Satomi Kohno

Sequencing three crocodilian genomes to illuminate the evolution of archosaurs and amniotes

The International Crocodilian Genomes Working Group (ICGWG) will sequence and assemble the American alligator (Alligator mississippiensis), saltwater crocodile (Crocodylus porosus) and Indian gharial (Gavialis gangeticus) genomes. The status of these projects and our planned analyses are described.

Molecular cloning of an anuran V2 type [Arg8] vasotocin receptor and mesotocin receptor: functional characterization and tissue expression in the Japanese tree frog (Hyla japonica)

In most amphibians, [Arg(8)] vasotocin (VT) has an antidiuretic effect that is coupled to the activation of adenylate cyclase. In contrast, mesotocin (MT) has a diuretic effect and acts via the inositol phosphate/calcium signaling pathway in amphibians. To further clarify the mechanisms of VT and MT activation, we report the molecular cloning of a VT receptor (VTR) and a MT receptor (MTR) from the Japanese tree frog, Hyla japonica. Tree frog VTR or MTR cDNA encoded 363 or 389 amino acids, and their amino acid sequences revealed close similarity to the mammalian vasopressin V(2) (51-52% identity) or toad MT (94% identity) receptors, respectively. Using CHO-K1 cells transfected with tree frog VTR, we observed elevated concentrations of intracellular cAMP following exposure of the cells to VT or other neurohypophysial hormones, whereas the cells transfected with MTR did not exhibit altered cAMP concentrations. The cells transfected with VTR exhibited the following efficiency for cAMP accumulation: VT = hydrin 1 > or = vasopressin > or = hydrin 2 > MT = oxytocin > isotocin. VTR or MTR mRNA exhibits a single 2.2- or 5.5-kb transcription band, respectively, and both are expressed in various tissues. VTR mRNA is clearly expressed in brain, heart, kidney, pelvic patch of skin, and urinary bladder, whereas brain, fat body, heart, kidney, and urinary bladder express MTR mRNA. Specifically, VTR mRNA in the pelvic patch or MTR mRNA in the dorsal skin is present at elevated levels in the skin. Characteristic distribution of VTR and MTR on osmoregulating organs indicates the ligands for these receptors would mediate a variety of functions. Further, the distribution of VTR in the skin would make the regional difference on cutaneous water absorption in response to VT in the Japanese tree frog.

Potential Contributions of Heat Shock Proteins to Temperature-Dependent Sex Determination in the American Alligator

Sex determination in the American alligator depends on the incubation temperature experienced during a thermo-sensitive period (TSP), although sex determination can be ‘reversed’ by embryonic exposure to an estrogenic compound. Thus, temperature and estrogenic signals play essential roles during temperature-dependent sex determination (TSD). The genetic basis for TSD is poorly understood, although previous studies observed that many of the genes associated with genetic sex determination (GSD) are expressed in species with TSD. Heat shock proteins (HSPs), good candidates because of their temperature-sensitive expression, have not been examined in regard to TSD but HSPs have the ability to modify steroid receptor function. A number of HSP cDNAs (HSP27, DNAJ, HSP40, HSP47, HSP60, HSP70A, HSP70B, HSP70C, HSP75, HSP90α, HSP90β, and HSP108) as well as cold-inducible RNA binding protein (CIRBP) and HSP-binding protein (HSPBP) were cloned, and expression of their mRNA in the gonadal-adrenal-mesonephros complex (GAM) was investigated. Embryonic and neonatal GAMs exhibited mRNA for all of the HSPs examined during and after the TSP. One-month-old GAMs were separated into 3 portions (gonad, adrenal gland, and mesonephros), and sexual dimorphism in the mRNA expression of gonadal HSP27 (male > female), gonadal HSP70A (male < female), and adrenal HSP90α (male > female) was observed. These findings provide new insights on TSD and suggest that further studies examining the role of HSPs during gonadal development are needed.

Water temperature and concomitant waterborne ethinylestradiol exposure affects the vitellogenin expression in juvenile brown trout (Salmo trutta)

Environmental estrogens have the potential to considerably affect the reproduction and development of aquatic vertebrates by interfering with the endocrine system. In addition to the potential risk of environmental estrogens, increasing water temperatures as a result of global warming have become a serious problem in many rivers and streams. To assess the degree of estrogenic exposure, the analysis of the estrogen-dependent protein vitellogenin (Vtg) is a frequently used biomarker in field studies. Little, however, is known regarding the potential interaction between ambient water temperature and the Vtg production induced by waterborne environmental estrogens. In order to test the influence of temperature on Vtg synthesis, we exposed juvenile brown trout to an environmentally relevant concentration of ethinylestradiol (EE(2)) and held them either at low or high temperatures (12 and 19 degrees C, respectively), but also at temperature cycles of 12-19 degrees C in order to simulate the field situation. The EE(2) exposure caused a 7-74-fold increase of hepatic Vtg mRNA. The synthesis of Vtg mRNA was clearly stimulated in fish held at higher water temperatures (12-19 degrees C and 19 degrees C, respectively). On the protein level, Vtg showed a similar pattern; the higher the temperature, the higher the concentration of Vtg in the plasma. The experiment further revealed a temperature-dependent increasing amount of hepatic estrogen receptor alpha mRNA (ERalpha) after exposure to waterborne EE(2). The gene expression of estrogen receptor beta-1 (ERbeta-1) and the glucocorticoid receptor (GR) in the liver of EE(2) exposed fish, however, showed no treatment-related alterations. In line with observed constant bile cortisol concentrations, our data do not indicate corresponding stress related effects on hepatic Vtg production. The present survey, however, clearly demonstrates that increased temperature significantly elevates the estrogen-induced expression of Vtg and therefore has to be considered when interpreting environmental monitoring studies.

Differential Incubation Temperatures Result in Dimorphic DNA Methylation Patterning of the SOX9 and Aromatase Promoters in Gonads of Alligator (Alligator mississippiensis) Embryos

ABSTRACT Environmental factors are known to influence sex determination in many nonmammalian vertebrates. In all crocodilians studied thus far, temperature is the only known determinant of sex. However, the molecular mechanisms mediating the effect of temperature on sex determination are not known. Aromatase (CYP19A1) and SOX9 play critical roles in vertebrate sex determination and gonadogenesis. Here, we used a variety of techniques to investigate the potential roles of DNA methylation patterning on CYP19A1 and SOX9 expression in the American alligator, an organism that relies on temperature-dependent sex determination. Our findings reveal that developing gonads derived from embryos incubated at a male-producing temperature (MPT) show elevated CYP19A1 promoter methylation and decreased levels of gene expression relative to incubation at a female-producing temperature (FPT). The converse was observed at the SOX9 locus, with increased promoter methylation and decreased expression occurring in embryonic gonads resulting from incubation at FPT relative to that of MPT. We also examined the gonadal expression of the three primary, catalytically active DNA methyltransferase enzymes and show that they are present during critical stages of gonadal development. Together, these data strongly suggest that DNA methylation patterning is a central component in coordinating the genetic cascade responsible for sexual differentiation. In addition, these data raise the possibility that DNA methylation could act as a key mediator integrating temperature into a molecular trigger that determines sex in the alligator.

Increased Posthatching Mortality and Loss of Sexually Dimorphic Gene Expression in Alligators (Alligator mississippiensis) from a Contaminated Environment

A previous study from our laboratory examining development in neonatal alligators from polluted Lake Apopka, Florida, found numerous differences relative to neonates from a reference site, Lake Woodruff National Wildlife Refuge. We postulated that the differences were the result of organizational changes derived from embryonic exposure to environmental contaminants and are related to the poor reproductive success reported in alligators from Lake Apopka. In this study we examine differences in alligators collected as eggs from these two populations and raised under similar conditions for 1 yr. Egg hatch rates did not differ between lake populations; however, posthatching mortality was much higher among Lake Apopka hatchlings. Snout-vent length and body mass were greater in Lake Apopka hatchlings, but no differences were detected between lake populations in thyroid, liver, and spleen mass corrected for body size or in plasma concentrations of testosterone and estradiol. Males from Lake Woodruff exhibited greater relative expression of gonadal mRNA for steroidogenic factor 1 (Nr5a1) and steroidogenic acute regulatory protein (Star) than males from Lake Apopka. Alligators from Lake Woodruff also expressed all genes examined in a sexually dimorphic pattern. In contrast, mRNA expression did not differ between males and females from Lake Apopka for Nr5a1, Star, cytochrome P450 11A1 (Cyp11a1), and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (Hsd3b1). Our results document persistent differences in development, survivorship, and gene expression in alligators from a contaminated environment. Because these animals were raised under similar laboratory conditions, the differences are most likely of embryonic origin and organizational in nature.

Gene expression patterns in juvenile American alligators (Alligator mississippiensis) exposed to environmental contaminants.

Reproductive and developmental abnormalities have been reported in the American alligator (Alligator mississippiensis) population from Lake Apopka, FL, that is chronically exposed to a complex mixture of environmental contaminants. To begin to understand the molecular mechanisms that could lead to the observed abnormalities of the reproductive and endocrine system, we quantified concentrations of the steroid hormones testosterone (T) and estradiol-17beta (E(2)) and expression of steroid hormone receptors and genes relating to steroidogenesis in gonadal tissue from juvenile alligators from three lakes in Florida using enzyme immunoassay and quantitative real-time polymerase chain reaction. Alterations of ESR2 (estrogen receptor beta) and SF1 (steroidogenic factor 1) mRNA expression in male gonadal tissue, without an observed difference in plasma concentrations of T, from the different lakes, begin to provide insight into potential mechanisms underlying the alterations of the reproductive system previously observed. Likewise, alterations in P450 aromatase and DAX1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita critical region on the X chromosome, gene 1) mRNA expression, with elevated plasma E(2) concentrations in females, provide leads to the potential mechanisms modifying folliculogenesis and ovarian development. The investigation of these genes also helps clarify normal endocrine and reproductive system function in the American alligator.

Developmental toxicity of estrogenic chemicals on rodents and other species

ABSTRACT  Antenatal sex‐hormone exposure induces lesions in mouse reproductive organs, which are similar to those in humans exposed in utero to a synthetic estrogen, diethylstilbestrol. The developing organisms including rodents, fish and amphibians are particularly sensitive to exposure to estrogenic chemicals during a critical window. Exposure to estrogens during the critical period induces long‐term changes in reproductive as well as non‐reproductive organs, including persistent molecular alterations. The antenatal mouse model can be utilized as an indicator of possible long‐term consequences of exposure to exogenous estrogenic compounds including possible environmental endocrine disrupters. Many chemicals released into the environment potentially disrupt the endocrine system in wildlife and humans, some of which exhibit estrogenic activity by binding to the estrogen receptors. Estrogen responsive genes, therefore, need to be identified to understand the molecular basis of estrogenic actions. In order to understand molecular mechanisms of estrogenic chemicals on developing organisms, we are identifying estrogen responsive genes using cDNA microarray, quantitative RT‐PCR, and differential display methods, and genes related to the estrogen‐independent vaginal changes in mice induced by estrogens during the critical window. In this review, discussion of our own findings related to endocrine distuptor issue will be provided.

Molecular Cloning, Characterization, and Chromosome Mapping of Reptilian Estrogen Receptors

In many vertebrates, steroid hormones are essential for ovarian differentiation during a critical developmental stage as well as promoting the growth and differentiation of the adult female reproductive system. Although studies have been extensively conducted in mammals and a few fish, amphibians, and bird species, the molecular mechanisms of sex steroid hormone (estrogens) action have been poorly examined in reptiles. Here, we evaluate hormone receptor and ligand interactions in two species of snake, the Okinawa habu (Protobothrops flavoviridis, Viperidae) and the Japanese four-striped rat snake (Elaphe quadrivirgata, Colubridae) after the isolation of cDNAs encoding estrogen receptor α (ESR1) and estrogen receptor β (ESR2). Using a transient transfection assay with mammalian cells, the transcriptional activity of reptilian (Okinawa habu, Japanese four-striped rat snake, American alligator, and Florida red-belly freshwater turtle) ESR1 and ESR2 was examined. All ESR proteins displayed estrogen-dependent activation of transcription via an estrogen-response element-containing promoter; however, the responsiveness to various estrogens was different. Further, we determined the chromosomal locations of the snake steroid hormone receptor genes. ESR1 and ESR2 genes were localized to the short and long arms of chromosome 1, respectively, whereas androgen receptor was localized to a pair of microchromosomes in the two snake species examined. These data provide basic tools that allow future studies examining receptor-ligand interactions and steroid endocrinology in snakes and also expands our knowledge of sex steroid hormone receptor evolution.

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.