SSC Niche and SSC In Zebrafish Figure 2. Label retaining cell (LRC) approach in zebrafish testes. A. BrdU labeling index of type Aund* and Aund during BrdU exposure (4, 10, and 30 h) and after 4, 11, 18 and 25 days of chase. Bars represent the percentage mean 6 SE (n = 5); different letters denote significant differences (p,0.05) in time. B. Distribution of BrdU-positive Aund* and Aund spermatogonia after 18 and 25 days of chase. Bars represent the mean 6 SE which are expressed as percentage, and different letters indicate significant differences (p,0.05) among the groups. C–H. BrdU immunodetection with PAS staining after 4 (C–E), 11 (F), 18 (G), and 25 (H) days of chase. Note that BrdU immunostaining is diluted as a consequence of the progression of spermatogenesis. Only the slow-cycling cells (stem cells candidates) are able to retain the BrdU label for long periods of time. LRCs are indicated by arrows. Some of LRC are near to blood vessels (arrowhead) (E). Insets are high magnification of the LRCs (type A undifferentiated spermatogonia). The interstitium is delimited by spotted lines. Scale bars = 10 mm. doi:10.1371/journal.pone.0012808.g002 spermatogenesis. Then, 60 zebrafish males were kept at 35uC for one week, and received a single intraperitoneal dose of 30 or 40 mg/Kg/BW of busulfan (Sigma). Testes from males (n = 6) were sampled 2, 4, 6, 10 and 12 days after the injection, weighed, fixed, embedded, sectioned and stained as above. As control group, males (n = 30) received a single intraperitoneal injection of dimethyl sulfoxide and sampled at the same reported periods. To evaluate the optimal dose and the best window of depletion, frequency of spermatogenic cysts, germ cell apoptosis, and Sertoli cell only phenotype were determined for each sampled period. Results were expressed as percentage of the total number of counted structures. To address the composition of type A undifferentiated spermatogonia (Aund* or Aund) in busulfan- depleted testes (n = 10), the percentage of both subtypes was determined by counting the number of Aund* and Aund out of 100– 200 type A undifferentiated spermatogonia in each male. As control, the percentage of both subtypes was also determined in normal testes (n = 7). Preparation of zebrafish female recipients for SSC transplantation Ovoposition was induced according to usual procedures (http://zfin.org). To evaluate if females after ovoposition may be suitable recipients for SSC transplantation, ovaries (n = 5) were fixed, embedded, and sectioned as above, and stained with PAS to determine the number of postovulatory follicles (POFs)/mm2 of tissue. POFs consisting for a great part of granulosa cells remaining after ovulation were considered as a space potentially available for transplanted SSCs. The number of POFs/mm2 from preovulatory females was also evaluated as a control. Donor cell preparation and SSC transplantation into male and female zebrafish recipients Testes from males (n = 10) were digested with 0.2% collagenase and 0.12% dispase [30]. The obtained cell suspension was immediately submitted to FACS (Fluorescence Activated Cell Sorting) using an inFlux cell sorter (BD Bioscience, San Jose, CA, USA, www.bdbiosciences.com). Vasa is highly expressed in type A undifferentiated spermatogonia, but expression decreases during meiosis and spermiogenesis [23]. Since type A undifferentiated spermatogonia are the largest germ cell type in zebrafish testes (,10 mm nuclear diameter [23]), FACS settings were adjusted to sort a cell population displaying large size and high intensity of fluorescence, which should enrich type A undifferentiated spermatogonia. To validate the enrichment, the unsorted and sorted cell fractions obtained by FACS was analyzed under fluorescence microscopy, or fixed, embedded, sectioned, and stained as above to determine the percentage of germ cell type. For SSC transplantation, the FACS-enriched cell fraction was suspended in L-15 medium (Sigma), 5% trypan blue and 10% calf serum. SSCs were transplanted into testes or ovaries through the genital pores, using a glass capillary needle coupled to a peristaltic pump (Figure S2). To optimize this procedure, zebrafish male and female genital pores were analyzed morphologically (diameter and angle) to adjust the settings for the glass capillary needle (Figure S2). The transplantation route was standardized and tested by injecting trypan blue (Figure S2). SSC transplantation analysis Recipients were sacrificed 2 and 3 weeks (males) or 3 and 4 weeks (females) after transplantation. The gonads were fixed in 2% buffered paraformaldehyde for 2 h, permeabilized, and stained with DAPI before analysis by CLSM, as described above. As positive control, Vasa protein expression was examined by GFP immunodection in vasa::egfp gonad sections or by Vasa immuno- cytochemistry in wild-type males [31]. The PCR detection of gfp DNA from donor-derived germ cells in male and female recipients was carried out as described previously [32]. 11-Ketotestosterone (11-KT) plasma levels in busulfan- depleted male zebrafish and 11-KT release by depleted testes in vitro Males (n = 19) were sampled 10 days after busulfan (40 mg/kg) treatment. A blood sample was collected for quantification of 11- ketotestosterone (11-KT) plasma levels, as described previously [33]. As controls, 11-KT plasma levels were also quantified in zebrafish kept at 27uC (n = 7), or at 35uC (n = 11). In other experiments, carried out at the same time, but published separately [33], 11-KT plasma levels were measured 2 h after a single injection of recombinant zebrafish Fsh or hCG. Results are expressed as ng 11-KT/ml of plasma. To evaluate testicular 11- KT release in tissue culture, testes were collected from adult zebrafish kept at 27uC (control) (n = 7), or from busulfan-treated zebrafish (n = 7). The two testes of a given fish were incubated in parallel, such that one of them (randomly chosen left or right) served as control (basal) for the contralateral one, which was incubated in the presence of 1 mM of the adenylate cyclase activator forskolin [33]. After incubation the medium was processed for the quantification of 11-KT [34]. Results were expressed as ng 11-KT/mg of tissue. Gene expression in spermatogenesis-depleted testes in busulfan-treated zebrafish Testes from males kept at 27uC (control) (n = 7), or at 35uC (n = 5), or treated with busulfan (n = 12) were snap frozen in liquid nitrogen and stored at 280uC until RNA extraction. Total RNA was extracted from testes using the RNAqueousH-Micro Kit (Ambion, Austin, TX, USA, http://www.ambion.com). Further processing to determine the threshold cycle (Cq) values of the reference endogenous control gene elongation factor 1-alpha (ef1a) and b-actin1, as well as of insulin-like 3 (insl3) [35], steroidogenic acute regulatory protein (star), and cytochrome P450, family 17, subfamily A, polypeptide 1 (cyp17a1), androgen receptor (ar), anti-Mu¨llerian hormone (amh), gonadal soma-derived growth factor (gsdf), insulin growth factor 1a (igf1a) and 1b (igf1b) [36], and germ cell genes piwil1 (spermato- gonia), and synaptonemal complex protein 3 (sycp3l) (spermatocytes) by qPCR analysis was performed as reported [33,37,38]. No significant differences (P.0.05) were found among the mean b- PLoS ONE | www.plosone.org 5 September 2010 | Volume 5 | Issue 9 | e12808