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

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.

Making gametes from alternate sources of stem cells: past, present and future

“…Patel and Bhartiya [ 27 ] proposed that VSELs are the most primitive stem cells in the testes that undergo asymmetric cell division to self-renew and give rise to the SSCs that further differentiate into germ cells, undergo meiosis to produce sperm. Anand et al [ 25 , 26 ] showed that VSELs and GFRA positive stem cells exist as two distinct populations of stem cells in the testis. VSELs are smaller in size, with a surface phenotype of LIN-CD45-SCA-1+ in mouse testis with nuclear OCT-4 whereas SSCs express cytoplasmic OCT-4.…”

Prolonged Growth Hormone/Insulin/Insulin-like Growth Factor Nutrient Response Signaling Pathway as a Silent Killer of Stem Cells and a Culprit in Aging

“…BM-isolated VSELs have been shown to remain as precursors of TCSCs for several types of cells, including hematopoietic cells, mesenchymal cells, endothelial cells, lung alveolar epithelial cells, and cardiomyocytes [11–18, 83, 84]. At the same time, VSELs isolated from murine and human gonads have been proposed to be precursors of male and female gametes [6, 8–10]. …”

<p>Role of stem cells in fertility preservation: current insights</p>

“…From the perspective of future clinical application, the question of whether SSC transplantation in a chemotherapy/radiotherapy-damaged niche may restore fertility arises, as Sertoli and Leydig cell defects have both been reported after gonadotoxic therapy. 130,131 As healthy Sertoli cells present in the TCSs were shown to enhance SSC engraftment and bring adequate signals to surviving endogenous SSCs, 132,133 the use of SCs as supporting cells was considered to improve SSC-transplantation outcomes (Figure 3). In this regard, MSCs can be considered deal candidates, since several studies have suggested that male fertility can be improved, thanks to their paracrine secretions (Table 2).…”