Androgens act through the nuclear androgen receptor (AR) to regulate gonad differentiation and development. In mice, AR is necessary for spermatogenesis, testis development, and formation of external genitalia in males and oocyte maturation in females. However, the extent to which these phenotypes are conserved in nonmammalian vertebrates is not well understood. Here, we generate zebrafish with a mutation in the ar gene (aruab105/105) and examine the role of AR in sexual determination and gonad development. We found that zebrafish AR regulates male sexual determination, because the majority of aruab105/105 mutant embryos developed ovaries and displayed female secondary sexual characteristics. The small percentage of mutants that developed testes displayed female secondary sexual characteristics, exhibited structurally disorganized testes, and were unable to release or produce normal levels of sperm, demonstrating that AR is necessary for zebrafish testis development and fertility. In females, we found that AR regulates oocyte maturation and fecundity. The aruab105/105 mutant females developed ovaries filled primarily with immature stage I oocytes and few mature stage III oocytes. Two genes whose expression is enriched in wild-type ovaries compared with testes (cyp19a1a, foxl2a) were upregulated in ar mutant testes, and two genes enriched in testes (amh, dmrt1) were upregulated in ar mutant ovaries. These findings demonstrate that AR regulates sexual determination, testis development, and oocyte maturation and suggest that AR regulates sexually dimorphic gene expression. The ar mutant we developed will be useful for modeling human endocrine function in zebrafish.

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The development of craniofacial skeletal structures requires well-orchestrated tissue interactions controlled by distinct molecular signals. Disruptions in normal function of these molecular signals have been associated with a wide range of craniofacial malformations. A pathway mediated by estrogens is one of those molecular signals that plays role in formation of bone and cartilage including craniofacial skeletogenesis. Studies in zebrafish have shown that while higher concentrations of 17-β estradiol (E2) cause severe craniofacial defects, treatment with lower concentrations result in subtle changes in head morphology characterized with shorter snouts and flatter faces. The molecular basis for these morphological changes, particularly the subtle skeletal effects mediated by lower E2 concentrations, remains unexplored. In the present study we address these effects at a molecular level by quantitative expression analysis of sets of candidate genes in developing heads of zebrafish larvae treated with two different E2 concentrations. To this end, we first validated three suitable reference genes, ppia2, rpl8 and tbp, to permit sensitive quantitative real-time PCR analysis. Next, we profiled the expression of 28 skeletogenesis-associated genes that potentially respond to estrogen signals and play role in craniofacial development. We found E2 mediated differential expression of genes involved in extracellular matrix (ECM) remodelling, mmp2/9/13, sparc and timp2a, as well as components of skeletogenic pathways, bmp2a, erf, ptch1/2, rankl, rarab and sfrp1a. Furthermore, we identified a co-expressed network of genes, including cpn1, dnajc3, esr1, lman1, rrbp1a, ssr1 and tram1 with a stronger inductive response to a lower dose of E2 during larval head development.

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