Functional distinctions associated with the diversity of sex steroid hormone receptors ESR and AR.

Sex steroid hormones including estrogens and androgens play fundamental roles in regulating reproductive activities and they act through estrogen and androgen receptors (ESR and AR). These steroid receptors have evolved from a common ancestor in association with several gene duplications. In most vertebrates, this has resulted in two ESR subtypes (ESR1 and ESR2) and one AR, whereas in teleost fish there are at least three ESRs (ESR1, ESR2a and ESR2b) and two ARs (ARα and ARß) due to a lineage-specific whole genome duplication. Functional distinctions have been suggested among these receptors, but to date their roles have only been characterized in a limited number of species. Sexual differentiation and the development of reproductive organs are indispensable for all animal species and in vertebrates these events depend on the action of sex steroid hormones. Here we review the recent progress in understanding of the functions of the ESRs and ARs in the development and expression of sexually dimorphic characteristics associated with steroid hormone signaling in vertebrates, with representative fish, amphibians, reptiles, birds and mammals.

Characterization of evolutionary trend in squamate estrogen receptor sensitivity.

Steroid hormones are a key regulator of reproductive biology in vertebrates, and are largely regulated via nuclear receptor families. Estrogen signaling is regulated by two estrogen receptor (ER) subtypes alpha and beta in the nucleus. In order to understand the role of estrogen in vertebrates, these ER from various species have been isolated and were functionally analyzed using luciferase reporter gene assays. Interestingly, species difference in estrogen sensitivity has been noted in the past, and it was reported that snake ER displayed highest estrogen sensitivity. Here, we isolated additional ER from three lizards: chameleon (Bradypodion pumilum), skink (Plestiodon finitimus), and gecko (Gekko japonicus). We have performed functional characterization of these ERs using reporter gene assay system, and found high estrogen sensitivity in all three species. Furthermore, comparison with results from other tetrapod ER revealed a seemingly uniform gradual pattern of ligand sensitivity evolution. In silico 3D homology modeling of the ligand-binding domain revealed structural variation at three sites, helix 2, and juncture between helices 8 and 9, and caudal region of helix 10/11. Docking simulations indicated that predicted ligand-receptor interaction also correlated with the reporter assay results, and overall squamates displayed highest stabilized interactions. The assay system and homology modeling system provides tool for in-depth comparative analysis of estrogen function, and provides insight toward the evolution of ER among vertebrates.

RNA-seq analysis of the gonadal transcriptome during Alligator mississippiensis temperature-dependent sex determination and differentiation.

BACKGROUND: The American alligator (Alligator mississippiensis) displays temperature-dependent sex determination (TSD), in which incubation temperature during embryonic development determines the sexual fate of the individual. However, the molecular mechanisms governing this process remain a mystery, including the influence of initial environmental temperature on the comprehensive gonadal gene expression patterns occurring during TSD. RESULTS: Our characterization of transcriptomes during alligator TSD allowed us to identify novel candidate genes involved in TSD initiation. High-throughput RNA sequencing (RNA-seq) was performed on gonads collected from A. mississippiensis embryos incubated at both a male and a female producing temperature (33.5 °C and 30 °C, respectively) in a time series during sexual development. RNA-seq yielded 375.2 million paired-end reads, which were mapped and assembled, and used to characterize differential gene expression. Changes in the transcriptome occurring as a function of both development and sexual differentiation were extensively profiled. Forty-one differentially expressed genes were detected in response to incubation at male producing temperature, and included genes such as Wnt signaling factor WNT11, histone demethylase KDM6B, and transcription factor C/EBPA. Furthermore, comparative analysis of development- and sex-dependent differential gene expression revealed 230 candidate genes involved in alligator sex determination and differentiation, and early details of the suspected male-fate commitment were profiled. We also discovered sexually dimorphic expression of uncharacterized ncRNAs and other novel elements, such as unique expression patterns of HEMGN and ARX. Twenty-five of the differentially expressed genes identified in our analysis were putative transcriptional regulators, among which were MYBL2, MYCL, and HOXC10, in addition to conventional sex differentiation genes such as SOX9, and FOXL2. Inferred gene regulatory network was constructed, and the gene-gene and temperature-gene interactions were predicted. CONCLUSIONS: Gonadal global gene expression kinetics during sex determination has been extensively profiled for the first time in a TSD species. These findings provide insights into the genetic framework underlying TSD, and expand our current understanding of the developmental fate pathways during vertebrate sex determination.

TRPV4 associates environmental temperature and sex determination in the American alligator.

Temperature-dependent sex determination (TSD), commonly found among reptiles, is a sex determination mode in which the incubation temperature during a critical temperature sensitive period (TSP) determines sexual fate of the individual rather than the individual's genotypic background. In the American alligator (Alligator mississippiensis), eggs incubated during the TSP at 33 °C (male producing temperature: MPT) yields male offspring, whereas incubation temperatures below 30 °C (female producing temperature: FPT) lead to female offspring. However, many of the details of the underlying molecular mechanism remains elusive, and the molecular link between environmental temperature and sex determination pathway is yet to be elucidated. Here we show the alligator TRPV4 ortholog (AmTRPV4) to be activated at temperatures proximate to the TSD-related temperature in alligators, and using pharmacological exposure, we show that AmTRPV4 channel activity affects gene expression patterns associated with male differentiation. This is the first experimental demonstration of a link between a well-described thermo-sensory mechanism, TRPV4 channel, and its potential role in regulation of TSD in vertebrates, shedding unique new light on the elusive TSD molecular mechanism.

Identification and Characterization of the Androgen Receptor From the American Alligator, Alligator mississippiensis.

Androgens are essential for the development, reproduction, and health throughout the life span of vertebrates, particularly during the initiation and maintenance of male sexual characteristics. Androgen signaling is mediated by the androgen receptor (AR), a member of the steroid nuclear receptor superfamily. Mounting evidence suggests that environmental factors, such as exogenous hormones or contaminants that mimic hormones, can disrupt endocrine signaling and function. The American alligator (Alligator mississippiensis), a unique model for ecological research in that it exhibits environment-dependent sex determination, is oviparous and long lived. Alligators from a contaminated environment exhibit low reproductive success and morphological disorders of the testis and phallus in neonates and juveniles, both associated with androgen signaling; thus, the alterations are hypothesized to be related to disrupted androgen signaling. However, this line of research has been limited because of a lack of information on the alligator AR gene. Here, we isolated A mississippiensis AR homologs (AmAR) and evaluated receptor-hormone/chemical interactions using a transactivation assay. We showed that AmAR responded to all natural androgens and their effects were inhibited by cotreatment with antiandrogens, such as flutamide, p,p'-dichlorodiphenyldichloroethylene, and vinclozolin. Intriguingly, we found a spliced form of the AR from alligator cDNA, which lacks seven amino acids within the ligand-binding domain that shows no response to androgens. Finally, we have initial data on a possible dominant-negative function of the spliced form of the AR against androgen-induced AmAR.

Gonadal differentiation in reptiles exhibiting environmental sex determination.

As temperature-dependent sex determination (TSD) and homozygote or heterozygote genetic sex determination (GSD) exist in multiple reptilian taxa, they represent sex determination systems that have emerged de novo. Current investigations have revealed that the genetic mechanisms used by various reptilian species are similar to those used by other vertebrates. However, the recent completion or near completion of various reptilian genome projects suggests that new studies examining related species with and without TSD could begin to provide additional insight into the evolution of TSD and GSD in vertebrate ancestors. Major questions still remain concerning germ cell migration and specification, the differentiation of gonadal accessory cells, such as the Sertoli cells and granulosa cells of the developing testis and ovary, respectively, and the mechanisms by which gene expression is regulated during TSD events. Further, reptilian sentinels and their mechanisms of gonadogenesis will likely remain important indicator species for environmental health. Thus, ongoing and new investigations need to tie molecular information to gonadal morphogenesis and function in reptiles. Such data will not only provide important information for an understanding of the evolution of these phenomena in vertebrates, but could also provide an important understanding of the health of the environment around us.

Molecular impact of juvenile hormone agonists on neonatal Daphnia magna.

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.

Changes in gonadal gene network by exogenous ligands in temperature-dependent sex determination.

We examined the expression of candidate sex-determining genes in the red-eared slider turtle (Trachemys scripta) during the temperature-sensitive period (TSP). Aromatase and Rspo1 were used as markers of ovarian differentiation and Sox9 was used as a marker of testicular differentiation. Eggs were incubated at a male-producing temperature (26 °C or MPT) and a female-producing temperature (31 °C or FPT). First, eggs at the beginning of the TSP (stage 16) were topically treated with the steroid hormones 17ß-estradiol (E2), testosterone in combination with aromatase inhibitor (AI+T), the E2 antagonist (ICI 182 780), and the androgen antagonist (flutamide). Secondly, gonads were removed at stage 16 and treated in vitro with E2, AI+T, or hormone antagonists. At the FPT, AI+T in ovo suppressed aromatase and Rspo1, while activating Sox9. At the MPT, E2 treatment rapidly increased aromatase and Rspo1, while suppressing Sox9. Treatment with the E2 antagonist in ovo decreased aromatase at the FPT. Treatment with the androgen antagonist in ovo increased aromatase and Rspo1 at early time points at MPT and decreased Sox9 at MPT at later developmental stages. Treatment of isolated gonads cultured in vitro with AI+T at FPT decreased aromatase and Rspo1 and E2 increased the expression of these genes at MPT. In vitro treatment with E2 antagonist suppressed aromatase and Rspo1 expression at FPT. Overall, our results suggest that exogenous ligands dictate gonadal development by redirecting the expression of candidate sex-determining genes within the genetic cascades induced by temperature.

Establishment of transactivation assay systems using fish, amphibian, reptilian and human thyroid hormone receptors.

Thyroid hormones are essential for the regulation of a wide range of biological processes associated with normal development and metabolism in vertebrates. For the screening of chemicals with a potential thyroid hormone and anti-thyroid hormone activities, we have established transient transactivation assay systems using thyroid hormone receptors (TRα and TRß) from three frog species (Xenopus laevis, Silurana tropicalis and Rana rugosa), a fish (Oryzias latipes), an alligator (Alligator mississippiensis) and a human (Homo sapiens). In all species examined, similar transcriptional activities were found for triiodothyronine (T3 : 10(-11) M in TRα and 10(-10) M in TRß) and thyroxine (T4 : 10(-9) M in TRα and 10(-8) M in TRß). Analogs of thyroid hormone (3,5,3',-triiodothyroacetic acid and 3,3',5,5'-tetraiodothyroacetic acid) exhibited weaker activity, requiring 10-fold higher concentrations for induction of activity when compared with T3 and T4 . These results provide support for the usefulness of in vitro screening assay systems as part of an approach to test chemicals for potential thyroid hormone receptor activity. In addition, we observed that T3 -stimulated transcriptional activity of the O. latipes TRα was inhibited by 10(-5) M tetrabromobisphenol A (TBBPA). In contrast, TR antagonist activities on TRα were not encountered in other species, even with TBBPA concentrations at 10(-5) M. In vitro transactivation assay systems using TRs from various species can be used for the screening of chemicals with thyroid-receptor agonist and antagonist activities. They also can be used for studies that examine evolutionary differences among species in the potency of TR activation.

Genetic network underlying temperature-dependent sex determination is endogenously regulated by temperature in isolated cultured Trachemys scripta gonads.

In reptiles with temperature-dependent sex determination, gonadogenesis is initially directed by the incubation temperature of the egg during the middle third of embryonic development. The mechanism by which temperature is transduced into a sex-determining molecular signal remains a mystery, and here we examine the molecular network underlying sex determination in gonads in vitro. We use a whole organ culture system to show that expression of putative members of the sex-determining network (Dmrt1, Sox9, Mis, and FoxL2) are regulated by temperature endogenously within cells in the bipotential gonad and do not require other embryonic tissues to be expressed in a normal pattern in the red-eared slider turtle, Trachemys scripta. Furthermore, following a change in temperature, these factors exhibit temperature-responsive expression patterns that last for the duration of gonadogenesis. Finally, mosaic misexpression of a fusion Sox9 construct demonstrates the ability to functionally manipulate the gonad at the molecular level.