BackgroundThe 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.ResultsOur 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.ConclusionsGonadal 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2396-9) contains supplementary material, which is available to authorized users.

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.

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.

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.

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 ARβ) 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.

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 8C or MPT) and a female-producing temperature (31 8C or FPT). First, eggs at the beginning of the TSP (stage 16) were topically treated with the steroid hormones 17b-estradiol (E 2 ), testosterone in combination with aromatase inhibitor (AICT), the E 2 antagonist (ICI 182 780), and the androgen antagonist (flutamide). Secondly, gonads were removed at stage 16 and treated in vitro with E 2 , AICT, or hormone antagonists. At the FPT, AICT in ovo suppressed aromatase and Rspo1, while activating Sox9. At the MPT, E 2 treatment rapidly increased aromatase and Rspo1, while suppressing Sox9. Treatment with the E 2 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 AICT at FPT decreased aromatase and Rspo1 and E 2 increased the expression of these genes at MPT. In vitro treatment with E 2 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.

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.

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 TRβ) 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 TRβ) and thyroxine (T4 : 10(-9) M in TRα and 10(-8) M in TRβ). 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.