SSC Niche and SSC In Zebrafish spotted lines). E is a high magnification of the square in D. Scale bars = 10 mm. F–I: Cryosections of fli::egfp testes stained with DAPI (nuclear staining) and analyzed under fluorescence microscopy. The arrow in F shows a blood vessel (green) surrounding the circumference of a seminiferous tubule (ST). Interstitium (delimited with red dotted lines), Leydig cells (LE) and group of type A spermatogonia (asterisks) are shown in G. H,I. Aund*/Aund are near the endothelial cell (EC) nuclei (n). Note a metaphase figure (M) in H. Nucleolus (nu) of type A undifferentiated spermatogonia is shown in I. Compare the similar morphology of Aund* (inset) with the cells found near the endothelial cell. Scale bars = 10 mm. doi:10.1371/journal.pone.0012808.g003 actin1 and ef1a Cq values in the different groups (Figure S3) thus validating b-actin1 and ef1a as suitable references for the current experiments. Then, relative mRNA levels of the selected genes were normalized to b-actin1 and ef1a, and expressed as fold of relative control (27uC) mRNA levels. Nomenclature of zebrafish proteins, mRNAs and genes are according to ZFIN (http://zfin. org) rules. Statistical analysis Significant differences between two groups were identified using Student test (paired and unpaired) (P,0.05). Comparisons of more than two groups were performed with one-way ANOVA followed by Student-Newman-Keuls test (P,0.05). Graph Pad Prism 4.0 (Graph Pad Software, Inc., San Diego, CA, USA, http://www. graphpad.com) was used for all statistical analysis. Results Characterization of SSC candidates and their niche in zebrafish testes Analyzing the topographical distribution of type A spermatogo- nia showed that 75% of these cells are situated adjacent to the interstitial compartment (Figure 1A,C,D) although this compart- ment represents only 1/3rd of the total perimeter of the spermatogenic tubules (Figure 1B). Quantification of the mitotic index of these cells was achieved by determining the BrdU labeling index during three successive 10 h long exposures to BrdU during three consecutive days. The results suggested that both subtypes of type A undifferentiated spermatogonia (Aund* and Aund) have a long cell cycle lasting at least 10 h but less than 30 h (Figure 2A). To confirm the existence of slow-cycling cells (stem cell candidates), testes were examined for BrdU-positive type Aund* and Aund spermatogonia at different periods (up to 25 days) after the final BrdU exposure; putative stem cells are considered to be part of the LRC population. During this chase period, two distinct patterns of BrdU staining were found among the two subtypes of type A spermatogonia (Figure 2A). Aund rapidly lost the label until the 18th day of chase, when the labeling index stabilized at ,10% for this cell type (Figure 2A). On the other hand, the percentage of BrdU- positive type Aund* spermatogonia varied in a statistically not significant manner around 30% during the complete chase period (Figure 2A). Apart from some somatic elements, type A undiffer- entiated spermatogonia (Aund* and Aund) were the only BrdU retaining germ cells after 18 and 25 days of chase. Intriguingly, most of the labeled spermatogonia (71% of Aund* and 80% of Aund) were situated adjacent to the interstitium (Figure 2B,C–H). A similar pattern was found in vasa::egfp testes examined under CLSM where the strongest expression of vasa was observed adjacent to the interstitium (Figure 3 and Video S1). To further study the spatial relation between the vasculature and undifferentiated spermatogo- nia, testes of transgenic zebrafish expressing GFP in endothelial cells (fli::egfp) were analyzed. Capillaries surround the seminiferous tubules (Figure 3F), and type A undifferentiated spermatogonia were often found in close association with endothelial cells (Figures 2E-inset; 3G–I). A combination of the above results is illustrated schematically in Figure S4, showing a hypothetical spermatogonial stem cell niche in zebrafish. Preparation of male and female recipients for SSC transplantation Zebrafish were exposed to different temperatures to optimize the treatment with busulfan (Figure 4A, Figure S1). Higher temperatures accelerated spermatogenesis (Figure S1), whereas at 35uC, spermatogenesis did not progress beyond metaphase I and showed abnormalities, such as massive apoptosis in particular amongst spermatocytes, absence of sperm, and a significant decrease in the GSI (Figure 4A,E; Figure S1). Both doses of busulfan (30 or 40 mg/Kg/BW) tested at 35uC induced further and a more general germ cell apoptosis (Figure 4D–H) and a progressive GSI decrease, which reached its lowest value 10 days after injection (Figure 4B,C). However, only the higher dose suppressed efficiently endogenous spermatogenesis, resulting in 88% of the spermatogenic tubules showing a Sertoli cell only appearance, i.e. all germ cells were missing (Figure 4D,I). Analyzing the composition of type A spermatogonia in the 12% of tubules where some germ cells remained and comparing this to normal testes demonstrated that the ratio between Aund* and Aund shifted from 1:1 to 1:3 in busulfan-depleted testes (Figure 1E). The preponderance of Aund might be associated with the observation that 12 days after injection, most tubules showed the first type B spermatogonia and/or spermatocytes again, i.e. showed a fast recovery of endogenous spermatogenesis after treatment with 40 mg/Kg/BW of busulfan at 35uC (Figure 4D). To prepare female zebrafish recipients for SSC transplantation, ovoposition was induced; a pilot study indicated a high mortality among females when applying the combination of high temperature and busulfan. The increased number of POFs showed that ovoposition created spaces and ‘‘free’’ somatic cells (follicle cells from POFs), potentially suitable for receiving injected germ cells, and to support transplanted SSCs development, respectively (Figure S5). Donor cell isolation and male and female transplantation Using vasa::egfp transgenic zebrafish as donors, a testicular cell suspension was obtained and subsequently submitted to FACS, in order to enrich transplantable SSC (Figure 4J,K) by sorting for big cells carrying high fluorescence (Figure 4L,M). Indeed, after sorting, we observed an enrichment of type A undifferentiated spermatogonia (Figure 4K–N). The sorted cells (8.10324.104 cells/ml) were injected through the genital pore (Figure 4O, Figure S2). Since females have a prominent belly, direct injections into ovaries (via lateral body wall) were also successfully performed (Figure S2). After transplantation, zebrafish recipient males and females were placed in water of 27uC. Male and female transplantation analysis After two weeks of transplantation, donor cells colonized the recipient’s seminiferous epithelium, and formed clusters, which were situated near the interstitial compartment (Figure 5A,B). Using CLSM, we found that these clusters were composed of ,8 cells/cyst (Figure 5C,D; Video S2). Three weeks after transplantation, donor- derived cysts had increased in number and size, and were found at different stages of spermatogenesis (e.g. differentiating type A spermatogonia, type B spermatogonia and spermatocytes) along the recipient’s seminiferous epithelium (Figure 5E,F; Video S3). Donor type A undifferentiated spermatogonia (Aund* and Aund) were also PLoS ONE | www.plosone.org 7 September 2010 | Volume 5 | Issue 9 | e12808