Experimental design
This study aimed to reveal the effects of histone acetylation perturbation on female germ cell development with a focus on embryonic events in which meiosis, the chromosome segregation process allowing the formation of haploid gametes, is initiated. Inbred C57BL/6J mice were treated with PB during the somatic-to-germline transition window, which corresponds to embryonic day (E) 6.5 to E15.5. Mice were dissected at several time points: E15.5, E17.5, E18.5, post-natal day (PND) 5 and 5 months. A schematic presentation of the experiments is presented in Additional file 1: Fig. S1. To analyse the effects of PB on female germ cells, we treated mice with a dose of PB equal to 5 mg/kg every other day. We observed a significant 13% increase in the body weight of the embryos at E15.5 but not at E17.5 (Additional file 1: Fig. S2). We did not observe significant changes in body or ovary weights in 5-day-old and adult mice, suggesting that the dose used was not grossly toxic (Additional file 1: Figs. S2, S3). No alterations in gestation parameters, such as litter size and gestation time, were observed in PB-treated animals compared to controls (Additional file 1: Fig. S4).
Increases in H3K9Ac, H4Ac, and H3K4me3 and a decrease in H3K9me3 after PB exposure
To reveal whether gestational exposure to PB affects histone modifications, we analysed the histone marks important for meiosis, H4Ac, H3K9Ac, H3K4me3 and H3K9me3. Defects in histone acetylation regulation may have an impact on histone methylation since methylation and acetylation are mutually exclusive on the lysine epsilon amino group. Therefore, histone acetylation and methylation on lysines must be coordinated, with methylation being able to take place only on deacetylated lysines, and vice versa. Since analyses of histone acetylation levels can provide clues on the efficiency of PB exposure, we immunostained ovarian surface spreads for H3K9Ac and costained the slides with SYCP3 to visualize the synaptonemal complex (SC). H3K9Ac normally appears as strong staining around all chromosomes except at compact heterochromatin (Fig. 1A). Quantitative analysis of H3K9Ac staining showed that in PB-treated oocytes, the intensity of H3K9Ac was increased 1.7-fold (Fig. 1B).
H4 acetylation has been reported to be important for both DNA repair and chromatin remodelling events [29]. Structurally preserved nuclei were immunostained with an anti-H4Ac antibody (Penta, acetylated at residues K5, K8, K12, and K16) (Additional file 1: Fig. S5A) in E15.5 ovaries. Quantitative analysis showed a 1.32-fold increase in H4Ac intensity in PB-exposed oocytes at E15.5 (Additional file 1: Fig. S5B).
We also examined H3K4me3, which is essential for HR, as it is required for the initiation of double-strand breaks. Meiotic spreads were immunostained for H3K4me3 and SYCP3 (Fig. 1C). Quantitative analysis showed that H3K4me3 intensity was increased 1.5-fold in embryonic oocytes from treated mice (Fig. 1D).
H3K9me3 plays a major role in the formation of heterochromatin where telomeres attach during meiosis. Quantification of this histone modification revealed a 1.2-fold decrease in the levels of H3K9me3 in embryonic ovaries (p = 0.051) (Additional file 1: Fig. S6A, B).
Thus, our data suggested that epigenetic marks associated with meiosis and DNA repair were altered upon PB exposure in embryonic oocytes.
The histone demethylase KDM5A is decreased in PB-exposed ovaries
Since it has been shown that the demethylase KDM5A is functionally linked to two histone deacetylase complexes [30], we investigated whether it could play a role in PB-exposed ovaries. We immunostained embryonic oocyte cells with antibodies against KDM5A and SYCP3 (Fig. 2A) and performed a quantitative analysis of the z-stack images. Our analysis revealed that KDM5A had strong nuclear staining in the control group but 2.7-fold lower intensity in the PB-exposed groups (Fig. 2B).
Meiotic defects are increased in PB-exposed ovaries
Since we observed alterations in major epigenetic marks involved in DNA repair at recombination hotspots and during meiosis, we investigated whether these epigenetic alterations could influence meiotic DSB repair efficiency. To this end, we immunostained E15.5 surface spreads for the DNA-binding recombination protein DMC1. SYCP3 was also labelled to visualize paired chromosomes (Fig. 3). DSB formation and repair are dynamic processes; new DSBs are formed and repaired simultaneously, so the number of DMC1-stained foci varies from cell to cell. We were not able to observe DSBs at embryonic day 15.5 in PB-exposed cells since the synaptonemal complex (SC) was not fully formed in exposed oocytes and the lateral component of SC, SYCP3, appeared as dots in a few cells (Fig. 3A). At E17.5 and E18.5, DMC1 foci were visible in PB-exposed oocytes (Fig. 3B, C). To evaluate the efficiency of meiotic DNA repair, we measured the average number of DMC1 foci per oocyte in the control and treated groups. We found that the number of DSB foci at E17.5 in the treated group (110 ± 6) was higher than that in the control group (44 ± 2, p < 0.05) (Fig. 3D). On embryonic day E18.5, most of the breaks were repaired in untreated control mice (16 ± 2, DMC1 foci per cell); however, in the PB-treated group, the number of DMC1 foci remained high (54 ± 3, p < 0.05) (Fig. 3D).
Thus, exposure to PB leads to a delay in the formation of the synaptonemal complex and the persistence of DSBs in embryonic ovaries.
Exposure to PB leads to gene expression alterations in embryonic ovaries
Next, to determine whether in utero PB exposure affects gene expression, we performed transcriptomics analysis by using strand-specific RNA sequencing. We identified 1169 transcripts whose expression was changed in comparison to the expression in control ovaries (FC ≥ 2, FDR ≤ 0.05). These transcripts represented 2.45% of the total transcriptome (47,623 transcripts expressed in all three biological replicates in the control group) (Additional file 1: Table S1). Of the differentially expressed genes (DEGs), the numbers of upregulated and downregulated genes were nearly equal (Additional file 1: Fig. S7).
To reveal the effects of PB treatment on molecular pathways, we performed functional annotation of differentially expressed genes (DEGs) using the DAVID tool. Several functions were overrepresented for the downregulated genes. Notably, we identified enrichment for 16 genes encoding WNT signalling pathway proteins (e.g., Cxx4, Dixdc1, Sox17, Axin2, Hic1, Lgr4, Rnf146, Wnt2) and spermatogenesis-related proteins (12 genes, e.g., Sohlh1, Tdrd9, Zfx, Rnf114, Nek1) (Fig. 4A). For the upregulated genes, we found several functions/characteristics that were overrepresented, including transcriptional regulation (70 genes), Zn-fingers (67 genes) and kinases (43 genes) (Fig. 4B). Thus, our analysis of DEGs showed that genes encoding Zn-finger proteins were among the most altered genes, suggesting that PB exposure mainly affects the expression of these genes.
We also performed an alternative functional annotation using the ChEA2016 database from Enrichr, and we detected upregulated genes that are known to be regulated by male-specific factors. According to the ChEA2016 database, 137 out of 1384 differentially expressed genes are regulated by SOX9, and 170 out of 2072 are regulated by DMRT1 (Fig. 4C).
Next, we also analysed DEGs using g:Profiler, a functional annotation tool. This analysis showed that a large group of genes were annotated to organism development processes. These genes fell into the cell cycle, DNA repair, and transcription factor categories (Fig. 4D).
Our RNA-seq data did not reveal any alterations in HDAC mRNA expression, except for that of Hdac7 (transcript variant X6, XM_006521208.3), which was expressed only in PB-exposed mice (FC = 875.93, FDR = 0.056), suggesting that the phenotypes observed following PB exposure were a consequence of direct HDAC inhibition rather than impaired transcriptional regulation of HDAC genes.
To determine whether the PB-induced defects observed in embryonic ovaries were restored after birth, we performed RT-qPCR of some target genes at PND5. We found that genes encoding histone-modifying enzymes, such as Fgfr1, Foxo3, Kat2a and Kat2b, were downregulated (Wilcoxon–Mann–Whitney, p-value = 0.057) (Additional file 1: Fig. S8), whereas genes encoding DNA repair or apoptosis proteins were not altered (except for the repair protein Mus81, which was downregulated). In summary, in utero PB exposure results in mostly transitory transcriptional deregulation of several key functional compartments in the embryonic ovaries.
DNA methylation tends to be increased at the promoters of the master regulator genes Dazl, Ddx4 and Hormad1
Alterations in gene expression could be a consequence of the effects of PB on the somatic-to-germline transition process, during which somatic cells lose methylation and establish germline lineage-specific marks. Since DNA demethylation at master regulator genes such as Ddx4, Hormad1 and Dazl is critical for maintaining the germ cell lineage [2], we analysed the promoter methylation of these genes in E15.5 ovaries. We found a tendency for increased DNA methylation at Ddx4, Dazl and Hormad1 (p = 0.057 for Ddx4 and Dazl, p = 0.1 for Hormad1) promoter CpG islands (Fig. 5), suggesting a possible impact of PB not only on histone modification levels, but also on DNA methylation levels. This DNA methylation increase could also reflect an alteration of the germline-to-somatic cell ratio, as these genes are normally highly methylated in somatic cells.
Decreased oocyte number at E15.5 in PB ovaries
Since SGT reprogramming is critical for the establishment of the germ cell lineage and because we observed alterations in DNA methylation at genes involved in the maintenance of the germ cell lineage, we counted germ cells in E15.5 ovary sections. We prepared paraffin sections from E15.5 ovaries and immunostained them for the germ cell marker DDX4 (Fig. 6A). Germ cell counting revealed a 30% decrease in oocyte number at E15.5 (p < 0.05) (Fig. 6B), suggesting a possible deleterious effect of PB exposure on the germline cell population.
Increased follicle growth at PND5 in PB-exposed ovaries
To check the follicle maturation efficiency, we immunostained PND5 ovary sections with an oocyte marker, MSY2, which is a cytoplasmic marker of germ cells. At this timepoint, MSY2 stained mainly primary and secondary follicles, which are oocytes surrounded by one or two layers of somatic granulosa cells (Fig. 7A). We detected an increase in MSY2-positive cells in the PB group (Fig. 7B), suggesting that more oocytes were undergoing maturation in the PB group than in the control group. To confirm our observation, we coimmunostained the ovaries for the proliferation marker PCNA and the mitosis marker phospho-histone H3 (serine 10) (Fig. 7C). We observed that both markers had increased signal intensity at PND5, confirming the proliferation of somatic cells.
To determine whether gestational exposure to PB affects the follicular population in adult mice, we analysed histological sections of ovaries from 5-month-old mice (Fig. 8A, B) and scored the different follicle types as described in the Methods section. Analysis of the follicle counts (Fig. 8C) showed a 3.6-fold decrease in primordial follicles (139 ± 24 control, 39 ± 24 treatment, *p < 0.05, t-test), a 2.2-fold decrease in primary follicles (191 ± 27 control, 88 ± 32, treatment, *p < 0.05, t-test) and a 1.7-fold decrease in secondary follicles (61 ± 8, control, 35 ± 6 treatment, *p < 0.05, t-test, minimum 4 animals for each group).
The transcriptional network is perturbed in adult mouse ovaries following gestational PB exposure
We performed RT-qPCR gene expression analysis on the ovaries of adult mice that were exposed to PB as embryos. Several candidate genes were selected, including HDACs, histone-modifying enzymes, germ cell markers, genes involved in the oestrogen signalling pathway and genes involved in the transcriptional regulation of ovarian development (Foxl2, Foxo3). We also examined the expression of genes that are involved in the oxidative stress response (Gpx1, Sod1) and DNA damage (H2afx). We found that the expression of the genes encoding Hdac1, Hdac5, Hdac6, Hdac11, Sirt2, Sirt3 and Sirt7 increased following in utero PB treatment (Fig. 9A). We also found that the Esr2, Lhchr, Fshr, Fst, Foxl2 and Foxo3 genes were upregulated in the ovaries of in utero-treated mice (Fig. 9B). The expression of other histone-modifying enzymes was not significantly affected. The expression of Gpx1, a gene encoding a detoxification enzyme, was reduced in exposed ovaries (Fig. 9C). Our data show that in utero exposure to PB leads to gene expression alterations.
Decreased H3K4me3 marks in adult oocytes following in utero PB exposure
To determine whether gene expression is associated with alterations in epigenetic marks in adult ovaries, we immunostained ovary sections using antibodies against the important histone marks H4Ac and H3K4me3 (Fig. 10A, C). Since the signal for both histone marks was detectable only in the nuclei of fully grown oocytes, we analysed this mark in fully grown oocytes and in surrounding granulosa cells. Quantitative analysis of immunofluorescence of H4Ac showed a significant 1.5-fold increase in this marker in surrounding granulosa cells of fully grown oocytes, but we did not observe significant changes in oocytes themselves (Fig. 10B).
Quantitative analysis of the H3K4me3 mark showed a 1.3-fold decrease in H3K4me3 marks in surrounding granulosa cells, but no significant changes could be observed in the oocytes of treated ovaries (Fig. 10D).
In conclusion, our analysis revealed that gestational exposure to PB results in changes in gene expression in adult ovaries that are accompanied by an increase in H4Ac and a decrease in H3K4me3 in granulosa cells that surround fully grown oocytes. However, these changes in the levels of key epigenetic marks are not observed in fully grown oocytes.