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Abstract
Study Objectives: Adult mammalian testes harbor a novel population of quiescent, pluripotent, very small embryonic-like stem cells (VSELs) along with spermatogonial stem cells (SSCs). Present study was undertaken to (i) characterize testicular VSELs (ii) investigate differential effect of chemotherapy on VSELs and SSCs and (iii) to restore the differentiation ability of surviving VSELs by providing healthy somatic microenvironment. Methods: Effect of busulphan (25 mg/Kg) was studied on mouse testes. Syngenic Sertoli cells(105 cells per testis) and bone marrow derived mesenchymal cells (104 cells per testis) were transplanted separately through intertubular route. Effect of niche reconstruction was studied two months later by histology and immuno-localization of germ cell markers MVH and PCNA. Caudal sperm were evaluated for their ability to fertilize oocytes in vitro. Results: VSELs were 2-6 μm in size, SCA-1+/CD45-/LIN-, had high nucleo-cytoplasmic ratio and comprised 0.03% of testicular cells whereas SSC specific marker GFRa localized on a distinct, larger cell population. Busulphan selectively destroyed SSCs and other germ cells however, nuclear OCT-4A, Nanog, Sox-2 and SCA-1 positive VSELs (0.06%) survived. Persisting VSELs were unable to differentiate possibly because chemotherapy also affected the ‘niche’ comprising Sertoli cells. Complete restoration of spermatogenesis was observed two months post transplantation of Sertoli and mesenchymal cells. Transplanted cells formed neo-tubules in the vicinity of surviving tubules and were possibly a source of growth factors essential for VSELs proliferation and differentiation. Both MVH and PCNA showed increased staining in the transplanted group. qRT-PCR studies revealed existence of a meiotic block in busulphan treated testis which was overcome after transplantation. Resulting sperm progressed to epididymis, showed normal motility and ability to fertilize in vitro. Conclusion: Results show that VSELs survive chemotherapy and can restore spermatogenesis in germ cells depleted mice. Results have direct relevance to address fertility issues of cancer survivors.
Highlights
The continuity of spermatogenesis throughout life in an adult mammal is facilitated by stem cells
Results show that very small embryonic-like stem cells (VSELs) survive chemotherapy and can restore spermatogenesis in germ cells depleted mice
The two stem cell populations in the gonads are distinguished by their ability to differentially express OCT-4A [1,3], transcription factor known to be involved in maintenance of pluripotency in stem cells [8]
Summary
The continuity of spermatogenesis throughout life in an adult mammal is facilitated by stem cells. Our group has earlier reported that adult human and mouse testes harbor two distinct stem cell populations that include quiescent very small ES-like stem cells (VSELs) with pluripotent characteristics and their active ‘descendants’ progenitors viz. Similar existence of two stem cell populations including VSELs and the progenitors (ovary germ stem cells, OGSCs) were previously identified in mammalian ovaries by our group [3,4]. Co-existence of quiescent and active stem cell populations is an emerging concept reported in other adult body tissues as well [5,6,7]. The two stem cell populations in the gonads are distinguished by their ability to differentially express OCT-4A [1,3], transcription factor known to be involved in maintenance of pluripotency in stem cells [8]. VSELs may be required to maintain tissue homeostasis similar to that seen in other body tissues [13,14]
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