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Underlying Mechanisms that Restore Spermatogenesis on Transplanting Healthy Niche Cells in Busulphan Treated Mouse Testis

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Abstract

Very small embryonic-like stem cells (VSELs) exist among spermatogonial stem cells and survive chemotherapy in both mice and human testes because of their relatively quiescent nature. Our earlier study revealed that inter-tubular transplantation of niche (Sertoli or bone marrow derived mesenchymal) cells can restore spermatogenesis from endogenous surviving VSELs. Present study was undertaken to delineate the effect of busulphan on testicular stem/germ/Sertoli cells and to comprehend the underlying mechanisms of how transplanted niche cells restore spermatogenesis. Ploidy analysis showed an increase in diploid cells on D7 and VSELs (2–6 μm; LIN−/CD45−/SCA-1+) were detected at all time-points studied and were maximum on D15 after busulphan treatment. They were visualized in cell smears, expressed nuclear NANOG and SOX2 and BrdU uptake on D15 suggested they were proliferating in response to stress induced by busulphan. Verapamil-sensitive side population detected comprised SCA-1 positive stem cells (5 ± 0.02 % in normal and 8.6 ± 2.02 % in chemoablated testis). Adverse effects of busulphan on Sertoli cells by transcriptome analysis included altered expression of Gdnf, Scf, Fgf, Bmp4, androgen binding protein, components of blood-testis-barrier and also stem cells related signaling pathways including Wnt. GFP positive transplanted cells aligned themselves as ‘neo-tubules’ and were visualized adjacent to ‘native’ germ cells depleted tubules. ‘Neo-tubules’ provide paracrine support to endogenous VSELs to undergo spermatogenesis. Quantitative analysis was done to track proliferation (PCNA) and differentiation (MVH) of stem cells by immuno-localization studies at different time intervals. Results provide an alternative strategy to restore spermatogenesis in cancer survivors from endogenous stem cells which needs to be further researched.

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Acknowledgments

We thank Harshada Modak and Smita Bhutda from the Stem Cell Biology Department for technical help. The help from Dr. Mukherjee, Ms. Gayatri and Ms. Sushma in flow cytometry studies and from Ms. Reshma and Ms. Shobha in confocal studies is also acknowledged. We also thank Indian Council of Medical Research and Department of Science and Technology, Government of India, New Delhi for providing financial support for the study.

Author Contributions

SA Conception and design, Collection of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript; DB : Conception and design, Data analysis and interpretation, Manuscript writing, Final approval of manuscript KS: Collection of data, Data analysis and interpretation, Manuscript writing, Final approval of manuscript. AM: Collection of data, Final approval of manuscript.

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Authors and Affiliations

  1. Stem Cell Biology Department, National Institute for Research in Reproductive Health, JM Street, Parel, Mumbai, 400 012, India

    Sandhya Anand, Deepa Bhartiya, Kalpana Sriraman & Alpna Mallick

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  1. Sandhya Anand
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  2. Deepa Bhartiya
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Correspondence to Deepa Bhartiya.

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Financial Support for the study was provided by Indian Council of Medical Research and Department of Science and Technology, Government of India, New Delhi, India.

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Anand, S., Bhartiya, D., Sriraman, K. et al. Underlying Mechanisms that Restore Spermatogenesis on Transplanting Healthy Niche Cells in Busulphan Treated Mouse Testis. Stem Cell Rev and Rep 12, 682–697 (2016). https://doi.org/10.1007/s12015-016-9685-1

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  • DOI: https://doi.org/10.1007/s12015-016-9685-1

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