Genetic Polymorphisms in Aromatase (cyp19a1) Are Not Associated with Gonadal Phenotypes in Red-Eared Slider Turtle Hatchlings Developed at a Pivotal Temperature.

Embryonic gonad sex in many reptilian species is determined by the incubation temperature of the egg, a differentiation process known as temperature-dependent sex determination (TSD). Incubation at the pivotal temperature (PvT) results in approximately an equal number of offspring of both sexes. We investigated the potential contribution of genetic variations that drives the gonadal differentiation into testes or ovaries under this temperature in the red-eared slider turtle (Trachemys scripta). Four male and 4 female hatchlings of eggs that had been incubated at the PvT were examined for polymorphisms at an approximately 23-kb region of the aromatase (cyp19a1) gene. By aligning the red-eared slider aromatase gene to a reference genome of the western painted turtle (Chrysemys picta bellii), we discovered 22 exonic and 1,268 intronic polymorphisms. Of these, 12 (55%) exonic polymorphisms were unique to the individuals of the red-eared slider; 10 were synonymous and 2 were nonsynonymous changes. We found no pattern in these genetic variants as well as intronic variants that are consistently different between male and female hatchlings at the PvT. Overall, our study suggests that polymorphisms within the aromatase gene - at least by themselves - do not constrain the gonad sex differentiation in embryos developed at the PvT.

Temperature Shift Alters DNA Methylation and Histone Modification Patterns in Gonadal Aromatase (cyp19a1) Gene in Species with Temperature-Dependent Sex Determination.

The environment surrounding the embryos has a profound impact on the developmental process and phenotypic outcomes of the organism. In species with temperature-dependent sex determination, gonadal sex is determined by the incubation temperature of the eggs. A mechanistic link between temperature and transcriptional regulation of developmental genes, however, remains elusive. In this study, we examine the changes in DNA methylation and histone modification patterns of the aromatase (cyp19a1) gene in embryonic gonads of red-eared slider turtles (Trachemys scripta) subjected to a temperature shift during development. Shifting embryos from a male-producing temperature (MPT) to a female-producing temperature (FPT) at the beginning of the temperature-sensitive period (TSP) resulted in an increase in aromatase mRNA expression while a shift from FPT to MPT resulted in decreased expression. DNA methylation levels at CpG sites in the promoter of the aromatase gene were high (70-90%) at the beginning of TSP, but decreased in embryos that were incubated at constant FPT and those shifted from MPT to the FPT. This decrease in methylation in the promoter inversely correlated with the expected increase in aromatase expression at the FPT. The active demethylation under the FPT was especially prominent at the CpG site upstream of the gonad-specific TATA box at the beginning of TSP and spread downstream of the gene including exon1 as the gonad development progressed. In embryos incubated at FPT, the promoter region was also labeled by canonical transcriptional activation markers, H3K4me3 and RNA polymerase II. A transcriptional repression marker, H3K27me3, was observed in temperature-shifted gonads of both temperature groups, but was not maintained throughout the development in either group. Our findings suggest that DNA hypomethylation and H3K4me3 modification at the aromatase promoter may be a primary mechanism that releases a transcriptional block of aromatase to initiate a cascade of ovarian differentiation.

Hormone-induced and DNA demethylation-induced relief of a tissue-specific and developmentally regulated block in transcriptional elongation.

Genome-wide studies have revealed that genes commonly have a high density of RNA polymerase II just downstream of the transcription start site. This has raised the possibility that genes are commonly regulated by transcriptional elongation, but this remains largely untested in vivo, particularly in vertebrates. Here, we show that the proximal promoter from the Rhox5 homeobox gene recruits polymerase II and begins elongating in all tissues and cell lines that we tested, but it only completes elongation in a tissue-specific and developmentally regulated manner. Relief of the elongation block is associated with recruitment of the elongation factor P-TEFb, the co-activator GRIP1, the chromatin remodeling factor BRG1, and specific histone modifications. We provide evidence that two mechanisms relieve the elongation block at the proximal promoter: demethylation and recruitment of androgen receptor. Together, our findings support a model in which promoter proximal pausing helps confer tissue-specific and developmental gene expression through a mechanism regulated by DNA demethylation-dependent nuclear hormone receptor recruitment.

Embryonic PCB exposure alters phenotypic, genetic, and epigenetic profiles in turtle sex determination, a biomarker of environmental contamination.

In species with temperature-dependent sex determination, embryonic gonadal differentiation can be modified by exposure to exogenous chemicals such as environmental contaminants. Although phenotypic outcomes of such events are well documented, the underlying molecular mechanisms are rarely described. Here we examine the genetic and epigenetic effect of the embryonic exposure to polychlorinated biphenyls (PCBs) on gonad differentiation in red-eared slider turtles (Trachemys scripta). Some PCB congeners are without effect whereas others synergize to alter sex determination in this species. Application of two potent PCB congeners alter the physiological processes of gonad development normally dictated by the male-producing temperature (MPT), resulting sex ratios significantly biased toward female hatchlings. Of these PCB-induced females, oviduct formation is prominently distorted regardless of ovary development. Further, gonadal expression of ovarian markers, aromatase, FoxL2, and Rspo1, is activated whereas testicular markers, Dmrt1 and Sox9, are suppressed compared with typical expression patterns observed at MPT. DNA methylation profiles of the aromatase promoter in PCB-treated gonads do not follow the typical methylation pattern observed in embryos incubating at female-producing temperature. Rather, the MPT-typical methylation profiles is retained despite the induced ovarian formation. Overall, our studies demonstrate that PCB exposure alters the transcriptional profiles of genes responsible for gonadal differentiation but does not re-establish the epigenetic marks of the aromatase promoter normally set by incubation temperatures in embryonic gonads.

Transdermal delivery of flurbiprofen from surfactant-based vesicles: particle characterization and the effect of water on in vitro transport.

Flurbiprofen loaded rigid and elastic vesicles comprising the bilayer-forming surfactant sucrose-ester laurate were prepared by the film rehydration and extrusion method. The charge-inducing agent sodium dodecyl sulfate, and the micelle-forming surfactants, sorbitan monolaurate, polyethylene glycol monolaurate, and polysorbate 20, were used to enhance elasticity. Vesicle formulations were evaluated for size, zeta potential, (1)H and (19)F nuclear magnetic resonance (NMR) spectra, and in vitro skin permeation across Yucatan micropig (YMP) skin. Vesicle formulations were stable for 2 weeks and their mean sizes were 95-135 nm. NMR spectroscopy showed that flurbiprofen molecular mobility was restricted by interaction with vesicle components because of entrapment in vesicle bilayers. Moreover, sorbitan monolaurate-containing vesicles strongly retained flurbiprofen molecules. After non-occlusive application to YMP skin, flurbiprofen transport from all vesicle formulations was superior to that of flurbiprofen alone and remarkably decreased after water vaporization. Polarization microscopy and small-angle X-ray diffraction analysis showed that the vesicle formulation was transferred to liquid crystalline state. Suppression of vesicle transition to the liquid crystalline state was observed with applications of both large quantities and diluted samples. The presence of water in the formulations was associated with maintenance of the vesicle structure and greater flurbiprofen transport across YMP skin.

Epigenetic control of gonadal aromatase (cyp19a1) in temperature-dependent sex determination of red-eared slider turtles.

In the red-eared slider turtle (Trachemys scripta), a species with temperature-dependent sex determination (TSD), the expression of the aromatase gene during gonad development is strictly limited to the female-producing temperature. The underlying mechanism remains unknown. In this study, we identified the upstream 5'-flanking region of the aromatase gene, gonad-specific promoter, and the temperature-dependent DNA methylation signatures during gonad development in the red-eared slider turtle. The 5'-flanking region of the slider aromatase exhibited sequence similarities to the aromatase genes of the American alligator, chicken, quail, and zebra finch. A putative TATA box was located 31 bp upstream of the gonad-specific transcription start site. DNA methylation at the CpG sites between the putative binding sites of the fork head domain factor (FOX) and vertebrate steroidogenic factor 1 (SF1) and adjacent TATA box in the promoter region were significantly lower in embryonic gonads at the female-producing temperature compared the male-producing temperature. A shift from male- to female-, but not from female- to male-, producing temperature changed the level of DNA methylation in gonads. Taken together these results indicate that the temperature, particularly female-producing temperature, allows demethylation at the specific CpG sites of the promoter region which leads the temperature-specific expression of aromatase during gonad development.

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.

Molecular mechanisms of temperature-dependent sex determination in the context of ecological developmental biology.

Temperature-dependent sex determination (TSD) is a prime example of phenotypic plasticity in that gonadal sex is determined by the temperature of the incubating egg. In the red-eared slider turtle (Trachemys scripta), the effect of temperature can be overridden by exogenous ligands, i.e., sex steroid hormones and steroid metabolism enzyme inhibitors, during the temperature-sensitive period (TSP) of development. Precisely how the physical signal of temperature is transduced into a biological signal that ultimately results in sex determination remains unknown. In this review, we discuss the sex determining pathway underlying TSD by focusing on two candidate sex determining genes, Forkhead box protein L2 (FoxL2) and Doublesex mab3- related transcription factor 1 (Dmrt1). They appear to be involved in transducing the environmental temperature signal into a biological signal that subsequently determines gonadal sex. FoxL2 and Dmrt1 exhibit gonad-typical patterns of expression in response to temperature during the TSP in the red-eared slider turtle. Further, the biologically active ligands regulate the expression of FoxL2 and Dmrt1 during development to modify gonad trajectory. The precise regulatory mechanisms of expression of these genes by temperature or exogenous ligands are not clear. However, the environment often influences developmental gene expression by altering the epigenetic status in regulatory regions. Here, we will discuss if the regulation of FoxL2 and Dmrt1 expression by environment is mediated through epigenetic mechanisms during development in species with TSD.

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.