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Estrogen exposure overrides the masculinizing effect of elevated temperature by a downregulation of the key genes implicated in sexual differentiation in a fish with mixed genetic and environmental sex determination



Understanding the consequences of thermal and chemical variations in aquatic habitats is of importance in a scenario of global change. In ecology, the sex ratio is a major population demographic parameter. So far, research that measured environmental perturbations on fish sex ratios has usually involved a few model species with a strong genetic basis of sex determination, and focused on the study of juvenile or adult gonads. However, the underlying mechanisms at the time of gender commitment are poorly understood. In an effort to elucidate the mechanisms driving sex differentiation, here we used the European sea bass, a fish species where genetics and environment (temperature) contribute equally to sex determination.


Here, we analyzed the transcriptome of developing gonads experiencing either testis or ovarian differentiation as a result of thermal and/or exogenous estrogen influences. These external insults elicited different responses. Thus, while elevated temperature masculinized genetic females, estrogen exposure was able to override thermal effects and resulted in an all-female population. A total of 383 genes were differentially expressed, with an overall downregulation in the expression of genes involved in both in testicular and ovarian differentiation when fish were exposed to Estradiol-17ß through a shutdown of the first steps of steroidogenesis. However, once the female phenotype was imposed, gonads could continue their normal development, even taking into account that some of the resulting females were fish that otherwise would have developed as males.


The data on the underlying mechanisms operating at the molecular level presented here contribute to a better understanding of the sex ratio response of fish species subjected to a combination of two of the most common environmental perturbations and can have implications in future conservational policies.


The sex ratio is an essential demographic parameter in population ecology, and its proper establishment is crucial for the perpetuation of all sexually-reproducing species [1]. In fish, the establishment of the primary sex ratio mostly depends on the genetic and environmental contribution to the process of sex determination and differentiation [2, 3], although other factors such as differential survival can also have an influence.

While downstream genes implicated in gonadal sex differentiation are conserved [4, 5], master sex-determining genes are not [6]. Importantly, and also in contrast to mammals, in all non-mammalian vertebrates estrogens are essential for proper ovarian differentiation [7]. Therefore, blockade of gonadal aromatase, the steroidogenic enzyme that irreversibly converts androgens into estrogens such as estradiol-17ß (E2) results in the masculinization of the gonads of genetic females in different species [8,9,10,11]. Conversely, exposure to E2 feminizes the gonads of genotypic males in many species [9, 12,13,14,15].

Temperature increases related to global change and pollution of water bodies, both ultimately due to human activity, greatly influence aquatic ecosystems, with opposing effects on sex ratios of many fish populations. In sensitive fish species, the sex ratio (gonadal differentiation) response to elevated temperatures is an increase in the number of males [16]. Thus, abnormally elevated temperatures often result in a severely male-biased population [17]. On the other hand, many chemicals present in the aquatic environment have a feminizing effect since they are able to disrupt the endocrine system by mimicking the effects of estrogens through binding to the estrogen receptor [18]. Consequently, even at low environmental concentrations, a sufficiently long exposure can lead to the feminization of the entire population [19].

Fish transcriptomes have been analyzed during sexual differentiation [20,21,22,23,24,25] and after E2 exposure [18, 26,27,28]. Tissue- and gender-specific responses, [29, 30] as well as biogeographical differences [31], have been shown. However, it is still not clear whether the exposure to exogenous steroids elicits changes similar to those occurring during natural physiological processes [32,33,34]. These exogenous steroids inhibit the expression of several steroidogenic enzymes, as observed in different species [34,35,36,37] and thus alter normal hormonal functions [38, 39]. However, most of the studies referred to above were conducted in species with a strong sexual determining system (XX/XY or ZW/ZZ) where sex is highly canalized and not easily influenced by environmental perturbations. This contrast with species with a polygenic sex determination system, where the final sex depends on a delicate balance between endogenous and external stimuli [24].

The European sea bass (Dicentrarchus labrax) is a gonochoristic species that lacks sex chromosomes and for which a polygenic system of sex determination involving a two-biallelic system has been proposed [40]. Furthermore, sex determination and differentiation are influenced by environment during early development [41], when temperatures just a few degrees above 17 °C applied during the thermosensitive period (0–60 days post hatch; dph [24, 42]) masculinize about half of the fish that under natural temperatures would develop as females. This masculinization is induced through the hypermethylation of the cyp19a1a promoter in females that prevents the synthesis of the E2 necessary for ovarian development [43]. It is also known that E2 administered during the hormone-sensitive period (HSP = 90–160 days post hatch [44]) can result in feminization of the whole population [14]. The study of European sea bass responses to environmental cues is also interesting because the nursing of this species takes place in coastal shallow waters of 10 m depth [45], that are more sensitive to thermal fluctuations as the ones predicted in current climate change models [46], and also likely containing xenoestrogens that can act as endocrine disruptors [47].

The goal of our study was to compare patterns of gene expression in a species with polygenic sex determination such as the European sea bass at the time when gonads were experiencing opposite pathways of differentiation as a consequence of the environmental cues to which they were exposed. For that we generated two European sea bass populations: 1) one male-biased (78% males) through exposure to elevated water temperature, and another female-biased (100% females) by exposing fish to E2 during the HSP. We then examined gene expression in gonads at 170 dph, i.e., during the sex differentiation period by a custom-made oligo microarray.


Rearing conditions, experimental design and basic data collection

Twenty-four hours post hatch European sea bass larvae from a commercial hatchery (St. Pere Pescador, Girona, Spain) were transferred to the Institute of Marine Sciences “Experimental Aquarium Facility” (ZAE). Larvae densities per tank, environmental and rearing conditions followed the protocols previously described [48]. Fish used for this article were reared and sacrificed in agreement with the European Convention for the Protection of Animals used in scientific experimentation (EST Nu 123, 01/01/91).

The male fish used in this study were siblings of males used in a previous study [24]. Briefly, fish were divided into four tanks and maintained at 17 °C during the first 20 dph. Then, water temperature was raised until 21 °C for two of the groups (Control “HT” and Estradiol “HT-E2”) for the remaining two groups; temperature was decreased to 15 °C (Control low temperature “LT” and Estradiol “LT-E2”), at a rate of 0.5 °C/day. At ~220 dph (fall) water was left to follow the natural fluctuations in temperature. From 90 to 154 dph, the HT and LT groups (n = 150 fish/group) were fed ad libitum two times a day with dry food sprayed with 96% Ethanol, while the other groups E2 groups (n = 150 fish/group) were fed with the same diet supplemented with Estradiol diluted in 96% Ethanol at 10 mg/Kg food (Additional file 1: Figure S3). No treatment-related mortality was observed.

Following the protocols already detailed in [24]; biometric data (standard length: SL, body weight: BW) was collected periodically and gonads samples for microarray and qPCR analysis of gene expression were taken at 170 dph and immediately frozen in liquid Nitrogen. At 170 dph gonads were fixed in 4% Paraformaldehyde to assess the female stages of oocyte maturation and male spermatogenesis progression [49]. Gonadosomatic (GSI), hepatosomatic (HSI) and carcass (CI) indices were determined to analyze the possible effects of temperature and hormonal exposure on fish maturation at 337 dph.

RNA extraction and cDNA synthesis

As previously described in [50], total RNA was obtained from 170 dph sea bass gonads using Trizol and a chloroform-isopropanol-ethanol protocol. RNA concentration was measured with a ND-1000 spectrophotometer, and quality was confirmed by examination on 1% agarose/formaldehyde gels. Total RNA was treated with RNase-free DNase, reverse transcribed to cDNA and checked using a Bioanalyzer 6000 Nano LabChip. Samples with a 100–200 ng/μl concentration and RIN values >7 were selected for microarray hybridizations.

Quantitative real-time PCR (qPCR)

As previously described in [24] qPCR was used to: 1) select high cyp19a1a expressors (presumably females) at 170 dph for microarray analysis (Additional file 2: Figure S7) and, 2) validate microarray results and check several genes related to sex differentiation (Additional file 3: Table S1 for a gene glossary). cDNA was always diluted 1:10 for target genes and 1:500 for the r18S housekeeping gene (previously validated in [47]). Briefly, primer design and quality checking was done using Primer 3 Plus, primer specificity and performance was checked with a melting curve analysis after amplification (Additional file 4: Table S2: E: efficiency between 1.99 and 2.27; slope ranging from −2.6 to −3.3 and R 2: linear correlations higher than 0.94) and a standard qPCR program was performed. Samples were run in triplicate on an ABI 7900HT in 384-wells plates in a final volume of 10 μl per well with negative controls lacking cDNA/primers always included in duplicate. Data were collected and analyzed using SDS 2.3 and RQ Manager 1.2 software. Primer E was used to adjust Ct values and the r18S housekeeping gene was used to correct for intra- and inter-assay variations [51].


Five individuals per group were individually hybridized and randomly distributed on different slides to avoid batch effects. Microarrays were hybridized at the Institute of Biotechnology and Biomedicine (UAB, Barcelona). Briefly, RNA was Cy3-labeled with Agilent’s One-Color Microarray-Based Gene Expression Analysis, along with Agilent’s One-Color RNA SpikeIn Kit), cRNA was purified, quantified on a ND-1000 Nanodrop, verified on a Bionalyazer 2100, hybridized in a custom sea bass array (Agilent ID 023790), washed and scanned (see detailed protocol in [24]). Intensities and control features were checked by Agilent’s Feature Extraction software version The platform that validates the array can be seen at Gene Expression Omnibus (GEO)-NCBI database (GPL13443). Datasets used in this article are accessible at GSE52307 for the LT and HT samples and at GSE52938 for LT-E2 and HT-E2 ones.

Statistical analysis of data

Briefly, data were checked for normality (Kolmogorov-Smirnov’s test), homoscedasticity of variance (Levene’s test) and log-transformed when needed. GSI, HSI and CI data were arcsine transformed before any statistical analysis. A two-step cluster analysis previously validated and described elsewhere [52] of 2DCt cyp19a1a values at 170 dph was used to select the highest cyp19a1a expressors per sample for the hybridizations based on the available expression data (Additional file 2: Figure S7). One-way analysis of variance (ANOVA) was used to determine differences between relative cyp19a1a mRNA levels resulting from qPCR high and low expressors and for length, weight, GSI, HSI and CI data sets. Post hoc multiple comparisons (Tukey’s HSD test) were done when statistical differences were present. Data are expressed as mean ± SEM (standard error of the mean). In this study, differences were accepted as significant when P < 0.05. Chi-square test with Yates’ correction [53] was used for sex ratio analysis and qPCR 2DCt values [51] were analyzed by Student’s t-test. Analyses were performed using IBM SPSS Statistics 19.

Briefly, microarray raw data from the Feature Extraction output files was corrected for background noise [54] and quantile normalized [55]. Limma [56] was used to analyze differential expression, and then corrected for multiple testing (False Discovery Rate method, FDR). Genes were considered to be differentially expressed genes (DEG) when the absolute fold change between the two compared groups, was higher than 1.5, the adjusted P-value was lower than 0.05 and were reliable in all samples. A Principal Component Analysis was performed to visualize the variability of the samples (Additional file 5: Figure S4). Statistical analysis was performed with the Bioconductor project ( ) in the R statistical environment ( ) [57].

Gene annotation enrichment analysis

Briefly, Genecards ( ) and Uniprot ( were used to assign gene names, gene symbols, synonyms and functions. The web based tool AMIGO ( ; [58]) was used to obtain sequences of the DEG, Blast2GO software [59], KEGG ( ) and DAVID (; [60, 61]) were used to assign GO terms as well as the pathways associated to these genes. In addition, Blast2GO was used with a reference set containing all the genes from the custom-made microarray to check GO term results by a two-tailed Fisher’s Exact Test with Multiple Testing Correction of FDR [62]. Physical and functional protein interactions of the genes were modeled with a web based tool STRING v9.1 (; [63]) using the STRING human database as a background.



Standard length (SL) and body weight (BW) were assessed at 170 (when samples for microarray hybridizations and qPCR were taken) and 337 dph (when sex ratio was assessed). At 170 dph, in both the low and the high cyp19a1a expressors, the Estradiol (E2) treated fish were shorter (P < 0.01) and lighter (P < 0.001) than those of the high temperature (HT) group indicating a negative effect of E2 on growth. These differences were also present when comparing low temperature (LT) vs. low temperature plus Estradiol (LT-E2) fish (Table 1). At 337 dph, sexual growth dimorphism (SGD) was not observed in the HT group, since there were no differences in BW between sexes (Table 2). In contrast to the situation observed at 170 dph, at 337 dph HT females, despite being slightly bigger and heavier than E2-exposed females, showed no differences for SL nor BW (Table 2) and neither for hepatosomatic (HIS) nor carcass index (CI) (data not shown).

Table 1 Growth of European sea bass juveniles at 170 days post hatch according to treatment and cyp19a1a expression levels by qPCR
Table 2 Growth of European sea bass juveniles at 337 days post hatch according to treatment and sex

Sex ratio and gonadosomatic index (GSI)

Sex ratio was female- (100% females) and male- biased (21% females) at the HT-E2 and HT groups, respectively (Fig. 1a) with statistically significant differences between them (P < 0.001) and between HT and LT, but not between HT-E2 and LT-E2 (P > 0.05, Additional file 6: Figure S1). At 337 dph, the GSI was significantly higher in HT females when compared to the HT-E2 females (P < 0.05) (Fig. 1b), but with no differences when compared to LT or LT-E2 (Additional file 6: Figure S1). Females were still immature, with ovaries replete with oocytes at the cortical alveolar stage (Additional file 7: Figure S2a-b). On the other hand, HT group males were fully mature and presented seminiferous tubules filled with sperm (Additional file 7: Figure S2c).

Fig. 1
figure 1

Effects of thermal and estrogen exposure on one-year-old European sea bass gonads. a Percent females in each group, with a difference from a Fisherian 1:1 balanced sex ratio. b Female gonadosomatic index. Data as mean + SEM. Males were not included since no males were present in the E2-treated group. * = P < 0.05; *** = P < 0.001

Transcriptomic analysis of gene expression in sexually differentiating gonads

Here we focus on the analysis of gonads undergoing different developmental pathways by using the masculinization effect of high temperature to force fish to develop as males and then either rescuing the phenotype by administering E2 (HT-E2 group) or studying the temperature-resistant females (HT group). A Principal Component Analysis showed how samples clustered on a treatment-manner (Additional file 5: Figure S4) and further microarray analysis yielded 383 significantly differentially expressed genes (DEG) where 92 were up- and 291 downregulated (Additional file 8: Table S3 and Additional file 9: Table S4). Hierarchical clustering based on the DEG showed that fish clustered in a treatment-related manner (Fig. 2). The associated pathways analyzed by DAVID (Additional file 9: Table S4), suggested that E2 exposure inhibited pathways processes related to DNA replication and repair, reproduction (progesterone-mediated oocyte maturation), hormonal-signaling (gonadotropin releasing hormone GnRH, epidermal growth factor erbB or Hedgehog), lipid metabolism or immunology. In contrast, pathways related to oocyte meiosis, steroid biosynthesis, sugar metabolism or cytokine receptor interactions were induced (Fig. 2, Additional file 10: Table S5). The comparison between LT (fish reared at low temperature allowing female development) and LT-E2 (fish reared at low temperature but forced to develop as females) had just 2 DEG, showing no differences between natural and artificial females (data not shown). On the other hand, when analyzing the common DEG between the double comparisons of the HT-E2 group vs. LT or vs. LT-E2 there were 91 upregulated and 93 downregulated common genes in the E2 group (Fig. 3, Additional file 11: Table S6). These genes were mainly involved in increased DNA repair, mitotic cell cycle and apoptosis; and in a reduction of the integration of energy metabolism, adherent and tight junctions, adipocytokine, epithelial cell signaling pathways and muscle contraction (data not shown).

Fig. 2
figure 2

Individual heatmap representation of the differentially expressed genes in European sea bass 170 dph gonads from HT-E2 vs. HT group fish. Low to high expression is shown by a gradation color from red to green. On the right, the hierarchical clustering of genes by DAVID is shown (for a more tailed list of the altered pathways see Additional file 8: Table S3)

Fig. 3
figure 3

Venn diagram representation of the common up- and down-regulated genes for the comparisons HT-E2 vs. LT and HT-E2 vs. LT-E2)

Fig. 4
figure 4

Quantitative real time PCR validation results for genes related to testis differentiation (a-c): anti-Müllerian hormone (amh), doublesex- and mab-3-related transcription factor 1 (dmrt1) and tescalcin (tesc); (d) cholesterol import: steroidogenic acute regulatory protein (star); ovarian differentiation (e-h): gonadal isoform of aromatase (cyp19a1a), SRY-related HMG-box transcription factor SOX17 (sox17), Wnt inducible signaling pathway protein 1 (wisp1), and vasa protein (vasa); (i) the neural isoform of aromatase (cyp19a1b), and (j) insulin-like growth factor 1 (igf1). Asterisks indicate significant statistical differences between groups (* = P < 0.05, ** = P < 0.01, *** = P < 0.001)

Fig. 5
figure 5

A KEGG pathway-based figure depicting the ovarian steroidogenesis pathway. Microarray results are marked with arrows (green arrows indicate a fold change (FC) higher than 1.5, while red arrows indicate a FC lower than 1.5). Yellow stars note genes in the microarray HT-E2 vs. HT comparison

Validation of microarray results by qPCR

qPCR of some genes relevant for growth and reproduction were used to validate the array (Fig. 4 and Table 3). E2 exposure significantly (P < 0.05) downregulated genes related to testis differentiation, such as the anti-Müllerian hormone (amh; P < 0.05), doublesex- and mab-3-related transcription factor 1 (dmrt1; P < 0.01) and tescalcin (tesc;P < 0.05) (Fig. 4a-c, respectively). It also downregulated cholesterol import-related genes such as the steroidogenic acute regulatory protein (star; P < 0.01; Fig. 4d) and two ovarian differentiation-related genes: gonadal aromatase (cyp19a1a; Fig. 4e) and the Wnt1-inducible-signaling pathway protein1 (wisp1; Fig. 4g). However, other genes related to ovarian differentiation such as the transcription factor sox17 (Fig. 4f) and a germ cell marker, vasa (Fig. 4h) were not affected. The neural isoform of aromatase (cyp19a1b) was significantly upregulated (P < 0.05) by E2 exposure (Fig. 4i), while insulin-like growth factor-1 (igf1) was not altered (Fig. 4j). For half of the analyzed genes, their expression was increased in the E2 group when comparing fold change changes (data not shown).

Table 3 Microarray validation by qPCR for the HT-E2 vs. HT comparison

Gene ontology enrichment analysis of genes regulated by exposure to estradiol

Blast2GO analysis enabled the identification of the associated GO terms for the DEG and related then to biological process (BP), molecular function (MF) or cell component (CC) and always showed more downregulated GO terms at any given comparison. The main subcategories were related to reproduction, signaling, responses to stimulus, growth, immune system and developmental processes. Binding and catalytic activities were the most abundant among the MF subcategories (Additional file 12: Table S7). A two-tailed Fisher’s exact test with multiple testing corrections for FDR (P-value filter of 0.05) was performed to assess the over-representation of the functions related to the downregulated genes due to E2 exposure. Specifically, there were 148 over-represented functions when taking the microarray as background (Additional file 13: Table S8). The most interesting enriched MF GO-terms (26) were related to nuclear hormone receptor binding, growth factor and NADP-retinol hydrogenase activity. Enriched GO-terms (97) were clearly related to reproduction, immunology and growth.

KEGG pathway enrichment analysis of genes regulated by estradiol exposure

There were 46 pathways affected by the E2 treatment (Additional file 14: Table S9), mainly related to metabolism (i.e. retinol), immunological signaling and steroid hormone biosynthesis. Furthermore, DAVID analysis on the GO-terms with the highest stringency showed that meiosis (two clusters with 1.47 and 1.06 enriched scores), reproduction (three clusters with 1.17, 0.81 and 0.4 enriched scores) and hormone regulation (one cluster with 0.33 enriched scores) were the most enriched ones. Since we found the genes involved in reproduction to be downregulated as a result of the E2 exposure, we further focused on the genes related to the ovarian steroidogenic pathway, which for most of them showed the same tendency towards downregulation. Among them, star and gnrh genes exhibited a significant downregulation (P < 0.05). On the other hand, igf1 exhibited an opposite significant increase in the expression (P < 0.05) due to the E2 exposure (Fig. 5).

Protein-protein interaction analysis

Proteins coded by the DEG analyzed using STRING, showed enrichment in interactions (P < 0.001). E2 exposure caused an increase in protein interactions (range of combined scores of interactions 0.400–0.999) for protein networks related to transcriptional activation, DNA repair, immunity, catabolism, oxidative phosphorylation and muscle contraction (Additional file 15: Figure S5). On the other hand, protein networks obtained from the downregulated genes (range of combined scores of interactions 0.402–0.999) were more related to: apoptosis, inflammation, histone demethylases and inhibition of histone acetylase 1, cell adhesion, morphology and motility, protein complex assembly, intracellular trafficking and secretion, Rho and Rac GTPases activators, response to hormonal stimulus and reproductive structure development (Additional file 16: Figure S6).

Genes related to epigenetic regulatory mechanisms

We found that demethylases, dicer1, helicases, most of the histone deacetylases, polycomb complex members, as well as DNA-methyltransferases 1 and 3 were downregulated in the HT-E2 group. In contrast, most histone acetyltransferases and methyltransferases were upregulated. Finally, histone acetylase 11 (hdac11) and euchromatic histone lysine N-methyltransferase (ehmt2), two genes previously analyzed by qPCR, showed a heat-related upregulation even under the E2 exposure (Additional file 17: Table S10).


In this study, European sea bass, a fish with mixed genetic and environmental influences [40, 41], was used to analyze the transcriptome of fish gonads at the time of sex differentiation after being exposed to thermal and chemical perturbations. Here we show how the exposure to estrogen at early juvenile development was able to completely feminize the population that otherwise would have developed as males due to the high temperature. Moreover, estrogen-exposed females showed a transcriptome reprograming that affected not only steroidogenesis but also pathways related to reproduction, immunity, growth, response to stimulus and the metabolism of lipids and xenobiotics. However, based on histological analysis, we observed that once the female phenotype is imposed gonads could proceed with apparent normal development.

The HT group was masculinized (21% females) by the elevated temperatures. However, this masculinizing effect was completely overridden by the E2 exposure (100% females at HT-E2 group) without affecting the histological structure of the immature ovary, where cortical alveolar oocytes in both groups predominated. However, GSI values indicate a reduction in ovarian growth as already described in other fish species [64,65,66]. GSI values of E2-exposed females are higher than those of control females, without any apparent effect on fat content, opposite to what was found by Saillant et al. [14], and with slightly higher HSI in E2-exposed females, in agreement with previous studies on fish subjected to the effects of xenoestrogens [67,68,69].

Microarray analysis showed that E2 exposure caused an alteration in the expression of 383 genes from gonads at the time of sex differentiation. The most important of those effects are discussed below.


It is well established that cyp19a1a gene expression and aromatase enzyme activity are necessary for normal ovarian differentiation and maintenance in all non-mammalian vertebrates including fishes [3]. E2 treatment caused a significant downregulation of cyp19a1a at 170 dph, as assessed both by analysis of microarray and qPCR data. This downregulation took place after the hormonal exposure finished, as has also been observed in rainbow trout and zebrafish [34, 70]. Nevertheless, since 1) E2 completely feminized the exposed fish, even after initial heat-induced masculinization, and 2) the European sea bass cyp19a1a promoter lacks estrogen response elements (EREs), as in other fish [71], these observations suggest that E2-induced feminization likely did not involve direct cyp19a1a regulation. This interpretation is supported by qPCR results showing that star, an upstream component of the steroidogenic pathway, also was significantly downregulated by E2, in agreement with previous results in zebrafish [70], suggesting that E2 shuts down the first steps of steroidogenesis by blocking star expression. Furthermore, the microarray analysis showed a whole downregulation of the ovarian steroidogenesis pathway and this seems to happen in a dose- and species-dependent manner. This is supported by the fact that E2 expression decreases in some species and increases in some others [38, 66, 70, 72]; and because other downstream genes such as cyp19a1a and 17β-hsd are affected in females but not in males [70, 72, 73]. However, studies in our lab have shown how the cyp19a1a expression downregulation does not involve changes in its promoter DNA methylation since unexposed and feminized females by E2 showed no differences in the gonadal aromatase promoter methylation levels [43]. Other genes related to ovarian differentiation (i.e., wisp1, cyp19a1a, 17β-hsd and star) as well as male-related genes (amh, dmrt1 and tesc) or cyp19a1b and sox17 were downregulated after E2 exposure, although in contrast to what has been found in other fish species [66].


Some studies have suggested the possibility that sex steroids affect the immune system [74]. The microarray we used has a good representation of immunity-related terms and contains probes for a group of genes constituting the signaling pathways responsible for generating the immune response, including the Toll-like, NOD-like, RIG-I-like and the T-cell receptor signaling pathways. Interestingly, the latter was downregulated in the E2 group. Also, many genes of the complement component, some cytokines and lysozymes were also downregulated after E2 exposure, as seen before for medaka [29, 30]. In contrast, several terms referring to response to stimulus were enriched, including response to estradiol, mechanical stimulus, lipopolysaccharids and regulation of response to stress.

Xenobiotic metabolism

Microarray DEG showed three pathways related to drug and xenobiotic metabolism through cytochrome P450 downregulation. Furthermore, some proteins that are able to metabolize xenobiotics like the glutathione S-transferase proteins (GSTs; [75]) were up- (gst and gstθ) and downregulated (gstα, gstk, gstm) in the microarray. The latter is in agreement with what have been seen for gstα in goldfish when exposed to a hepatotoxin (Carassius auratus; [76]) or to gstπ in Atlantic salmon when exposed to tributyltin (Salmo salar; [77]).


Sex steroids can influence fish growth by altering the GH-IGF system [78]. Furthermore, during the sexual maturation of some fish species [79,80,81], plasma levels of sex steroids and GH correlate indicating a crosstalk between reproduction and growth-related pathways. In this study, all genes related to growth hormone and its receptor were downregulated. The same occurred with the insulin-like growth factor II gene, its receptors, igf1r, and its associated binding proteins in opposition to what has been described for the fathead minnow [38, 66] when exposed only to E2. Further studies are needed since, at present, it is not possible to discern if these differences are species-specific or the result of the combination of both thermal and E2 exposures.

Lipid metabolism

After E2 exposure, terms related to lipid metabolism such as white fat cell differentiation, regulation of fat cell differentiation, plasma lipoprotein clearance, apolipoprotein binding or high and low density lipoprotein particle remodeling were over represented. Similar downregulations of apolipoproteins have been shown in different fish species [82,83,84], but not in the mummichog (Fundulus heteroclitus; [39]). Moreover, apoe, a protein related to lipid uptake by oocytes, was also downregulated in our study, in agreement with what has been previously shown for zebrafish [85].

Epigenetic regulatory mechanisms-related genes

Although it is known that epigenetic mechanisms are responsible for the acquisition and maintenance of cell identity, they have been only marginally explored in an ecological context. Thus, we have analyzed by qPCR the behavior of seven genes related to epigenetic regulatory mechanisms present in our microarray. Among them, dicer1, jarid2a, pcgf2, suz12 and mettl22 were downregulated by E2, overriding temperature effects. In contrast, the expression of ehmt2 and hdac11 was unchanged. Interestingly, we have also observed that the expression of six heat shock proteins (hrsp12, hsbp1, hsp10, hsp60, hspa14 and hsp70) was upregulated, implying that early exposure to elevated temperatures had persistent effects on the gonadal transcriptome, effects that were not overridden by the subsequent E2 exposure.


Taken together, these results show how at the population level all fish developed as females since estrogen exposure during early juvenile development is able to completely override the masculinizing effect of elevated temperatures. However, these fish developed as females despite showing at the time of sex differentiation a downregulation of key genes in steroidogenesis. This blockage happened not only at upstream genes of the pathway such as star, but also at downstream genes such as cyp19a1a and 17β-hsd. Furthermore, estradiol administration also affected pathways related to reproduction, immunity, xenobiotic and lipid metabolism, signaling, responses to stimulus and growth. Thus, exposure to exogenous estrogens of sexually differentiating fish had a profound reprogramming effect on their gonadal transcriptome, causing not only a complete feminization of the population but an inhibition of steroidogenesis in developing females. It should be noted that some of the resulting females will be fish that otherwise would have developed as males. However, at 1 year of age, feminized fish exhibited a normal gonadal histology suggesting that once the female phenotype is imposed gonads can apparently continue their normal development. Although the impact of sex-reversed fish on natural populations has been simulated in species with simple chromosomal sex determining systems [86], the situation in species with more complex systems like the one used in this study remains unexplored. The data shown in this study helps to fill in the gap on the underlying mechanisms operating at the molecular level.


amh :

Anti-Müllerian hormone

aqp1 :

Aquaporin 1


Biological process


Body weight


Cell component


Carcass index

col18a1 :

Collagen alpha-1 (XVIII) chain

cyp19a1a :

Cytochrome P450, family 19, subfamily A, polypeptide 1a

cyp19a1b :

Cytochrome P450, family 19, subfamily A, polypeptide 1b


Differentially expressed genes

dicer1 :

Endoribonuclease Dicer

dmrt1 :

Doublesex- and mab-3- related transcription factor I


Days post hatch

E2 :


ehmt2 :

Euchromatic histone-lysine N-methyltransferase 2


Epidermal growth factor receptor


Estrogen response elements


Fold change


False discovery rate


Gene expression Omnibus

gnrh :

Gonadotropin-releasing hormone


Gonadosomatic index


Glutathione S-transferase proteins

hdac11 :

Histone deacetylase 11


Hepatosomatic index


Heat responsive protein 12


Heat shock binding protein


Hormone-sensitive period


Heat shock protein 10


Heat shock protein 60


Heat shock protein 14


Heat shock protein 70


Control high temperature

HT-E2 :

Estradiol high temperature

igf1 :

Insulin-like growth factor I

jarid2a :

Protein Jumonji


Control low temperature

LT-E2 :

Estradiol low temperature

mettl22 :

Methyltransferase-like protein 22


Molecular function


Principal component analysis

pcgf2 :

Polycomb group ring finger 2

prl :



Quantitative real-time PCR

r18S :



Sexual growth dimorphism


Standard length

sox17 :

HMG-box transcription factor SOX17

star :

Steroidogenic acute regulatory protein

suz12 :

Suppressor of zeste 12 homolog

tesc :


vasa :

Vasa protein

wisp1 :

WNT1 inducible signaling pathway protein 1


  1. Werren JH, Beukeboom LW. Sex determination, sex ratios, and genetic conflict. Annu Rev Ecol Syst. 1998;29:233–61.

    Article  Google Scholar 

  2. Penman DJ, Piferrer F. Fish gonadogenesis. Part 1. Genetic and environmental mechanisms of sex determination. Rev Fish Sci. 2008;16(S1):16–34.

    Article  CAS  Google Scholar 

  3. Piferrer F, Guiguen G. Fish Gonadogenesis. Part II: molecular biology and genomics of sex differentiation. Rev Fish Sci. 2008;16(S1):35–55.

    Article  CAS  Google Scholar 

  4. Cutting A, Chue J, Smith CA. Just how conserved is vertebrate sex determination? Dev Dynam. 2013;242:380–7.

    Article  CAS  Google Scholar 

  5. Ravi P, Jiang J, Liew WC, Orban L. Small-scale transcriptomics reveals differences among gonadal stages in Asian seabass (Lates calcarifer). Reprod Biol Endocrin. 2014;12:5.

    Article  Google Scholar 

  6. Kikuchi K, Hamaguchi S. Novel sex-determining genes in fish and sex chromosome evolution. Dev Dynam. 2013;242:339–53.

    Article  CAS  Google Scholar 

  7. Nagahama Y. Molecular mechanisms of sex determination and gonadal sex differentiation in fish. Fish Physiol Biochem. 2006;31:105–9.

    Article  Google Scholar 

  8. Piferrer F, Zanuy S, Carrillo M, Solar I, Devlin RH, Donaldson EM. Brief treatment with an aromatase inhibitor during sex differentiation causes chromosomally female salmon to develop as normal, functional males. J Exp Zool. 1994;270:255–62.

    Article  Google Scholar 

  9. Navarro-Martín L, Blázquez M, Piferrer F. Masculinization of the European sea bass (Dicentrarchus labrax) by treatment with an androgen or aromatase inhibitor involves different gene expression and has distinct lasting effects on maturation. Gen Comp Endocr. 2009a;160:3–11.

    Article  PubMed  Google Scholar 

  10. Paul-Prasanth B, Bhandari RK, Kobayashi T, Horiguchi R, Kobayashi Y, Nakamoto M, et al. Estrogen oversees the maintenance of the female genetic program in terminally differentiated gonochorists. Sci Rep-UK. 2014;3:2862.

    Article  Google Scholar 

  11. Sun LN, Jiang XL, Xie QP, Yuan J, Huang BF, Tao WJ, et al. Transdifferentiation of differentiated ovary into functional testis by long-term treatment of aromatase inhibitor in Nile tilapia. Endocrinology. 2013;155:1476–88.

    Article  Google Scholar 

  12. Devlin RH, Nagahama Y. Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences. Aquaculture. 2002;208:191–364.

    Article  CAS  Google Scholar 

  13. Piferrer F. Endocrine sex control strategies for the feminization of teleost fish. Aquaculture. 2001;197:229–81.

    Article  CAS  Google Scholar 

  14. Saillant E, Fostier A, Menu B, Haffray P, Chatain B. Sexual growth dimorphism in sea bass Dicentrarchus labrax. Aquaculture. 2001;202:371–87.

    Article  Google Scholar 

  15. Pandian TJ, Kirankumar S. Recent advances in hormonal induction of sex reversal in fish. J Appl Aquaculture. 2003;13:205–30.

    Article  Google Scholar 

  16. Ospina-Alvárez N, Piferrer F. Temperature-dependent sex determination in fish revisited: prevalence, a single sex ratio response pattern, and possible effects of climate change. PLoS One. 2008;3:e2837.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Strüssmann CA, Conover DO, Somoza GM, Miranda LA. Implications of climate change for the reproductive capacity and survival of new world silversides (family Atherinopsidae). J Fish Biol. 2010;77:1818–34.

    Article  PubMed  Google Scholar 

  18. Schiller V, Wichmann A, Kriehuber R, Schafers C, Fischer R, Fenske M. Transcriptome alterations in zebrafish embryos after exposure to environmental estrogens and anti-androgens can reveal endocrine disruption. Reprod Toxicol. 2013;42:210–23.

    Article  CAS  PubMed  Google Scholar 

  19. Kidd KA, Blanchfield PJ, Mills KH, Palace VP, Evans RE, Lazorchak JM, et al. Collapse of a fish population after exposure to a synthetic estrogen. PNAS. 2007;104:8897–901.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kishi K, Kitagawa E, Onikura N, Nakamura A, Iwahashi H. Expression analysis of sex-specific and 17 beta-estradiol-responsive genes in the Japanese medaka, Oryzias latipes, using oligonucleotide microarrays. Genomic. 2006;88:241–51.

    Article  CAS  Google Scholar 

  21. Tao W, Yuan J, Zhou L, Sun L, Sun Y, Yang S, et al. Characterization of gonadal transcriptomes from Nile tilapia (Oreochromis niloticus) reveals differentially expressed genes. PLoS One. 2013;8:e63604.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Zheng W, Xu H, Lam SH, Luo H, Karuturi RKM, Gong A. Transcriptomic analyses of sexual dimorphism of the zebrafish liver and the effect of sex hormones. PLoS One. 2013;8:e53562.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Shao CW, Li QY, Chen SL, Zhang O, Kian J, Hu Q, et al. Epigenetic modification and inheritance in sexual reversal of fish. Genome Res. 2014;24:604–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Díaz N, Piferrer F. 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. BMC Genomics. 2015;16:679.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Ribas L, Robledo D, Gómez-Tato A, Viñas A, Martínez P, Piferrer F. Comprehensive transcriptomic analysis of the process of gonadal sex differentiation in the turbot (Scophthalmus maximus). Mol Cell Endocrinol. 2015;422:132–49.

    Article  PubMed  Google Scholar 

  26. Gunnarsson L, Kristiansson E, Forlin L, Nerman O, Larsson DGJ. Sensitive and robust gene expression changes in fish exposed to estrogen - a microarray approach. BMC Genomics. 2007;8:149.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Geoghegan F, Katsiadaki I, Williams TD, Chipman JK. A cDNA microarray for the three-spined stickleback, Gasterosteus aculeatus L., and analysis of the interactive effects of oestradiol and dibenzanthracene exposures. J Fish Biol. 2008;72:2133–53.

    Article  CAS  Google Scholar 

  28. Hao R, Bonsson M, Singh AV, Riu A, McCollum CW, Knudsen TB, et al. Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis. PLoS One. 2013;8:e79020.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Sun L, Shao X, Wu Y, Li J, Zhou Q, Lin B, et al. Ontogenetic expression and 17β-estradiol regulation of immune-related genes in early life stages of Japanese medaka (Oryzias latipes). Fish Shellfish Immun. 2011a;30:1131–7.

    Article  CAS  Google Scholar 

  30. Sun LW, Shao XL, Hu XH, Chi J, Jin YX, Ye WH, et al. Transcriptional responses in Japanese medaka (Oryzias latipes) exposed to binary mixtures of an estrogen and anti-estrogens. Aquat Toxicol. 2011b;105:629–39.

    Article  CAS  PubMed  Google Scholar 

  31. Baker ME, Vidal-Dorsch DE, Ribecco C, Sprague LJ, Angert M, Lekmine N, et al. Molecular analysis of endocrine disruption in hornyhead turbot at wastewater outfalls in Southern California using a second generation multi-species microarray. PLoS One. 2013;8:e75553.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Baron D, Montfort J, Houlgatte R, Fostier A, Guiguen Y. Androgen-induced masculinization in rainbow trout results in a marked dysregulation of early gonadal gene expression profiles. BMC Genomics. 2007;8:357.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Vizziano D, Randuineau G, Mahe S, Cauty C, Guiguen Y. Comparison of gonadal gene expression patterns after masculinization of female rainbow trout with an androgen or an aromatase inhibitor. Cybium. 2008;32:83–5.

    Google Scholar 

  34. Vizziano-Cantonnet D, Baron D, Mahe S, Cauty C, Fostier A, Guiguen Y. Estrogen treatment up-regulates female genes but does not suppress all early testicular markers during rainbow trout male-to-female gonadal transdifferentiation. J Mol Endocrin. 2008;41:277–88.

    Article  CAS  Google Scholar 

  35. Govoroun M, McMeel OM, Mecherouki H, Smith TJ, Guiguen Y. 17 beta-estradiol treatment creases steroidogenic enzyme messenger ribonucleic acid levels in the rainbow trout testis. Endocrinology. 2001;142:1841–8.

    Article  CAS  PubMed  Google Scholar 

  36. Santos EM, Paull GC, Van Look KJW, Workman VL, Holt WV, van Aerle R, Kille P, Tyler CR. Gonadal transcriptome responses and physiological consequences of exposure to oestrogen in breeding zebrafish (Danio rerio). Aquat Toxicol. 2007;83:134–42.

    Article  CAS  PubMed  Google Scholar 

  37. Santos EM, Workman VL, Paull GC, Filby AL, Van Look KJW, Kille P, et al. Molecular basis of sex and reproductive status in breeding zebrafish. Physiol Genomics. 2007;30:111–22.

    Article  CAS  PubMed  Google Scholar 

  38. Filby AL, Thorpe KL, Tyler CR. Multiple molecular effect pathways of an environmental oestrogen in fish. J Mol Endocrin. 2006;37:121–34.

    Article  CAS  Google Scholar 

  39. Doyle MA, Boskera T, Martyniuk CJ, MacLatchy DL, Munkittrick KR. The of 17-alpha-ethinylestradiol (EE2) on molecular signaling cascades in mummichog (Fundulus heteroclitus). Aquat Toxicol. 2013;134:34–46.

    Article  PubMed  Google Scholar 

  40. Vandeputte M, Dupont-Nivet M, Chavanne H, Chatain B. A polygenic hypothesis for sex determination in the European sea bass (Dicentrarchus labrax). Genetic. 2007;176:1049–57.

    Article  Google Scholar 

  41. Piferrer F, Blázquez M. Aromatase distribution and regulation in fish. Fish Physiol Biochem. 2005;31:215–26.

    Article  CAS  PubMed  Google Scholar 

  42. Navarro-Martín L, Blázquez M, Viñas J, Joly S, Piferrer F. Balancing the effects of rearing at low temperature during early development on sex ratios, growth and maturation in the European sea bass (Dicentrarchus labrax). Limitations and opportunities for the production of highly female-biased stocks. Aquaculture. 2009b;296:347–58.

    Article  Google Scholar 

  43. Navarro-Martín L, Viñas J, Ribas L, Díaz N, Gutierrez A, Di Croce L, et al. DNA methylation of the gonadal aromatase (cyp19a) promoter is involved in temperature-dependent sex ratio shifts in the European sea bass. PLoS Genet. 2011;7:e1002447.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Blázquez M, Felip A, Zanuy S, Carrillo M, Piferrer F. Critical period of androgen-inducible sex differentiation in a teleost fish, the European sea bass. J Fish Biol. 2001;58:342–58.

    Article  Google Scholar 

  45. Frimodt C. Fishing multilingual illustrated guide to the world's commercial coldwater fish. Osney Mead: Publ., Oxford; 1995.

    Google Scholar 

  46. Referencing this report IPCC. Climate Change 2014: Synthesis report. Contribution of working groups I, II and III to the fifth assessment report of the intergovernmental panel on climate change. Core writing team, Pachauri RK, Meyer LA, (eds.). Geneva: IPCC; 2014. pp. 151.

  47. Ankley GT, Feifarek D, Blackwell B, Cavallin JE, Jensen KM, Kahl MD, Poole S, Rnadolph E, Saari R, Villeneuve DL. Re-evaluating the significance of Estrone as an environmental estrogen. Environ Sci Technol. 2017;51:4705–13.

    Article  CAS  PubMed  Google Scholar 

  48. Díaz N, Ribas L, Piferrer F. The relationship between growth and sex differentiation in the European sea bass (Dicentrarchus labrax). Aquaculture. 2013;408-409:191–202.

    Article  Google Scholar 

  49. Brown-Peterson NJ, Wyanski DM, Saborido-Rey F, Macewicz BJ, Lowerre-Barbieri SK. A standardized terminology for describing reproductive development in fishes. Mar Coast Fish: Dyn, Manage Ecosystem Sci. 2011;3:52–70.

    Article  Google Scholar 

  50. Díaz N, Ribas L, Piferrer F. Effects of changes in food supply at the time of sex differentiation on the gonadal transcriptome of juvenile fish. Implications for natural and farmed populations. PLoS One. 2014;9(10):e111304.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc. 2008;3:1101–8.

    Article  CAS  PubMed  Google Scholar 

  52. Blázquez M, Navarro-Martín L, Piferrer F. Expression profiles of sex differentiation-related genes during ontogenesis in the European sea bass acclimated to two different temperatures. J Exp Zool B Mol Dev Evol. 2009;312:686–700.

    Article  PubMed  Google Scholar 

  53. Fowler J, Cohen L, Jarvis P. Practical statistics for field biology. Hoboken: John Wiley and Sons Inc.; 2008.

    Google Scholar 

  54. Ritchie ME, Silver J, Oshlack A, Holmes M, Diyagama D, Holloway A, et al. A comparison of background correction methods for two-colour microarrays. Bioinformatics. 2007;23:2700–7.

    Article  CAS  PubMed  Google Scholar 

  55. Bolstad, B. Probe level quantile normalization of high density oligonucleotide array data. ( 2001.

  56. Smyth G. Limma: linear models for microarray data. Bioinformatics and computational biology solutions using R and bioconductor; 2005. p. 397–420.

    Book  Google Scholar 

  57. Gentleman R, Carey V, Bates D, Bolstad B, Dettling M, Dudoit S, et al. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol. 2004;5:R80.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Carbon S, Ireland A, Mungall CJ, Shu S, Marshall B, Lewis S. AmiGO: online access to ontology and annotation data. Bioinformatics. 2009;25:288–9.

    Article  CAS  PubMed  Google Scholar 

  59. Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics. 2005;21:3674–6.

    Article  CAS  PubMed  Google Scholar 

  60. Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009a;37:1–13.

    Article  Google Scholar 

  61. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009b;4:44–57.

    Article  CAS  Google Scholar 

  62. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc B. 1995;57:289–300.

    Google Scholar 

  63. Franceschini A, Szklarczyk D, Frankild S, Kuhn M, Simonovic M, Roth A, et al. STRING v9.1: protein-protein interaction networks, with increased coverage and integration. Nucleic Acids Res. 2013;41:D808–15.

    Article  CAS  PubMed  Google Scholar 

  64. Brion F, Tyler CR, Palazzi X, Laillet B, Porcher JM, Garric J, Flammarion P. Impacts of 17beta-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish (Danio rerio). Aquat Toxicol. 2004;68:193–217.

    Article  CAS  PubMed  Google Scholar 

  65. Pawlowski S, Van Aerle R, Tyler CR, Braunbeck T. Effects of 17alpha-ethinylestradiol in a fathead minnow (Pimephales promelas) gonadal recrudescence assay. Ecotox Environ Safe. 2004;57:330–45.

    Article  CAS  Google Scholar 

  66. Filby AL, Thorpe KL, Maack G, Tyler CR. Gene expression profiles revealing the mechanisms of anti-androgen- and estrogen-induced feminization in fish. Aquat Toxicol. 2007;81:219–31.

    Article  CAS  PubMed  Google Scholar 

  67. Schultz IR, Skillman A, Nicolas JM, Cyr DG, Nagler JJ. Short-term exposure to 17a-ethynylestradiol creases the fertility of sexually maturing male rainbow trout (Oncorhynchus mykiss). Environ Toxicol Chem. 2003;22:1272–80.

    Article  CAS  PubMed  Google Scholar 

  68. Parrott JL, Blunt CR. Life-cycle exposure of fathead minnows (Pimephales promelas) to an ethinylestradiol concentration below 1 ng/L reduces egg fertilization success and masculinizes males. Environ Toxicol. 2005;20:131–41.

    Article  CAS  PubMed  Google Scholar 

  69. Osachoff HL, Shelley LK, Furtula V, Aggelen GC, Kennedy CJ. Induction and recovery of estrogenic effects after short-term 17β-Estradiol exposure in juvenile rainbow trout (Oncorhynchus mykiss). Arch Environ Con Tox. 2013;65:276–85.

    Article  CAS  Google Scholar 

  70. Urbatzka R, Rocha E, Reis B, Cruzeiro C, Monteiro MJ. Effects of ethinylestradiol and of an environmentally relevant mixture of xenoestrogens on steroidogenic gene expression and specific transcription factors in zebrafish. Environ Pollut. 2012;164:28–35.

    Article  CAS  PubMed  Google Scholar 

  71. Galay-Burgos M, Gealy C, Navarro-Martin L, Piferrer F, Zanuy S, Sweeney GE. Cloning of the promoter from the gonadal aromatase gene of the European sea bass and identification of single nucleotide polymorphisms. Comp Biochem Physiol A Mol Integr Physiol. 2006;145:47–53.

    Article  PubMed  Google Scholar 

  72. Nakamura I, Kusakabe M, Young G. Differential suppressive effects of low physiological doses of estradiol-17beta in vivo on levels of mRNAs encoding steroidogenic acute regulatory protein and three steroidogenic enzymes in previtellogenic ovarian follicles of rainbow trout. Gen Comp Endocr. 2009;163:318–23.

    Article  CAS  PubMed  Google Scholar 

  73. Sawyer SJ, Gerstner KA, Callard GV. Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation. Gen Comp Endocr. 2006;147:108–17.

    Article  CAS  PubMed  Google Scholar 

  74. Cuesta A, Rodríguez A, Salinas I, Meseguer J, Esteban MA. Early local and systemic innate immune responses in the teleost gilthead seabream after intraperitoneal injection of whole yeast cells. Fish Shellfish Immun. 2007;22:242–51.

    Article  CAS  Google Scholar 

  75. Blanchette B, Feng X, Sing B. Marine glutathione S-transferases. Mar Biotechnol. 2007;9:513–42.

    Article  CAS  PubMed  Google Scholar 

  76. Li GY, Xie P, Fu J. Microcystin-induced variations in transcription of GSTs in an omnivorous freshwater fish, goldfish. Aquat Toxicol. 2008;88:75–80.

    Article  CAS  PubMed  Google Scholar 

  77. Mortensen AS, Arukwe A. Modulation of xenobiotic biotransformation system and hormonal responses in Atlantic salmon (Salmo salar) after exposure to tributyltin (TBT). Comp Biochem Physiol C Toxicol Pharmacol. 2007;145:431–41.

    Article  PubMed  Google Scholar 

  78. Carnevali O, Cardinali M, Maradonna F, Parisi M, Olivotto I, Polzonetti-Magni AM, et al. Hormonal regulation of hepatic IGF-I and IGF-II gene expression in the marine teleost Sparus aurata. Mol Reprod Dev. 2005;71:12–8.

    Article  CAS  PubMed  Google Scholar 

  79. Truau V, Somoza GM, Nahorniak CS, Peter RE. Interactions of estradiol with gonadotropin-releasing hormone and thyrotropin-releasing hormone in the control of growth hormone secretion in the goldfish. Neuroendocrinology. 1992;56:483–90.

    Article  Google Scholar 

  80. Björnsson BT, Taranger GL, Hansen T, Stefansson SO, Haux C. The interrelation between photoperiod, growth hormone, and sexual maturation of adult Atlantic salmon (Salmo salar). Gen Comp Endocrin. 1994;93:70–81.

    Article  Google Scholar 

  81. Norbeck LA, Sheridan MA. An in vitro model for evaluating peripheral regulation of growth in fish: effects of 17β-estradiol and testosterone on the expression of growth hormone receptors, insulin-like growth factors, and insulin-like growth factor type 1 receptors in rainbow trout (Oncorhynchus mykiss). Gen Comp Endocr. 2011;173:270–80.

    Article  CAS  PubMed  Google Scholar 

  82. Moens LN, van Ven K, Van Remortel P, Favero J, Coen WM. Expression profiling of endocrine-disrupting compounds using a customized Cyprinus carpio cDNA microarray. J Toxicol Sci. 2006;93:298–310.

    Article  CAS  Google Scholar 

  83. Skillman AD, Nagler JJ, Hook SE, Small JA, Schultz IR. Dynamics of 17α-Ethynylestradiol exposure in rainbow trout (Oncorhynchus mykiss): absorption, tissue distribution, and hepatic gene expression pattern. Environ Toxicol Chem. 2006;25:2997–3005.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Hoffmann JL, Thomason RG, Lee DM, Brill JL, Price BB, Carr GJ, et al. Hepatic gene expression profiling using GeneChips in zebrafish exposed to 17α-methyldihydrotestosterone. Aquat Toxicol. 2008;87:69–80.

    Article  CAS  PubMed  Google Scholar 

  85. Wit MD, Keil D, Ven KVD, Vandamme S, Witters E, Coen WD. An integrated transcriptomic and proteomic approach characterizing estrogenic and metabolic effects of 17 α-ethinylestradiol in zebrafish (Danio rerio). Gen Comp Endocrin. 2010;167:190–201.

    Article  Google Scholar 

  86. Cotton S, Wedekind K. Population consequences of environmental sex reversal. Conserv Biol. 2008;23:196–206.

    Article  PubMed  Google Scholar 

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Thanks are due to S. Joly for assistance with different aspects of this work and to the fish maintenance staff at our Experimental Aquarium facility (ZAE).


N.D. was supported first by a scholarship from the Government of Spain (BES-2007-14,273) and then by a contract from the Epigen-Aqua (AGL2010–15939) project granted to F.P. Research was supported by the Spanish Ministry of Economy and Competitiveness grants Consolider Aquagenomics (CDS2007–002) and Epifarm (AGL2013–41047-R) to F.P.

Availability of data and materials

As stated in the Methods section, the platform that validates the array can be accessed at Gene Expression Omnibus (GEO)-NCBI database (GPL13443). Datasets used in this article are accessible at GSE52307 for the LT and HT samples and at GSE52938 for LT-E2 and HT-E2 ones.

Author information

Authors and Affiliations



N.D. conceived the experiment, reared the fish, performed the experiments, analyzed the data and wrote the manuscript. FP conceived the experiment, analyzed the results and wrote the paper. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Francesc Piferrer.

Ethics declarations

Ethics approval and consent to participate

Our facilities are approved for animal experimentation by the Ministry of Agriculture and Fisheries (certificate number 08039–46–A) in accordance with the Spanish law (R.D. 223 of March 1988). As stated in the Methods section, fish used in this experiment were treated in agreement with the European Convention for the Protection of Animals used for Experimental and Scientific Purposes (EST Nu 123, 01/01/91). The experimental protocol was approved by the Spanish National Research Council (CSIC) Ethics Committee within the project AGL2013–41047-R. Fish were sacrificed using an overdose of 2-phenoxyethanol (2PE).

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Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Additional files

Additional file 1: Figure S3.

Experimental design. Upper graph depicts thermal differences. High/low temperatures (21 °C/15 °C) are marked in red/orange and blue/purple, respectively. The duration of the Estradiol treatment (90 to 154 dph) is marked with a light orange box. The bottom panel marks the key events related to sex differentiation and maturation in the European sea bass, as well as the main sampling points. (TIFF 2702 kb)

Additional file 2: Figure S7.

Two-step cluster analysis of 2DCt values to divide among high and low cyp19a1a expressors. a) LT (blue circles) and HT(red squares) cyp19a1a 2DCt values. b) LT-E2 (purple circles) and HT-E2 (orange squares) cyp19a1a 2DCt values. The black line denotes the median. (TIFF 2702 kb)

Additional file 3: Table S1.

Gene abbreviation glossary of the genes analyzed by qPCR or selected from the list of differentially expressed genes. (XLSX 9.44 kb)

Additional file 4: Table S2.

Quantitative PCR primer characteristics. (XLSX 11.3kb)

Additional file 5: Figure S4.

Predicted protein-protein interactions by STRING for the 92 upregulated genes in the HT-E2 vs. HT comparison. (TIFF 2702 kb)

Additional file 6: Figure S1.

Sex ratio and GSI. a) Female percent is shown in pink for all the four studied groups. b) GSI for females (pink) and males (blue) at 337 dph. Letters mark statistical differences between groups (females: uppercase; males: lowercase). (TIFF 2702 kb)

Additional file 7: Figure S2.

Histological images of one-year-old European sea bass. a) HT females; b) HT-E2 females and; c) HT males. Scale bar = 50 μm. (TIFF 2702 kb)

Additional file 8: Table S3.

Number of differentially expressed genes in 170 dph European sea bass gonads when comparing fishes from the HT-E2- vs. HT group during sex differentiation. (XLSX 9 kb)

Additional file 9: Table S4.

Differentially expressed genes for the HT-E2 vs. HT comparison. (XLSX 29 kb)

Additional file 10: Table S5.

Pathway analysis by DAVID of clusters of DEG for the HT-E2 vs. HT comparison. (XLSX 14 kb)

Additional file 11: Table S6.

Common DEG when comparing HT-E2 (artificial) females vs. LT and LT-E2 (natural) females. (XLSX 18 kb)

Additional file 12: Table S7.

GO results subdivided into three categories: biological process (BP), molecular function (MF) and cell component (CC) for the HT-E2 vs. HT comparison. (XLSX 12.3 kb)

Additional file 13: Table S8.

Fisher’s exact test with multiple testing corrections of FDR results. (XLSX 17.1 kb)

Additional file 14: Table S9.

KEGG pathways derived from the differentially expressed genes (DEG) that were either up- or downregulated in the HT-E2 vs. HT comparison. (XLSX 11.3 kb)

Additional file 15: Figure S5.

Principal Component Analysis (PCA). Individuals from each group are marked with red squares (HT), orange squares (HT-E2), blue circles (LT) or purple circles (LT-E2). (TIFF 2702 kb)

Additional file 16: Figure S6.

Predicted protein-protein interactions by STRING for the 291downregulated genes in the HT-E2 vs. HT comparison. (TIFF 2702 kb)

Additional file 17: Table S2.

List of the genes related to epigenetic regulatory mechanisms discussed in the text for the HT-E2 vs. HT comparison. (XLSX 12 kb)

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Díaz, N., Piferrer, F. Estrogen exposure overrides the masculinizing effect of elevated temperature by a downregulation of the key genes implicated in sexual differentiation in a fish with mixed genetic and environmental sex determination. BMC Genomics 18, 973 (2017).

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