Spermatogonial Stem Cell Niche and Spermatogonial Stem Cell Transplantation in Zebrafish Rafael Henrique No´ brega1,2, Caaj Douwe Greebe1, Henk van de Kant1, Jan Bogerd1, Luiz Renato de Fran c¸a2*, Ru¨ diger W. Schulz1,3* 1 Division of Developmental Biology, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands, 2 Laboratory of Cellular Biology, Department of Morphology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil, 3 Research Group Reproduction and Growth in Fish, Institute of Marine Research, Bergen, Norway Abstract Background: Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis, and reside within a specific microenvironment in the testes called ‘‘niche’’ which regulates stem cell properties, such as, self-renewal, pluripotency, quiescence and their ability to differentiate. Methodology/Principal Findings: Here, we introduce zebrafish as a new model for the study of SSCs in vertebrates. Using 59-bromo-29-deoxyuridine (BrdU), we identified long term BrdU-retaining germ cells, type A undifferentiated spermatogonia as putative stem cells in zebrafish testes. Similar to rodents, these cells were preferentially located near the interstitium, suggesting that the SSC niche is related to interstitial elements and might be conserved across vertebrates. This localization was also confirmed by analyzing the topographical distribution of type A undifferentiated spermatogonia in normal, vasa::egfp and fli::egfp zebrafish testes. In the latter one, the topographical arrangement suggested that the vasculature is important for the SSC niche, perhaps as a supplier of nutrients, oxygen and/or signaling molecules. We also developed an SSC transplantation technique for both male and female recipients as an assay to evaluate the presence, biological activity, and plasticity of the SSC candidates in zebrafish. Conclusions/Significance: We demonstrated donor-derived spermato- and oogenesis in male and female recipients, respectively, indicating the stemness of type A undifferentiated spermatogonia and their plasticity when placed into an environment different from their original niche. Similar to other vertebrates, the transplantation efficiency was low. This might be attributed to the testicular microenvironment created after busulfan depletion in the recipients, which may have caused an imbalance between factors regulating self-renewal or differentiation of the transplanted SSCs. Citation: N o´brega RH, Greebe CD, van de Kant H, Bogerd J, de Franc¸a LR, et al. (2010) Spermatogonial Stem Cell Niche and Spermatogonial Stem Cell Transplantation in Zebrafish. PLoS ONE 5(9): e12808. doi:10.1371/journal.pone.0012808 Editor: David S. Milstone, Brigham and Women’s Hospital, United States of America Received April 7, 2010; Accepted August 20, 2010; Published September 20, 2010 Copyright: 2010 N o´brega et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Norwegian Research Council, Utrecht University, the National Council for Scientific and Technological Development (CNPq), and Research Foundation of the State of Minas Gerais (FAPEMIG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: lrfranca@icb.ufmg.br (LRdF); r.w.schulz@uu.nl (RWS) Introduction Spermatogenesis is a cellular developmental process by which self-renewing spermatogonial stem cells (SSCs) differentiate into millions of sperm daily [1,2]. To sustain this process continuously throughout the male reproductive life span, SSCs reside within a specific microenvironment in the testes called ‘‘niche’’ which regulates their properties, such as, self-renewal, pluripotency, quiescence and their ability to differentiate [3–5]. Despite of its crucial importance on SSC fate, the cellular and molecular composition of SSC niche remain unknown for several species of vertebrates. In rodents, the SSC niche has recently been identified within regions of the seminiferous tubules which are adjacent to the interstitial compartment [6,7], preferentially along the branches of the interstitial blood vessels [8]. It has been hypothesized that the cellular and molecular environment near the interstitial compart- ment promotes SSC renewal, and when SSCs leave these areas, the associated changes in their environment promote SSC differenti- ation [5]. The proximity of SSC niche to the interstitium perhaps reflects the vascular supply of oxygen, nutrients, or hormones, such as follicle-stimulating hormone (FSH) or luteinizing hormone (LH) which influence Leydig and Sertoli cell functions on SSC self- renewal and also on SSC retention and homing in the niche [5,9– 11]. For example, FSH induces the secretion of GDNF (glial cell- line derived neurotrophic factor), an extrinsic stimulator of SSC self- renewal, produced by Sertoli cells [3,12]. Currently, the only means to study SSCs and their niche is by exploiting the stem cells’ functional properties, such as slow-cycling and quiescent nature through the label-retaining cell (LRC) approach [13], or by studying SSC functionality and plasticity by transplantation assays. In this context, transplantation techniques developed by Brinster and collaborators [14,15] has enabled tremendous progress in the phenotypic and functional investiga- tions of SSCs. Nowadays, SSC transplantation approaches have PLoS ONE | www.plosone.org 1 September 2010 | Volume 5 | Issue 9 | e12808