Unraveling the genotype by environment interaction in a thermosensitive fish with a polygenic sex determination system.

In most animals, sex determination occurs at conception, when sex chromosomes are segregated following Mendelian laws. However, in multiple reptiles and fishes, this genetic sex can be overridden by external factors after fertilization or birth. In some species, the genetic sex may also be governed by multiple genes, further limiting our understanding of sex determination in such species. We used the European sea bass (Dicentrarchus labrax) as a model and combined genomic (using a single nucleotide polymorphism chip) and transcriptomic (RNA-Sequencing) approaches to thoroughly depict this polygenic sex determination system and its interaction with temperature. We estimated genetic sex tendency (eGST), defined as the estimated genetic liability to become a given sex under a liability threshold model for sex determination, which accurately predicts the future phenotypic sex. We found evidence that energetic pathways, concerning the regulation of lipids and glucose, are involved in sex determination and could explain why females tend to exhibit higher energy levels and improved growth compared to males. Besides, early exposure to high-temperature up-regulated sox3, followed by sox9a in individuals with intermediate eGST, but not in individuals showing highly female-biased eGST, providing the most parsimonious explanation for temperature-induced masculinization. This gonadal state was maintained likely by DNA methylation and the up-regulation of several genes involved in histone modifications, including jmjd1c Overall, we describe a sex determination system resulting from continuous genetic and environmental influences in an animal. Our results provide significant progress in our understanding of the mechanisms underlying temperature-induced masculinization in fish.

Improved biomarker discovery through a plot twist in transcriptomic data analysis.

BACKGROUND: Transcriptomic analysis is crucial for understanding the functional elements of the genome, with the classic method consisting of screening transcriptomics datasets for differentially expressed genes (DEGs). Additionally, since 2005, weighted gene co-expression network analysis (WGCNA) has emerged as a powerful method to explore relationships between genes. However, an approach combining both methods, i.e., filtering the transcriptome dataset by DEGs or other criteria, followed by WGCNA (DEGs + WGCNA), has become common. This is of concern because such approach can affect the resulting underlying architecture of the network under analysis and lead to wrong conclusions. Here, we explore a plot twist to transcriptome data analysis: applying WGCNA to exploit entire datasets without affecting the topology of the network, followed with the strength and relative simplicity of DEG analysis (WGCNA + DEGs). We tested WGCNA + DEGs against DEGs + WGCNA to publicly available transcriptomics data in one of the most transcriptomically complex tissues and delicate processes: vertebrate gonads undergoing sex differentiation. We further validate the general applicability of our approach through analysis of datasets from three distinct model systems: European sea bass, mouse, and human. RESULTS: In all cases, WGCNA + DEGs clearly outperformed DEGs + WGCNA. First, the network model fit and node connectivity measures and other network statistics improved. The gene lists filtered by each method were different, the number of modules associated with the trait of interest and key genes retained increased, and GO terms of biological processes provided a more nuanced representation of the biological question under consideration. Lastly, WGCNA + DEGs facilitated biomarker discovery. CONCLUSIONS: We propose that building a co-expression network from an entire dataset, and only thereafter filtering by DEGs, should be the method to use in transcriptomic studies, regardless of biological system, species, or question being considered.

Transposon-induced epigenetic silencing in the X chromosome as a novel form of dmrt1 expression regulation during sex determination in the fighting fish.

BACKGROUND: Fishes are the one of the most diverse groups of animals with respect to their modes of sex determination, providing unique models for uncovering the evolutionary and molecular mechanisms underlying sex determination and reversal. Here, we have investigated how sex is determined in a species of both commercial and ecological importance, the Siamese fighting fish Betta splendens. RESULTS: We conducted association mapping on four commercial and two wild populations of B. splendens. In three of the four commercial populations, the master sex determining (MSD) locus was found to be located in a region of ~ 80 kb on LG2 which harbours five protein coding genes, including dmrt1, a gene involved in male sex determination in different animal taxa. In these fish, dmrt1 shows a male-biased gonadal expression from undifferentiated stages to adult organs and the knockout of this gene resulted in ovarian development in XY genotypes. Genome sequencing of XX and YY genotypes identified a transposon, drbx1, inserted into the fourth intron of the X-linked dmrt1 allele. Methylation assays revealed that epigenetic changes induced by drbx1 spread out to the promoter region of dmrt1. In addition, drbx1 being inserted between two closely linked cis-regulatory elements reduced their enhancer activities. Thus, epigenetic changes, induced by drbx1, contribute to the reduced expression of the X-linked dmrt1 allele, leading to female development. This represents a previously undescribed solution in animals relying on dmrt1 function for sex determination. Differentiation between the X and Y chromosomes is limited to a small region of ~ 200 kb surrounding the MSD gene. Recombination suppression spread slightly out of the SD locus. However, this mechanism was not found in the fourth commercial stock we studied, or in the two wild populations analysed, suggesting that it originated recently during domestication. CONCLUSIONS: Taken together, our data provide novel insights into the role of epigenetic regulation of dmrt1 in sex determination and turnover of SD systems and suggest that fighting fish are a suitable model to study the initial stages of sex chromosome evolution.

Exploring the Effects of Rearing Densities on Epigenetic Modifications in the Zebrafish Gonads.

Rearing density directly impacts fish welfare, which, in turn, affects productivity in aquaculture. Previous studies have indicated that high-density rearing during sexual development in fish can induce stress, resulting in a tendency towards male-biased sex ratios in the populations. In recent years, research has defined the relevance of the interactions between the environment and epigenetics playing a key role in the final phenotype. However, the underlying epigenetic mechanisms of individuals exposed to confinement remain elucidated. By using zebrafish (Danio rerio), the DNA methylation promotor region and the gene expression patterns of six genes, namely dnmt1, cyp19a1a, dmrt1, cyp11c1, hsd17b1, and hsd11b2, involved in the DNA maintenance methylation, reproduction, and stress were assessed. Zebrafish larvae were subjected to two high-density conditions (9 and 66 fish/L) during two periods of overlapping sex differentiation of this species (7 to 18 and 18 to 45 days post-fertilization, dpf). Results showed a significant masculinization in the populations of fish subjected to high densities from 18 to 45 dpf. In adulthood, the dnmt1 gene was differentially hypomethylated in ovaries and its expression was significantly downregulated in the testes of fish exposed to high-density. Further, the cyp19a1a gene showed downregulation of gene expression in the ovaries of fish subjected to elevated density, as previously observed in other studies. We proposed dnmt1 as a potential testicular epimarker and the expression of ovarian cyp19a1a as a potential biomarker for predicting stress originated from high densities during the early stages of development. These findings highlight the importance of rearing densities by long-lasting effects in adulthood conveying cautions for stocking protocols in fish hatcheries.

Genomic Basis of Striking Fin Shapes and Colors in the Fighting Fish.

Resolving the genomic basis underlying phenotypic variations is a question of great importance in evolutionary biology. However, understanding how genotypes determine the phenotypes is still challenging. Centuries of artificial selective breeding for beauty and aggression resulted in a plethora of colors, long-fin varieties, and hyper-aggressive behavior in the air-breathing Siamese fighting fish (Betta splendens), supplying an excellent system for studying the genomic basis of phenotypic variations. Combining whole-genome sequencing, quantitative trait loci mapping, genome-wide association studies, and genome editing, we investigated the genomic basis of huge morphological variation in fins and striking differences in coloration in the fighting fish. Results revealed that the double tail, elephant ear, albino, and fin spot mutants each were determined by single major-effect loci. The elephant ear phenotype was likely related to differential expression of a potassium ion channel gene, kcnh8. The albinotic phenotype was likely linked to a cis-regulatory element acting on the mitfa gene and the double-tail mutant was suggested to be caused by a deletion in a zic1/zic4 coenhancer. Our data highlight that major loci and cis-regulatory elements play important roles in bringing about phenotypic innovations and establish Bettas as new powerful model to study the genomic basis of evolved changes.

Environmentally-induced sex reversal in fish with chromosomal vs. polygenic sex determination.

Sex ratio depends on sex determination mechanisms and is a key demographic parameter determining population viability and resilience to natural and anthropogenic stressors. There is increasing evidence that the environment can alter sex ratio even in genetically sex-determined species (GSD), as elevated temperature can cause female-to-male sex reversal (neomales). Alarmingly, neomales are being discovered in natural populations of several fish, amphibian and reptile species worldwide. Understanding the basis of neomale development is important for conservation biology. Among GSD species, it is unknown whether those with chromosomal sex determination (CSD), the most common system, will better resist the influence of high temperature than those with polygenic sex determination (PSD). Here, we compared the effects of elevated temperature in two wild zebrafish strains, Nadia (NA) and Ekkwill (EKW), which have CSD with a ZZ/ZW system, against the AB laboratory strain, which has PSD. First, we uncovered novel sex genotypes and the results showed that, at control temperature, the masculinization rate roughly doubled with the addition of each Z chromosome, while some ZW and WW fish of the wild strains became neomales. Surprisingly, we found that at elevated temperatures WW fish were just as likely as ZW fish to become neomales and that all strains were equally susceptible to masculinization. These results demonstrate that the Z chromosome is not essential for male development and that the dose of W buffers masculinization at the control temperature but not at elevated temperature. Furthermore, at the elevated temperature the testes of neomales, but not of normal males, contained more spermatozoa than at the control temperature. Our results show in an unprecedented way that, in a global warming scenario, CSD species may not necessarily be better protected against the masculinizing effect of elevated temperature than PSD species, and reveal genotype-by-temperature interactions in male sex determination and spermatogenesis.

The first mitochondrial 5-methylcytosine map in a non-model teleost (Oreochromis niloticus) reveals extensive strand-specific and non-CpG methylation.

DNA methylation is one of the main epigenetic mechanisms that regulate gene expression in a manner that depends on the genomic context and varies considerably across taxa. This DNA modification was first found in nuclear genomes of eukaryote several decades ago and it has also been described in mitochondrial DNA. It has recently been shown that mitochondrial DNA is extensively methylated in mammals and other vertebrates. Our current knowledge of mitochondrial DNA methylation in fish is very limited, especially in non-model teleosts. In this study, using whole-genome bisulfite sequencing, we determined methylation patterns within non-CpG (CH) and CpG (CG) contexts in the mitochondrial genome of Nile tilapia, a non-model teleost of high economic importance. Our results demonstrate the presence of mitochondrial DNA methylation in this species predominantly within a non-CpG context, similarly to mammals. We found a strand-specific distribution of methylation, in which highly methylated cytosines were located on the minus strand. The D-loop region had the highest mean methylation level among all mitochondrial loci. Our data provide new insights into the potential role of epigenetic mechanisms in regulating metabolic flexibility of mitochondria in fish, with implications in various biological processes, such as growth and development.

Epigenetic mapping of the somatotropic axis in Nile tilapia reveals differential DNA hydroxymethylation marks associated with growth.

In vertebrates, the somatotropic axis comprising the pituitary gland, liver and muscle plays a major role in myogenesis. Its output in terms of muscle growth is highly affected by nutritional and environmental cues, and thus likely epigenetically regulated. Hydroxymethylation is emerging as a DNA modification that modulates gene expression but a holistic characterization of the hydroxymethylome of the somatotropic axis has not been investigated to date. Using reduced representation 5-hydroxymethylcytosine profiling we demonstrate tissue-specific localization of 5-hydroxymethylcytosines at single nucleotide resolution. Their abundance within gene bodies and promoters of several growth-related genes supports their pertinent role in gene regulation. We propose that cytosine hydroxymethylation may contribute to the phenotypic plasticity of growth through epigenetic regulation of the somatotropic axis.

A genome-wide association study, supported by a new chromosome-level genome assembly, suggests sox2 as a main driver of the undifferentiatiated ZZ/ZW sex determination of turbot (Scophthalmus maximus).

BACKGROUND: Understanding sex determination (SD) across taxa is a major challenge for evolutionary biology. The new genomic tools are paving the way to identify genomic features underlying SD in fish, a group frequently showing limited sex chromosome differentiation and high SD evolutionary turnover. Turbot (Scophthalmus maximus) is a commercially important flatfish with an undifferentiated ZW/ZZ SD system and remarkable sexual dimorphism. Here we describe a new long-read turbot genome assembly used to disentangle the genetic architecture of turbot SD by combining genomics and classical genetics approaches. RESULTS: The new turbot genome assembly consists of 145 contigs (N50 = 22.9 Mb), 27 of them representing >95% of its estimated genome size. A genome wide association study (GWAS) identified a ~ 6.8 Mb region on chromosome 12 associated with sex in 69.4% of the 36 families analyzed. The highest associated markers flanked sox2, the only gene in the region showing differential expression between sexes before gonad differentiation. A single SNP showed consistent differences between Z and W chromosomes. The analysis of a broad sample of families suggested the presence of additional genetic and/or environmental factors on turbot SD. CONCLUSIONS: The new chromosome-level turbot genome assembly, one of the most contiguous fish assemblies to date, facilitated the identification of sox2 as a consistent candidate gene putatively driving SD in this species. This chromosome SD system barely showed any signs of differentiation, and other factors beyond the main QTL seem to control SD in a certain proportion of families.

Footprints of global change in marine life: Inferring past environment based on DNA methylation and gene expression marks.

Ocean global warming affects the distribution, life history and physiology of marine life. Extreme events, like marine heatwaves, are increasing in frequency and intensity. During sensitive stages of early fish development, the consequences may be long-lasting and mediated by epigenetic mechanisms. Here, we used European sea bass as a model to study the possible long-lasting effects of a marine heatwave during early development. We measured DNA methylation and gene expression in four tissues (brain, muscle, liver and testis) and detected differentially methylated regions (DMRs). Six genes were differentially expressed and contained DMRs three years after exposure to increased temperature, indicating direct phenotypic consequences and representing persistent changes. Interestingly, nine genes contained DMRs around the same genomic regions across tissues, therefore consisting of common footprints of developmental temperature in environmentally responsive loci. These loci are, to our knowledge, the first metastable epialleles (MEs) described in fish. MEs may serve as biomarkers to infer past life history events linked with persistent consequences. These results highlight the importance of subtle phenotypic changes mediated by epigenetics to extreme weather events during sensitive life stages. Also, to our knowledge, it is the first time the molecular effects of a marine heatwave during the lifetime of individuals are assessed. MEs could be used in surveillance programs aimed at determining the footprints of climate change on marine life. Our study paves the way for the identification of conserved MEs that respond equally to environmental perturbations across species. Conserved MEs would constitute a tool of assessment of global change effects in marine life at a large scale.

Epimutations in Developmental Genes Underlie the Onset of Domestication in Farmed European Sea Bass.

Domestication of wild animals induces a set of phenotypic characteristics collectively known as the domestication syndrome. However, how this syndrome emerges is still not clear. Recently, the neural crest cell deficit hypothesis proposed that it is generated by a mildly disrupted neural crest cell developmental program, but clear support is lacking due to the difficulties of distinguishing pure domestication effects from preexisting genetic differences between farmed and wild mammals and birds. Here, we use a farmed fish as model to investigate the role of persistent changes in DNA methylation (epimutations) in the process of domestication. We show that early domesticates of sea bass, with no genetic differences with wild counterparts, contain epimutations in tissues with different embryonic origins. About one fifth of epimutations that persist into adulthood are established by the time of gastrulation and affect genes involved in developmental processes that are expressed in embryonic structures, including the neural crest. Some of these genes are differentially expressed in sea bass with lower jaw malformations, a key feature of domestication syndrome. Interestingly, these epimutations significantly overlap with cytosine-to-thymine polymorphisms after 25 years of selective breeding. Furthermore, epimutated genes coincide with genes under positive selection in other domesticates. We argue that the initial stages of domestication include dynamic alterations in DNA methylation of developmental genes that affect the neural crest. Our results indicate a role for epimutations during the beginning of domestication that could be fixed as genetic variants and suggest a conserved molecular process to explain Darwin's domestication syndrome across vertebrates.

Exposure of zebrafish to elevated temperature induces sex ratio shifts and alterations in the testicular epigenome of unexposed offspring.

Accumulating evidence shows that environmental changes can affect population sex ratios through epigenetic regulation of gene expression in species where sex depends on both genetic and environmental cues. Sometimes, altered sex ratios persist in the next generation even when the environmental cue is no longer present (a multigenerational effect). However, evidence of transgenerational effects (i.e., beyond the first non-exposed generation), which tend to be paternally transmitted, is scarce and a matter of debate. Here, we used the AB strain of zebrafish, where sex depends on both genetic and environmental influences, to study possible multi- (to the F1) and transgenerational (to the F2) effects of elevated temperature during the critical period of sex differentiation. From eight initial different families, five were selected in order to capture sufficient variation between the sex ratio of the control group (28 °C) and the group exposed to elevated (35 °C) temperature only at the parental (P) generation. Results showed a consistent increase in the proportion of males in the P generation in all five families as a result of heat treatment. Sex ratios were then determined in the F1 and F2 offspring derived from both above groups, which were all raised at 28 °C. A persisting male-skewed sex ratio in the 35°C-derived, unexposed offspring of the F1 generation was observed in three families, denoting family-dependent multigenerational effects. However, no transgenerational effects were observed in the F2 generation of any family. DNA methylation was also assessed in the testis of P, F1 and F2 males derived from exposed and non-exposed fathers and grandfathers. DNA methylation was significantly decreased only in the testis of the 35°C-derived males in the F1 generation but not of the F2 generation and, surprisingly, neither in the 35°C-exposed males of the P generation. Taken together, our results show great interfamily variation, not only in sex ratio response to elevated temperature, but also on its multigenerational effects, denoting a strong influence of genetics. Alterations in the testicular epigenome in F1 males calls for attention to possible, previously unnoticed, effects of temperature in the unexposed offspring of heat-exposed parents in a global warming scenario.

Heat-induced masculinization in domesticated zebrafish is family-specific and yields a set of different gonadal transcriptomes.

Understanding environmental influences on sex ratios is important for the study of the evolution of sex-determining mechanisms and for evaluating the effects of global warming and chemical pollution. Fishes exhibit sexual plasticity, but the underlying mechanisms of environmental effects on their reproduction are unclear even in the well-established teleost research model, the zebrafish. Here we established the conditions to study the effects of elevated temperature on zebrafish sex. We showed that sex ratio response to elevated temperature is family-specific and typically leads to masculinization (female-to-male sex reversal), resulting in neomales. These results uncovered genotype-by-environment interactions that support a polygenic sex determination system in domesticated (laboratory) zebrafish. We found that some heat-treated fish had gene expression profiles similar to untreated controls of the same sex, indicating that they were resistant to thermal effects. Further, most neomales had gonadal transcriptomes similar to that of regular males. Strikingly, we discovered heat-treated females that displayed a normal ovarian phenotype but with a "male-like" gonadal transcriptome. Such major transcriptomic reprogramming with preserved organ structure has never been reported. Juveniles were also found to have a male-like transcriptome shortly after exposure to heat. These findings were validated by analyzing the expression of genes and signaling pathways associated with sex differentiation. Our results revealed a lasting thermal effect on zebrafish gonads, suggesting new avenues for detection of functional consequences of elevated temperature in natural fish populations in a global warming scenario.

Ovarian transcriptomic signatures of zebrafish females resistant to different environmental perturbations.

Sex is remarkably plastic in fish and can be easily influenced by environmental cues, in which temperature has been the most studied abiotic factor. However, it has been shown that elevated population densities can increase the number of males in several species but little is known about the underlying molecular mechanisms and whether general patterns exist. Here, we studied the long-term effects of population density on the gene expression program in zebrafish gonads. The ovarian transcriptome of females exposed to high versus low population densities contained 4,634 differentially expressed genes. Among them, a set of promale genes (amh, sypc3, spata6, and sox3) were upregulated in the high-population density group. Next, we compared the transcriptomes of ovaries of female zebrafish resistant to the masculinizing effects of either high density or elevated temperature. Results showed a set of 131 and 242 common upregulated and downregulated genes, respectively, including the upregulation of known male-related genes (e.g., amh and sycp3) but also genes involved in other functions (e.g., faima, ccm21, and ankrd6b) and a downregulation of cyp19a1a together with other genes (e.g., lgals9l1 and ubxn2a). We identified the common Gene Ontology terms involved in the reproduction and sexual development that were consistently affected in both environmental factors. These results show that regardless of the environmental perturbation there are common genes and cellular functions involved in the resistance to masculinization. These altered gene-expression profiles can be used as markers indicative of previous exposure to environmental stress independent of conspicuous alterations in sex ratios or gonadal morphology.

Appropriate rearing density in domesticated zebrafish to avoid masculinization: links with the stress response.

The zebrafish (Danio rerio) has become a well-established experimental model in many research fields but the loss of the primary sex-determining region during the process of domestication renders laboratory strains of zebrafish susceptible to the effects of environmental factors on sex ratios. Further, an essential husbandry aspect - the optimal rearing density to avoid stress-induced masculinization - is not known. We carried out two experiments: the first focusing on the effects of density on survival, growth and sex ratio by rearing zebrafish at different initial densities (9, 19, 37 and 74 fish per litre) for 3 months (6-90 days post-fertilization, dpf), and the second focusing on the effects of cortisol during the sex differentiation period (15-45 dpf) for zebrafish reared at low density. The results showed an increase in the number of males in groups subjected to the two highest initial rearing densities; we also observed a reduction of survival and growth in a density-dependent manner. Furthermore, zebrafish treated with cortisol during the sex differentiation period showed a complete masculinization of the population; treatment with the cortisol synthesis inhibitor metyrapone negated the effects of exogenous cortisol. Our results indicate that the process of sex differentiation in domesticated zebrafish can be perturbed by elevated stocking density and that this effect is likely to be mediated by an increase in cortisol through the stress response. However, the underlying mechanism needs further study.

Lasting effects of early exposure to temperature on the gonadal transcriptome at the time of sex differentiation in the European sea bass, a fish with mixed genetic and environmental sex determination.

BACKGROUND: Sex in fish is plastic and in several species can be influenced by environmental factors. In sensitive species, elevated temperatures have a masculinizing effect. Previous studies on the effects of temperature on gene expression have been restricted to a few cognate genes, mostly related to testis or ovarian development, and analyzed in gonads once they had completed the process of sex differentiation. However, studies on the effect of temperature at the whole gonadal transcriptomic level are scarce in fish and, in addition, temperature effects at the time of sex differentiation at the transcriptomic level are also unknown. Here, we used the European sea bass, a gonochoristic teleost with a polygenic sex determination system influenced by temperature, and exposed larvae to elevated temperature during the period of early gonad formation. Transcriptomic analysis of the gonads was carried out about three months after the end of temperature exposure, shortly after the beginning of the process of sex differentiation. RESULTS: Elevated temperature doubled the number of males with respect to untreated controls. Transcriptomic analysis of early differentiating female gonads showed how heat caused: 1) an up-regulation of genes related to cholesterol transport (star), the stress response (nr3c1) and testis differentiation (amh, dmrt, etc.), 2) a decrease in the expression of genes related to ovarian differentiation such as cyp19a1a, and 3) an increase in the expression of several genes related to epigenetic regulatory mechanisms (hdac11, dicer1, ehmt2, jarid2a, pcgf2, suz12, mettl22). CONCLUSIONS: Taken together, the results of this study contribute to the understanding of how the early environment sets permanent changes that result in long-lasting consequences, in this case in the sexual phenotype. Results also show the usefulness of comparing the effects of heat on the behavior of cognate genes related to sex differentiation as well as that of genes involved in establishing and maintaining cell identity through epigenetic mechanisms.

Gene expression analysis at the onset of sex differentiation in turbot (Scophthalmus maximus).

BACKGROUND: Controlling sex ratios is essential for the aquaculture industry, especially in those species with sex dimorphism for relevant productive traits, hence the importance of knowing how the sexual phenotype is established in fish. Turbot, a very important fish for the aquaculture industry in Europe, shows one of the largest sexual growth dimorphisms amongst marine cultured species, being all-female stocks a desirable goal for the industry. Although important knowledge has been achieved on the genetic basis of sex determination (SD) in this species, the master SD gene remains unknown and precise information on gene expression at the critical stage of sex differentiation is lacking. In the present work, we examined the expression profiles of 29 relevant genes related to sex differentiation, from 60 up to 135 days post fertilization (dpf), when gonads are differentiating. We also considered the influence of three temperature regimes on sex differentiation. RESULTS: The first sex-related differences in molecular markers could be observed at 90 days post fertilization (dpf) and so we have called that time the onset of sex differentiation. Three genes were the first to show differential expression between males and females and also allowed us to sex turbot accurately at the onset of sex differentiation (90 dpf): cyp19a1a, amh and vasa. The expression of genes related to primordial germ cells (vasa, gsdf, tdrd1) started to increase between 75-90 dpf and vasa and tdrd1 later presented higher expression in females (90-105 dpf). Two genes placed on the SD region of turbot (sox2, fxr1) did not show any expression pattern suggestive of a sex determining function. We also detected changes in the expression levels of several genes (ctnnb1, cyp11a, dmrt2 or sox6) depending on culture temperature. CONCLUSION: Our results enabled us to identify the first sex-associated genetic cues (cyp19a1a, vasa and amh) at the initial stages of gonad development in turbot (90 dpf) and to accurately sex turbot at this age, establishing the correspondence between gene expression profiles and histological sex. Furthermore, we profiled several genes involved in sex differentiation and found specific temperature effects on their expression.

DNA methylation of the gonadal aromatase (cyp19a) promoter is involved in temperature-dependent sex ratio shifts in the European sea bass.

Sex ratio shifts in response to temperature are common in fish and reptiles. However, the mechanism linking temperature during early development and sex ratios has remained elusive. We show in the European sea bass (sb), a fish in which temperature effects on sex ratios are maximal before the gonads form, that juvenile males have double the DNA methylation levels of females in the promoter of gonadal aromatase (cyp19a), the enzyme that converts androgens into estrogens. Exposure to high temperature increased the cyp19a promoter methylation levels of females, indicating that induced-masculinization involves DNA methylation-mediated control of aromatase gene expression, with an observed inverse relationship between methylation levels and expression. Although different CpGs within the sb cyp19a promoter exhibited different sensitivity to temperature, we show that the increased methylation of the sb cyp19a promoter, which occurs in the gonads but not in the brain, is not a generalized effect of temperature. Importantly, these effects were also observed in sexually undifferentiated fish and were not altered by estrogen treatment. Thus, methylation of the sb cyp19a promoter is the cause of the lower expression of cyp19a in temperature-masculinized fish. In vitro, induced methylation of the sb cyp19a promoter suppressed the ability of SF-1 and Foxl2 to stimulate transcription. Finally, a CpG differentially methylated by temperature and adjacent to a Sox transcription factor binding site is conserved across species. Thus, DNA methylation of the aromatase promoter may be an essential component of the long-sought-after mechanism connecting environmental temperature and sex ratios in vertebrate species with temperature-dependent sex determination.

Epigenetics of sex determination and gonadogenesis.

Epigenetics is commonly defined as the study of heritable changes in gene function that cannot be explained by changes in DNA sequence. The three major epigenetic mechanisms for gene expression regulation include DNA methylation, histone modifications, and non-coding RNAs. Epigenetic mechanisms provide organisms with the ability to integrate genomic and environmental information to modify the activity of their genes for generating a particular phenotype. During development, cells differentiate, acquire, and maintain identity through changes in gene expression. This is crucial for sex determination and differentiation, which are among the most important developmental processes for the proper functioning and perpetuation of species. This review summarizes studies showing how epigenetic regulatory mechanisms contribute to sex determination and reproductive organ formation in plants, invertebrates, and vertebrates. Further progress will be made by integrating several approaches, including genomics and Next Generation Sequencing to create epigenetic maps related to different aspects of sex determination and gonadogenesis. Epigenetics will also contribute to understand the etiology of several disorders of sexual development. It also might play a significant role in the control of reproduction in animal farm production and will aid in recognizing the environmental versus genetic influences on sex determination of sensitive species in a global change scenario.