we used ImageJ (RRID:SCR_003070) and GraphPad Prism (RRID:SCR_002798) software programs. We also used anti-Ddx4 (Abcam Cat# ab13840, RRID: AB_443012), anti-Id4 polyclonal (Thermo Fisher Scientific Cat# PA5-26,976, RRID:AB_2544476), anti-Sycp3 (Abcam Cat# ab15093, RRID: AB_301639), anti-C-Kit (Abcam Cat# ab5506, RRID:AB_304943), goat anti-rabbit IgG-H&L polyclonal HRP conjugate (Abcam Cat# ab721, RRID: AB_955447), goat anti-mouse IgG (H&L)-HRP conjugate (Bio-Rad Cat# 170–6516, RRID: AB_11125547), goat anti-rabbit IgG-H&L polyclonal FITC conjugate (Abcam Cat# ab6717, RRID: AB_955238), rabbit anti-GFR alpha-1 polyclonal antibody (Bioss Cat# bs-0201R-FITC, RRID:AB_11108563), rat polyclonal anti-α6-integrin (BD Biosciences Cat# 555,736, RRID:AB_396079), and rat polyclonal anti-β1-integrin (Sigma-Aldrich, USA) (FITC) (BD Biosciences Cat# 555,005, RRID:AB_395639) antibodies.

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Acknowledgements

The authors thank the Tehran University of Medical Sciences and Health Services, Tehran, Iran.

Funding

This study was supported in part by a grant received from the Tehran University of Medical Sciences and Health Services, Tehran, Iran (grant number: 37741).

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Authors and Affiliations

  1. Department of Anatomy, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Mehrnoush Malekzadeh, Nasrin Takzaree, Shokoofeh Kazemzadeh, Nasrin Khanmohammadi, Ghazaleh Sadeghiani, Maryam Shabani & Tayebeh Rastegar

  2. Department of Anatomy, School of Medicine, Shahroud University of Medical Sciences, Semnan, Iran

    Heidar Toolee

  3. Department of Anatomy, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran

    Somayeh Solhjoo

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  1. Mehrnoush Malekzadeh
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Contributions

All authors participated in the design, interpretation of the studies, analysis of the data, and review of the manuscript. Mehrnoush Malekzadeh and Tayebeh Rastegar designed the study, Shokoofeh Kazemzadeh supervised the data collection, Ghazaleh Sadeghiani analyzed the data, Somayeh Solhjoo and Heidar Toolee interpreted the data and prepared the manuscript for publication, Nasrin Takzaree and Nasrin Khanmohammadi supervised the data collection and analyzed the data, and Maryam Shabani reviewed the draft of the manuscript. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Tayebeh Rastegar.

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Ethics Approval and Consent to Participate

This article does not contain any studies with human participants performed by any of the authors. All experiments involving the use of animals were approved by the Institutional Animal Care and Use Committee (IACUC) of the Tehran University of Medical Science. All applicable international, national, and institutional guidelines for the care and use of animals were followed. All participants signed informed consent forms approved by the Ethical Committee of Tehran University of Medical Sciences (IR.TUMS.MEDICINE.REC.1397.210).

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The authors declare no competing interests.

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Malekzadeh, M., Takzaree, N., Toolee, H. et al. Cryoprotective Effect of Pentoxifylline on Spermatogonial Stem Cell During Transplantation into Azoospermic Torsion Mouse Model. Reprod. Sci. 29, 526–539 (2022). https://doi.org/10.1007/s43032-021-00729-6

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  • DOI: https://doi.org/10.1007/s43032-021-00729-6

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