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# Epigenetic regulation of sex: the role of DNA methylation and zbtb38 in zebrafish sex differentiation and heat-induced masculinization

Fabien Pierron, Débora Heroin, Flore Daramy

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Fabien Pierron, Débora Heroin, Flore Daramy. Epigenetic regulation of sex: the role of DNA methylation and zbtb38 in zebrafish sex differentiation and heat-induced masculinization. Molecular and Cellular Endocrinology, 2025, 609, pp.112636. 10.1016/j.mce.2025.112636. hal-05343075

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Submitted on 3 Nov 2025

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# Epigenetic regulation of sex: the role of DNA methylation and *zbtb38* in zebrafish sex differentiation and heat-induced masculinization

Fabien Pierron* ID, Débora Heroin ID, Flore Daramy ID

Univ. Bordeaux, CNRS, Bordeaux INP, EPOC, UMR 5805, F-33600, Pessac, France

<figure><img src="image_4.png" /></figure>

## ARTICLE INFO

### Keywords:

- Sex differentiation
- DNA methylation
- Mutual antagonism
- Temperature
- *zbtb38*
- Aromatase

## ABSTRACT

There is increasing evidence that global change can threaten biodiversity by inducing skewed sex ratios. Accumulating evidences support a role of epigenetics, mainly DNA methylation, in sex differentiation. The aim of the present work was to investigate the potential role of *zbtb38*, a transcriptional factor that binds to methylated promoters, in sex differentiation and/or maintenance in zebrafish. We analyzed the methylation and transcription level of *zbtb38* in males, females and undifferentiated individuals raised at standard or high temperature, a masculinizing factor. Results were compared to those obtained for genes already known to be involved in sex differentiation/maintenance (*cyp19a1a*, *foxl2a*, *dmrt1*). All genes presented a sex-specific pattern of DNA methylation and transcription but the most significant differences between sexes were observed for *zbtb38*. Moreover, a highly significant positive correlation was observed between the methylation level of *zbtb38* and *cyp19a1a*, which encodes an enzyme that converts androgens into estradiol. However, while the hypermethylation of *cyp19a1a* was associated with its down-regulation, an inverse relationship was observed for *zbtb38*, providing a basis for mutual antagonism. Furthermore, *zbtb38* was the only gene for which its transcription level was affected by temperature, being up-regulated in females that escaped to masculinization. Finally, despite embryos presented a paternal methylome, *zbtb38* was the only gene for which its methylation level rapidly changed during early development to reach intermediate values between males and females at the larval stage, ie a bi-potential state. Our results strongly support a strategic role of DNA methylation and *zbtb38* in sex differentiation and maintenance.

## 1. Introduction

Population maintenance and, by extension, species perpetuation in sexually reproducing organisms critically depends on sex differentiation, ie the process by which an organism initiates its development as a male (testicle) or female (ovary). In many species, sex differentiation occurs during early development and at adulthood the individual can produce either female (oocytes) or male (spermatozoids) gametes (Aharon and Marlow, 2021; Capel, 2017). For several decades now, scientists have issued warnings about the influence of human activities on sex ratios, like the increasing threat of sea turtle populations due to climate change (Carter and Hopkins, 2022; Edmonds, 2021). Thus, in so called temperature-dependent sex determination (TSD) species like sea turtles, exposure to elevated temperatures at the embryo-larval stage can influence the sex differentiation process, determining the gonadal/phenotypic sex of animals at adulthood (Schwanz and Georges,

2021). In sea turtles, exposure to high temperatures during the egg incubation period leads to female-biased sex ratios in new cohorts. As global temperatures rise, sea turtles are at risk of decline and extinction (Simantiris, 2024). Despite temperature is the most striking example, other environmental factors such as pollutants, hypoxia, photoperiod or population density are also known to affect sex ratios (Capel, 2017; Santos et al., 2017). Thus, the risk of altered sex ratios extends beyond species with TSD and effects of global change on sex ratios can affect organisms of diverse taxa, from plants to mammals. Finally, it is important to underline that such mechanisms can occur not only in species with an environmental sex determination system (ESD, sex determination is driven by environmental factors) but also in species for which their sex is normally determined by heritable genetic components, leading to genotype-phenotype mismatches (ie individuals with a male genotype but presenting a female phenotype (ovaries), and vice versa) that can have long lasting effects on population demography,

<footnote>* Corresponding author. UMR 5805, EPOC, OASU, Station Marine d'Arcachon Université de Bordeaux, Place du Docteur Bertrand Peyneau, 33120, ARCACHON, France.</footnote>

<footnote>E-mail address: fabien.pierron@u-bordeaux.fr (F. Pierron).</footnote>
genetics and viability (Wedekind, 2017). Understanding the mechanisms involved in sex differentiation is a long-standing challenge in biology with important implications for populations, biodiversity and conservation biology. This also represents an interesting model to explore the various levels of regulation involved in cell fate commitment, sex maintenance and hormonal control.

Recently, a particular attention was paid to the potential role of epigenetics in sex differentiation (from plants to mammals, Budd et al., 2022; Garcia-Moreno et al., 2018; Kuroki and Tachibana, 2018; Li et al., 2021; Ribas et al., 2017a; Sun et al., 2022; Wang et al., 2021). Epigenetic marks are indeed sensitive to environmental factors and are known to play a key role in cell differentiation (Wu and Sun, 2006) and phenotypic plasticity (from plants to mammals, Cubas et al., 1999; Dolinoy et al., 2007; Duncan et al., 2022), ie the process by which a given genome can give rise to different phenotypes. DNA methylation refers to the addition of methyl groups to cytosines and is generally associated with gene repression (Dhar et al., 2021). Several authors notably emphasized the role of the methylation level of the aromatase (also called cytochrome P450 19A1, cyp19a1) gene in sex differentiation and/or maintenance in fish, its promoter being hypermethylated in male gonads and hypomethylated in female gonads (Navarro-Martín et al., 2011; Pierron et al., 2021; Wang et al., 2017). CYP19a catalyzes the conversion of androgens into estradiol and mutants lacking gonadal aromatase develop as males (Lau et al., 2016). However, how the methylation level of cyp19a1 is regulated and how it could be influenced by the environment remains an enigma. Using a whole methyme approach, we recently reported in zebrafish (Danio rerio) that the methylation level of the exon 3 (ex3, non-coding exon) of the gene encoding the zinc finger and BTB domain-containing protein 38 (zbtb38) also significantly differs between the gonads of males and females. Moreover, the transcription level in gonads of parents of a natural antisense RNA (ie an epigenetic mechanism) encoded by this exon was found to be highly correlated with offspring sex ratios, its up-regulation induced by cadmium or high density leading to offspring female-biased sex ratios (Pierron et al., 2023). Two other studies (Pan et al., 2022; Yang et al., 2022) also reported a potential role of ztbtb38 in sex determination in the catfish (Ictalus punctatus) since some male-specific single nucleotide polymorphisms (SNPs) were identified in the coding region of the ztbtb38 gene. Moreover, ztbtb38 itself is involved in the regulation of genes by DNA methylation as it encodes a zinc finger transcription factor that binds to methylated DNA (Marchal et al., 2022). Some authors have even proposed that ZBTB38 (previously known as CIBZ) can be involved in the regulation and/or maintenance of DNA methylation marks at specific sites (Reddington and Meehan, 2011; Vural et al., 2021). Since (i) sex differentiation and maintenance proceed by activating pro-female genes (eg cyp19a) while repressing pro-male genes (eg Doublesex- and mab-3-related transcription factor 1, dmrt1), and reciprocally, and (ii) genes involved in sex determination (eg cyp19a) present differing DNA methylation levels between males and females, it is tempting to speculate that ztbtb38 could play a strategic role in sex differentiation and/or maintenance. The aim of the present work was thus to get more insights into the role of ztbtb38 in sex differentiation and/or maintenance in zebrafish. Zebrafish is a gonochoric species with a genetic sex determination system. Wild zebrafish utilize a WZ/ZZ chromosomal method for sex determination. Sex determination in domesticated (wild-type) strains is more complex. Indeed, previous investigations have shown that wild-type zebrafish not only lack classic ESD but also sex chromosomes. Accumulating evidences support the fact that the sex of wild-type zebrafish is primarily determined by genetic factors and more precisely by a polygenic system, where sex determining genes are distributed throughout the genome (Liew et al., 2012; Wilson et al., 2014; Ribas et al., 2017b). However, despite zebrafish sex is genetically determined, some influences from the environment were reported in both wild and wild-type zebrafish (see Santos et al., 2017; Aharon and Marlow, 2021 for reviews). Particularly, using a wild-type strain and genetic all-females, Uchida et al. (2004) have shown that

rearing zebrafish with a female genotype at high temperatures induces a male-biased sex-ratio. In wild strains, despite heterogametic ZW individuals normally develop as females, some of them can develop as males under certain conditions (Wilson et al., 2014). These sex-reversed females are called neomales or pseudomales. We analyzed the methylation and transcription level of ztbtb38 in adult gonads and in whole embryos and larvae before comparing these results to those obtained for genes already known to be involved in sex differentiation and/or maintenance (see Pierron et al., 2021): cyp19a1a (ovarian differentiation and development, Lau et al., 2016), foxl2a (Forkhead box protein L2, ovary maintenance, Yang et al., 2017) and dmrt1 (testis development, Webster et al., 2017). We also investigated the response of ztbtb38 to high temperature, a factor that directly induces masculinization of genetic female zebrafish (Uchida et al., 2004). For all genes, methylation analyses were carried out in the promoter region. For ztbtb38, analyses were carried both in its promoter (pr) and ex3 and the transcription level of its exon 4 (ie coding exon) was analyzed.

## 2. Methods

### 2.1. Fish samples

In order to limit the number of animals used in scientific research, we used fish samples (domesticated, TU strain, Danio rerio) from a previous transgenerational experiment (4 generations, a full description is available in Pierron et al., 2021). Briefly, at the first generation, half of the individuals were raised throughout their life in cadmium-contaminated water (Cd, 1 µg/L) while the other half was raised in Cd-free water (control condition). In the subsequent generations, fish were no more exposed to Cd. During the experiment, a nominal temperature of 28 °C was used. Fish were held in four large aquariums (75 L) per condition at a density that varied from 1.01 to 2.21 fish.L⁻¹ among generations. However, at the first and third generation, 75–80 individuals from both groups (control and Cd-exposed fish) were raised from 14 dpf at an elevated temperature, 34 °C, in two large aquariums (75 L). Heat-exposed fish were not mated. To limit a potential genetic/family bias, each aquarium was initially filled with juveniles obtained from 3 to 5 different mating pairs. Throughout the experiment, the light-dark cycle was fixed at 14-h light and 10-h dark and fish were fed two times daily with live brine shrimps and one time with dry granules (Tetramin). Each aquarium was equipped with a pump and an UV lamp. Moreover, to maintain water quality throughout the experiment among aquariums, all aquariums were permanently supplied with water providing from a single reservoir (the water was filtered, aerated, heated and pH was regulated by adding CO₂) at a rate of 6.5 ml min⁻¹. At the adult stage, mortality was less than 1 % at 28 °C and ranged from 4 to 7.5 % at 34 °C. Total duration of the experiment was 643 days.

Fish were sampled at the larval stage at 5 days post-fertilization (dpf) and sexually mature individuals (both males and females) were sampled, dissected and sexed by visual inspection at 124–130 dpf. In addition, some embryos were sampled at 2–3 h post-fertilization (hpf). All samples were preserved at –20 °C in RNA later until analyses.

The experimental procedure was approved by the Aquitaine fish-birds ethic committee (APAFIS#7535–2016111009351504).

### 2.2. RNA/DNA extraction and processing steps

At each generation (F0-F3) and for each condition (Control or Cd), total RNA and DNA were individually purified from gonads of 6 males and 6 females or 6 whole larvae using the AllPrep DNA/RNA kit (Qiagen). Thus, DNA/RNA were extracted from 48 females (24 females per condition), 48 males (24 males per condition) and 48 larvae (24 larvae per condition) raised at 28 °C. For fish raised at 34 °C, considering that temperature significantly increased the proportion of males, only 5 control females per generation could be obtained. In contrast, for males and Cd-exposed females, 8 heat-treated animals per generation and
condition were used. Thus, DNA/RNA were extracted from 26 females (10 controls and 16 Cd) and 32 males (16 fish per condition) raised at 34 °C. An amount of 500 ng of DNA was then submitted to bisulfite treatment by means of the MethylEdge bisulfite conversion system (Promega). First-strand cDNA was synthesized from 250 ng of total RNA for larval and male samples and from 500 ng of total RNA for females using the GoScript Reverse Transcription System (Promega).

For embryos, considering the low amount of genetic material, individual embryos were directly "picked" into 20 µl of Tris-EDTA buffer (Tris-HCl 6.7 mM, EDTA 0.3 mM, pH 9). The solution was then directly submitted to bisulfite conversion as described above.

## 2.3. DNA methylation analysis

For all the sequences under analysis (promoter of *cyp19a1a*, *foxl2a*, *dmrt1* and *zbtb38* and exon 3 of *zbtb38*), their methylation level was analyzed by bisulfite-pyrosequencing using the PyroMark Q48 instrument (Qiagen) as previously described in Pierron et al. (2021). Specific primers used as well as the coordinates of the regions amplified and sequenced are available in Table S1. For *zbtb38*, we also carried out an allele-specific analysis. Indeed, in our previous paper (Pierron et al., 2023), we reported the presence of two alleles for the *zbtb38* gene in our samples. The gene methylation level as well as the methylation level of each allele were analyzed. The two alleles are respectively called REF (reference) and alt (alternative). The first one (REF) is 100% identical to the sequence available in the zebrafish reference genome GRCz11. The second one (alt) presents a high homology with the REF sequence but presents comparatively a higher similarity with the sequence from the wild-derived zebrafish strain Nadia. Thus, by comparing the sequence of *zbtb38* from the TU (Reference genome GRCz11) and Nadia strains, we identified some single nucleotide polymorphisms (SNPs) in the regions of interest (ie promoter and ex3). The presence of these SNPs was confirmed by PCR and Sanger sequencing. We then used these SNPs to design allele-specific primers. For the two regions analyzed (ie promoter and ex3), the reverse primer was common to the two alleles. Instead, the forward primer was designed in a region presenting one (ex3) or three (promoter) SNPs. As one SNP (ex3) appeared to be insufficient to obtain satisfactory results, we used a strategy described by Chen and Schedl (2021). This strategy employs the differential efficiency of PCR using a primer that has a single mismatch with the REF allele versus two mismatches with the corresponding alt allele, and reciprocally (Fig. S1). Finally, to further increase specificity and in order to increase PCR yield, for the two regions analyzed, we also used a touch-down strategy. Cycling parameters are described in Text S1. In order to validate the specificity of the method, some homozygote (REF/REF or alt/alt) individuals were analyzed. Results (ie pyrograms) are presented in Fig. S2. In addition, for each region, a forward primer common to the two alleles was designed to determine the methylation level of the gene (Fig. S1). The difference between the mean methylation level of the two alleles and the methylation level of the gene was inferior to 6 %, indicating good results.

## 2.4. Quantitative analysis of transcription levels

Complementary DNA was diluted 6-, 8- and 16-fold for larvae, males and females, respectively. PCR reactions were performed using a LC480 (Roche), 95 °C for 2 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 1 min. Each 20 µl reaction contained 12.5 µl GoTaq qPCR Master mix (Promega), 5 µl template and the specific primer pairs at a final concentration of 250 nM each. The reaction specificity was determined for each reaction from the dissociation curve of the PCR product. Moreover, amplification efficiencies for all primer sets were calculated and were found be sufficient to allow direct comparison (see Table S1 for primer sequences). The *actb1*, *eef1a1a* and *rpl13a* genes were initially used as controls. However, the *eef1a1a* gene was finally used as the only reference as the transcription level of *actb1* was found to be affected by

sex and *rpl13a* by the stage of development.

## 2.5. Statistical analyses

Considering the binary distribution (sex-specific) of the data and consequently the absence of normality (Kolmogorov-Smirnov), comparisons among fish groups were performed by the nonparametric Kruskal-Wallis test. When significant effects were detected, pairwise comparisons between each group were then carried out by means of the Dunn-Bonferroni post-hoc test. A Principal Component Analysis (PCA) was used to visually discriminate males from females based on gene methylation and transcription levels and investigate the relative contribution of each variable in the phenotypic sex of animals. Computations were performed using XLSTAT (Addinsoft version 2020.1.1).

# 3. Results and discussion

In order to increase sample size and to use more accurate results, results from the different generations (F0-F3) were combined. In the same way, as Cd did not significantly impact the variables under study, data from control and Cd fish were also combined. Indeed, all generations considered, no significant difference was observed between control and Cd-exposed males or between control and Cd-exposed females (Table S2). Moreover, Cd exposure did not directly affect the sex ratio of the population but instead led to a progressive and transgenerational feminization of the subsequent unexposed generations. This transgenerational effect of Cd was already addressed in our previous articles (refer to Pierron et al., 2021, 2023). Thus, only the phenotypic sex of animals was considered in the present work. Our aim was indeed here to investigate the relationship between *zbtb38* and the phenotypic sex of animals, whether or not fish had been exposed to Cd, in order to get further insight into the potential role of *zbtb38* in sex differentiation and/or maintenance. However, as temperature directly induced the masculinization of zebrafish (see Pierron et al., 2021), data from fish raised at the high temperature (HT) were analyzed separately. Thus, four groups of animals were considered: phenotypic males raised at 28 °C, phenotypic males raised at 34 °C (this group being constituted both by males and neomales), phenotypic females raised at 28 °C and phenotypic females raised at 34 °C (ie a group constituted by the low number of females that escaped heat-induced masculinization). Each group was constituted by an equivalent number of (i) control and Cd fish and (ii) fish from each generation (Table S2).

### 3.1. Sex-specific DNA methylation patterns in gonads of sexually mature fish

We have first to note that a highly significant positive correlation was observed between the methylation level of the promoter and exon 3 (ex3) of the *zbtb38* gene (r = 0.92, P < 0.0001). As can be observed in Fig. 1A, a similar methylation pattern was observed for the promoter and ex3 of the *zbtb38* gene.

For all genes under study and, at both 28 °C and 34 °C, highly significant differences in term of DNA methylation levels were observed between the gonads of sexually mature (124-130 dpf) males and females (Fig. 1A). The methylation level of *cyp19a1a* and *zbtb38* (both the ex3 and pr) were significantly higher in males in comparison to females. An inverse pattern was observed for *foxl2a* and *dmrt1*, their methylation levels being significantly more important in females in comparison to males. In response to HT (34 °C), the sex-ratio (males %) increased in mean from 55.2 % to 78.6 % (see Pierron et al., 2021 for details). Thus, some of females developed as males (ie testes) in response to HT, ie neomales. The fact that a sex-specific pattern was observed at 34 °C indicates that the methylation level of the 4 genes was associated with the phenotypic sex of animals. However, we have to nuance this assumption.

Indeed, despite no significant effect of temperature was observed on
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** *Zbtb38* is firmly associated with the phenotypic sex of animals. Distribution of (A) DNA methylation and (B) transcription levels of the different genes in gonads of sexually mature (124–130 dpf) males (M, blue) and females (F, pink), and in whole larvae (5 dpf, L, black) raised at 28 (solid lines or full dots) or 34 °C (hashed lines or empty dots). Different letters denote significant differences (Dunn-Bonferroni, *P* < 0.05; F28 n = 45–47; M28 n = 47–48; L28 n = 35–48; F34 n = 25–26; M34 n = 30–31). For *zbtb38*, *ex3* refers to exon3 and *pr* to promoter; ND, non-detectable.</figcaption></figure>
the mean methylation levels of the 4 genes in males or females, HT significantly increased the variance of data, especially in the case of males and for the *foxl2a* and *dmrt1* genes. As previously described in Pierron et al. (2021), some males presented methylation levels similar to females (and vice versa) in response to HT for these two genes. However, this was not the case for *cyp19a1a* and *zbtb38* and the most discriminant results between males and females were observed for the ex3 of *zbtb38*. Its methylation level ranged from 14.0 to 44.0 % in phenotypic females (including females raised at 28 and 34 °C) and from 63.7 % to 94.4 % in phenotypic males (including males raised at 28 and 34 °C). Furthermore, a highly significant positive correlation was observed between the methylation level of *cyp19a1a* and *zbtb38* (r = 0.92, P < 0.0001, further described in section 3.4). Thus, our results highlight the fact that the methylation level of *zbtb38* and *cyp19a1a* are firmly associated with the phenotypic sex of animals, even in animals raised at HT, and support the existence of an interconnection between them. Finally, we have to note that despite HT had no significant effect on the methylation level of *zbtb38* or *cyp19a1a* in each sex, slight variations were observed. Thus, in the case of females, a slight increase in their methylation levels was observed in response to HT. Inversely, HT induced a slight decrease in males while increasing the interindividual variably. For the ex3 of *zbtb38*, its methylation level in females increased in mean from 23.6 to 33.1 % in response to HT. In males, its methylation level decreased in mean from 89.9 to 83.3 % in response to HT while the variance increased from 10.2 to 51.9 % (Fig. 1A). A similar pattern was observed at the promoter level. The methylation level of the *zbtb38* promoter decreased in mean from 74.7 to 65.5 % in males in response to HT while the variance increased from 50.4 to 164.8 %. This result could be explained by the fact that the group of phenotypic males at HT was constituted by both males and sex-reversed females. Thus, it could be hypothesized that temperature led to an increase in the methylation level of *zbtb38* in females and that above a certain threshold, genetic females developed as males. This obviously requires further investigations.

## 3.2. Sex-specific transcription patterns in gonads of sexually mature fish

In accordance to the results obtained at the DNA methylation level, for the 4 genes under study, significant differences were observed between males and females at the transcriptional level (Fig. 1B). *Foxl2a* and *cyp19a1a* were significantly more transcribed in females while *zbtb38* and *dmrt1* were significantly more transcribed in males. Thus, *foxl2a* and *cyp19a1a* are usually considered as pro-female genes, being more transcribed in females (Hosseini et al., 2019). Inversely, *zbtb38*, as it is the case for *dmrt1*, can be considered as a pro-male gene. No significant effect of temperature was observed on the transcription level of *cyp19a1a*, *foxl2a* and *dmrt1* (Fig. 1B). In contrast, the transcription level

of *zbtb38* was significantly up-regulated by HT in females (3.6-fold more transcribed in HT females in comparison to females raised at 28 °C). However, its transcription level in HT females remained significantly lower than that in males (3.8-fold less transcribed in HT females in comparison to males raised at either 28 or 34 °C). In other words, HT females presented intermediate values between females raised at 28 °C and males (Fig. 1B). In contrary to the other genes under study, *zbtb38* was thus the only gene for which its transcription level was significantly influenced by temperature. We have to recall that exposure to HT led to a masculinization of the population with the emergence of pseudomales. It is thus tempting to speculate that the up-regulation of *zbtb38* induced by temperature contributed to the masculinization of genetic females. In other words, above a certain threshold of transcription, genetic females would become phenotypic males. This obviously requires further investigations. In this optic, it could be interesting to study the ability of ZBTB38 to bind to its own promoter or ex3 in order to investigate if changes in its expression level could affect its methylation level.

## 3.3. The key role of DNA methylation and zbtb38 in the phenotypic sex of sexually mature fish

In order to gain some insight into the relative influence of each gene and variable on the phenotypic sex of animals we next carried out a multifactorial analysis (Principal Component Analysis, PCA) combining both DNA methylation and transcription levels. Males and females were clearly separated along the principal component 1 (PC1) which explained 74.4 % of the variance (Fig. 2A). Generally, contribution of the variables to the PC1 were more important for DNA methylation levels than for transcription levels (Fig. 2B), highlighting the primary role of DNA methylation in the phenotypic sex. The variable that contributed the most to the PC1 was the methylation level of *zbtb38* followed by the methylation level of *cyp19a1a*. Thus, the methylation level of *zbtb38* appeared to be the most discriminating variable between males and females, reinforcing the hypothesis that *zbtb38* plays an important role, at least, in sex maintenance.

## 3.4. The relationship between DNA methylation and gene transcription as a basis of mutual antagonism?

As previously evoked, a highly significant positive correlation was observed between the methylation level of *cyp19a1a* and *zbtb38* (Fig. 3A). However, such a correlation was not observed at the transcription level (Fig. 3B). Indeed, an inverse relationship between DNA methylation and gene transcription was observed for the two genes. The hypermethylation of

*cyp19a1a* in males was associated with its down-regulation at the transcription level (Fig. 1A and B), ie the gene was significantly more

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** DNA methylation and *zbtb38* are the most sex-discriminating variables (A) Principal component analysis (PCA) showing clustering of sex based on both methylation and transcription levels of the four genes under study. Data from gonads of males (blue) and females (pink) raised at 28 (full dots) or 34 °C (empty triangles) were used (n = 144). (B) Contribution (%) of each variable to the principal component 1 (PC1).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Co-methylation of *zbtb38* and *cyp19a1a* is associated with divergent transcription profiles between males and females, Relationship between *zbtb38* and *cyp19a1a* at the (A) DNA methylation (%) and (B) transcriptional level (a.u.). The Pearson correlation coefficient (r) and the associated P value are reported, n = 149. Data from males (blue) and females (pink) raised at 28 (full dots) or 34 °C (empty triangles) were used. (C) Schematic representation of the relationship between DNA methylation and gene transcription for the couples *cyp19a1a/zbtb38* and *dmrt1/foxl2a*.</figcaption></figure>

transcribed in females in comparison to males. Such a relationship appears in agreement with the fact that DNA methylation is generally associated with transcriptional repression (Newell-Price et al., 2000). However, in the case of *zbtb38*, its hypermethylation in males in comparison to females was associated with higher transcription levels in males (Fig. 1A and B). We have to note that such a relationship was also observed for *foxl2a* and *dmrt1* (previously described in Pierron et al., 2021). The methylation level of *foxl2a* and *dmrt1* were positively correlated (r = 0.90, P < 0.001). Despite the hypermethylation of *dmrt1* in females was associated with its down-regulation, the hypermethylation of *foxl2a* in females was associated with its up-regulation. Interestingly, in both cases, ie for the couple *foxl2a/dmrt1* or *cyp19a1a/zbtb38*, (i) each couple of genes is constituted by a gene involved in the pro-female pathway (*foxl2a*, *cyp19a1a*) and by a gene involved in the pro-male pathway (*dmrt1*, *zbtb38*) and (ii) the methylation level of the two genes is highly correlated but the fact that methylation is associated with opposite effects on their respective transcription levels leads to gender-specific transcription profiles (Fig. 3B and C). Such a regulation mechanism appears in agreement with the fact that sex differentiation and maintenance proceeds by activating the female pathway (pro-female genes) while repressing the male pathway (pro-male genes), and *vice versa*, a mechanism called mutual antagonism (Aharon and Marlow, 2021; Garcia-Moreno et al., 2018, Fig. 3C). In the present case, one signal, ie co-methylation of *zbtb38* and *cyp19a1a*.

would allow activating the transcription of a pro-male gene (*zbtb38*) while repressing the transcription of a pro-female gene (*cyp19a1a*), leading to male phenotype, and *vice versa*.

Before to conclude this paragraph, we have to note that if DNA methylation was believed for many years to play a crucial role in gene silencing, recent works suggest that regulation of transcription by DNA

methylation is more complex than previously expected. Several works indeed reported up-regulation from methylated promoters (de Mendoza et al., 2022; Harris et al., 2018; Rauluseviciute et al., 2020), notably in zebrafish gonads (Skvortsova et al., 2019) and mammal germ cells (Hammoud et al., 2014). An example can be provided by *zbtb38* itself. Despite little is known about ZBTB38, recent investigations (which remain restricted to mammalian models) have shown that ZBTB38 is a transcriptional activator that binds to methylated promoters. More precisely, ZBTB38 is a methyl-DNA binder transcription factor that presents two independent sets of C2H2 zinc fingers (ZNFs) in the central and C-terminal section of the protein which recognize two different methylated consensus sequences, called M2 and M1 motifs, respectively (Marchal et al., 2022). We identified only one cluster of ZNFs in the zebrafish ZBTB38 protein, corresponding to the central ZNFs 3, 4 and 5 that binds to the M2 motif. The sequence of this cluster is exceptionally conserved between zebrafish and humans (78 aa, identity = 90 %, evalue = 2⁻⁴⁸). Using a human cell line, Marchal et al. (2022) showed that promoters targeted by ZBTB38, more precisely at the M2 motif, are transcribed at higher levels compared to the bulk genome. In addition, they showed that the M2 motif is quite similar to the binding site of the transcription factor E2F4, a transcriptional regulator of cell cycle genes. However, in contrast to ZBTB38, E2F4 only binds promoters presenting an unmethylated M2 motif. Finally, they showed that genes are no more cell cycle regulated when their promoters are methylated and bound to ZBTB38. In other words, if both methylated and unmethylated genes are expressed, when promoters are methylated and bound to ZBTB38, genes become insensitive to cell cycle regulators. This illustrates how DNA methylation can be associated with high expression levels while affecting the sensitivity of promoters to other transcription factors and thus, cellular or environmental signals. More generally, mounting evidences suggest that DNA methylation affects the activity of genes by

<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Embryos present a paternal methylome but the methylation level of *zbtb38* rapidly evolves to a bi-potential state (A) Change in the DNA methylation level (mean ± SE) of the different genes during zebrafish development. Analysis at the adult stage were carried in gonads of females (pink, n = 70–72) and males (blue, n = 74–79) raised at either 28 or 34 °C (data from the two temperatures were combined). At the embryo (n = 7–12) or larval (n = 35–48) stage, analyses were carried out on the whole organism. (B) Distribution of the methylation levels of *zbtb38* ex3 and pr in gonads of males (blue) and females (pink) raised at 28 (full dots) or 34 °C (empty triangles) and in whole embryos (E) and larvae (L) raised at 28 °C.</figcaption></figure>
affecting the binding capacity of transcription factors to DNA. Recip-
rocally, transcription factor binding could also affect the methylation
state of DNA (Héberlé and Bardet, 2019).

3.5. DNA methylation dynamics during early development

As previously described, for all genes under study, significant
methylation differences were observed between the gonads of sexually
mature males and females, ie between male and female gametes. Thus,
when fertilization occurs, embryos inherit both an hypermethylated and
hypomethylated allele from their parents. It thus appears interesting to
investigate the dynamic of DNA methylation during early steps of
development, knowing that embryos or larvae are bi-potential, ie in-
dividuals can either develop as males or females (Aharon and Marlow,
2021). In this optic, we analyzed whole embryos obtained just after
fertilization and whole larvae at 5 dpf. Individuals at 5 dpf indeed
present a histologically evident gonad that is sexually undifferentiated
and bi-potential. The gonad remains differentiated and bi-potential
until 12 dpf. From 13 dpf, all individuals develop a juvenile ovary,
although the gonad remains bi-potential. The period of transitioning
that leads to the appearance of a mature ovaries in females or testes in
males occurs later, between 25 and 50 dpf (Aharon and Marlow, 2021).
Afterwards, no sex reversal or intersex is normally observed. We have to
note that even if the period of sex differentiation occurs at the juvenile
stage (25-50 dpf), previous works have shown that exposure to HT at the
embryo (0-3 dpf) or larval (3-25 dpf) stage induces a male-biased sex
ratio at adulthood (Abozaid et al., 2011; Ribas et al., 2017b).

We first analyzed the methylation levels of the four genes in some
embryos sampled at 2-3 h post-fertilization (hpf). For all sequences, no
significant difference was observed between male gonads and embryos
(Fig. 4A). These results are in accordance with previous works that re-
ported that zebrafish embryos "inherit" the methylome of their father, at
both the whole organism level or in primordial germ cells (from 4 to 36
hpf; Skvortsova et al., 2019; Jiang et al., 2013; Potok et al., 2013). In our
case, these results imply that the methylation level of the maternal allele
evolves quickly after fertilization. For *cyp19a1a* and *zbtb38*, the
maternal

allele is rapidly methylated so that the methylation level of the gene
reaches a methylation level close to that of male gonads. Inversely, for
*foxl2a/dmrt1*, the maternal allele is rapidly demethylated. However, we
have to nuance this assumption. Despite no significant difference was
observed between embryos and male gonads, a high interindividual
variability was observed in the case of the *zbtb38* promoter (Fig. 4B) and
its methylation level in embryos was close to that of the parental gonads,
reaching in mean 48.9 % in embryos (raised at 28 °C, exposure to HT
began at 15 dpf) and 43.4 % in parents; ie 74.7 % in male gonads (males
raised at 28 °C) and 12.1 % in female gonads (females raised at 28 °C).
Remarkably, this high interindividual variability was not observed in
the case of the ex3 (Fig. 4B).

We then carried out analyses on whole larvae at 5 dpf (Figs. 1A and 4A). For both *cyp19a1a* and *dmrt1*, larvae presented methylation levels comparable to that of male gonads. For *dmrt1*, its DNA methylation level in larvae did not differ significantly from males raised at either 28 or 34 °C. For *cyp19a1a*, larvae did not differ significantly from embryos or males raised at 34 °C. Thus, for these two genes, only slightly variations were observed during early development, ie from fertilization to the larval stage (5 dpf), and their methylation levels remained close to that of male gonads. The methylation level of *foxl2a* significantly increased (Fig. 4A), reaching a value that did not differ significantly from females raised at 34 °C (Fig. 1A). For both its promoter or ex3, *zbtb38* was the only gene for which its methylation level in larvae significantly differed from both males and females, presenting intermediate levels between males and females, ie between their parents (Figs. 1A and 4A). For the promoter, the methylation level of *zbtb38* in larvae reached in mean 32.2 % while it reached 12.1 % in mothers and 74.7 % in fathers. For the ex3, it reached 51.0 % in larvae and 56.8 % in parents; 89.9 % in males

and 23.6 % in females. We then measured the methylation level of each
allele (allele-specific approach) in a limited number of males, females
and larvae to test whether methylation patterns can be inherited in a
parent-of-origin-specific manner, ie in a fashion similar to DNA
methylation imprinting in mammals (Barlow and Bartolomei, 2014). No
difference was observed between the two alleles at either the promoter
or ex3 level (Fig. S3). This result appears in agreement with previous
works that also reported no evidence of imprinting in zebrafish (Murphy
and Cairns, 2016). As only three possible methylation states can occur at
a single CpG site in each cell: unmethylated (0 %), methylated on one
allele (50 %) or methylated on both alleles (100 %), intermediate levels
of DNA methylation in somatic cells represent the cell population
average (Hay et al., 2023). Thus, our results suggest that the methylation
state of *zbtb38* in larvae is hypermethylated (ie presenting a male epi-
genotype) in half of their cells and hypomethylated (female epi-
genotype) in the other half. Such a methylation pattern would be in
accordance with the fact that larvae at 5 dpf present a sexually undif-
ferentiated gonad and are bi-potential. Interestingly, the transcription
level of *zbtb38* in larvae also presented intermediate values between
males and females (Fig. 1B). However, it was not significantly different
from females raised at HT, ie from the low number of females that
escaped to masculinization. The transcription level of *foxl2a* also pre-
sented intermediate values between males and females. In contrast, the
transcription level of *cyp19a1a* and *dmrt1* were near undetectable in
larvae (Ct > 38 in RT-qPCR), underlining the fact that the transcriptional
activity of *zbtb38* and *foxl2a* during early development is more impor-
tant and precocious than *cyp19a1a* and *dmrt1*.

To summary, for the four genes under study, the methylation level of
the maternal allele changed very quickly after fertilization to reach a
value close to the paternal allele. However, in contrast to the other genes
under study, the methylation level of the two alleles of *zbtb38* rapidly
decreased during early development to reach an intermediate value
between males and females at the larval stage. Thus, *zbtb38* rapidly
reached a methylation status that appears compatible with the undif-
ferentiated and bi-potential state of larvae, supporting a potential role of
*zbtb38* not only in sex maintenance (ie adults) but also in sex differen-
tiation. However, further researches are needed. Mainly, considering the
small size of individuals, analyses on early stages of development were
carried out on whole organisms. Thus, it is necessary to analyze the
gonads of juveniles during the transitioning period (25-50 dpf) as both
methylation and transcription levels differ among organs (ie organ-
specific patterns). Considering the size of juveniles and the fact that is
not possible to pool samples, this could be a challenging but necessary
task. In addition, other epigenetic marks such as histone modifications
should be investigated. For example, while the methylation levels of
*cyp19a1a* and *zbtb38* were highly correlated in gonads of adults, this
relationship was not observed during early development, suggesting
that other mechanisms are involved.

4. Conclusion

Among the different genes and variables analyzed, the methylation
level of *zbtb38* was found to be the most discriminating variable between
males and females. Highly significant differences in term of DNA
methylation and transcription levels were observed for *zbtb38* between
the gonads of phenotypic males and females, even in animals raised at
HT. Moreover, a highly significant correlation was observed between the
methylation level of *zbtb38* and *cyp19a1a*, ie a gene that is considered to
play a central, if not the key, role in sex differentiation and/or mainte-
nance in fish. In addition, *zbtb38* was the only gene for which its tran-
scription level in females was affected by high temperature, a known
masculinizing factor. Finally, in contrast to the other genes under study,
*zbtb38* was the only gene for which sexually undifferentiated and bi-
potential individuals presented intermediate levels of DNA methyl-
ation and transcription between males and females. Thus, our results
support a non-negligible role of *zbtb38* in sex determination and
maintenance. At least, zbtb38 appears as an interesting candidate gene to investigate and understand how environmental factors can affect the sex differentiation process. More generally, although multiple mechanisms are involved and interwoven in the process of cell fate commitment, sex differentiation and gonad development, our results support the fact that epigenetic mechanisms play an important role in these processes. In this optic, considering the pressing need to protect and restore nature, epigenetics represents a way to (i) link stress-induced molecular events to individual and population-level effects and (ii) develop new tools to detect and assess molecules with endocrine disruptive properties.

## CRediT authorship contribution statement

**Fabien Pierron:** Writing – original draft, Validation, Supervision, Methodology, Funding acquisition, Formal analysis. **Débora Heroin:** Investigation. **Flore Daramy:** Investigation.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

This work was supported by the (i) Agence Nationale de la Recherche of France (ANR JCJC TRACE ANR-16-CE34-0008), (ii) French National program EC2CO (Ecosphère Continentele et Côtière) and (iii) Réseaux de Recherche Impulsion, RRI Tackling Global Change, University of Bordeaux. Christian Portier, Kévin Lebaillif and Jacques Martin are thanked for their technical help with the experimental setup. Emilie Léglise and Lydia Bézard are thanked for their help with administrative tasks.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.mce.2025.112636.

## Data availability

Data will be made available on request.

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