Gene Duplication of Androgen Receptor As An Evolutionary Driving Force Underlying the Diversity of Sexual Characteristics in Teleost Fishes.

Sexual dimorphism allows species to meet their fitness optima based on the physiological availability of each sex. Although intralocus sexual conflict appears to be a genetic constraint for the evolution of sex-specific traits, sex-linked genes and the regulation of sex steroid hormones contribute to resolving this conflict by allowing sex-specific developments. Androgens and their receptor, androgen receptor (Ar), regulate male-biased phenotypes. In teleost fish, ar ohnologs have emerged as a result of teleost-specific whole genome duplication (TSGD). Recent studies have highlighted the evolutionary differentiation of ar ohnologs responsible for the development of sexual characteristics, which sheds light on the need for comparative studies on androgen regulation among different species. In this review, we discuss the importance of ar signaling as a regulator of male-specific traits in teleost species because teleost species are suitable experimental models for comparative studies owing to their great diversity in male-biased morphological and physiological traits. To date, both in vivo and in vitro studies on teleost ar ohnologs have shown a substantial influence of ars as a regulator of male-specific reproductive traits such as fin elongation, courtship behavior, and nuptial coloration. In addition to these sexual characteristics, ar substantially influences immunity, inducing a sex-biased immune response. This review aims to provide a comprehensive understanding of the current state of teleost ar studies and emphasizes the potential of teleost fishes, given their availability, to find molecular evidence about what gives rise to the spectacular diversity among fish species.

The Conflict between Regulatory Agencies over the 20,000-Fold Lowering of the Tolerable Daily Intake (TDI) for Bisphenol A (BPA) by the European Food Safety Authority (EFSA).

BACKGROUND: The European Food Safety Authority (EFSA) recommended lowering their estimated tolerable daily intake (TDI) for bisphenol A (BPA) 20,000-fold to 0.2 ng/kg body weight (BW)/day. BPA is an extensively studied high production volume endocrine disrupting chemical (EDC) associated with a vast array of diseases. Prior risk assessments of BPA by EFSA as well as the US Food and Drug Administration (FDA) have relied on industry-funded studies conducted under good laboratory practice protocols (GLP) requiring guideline end points and detailed record keeping, while also claiming to examine (but rejecting) thousands of published findings by academic scientists. Guideline protocols initially formalized in the mid-twentieth century are still used by many regulatory agencies. EFSA used a 21st century approach in its reassessment of BPA and conducted a transparent, but time-limited, systematic review that included both guideline and academic research. The German Federal Institute for Risk Assessment (BfR) opposed EFSA's revision of the TDI for BPA. OBJECTIVES: We identify the flaws in the assumptions that the German BfR, as well as the FDA, have used to justify maintaining the TDI for BPA at levels above what a vast amount of academic research shows to cause harm. We argue that regulatory agencies need to incorporate 21st century science into chemical hazard identifications using the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) nonguideline academic studies in a collaborative government-academic program model. DISCUSSION: We strongly endorse EFSA's revised TDI for BPA and support the European Commission's (EC) apparent acceptance of this updated BPA risk assessment. We discuss challenges to current chemical risk assessment assumptions about EDCs that need to be addressed by regulatory agencies to, in our opinion, become truly protective of public health. Addressing these challenges will hopefully result in BPA, and eventually other structurally similar bisphenols (called regrettable substitutions) for which there are known adverse effects, being eliminated from all food-related and many other uses in the EU and elsewhere. https://doi.org/10.1289/EHP13812.

Iopanoic acid alters thyroid hormone-related gene expression, thyroid hormone levels, swim bladder inflation, and swimming performance in Japanese medaka.

Disruption of the thyroid hormone system by synthetic chemicals is gaining attention owing to its potential negative effects on organisms. In this study, the effects of the dio-inhibitor iopanoic acid (IOP) on the levels of thyroid hormone and related gene expression, swim bladder inflation, and swimming performance were investigated in Japanese medaka. Iopanoic acid exposure suppressed thyroid-stimulating hormone ß (tshß), tshß-like, iodotyronin deiodinase 1 (dio1), and dio2 expression, and increased T4 and T3 levels. In addition, IOP exposure inhibited swim bladder inflation, reducing swimming performance. Although adverse outcome pathways of thyroid hormone disruption have been developed using zebrafish, no adverse outcome pathways have been developed using Japanese medaka. This study confirmed that IOP inhibits dio expression (a molecular initiating event), affects T3 and T4 levels (a key event), and reduces swim bladder inflation (a key event) and swimming performance (an adverse outcome) in Japanese medaka.

European Medicines Agency Conflicts With the European Food Safety Authority (EFSA) on Bisphenol A Regulation.

The European Food Safety Authority (EFSA) has revised their estimate of the toxicity of bisphenol A (BPA) and, as a result, have recommended reducing the tolerable daily intake (TDI) by 20 000-fold. This would essentially ban the use of BPA in food packaging such as can liners, plastic food containers, and in consumer products. To come to this conclusion, EFSA used a systematic approach according to a pre-established protocol and included all guideline and nonguideline studies in their analysis. They found that Th-17 immune cells increased with very low exposure to BPA and used this endpoint to revise the TDI to be human health protective. A number of regulatory agencies including the European Medicines Agency (EMA) have written formal disagreements with several elements of EFSA's proposal. The European Commission will now decide whether to accept EFSA's recommendation over the objections of EMA. If the Commission accepts EFSA's recommendation, it will be a landmark action using knowledge acquired through independent scientific studies focused on biomarkers of chronic disease to protect human health. The goal of this Perspective is to clearly articulate the monumental nature of this debate and decision and to explain what is at stake. Our perspective is that the weight of evidence clearly supports EFSA's proposal to reduce the TDI by 20 000-fold.

Comparative analysis of gonadal transcriptomes between turtle and alligator identifies common molecular cues activated during the temperature-sensitive period for sex determination.

The mode of sex determination in vertebrates can be categorized as genotypic or environmental. In the case of genotypic sex determination (GSD), the sexual fate of an organism is determined by the chromosome composition with some having dominant genes, named sex-determining genes, that drive the sex phenotypes. By contrast, many reptiles exhibit environmental sex determination (ESD), whereby environmental stimuli drive sex determination, and most notably temperature. To date, temperature-dependent sex determination (TSD) has been found in most turtles, some lizards, and all crocodylians, but commonalities in the controlling processes are not well established. Recent innovative sequencing technology has enabled investigations into gonadal transcriptomic profiles during temperature-sensitive periods (TSP) in various TSD species which can help elucidate the controlling mechanisms. In this study, we conducted a time-course analysis of the gonadal transcriptome during the male-producing temperature (26℃) of the Reeve's turtle (Chinese three-keeled pond turtle) Mauremys reevesii. We then compared the transcriptome profiles for this turtle species during the TSP with that for the American alligator Alligator mississippiensis to identify conserved reptilian TSD-related genes. Our transcriptome-based findings provide an opportunity to retrieve the candidate molecular cues that are activated during TSP and compare these target responses between TSD and GSD turtle species, and between TSD species.

The role of mesenchymal estrogen receptor 1 in mouse uterus in response to estrogen.

Estrogens play important roles in uterine growth and homeostasis through estrogen receptors (ESR1 and ESR2). To address the role of ESR1-mediated tissue events in the murine uterus, we analyzed mice with a mesenchymal tissue-specific knockout of Esr1. Isl1-driven Cre expression generated Esr1 deletion in the uterine stroma and endometrium (Isl-Esr1KO). We showed that overall structure of the Isl1-Esr1KO mouse uterus developed normally, but estrogen responsiveness and subsequent growth were defective, suggesting that mesenchymal ESR1 is necessary for both epithelial and mesenchymal cell proliferation. Furthermore, RNA-seq analysis revealed that the majority of estrogen-induced genes were regulated by stromal ESR1. In control mice, E2 administration induced 9476 up-regulated differentially expressed genes (DEGs), whereas only 1801 up-regulated DEGs were induced by E2 in Isl1-Esr1KO mice. We further showed that stromal ESR1-regulated genes in the mouse uterus included several growth factors and cytokines, which are potential factors that regulate epithelial and stromal tissue interaction, and also genes involved in lipid homeostasis. Therefore, we infer that stromal ESR1 expression is indispensable for most estrogen actions in the mouse uterus and the current results provide new insights into estrogen-mediated homeostasis in female reproductive organs.

Evolutionary differentiation of androgen receptor is responsible for sexual characteristic development in a teleost fish.

Teleost fishes exhibit complex sexual characteristics in response to androgens, such as fin enlargement and courtship display. However, the molecular mechanisms underlying their evolutionary acquisition remain largely unknown. To address this question, we analyse medaka (Oryzias latipes) mutants deficient in teleost-specific androgen receptor ohnologs (ara and arb). We discovered that neither ar ohnolog was required for spermatogenesis, whilst they appear to be functionally redundant for the courtship display in males. However, both were required for reproductive success: ara for tooth enlargement and the reproductive behaviour eliciting female receptivity, arb for male-specific fin morphogenesis and sexual motivation. We further showed that differences between the two ar ohnologs in their transcription, cellular localisation of their encoded proteins, and their downstream genetic programmes could be responsible for the phenotypic diversity between the ara and arb mutants. These findings suggest that the ar ohnologs have diverged in two ways: first, through the loss of their roles in spermatogenesis and second, through gene duplication followed by functional differentiation that has likely resolved the pleiotropic roles derived from their ancestral gene. Thus, our results provide insights into how genome duplication impacts the massive diversification of sexual characteristics in the teleost lineage.

Adverse effects of thyroid-hormone-disrupting chemicals 6-propyl-2-thiouracil and tetrabromobisphenol A on Japanese medaka (Oryzias latipes).

Thyroid-hormone-disrupting chemicals are increasingly attracting attention because of their potential harmful effects on animal health, including on fishes. Here, we investigated the effects of exposure to the thyroid-hormone-disrupting chemicals 6-propyl-2-thiouracil (PTU) and tetrabromobisphenol A (TBBPA) on swim bladder inflation, eye development, growth, swimming performance, and the expression of thyroid-related genes in Japanese medaka (Oryzias latipes). PTU exposure resulted in reductions in eye size, growth, and swim bladder inflation, and these effects led to poorer swimming performance. These phenotypic effects were accompanied by increased expression of the thyroid-stimulating hormone subunit beta (tshß) paralog tshß-like, but there were no significant changes in expression for tshß, deiodinase 1 (dio1), deiodinase 2 (dio2), and thyroid hormone receptor alpha (trα) and beta (trß). For PTU exposure, we identified the key event (swim bladder inflation reduction) and an adverse outcome (swimming performance reduction). No significant effects from TBBPA exposure were seen on swim bladder inflation, eye development, growth, or swimming performance. However, expression of tshß-like and tshß (significantly enhanced) and trα and trß (significantly reduced) were affected by TBBPA exposure albeit not in dose-dependent manners. There were no effects of TBBPA on the expression of dio1 and dio2. We thus show that the two thyroid-hormone-disrupting chemicals PTU and TBBPA differ in their effect profiles with comparable effects on the studied phenotypes and thyroid-related gene expression to those reported in zebrafish.

Gonadal Soma-Derived Factor Expression is a Potential Biomarker for Predicting the Effects of Endocrine-Disrupting Chemicals on Gonadal Differentiation in Japanese Medaka (Oryzias Latipes).

Chemicals with androgenic or estrogenic activity induce the sex reversal and/or intersex condition in various teleost fish species. Previously, we reported that exposure to 17α-methyltestosterone, bisphenol A, or 4-nonylphenol induces changes in expression of the gonadal soma-derived factor (gsdf) gene accompanied by disruption of gonadal differentiation in Japanese medaka (Oryzias latipes). These findings suggest that gsdf expression might be a useful biomarker for predicting the potential effect of chemicals on gonadal differentiation. We examined the gsdf expression in Japanese medaka exposed to chemicals with estrogenic or androgenic activity. Exposure to the androgenic steroid 17ß-trenbolone at 0.5-22.1 µg/L induced the development of ovotestis (presence of ovarian tissue with testicular tissue) and female-to-male sex reversal in XX embryos, and exposure at 6.32 and 22.1 µg/L significantly increased gsdf expression in XX embryos compared with controls at developmental stage 38 (1 day before hatching). In the present study, no statistically significant difference in gsdf mRNA expression was observed after exposure to 17ß-estradiol, 17α-ethinylestradiol, and 4-t-octylphenol, which have estrogenic activity. In addition, antiandrogenic chemicals or chemicals without endocrine-disrupting activity did not induce changes in gsdf expression in XX or XY embryos. Thus, an increase in gsdf expression after androgen exposure was observed in XX embryos. Together, these findings indicate that gsdf expression might be useful for predicting the adverse effect of chemicals on gonadal differentiation. Environ Toxicol Chem 2022;41:1875-1884. © 2022 SETAC.

Laterally biased diffusion of males of the water flea Daphnia magna.

The water flea, Daphnia magna, is a representative zooplankton that lives in freshwater environments. It primarily propagates via asexual reproduction in normal and healthy environmental conditions. Unsuitable environmental conditions induce D. magna to change its mode of reproduction from asexual to sexual reproduction. During sexual reproduction, D. magna produces special tough eggs (resting eggs) that can survive severe environmental conditions. Despite an increase in our understanding of their mating behavior, the sex-specific characteristics of swimming behavior among daphnid species are poorly understood. In this study, we analysed the swimming patterns and dynamics of female and male adult D. magna using computer modeling. Males displayed laterally biased diffusion in contrast to the homogeneous, nondirectional diffusion of females. Computer modeling analysis using a discrete-time Markov chain simulation, in which the frequencies of turning behavior were evaluated as probability distributions, explained the greater diffusion of males in the horizontal direction. We presumed that high diffusion in the horizontal direction would increase the probability of encountering a distant mate. Our findings suggest that male D. magna increases genotypic heterogeneity by effectively selecting certain motion parameters.

Preself-Feeding Medaka Fry Provides a Suitable Screening System for in Vivo Assessment of Thyroid Hormone-Disrupting Potential.

Endocrine-disrupting chemicals are assessed based on their physiological potential and their potential associated adverse effects. However, suitable end points for detection of chemicals that interfere with the thyroid hormone (TH) system have not been established in nonmammals, with the exception of amphibian metamorphosis. The aims of the current study were to develop an in vivo screening system using preself-feeding medaka fry (Oryzias latipes) for the detection of TH-disrupting chemicals and elucidate the underlying molecular mechanism. 17α-Ethinylestradiol (EE2: <100 ng/L) did not induce mRNA expression of estrogen-responsive genes, vitellogenins (vtgs) mRNA. Meanwhile, coexposure with thyroxin (T4) induced an increase of vtg expression. TH-disrupting chemicals (thiourea (TU), perfluorooctanoic acid (PFOA), and tetrabromobisphenol A (TBBPA)) significantly suppressed EE2 (1,000 ng/L)-induced vtg1 expression, while T4 rescued their expression as well as that of thyroid hormone receptor α (tRα) and estrogen receptors (esrs). These results were supported by in silico analysis of the 5'-transcriptional regulatory region of these genes. Furthermore, the esr1 null mutant revealed that EE2-induced vtg1 expression requires mainly esr2a and esr2b in a TH-dependent manner in preself-feeding fry. Application of preself-feeding medaka fry as a screening system might help decipher the in vivo mechanisms of action of TH-disrupting molecules, while providing an alternative to the traditional animal model.

Effect of thyroid hormone-disrupting chemicals on swim bladder inflation and thyroid hormone-related gene expression in Japanese medaka and zebrafish.

We compared the influence of thyroid hormone-disrupting chemicals (heptafluorobutanoic acid, PFBA and tris[1,3-dichloro-2-propyl] phosphate, TDCPP) and thyroid hormone (3,3',5-triiodo-L-thyronine, T3) on swim bladder inflation and thyroid hormone-related gene expression in Japanese medaka and zebrafish. The swim bladder of most larvae had inflated at 4 h post hatching (hph) in Japanese medaka and at 48 hph in zebrafish in controls. In both fish species, the swim bladder inflation was inhibited in larvae exposed to PFBA (lowest observed effect concentration [LOEC] in medaka: 40 mg/L; in zebrafish: 80 mg/L), TDCPP (LOEC in medaka: 1 mg/L; in zebrafish: 0.5 mg/L), and T3 (no inhibition in Japanese medaka; LOEC in zebrafish: 7.5 µg/L). We also examined the influence of PFBA, TDCPP, and T3 on the expression of thyroid stimulating hormone subunit beta (tshß) or thyroid hormone receptor alpha (trα) and beta (trß). No changes were observed in the expression of genes after PFBA and TDCPP exposure; however, T3 exposure upregulated trα and trß expression in both fish species. When the results were compared between Japanese medaka and zebrafish, swim bladder inflation in both species was found to be inhibited by exposure to thyroid hormone-disrupting chemicals. Our results show that inhibition of the swim bladder inflation at 4 hph in Japanese medaka and 48 hph in zebrafish is a potential indicator of thyroid hormone-disturbing activity of chemicals.

Summary of 17 chemicals evaluated by OECD TG229 using Japanese Medaka, Oryzias latipes in EXTEND 2016.

In June 2016, the Ministry of the Environment of Japan announced a program "EXTEND2016" on the implementation of testing and assessment for endocrine active chemicals, consisting of a two-tiered strategy. The aim of the Tier 1 screening and the Tier 2 testing is to identify the impacts on the endocrine system and to characterize the adverse effects to aquatic animals by endocrine disrupting chemicals detected in the aquatic environment in Japan. For the consistent assessment of the effects on reproduction associated with estrogenic, anti-estrogenic, androgenic, and/or anti-androgenic activities of chemicals throughout Tier 1 screening to Tier 2 testing, a unified test species, Japanese medaka (Oryzias latipes), has been used. For Tier 1 screening, the in vivo Fish Short-Term Reproduction Assay (OECD test guideline No. 229) was conducted for 17 chemicals that were nominated based on the results of environmental monitoring, existing knowledge obtained from a literature survey, and positive results in reporter gene assays using the estrogen receptor of Japanese medaka. In the 17 assays using Japanese medaka, adverse effects on reproduction (i.e., reduction in fecundity and/or fertility) were suggested for 10 chemicals, and a significant increase of hepatic vitellogenin in males, indicating estrogenic (estrogen receptor agonistic) potency, was found for eight chemicals at the concentrations in which no overt toxicity was observed. Based on these results, and the frequency and the concentrations detected in the Japanese environment, estrone, 4-nonylphenol (branched isomers), 4-tert-octylphenol, triphenyl phosphate, and bisphenol A were considered as high priority candidate substances for the Tier 2 testing.

Juvenile hormone synthesis and signaling disruption triggering male offspring induction and population decline in cladocerans (water flea): Review and adverse outcome pathway development.

Juvenile hormone (JH) are a family of multifunctional hormones regulating larval development, molting, metamorphosis, reproduction, and phenotypic plasticity in arthropods. Based on its importance in arthropod life histories, many insect growth regulators (IGRs) mimicking JH have been designed to control harmful insects in agriculture and aquaculture. These JH analogs (JHAs) may also pose hazards to nontarget species by causing unexpected endocrine-disrupting (ED) effects such as molting and metamorphosis defects, larval lethality, and disruption of the sexual identity. This critical review summarizes the current knowledge of the JH-mediated effects in the freshwater cladoceran crustaceans such as Daphnia species on JHA-triggered endocrine disruptive outputs to establish a systematic understanding of JHA effects. Based on the current knowledge, adverse outcome pathways (AOPs) addressing the JHA-mediated ED effects in cladoceran leading to male offspring production and subsequent population decline were developed. The weight of evidence (WoE) of AOPs was assessed according to established guidelines. The review and AOP development aim to present the current scientific understanding of the JH pathway and provide a robust reference for the development of tiered testing strategies and new risk assessment approaches for JHAs in future ecotoxicological research and regulatory processes.

Sex Determination and Differentiation in Decapod and Cladoceran Crustaceans: An Overview of Endocrine Regulation.

Mechanisms underlying sex determination and differentiation in animals are known to encompass a diverse array of molecular clues. Recent innovations in high-throughput sequencing and mass spectrometry technologies have been widely applied in non-model organisms without reference genomes. Crustaceans are no exception. They are particularly diverse among the Arthropoda and contain a wide variety of commercially important fishery species such as shrimps, lobsters and crabs (Order Decapoda), and keystone species of aquatic ecosystems such as water fleas (Order Branchiopoda). In terms of decapod sex determination and differentiation, previous approaches have attempted to elucidate their molecular components, to establish mono-sex breeding technology. Here, we overview reports describing the physiological functions of sex hormones regulating masculinization and feminization, and gene discovery by transcriptomics in decapod species. Moreover, this review summarizes the recent progresses of studies on the juvenile hormone-driven sex determination system of the branchiopod genus Daphnia, and then compares sex determination and endocrine systems between decapods and branchiopods. This review provides not only substantial insights for aquaculture research, but also the opportunity to re-organize the current and future trends of this field.

Summary of reference chemicals evaluated by the fish short-term reproduction assay, OECD TG229, using Japanese Medaka, Oryzias latipes.

Under the Organisation for Economic Co-operation and Development (OECD), the Ministry of the Environment of Japan (MOE) added Japanese medaka (Oryzias latipes) to the test guideline fish short-term reproduction assay (FSTRA) developed by the United States Environmental Protection Agency (US EPA) using fathead minnow (Pimephales promelas). The FSTRA was designed to detect endocrine disrupting effects of chemicals interacting with the hypothalamic-pituitary-gonadal axis (HPG axis) such as agonists or antagonists on the estrogen receptor (Esr) and/or the androgen receptor (AR) and steroidogenesis inhibitors. We conducted the FSTRA with Japanese medaka, in accordance with OECD test guideline number 229 (TG229), for 16 chemicals including four Esr agonists, two Esr antagonists, three AR agonists, two AR antagonists, two steroidogenesis inhibitors, two progesterone receptor agonists, and a negative substance, and evaluated the usability and the validity of the FSTRA (TG229) protocol. In addition, in vitro reporter gene assays (RGAs) using Esr1 and ARß of Japanese medaka were performed for the 16 chemicals, to support the interpretation of the in vivo effects observed in the FSTRA. In the present study, all the test chemicals, except an antiandrogenic chemical and a weak Esr agonist, significantly reduced the reproductive status of the test fish, that is, fecundity or fertility, at concentrations where no overt toxicity was observed. Moreover, vitellogenin (VTG) induction in males and formation of secondary sex characteristics (SSC), papillary processes on the anal fin, in females was sensitive endpoints to Esr and AR agonistic effects, respectively, and might be indicators of the effect concentrations in long-term exposure. Overall, it is suggested that the in vivo FSTRA supported by in vitro RGA data can adequately detect effects on the test fish, O. latipes, and probably identify the mode of action (MOA) of the chemicals tested.

Methyl farnesoate regulatory mechanisms underlying photoperiod-dependent sex determination in the freshwater crustacean Daphnia magna.

Freshwater zooplankton Daphnia magna has been widely used in ecotoxicology studies. During the last 20 years, it has been demonstrated that the topical application of juvenile hormone (JH) or JH analogs to mother daphnids induce male offspring production. Based on this finding, an in vivo screening validation method for chemicals with JH agonistic effect has developed. Although this screening system successfully identified a number of JH-like chemicals, molecular mechanisms underlying the male sex-determining process remain largely unknown. To address this issue, we established a reliable male- or female-producing system using Daphnia pulex WTN6 strain by changing the rearing photoperiod. Taking advantage of this rearing system, we successfully found several factors involving male sex determination such as ionotropic glutamate receptors, protein kinase C and pantothenate. Here, we used two D. magna strains that can also control the production of female or male offspring by photoperiod differences as model species for ecotoxicology studies. We demonstrated that either treatment of antagonist of ionotropic glutamate receptors or inhibitor of protein kinase C strongly suppressed male offspring production even under male-producing conditions. Moreover, we revealed that male sex-determining processes are likely diverged between D. magna and D. pulex based on the current experiment. This study provides a fine experimental method for in vivo screening not only JH agonists but also JH antagonists. Moreover, using daphnids with photoperiod-dependent sex determination manner will hugely contribute to understanding the mode-of-action of JH in daphnids.

Exposure to 4-nonylphenol induces a shift in the gene expression of gsdf and testis-ova formation and sex reversal in Japanese medaka (Oryzias latipes).

The branched isomer mixture 4-nonylphenol (4-NP) has been used worldwide as a surfactant, and can have endocrine-disrupting effects on aquatic organisms. For instance, 4-NP induces the formation of testis-ova (i.e., testicular and ovarian tissue in the same gonad) or male to female sex reversal of various teleost fishes. Recently, our group revealed that altered gsdf gene expression is associated with disruption of gonadal differentiation in Japanese medaka (Oryzias latipes) embryos exposed to methyltestosterone or bisphenol A, suggesting that gsdf might be useful as a biomarker for predicting the impact of endocrine-disrupting chemicals (EDCs) on gonadal differentiation. Here, we used 4-NP to examine further whether gsdf expression at the embryo stage is useful for predicting EDC impact on gonadal sex differentiation. When fertilized medaka eggs were exposed to 32 or 100 µg/L 4-NP, testis-ova in genetic males and sex reversal from genetic male to phenotypic female were observed. At stage 38 (just before hatching), 4-NP exposure at 1-100 µg/L did not affect gsdf expression in XX embryos compared with the nontreated control; however, in XY embryos, the gsdf expression in the 100 µg/L-exposed group was significantly lower than that in the controls. The 4-NP concentration at which gsdf expression was suppressed was equal to that at which testis-ova and sex reversal were induced. These results indicate that expression of the gsdf gene at the embryonic stage in medaka is a useful biomarker for predicting the impact of EDCs on sexual differentiation.

Characterization of G protein-coupled estrogen receptors in Japanese medaka, Oryzias latipes.

The G protein-coupled estrogen receptor 1 (Gper1) is a membrane-bound estrogen receptor that mediates non-genomic action of estrogens. A Gper1-mediating pathway has been implicated in reproductive activities in fish, including oocyte growth, but Gper1 has been characterized in only a very limited number of fish species. In this study, we cloned and characterized two genes encoding medaka (Oryzias latipes) Gper1s, namely, Gper1a and Gper1b, and phylogenic and synteny analyses suggest that these genes originate through a teleost-specific whole genome duplication event. We found that Gper1a induced phosphorylation of mitogen-activated protein kinase (MAPK) in 293T cells transfected with medaka Gper1s on exposure to the natural estrogen, 17ß-estradiol (E2) and a synthetic Gper1 agonist (G-1), and treatment with both E2 and G-1 also decreased the rate of spontaneous maturation in medaka oocytes. These findings show that the processes for oocyte growth and maturation are sensitive to estrogens and are possibly mediated through Gper1a in medaka. We also show that 17α-ethinylestradiol (EE2), one of the most potent estrogenic endocrine-disrupting chemicals, and bisphenol A (BPA, a weak environmental estrogen) augmented phosphorylation of MAPK through medaka Gper1s in 293T cells. Interestingly, however, treatment with EE2 or BPA did not attenuate maturation of medaka oocytes. Our findings support that Gper1-mediated effects on oocytes are conserved among fish species, but effects of estrogenic endocrine-disrupting chemicals on oocytes acting through Gper1 may be divergent among fish species.

New frontiers of developmental endocrinology opened by researchers connecting irreversible effects of sex hormones on developing organs.

In the early 1960's, at Professor Bern's laboratory, University of California, Berkeley) in the US, Takasugi discovered ovary-independent, persistent vaginal changes in mice exposed neonatally to estrogen, which resulted in vaginal cancer later in life. Reproductive abnormalities in rodents were reported as a result of perinatal exposure to various estrogenic chemicals. Ten years later, vaginal cancers were reported in young women exposed in utero to the synthetic estrogen diethylstilbestrol (DES) and this has been called the "DES syndrome". The developing organism is particularly sensitive to developmental exposure to estrogens inducing long-term changes in various organs including the reproductive organs. The molecular mechanism underlying the persistent vaginal changes induced by perinatal estrogen exposure was partly demonstrated. Persistent phosphorylation and sustained expression of EGF-like growth factors, lead to estrogen receptor α (ESR1) activation, and then persistent vaginal epithelial cell proliferation. Agents which are weakly estrogenic by postnatal criteria may have major developmental effects, especially during a critical perinatal period. The present review outlines various studies conducted by four generations of investigators all under the influence of Prof. Bern. The studies include reports of persistent changes induced by neonatal androgen exposure, analyses of estrogen responsive genes, factors determining epithelial differentiation in the Müllerian duct, ESR and growth factor signaling, and polyovular follicles in mammals. This review is then expanded to the studies on the effects of environmental estrogens on wildlife and endocrine disruption in Daphnids.