been developed for a number of species including also teleost fish [16,17]. These data have broad implications for understanding the regulation of spermatogenesis, stem cell biology, etiology of male infertility [18–22], and also for advancing biotechnologies such as conservation of valuable genetic stocks, preservation of endan- gered species, and also as new option for transgenesis [16,17,19]. In anamniote vertebrates (fishes and amphibians), we find the cystic type of spermatogenesis [2]. There are two main differences compared to higher vertebrates. First, within the spermatogenic tubules, cytoplasmic extensions of Sertoli cells form cysts that envelope a single, clonally and hence synchronously developing group of germ cells deriving from a single spermatogonium. Second, the cyst-forming Sertoli cells retain their capacity to proliferate also in adult fish [23,24]. Hence, the basic functional unit of the spermatogenic epithelium in fish is a spermatogenic cyst formed by a dynamic group of Sertoli cells surrounding and nursing one synchronously developing germ cell clone. Different clones being in different stages of development generate the typical histological picture of fish testes, where the tubular compartment contains cysts of different sizes with groups of germ cells in different stages of spermatogenesis (see Figures 1, 2 and 3). Detailed histological studies in zebrafish (Danio rerio) have described two subtypes of type A undifferentiated spermatogonia in the testes, designated as Aund* and Aund [23]. It is not known if these two subtypes are separated by mitosis, or represent different stages of the same cell cycle, and moreover, there is no information on the spermatogonial stem cell niche in the zebrafish testis, or on the stemness of Aund* and Aund. In the current study, we identified the putative SSCs and their niche by identifying the LRCs in zebrafish testes and using a transgenic zebrafish expressing enhanced green fluorescent protein under the control of the germ cell specific vasa promoter (vasa::egfp) [25]. Furthermore, we evaluated the spatial relationship between blood vessels and the SSC niche, using testes expressing enhanced green fluorescent protein under the control of the endothelial cell specific fli promoter (fli::egfp) [26]. To confirm the biological activity (re-establishment of function and plasticity) of the potential SSCs, we developed a transplantation assay in zebrafish. Finally, we studied the recipient’s testicular microenvironment prior to transplantation as it might influence the behavior of transplanted SSCs. Materials and Methods Animals Sexually mature zebrafish males and females, and sexually mature transgenic zebrafish males expressing enhanced green fluorescent protein under the control of the germ cell-specific vasa promoter (vasa::egfp) [25] or the endothelial cell-specific fli promoter (fli::egfp) [26] were used. Animal housing and experi- mentation were consistent with Dutch and Brazilian national regulations and were approved by the Utrecht University and Federal University of Minas Gerais animal use and care committees, respectively. Topographical distribution of type A undifferentiated spermatogonia in zebrafish seminiferous tubules Testes from males (n = 5) were fixed in 4% buffered glutaral- dehyde at 4uC overnight, dehydrated, and embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany, http://www.kulzer- technik.de), sectioned and stained according to conventional histological procedures [23,27]. The topographical distribution of A spermatogonia was recorded by examining if type A undifferentiated spermatogonia (Aund* or Aund) were adjacent to SSC Niche and SSC In Zebrafish the interstitial compartment, or contacted one or more tubules (intertubule). The position of 500 type A undifferentiated spermatogonia was counted per animal and expressed as percentage of the total number evaluated; to determine if the distribution of these cells follows a random pattern, the tubular perimeters adjacent to the interstitium, or intertubule were measured using Image J software (National Institutes of Health, Bethesda, Maryland, USA, http://rsbweb.nih.gov/ij), and the values were expressed as percentage of the total tubular perimeter (n = 50 tubules/animal). Identification and quantification of label retaining cells (LRCs) in zebrafish testes To estimate the cell cycle duration of type A undifferentiated spermatogonia (Aund* and Aund), 10 males were exposed to BrdU (Sigma-Aldrich, St. Louis, MO, USA, http://www.sigmaaldrich. com) dissolved in water (4 mg/ml) for approximately 4 and 10 h. To identify the LRC population, 25 males were pulsed with BrdU dissolved in water (4 mg/ml) for 10 h/day during 3 consecutive days. Animals (n = 5) were sacrificed immediately after the third BrdU pulse, and after 4, 11, 18 and 25 days of chase. Testes were fixed, embedded, and sectioned as described above. BrdU incorporation was detected by immunohistochemistry as described previously [28]. The labeling index of both types of A undifferentiated spermatogonia (Aund* or Aund) was determined by counting the total number of labeled cells out of 300 cells (labeled and non-labeled) during the different periods after chase. After 18 and 25 days of chase, the spatial distribution of labeled Aund* or Aund (LRCs) was determined by counting the number of labeled cells situated near the interstitium, or in the intertubule, or near the testicular capsule. The values were expressed as percentage of positive-BrdU cells in the mentioned regions. Whole-mount analysis of vasa::egfp testes under confocal laser scanning microscopy (CLSM) and examination of fli::egfp testes Testes from transgenic vasa::egfp [25] or fli::egfp [26] zebrafish were fixed in 2% buffered paraformaldehyde for 2 h. For whole- mount examination, vasa::egfp testes were permeabilized with 0.2% PBT (0.2% Triton X-100 in PBS) for 10 min, and subsequently stained in DAPI (Invitrogen Molecular Probes, Carlsbad, CA, USA, www.invitrogen.com) for 5 min, followed by two rinses in PBS for 10 min. vasa::egfp testes (n = 5) were analyzed by CLSM 510 Meta (Zeiss, Jena, Germany, www.zeiss.de/lsm) using 358 nm and 488 nm as excitation wavelengths for DAPI and GFP, respectively. fli::egfp testes were frozen in Tissue-Tek (Sakura Finetek Europe B.V., Leiden, Netherlands, http://www.sakura.eu) and cryosectioned at 10 mm, permeabilized in 0.2% PBT for 10 min, stained with DAPI (Invitrogen) for 5 min, and mounted with a coverslip using an anti-fading Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA, http:// www.vectorlabs.com). Sections were examined under fluorescence microscopy using filters for DAPI and FITC visualization. Depletion of endogenous spermatogenesis in zebrafish male recipients for SSC transplantation To deplete endogenous spermatogenesis, we first examined the effect of different temperatures. Overall, the speed of spermato- genesis in teleost fish is significantly influenced by temperature [29]. Hence, males were kept in water at 20uC (n = 15), 27uC (n = 12), 30uC (n = 7), or 35uC (n = 12) for at least one week. Then, animals received one single intraperitoneal injection of 3H- thymidine (Amersham/GE Healthcare, Piscataway, NJ, USA, PLoS ONE | www.plosone.org 2 September 2010 | Volume 5 | Issue 9 | e12808