# Bisphenol A exposure during gestation and lactation in mice: Sex-specific consequences on oligodendrocytes and myelination

Vanessa Naffaa, Juliette van Steenwinckel, Romain Magny, Anne Regazzetti, Céline Keime, Pierre Gressens, Olivier Laprévote, Nicolas Auzeil, Anne-Laure Schang

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Vanessa Naffaa, Juliette van Steenwinckel, Romain Magny, Anne Regazzetti, Céline Keime, et al. Bisphenol A exposure during gestation and lactation in mice: Sex-specific consequences on oligodendrocytes and myelination. Toxicology, 2025, 512, pp.154041. 10.1016/j.tox.2024.154041. hal-05015310

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# Bisphenol A exposure during gestation and lactation in mice: Sex-specific consequences on oligodendrocytes and myelination

Vanessa Naffaa<sup>a</sup>, Juliette Van Steenwinckel<sup>b</sup>, Romain Magny<sup>a</sup>, Anne Regazzetti<sup>a</sup>, Céline Keime<sup>c</sup>, Pierre Gressens<sup>b</sup>, Olivier Laprévote<sup>a</sup>, Nicolas Auzeil<sup>a,1</sup>, Anne-Laure Schang<sup>d, *, 1</sup>

<figure><img src="image_2.png" /></figure>

## ARTICLE INFO

Handling Editor: Mathieu Vinken

**Keywords:**
Bisphenol A
Oligodendrocytes
Myelin
Myelination
Lipidome
Transcriptome

## ABSTRACT

Bisphenol A (BPA), a ubiquitous environmental endocrine disruptor, is suspected of disturbing brain development through largely unknown cellular and molecular mechanisms. In the central nervous system, oligodendrocytes are responsible for forming myelin sheaths, which enhance the propagation of action potentials along axons. Disruption of axon myelination can have lifelong consequences, making oligodendrocyte differentiation and myelination critical stages of brain development. In the present study, mice were exposed to BPA during gestation and lactation through drinking water at concentrations of 25 and 250 µg.L⁻<fnref n="1" />. These doses, corresponding to estimated exposures of 4 µg.kg⁻<fnref n="1" />.d⁻<fnref n="1" /> and 40 µg.kg⁻<fnref n="1" />.d⁻<fnref n="1" />, respectively, led to disturbances in lipid remodeling associated with myelination in the offspring. Importantly, changes in myelin lipid composition were selectively observed in female mice and were transient, being visible only at post-natal day P15 but not at later stages (P30 and P60). In females exposed to BPA, myelin exhibited a lower proportion of phosphatidylcholines and higher proportions of other glycerophospholipid subclasses, thus resembling more mature myelin. Conversely, male myelin was not affected, likely due to its already more mature lipid composition. Additionally, transcriptomic analysis of female oligodendrocytes at P15 did not reveal any transcriptional changes in genes related to lipid metabolism, further suggesting post-transcriptional effects of BPA *via* chaperone-mediated protein folding and RNA splicing. In males, the altered genes were mainly associated with synaptic transmission. Finally, alterations in chromatin accessibility were also largely sex dependent and did not correlate with transcription, with the exception of the *Cwc22*. At this locus, BPA exposure increased chromatin accessibility in half of mice of both sexes, leading to an “unchanged/open” bimodal profile correlated with “unchanged/upregulated” gene expression. Together, these results open new insights into the sex-dependent mechanisms of BPA’s effects on brain development.

## 1. Introduction

Bisphenol A (BPA) has been widely used for more than 50 years, mainly in the manufacture of polycarbonate plastics and epoxy resins, but also as a component of other polymers and resins, in the synthesis of flame retardants, and as a developer in thermal papers. This established endocrine disruptor (EDC) has thus become pervasive in the environment and, as a result, in the human body (Fillol et al., 2021). BPA enters the organism primarily through food, easily crosses biological barriers,

including the digestive, placental and blood-brain barriers, can be secreted in breast milk, and has the ability to impact multiple signaling pathways (Nesan et al., 2018). Although not accumulated in the body, BPA represents a health risk from daily exposure. In 2017, this EDC was classified as a "substance of very high concern" under the REACH regulation. Indeed, strong epidemiological and experimental evidence has linked BPA exposure to a variety of health outcomes, including disruption of reproductive, metabolic, immunological, and neurological functions (Ma et al., 2019; Prins et al., 2019). In several countries,

<footnote><sup>a</sup> Université Paris Cité, CNRS, CITCoM, Paris 75006, France</footnote>

<footnote><sup>b</sup> Université Paris Cité, Inserm, NeuroDiderot, Paris 75019, France</footnote>

<footnote><sup>c</sup> Université de Strasbourg, IGBMC, Inserm U1258, CNRS UMR7104, Illkirch 67404, France</footnote>

<footnote><sup>d</sup> Université Paris Cité, Inserm, HERA Team, CRESS UMR 1153, Paris 75006, France</footnote>

<footnote>* Corresponding author.
E-mail address: anne-laure.schang@u-paris.fr (A.-L. Schang).</footnote>

<footnote n="1">¹ These authors contributed equally to this work.</footnote>
regulations have been passed to restrict the use of BPA in thermal paper and food containers, especially in baby bottles (Commission Regulation EU 2016/2235, 2016; Kassotis et al., 2020). Despite these restrictions, BPA is still ubiquitous worldwide in human biological fluids, including blood, urine, umbilical cord blood, and breast milk, exposing the human body from conception, during development and throughout life (Fillol et al., 2021; Gély et al., 2021; Niu et al., 2021).

Of particular concern are the reported or suspected effects of BPA on brain development (Patisaul, 2020). In humans, BPA exposure during fetal life and/or childhood, typically assessed by quantification of BPA in urine, has been correlated with several neurodevelopmental disorders, including autism spectrum disorders (ASD), attention deficit with or without hyperactivity disorders (ADHD), anxiety, depression, and aggressive behavior. In experimental models, numerous studies have demonstrated several effects of early BPA exposure on memory, anxiety, locomotor and exploratory activities, and social and reproductive behavior (Mhaouty-Kodja et al., 2018; Patisaul, 2020). Like many EDCs, BPA most often exerts sex-dependent effects; some have been attributed to its xenoestrogenic activity. However, the cellular and molecular mechanisms by which BPA exerts its effects in the developing brain remain largely unknown.

In the central nervous system (CNS), oligodendrocytes are responsible for the formation of myelin sheaths around axons. From the third trimester of pregnancy, oligodendrocyte precursor cells (OPC) proliferate, migrate and differentiate into immature and then mature oligodendrocytes, which myelinate the CNS by wrapping plasma membrane extensions around axons. By isolating nerve fibers, myelin sheaths improve propagation of action potentials. Myelinated axons are the main constituents of the white matter providing the functional interconnection between brain regions. Abnormal myelination is associated with neurological deficits, including ADHD and ASD (Deoni et al., 2015; Wu et al., 2017). Thus, oligodendrocyte differentiation and myelination represent critical stages of brain development whose disruption may have lifelong consequences. Despite this, there is little data on the effects of EDCs on these processes, far fewer than studies focusing on neural stem cell fate or synaptic plasticity (Welch and Mulligan, 2022). It should be noted that myelination is tightly controlled by hormonal signals, especially steroid and thyroid hormones, suggesting that EDCs may interfere with their completion (Naffaa et al., 2021). In this sense, exposure to BPA during gestation and lactation in rat led to a reduction in myelin compactness, changes in g-ratio, and lower levels of several myelin proteins in the hippocampus in adult (Tiwari et al., 2015). Additionally, early exposure to BPA induced hypomyelination and oligodendrocyte loss in the hippocampus, both of which were associated with a contextual fear memory deficit (Xu et al., 2017). Although these studies do indicate that BPA may disrupt myelination, the evidence is still scant and further examination is needed.

To improve the assessment of EDCs' effects on myelination, we have previously developed an experimental strategy based on ultra-performance liquid chromatography coupled to mass spectrometry, providing an exhaustive description of myelin lipid composition (Naffaa et al., 2022b). Indeed, lipids are the main constituents of myelin, representing 70–85 % of its dry weight (Morell and Quarles, 1999). These biomolecules are therefore valuable targets for assessing myelination. By comparing the myelin lipidome in female mice at a poorly myelinated stage, postnatal day (P) 15, and at an advanced myelination stage, P40, we have described changes of myelin lipidome during this post-natal developmental period. Note that in rodents, myelination fully takes place during postnatal development, contrasting with its *in utero* initiation in human (Semple et al., 2013). In addition to the expected enrichment in characteristic myelin lipids such as plasmalogens and sulfatides, our previous study uncovered a significant remodeling in the composition of glycerophospholipid subclasses during myelination (Naffaa et al., 2022b). It included an important reduction in phosphatidylcholine (PC) levels associated with an elevation in phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol

(PI) contents. In these subclasses, an increased proportion of monounsaturated fatty acids (MUFA) was also evidenced. The present study aimed to assess the effect of early BPA exposure on myelination by investigating myelin lipidome at three stages: juvenile (P15), adolescent (P30) and adult (P60). In rodents, myelination begins after birth, with oligodendrocyte maturation occurring during the first 10 postnatal days, and the myelination rate peaking around P18–P20 (Baumann and Pham-Dinh, 2001; Semple et al., 2013). P15 corresponds to an early stage of myelination, prior to this peak. By P30, the myelination process has significantly slowed, marking a phase where myelin composition tends to stabilize. Finally, P60 represents a late stage, capturing the myelin composition in young post-pubertal adults. To gain insight into the mechanisms underlying BPA effects, transcriptome and chromatin accessibility were also explored in oligodendrocytes of juvenile mice. This study, performed in both male and female mice, highlighted sex-specific effects of BPA. Indeed, at P15, BPA induced a transient alteration in the lipid composition of myelin, mainly targeting glycerophospholipids and observed selectively in females. In oligodendrocytes, BPA also altered the transcriptome and chromatin accessibility in both males and females, albeit in striking different ways. Finally, we identified CWC22 Spliceosome Associated Protein Homolog gene as upregulated by BPA whatever the sex yet in a heterogenous “all-or-none” manner. Altogether, these results could improve our understanding of consequences of early BPA exposure on myelination.

## 2. Materials and methods

The experimental design is displayed in Fig. 1.

### 2.1. Animals

Animal experimentation was performed in Inserm U1141 facilities (agreement B751901) according to the guidelines of Inserm and directive 2010/63/EU. Protocols were approved by the ethics committee of Université Paris Cité and authorized by the Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation (APAFIS #23723-2019121114392342 and #35036-2022012909337464). Experiments were performed on OF1 mice. Pregnant mice were purchased from Charles River (L'Arbresle, France). Animals were housed under constant photoperiod cycle (12 h light / 12 h darkness) and controlled room temperature (22 +/- 2°C) in BPA-free cages (Tecniplast France, Decines-Charpieu, France) equipped with BPA-free bottles, with *ad libitum* access to low phytoestrogen (soy protein-free) chow (SAFE® 150, Augy, France) and BPA-free water originally packaged in polyethylene terephthalate (PET) bottles (Cristaline®, source Noémie, Chelles, France). At arrival, pregnant mice were randomly housed by pairs. Pups were born in our animal facilities. On post-natal day 1, pups were sexed and litters were culled to 12 pups per mother, i.e. 24 pups per cage (12 males and 12 females). Juvenile mice were weaned at P22. Measures were taken to minimize the litter effect. At least 6 pregnant females were assigned to each experimental group simultaneously. At P1, pups were randomly distributed among different cages within the same experimental group. For experiments, mice from different cages were systematically used, also ensuring a comparable number of animals in all cages. The sample sizes were determined using the minimum number of animals and, according to a previous study, allowing robust statistical analysis (Naffaa et al., 2022b). No animals were excluded.

### 2.2. BPA exposure

BPA exposure through drinking water started at G7 and ended at P22. Drinking water containing BPA (Sigma, Saint-Quentin-Fallavier, Frane) was prepared from BPA stock solutions in ethanol at 2.5 and 25 g.L⁻¹, diluted in drinking water to provide BPA final concentrations of 25 and 250 µg.L⁻¹. The control group, not exposed to BPA, received water containing 0.001 % ethanol. Based on a 0.16 mL.g⁻¹ average
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Experimental design of the study. The figure recapitulates the experimental approaches performed, indicates the biological material used in each case and the corresponding figure. G: gestational day, P: post-natal day.</figcaption></figure>

daily water consumption estimated between G7 and P3, an exposure of 4 µg.kg⁻¹.d⁻¹ and 40 µg.kg⁻¹.d⁻¹ was expected to be achieved during gestation and early lactation, being close to the temporary tolerable daily intake (TDI) established by EFSA prior to 2023 (4 µg.kg⁻¹) and 2014 (50 µg.kg⁻¹), respectively. Although maternal water consumption subsequently increased to meet the growing needs of the pups which themselves began to drink some water at the end of lactation, BPA concentrations in drinking water remained constant until weaning.

## 2.3. Cerebrum dissociation and myelin magnetic sorting

The entire experimental procedure described below was carried out at 4°C to prevent lipid degradation. Brains were weighted and tissues mechanically dissociated in phosphate buffered saline (PBS) using a glass potter. The brain homogenates were then filtered through a 100 µm stainer (Miltenyi Biotec, Paris, France). Brain suspension volumes corresponding to 100–200 mg (P15) or 50 mg (P30 and P60) were used for myelin enrichment. Samples were centrifuged at 4°C/300 g for 10 min and the supernatant was discarded. Pellets were resuspended in 900 µL (P15) or 225 µL (P30 and P60) of PBS containing 0.5 % bovine serum albumin (BSA), added of 100 µL (P15) or 25 µL (P30 and P60) of anti-myelin antibodies (9:1) coupled to magnetic beads (Myelin Isolation Beads, Miltenyi Biotec, Paris, France) and incubated 15–20 min at 4°C. Columns (MultiMACS™ Cell24, Miltenyi Biotec, Paris, France), placed in a magnetic field, were equilibrated with 2 mL of PBS + 0.5 % BSA and added with cell suspensions. The columns were rinsed three times with 1 mL of PBS then removed from the magnetic field and the myelin-enriched fractions were eluted with 1 mL of PBS.

## 2.4. Cerebrum dissociation and oligodendrocyte magnetic sorting

Brain dissociation was performed using gentleMACS™ C tubes, gentleMACS™ dissociator, Neural Tissue Dissociation Kit (NTDK, P) for P4 mice and P10 and Adult Brain Dissociation Kit (ABDK) for P15, P30 and P60 mice, according to the manufacturer's protocol (Miltenyi Biotec, Paris, France). Isolation of O4 + oligodendrocytes was performed using anti-O4 antibodies coupled to magnetic microbeads, Multi-24 column blocks and MultiMACS™ Cell24 Separator Plus according to the manufacturer's protocol (Miltenyi Biotec, Paris, France). The final suspensions were centrifuged at 300 g at 4°C for 10 min and pellets were stored at -80°C.

## 2.5. Lipid analysis

Lipid extraction was performed as previously described (Naffaa et al., 2022b). The extraction was carried out with a hexane-methanol-water mixture (7.5:2.25:1 v/v/v). All organic solvents used contain 0.01 % (w/v) butylated hydroxytoluene to prevent lipid oxidation. Briefly, the volume of myelin-enriched fractions corresponding to 10 mg of brain tissue was added with 200 µL of water (Atlantic Laboratoire, Mauves sur Loire, France), 15 µL of a mixture of 11 lipid internal standards (Supplementary table 1) (Avanti Polar Lipids, Inc., Alabaster, AL, USA), 450 µL of methanol (J.T. Baker, Phillipsburg, NJ, USA), and 1.5 mL of hexane (Sigma, Saint-Quentin-Fallavier, France). The mixtures were agitated for 30 min, centrifuged at 2500 rpm for 10 min at 15°C, the upper organic phase was collected and evaporated under reduced pressure at 45°C in a SpeedVac (Thermo Scientific™ Savant™ SpeedVac™ Concentrator, Altrincham, UK). The lipid extracts were resuspended in 300 µL of chloroform (Sigma, Saint-Quentin-Fallavier, France) and evaporated again under reduced pressure at 45°C. Lipid extracts were finally resuspended in 150 µL of acetonitrile (J.T. Baker, Phillipsburg, NJ, USA), isopropanol (Atlantic Laboratoire, Mauves-sur-Loire, France), chloroform and water mixture (ACN/IPr/CHCl₃/H₂O [35:35:20:10 v/v/v/v]) and transferred to screw top vials containing a 200 µL insert (Chromoptic, Courtaboeuf, France).

Lipid analysis was performed by UHPLC-HRMS/MS, including a UPLC system (Waters, Manchester, UK) combined with a SynaptG32 (Q-TOF) mass spectrometer (Waters, Manchester, UK), as previously described (Magny et al., 2020). Ionization was achieved using an electrospray ionization source. Data were acquired in both positive (ESI+) and negative (ESI-) ion mode.

Data-dependent acquisition was used to obtain tandem mass spectra of the five most intense ions. The determination of lipid structure is based on the data obtained in MS and MS/MS, using a tolerance window on the values of *m/z* and *t*<sub>R</sub> of 5 and 15 ppm, respectively. The identification was reinforced by the comparison of the experimental *t*<sub>R</sub> values with the theoretical *t*<sub>R</sub> values obtained using prediction models previously developed in the laboratory (Magny et al., 2020). The identification of lipids included the determination of the polar head for glycerophospholipids, the characterization of sphingosine for sphingolipids and that of the alkyl side chains. In addition, for phospholipids, the relative intensity of the carboxylate product ions present on the MS/MS spectra has been used to provide information on the nature of the alkyl chains present at the *sn*-1 and *sn*-2 position (Hsu and Turk, 2009). The present study always refers to the major regioisomer of lipid
species. However, the presence of the minor regioisomer cannot be excluded.

The amount of lipid species was calculated using corresponding internal standards by normalizing the signal intensity of each lipid specie to that of the internal standard belonging to the same subclass. It should be noted that the lipid species used as internal standard contain one or two alkyl chains whose total carbon number is odd or whose certain hydrogens are replaced by deuterium and therefore cannot be endogenous lipid species. The lipid content was then expressed as a molar percentage (mol% or percent of total lipids) thus allowing to compare lipid distribution between samples regardless of their individual lipid content (Fitzner et al., 2020; Naffaa et al., 2022b).

## 2.6. Cholesterol quantification

The Amplex™ Red cholesterol assay kit (Thermofisher scientific, Courtaboeuf, France) was used according to the manufacturer's protocol and was performed on 1:25 diluted samples, except for P15 female samples which were diluted at 1:5.

## 2.7. Targeted gene expression analysis

### 2.7.1. RNA extraction

RNA extraction was performed with the NucleoSpin RNA Plus XS Kit (Macherey Nagel, Hoerdt, France) according to supplier's recommendations. RNAs were eluted in 20 µL of RNase-free water and quantified using the Qubit™ Broad Range (BR) RNA Assay Kit (Thermofisher scientific, Courtaboeuf, France) in a DeNovix spectrofluorimeter.

### 2.7.2. Reverse transcription (RT)

The synthesis of complementary DNA (cDNA) strands was carried out from an identical quantity of RNA for each exposure condition (500 ng – 1 µg) using the IScript cDNA Synthesis reverse transcription kit according to the manufacturer's protocol (Bio-rad, Marnes-la-Coquette, France). The reaction was carried out in a thermocycler (CFX Connect® Bio-rad, Marnes-la-Coquette, France) under the following conditions: (1) 25°C for 5 min, (2) 46°C (temperature at which the enzyme is active) for 20 min, and finally (3) 85°C for 5 min to inactivate the enzyme. Samples were then diluted to the same concentration in all conditions and immediately stored at –20°C until further use.

### 2.7.3. Quantitative polymerase chain reaction (qPCR)

Quantitative PCR was performed in duplicates using SYBR® Green Supermix in CFX96 Real-Time PCR machine (Bio-Rad, Marnes la Coquette, France), using a 3-step program (5 s of denaturation at 95°C, 10 s of annealing at 60°C and 10 s of elongation at 72°C) and Gapdh (glyceraldehyde-3-phosphate dehydrogenase gene) as a reference gene (Naffaa et al., 2022b). Specific primer sequences are given in supplementary table 2.

## 2.8. High-throughput mRNA sequencing

Brain oligodendrocytes were isolated by magnetic sorting at P16 from male and female mice either control or exposed to BPA from G7 (250 µg.L⁻¹). RNAs were extracted using the Nucleospin XS Plus kit (Macherey Nagel, Hoerdt, France). 4 samples per group were sequenced by the GENOMEAST platform of the Institute of Genetics, Molecular and Cellular Biology (IGBMC, Strasbourg). The preparation of the libraries was carried out by ligation stranded mRNA-seq (Illumina, San Diego, California, USA), and the sequencing (1 × 50 bases) was performed on a Hiseq 4000 sequencer (Illumina, San Diego, California, USA). Raw data are available in NCBI Gene Expression Omnibus, GEO accession no: GSE280176, subseries GSE280174. After preprocessing, sequence alignment to the mm39 murine genome using STAR version 2.5.3a, and gene expression quantification using htseq-count version 0.6.1p1,

comparisons were performed using Wald's differential expression test and Benjamini Hochberg's correction (FDR) using the Bioconductor package DESeq2 version 1.16.1. Lists of genes significantly up or down regulated in males or females after BPA exposure were finally subjected to functional annotation by enrichment analysis using DAVID (Sherman et al., 2022).

## 2.9. Assay for transposase-accessible chromatin with high throughput sequencing

The ATAC-seq procedure was conducted following previously established protocols (Buenrostro et al., 2013; Schang et al., 2022). Following MACS-based O4 + cell sorting at P16, 50,000 nuclei underwent Tn5 transposition (Illumina, San Diego, California, USA) for 30 min, producing “tagmented” DNA fragments. These fragments were then purified using MinElute columns (Qiagen, Courtaboeuf, France) and subjected to a two-step amplification and tagging process with NEBnext High-Fidelity 2 × PCR master mix (New England Biolabs, Evry, France). The resulting amplified DNA underwent double purification using 1.8 volumes of NucleoMag NGS Clean-up and Size Select beads (Macherey Nagel, Hoerdt, France). The libraries were multiplexed and sequenced on an Illumina NextSeq500 system (Illumina, San Diego, California, USA) at IGenSeq Platform (ICM, Paris, France). Raw data are available in NCBI Gene Expression Omnibus, GEO accession no: GSE280176, subseries GSE280169. Initial bioinformatics analyses were performed on the Galaxy platform (The Galaxy community, 2022). Following quality check (Fastqc) and trimming (Trimmomatic), the reads were aligned to the mm10 genome using Bowtie 2 (default settings) and duplicates were removed with RmDup. Peak calling was carried out with MACS2 for each group using pooled (n = 4) bam files. The resulting four bed files were then merged. After removing the mm10 blacklist (mitra.stanford.edu, 2016), 151,033 DNA regions (peaks) detected in at least one group were identified. The number of reads per peak for each sample was determined using Bedtools MultiCovBed and normalized to the library sizes. Differential peak analysis between control and BPA-exposed samples was performed with the Bioconductor software package EdgeR v 3.38.4 (Robinson et al., 2010) in RStudio v4.2.1 (http://www.rstudio.com) and statistical comparison was carried out using the exact test function followed by False Discovery Rate (FDR) determination via the Benjamini-Hochberg method. Peak annotation was performed with ChipSeeker v1.32.1 (Yu et al., 2015) using TxDb. Mmusculus.UCSC.mm10.knownGene annotation package.

## 2.10. Graphical representations and statistical analyses

Principal component analysis score plots were generated using Statskingdom PCA calculator (Statistics kingdom, 2017). Bar plots, whisker and box plots, pie charts and statistical analyzes (except for RNA-seq and ATAC-seq, see 2.8. and 2.9. sections) were completed using GraphPad Prism Software 8.0 (Graph-Pad Software, San Diego, CA, USA). Data were expressed as mean value ± standard deviation (SD). Due to unequal variances and the non-normal distribution of data in some groups, non-parametric statistical tests were chosen. Mann-Whitney test was used to compare the mean of two groups (BPA vs control, males vs females). When more than 2 groups were compared (i. e. 3 stages or 3 treatment groups), Kruskal-Wallis ANOVA test followed by Dunn's post-test was used. The Spearman correlation coefficient was used to evaluate the association between gene expression and chromatin accessibility. mol% of lipid subclasses were compared by multiple t-tests followed by FDR correction. The value of *p* < 0.05 was considered as statistically significant.

Heatmaps were created with Morpheus (https://software.broadinstitute.org/morpheus). For RNA-seq and ATAC-seq, relative color scheme and row hierarchical clustering (one minus Pearson correlation, linkage method: average) were used. Lipidomic changes at P15 were represented with a non-clustered heatmap using a fixed color scheme.
Normalized read density from ATAC-seq analysis was visualized using IGV (Robinson et al., 2011).

# 3. Results

## 3.1. Myelin protein gene expression following BPA exposure

Prior to the lipidomic analysis of myelin, the expression of four myelination markers was analyzed in the different groups by RT-qPCR from forebrain RNA (Fig. 2). In control animals, both male and female, expression of myelin genes increased between P15 and P30 and decreased at P60 to a level close to P15. This result was expected since the P15-P30 period encompasses the myelination peak which occurs at P18 in mouse (Baumann and Pham-Dinh, 2001). Exposure to BPA did not change the expression of myelin genes in females at all studied stages. In contrast, in males, at P60, BPA exposure (250 µg.L⁻¹) led to a decrease of more than 30 % in the expression of *Cnp*, *Mbp*, *Plp1*, and *Mog* (*p* < 0.05).

## 3.2. Lipid composition of myelin accompanying development in male and female mice

Characterization of myelin lipid composition was based on an untargeted lipidomic analysis, including two main steps. Indeed, a myelin enrichment was performed using magnetically-labeled anti-myelin antibodies and was followed by a lipid analysis through ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry with electrospray ionization in positive and negative modes (UHPLC-HRMS-ESI+/-). Using this analytical approach, 410 lipids were identified and semi-quantified. They belong to 14 lipid subclasses and include 26 phosphatidylethanolamines (PE), 43 ethanolamine plasmalogens (PE-O), 77 phosphatidylcholines (PC), comprising 4 lysophosphatidylcholines LPC), 19 choline plasmalogens (PC-O), 11 phosphoglycerides (PG), 31 phosphatidylserines (PS), comprising 6 plasmalogens PS-O), 17 phosphatidylinositols (PI), comprising 2 plasmalogens PI-O), 21 diacylglycerols (DG), 51 triacylglycerols (TG), 23 free fatty acids (FFA), 22 ceramides (Cer), 14 sphingomyelins (SM), 30 hexosylceramides (HexCer), and 25 sulfatides (ST) (Supplementary table 3). In addition, for 126 glycerophospholipids, the *sn*-1 *sn*-2 position of the alkyl side chains was determined for the predominant regioisomer. Cholesterol contents were also determined using a fluorometric method. Prior to investigation of BPA-induced alterations on myelin lipidome, we characterized changes in the lipid composition of myelin between P15, P30 and P60 postnatal stages including male-female comparisons.

Principal component analysis (PCA) score plot comparing myelin lipid distribution expressed in molar percentage (mol%) revealed 3 clusters, P15 females, P15 males, and a third one encompassing P30 and P60 males and females (Fig. 3A, Supplementary Fig. 1). In both males and females, the clear separation between P15 and P30 clusters indicate important changes in lipid distribution around myelination peak. PCA score plot also revealed sex-dependent clustering at P15 and to a lesser extent at P60, but not at P30, suggesting different time-courses of myelination based on gender (Fig. 3A, Supplementary Fig. 1).

In males, significant lipid changes occurred between P15 and P60, including an increase in HexCer from 2.7 % to 6.2 % (*p* < 0.05), ST from 0.68 % to 5.3 % (*p* < 0.001), and ePE from 17 % to 29 % (*p* < 0.001) (Fig. 3B, supplementary table 3). From P15 to P30, PC decreased from 28 % to 17 % (*p* < 0.01) and PI from 8.9 % to 6.2 % (*p* < 0.01), with ePE increasing from 17 to 25 % (*p* < 0.05) and ST from 0.68 % to 4.1 % (*p* < 0.001). Between P30 and P60, no significant changes were observed.

In females, significant changes in lipid levels were also observed between P15 and P60 in almost all subclasses including an increase in characteristic lipids of myelin (Fig. 3B, supplementary table 3): HexCer (from 3.4 % to 9.6 %, *p* < 0.01), ST (from 0.54 % to 4.6 %, *p* < 0.001)

and ePE from 3.5 % to 20.1 % (*p* < 0.001). Between P15 and P30, a dramatic decrease in PC from 66 % to 17 % (*p* < 0.001), an increase in PE from 8.1 % to 24.5 % (*p* < 0.001) and in ePE from 3.5 % to 25 % (*p* < 0.001) were observed. In addition, a marked increase in cholesterol (*p* < 0.05) was displayed, not exhibited in males (Fig. 3C). Between P30 and P60, the content in PI decreased from 6.6 % to 3.5 % (*p* < 0.01) (Fig. 3B, supplementary table 3).

Differences between males and females in lipid distribution during myelination were mainly observed at P15, primarily due to differences in glycerophospholipid and cholesterol contents (Fig. 3B, supplementary table 3). Indeed, at this early stage, the relative PC content was higher in females (66 % vs 28 % in males, *p* < 0.001), whereas PE (8.1 % vs 24 %, *p* < 0.001), ePE (3.5 % vs 17 %, *p* < 0.001), and PI (2.8 % vs 8.9 % *p* < 0.001) were lower. Cholesterol content was also 14.7 times lower in females than males at P15 (*p* < 0.01) (Fig. 3C). At P30, the lipid distribution was very similar in both sexes. At P60, differences reemerged mostly in glycerophospholipids: PC were higher in females (27 % vs 14 % in males, *p* < 0.05), while PE and ePE were lower (respectively 17 % vs 25 %, *p* < 0.05 and 20 % vs 29 %, *p* < 0.05).

A systematic identification of phospholipid alkyl side chains was performed, allowing glycerophospholipids to be classified according to their contents in saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids (Supplementary table 4). Hereinafter, phospholipids containing SFA, MUFA and PUFA will refer to lipid species containing two SFA, one MUFA associated with one SFA or one MUFA, and at least one PUFA, respectively. The distribution of SFA-, MUFA- and PUFA-containing phospholipids in PC, PS, PI, ePE and PE subclasses was thus investigated at all stages (Fig. 3D, supplementary table 4). Between P15 and P30, in both sexes, a marked increase in proportion of MUFA-containing species was evidenced for PS, PI, ePE, and PE (ePE not included) subclasses, as previously described in female myelin (Naffaa et al., 2022b). Indeed, in females, this proportion increased from 0.72 % to 2.3 % (*p* < 0.001) in PS, 0.04–0.20 % (*p* < 0.001) in PI, 0.33–7.1 % (*p* < 0.001) in ePE and 0.64–3.36 % (*p* < 0.001) in PE. In male, this proportion increased from 1.3 % to 2.1 % (*p* < 0.001) in PS, 0.1–0.2 % (*p* < 0.01) in PI, 1.7–7.1 % (*p* < 0.001) in ePE and 2.2–3.4 % (*p* < 0.01) in PE. In contrast, MUFA-containing PC proportion decreased in both male (18.1–11.4 %, *p* < 0.05) and female (49 % to 11.5 %, *p* < 0.001) mice. The above results also revealed a strong difference at P15 between male and female myelin in MUFA-containing PE, ePE, PI and PC (*p* < 0.001) (Supplementary table 4). Similar to MUFA-containing phospholipids, in females, between P15 and P30, proportion of SFA- and PUFA-containing PE, ePE and PI increased while proportion of SFA and PUFA-containing PC decreased. In male, changes only affected a few subclasses without any trend emerging (Fig. 3D, supplementary table 4).

## 3.3. Kennedy pathway activation during development

The differences between males and females at P15 in glycerophospholipid distribution and FA content prompted us to explore the expression of genes related to the Kennedy pathway (synthesis of PE and PC from CDP-ethanolamine and CDP-choline) and MUFA synthesis in O4 + oligodendrocytes during early myelination (P4, P10 and P15) (Naffaa et al., 2022b). A strong increase in the expression of several genes coding for PE and MUFA synthesis enzymes was displayed between P4 and P15 in both male and female mice (Supplementary Fig. 2). Interestingly, this increase appeared to start earlier in males, with Selenoi level being significantly higher at P10 compared to P4 (1.7-fold, *p* < 0.01). In females, a significant change for this gene occurred only at P15. A similar trend was observed in *Etnk2* and *Pcyt2* expressions.

## 3.4. Myelin lipid redistribution following BPA exposure

Effects of BPA exposure (25 and 250 µg.L⁻¹) on the lipid composition of myelin at P15, P30 and P60 were investigated using multivariate
Male

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

Relative expression

Cnp

<figure><img src="image_5.png" /></figure>

Female

<figure><img src="image_6.png" /></figure>

Relative expression

Cnp

<figure><img src="image_7.png" /></figure>

Mbp

<figure><img src="image_8.png" /></figure>

Relative expression

Mbp

<figure><img src="image_9.png" /></figure>

Mog

<figure><img src="image_10.png" /></figure>

Relative expression

Mog

<figure><img src="image_11.png" /></figure>

Plp1

<figure><img src="image_12.png" /></figure>

Relative expression

Plp1

<figure><img src="image_13.png" /></figure>

(caption on next page)
**Fig. 2.** Expression of genes encoding myelin proteins in the brain at P15, P30 and P60 after early exposure to BPA. Evaluation of the expression of 4 genes encoding myelin proteins: 2',3'-cyclic nucleotide 3'-phosphodiesterase (Cnp), myelin basic protein (Mbp), myelin oligodendrocyte glycoprotein (Mog) and proteolipid protein (Plp1) in the forebrain at P15, P30 and P60 in animals exposed to BPA through maternal drinking water at 25 µg.L⁻¹ (BPA-25) and 250 µg.L⁻¹ (BPA-250) during gestation and lactation (from G7 to P22), and control animals (BPA-0), *n* = 6 per group. The expression was determined by RT-qPCR and normalized to the expression of Gapdh. Results are expressed as mean ± SD. Statistical analysis was performed at each stage (treatments compared to control group) and between the 3 control groups by Kruskal-Wallis test followed by Dunn's post-test (*p* < 0.05, **p** < 0.01, ***p** < 0.001).

statistical analysis. Regardless of the stage studied or the sex, exposure to the lowest concentration of BPA did not result in any clustering or separation on the PCA score plot between the control and the exposed groups, suggesting the absence of modification of the lipid composition of the myelin (Supplementary Fig. 3). In contrast, exposure to the highest concentration (250 µg.L⁻¹) led to an almost complete separation at P15, however only observed in female mice. At P30 and P60, no clustering or separation was found (Fig. 4A).

In P15 female mice, the clustering and separation due to BPA exposure mainly originated in glycerophospholipids (Figs. 4B, 4C). Indeed, BPA induced a decrease in PC and ePC, from 66% to 55% (*p* < 0.05) and from 2.0 to 1.4% (*p* < 0.05), respectively. In contrary, an increase was observed in the levels of PE from 8.1% to 13% (*p* < 0.05), ePE from 3.5% to 5.4% (*p* < 0.05), PI from 2.8% to 4.3% (*p* = 0.062) and PS from 6.9% to 9.6% (*p* < 0.001). The increase in PE, ePE, PI and PS, and the decrease in PC was observed regardless of their content in FA (Supplementary table 4, Supplementary Fig. 4A). Consequently, the relative proportion of SFA-, MUFA- and PUFA-containing species within each subclass was comparable between control and BPA-exposed females (Supplementary Fig. 4B). In addition, Cer, DG, TG and FFA, which represent less than 8% of total myelin lipids identified and semi-quantified, displayed the same behavior as PE, ePE, PI and PS (Figs. 4B, 4C). Indeed, in P15 female mice, they experienced an increase in their levels following BPA exposure. The same trend was also followed by cholesterol.

### 3.5. Transcriptomic and epigenetic modifications after BPA exposure

Changes in the myelin lipidome induced by BPA at P15 in female mice prompted us to examine the transcriptome and chromatin accessibility of O4 + oligodendrocytes isolated at the same stage (P16) by RNA-seq and ATAC-seq, respectively. Despite the lack of changes in myelin lipidome, these analyses were also conducted in male mice. Sequencing RNA of O4 + oligodendrocytes made it possible to identify differentially expressed genes (DEG) following BPA exposure of pregnant/lactating mice at 250 µg.L⁻¹. It should be noted that all identified DEG were gender specific, as none were significantly modulated by BPA (with FDR<0.05) in both males and females. In females, exposure to BPA altered the expression of 26 genes (FDR<0.05), including 10 upregulated and 16 downregulated genes (Fig. 5A, Supplementary table 5). Among the 10 upregulated genes, functional analysis using DAVID database highlighted significant enrichment of several pathways related to chaperones (Fig. 5B, Supplementary table 5). Indeed, 3 of these 10 genes encode chaperone proteins assisting protein folding in the endoplasmic reticulum (Dnajb1, Hspa5, Dnajb4). Regarding the 16 downregulated genes, an enrichment in genes belonging to the GOTERM_CC_DIRECT "GO:0016020~membrane" was evidenced (Fig. 5B, Supplementary table 5). In males, BPA altered the expression of 84 genes (FDR<0.05): 25 upregulated and 59 downregulated (Fig. 5C, supplementary table 5). Among the 59 repressed genes, functional analysis revealed an enrichment in genes belonging to several categories related to synapses (Fig. 5D, supplementary table 5). Indeed, 5 of these 59 genes encode proteins involved in synaptic transmission (Nlgn2, Tnr, Fyn, Adcy1, Lrrc4c). In contrast, for the 25 upregulated genes, no significant gene enrichment was evidenced (Fig. 5D, supplementary table 5).

Finally, this analysis also highlighted 16 genes differentially expressed between control males and females including, as expected, 9

genes located on sex chromosomes such as *Ddx3y* and *Eif2s3y*, overexpressed in males and *Xist*, *Ddx3x*, and *Eif2s3x*, overexpressed in females. Among the 7 genes located on autosome chromosomes, the gene encoding fibroblast growth factor *Fgf1* was twice as expressed in males compared to females (Supplementary table 5).

### 3.6. Modifications of chromatin accessibility after BPA exposure

Using the same O4 + oligodendrocytes samples, chromatin accessibility was also explored in 151,033 DNA regions. Early BPA exposure modified the accessibility of 33 regions in females (Fig. 6A, supplementary table 6) and 69 DNA regions in males (FDR<0.05) (Fig. 6B, supplementary table 6); hereafter, these regions will be designated as differentially accessible regions (DAR). In females, 29 DAR displayed higher accessibility (open DAR) and 4 DAR showed reduced accessibility (closed DAR). In males, BPA exposure was associated with 34 open DAR and 35 closed DAR. With one exception described below, differences in chromatin accessibility were sex dependent, and genes within or adjacent to these regions were not altered at the transcriptional level. Furthermore, these genes were not directly linked to myelination and functional analysis did not identify any notable signaling pathways. One open DAR, common to males and females, was located within the intron 13 of *Cwc22* gene (chr2:77913829~77914445). In females, 4 open DAR were located in *Cwc22* gene, including a region encompassing the transcription start site (TSS) (Chr2:77945335~77946640). In males, 2 DAR were located in *Cwc22* gene. This prompted us to focus on the impact of BPA exposure on *Cwc22* chromatin accessibility and gene expression. Regarding DAR located at the *Cwc22* locus, it must be emphasized that only 2 males (out of 5) and 3 females (out of 5) displayed chromatin opening after BPA exposure whereas the remaining samples were almost unaffected; this is indicative of a heterogeneity as to BPA effect on *Cwc22* chromatin accessibility (Fig. 6A, 6B, 6C). The response to BPA being observed in both males and females but with a strong heterogeneity, the impacts of BPA on *Cwc22* were next analyzed regardless of sex. *Cwc22* expression was first examined based on RNA-seq data (Fig. 6D). Although *Cwc22* did not belong to identified DEG neither in males nor in females, its expression was strongly upregulated after BPA exposure in 5 samples, including 3 (out of 4) female and 2 (out of 4) male samples, while being unaffected in the 3 other samples. Based on all samples regardless of sex, we highlighted a significant upregulation of *Cwc22* expression (*p* < 0.05). Importantly, 3 samples from BPA exposed animals (1 female and 2 males) did not display any change in chromatin accessibility nor in gene expression. This heterogenous "all-or-none" response to BPA at the *Cwc22* locus was further reinforced by analyzing *Cwc22* expression by RT-qPCR from the same samples. Indeed, a heterogenous upregulation of *Cwc22* expression was evidenced in half samples from BPA-exposed mice (Fig. 6E, *p* < 0.01). Finally, because RNA-seq and ATAC-seq were performed on the same samples, we were able to show a significant association between *Cwc22* expression and chromatin accessibility at its locus (Fig. 6F, *p* = 0.0021). Indeed, increased expression of *Cwc22* was correlated with increased chromatin accessibility in 5 BPA samples. Similarly, in 8 controls and 3 BPA samples, lower *Cwc22* expression was associated with lower chromatin accessibility.

## 4. Discussion

BPA, an endocrine disruptor widely distributed in the environment,
A

<figure><img src="image_14.png" /></figure>

B

<figure><img src="image_15.png" /></figure>

C

<figure><img src="image_16.png" /></figure>

D

<table><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th colspan="3">Males</th><th colspan="3">Females</th></tr><tr><th>P15</th><th>P30</th><th>P60</th><th>P15</th><th>P30</th><th>P60</th></tr></thead><tbody><tr><td rowspan="5">SFA</td><td>PE</td><td>2,10</td><td>1,50</td><td>1,36</td><td>0,67</td><td>1,59</td><td>1,11</td></tr><tr><td>ePE</td><td>0,17</td><td>0,14</td><td>0,09</td><td>0,04</td><td>0,16</td><td>0,09</td></tr><tr><td>PC</td><td>0,35</td><td>0,26</td><td>0,21</td><td>0,69</td><td>0,28</td><td>0,30</td></tr><tr><td>PI</td><td>0,06</td><td>0,06</td><td>0,06</td><td>0,02</td><td>0,07</td><td>0,06</td></tr><tr><td>PS</td><td>0,29</td><td>0,40</td><td>0,45</td><td>0,21</td><td>0,40</td><td>0,37</td></tr><tr><td rowspan="5">MUFA</td><td>PE</td><td>2,22</td><td>3,38</td><td>3,87</td><td>0,64</td><td>3,36</td><td>2,70</td></tr><tr><td>ePE</td><td>1,74</td><td>7,13</td><td>9,77</td><td>0,33</td><td>7,10</td><td>6,79</td></tr><tr><td>PC</td><td>18,08</td><td>11,38</td><td>9,63</td><td>48,98</td><td>11,53</td><td>19,74</td></tr><tr><td>PI</td><td>0,10</td><td>0,20</td><td>0,31</td><td>0,04</td><td>0,20</td><td>0,14</td></tr><tr><td>PS</td><td>1,26</td><td>2,07</td><td>2,52</td><td>0,72</td><td>2,31</td><td>2,48</td></tr><tr><td rowspan="5">PUFA</td><td>PE</td><td>19,63</td><td>19,17</td><td>19,59</td><td>6,79</td><td>19,50</td><td>13,64</td></tr><tr><td>ePE</td><td>14,90</td><td>17,57</td><td>18,49</td><td>3,16</td><td>17,88</td><td>13,12</td></tr><tr><td>PC</td><td>10,13</td><td>5,16</td><td>4,36</td><td>16,75</td><td>5,52</td><td>6,59</td></tr><tr><td>PI</td><td>8,29</td><td>5,34</td><td>4,65</td><td>2,66</td><td>5,93</td><td>3,16</td></tr><tr><td>PS</td><td>6,75</td><td>5,75</td><td>4,46</td><td>5,37</td><td>5,81</td><td>4,41</td></tr></tbody></table>

(caption on next page)
**Fig. 3.** Comparison of myelination in female and male mice. (A) Principal component analysis (PCA) comparing lipid composition at P15, P30 and P60 between males and females, (B) Distribution of lipid categories in myelin female and male samples at P15, P30 and P60 (n = 6 samples per group). PC: phosphatidylcholine, PC-O (also designated as ePC): phosphatidylcholine plasmalogen, PE: phosphatidylethanolamine, PE-O (also designated as ePE): phosphatidylethanolamine plasmalogen, PI: phosphatidylinositol, PS: phosphatidylserine, PG: phosphoglyceride, ST: sulfatide, SM: sphingomyelin, HexCer: hexosylceramide, Cer: ceramide, DG: diacylglycerol, TG: triacylglycerol, FFA: free fatty acid, (C) Cholesterol levels in myelin samples expressed in µg/mg of brain, represented as whiskers (min to max) and boxes (25–75 percentile). Statistical analysis was performed using Mann-Whitney test for male-female comparison at P15, and by Kruskal-Wallis test followed by Dunn's post-test for P15-P30-P60 comparison in each sex (*P* < 0.05, **p** < 0.01), (D) Levels (mol%) of glycerophospholipid subclasses in males and females at P15, P30 and P60 taking into account their fatty acid content.

has for around fifteen years been suspected to be involved in the
occurrence of neurodevelopmental disorders. Due to the importance of
myelination during neurodevelopment and the large abundance of lipids
in myelin, the present study deals with the effect of early BPA exposure
on myelination by carefully investigating myelin lipidome in mice.
During a preliminary study in female mice, we demonstrated that
myelination of brain tissue is accompanied by profound changes in lipid
composition (Naffaa et al., 2022b). These results were confirmed in the
present study, in which males were also included. Indeed, between P15
and P60, myelination induced the progressive increase in the relative
proportion (mol%) of HexCer and ST, characteristic lipids of myelin. In
addition, between P15 and P30, the relative proportion of PC decreased
while that of PE, ePE, PS and PI increased, as did their MUFA content.
However, it must be emphasized that the magnitude of these changes
differs between females and males. For example, the PC rate experienced
a 5-fold decrease in females (from 65 % to 14 %) whereas it was only
halved in male (from 23 % to 13 %). This difference originated in a
significantly higher proportion of PC in females than males at P15 while
it is approximately the same at P30. A difference in magnitude change
was also exhibited between males and females regarding the increase in
PE, ePE and PI. Thus, the level of ePE, another characteristic lipid
of myelin, experienced a 6-fold increase in females (3.5–20 %) vs a 1.3
increase in males (from 14 % to 18 %), also due to a marked difference
between males and females in ePE rate at P15 while being almost similar
at P30. Similarly, cholesterol content, lower in females than males at
P15, increased to reach a comparable level at P30. These results high-
light that, between P15 and P30, myelination is characterized by a clear
lipid remodeling more marked in females, reflecting a delay in myeli-
nation at P15. Our results thus indicate a temporal shift between males
and females in the myelination process during early postnatal life. In
oligodendrocytes, this shift was supported by the earlier expression of
several genes coding for PE synthesis enzymes in males (Kennedy
Pathway). Despite this, at P30, the myelin lipid composition is almost
identical regardless of sex. Finally, at P60, moderate differences reap-
pear. Consistent with this, sex-related differences have previously been
reported regarding oligodendrocytes and myelin in adult mice; it in-
cludes greater oligodendrocyte density, thicker myelin sheaths, and
higher expression of myelin genes and proteins in male brain (Abi
Ghanem et al., 2017; Cerghet et al., 2009). In human, sexual dimor-
phism was also evidenced since white matter volume was found to be
greater in men (Goldstein et al., 2001). These differences could originate
in hormonal impregnation accompanying puberty since sexual hor-
mones including progesterone, estradiol and androgens have been
involved in the myelination process (Naffaa et al., 2021). Finally, our
transcriptomic analysis of isolated oligodendrocytes revealed that, at
P15, males display twice as much *Fgf1* expression as females. Since *FGF1*
is known to promote myelination, our results suggest that this growth
factor plays a role in myelin lipid remodeling (Mohan et al., 2014).

BPA exposure (250 µg.L⁻¹ corresponding to an estimated exposure of
40 µg.kg⁻¹.d⁻¹) during gestation and lactation led to perturbations of
lipid remodeling accompanying myelination. Importantly, changes in
myelin lipid composition were observed selectively in female mice and
were transient, as they were only visible at P15. It mainly involved a
lower proportion of PC alongside higher proportions of PS, PE, ePE and
PI, resulting in a lipid composition which approximates that of more
mature myelin and therefore that of male myelin at this stage. This
suggests that, in females, exposure to BPA induced an earlier onset of

lipid remodeling accompanying myelination, not observed in males
since they exhibit a more advanced myelination at P15. Nevertheless, a
comparable effect of BPA in males before P15 cannot be ruled out.
Interestingly, previous studies have shown that BPA can accelerate
neuronal differentiation and migration suggesting that similar mecha-
nisms might be involved in the accelerated lipid remodeling observed in
juvenile females (Nakamura et al., 2006). Surprisingly, in female mice,
BPA did not disrupt brain expression of the myelin marker genes *Cnp*,
*Mbp*, *Mog* and *Plp1* at no stage studied while their expressions were
decreased in males at P60. Previous studies also indicate disruption of
myelin marker gene expression in rodents exposed to BPA during
development (Tiwari et al., 2015; Xu et al., 2017). However, these
studies, conducted in rats, focused on the hippocampus and differed in
methodology. In one study, dams were exposed to BPA *via* gavage at
40 µg.kg⁻¹.d⁻¹ from G6 to P21, and MBP expression was assessed at P21
and P90 without specifying the sex of the animals (Tiwari et al., 2015).
In another study, exposure was initiated on the last day of gestation until
weaning, with BPA diluted at 100 µg.L⁻¹ in water, and MBP expression
was analyzed in males at P70 (Xu et al., 2017). In our study, we also
observed a slight but significant decrease in the expression of several
myelin markers in males at P60, while no effects were observed on the
myelin lipidome. These results underscore the importance of consid-
ering factors such as sex, developmental stage, and methodology in
assessing BPA's effects on myelination and emphasize the value of
integrating both gene expression and lipidomic profiles to gain a more
comprehensive understanding of BPA-induced disruptions.

Since oligodendrocytes form the myelin sheath, we next evaluated
the impact of BPA exposure on the transcriptome of
O4 + oligodendrocytes at P15. BPA dysregulated a moderate number of
genes, 26 in females and 84 in males, and none were common to both
males and females, further confirming the sex-dependent BPA effects. In
males, among the 59 downregulated genes, five encode proteins related
to synaptic transmission, suggesting an involvement of oligodendrocytes
in the known effects of BPA on this process (Welch and Mulligan, 2022).
In females, 13 of the 16 downregulated genes belong to the GO-CC
"membrane", consistent with an alteration of the oligodendrocyte
plasma membrane that continuously extends to create the myelin
sheath. Additionally, 3 of the 10 upregulated genes encode chaperone
proteins which assist protein folding in the endoplasmic reticulum:
*Hspa5*, *Dnajb1* and *Dnajb4*. The former, encoding a member of the HSP70
family, plays a critical role for oligodendrocyte survival. Indeed, *Hspa5*
inactivation in oligodendrocytes during development or adulthood re-
sults in oligodendrocyte loss and severe myelin abnormalities in mice
(Hussien et al., 2015). *Dnajb1* and *Dnajb4* encode members of the HSP40
family. The upregulation of these 3 genes strongly supports the induc-
tion by BPA of endoplasmic reticulum stress due to the accumulation of
misfolded proteins, as previously reported in several experimental
models (Asahi et al., 2010; Kita et al., 2009; Tabuchi et al., 2006).

Strikingly, BPA exposure did not modify the expression of genes
directly linked to lipid metabolism in oligodendrocytes at P15 in either
sex. In females, BPA may act on these genes at an earlier stage. Another
plausible hypothesis involves post-transcriptional effects of BPA
including the modification of enzymatic activity and/or stability
through effects on protein folding, as suggested by the upregulation of
chaperone-encoding genes in females. BPA could also exert post-
transcriptional effects through modifications of RNA splicing, consis-
tent with a recent *in vitro* study (Kim et al., 2021). Indeed, in females, our
<figure><img src="image_17.png" /><figcaption>**Fig. 4.** Myelin lipid composition at P15, P30 and P60 after early exposure to BPA. (A) Principal component analysis based on myelin lipid composition (mol%) in male and female mice exposed to BPA through drinking water at 250 µg.L⁻¹ (M-BPA and F-BPA) and controls (M-control and F-control) at P15, P30 and P60, *n* = 6/group, (B) Heatmap representation (Morpheus, fixed color scheme) of lipid distribution alterations (fold-change) at P15 following BPA exposure at 250 µg.L⁻¹ in male and female mice, (C) whisker and box plots representing selected lipid subclasses levels determined by UHPLC-HRMS-ESI+/- analysis (mol%) and cholesterol contents (µg/mg of brain) at P15. Statistical analysis was performed using unpaired *t*-test followed by 5 % FDR correction for UHPLC-HRMS-ESI+/- data, and using Mann-Whitney test for cholesterol contents, *n* = 6/group (*p* < 0.05, **p** < 0.01, ***p** < 0.001).</figcaption></figure>
<figure><img src="image_18.png" /><figcaption>B</figcaption></figure>

Up-regulated in female (10)

<table><thead><tr><th>Category</th><th>Term</th><th>Count</th><th>PValue</th><th>Genes</th><th>FDR</th></tr></thead><tbody><tr><td>GOTERM_BP_DIRECT</td><td>GO:0051085~Chaperone mediated protein folding requiring cofactor</td><td>3</td><td>7,06E-05</td><td>DNAJB1, HSPA5, DNAJB4</td><td>8,55E-03</td></tr><tr><td>GOTERM_MF_DIRECT</td><td>GO:0051082~unfolded protein binding</td><td>3</td><td>6,80E-04</td><td>DNAJB1, HSPA5, DNAJB4</td><td>3,20E-02</td></tr><tr><td>INTERPRO</td><td>IPR002939:Chaperone DnaJ, C-terminal</td><td>2</td><td>3,46E-03</td><td>DNAJB1, DNAJB4</td><td>3,63E-02</td></tr><tr><td>INTERPRO</td><td>IPR008971:HSP40/DnaJ peptide-binding</td><td>2</td><td>3,46E-03</td><td>DNAJB1, DNAJB4</td><td>3,63E-02</td></tr><tr><td>UP_CWCellular_component</td><td>KW-0963~Cytoplasm</td><td>8</td><td>1,12E-03</td><td>DNAJB1, HSPA5, DCTN4, DNAJB4, DDIT3, CCDC117, DZIP3, RBM22</td><td>9,00E-03</td></tr><tr><td>UP_CW_Molecular_function</td><td>KW-0143~Chaperone</td><td>3</td><td>4,31E-03</td><td>DNAJB1, HSPA5, DNAJB4</td><td>3,45E-02</td></tr></tbody></table>

Down-regulated in female (16)

<table><thead><tr><th>Category</th><th>Term</th><th>Count</th><th>PValue</th><th>Genes</th><th>FDR</th></tr></thead><tbody><tr><td>GOTERM_CC_DIRECT</td><td>GO:0016020~membrane</td><td>13</td><td>2,56E-04</td><td>MTCH1, DST, SMURF1, SHISA4, ATP10B, H13, PRICKLE1, NYNRIN, RAB44, TMEM178, GPC1, MGAT5, RELL1</td><td>1,79E-02</td></tr></tbody></table>

<figure><img src="image_19.png" /><figcaption>D</figcaption></figure>

Up-regulated in male (25)

No significant enrichment

Down-regulated in male (59)

<table><caption>**Fig. 5.** Transcriptomic changes in O4 + oligodendrocytes at P16 after early exposure to BPA at 250 µg.L⁻¹ in drinking water. Transcriptomic analysis was performed by RNA-seq (n = 4/group) on RNA purified from O4 + oligodendrocytes isolated at P16 by magnetic activated cell sorting. (A) Heatmap representation (Morpheus, relative color scheme, row hierarchical clustering) of differentially expressed genes (DEG) identified in females and (B) functional annotation (https://david.ncifcrf.gov/), (C) DEG identified in males and (D) functional annotation. Although DEG were selectively identified in females or males, (A) and (C) heatmaps include the expression levels of all groups studied.</caption><thead><tr><th>Category</th><th>Term</th><th>Count</th><th>PValue</th><th>Genes</th><th>FDR</th></tr></thead><tbody><tr><td>GOTERM_CC_DIRECT</td><td>GO:0098685~Schaffer collateral - CA1 synapse</td><td>6</td><td>2,34E-05</td><td>CADM1, TNR, CASK, FYN, ADCY1, LRRC4C</td><td>3,51E-03</td></tr><tr><td>GOTERM_CC_DIRECT</td><td>GO:0045202~synapse</td><td>10</td><td>5,42E-04</td><td>CYFIP2, MPST, NLGN2, CADM1, RPS19, CADM2, MAP1B, CASK, CDH13, LRRC4C</td><td>4,06E-02</td></tr><tr><td>UP_CW_BIOLOGICAL_PROCESS</td><td>KW-0130~Cell adhesion</td><td>7</td><td>5,61E-04</td><td>CYFIP2, NLGN2, CADM1, CADM2, CNTN1, TNR, CDH13</td><td>1,74E-02</td></tr><tr><td>UP_CWCellular_component</td><td>KW-0770~Synapse</td><td>8</td><td>4,32E-04</td><td>CYFIP2, MPST, NLGN2, CADM1, CADM2, MAP1B, SLC35F1, LRRC4C</td><td>9,07E-03</td></tr><tr><td>UP_CWCellular_component</td><td>KW-1003~Cell membrane</td><td>20</td><td>2,07E-03</td><td>ARRDC4, SLC44AS, DOCK4, NLGN2, CADM1, CADM2, CASK, ADCY1, PTPRE, GJB4, EPB41L2, CNTN1, KCNQ3, CDH13, FYN, PLEKHN1, LRRC4C, STEAP2, RHBDL2, CORIN</td><td>2,18E-02</td></tr><tr><td>UP_seq_FEATURE</td><td>REGION:Disordered</td><td>46</td><td>1,20E-04</td><td>ZFP469, SLC44AS, CYFIP2, DOCK4, NLGN2, SLC35F1, CHD3, ADCY1, NHS12, FAM83F, DHX33, RPS19, SERTAD2, EPB41L2, VT11A, TNR, BEND4, JHY, FYN, PLEKHN1, SOX6, LRRC4C, RHBDL2, TNS3, MPZL1, SOX4, UTP14B, CORIN, TMEM132D, TCF7L2, PLK5, PARP1, CADM2, TBC1D8, CASK, FRMD4A, GPCPD1, PTPRE, MEX3B, TMEM158, KIF26A, MAP1B, UST, CNTN1, KCNQ3, CDH13</td><td>3,01E-02</td></tr></tbody></table>

study identified the upregulation of *Rbm22*, a gene encoding an essential RNA-binding protein involved in pre-mRNA splicing. Altogether, our transcriptomic analysis provides numerous insights into understanding the mechanisms by which BPA affects immature oligodendrocytes in a sex-specific manner.

In addition to transcriptomic analysis, an ATAC-seq analysis performed in the same oligodendrocyte samples identified differences between control and BPA-exposed animals. Two main observations can be made: first, and as previously observed all along our study, most differences in chromatin accessibility were sex dependent. Second, the genes located within or next to these regions were largely not affected at the transcriptional level. Based on these nearby genes, none of these regions appeared directly associated with myelination, and no signaling

pathway was highlighted by functional analysis. The modifications of chromatin accessibility could thus induce delayed effects on oligodendrocyte function or modify later cellular responses. Conversely, the transcriptional effects highlighted by RNA-seq do not seem to result from chromatin remodeling, as previously reported in mouse oligodendrocytes exposed to perinatal inflammation (Schang et al., 2022).

The locus of CWC22 spliceosome associated protein homolog gene constitutes an exception to the previous observations since chromatin accessibility was affected by BPA in both sexes and was correlated with Cwc22 transcription. Indeed, while in half of the samples, chromatin accessibility was strongly increased, leading to upregulation of Cwc22 expression, both were unchanged in the other samples. This “all-or-none” response to BPA resulted in a quite unusual bimodal expression
<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /><figcaption>**Fig. 6.** Modification of chromatin accessibility in O4 + oligodendrocytes at P16 after early exposure to BPA at 250 µg.L⁻¹ in drinking water. Analysis of chromatin accessibility was performed by ATAC-seq (n = 5/group for M-Co, M-BPA and F-BPA, n = 4 for F-Co) on nuclei purified from O4 + oligodendrocytes isolated at P16 by magnetic activated cell sorting. Chromatin accessibility was assessed by comparing normalized read counts within 151,033 genomic regions, with coordinates corresponding to the mm10 reference genome. (A) Differentially accessible regions (DAR) identified in females, (B) DAR identified in males. Although DAR were selectively identified in females or males (A) and (B) heatmaps represent the relative chromatin accessibility of all groups studied. (C) visualization of normalized read density at Cwc22 locus (peak within intron 13 and at transcription start site, TSS) and Mbp locus (open reading frame, TSS). Cwc22 expression assessed by (D) RNA-seq and (E) RT-qPCR. Statistical analysis was performed using Mann-Whitney test (*p* < 0.05, **p** < 0.01). (F) Correlation between chromatin accessibility at chr2:77913829-77914445 and Cwc22 expression (RNA-seq).</figcaption></figure>
profile. In a study carried out in mouse gonadal adipose tissue following prenatal exposure to BPA, the *Fggy* gene also showed bimodal expression (Taylor et al., 2017). However, unlike what we observed for *Cwc22*, it was exhibited in the control group and suppressed by BPA exposure. Indeed, BPA induced strong *Fggy* expression in all male mice, this effect being mediated through DNA hypomethylation, whereas in control animals this gene was either hypermethylated/lowly expressed or hypo-methylated/highly expressed. In the same study, BPA also induced *Cwc22* hypomethylation in males (Taylor et al., 2017). In our study, chromatin decompaction and upregulation of *Cwc22* expression in oligodendrocytes upon BPA exposure might also originate in DNA hypomethylation, which nevertheless occurred only in half of animals according to a yet unknown mechanism. It is noteworthy that both studies were conducted in outbred mice thus displaying inter-individual genetic variation which could lead to heterogenous methylation and expression status. In the present study, the “all-or-none” response to BPA could thus result from a “gene x exposure” interaction in which the genetic component remains to be documented. Finally, the disruption of *Cwc22* expression would further support an effect of BPA on RNA splicing since, like *Rbm22*, *Cwc22* encodes a spliceosome protein.

The question arises of knowing by what cellular and molecular mechanisms BPA affects oligodendrocytes. For this purpose, we have recently analyzed the effects of BPA on the differentiation of the murine oligodendrocyte cell line Oli-Neu (Naffaa et al., 2022a). In vitro, BPA counteracted the lipid remodeling accompanying differentiation, whereas *in vivo* it accelerated this process in females. This opposite result favors indirect effects of BPA *in vivo*. Supporting this, we did not detect any expression of genes encoding estrogen receptors in oligodendrocytes at P15 by RNA-seq whereas BPA is known to exert its effects mainly through activation of these receptors. However, the absence or low expression of estrogen receptors in oligodendrocytes does not rule out a direct effect of BPA in these cells, since many other mechanisms have been described in the literature (Gassman, 2017; Nesan et al., 2018). In addition to endoplasmic reticulum stress and RNA splicing, another hypothesis involves the disruption of thyroid hormone signaling. Indeed, thyroid hormones are crucial regulators of oligodendrocyte differentiation and myelination (Naffaa et al., 2021). BPA has been shown to interfere with thyroid hormone levels and signaling pathways both in experimental models and epidemiological studies (Kim and Park, 2019). Altogether, further studies are needed to elucidate the precise cellular and molecular mechanisms by which BPA affects oligodendrocytes.

The present study highlights sex-specific effects of BPA early exposure on oligodendrocytes and myelination in mice namely a transient alteration in the myelin lipid composition in females characterized by disturbances in glycerophospholipids. This effect selectively observed in females might be due to the more advanced myelin lipid remodeling in juvenile males also evidenced in our study. Although sexual dysmorphism of myelin has been described previously, to our knowledge, it is the first time that such temporal shift is described. Moreover, regarding transcriptome and chromatin accessibility of oligodendrocytes, BPA sex-specific effects were also evidenced. Even though integrating all these levels of analysis represents a real challenge, we believe that multi-omics analyzes provide a valuable approach to understand the complex effects of BPA and other chemical pollutants. The present study opens new insights in understanding the mechanisms of BPA's effects, including the involvement of oligodendrocytes on synaptic disruption, the role of endoplasmic reticulum stress and chaperones, and possible effects on RNA splicing. Finally, we identified *Cwc22* as a potential marker of BPA exposure in oligodendrocytes. Importantly, this ubiquitously expressed gene was also affected by BPA in other cells. Knowing whether chromatin and *Cwc22* expression disruptions are specific to certain cell types or occur more generally would be interesting to address in future studies. These findings also have broader implications regarding BPA's impact on the nervous system. By targeting myelination process, BPA exposure may contribute to disturbances

in white matter structure, a characteristic feature of neuro-developmental disorders such as ASD and ADHD. This emphasizes the importance of further research to elucidate the mechanisms driving this susceptibility, including potential hormone involvement. Such insights could inform strategies to mitigate the risks associated with BPA and other endocrine disruptors.

## ARRIVE statement

All animal experiments, data description and statistical analyses complied with the ARRIVE guidelines.

## Declaration of Generative AI and AI-assisted technologies in the writing process

During the preparation of this work the authors used ChatGPT-OpenAI 2024 in order to improve the readability of the manuscript. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the content of the published article.

## Disclosure

All authors have approved the final article.

## Funding

This research received support from the French National Research Program for Environmental and Occupational Health (Anses, 2019/1/157). It was further funded by grants from the University Paris Cité, the French National Center for Scientific Research (CNRS), and the French National Institute for Health and Medical Research (Inserm), along with an additional grant from the "Investissement d'Avenir" program (ANR-11-INBS-0011) NeurATRIS. Sequencing was performed by the GenomEast and IGenSeq platforms, members of the "France Génomique" consortium (ANR-10-INBS-0009). VN received a PhD Fellowship from the French Ministry of Higher Education, Research, and Innovation. The funding organizations had no role in the preparation of the report or the decision to submit the article for publication.

## CRediT authorship contribution statement

**Vanessa Naffaa:** Writing – original draft, Visualization, Investigation, Formal analysis. **Juliette Van Steenwinckel:** Writing – original draft, Methodology, Formal analysis, Conceptualization. **Romain Magny:** Writing – original draft, Methodology, Investigation, Formal analysis. **Anne Regazzetti:** Writing – original draft, Methodology, Investigation. **Céline Keime:** Writing – review & editing, Formal analysis, Data curation. **Pierre Gressens:** Writing – review & editing, Supervision, Funding acquisition, Conceptualization. **Olivier Laprévote:** Writing – review & editing, Supervision, Funding acquisition, Conceptualization. **Nicolas Auzeil:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. **Anne-Laure Schang:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## 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.
## Acknowledgment

We acknowledge Marielle Young-Ten for her valuable assistance in the care and handling of the animals.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.tox.2024.154041.

## Data availability

I have shared the link of my data/code at the Attach File step

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