Abstract
Research on spermatogonia is hampered by complex architecture of the seminiferous tubule, poor viability of testicular tissue ex vivo and lack of physiologically relevant long-term culture systems. Therefore there is a need for an in vitro model that would enable long term survival and propagation of spermatogonia. We aimed at the most simplified approach to enable all different cell types within the seminiferous tubules to contribute to the creation of a niche for spermatogonia. In the present study we describe the establishment of a co-culture of mouse testicular cells that is based on proliferative and migratory activity of seminiferous tubule cells and does not involve separation, purification or differential plating of individual cell populations. The co-culture is composed of the constituents of testicular stem cell niche: Sertoli cells [identified by expression of Wilm's tumour antigen 1 (WT1) and secretion of glial cell line-derived neurotrophic factor, GDNF], peritubular myoid cells (expressing alpha smooth muscle actin, αSMA) and spermatogonia [expressing MAGE-B4, PLZF (promyelocytic leukaemia zinc finger), LIN28, Gpr125 (G protein-coupled receptor 125), CD9, c-Kit and Nanog], and can be maintained for at least five weeks. GDNF was found in the medium at a sufficient concentration to support proliferating spermatogonial stem cells (SSCs) that were able to start spermatogenic differentiation after transplantation to an experimentally sterile recipient testis. Gdnf mRNA levels were elevated by follicle-stimulating hormone (FSH) which shows that the Sertoli cells in the co-culture respond to physiological stimuli. After approximately 2–4 weeks of culture a spontaneous formation of cord-like structures was monitored. These structures can be more than 10 mm in length and branch. They are formed by peritubular myoid cells, Sertoli cells, fibroblasts and spermatogonia as assessed by gene expression profiling. In conclusion, we have managed to establish in vitro conditions that allow spontaneous reconstruction of testicular cellular microenvironments.
Highlights
Spermatogenic potential depends on a small population of spermatogonia called spermatogonial stem cells (SSCs)
The tubules were cut into small fragments and 50–100 approximately 1-mm-long segments of mouse seminiferous tubule were pipetted onto a culture dish in small volume of culture medium (100, 200 and 350 ml of suspension was plated onto 24, 12, and 6-wells plates, respectively)
Even though SSCs can be maintained for extended periods of time ex vivo, co-culture with other stem cell niche contributors would provide the most physiologically relevant environment to study their biology
Summary
Spermatogenic potential depends on a small population of spermatogonia called spermatogonial stem cells (SSCs). These cells are responsible for the life-long ability of sperm production in mammals and they are able to reconstitute spermatogenesis to recipients that are rendered experimentally sterile [1,2]. A very small fraction of cells in SSC cultures behave like SSCs in vivo and are able to reconstitute spermatogenesis after transplantation [6]. Cells in SSC cultures are a heterogenous population and it is not clear how closely they resemble undifferentiated spermatogonia in vivo. Their suitability to study germ cell biology can be justifiably questioned
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