Temperature-dependent expression of turtle Dmrt1 prior to sexual differentiation

Vertebrates employ varied strategies, both chromosomal and nonchromosomal, to determine the sex of the developing embryo. Among reptiles, temperature-dependent sex determination (TSD) is common. The temperature of incubation during a critical period preceding sexual differentiation determines the future sex of the embryo, presumably by altering the activity or expression of a temperature-dependent regulatory factor(s). Here we examine the expression of the Dmrt1 gene, a candidate regulator of mammalian and avian sexual development, in the turtle. During the sex-determining period, Dmrt1 mRNA is more abundant in genital ridge/mesonephros complexes at male-promoting than at female-promoting temperatures. Dmrt1 is the first gene found to show temperature-dependent expression prior to sexual differentiation, and may play a key role in sexual development in reptiles. genesis 26:174-178, 2000.     

Characterisation and expression of Sox9 in the Leopard gecko, Eublepharis macularius

Since the discovery of the sex-determining gene, Sry, a number of genes have been identified which are involved in sex determination and gonadogenesis in mammals. Although Sry is known to be the testis-determining factor in mammals, this is not the case in non-mammalian vertebrates. Sox9 is another gene that has been shown to have a male-specific role in sex determination, but, unlike Sry, Sox9 has been shown to be involved in sex determination in mammals, birds, and reptiles. This is the first gene to be described that has a conserved role in sex determination in species with either chromosomal or environmental sex-determining mechanisms. Many reptiles do not have sex chromosomes but exhibit temperature-dependent sex determination (TSD). Sox9 has been shown to be expressed in both turtle and alligator during gonadogenesis. To determine if Sox9 also has a role in a gecko species with TSD, we studied gonadal expression of Sox9 during embryonic development of the Leopard gecko (Eublepharis macularius). Gecko Sox9 was found to be highly conserved at the nucleotide level when compared to other vertebrate species including human, chick, alligator, and turtle. Sox9 was found to be expressed in embryos incubated at the male-producing temperature (32.5 degrees C) as well as in embryos incubated at the female-producing temperatures (26 and 34 degrees C), Northern blot analysis showed that Sox9 was expressed at both temperatures from morphological stages 31 to 37. mRNA in situ hybridisation on isolated urogenital systems showed expression at both female- and male-producing temperatures up to stage 36. After this stage, no expression was seen in the female gonads but expression remained in the male. These data provide further evidence that Sox9 is an essential component of a testis-determining pathway that is conserved in species with differing sex-determining mechanisms.     

Temperature-dependent sex determination in reptiles: Proximate mechanisms,ultimate outcomes,and practical applications

In many egg-laying reptiles, the incubation temperature of the egg determines the sex of the offspring, a process known as temperature-dependent sex determination (TSD). In TSD sex determination is an “all or none” process and intersexes are rarely formed. How is the external signal of temperature transduced into a genetic signal that determines gonadal sex and channels sexual development? Studies with the red-eared slider turtle have focused on the physiological, biochemical, and molecular cascades initiated by the temperature signal. Both male and female development are active processes—rather than the crganized/default system characteristic of vertebrates with genotypic sex determination—that require simultaneous activation and suppression of testis- and ovary-determining cascades for normal sex determination. It appears that temperature accomplishes this end by acting on genes encoaing for steroidogenic enzymes and steroid hormone receptors and modifying the endocrine microenvironment in the embryo. The temperature experienced in development also has long-term functional outcomes in addition to sex determination. Research with the leopard gecko indicates that incubation temperature as well as steroid hormones serve as organizers in shaping the adult phenotype, with temperature modulating sex hormone action in sexual differentiation. Finally, practical applications of this research have emerged for the conservation and restoration of endangered egg-laying reptiles as well as the embryonic development of reptiles as biomarkers to monitor the estrogenic effects of common environmental contaminants. © 1994 Wiley-Liss, Inc.     

Environmental sex determination in reptiles: ecology, evolution, and experimental design

Sex-determining mechanisms in reptiles can be divided into two convenient classifications: genotypic (GSD) and environmental (ESD). While a number of types of GSD have been identified in a wide variety of reptilian taxa, the expression of ESD in the form of temperature-dependent sex determination (TSD) in three of the five major reptilian lineages has drawn considerable attention to this area of research. Increasing interest in sex-determining mechanisms in reptiles has resulted in many data, but much of this information is scattered throughout the literature and consequently difficult to interpret. It is known, however, that distinct sex chromosomes are absent in the tuatara and crocodilians, rare in amphisbaenians (worm lizards) and turtles, and common in lizards and snakes (but less than 20% of all species of living reptiles have been karyotyped). With less than 2 percent of all reptilian species examined, TSD apparently is absent in the tuatara, amphisbaenians and snakes; rare in lizards, frequent in turtles, and ubiquitous in crocodilians. Despite considerable inter- and intraspecific variation in the threshold temperature (temperature producing a 1:1 sex ratio) of gonadal sex determination, this variation cannot confidently be assigned a genetic basis owing to uncontrolled environmental factors or to differences in experimental protocol among studies. Laboratory studies have identified the critical period of development during which gonadal sex determination occurs for at least a dozen species. There are striking similarities in this period among the major taxa with TSD. Examination of TSD in the field indicates that sex ratios of hatchlings are affected by location of the nests, because some nests produce both sexes whereas the majority produce only one sex. Still, more information is needed on how TSD operates under natural conditions in order to fully understand its ecological and conservation implications. TSD may be the ancestral sex-determining condition in reptiles, but this result remains tentative. Physiological investigations of TSD have clarified the roles of steroid hormones, various enzymes, and H-Y antigen in sexual differentiation, whereas molecular studies have identified several plausible candidates for sex-determining genes in species with TSD. This area of research promises to elucidate the mechanism of TSD in reptiles and will have obvious implications for understanding the basis of sex determination in other vertebrates. Experimental and comparative investigations of the potential adaptive significance of TSD appear equally promising, although much work remains to be performed. The distribution of TSD within and among the major reptilian lineages may be related to the life span of individuals of a species and to the biogeography of these species.(ABSTRACT TRUNCATED AT 400 WORDS)     

AMONG-FAMILY VARIATION FOR ENVIRONMENTAL SEX DETERMINATION IN REPTILES

Unlike birds and mammals, in many reptiles the temperature experienced by a developing embryo determines its gonadal sex. To understand how temperature-dependent sex determination (TSD) evolves, we must first determine the nature of genetic variation for sex ratio. Here, we analyze among-family variation for sex ratio in three TSD species: the American alligator (Alligator mississipiensis), the common snapping turtle (Chelydra serpentina) and the painted turtle (Chrysemys picta). Significant family effects and significant temperature effects were detected in all three species. In addition, family-by-temperature interactions were evident in the alligator and the snapping turtle, but not in the painted turtle. Overall, the among-family variation detected in this study indicates potential for sex-ratio evolution in at least three reptiles with TSD. Consequently, climate change scenarios that are posited on the presumption that sex-ratio evolution in TSD reptiles is genetically constrained may require reevaluation.     

Sexual differentiation in the spiny softshell turtle (Apalone spinifera), a species with genetic sex determination

It is hypothesized on the basis of sex determination theory that species exhibiting genetic sex determination (GSD) may undergo sexual differentiation earlier in development than species with environmental sex determination (ESD). Most turtle species exhibit a form of ESD known as temperature-dependent sex determination (TSD), and in such species the chronology of sex differentiation is well studied. Apalone spinifera is a species of softshell turtle (Trionychidae) that exhibits GSD. We studied sexual differentiation in this species in order to facilitate comparison to TSD species. Eggs were incubated at two different temperatures and embryos were harvested at various stages of mid to late development. Gonad length was measured with image analysis software, then prepared histologically. Indifferent gonads have differentiated in stage 19 embryos. Histological details of gonadogenesis follow the same pattern as described for other reptiles. Regression of the male paramesonephric duct closely follows testicular differentiation. Gonad lengths are longer at the warmer incubation temperature, and ovaries are generally longer than testes at each stage and for each temperature. Although sexual differentiation takes place at about the same stage as in other turtles with TSD (18-20), in A. spinifera this differentiation is irreversible at this stage, while in some of the TSD species sex is reversible until about stage 22. This immutable, definitive sexual differentiation may support the hypothesis of an accelerated chronology of sex differentiation for this species. We also note that sexual dichromatism at hatching is known in this species and may provide additional evidence of early differentiation. J. Exp. Zool. 290:190-200, 2001.     

Cortisol-Induced Masculinization: Does Thermal Stress Affect Gonadal Fate in Pejerrey,a Teleost Fish with Temperature-Dependent Sex Determination?

Background

Gonadal fate in many reptiles, fish, and amphibians is modulated by the temperature experienced during a critical period early in life (temperature-dependent sex determination; TSD). Several molecular processes involved in TSD have been described but how the animals “sense” environmental temperature remains unknown. We examined whether the stress-related hormone cortisol mediates between temperature and sex differentiation of pejerrey, a gonochoristic teleost fish with marked TSD, and the possibility that it involves glucocorticoid receptor- and/or steroid biosynthesis-modulation.

Methodology/Principal Findings

Larvae maintained during the period of gonadal sex differentiation at a masculinizing temperature (29°C; 100% males) consistently had higher cortisol, 11-ketotestoterone (11-KT), and testosterone (T) titres than those at a feminizing temperature (17°C; 100% females). Cortisol-treated animals had elevated 11-KT and T, and showed a typical molecular signature of masculinization including amh upregulation, cyp19a1a downregulation, and higher incidence of gonadal apoptosis during sex differentiation. Administration of cortisol and a non-metabolizable glucocorticoid receptor (GR) agonist (Dexamethasone) to larvae at a “sexually neutral” temperature (24°C) caused significant increases in the proportion of males.

Conclusions/Significance

Our results suggest a role of cortisol in the masculinization of pejerrey and provide a possible link between stress and testicular differentiation in this gonochoristic TSD species. Cortisol role or roles during TSD of pejerrey seem(s) to involve both androgen biosynthesis- and GR-mediated processes. These findings and recent reports of cortisol effects on sex determination of sequential hermaphroditic fishes, TSD reptiles, and birds provide support to the notion that stress responses might be involved in various forms of environmental sex determination.     

Molecular evolution of Dmrt1 accompanies change of sex-determining mechanisms in reptilia

In reptiles, sex-determining mechanisms have evolved repeatedly and reversibly between genotypic and temperature-dependent sex determination. The gene Dmrt1 directs male determination in chicken (and presumably other birds), and regulates sex differentiation in animals as distantly related as fruit flies, nematodes and humans. Here, we show a consistent molecular difference in Dmrt1 between reptiles with genotypic and temperature-dependent sex determination. Among 34 non-avian reptiles, a convergently evolved pair of amino acids encoded by sequence within exon 2 near the DM-binding domain of Dmrt1 distinguishes species with either type of sex determination. We suggest that this amino acid shift accompanied the evolution of genotypic sex determination from an ancestral condition of temperature-dependent sex determination at least three times among reptiles, as evident in turtles, birds and squamates. This novel hypothesis describes the evolution of sex-determining mechanisms as turnover events accompanied by one or two small mutations.     

Evolution of the gene network underlying gonadogenesis in turtles with temperature-dependent and genotypic sex determination

The evolution of sex determination has long fascinated biologists, as it has paramount consequences for the evolution of a multitude of traits, from sex allocation to speciation and extinction. Explaining the diversity of sex-determining systems found in vertebrates (genotypic or GSD and temperature-dependent or TSD) requires a comprehensive and integrative examination from both a functional and an evolutionary perspective. Particularly revealing is the examination of the gene network that regulates gonadogenesis. Here, I review some advances in this field and propose some additional hypotheses about the composition of the gene network underlying sexual development, the functional links among some of its elements and their evolution in turtles. I focus on several pending questions about: (1) What renders TSD systems thermo-sensitive? (2) Is there one developmentally conserved or multiple TSD mechanisms? (3) Have evolutionarily derived GSD species lost all ancestral thermal-sensitivity? New data are presented on embryonic expression of Dax1 (the dosage-sensitive sex-reversal adrenal hypoplasia congenital on the X chromosome gene in the turtles Chrysemys picta (TSD) and Apalone mutica (GSD). No differential Dax1 expression was detected in C. picta at any of the stages examined, consistent with reports on two other TSD turtles and alligators. Notably, significantly higher Dax1 expression was found at 30°C than at 25°C at stage 15 in A. mutica (GSD), likely caused by Wt1's identical expression pattern previously reported. Because Sf1 is an immediate downstream target of Dax1 and its expression is not affected by temperature, it is proposed that Sf1 renders Dax1's differential signal ineffective to induce biased sex ratios in A. mutica, as previously proposed for Wt1's thermosensitive expression. Thus, it is hypothesized that Sf1 plays a major role in the lack of response of sex ratio to temperature of A. mutica, and may function as a sex-determining gene in this GSD species. These and previous data permit formulating several mechanistic hypotheses: (1) the postulation of Wt1 as a candidate thermal master switch alone, or in combination with Sf1, in the TSD turtle C. picta; (2) the proposition of Sf1 as a sex-determining gene in the GSD turtle A. mutica; and (3) the hypothesis that differing patterns of gene expression among TSD taxa reflect multiple traits from a developmental perspective. Moreover, the recent finding of relic differential Wt1 expression in A. mutica and the results for Dax1 in this species provide empirical evidence that GSD taxa can harbor thermal sensitivity at the level of gene expression, potentially co-optable during TSD evolution.     

Embryological ontogeny of aromatase gene expression in Chrysemys picta and Apalone mutica turtles: comparative patterns within and across temperature-dependent and genotypic sex-determining mechanisms

Although the role of aromatase in many estrogen-dependent reproductive and metabolic functions is well documented in vertebrates, its involvement in the ovarian development of species exhibiting temperature-dependent sex determination (TSD) is incompletely understood. This is partly due to the conflicting temporal and spatial pattern of aromatase expression and activity across taxa. To help resolve this ongoing debate, we compared for the first time the embryological ontogeny of aromatase expression in turtles possessing genotypic sex determination (GSD) (Apalone mutica) and TSD (Chrysemys picta) incubated under identical conditions. As anticipated, we found no significant thermal differences in aromatase expression at any stage examined (prior to until the end of the thermosensitive period) in A. mutica. Surprisingly, the same was true for C. picta. When placed in a phylogenetic context, our results suggest that aromatase expression is evolutionarily plastic with respect to sex determination in reptiles, and that differences between reptilian TSD and GSD are not aromatase-driven. Further research across TSD and GSD species is warranted to fully decipher the evolution of functional differences among sex-determining mechanisms.     

中华鳖Dmrt1基因的克隆、表达及在性逆转中的变化分析

在大部分脊椎动物中,Dmrt1基因在雄性性别决定和性腺分化中起重要的调控作用.本文从m RNA和蛋白水平分析Dmrt1基因的组织差异性表达、在不同发育阶段性腺中的细胞定位及在性逆转中的表达变化,研究Dmrt1基因在中华鳖性别分化中的调控作用.Rapid-amplification of c DNA ends(RACE)结果显示,Dmrt1基因c DNA序列全长2409 bp,其中5′非编码区为230 bp,3′非编码区为1072 bp,开放阅读框为1107 bp,编码368个氨基酸,具有一个高度保守的DM结构域.荧光定量PCR和免疫组化结果显示,Dmrt1在性腺分化之前的第16期雄性性腺中开始表达,先于Amh和Sox9基因表达.随着性腺的发育,Dmrt1蛋白主要定位于性腺Sertoli细胞的细胞核上,在雌性性腺发育过程中并未见其表达.此外,在雌二醇诱导的雄性转雌性性逆转胚胎性腺中,Dmrt1表达显著下调;在芳香化酶抑制剂诱导的雌性转雄性性腺中,Dmrt1表达则显著上升.上述研究表明,Dmrt1基因是中华鳖雄性特异性基因,参与雄性性腺的发育过程,可能在中华鳖早期性别决定中起重要的调控作用.