Found at: doi:10.1371/journal.pone.0012808.s007 (0.82 MB TIF) Video S1 Z stack from vasa::egfp testis showing the distribution of vasa positive cells in the zebrafish seminiferous tubules. Found at: doi:10.1371/journal.pone.0012808.s008 (1.63 MB WMV) Video S2 Z stack from wild-type zebrafish testis 2 weeks after SSC transplantation. Note a donor-derived cyst composed of ,8 cells in the seminiferous epithelium. Found at: doi:10.1371/journal.pone.0012808.s009 (0.83 MB WMV) Video S3 Z stack from wild-type zebrafish testis 3 weeks after SSC transplantation. SSCs were able to colonize recipient testis and differentiate into daughter cells committed with the spermatogenic process. Donor-derived cysts at different stages of zebrafish spermatogenesis were found in recipient seminiferous epithelium. Found at: doi:10.1371/journal.pone.0012808.s010 (0.81 MB WMV) Video S4 Z stack from ovaries fragments 3 weeks after SSC transplantation into female zebrafish ovaries. Small cell clusters- derived from SSC were seen in female ovaries. References 1. Hess RA, Franc¸a LR (2008) Spermatogenesis and cycle of the seminiferous epithelium. Adv Exp Med Biol 636: 1–15. 10.1007/978-0-387-09597-4_1. 2. Schulz RW, Franc¸a LR, Lareyre JJ, LeGac F, Chiarini-Garcia H, et al. (2010) Spermatogenesis in fish. Gen Comp Endocrinol 165(3): 390–411. 10.1016/ j.ygcen.2009.02.013. 3. Oatley JM, Brinster RL (2008) Regulation of spermatogonial stem cell self- renewal in mammals. Annu Rev Cell Dev Biol 24: 263–286. 10.1146/ annurev.cellbio.24.110707.175355. 4. de Rooij DG (2006) Regulation of spermatogonial stem cell behavior in vivo and in vitro. Anim Reprod v.3(n.2): 130–134. 5. de Rooij DG (2009) The spermatogonial stem cell niche. Microsc Res Tech 72(8): 580–585. 10.1002/jemt.20699. 6. Chiarini-Garcia H, Hornick JR, Griswold MD, Russell LD (2001) Distribution of type A spermatogonia in the mouse is not random. Biol Reprod 65(4): 1179–1185. 7. Chiarini-Garcia H, Raymer AM, Russell LD (2003) Non-random distribution of spermatogonia in rats: Evidence of niches in the seminiferous tubules. Reproduction 126(5): 669–680. 8. Yoshida S, Sukeno M, Nabeshima Y (2007) A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis. Science 317(5845): 1722–1726. 10.1126/science.1144885. 9. Meistrich ML, Shetty G (2003) Suppression of testosterone stimulates recovery of spermatogenesis after cancer treatment. Int J Androl 26(3): 141–146. 10. Oatley JM, Oatley MJ, Avarbock MR, Tobias JW, Brinster RL (2009) Colony stimulating factor 1 is an extrinsic stimulator of mouse spermatogonial stem cell self-renewal. Development 136(7): 1191–1199. 10.1242/dev.032243. 11. Hofmann MC (2008) Gdnf signaling pathways within the mammalian spermatogonial stem cell niche. Mol Cell Endocrinol 288(1-2): 95–103. 10.1016/j.mce.2008.04.012. 12. Simon L, Ekman GC, Tyagi G, Hess RA, Murphy KM, et al. (2007) Common and distinct factors regulate expression of mRNA for ETV5 and GDNF, Sertoli cell proteins essential for spermatogonial stem cell maintenance. Exp Cell Res 313(14): 3090–3099. 10.1016/j.yexcr.2007.05.002. 13. Braun KM, Niemann C, Jensen UB, Sundberg JP, Silva-Vargas V, et al. (2003) Manipulation of stem cell proliferation and lineage commitment: Visualisation of label-retaining cells in wholemounts of mouse epidermis. Development 130(21): 5241–5255. 10.1242/dev.00703. 14. Brinster RL, Zimmermann JW (1994) Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci U S A 91(24): 11298–11302. 15. Brinster RL, Avarbock MR (1994) Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci U S A 91(24): 11303–11307. 16. Lacerda SM, Batlouni SR, Costa GM, Segatelli TM, Quirino BR, et al. (2010) A new and fast technique to generate offspring after germ cells transplantation in adult fish: The nile tilapia (Oreochromis niloticus) model. PLoS One 5(5): e10740. 10.1371/journal.pone.0010740. 17. Majhi SK, Hattori RS, Yokota M, Watanabe S, Strussmann CA (2009) Germ cell transplantation using sexually competent fish: An approach for rapid propagation of endangered and valuable germlines. PLoS One 4(7): e6132. 10.1371/journal.pone.0006132. SSC Niche and SSC In Zebrafish Found WMV) at: doi:10.1371/journal.pone.0012808.s011 (1.10 MB Video S5 Z stack from ovaries fragments 1 month after SSC transplantation. SSCs were able to colonize female ovaries and differentiate into female germ cell line. Note a male-derived oocyte in the female recipient ovary. Found at: doi:10.1371/journal.pone.0012808.s012 (0.60 MB WMV) Acknowledgments The authors would like to acknowledge Wytske van Dijk for technical assistance during radio immuno assays (RIAs); Ger Arkesteijn for technical assistance with FACS; Henk Schriek and Co Rootselaar for assistance during the in vivo experiments and maintaining the zebrafish stocks; and Teresa Bowman for the donation of fli::egfp transgenic zebrafish. Author Contributions Conceived and designed the experiments: RHN CDG JB LRdF RWS. Performed the experiments: RHN CDG HvdK. Analyzed the data: RHN CDG HvdK JB LRdF RWS. Contributed reagents/materials/analysis tools: RHN LRdF RWS. Wrote the paper: RHN JB LRdF RWS. Molecular biology, primers design, qPCR, PCR: JB. 18. McLean DJ (2005) Spermatogonial stem cell transplantation and testicular function. Cell Tissue Res 322(1): 21–31. 10.1007/s00441-005-0009-z. 19. Dobrinski I (2006) Transplantation of germ cells and testis tissue to study mammalian spermatogenesis. Anim Reprod v.3(n.2): 135–145. 20. Krawetz SA, de Rooij DG, Hedger MP (2009) Molecular aspects of male fertility. international workshop on molecular andrology. EMBO Rep 10(10): 1087–1092. 10.1038/embor.2009.211. 21. Wu X, Schmidt JA, Avarbock MR, Tobias JW, Carlson CA, et al. (2009) Prepubertal human spermatogonia and mouse gonocytes share conserved gene expression of germline stem cell regulatory molecules. Proc Natl Acad Sci U S A 106(51): 21672–21677. 10.1073/pnas.0912432106. 22. Sadri-Ardekani H, Mizrak SC, van Daalen SK, Korver CM, Roepers- Gajadien HL, et al. (2009) Propagation of human spermatogonial stem cells in vitro. JAMA 302(19): 2127–2134. 10.1001/jama.2009.1689. 23. Leal MC, Cardoso ER, No´brega RH, Batlouni SR, Bogerd J, et al. (2009) Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations. Biol Reprod 81(1): 177–187. 10.1095/biolreprod.109.076299. 24. Schulz RW, Menting S, Bogerd J, Franc¸a LR, Vilela DA, et al. (2005) Sertoli cell proliferation in the adult testis—evidence from two fish species belonging to different orders. Biol Reprod 73(5): 891–898. 10.1095/biolreprod.105.039891. 25. Krovel AV, Olsen LC (2002) Expression of a vas::EGFP transgene in primordial germ cells of the zebrafish. Mech Dev 116(1-2): 141–150. 26. Lawson ND, Weinstein BM (2002) In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol 248(2): 307–318. 27. Quintero-Hunter I, Grier H, Muscato M (1991) Enhancement of histological detail using metanil yellow as counterstain in periodic acid schiff’s hematoxylin staining of glycol methacrylate tissue sections. Biotech Histochem 66(4): 169–172. 28. van de Kant HJ, de Rooij DG (1992) Periodic acid incubation can replace hydrochloric acid hydrolysis and trypsin digestion in immunogold—silver staining of bromodeoxyuridine incorporation in plastic sections and allows the PAS reaction. Histochem J 24(3): 170–175. 29. No´brega RH, Batlouni SR, Franc¸a LR (2009) An overview of functional and stereological evaluation of spermatogenesis and germ cell transplantation in fish. Fish Physiol Biochem 35(1): 197–206. 10.1007/s10695-008-9252-z. 30. Sakai N (2006) In vitro male germ cell cultures of zebrafish. Methods 39(3): 239–245. 10.1016/j.ymeth.2005.12.008. 31. Braat AK, van de Water S, Goos H, Bogerd J, Zivkovic D (2000) Vasa protein expression and localization in the zebrafish. Mech Dev 95(1-2): 271–274. 32. Rehbein H, Bogerd J (2007) Identification of genetically modified zebrafish (Danio rerio) by protein- and DNA-analysis. Journal fu¨r Verbraucherschutz und Lebensmittelsicherheit 2(2): 122–125. Available: http://dx.doi.org/10.1007/ s00003-007-0179-6 via the Internet.. 33. Garc´ıa-Lo´pez A, de Jonge H, No´brega RH, de Waal PP, van Dijk W, et al. (2010) Studies in zebrafish reveal unusual cellular expression patterns of gonadotropin receptor messenger ribonucleic acids in the testis and unexpected functional differentiation of the gonadotropins. Endocrinology 151(5): 2349–2360. 10.1210/en.2009-1227. 34. Schulz RW, van der Sanden MC, Bosma PT, Goos HJ (1994) Effects of gonadotrophin-releasing hormone during the pubertal development of the male PLoS ONE | www.plosone.org 15 September 2010 | Volume 5 | Issue 9 | e12808