SSC Niche and SSC In Zebrafish Figure 5. SSC transplantation into male zebrafish recipients. A,B. Recipient testes after two weeks (2w) of transplantation analyzed by light (A) and fluorescence (B) microscopies. Seminiferous tubules are delimited by stippled lines. Arrows indicate the same area in A and B. Donor cells formed clusters situated near the interstitium in a similar way as observed in vasa::egfp testes (see arrowheads in the inset). Nuclei (blue) are stained with DAPI. Scale bars = 50 mm. C,D. CLSM analysis of recipient testes after two weeks (2w) of transplantation. Arrowheads indicate a donor-derived cyst composed of ,8 cells. Inset. High magnification of donor-derived cyst. Nuclei (blue) are stained with DAPI. E,F. Recipient testes after 3 weeks (3w) of transplantation analyzed under CLSM. Donor-derived cysts increased their number and size, being found at different stages of spermatogenesis. Type A undifferentiated spermatogonia (Aund*/Aund), type A differentiating spermatogonia (Adiff), type B spermatogonia (B), and spermatocytes (SC). Inset shows a high magnification of type A undifferentiated spermatogonia. Nuclei (blue) are stained with DAPI. doi:10.1371/journal.pone.0012808.g005 of two types of template DNA strand segregation, one random type, possibly reflecting the rapid loss of the label up to the 18th day of chase, and one non-random type (asymmetrical division), which could explain the retention of BrdU after 18 and 25 days of chase. We speculate that the asymmetrical division of Aund might prevent its exhaustion during spermatogenesis, maintaining, therefore, a subset of immature Aund. An asymmetrical division of type A undifferentiated spermatogonia was suggested previously in zebrafish based on morphological evidence [2]. The LRC assay also revealed the precise location of stem cells, and consequently the characterization of the neighboring cells that form the stem cell niche [46–48]. As in rodents [6,7], we found that slow-cycling BrdU-labeled type A undifferentiated spermato- gonia (Aund* and Aund) were preferentially situated in regions of the seminiferous tubules adjacent to the interstitial compartment. This observation suggests that elements of the SSC niche are probably conserved across vertebrates. The preferential location of SSC close to the interstitial compartment in rodents may be related to a locally high concentration of androgens which inhibit spermato- gonial differentiation [49]. 11-KT is the main androgen in zebrafish [37] and other teleost fish, and is able to support full spermatogenesis in testis tissue culture of juvenile Japanese eel (Anguilla japonica) [50] or adult zebrafish [38]. Moreover, induction of androgen insufficiency in adult male zebrafish inhibited the differentiation of type A to type B spermatogonia [51]. Although the possible role(s) of androgen signaling in regulating zebrafish PLoS ONE | www.plosone.org 10 September 2010 | Volume 5 | Issue 9 | e12808