SSC Niche and SSC In Zebrafish t-test). C. Relative mRNA levels of gsdf, star, amh, insl3, cyp17a, igf1a, igf1b, piwil1 and sycp3l from control (27uC) (n = 7), 35uC (n = 4), and busulfan- depleted testes [35uC + Bu (40 mg/Kg/BW)] (n = 12). Bars represent the mean6SE of relative mRNA levels normalized to b-actin1 or ef1a, and expressed as fold of relative control (27uC) mRNA levels. * and # are significantly different (p,0.05) from control levels (Student unpaired t-test) and among the groups (ANOVA), respectively. doi:10.1371/journal.pone.0012808.g007 germ cells. Another important application of germ cell transplan- tation is transgenesis through the male germ line using transplantation of transfected germ cells [3,19]. This might be an option to create transgenic animals in a shorter time than is possible with conventional methods [19]. Finally, we have demonstrated the plasticity of the transplanted spermatogonia by placing the SSC candidates into a different microenvironment, i.e. into an ovary. SSC candidates were able to colonize recipient ovaries and differentiate into female germ line cells. We present in the current work characteristics of zebrafish SSC candidates (Aund* and Aund), their niche, and their functional capability to both self-renew and differentiate in recipient testes, as well as their remarkable plasticity in recipient ovaries. To our knowledge, this is the first work to report on a SSC niche in fish. As in rodents, SSC location in zebrafish was characterized within areas of the seminiferous tubules which are opposite to the interstitial compartment, suggesting the influence of interstitial elements on SSC self-renewal and maintenance. We developed and standardized SSC transplantation techniques in male and female zebrafish, showing donor-derived spermatogenesis and male-derived oocytes, respectively, after transplantation. Thus, we introduced SSC transplantation in zebrafish as a promising technique for the study of SSCs. Supporting Information Figure S1 Effects of different temperatures on zebrafish spermatogenesis. A. More advanced labeled germ cell after 2 h, 12 h, 1d, 2d, 3d, 4d, 5d, 6d of 3H-thymidine injection in zebrafish males kept at 20uC, 27uC, 30uC, and 35uC. Leptotene/zygotene (L/Z) and pachytene (P) spermatocytes, metaphase I (MI), initial spermatids (E1), intermediate spermatids (E2), final spermatids (E3) and spermatozoa (Z). Scale bars = 10 mm. Cells were considered labeled when four to five or more grains were present over the nucleus in the presence of low-to-moderate background (i.e., very few grains per histological field observed under oil immersion). Black squares indicate the time in which labeled spermatozoa were found in the lumen of zebrafish testes at different temperatures. B. Histogram showing the combined duration of meiotic and spermiogenic phases at different temperatures. Spermatogenesis did not progress beyond the first meiotic division at 35uC. X axis represents the time, whereas y axis the 3H-thymidine labeled germ cell [Leptotene/zygotene (L/Z), pachytene (P) and diplotene (D) spermatocytes, metaphase I (MI), initial spermatids (E1), intermediate spermatids (E2), final spermatids (E3) and spermatozoa (Z)]. C–E. Histological sections of zebrafish testes at 27uC, 30uC, and 35uC. Sperm free and a massive germ cell apoptosis is seen in zebrafish seminiferous tubules at 35uC. Scale bars = 50 mm. Found at: doi:10.1371/journal.pone.0012808.s001 (4.05 MB TIF) Figure S2 Standardization of SSC transplantation techniques. A. Transplantation device, in which a glass capillary needle is coupled to a peristaltic pump. B,C. Standardization of transplan- tation via using trypan blue to monitor the efficiency of the injections. Note trypan blue inside the testis (B) and ovaries (C). D,E. Genital pore histological sections stained with toluidine blue (male - D, female - E). Genital pore measurements: 120680 mm dimensions, 63u angle (female); 75650 mm dimensions, 60u angle (male). Anus (A), genital pore (G, arrow), and urethra (U). Scale bars = 100 mm. F. Ovary cell transplantation throughout injections into the lateral body wall (indicated by stippled line). G. Trypan blue was used to monitor the specificity of the injection. Note trypan blue inside the ovaries. Found at: doi:10.1371/journal.pone.0012808.s002 (2.98 MB TIF) Figure S3 Scatter plot to check the stability of 18S rRNA, b- actin1 and ef1a mRNAs as a housekeeping gene in 35uC (n = 4), and depleted testes (35uC + busulfan 40 mg/Kg/BW) (n = 12). Each dot in the scatter plot represents the average Cq-value of duplicate measurements for each fish in the different experimental condition. Stability was seen only between b-actin1 and ef1a. Control (27uC) was not shown, but followed the same pattern. Found at: doi:10.1371/journal.pone.0012808.s003 (0.26 MB TIF) Figure S4 The hypothetical SSC niche in zebrafish testes. SSC niche is indicated by a red circled line. The niche is constituted by elements of the tubular and interstitial compartments (IC) such as: Sertoli cells (SE); basement membrane (BM); peritubular myoid cells (PM); Leydig cells (LE), blood vessels (BV); and other interstitial elements. Type A undifferentiated spermatogonia (Aund*/Aund), type A differentiated spermatogonia (Adiff), type B early spermatogonia (B early) and type B late spermatogonia (B late) and spermatozoa (SZ) are illustrated. Found at: doi:10.1371/journal.pone.0012808.s004 (2.57 MB TIF) Figure S5 Preparation of female zebrafish recipients for SSC transplantation. A–C. Ovaries histological sections of ovoposition- induced females stained with PAS (Periodic acid Schiff). Arrows indicate postovulatory follicles after ovoposition. Note in C the available follicle cells which can support the transplanted SSC development. Scales bars = 250 mm (A), 100 mm (B), 25 mm (C). D. Number of postovulatory follicles (POFs)/mm2 of ovary in control (n = 5) and ovopositioned females (n = 5). Bars represent the mean6 SE. * means significant differences (p,0.05) between the groups (Student unpaired t-test). Found at: doi:10.1371/journal.pone.0012808.s005 (1.53 MB TIF) Figure S6 Vasa expression pattern during male germ cell development using gfp immunodetection in sections of vasa::egfp testes (A), or vasa immunodetection in cryosections of wild-type testes (B). Type A undifferentiated spermatogonia (Aund*/Aund), type A differentiating spermatogonia (Adiff), type B spermatogonia (B), spermatocytes (SC), spermatids (ST), spermatozoa (SZ). Scale bars = 25 mm (A) and 10 mm (B). Found at: doi:10.1371/journal.pone.0012808.s006 (2.83 MB TIF) Figure S7 PCR analysis for GFP detection using primers located in the YFP gene, 2% agarose gel containing ethidium bromide, showing the detection of a faint background band. A. vasa::egfp testes were used as a positive control. Transplanted male after 3 weeks of transplantation, transplanted female after 1 month of transplantation, H2O water as negative control. Bands at left side are DNA markers from SMART ladder (Eurogentec). B. PCR detection using primers located in the PD-ECGF (plated-derived endothelial cell growth factor) gene as positive control for genomic DNA in the different individuals, 2% agarose gel containing ethidium bromide, showing the detection of a faint background band. Bands at left side are DNA markers from SMART ladder. PLoS ONE | www.plosone.org 14 September 2010 | Volume 5 | Issue 9 | e12808