Thyroid hormones participate in regulating growth and homeostatic processes in vertebrates, including development and adult functioning of the reproductive system. Here we report a new stimulatory role of thyroid hormone on the proliferation of Sertoli cells (SCs) and single, type A undifferentiated spermatogonia (A(und)) in adult zebrafish testes. A role for T-3 in zebrafish testis is suggested by in situ hybridization studies, which localized thyroid receptor alpha (thr alpha) in SCs and the beta (thr beta) mRNA in Sertoli and Leydig cells. Using a primary zebrafish testis tissue culture system, the effect of T-3 on steroid release, spermatogenesis, and the expression of selected genes was evaluated. Basal steroid release and Leydig cell gene expression did not change in response to T-3. However, in the presence of FSH, T-3 potentiated gonadotropin-stimulated androgen release as well as androgen receptor (ar) and 17 alpha-hydroxylase/17,20 lyase (cyp17a1) gene expression. Moreover, T-3 alone stimulated the proliferation of both SCs and A(und), potentially resulting in newly formed spermatogonial cysts. Additional tissue culture studies demonstrated that Igf3, a new, gonad-specific member of the IGF family, mediated the stimulatory effect of T-3 on the proliferation of A(und) and SCs. Finally, T-3 induced changes in connexin 43 mRNA levels in the testis, a known T-3-responsive gene. Taken together, our studies suggest that T-3 expands the population of SCs and A(und) involving Igf signaling and potentiates gonadotropin-stimulated testicular androgen production as well as androgen sensitivity.Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)European Union LIFE CYCLE projectConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Univ Fed Minas Gerais, Lab Cellular Biol, Dept Morphol, Inst Biol Sci, Belo Horizonte, MG, BrazilUniv Utrecht, Dept Biol, Reprod Biol Grp, Div Dev Biol,Fac Sci, NL-3584 CH Utrecht, NetherlandsState Univ Sao Paulo, Dept Morphol, Botucatu, SP, BrazilState Univ Sao Paulo, Dept Morphol, Botucatu, SP, BrazilEuropean Union LIFE CYCLE projectFP7/222719