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The relationship between CYP19A1 expression and methylation status at individual CpG dinucleotides in gonads from stage 27 embryos is shown. CYP19A1 expression levels were determined by qPCR and were plotted against the methylation status of each CpG for each individual. A and B) Linear regression analysis, including embryos incubated at both MPT and FPT (A; n ¼ 14) or only those embryos incubated at FPT, are shown (B; n ¼ 9). r 2 and P values are reported.
Source publication
Environmental factors are known to influence sex determination in many non-mammalian 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 verteb...
Contexts in source publication
Context 1
... next investigated the relationship between the expres- sion of CYP19A1 and the methylation status of individual CpGs. RNA was simultaneously extracted from those gonads used in the previous analysis and CYP19A1 transcript abundance was measured with qPCR. We performed a linear regression analysis between the methylation status at each CpG dinucleotide within the 500 base pairs upstream of the TSS and the transcript abundance for each individual (Fig. ...
Context 2
... using alligator as a sentinel species have made considerable contributions to our understanding of how chronic exposure to environmental contaminants influences reproduc- tive health [55,56]. Alligators from environments contaminat- ed with endocrine-disrupting compounds display disorders of the reproductive system that resemble well-documented human reproductive disorders including alterations in circulating sex hormone levels, morphological abnormalities of ovarian follicles, and a decreased robustness of sexually dimorphic gene expression [57][58][59]. Interestingly, ovarian expression of CYP19A1 is decreased in hatchlings originating from a contaminated site compared to those from a relatively pristine site [60]. These alterations in gene expression likely have epigenetic underpinnings as eggs removed from contaminated environments and placed in the laboratory yield juvenile alligators in which the aforementioned abnormalities persist [61]. In light of the current study, an attractive explanation for these observations is that developmental exposures to endo- crine-disrupting compounds during TSP disrupts sexually dimorphic DNA methylation patterning, resulting in long- lasting changes to the gonadal transcriptome. Could the alteration of sexually dimorphic methylated regions, in part, explain some of the hormonal and morphological abnormalities observed in animals from contaminated sites? A considerable challenge in addressing this question is analyzing DNA methylation across a population. The combined analysis of gene expression and DNA methylation within the same tissue (Fig. 3) across many individuals should reveal individual CpG dinucleotides whose methylation status is tightly correlated to the expression of a given gene. Those CpG dinucleotides identified as being highly predictive of transcriptional activity at a specific locus could then be probed at the population level by using less costly, highly targeted approaches aimed at determining the methylation status of individual ...
Context 3
... while the lines of best fit for each CpG trended negatively, there appeared to be variations among the degrees to which the methylation status of individual CpG dinucleo- tides were related to the expression level of CYP19A1. Little to no CYP19A1 expression was observed in gonads incubated at MPT; thus, to examine this relationship between DNA methylation and CYP19A1 expression at a higher resolution beyond the on/off observed between presumptive males and females we analyzed only those gonads resulting from incubation at FPT (Fig. 3B). The negative trends observed from examination of those gonads from embryos incubated at both MPT and FPT were still present. However, a linear regression analysis revealed no significant correlations between CpG methylation and the transcriptional activity of CYP19A1, when only those gonads from embryos incubated at FPT were analyzed. These data suggest that methylation status at some CpG dinucleotides is linked to transcriptional activity at the CYP19A1 locus and that methylation at some CpG dinucleo- tides might be more tightly associated with gonadal expression of CYP19A1 than ...
Citations
... Parrott et al. [76] discovered that the methylation patterns in the CYP19A1 promoter region of the SOX9 gene demonstrate sexual dimorphism and suggested that the methylation occurs due to temperature changes. They also observed sexually dimorphic epialleles, which revealed a new differentially methylated region (DMR) upstream in an organism that undergoes temperature-dependent sex determination [76]. ...
... Parrott et al. [76] discovered that the methylation patterns in the CYP19A1 promoter region of the SOX9 gene demonstrate sexual dimorphism and suggested that the methylation occurs due to temperature changes. They also observed sexually dimorphic epialleles, which revealed a new differentially methylated region (DMR) upstream in an organism that undergoes temperature-dependent sex determination [76]. The promoter region is predicted to have a regulation role in aromatase production, but it was only described in turtles [40]. ...
... A dimorphic expression of the SOX9 gene and the AMH could be measured in the embryonic stages 21-24 in American alligators [77], so the methylation was assumed to happen before this period. The AMH and the aromatase from the hormonal determination factors do not have any CpG islands on their promoters, so the regulation of these components could not happen due to methylation [76]. ...
The sex of crocodilians is determined by the temperature to which the eggs, and hence the developing embryo are exposed during critical periods of development. Temperature-dependent sex determination is a process that occurs in all crocodilians and numerous other reptile taxa. The study of artificial incubation temperatures in different species of crocodiles and alligators has determined the specific temperature ranges that result in altered sex ratios. It has also revealed the precise temperature thresholds at which an equal number of males and females are generated, as well as the specific developmental period during which the sex of the hatchlings may be shifted. This review will examine the molecular basis of the sex-determination mechanism in crocodilians elucidated during recent decades. It will focus on the many patterns and theories associated with this process. Additionally, we will examine the consequences that arise after hatching due to changes in incubation temperatures, as well as the potential benefits and dangers of a changing climate for crocodilians who display sex determination based on temperature.
... Knockdown of Kdm6b triggers male embryos to revert to females, showing the direct implication of this gene and the resulting enzyme. Another gene involved in TSD in multiple species is the CYP19 gene coding for the gonadal aromatase, an enzyme crucial for sexual development(Matsumoto et al., 2016;Navarro- Martín et al., 2011;Parrott et al., 2014).Anastasiadi et al. (2018) used a novel machine-learning predictive approach based on selected CpG sites to screen aromatase methylation on the European sea bass (Dicentrarchus labrax), while Valdivieso et al. (2023) examined the methylation of 15 CpGs in the aromatase gene of zebrafish, ...
Ongoing climatic shifts and increasing anthropogenic pressures demand an efficient delineation of conservation units and accurate predictions of populations' resilience and adaptive potential. Molecular tools involving DNA sequencing are nowadays routinely used for these purposes. Yet, most of the existing tools focusing on sequence‐level information have shortcomings in detecting signals of short‐term ecological relevance. Epigenetic modifications carry valuable information to better link individuals, populations, and species to their environment. Here, we discuss a series of epigenetic monitoring tools that can be directly applied to various conservation contexts, complementing already existing molecular monitoring frameworks. Focusing on DNA sequence‐based methods (e.g. DNA methylation, for which the applications are readily available), we demonstrate how (a) the identification of epi‐biomarkers associated with age or infection can facilitate the determination of an individual's health status in wild populations; (b) whole epigenome analyses can identify signatures of selection linked to environmental conditions and facilitate estimating the adaptive potential of populations; and (c) epi‐eDNA (epigenetic environmental DNA), an epigenetic‐based conservation tool, presents a non‐invasive sampling method to monitor biological information beyond the mere presence of individuals. Overall, our framework refines conservation strategies, ensuring a comprehensive understanding of species' adaptive potential and persistence on ecologically relevant timescales.
... The Ia pattern is found only in turtles, and shows a preference for females at high incubation temperatures and males at low temperatures [10]. The Ib pattern produces females at low temperature and males at high temperature, as in Alligator mississippiensis [11]. Type II produces females at both low and high temperature, males at intermediate temperature, and is found in all groups of reptiles (turtles, crocodiles, lizards) [12]. ...
Background
The Asian yellow pond turtle (Mauremys mutica) is an important commercial freshwater aquaculture species in China. The Asian yellow pond turtle is a highly sexual dimorphic species, with males growing at a faster rate than females. The Asian yellow pond turtle exhibits temperature-dependent sex determination (TSD), in which the incubation temperature during embryonic development determines the sexual fate. However, the mechanisms of the sex determination or sex differentiation of the Asian yellow pond turtle are remain a mystery.
Results
Here, we performed temperature-specific gonadal transcriptomics of the Asian yellow pond turtles during the thermosensitive period (stage 15) to identify candidate genes that initiate gonadal differentiation using RNA-seq technology. We uncover candidates that were the first to respond to temperature, they were sexually dimorphic in expression, reflecting differences in gonadal (Cirbp, Runx1) and germline differentiation (Vasa, Nanos1, Piwil2), gametogenesis (Hmgb3, Zar1, Ovoinhibitor-like, Kif4), steroid hormone biosynthesis (Hsd17b5, Hsd17b6), heat shock (Dnajb6, Hsp90b1, Hsp90aa1) and transient receptor potential channel genes (Trpm1, Trpm4, Trpm6, Trpv1).
Conclusions
Our work will provide crucial genetic information to elucidate the mechanisms of sex control in the Asian yellow pond turtles, and will contribute important genetic resources for further studies of temperature-dependent sex determination in turtles.
... In European seabass, elevated temperatures during a critical period of early development increased DNA methylation levels at the aromatase promoter, which prevented the expression of CYP19A1 [9]. In alligators, elevated CYP19A1 promoter methylation levels and decreased gene expression levels were observed in gonads when embryos were incubated at male production temperatures compared to those incubated at female production temperatures [10]. Expression of the mammalian sex-determining gene Sry has also been reported to be regulated by DNA methylation in the promoter region [11]. ...
DNA methylation plays a key role in sex determination and differentiation in vertebrates. However, there are few studies on DNA methylation involved in chicken gonad development, and most focused on male hypermethylated regions (MHM). It is unclear whether there are specific differentially methylated regions (DMRs) in chicken embryonic gonads regulating sex determination and differentiation. Here, the DNA methylation maps showed that the difference of DNA methylation level between sexes was much higher at embryonic day 10 (E10) than that at embryonic day 6 (E6), and the significant differentially methylated regions at both stages were mainly distributed on the Z chromosome, including MHM1 and MHM2. The results of bisulphite sequencing PCR (BSP) and qRT-PCR showed hypomethylation of female MHM and upregulation of long non-coding RNAs (lncRNAs) whose promoter in the MHM region was consistent with the sequencing results, and similar results were in brain and muscle. In female sex-reversed gonads, the methylation pattern of MHM remained unchanged, and the expression levels of the three candidate lncRNAs were significantly decreased compared with those in females, but were significantly increased compared to males. The fluorescence in situ hybridization (FISH) results also showed that these lncRNAs were highly expressed in female embryonic gonads. The results of methyltransferase inhibitor and dual-luciferase reporter assay suggest that lncRNA expression may be regulated by DNA methylation within their promoters. Therefore, we speculated that MHM may be involved in cell-autonomous sex identity in chickens, and that lncRNAs regulated by MHM may be involved in female sexual differentiation.
... The role of DNA methylation in regulating gene expression has been described under diverse contexts including toxicology (i.e. Kamstra et al., 2015) and sex change (Navarro-Martín et al., 2011;Parrott et al., 2014;Sun et al., 2016). The gene expression of a particular gene can increase or decrease based on the location of the DNA methylation. ...
Understanding the molecular mechanisms underlying individual responses to environmental changes is crucial for species conservation and management. Pelagic fishes including Atlantic herring ( Clupea harengus ) are of particular interest because of their key ecological and economic roles and their susceptibility to a changing ocean from global warming. Temperature and photoperiod have been linked with spawning time and location in adult herring, but no study has thus far investigated the role of environmental factors on gene regulation during the vulnerable early developmental stages. Here, we examine DNA methylation patterns of larval herring bred under two temperatures (11°C and 13°C) and photoperiod (6 and 12 h) regimes in a 2 × 2 factorial design. We found consistently high levels of global methylation across all individuals and a decline in global methylation with increased developmental stage that was more pronounced at 13°C ( p ≤ 0.007) than at 11°C ( p ≥ 0.21). Most of the differentially methylated sites were in exon and promoter regions for genes linked to metabolism and development, some of which were hypermethylated at higher temperature. These results demonstrate the important role of DNA methylation during larval development and suggest that this molecular mechanism might be key in regulating early‐stage responses to environmental stressors in Atlantic herring.
... As methylated cytosines undergo spontaneous deamination resulting in C to T (thymine) mutations, the abundance of CpG dinucleotides is reduced over evolutionary time from the expectation based on the frequency of Cs and Gs in the genome [6,7]. Various studies demonstrated that DNA methylation is sexually dimorphic in the developing or posthatching gonads of vertebrates with temperature-dependent sex determination (TSD), including turtles [6,[8][9][10][11] and alligator [12], and in fish with a mixed system of genotypicsex determination susceptible to thermal effects (GSD + TE) [13][14][15]. These observations raise the possibility that DNA methylation, if it were sexually dimorphic in somatic tissues, could be used as a non-lethal sex diagnostic. ...
Background: The gonads of Chrysemys picta, a turtle with temperature-dependent sex determination (TSD), exhibit differential DNA methylation between males and females, but whether the same is true in somatic tissues remains unknown. Such differential DNA methylation in the soma would provide a non-lethal sex diagnostic for TSD turtle hatchings who lack visually detectable sexual dimorphism when young. Methods: Here, we tested multiple approaches to study DNA methylation in tail clips of Chrysemys picta hatchlings, to identify differentially methylated candidate regions/sites that could serve as molecular sex markers To detect global differential methylation in the tails we used methylation-sensitive ELISA, and to test for differential local methylation we developed a novel hybrid method by sequencing immunoprecipitated and bisulfite converted DNA (MeDIP-BS-seq) followed by PCR validation of candidate regions/sites after digestion with a methylation-sensitive restriction enzyme. Results: We detected no global differences in methylation between males and females via ELISA. While we detected inter-individual variation in DNA methylation in the tails, this variation was not sexually dimorphic, in contrast with hatchling gonads. Conclusions: Results highlight that differential DNA methylation is tissue-specific and plays a key role in gonadal formation (primary sexual development) and maintenance post-hatching, but not in the somatic tail tissue.
... Epigenetic control was also reported for TSD of both plants and animals (47), but not for GSD. In fish and reptiles, DNA methylation-mediated control plays essential roles in TSD (48)(49)(50). In European sea bass, juvenile males have doubled DNA methylation levels in the promoter of gonadal aromatase (cyp19a) as compared to females; when exposed to high temperature, DNA methylation in the promoter region of gonadal aromatase cyp19a was increased in females (48). ...
... Recently, the epigenetic control of key genes for sexual development (cyp19a1a and dmrt1) in European sea bass has been reported (51). Considering the evolutionary position of channel catfish as a lower teleost, it is possible that such a sex determination mechanism could be operating in other lower vertebrates as well, especially those whose sex is highly vulnerable to environmental factors, such as temperature, which could function through their effects on genome methylation (28,48,50,(52)(53)(54). Our study here has implications to connecting control mechanisms of GSD and ESD (especially TSD), in many lower vertebrates, and environmental modulation of sex differentiation in various organisms. ...
The X and Y chromosomes of channel catfish have the same gene contents. Here, we report allelic hypermethylation of the X chromosome within the sex determination region (SDR). Accordingly, the X-borne hydin-1 gene was silenced, whereas the Y-borne hydin-1 gene was expressed, making monoallelic expression of hydin-1 responsible for sex determination, much like genomic imprinting. Treatment with a methylation inhibitor, 5-aza-dC, erased the epigenetic marks within the SDR and caused sex reversal of genetic females into phenotypic males. After the treatment, hydin-1 and six other genes related to cell cycle control and proliferative growth were up-regulated, while three genes related to female sex differentiation were down-regulated in genetic females, providing additional support for epigenetic sex determination in catfish. This mechanism of sex determination provides insights into the plasticity of genetic sex determination in lower vertebrates and its connection with temperature sex determination where DNA methylation is broadly involved.
... In C. serpentina, CYP19A1 exhibits delayed upregulation in response to female-promoting temperatures suggesting the influence of estrogen may be limited to ovarian differentiation rather than initial sex determination [Rhen et al., 2007;Rhen and Schroeder, 2010]. Similarly, CYP19A1 expression and aromatase activity is not upregulated until late in the thermosensitive period (stage 23-24) in A. mississippiensis [Milnes et al., 2002;Parrott et al., 2014]. In T. scripta, CYP19A1 expression is upregulated in the middle of the thermosensitive period Sex Dev DOI: 10.1159/000526009 (stage 18), though in some studies its expression appears to precede that of FOXL2 [Ramsey et al., 2007;Shoemaker et al., 2007;Bieser and Wibbels, 2014;Czerwinski et al., 2016]. ...
... This suggests that the function and localization of these chromatin modifiers likely depends upon the genomic context in which they operate, though the target loci of JARID2 and KDM6B have yet to be elucidated across different TSD species. Other epigenetic processes, including DNA methylation, have also been implicated in TSD [Navarro-Martín et al., 2011;Matsumoto et al., 2013;Parrott et al., 2014], and it is likely these mechanisms operate in a coordinated manner to shape the dynamic epigenome during sex determination, as has been demonstrated in other key developmental processes [DiGiacomo et al., 2013;Potok et al., 2013]. ...
Background:
Reptiles and amphibians provide untapped potential for discovering how a diversity of genetic pathways and environmental conditions are incorporated into developmental processes that can lead to similar functional outcomes. These groups display a multitude of reproductive strategies, and whereas many attributes are conserved within groups and even across vertebrates, several aspects of sexual development show considerable variation.
Summary:
In this review, we focus our attention on the development of the reptilian and amphibian ovary. First, we review and describe the events leading to ovarian development, including sex determination and ovarian maturation, through a comparative lens. We then describe how these events are influenced by environmental factors, focusing on temperature and exposure to anthropogenic chemicals. Lastly, we identify critical knowledge gaps and future research directions that will be crucial to moving forward in our understanding of ovarian development and the influences of the environment in reptiles and amphibians.
Key messages:
Reptiles and amphibians provide excellent models for understanding the diversity of sex determination strategies and reproductive development. However, a greater understanding of the basic biology of these systems is necessary for deciphering the adaptive and potentially disruptive implications of embryo-by-environment interactions in a rapidly changing world.
... Perhaps one of the most dramatic developmentally derived phenotypes is temperature dependent sex determination in some fish and reptiles. Research across several species indicates temperature determines sex via differential methylation patterns in the promoters of the cyp19a and SOX9 genes [92][93][94]. Other developmental environments have also been shown to have lasting phenotypic effects through impacts on methylation such as parental care, rainfall, and nutritional environment [60,65,66,95]. ...
DNA methylation is an epigenetic modification with wide-ranging consequences across the life of an organism. This modification can be stable, persisting through development despite changing environmental conditions. However, in other contexts, DNA methylation can also be flexible, underlying organismal phenotypic plasticity. One underappreciated aspect of DNA methylation is that it is a potent mutagen; methylated cytosines mutate at a much faster rate than other genetic motifs. This mutagenic property of DNA methylation has been largely ignored in eco-evolutionary literature, despite its prevalence. Here, we explore how DNA methylation induced by environmental and other factors could promote mutation and lead to evolutionary change at a more rapid rate and in a more directed manner than through stochastic genetic mutations alone. We argue for future research on the evolutionary implications of DNA methylation driven mutations both within the lifetime of organisms, as well as across timescales.
... More often, methylated cytosines physically block access of the transcription machinery to the DNA [12], silencing methylated genes. In general, DNA methylation is an important molecular mechanism that organisms use to respond to environmental stimuli [13,14], and it is critical for sex determination in turtles and other reptiles with TSD [15][16][17][18]. For instance, in Alligator mississipiensis, the promoter region of Sox9, a gene involved in male sexual development, is methylated at female-producing temperatures, repressing its expression in developing ovaries [15]. ...
... In general, DNA methylation is an important molecular mechanism that organisms use to respond to environmental stimuli [13,14], and it is critical for sex determination in turtles and other reptiles with TSD [15][16][17][18]. For instance, in Alligator mississipiensis, the promoter region of Sox9, a gene involved in male sexual development, is methylated at female-producing temperatures, repressing its expression in developing ovaries [15]. Another example of a male development gene regulated by methylation is Dmrt1, which exhibits hypermethylation (and thus is downregulated) in developing females compared to males in TSD and GSD fish [19][20][21][22]. ...
... Concordantly, we observed quantitative changes in gene expression that match this mode of action for cadmium. For instance, cadmium exposure can alter gene expression when gene promoters are regulated by DNA methylation, as is the case for Dmrt1 in fish [13,[20][21][22], aromatase in TSD turtles and fish [19,24], and Sox9 in alligators [15]. These genes are differentially transcribed in the developing embryo of C. picta turtles [28,29,38]. ...
Temperature-dependent sex determination (TSD) decides the sex fate of an individual based on incubation temperature. However, other environmental factors, such as pollutants, could derail TSD sexual development. Cadmium is one such contaminant of soils and water bodies known to affect DNA methylation, an epigenetic DNA modification with a key role in sexual development of TSD vertebrate embryos. Yet, whether cadmium alters DNA methylation of genes underlying gonadal formation in turtles remains unknown. Here, we investigated the effects of cadmium on the expression of two gene regulators of TSD in the painted turtle, Chrysemys picta, incubated at male-producing and female-producing temperatures using qPCR. Results revealed that cadmium alters transcription of Dmrt1 and aromatase, overriding the normal thermal effects during embryogenesis, which could potentially disrupt the sexual development of TSD turtles. Results from a preliminary DNA methylation-sensitive PCR assay implicate changes in DNA methylation of Dmrt1 as a potential cause that requires further testing (aromatase methylation assays were precluded).
