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Estrogens play fundamental roles in regulating reproductive activities and they act through estrogen receptor ( ESR) in all vertebrates. Most vertebrates have two ESR subtypes ( ESR1 and ESR2), whereas teleost fish have at least three (Esr1, Esr2a and Esr2b). Intricate functionalization has been suggested among the Esr subtypes, but to date, distinct roles of Esr have been characterized in only a limited number of species. Study of loss-of-function in animal models is a powerful tool for application to understanding vertebrate reproductive biology. In the current study, we established esr1 knockout ( KO) medaka using a TALEN approach and examined the effects of Esr1 ablation. Unexpectedly, esr1 KO medaka did not show any significant defects in their gonadal development or in their sexual characteristics. Neither male or female esr1 KO medaka exhibited any significant changes in sexual differentiation or reproductive activity compared with wild type controls. Interestingly, however, estrogen-induced vitellogenin gene expression, an estrogen-responsive biomarker in fish, was limited in the liver of esr1 KO males. Our findings, in contrast to mammals, indicate that Esr1 is dispensable for normal development and reproduction in medaka. We thus provide an evidence for estrogen receptor functionalization between mammals and fish. Our findings will also benefit interpretation of studies into the toxicological effects of estrogenic chemicals in fish. [ABSTRACT FROM AUTHOR]

ABSTRACT Daphnia magna has been used extensively to evaluate organism- and population-level responses to pollutants in acute toxicity and reproductive toxicity tests. We have previously reported that exposure to juvenile hormone (JH) agonists results in a reduction of reproductive function and production of male offspring in a cyclic parthenogenesis, D. magna. Recent advances in molecular techniques have provided tools to understand better the responses to pollutants in aquatic organisms, including D. magna. DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to JH agonists: methoprene (125, 250 and 500 ppb), fenoxycarb (0.5, 1 and 2 ppb) and epofenonane (50, 100 and 200 ppb). Exposure to these JH analogs resulted in chemical-specific patterns of gene expression. The heat map analyses based on hierarchical clustering revealed a similar pattern between treatments with a high dose of methoprene and with epofenonane. In contrast, treatment with low to middle doses of methoprene resulted in similar profiles to fenoxycarb treatments. Hemoglobin and JH epoxide hydrolase genes were clustered as JH-responsive genes. These data suggest that fenoxycarb has high activity as a JH agonist, methoprene shows high toxicity and epofenonane works through a different mechanism compared with other JH analogs, agreeing with data of previously reported toxicity tests. In conclusion, D. magna DNA microarray is useful for the classification of JH analogs and identification of JH-responsive genes. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: The gene doublesex (dsx) is known as a key factor regulating genetic sex determination in many organisms. We previously identified two dsx genes (DapmaDsx1 and DapmaDsx2) from a freshwater branchiopod crustacean, Daphnia magna, which are expressed in males but not in females. D. magna produces males by parthenogenesis in response to environmental cues (environmental sex determination) and we showed that DapmaDsx1 expression during embryonic stages is responsible for the male trait development. The D. magna dsx genes are thought to have arisen by a cladoceran-specific duplication; therefore, to investigate evolutionary conservation of sex specific expression of dsx genes and to further assess their functions in the environmental sex determination, we searched for dsx homologs in four closely related cladoceran species. Results: We identified homologs of both dsx genes from, D. pulex, D. galeata, and Ceriodaphnia dubia, yet only a single dsx gene was found from Moina macrocopa. The deduced amino acid sequences of all 9 dsx homologs contained the DM and oligomerization domains, which are characteristic for all arthropod DSX family members. Molecular phylogenetic analysis suggested that the dsx gene duplication likely occurred prior to the divergence of these cladoceran species, because that of the giant tiger prawn Penaeus monodon is rooted ancestrally to both DSX1 and DSX2 of cladocerans. Therefore, this result also suggested that M. macrocopa lost dsx2 gene secondarily. Furthermore, all dsx genes identified in this study showed male-biased expression levels, yet only half of the putative 5' upstream regulatory elements are preserved in D. magna and D. pulex. Conclusions: The all dsx genes of five cladoceran species examined had similar amino acid structure containing highly conserved DM and oligomerization domains, and exhibited sexually dimorphic expression patterns, suggesting that these genes may have similar functions for environmental sex determination in cladocerans. [ABSTRACT FROM AUTHOR]

Abstract: The Eastern and Western mosquitofish (Gambusia holbrooki and G. affinis) are potential bioindicator organisms for endocrine disruptors. Male mosquitofish have an elongated anal fin (gonopodium) used for internal fertilization whose formation is driven by androgens. Normal female mosquitofish have a normal, rounded anal fin which undergoes elongation into a gonopodium structure when female mosquitofish are exposed to androgenic chemicals. Significant issues with using mosquitofish as a bioindicator include the lack of knowledge on how anal fin growth in females corresponds to endpoints relevant to biological integrity and the lack of information on the molecular pathways that regulate anal fin growth. The objectives of this study were to understand how androgen-induced anal fin elongation relates to changes in endpoints related to the female reproductive system and to understand how anal fin elongation occurs in androgen-exposed female mosquitofish. To achieve these objectives, adult female G. holbrooki were exposed to a vehicle control or one of three doses of the androgen 17β-trenbolone (TB) at nominal concentrations of 0.1, 1 or 10μg TB/L. Anal fin measurements were taken and livers were used for quantitative polymerase chain reaction analysis of vitellogenin (vtg) mRNA expression at multiple time points. 10μg TB/L induced anal fin elongation after 7 days of treatment (one-way ANOVA, p <0.05) as did 0.1 and 1μg TB/L at later time points (one-way ANOVA, p <0.05). 10μg TB/L significantly reduced hepatic vtg gene expression at all time points assessed (one-way ANOVA, p <0.05). There was no correlation between anal fin elongation levels and vtg gene expression (Spearman''s ρ, p >0.05). In a separate experiment, female G. holbrooki and G. affinis were exposed to the vehicle control or 1μg TB/L. Anal fins were used for qualitative gene expression analysis of the genes sonic hedgehog (shh), muscle segment homeobox C (msxC), and fibroblast growth factor receptor 1 (fgfr1) by in situ hybridization. Shh was expressed in the distal tip of the gonopodium while msxC and fgfr1 were more widely expressed along the same anal fin rays during androgen exposure. These data provide insight into the molecular pathways involved in anal fin elongation and pave the way for future work toward developing the mosquitofish into a bioindicator organism for endocrine disruptors. [Copyright &y& Elsevier]

Abstract: We cloned a full-length androgen receptor (AR) cDNA from chicken (Gallus gallus) gonads. The cDNA sequence has an open reading frame of 2109bp encoding 703 amino acids. The chicken AR (cAR) shares high homology with ARs from other species in its amino acid sequences, in particular DNA binding domain (DBD) and ligand binding domain (LBD). RT-PCR analysis revealed that cAR mRNA is expressed in several embryonic tissues of both sexes, and relatively higher expression was observed in left ovary compared with testis. The immunoreactive signal of AR was co-localized within the ovarian cell nucleus, while such nuclear localization was not detected in those of testis. To get insight on the possible role of androgen–AR signaling during gonadal development, non-steroidal AR antagonist, flutamide, was administrated in ovo. The treatment induced the disorganization of sex cords in ovarian cortex at day 12 of incubation. The effect was restored by testosterone co-treatment, implying the possibility that AR mediated signaling may be involved in ovarian morphogenesis. Furthermore, co-treatment of flutamide with estradiol-17β (E2) also restored the phenotype, suggesting androgen–AR signaling might activate aromatase expression that is necessary for estrogen synthesis. These findings suggest androgen–AR signaling might contribute to chicken embryonic ovarian development. [Copyright &y& Elsevier]

Abstract: The occurrence of oocytes in the testis (testis–ova) of several fish species is often associated with exposure of estrogenic chemicals. However, induction mechanisms of the testis–ova remain to be elucidated. To develop marker genes for detecting testis–ova in the testis, adult male medaka were exposed to nominal concentration of 100ngL−1 of 17α-ethinylestradiol (EE2) for 3–5weeks, and 800ng estradiol benzoate (EB) for 3weeks (experiment I), and a measured concentration of 20ngL−1 EE2 for 1–6weeks (experiment II). Histological analysis was performed for the testis, and microarray analyses were performed for the testis, liver and brain. Microarray analysis in the estrogen-exposed medaka liver showed vitellogenin and choriogenin as estrogen responsive genes. Testis–ova were induced in the testis after 4weeks of exposure to 100ngL−1 EE2, 3weeks of exposure to 800ngEB, and 6weeks of exposure to 20ngL−1 EE2. Microarray analysis of estrogen-exposed testes revealed up-regulation of genes related to zona pellucida (ZP) and the oocytes marker gene, 42Sp50. Using quantitative RT-PCR we confirmed that Zpc5 gene can be used as a marker for the detection of testis–ova in male medaka. [Copyright &y& Elsevier]