BACKGROUND
environmental factors can alter dna methylation and epigenetic modifications in the germline  <cit> .
we studied the effects of the widely used herbicide atrazine  on meiosis.
in many countries, atz is the most common contaminant detected in rivers  <cit> .
atz residues are even detected in soil and aqueous environments in countries where it has been banned  <cit> .
low levels of atz metabolites in pregnant women are associated with low birth weight  <cit> .
in mammals, atz is metabolized by several groups of xenobiotic-metabolizing enzymes.
the metabolism of atz interferes with oxidative phosphorylation and cytochrome p <dig> function, resulting in decreased oxygen consumption  <cit> .
atz is known to alter reproductive processes in rodents  <cit> , reptiles  <cit> , birds  <cit> , goats  <cit> , amphibians  and fish  <cit> .
atz targets many tissues, including the testes  <cit> , ovaries  <cit> , brain  <cit> , liver  <cit> .
the effects of atz on reproduction have been extensively studied; however, the effect of atz on meiosis has not been elucidated.
meiosis is the division of germ cells and is essential for the production of haploid gametes.
in most organisms, homologous recombination  is important for the proper segregation of chromosomes during meiosis.
hr predominantly occurs in discrete areas of the genome known as recombination hotspots.
histone h <dig> trimethylation at lysine  <dig>  by prdm <dig> methyltransferase is indispensable for the proper formation of double-strand breaks  throughout the genome, except in the pseudoautosomal region  of x chromosome  <cit> .
germ cells lacking prdm <dig> are unable to repair dsbs at the pachytene stage of gametogenesis, and knock-out mice are infertile in both sexes  <cit> .
it has also been shown that environmental pollutants affect hr both in male and female.
the first study illustrating the effects of environmental exposure on meiosis was the bisphenol a  treatment study, in which the authors showed that embryonic exposure to bpa causes meiotic anomalies, which lead to aneuploidy in adult female mice  <cit> .
importantly, meiotic effects in animals were induced by environmentally relevant doses of bpa  <cit> .
bpa exposure of adult rat delays meiosis initiation and induces accumulation of meiotic dna dsbs in the late meiotic stage  <cit> .
in nematodes bpa exposure also results in impaired chromosome synapsis and disruption of meiotic double-strand break repair  progression of c. elegans  <cit> .
we hypothesized that atz may also alter meiosis because it reduces sperm number and changes testis morphology in animals  <cit> .
here, we studied the mechanisms by which atz affects meiosis in mice.
we showed that exposure to atz increases testosterone levels and reduces the number of spermatozoa in epididymis, causing a delay of meiosis.
using gene-chip and chip-seq analysis of h3k4me <dig> marks, we found that a wide range of cellular functions, including gtpase activity, mitochondrial function and steroid-hormone metabolism, are affected by atz.
furthermore, histone h3k4me <dig> marks are enriched in treated mice in regions of strong recombination  within very large genes and reduced at the par region of the x chromosome.
we therefore propose that atz disrupts normal meiosis.
RESULTS
experimental design
we treated adult male mice with atz at a concentration of 100 mg/l in water for three weeks and followed with two weeks without treatment to cover one full round of spermatogenesis .
given that mice drink approximately 5 ml of water per day, we estimate that the average intake of atz was approximately 25 mg/kg/day.
this dose is relatively low compared to that used previously  <cit> .
we chose this dose of atz because doses higher than 50 mg/kg/day are known to change the weight of internal organs and are considered as toxic  <cit> .
to study the effect of atz on the first wave of spermatogenesis, we treated newborn animals from postnatal day  <dig> through day  <dig> dpp by administering atz suspended in oil to the lactating dams at a dose of 25 mg/kg/day.
low levels of atrazine do not affect testicular morphology and cell viability
after treatment, the mice were euthanized, and reproductive organs were dissected.
we measured the organs and body weights of treated and control animals.
we found that the body weight and reproductive organs  were not affected at this dose .
to evaluate morphological changes in seminiferous tubules caused by atz, we analyzed paraffin-embedded sections stained with hematoxylin and eosin and compared matching stages from treated and control animals.
the morphological analysis did not reveal any significant changes in either the seminiferous tubules or the epididymis ).
however, we could not exclude small changes in the proportion of cells, which are difficult to distinguish by this method.
we verified cell populations by staining with propidium iodide  and sorting by facs and we comcluded that cell populations were not significantly affected by this treatment ).
to examine whether the number of germ or sertoli cells had changed, we immunostained testicular tissue with antibodies specific for undifferentiated spermatogonial stem cells  and sertoli cells  ).
we found that treatment did not significantly alter the number or proportion of those cell types .
to evaluate the rate of apoptosis, we performed tunel assays using the in situ cell-death detection kit.
we did not detect significant changes in the rate of apoptosis in adult atz-treated mice compared to control .
thus, treatment with a relatively low dose of atrazine does not affect organ weight or the cell populations within seminiferous tubules.
decreased sperm count and serum testosterone levels in atz-treated mice
one essential metric of reproductive state is the number of spermatozoa per epididymis.
to evaluate the effect of atz, we counted spermatozoa in the epididymis of treated and control mice.
we found that atz-treated mice had a significantly lower sperm count  than untreated animals .
to assess endocrine function after treatment, we measured serum levels of testosterone and follicle-stimulating hormone .
we found that serum testosterone was  <dig>  times lower in treated animals , while the level of fsh was not significantly affected .
our data show that a relatively low dose of atrazine decreases sperm counts and serum testosterone levels.fig.
<dig> low-dose atz treatment decreases spermatozoa numbers and testosterone levels.
a the number of spermatozoa in the epididymis decreased.
b the amount of testosterone in the serum decreased; n = 10/group; values are means +/−sd, while fsh was not significantly affected
analysis of the synaptonemal complex reveals delayed meiosis
we hypothesized that a decrease in sperm count might reflect defects in meiotic progression or spermiogenesis.
we thus asked whether atz negatively affects the meiotic stages of spermatogenesis.
to analyze meiotic progression in treated mice, we performed surface spreads from testis tissue and analyzed synaptonemal-complex  formation.
the sc is formed exclusively during prophase  <dig> of meiosis, and its major role is to bring the chromosomes together  <cit> .
prophase i is subdivided into several sub-stages .
we immunostained the surface cell spreads with antibodies against sycp <dig>  and γh2ax  and then classified cells into meiotic substages  as previously described  <cit> , .
during the leptotene phase, there is an increase in the number and intensity of γh2ax-positive domains throughout the nucleus.
in the zygotene stages, the number of γh2ax domains and the intensity of γh2ax staining decreased, as the staining was concentrated over the chromatin.
at the early pachytene stage, γh2ax had almost disappeared from the chromatin of the autosomes, but there were still some visible traces of marks on autosomes, and marks appeared to cover the sex chromosomes.
at the late pachytene stage, γh2ax was detected only at the sex chromosomes.
the proportions of prophase i substages detected in control mice were similar to those detected by other authors  <cit> .
however, we found that the number of early stage spermatocytes increased in atz-treated animals , while the late stages decreased   .
in control mice, the largest fraction of spermatocytes was in the late pachytene stage, where γh2ax markers were detectable only in sex chromosomes .
in atz-treated animals, the majority of spermatocytes showed patterns of γh2ax staining typical of early stages .
we performed quantitative analysis of early pachytene cell fractions in control and treated mice, and we detected three major defects in atz-treated mice:  incomplete synapsing of one or two chromosomes, which was often detected together with the formation of branched chromosome structures;  sex-body defects .
we found that the number of cells with incomplete synapses increased  <dig>  times in treated cells compared to untreated cells, the number of cells with sex-chromosome defects increased  <dig>  times .fig.
<dig> atrazine causes delayed meiosis in mice.
a γh2ax staining changes during meiotic prophase i substages.
b the distribution of meiotic substages in control  and atz-treated  mice;  <dig> cells from control and treated mice were classified according to γh2ax and sycp <dig> staining.
note that the number of spermatocytes in the zygotene and early pachytene stages is increased in atz-treated animals, while the number of spermatocytes in the late stages decreased.
c surface spreads from control and atz-treated testis stained with anti-γh2ax  and anti-sycp <dig>  antibodies.
in control mice, the majority of meiotic cells have γh2ax marks at the sex bodies only.
in atz-treated mice, the majority of meiotic cells have γh2ax marks at many chromosomes
the performed analysis of immunostaining pattern of γh2ax marks helped us to evaluate the meiotic progression, however, we do not exclude the fact that our observation is not perfect due to the possibility that meiosis in some cells is deregulated and that the stages could not be defined exactly.
our analysis is designed to observe the general view of meiotic progress.
we complete this finding by analyzing meiotic progress during the first wave of spermatogenesis .
taken together, our data show that atz has an effect on meiotic progression and increases the number of synapsing defects.
atz treatment delays meiotic progression due to persistence of dsbs
we next asked whether the increase in early stage meiotic cells occurred due to the persistence of dsbs breaks.
chromosomes undergo dsb formation at the leptotene-zygotene stage.
at this stage, a maximal number of dsbs can be detected by staining surface spreads with an antibody against dmc <dig>  which is a dna-binding protein of dsbs.
we found that the number of cells with dmc <dig> foci increased with atz treatment .
most of the cells positive for dmc <dig> in atz-treated animals had foci at the sex chromosomes.
the increase in the number of dmc1-positive cells with treatment could be the result of inefficient repair of dsbs in sex chromosomes.fig.
<dig> the proportions of dmc1- and atr-positive cells increase upon treatment.
a spreads from control and atz-treated mice were stained with anti-dmc <dig>  and anti-sycp <dig> .
in control mice, the dsbs are repaired at this stage, such that no dmc <dig> foci are detectable.
in atz-treated samples , several dmc <dig> foci are detectable at the pachytene stage.
b spreads stained with anti-atr  and anti-sycp <dig> .
in control mice, the atr signal is detected in sex bodies only, while atr staining is detected at several chromosomes in atz-treated mice.
c quantitative analysis of atr staining.
the spreads were costained with atr and sycp <dig> antibodies and classified according to pattern: sex body only  and sex body with staining of autosomal chromosomes
to further examine synapsing efficiency in atz-treated mice, we stained chromosomes with an antibody against atr , a marker of asynapsed chromosomes and silenced chromatin  <cit>  .
atr acts stage-specifically to regulate multiple aspects of mammalian meiotic silencing  <cit> .
if asynapsis persists at the pachytene stage, atr leads to the induction of repressive post-translational modifications and irreversible gene silencing over many megabases  <cit> .
to analyze the role of atr in atz-treated mice, we counted the number of cells that showed staining of atr at the sex body only or that showed simultaneous atr staining of both xy bivalents and autosomes.
in atz-treated mice, there was  <dig>  times more atr staining of autosomes .
consistent with previous data on the role of atr in chromatin silencing  <cit> , our experiments showed that atz treatment increases the number of chromosomes with incomplete synapsing.
delayed meiotic progression and increased apoptosis in young prepubertal animals
to analyze the effect of the atz on meiotic progression during the first wave of spermatogenesis, we treated newborn males with atz.
young prepubertal animals have a higher proportion of cells at any given meiotic stage because the first wave of spermatogenesis is generally synchronous.
the analysis of the testicular sections of treated animals revealed that the number of diplotene cells decreased  <dig>  times compared to untreated animals, while the numbers of early pachytene and zygotene cells increased  <dig>  times .
these data are consistent with the results obtained from treated adult mice showing that atz has a profound effect on meiosis.
as the first wave of spermatogenesis involves germ-cell apoptosis to regulate the number of germ cells under normal conditions  <cit> , we assayed this process following atz treatment.
atz treatment of young mice increased the number of apoptotic cells  compared to controls.
the increased apoptosis rate in young mice is the result of an elevated number of germ cells with meiotic defects, which are eliminated during the first wave of spermatogenesis.
thus, atz exposure of young prepubertal mice causes a delay in meiosis similar to that observed in adult males.fig.
<dig> atrazine delays meiosis in prepubertal mice.
h&e straining of histological sections of a control and  atz-treated mice.
the atz-treated mice have reduced numbers of cells in the seminiferous tubules.
c the percentages of seminiferous tubule cross-sections in which preleptotene and leptotene  cells, zygotene and early pachytene  cells, pachytene  cells, diplotene d spermatocytes and round spermatids  represent the most advanced types of germ cells in control  and atz-treated  testes at postnatal day  <dig>  the percentage of empty tubules is also indicated.
d tunel assays on testis histological sections from 20-day-old control and atz-treated mice.
the results are presented as a percent of tubules containing apoptotic cells, and the data of at least five mice are plotted
atz treatment affects the expression of genes involved in mitochondrial function, steroid-hormone function and gtpase activity
to assess how atz affects spermatogenesis at the molecular level, we performed a comparative analysis of gene expression using gene-chip analysis.
using a fold-change cutoff value of  <dig>  and a p-value cutoff of < <dig> , we identified  <dig> genes that were differentially expressed between atz-treated and control mice .
the cellular heterogeneity of seminiferous tubules limits the sensitivity of this approach, as patterns of gene expression may vary by cell type.
we confirmed differential expression by qpcr .
some differentially expressed genes  were transcription factors, and their roles in spermatogenesis have not yet been elucidated.
the expression of oxidative phosphorylation-related genes was increased .
these genes might be activated due to oxidative stress.
oxidative stress is the result of increased production of oxidizing species  <cit>  caused by the metabolism of atz  <cit> .
we confirmed that oxidative phosphorylation gene expression was associated with increased ros by measuring hydrogen peroxide in the serum .fig.
<dig> atrazine affects mrna transcription and increases production of hydrogen peroxide.
a qpcr analysis of rna samples was performed in three independent experiments.
qpcr primer sequences for each gene are shown in the materials and methods.
the data are presented as fold change in treated samples compared to the controls, *p <  <dig> , **p <  <dig> .
b hydrogen peroxide levels increase in the serum of treated animals.
the measurement of hydrogen peroxide in the serum of at least three independent experiments is shown.
the data are presented as average luminescence values, *p <  <dig>
the transport of cholesterol across the mitochondrial membrane is a key rate-limiting step in the regulation of steroid-hormone biosynthesis  <cit> .
we found decreased levels of mrnas encoding star, a major protein responsible for cholesterol transport.
the level of ldlr  also decreased, which affects cholesterol metabolism  <cit> .
other target genes included small gtpase regulatory proteins, such as g3bp <dig>  nmt <dig>  sar1a , xpo <dig> , gbf <dig>  and arfgef <dig> .
arf <dig> gtpase is also required for the maintenance of mitochondrial functionality and dynamics  <cit> .
we performed the analysis by using fold change cut-off value ≥ <dig>  the short list includes  <dig> differentially expressed genes  .
these are genes, which play role in anti-oxidative stress response , xenobiotic metabolism , cell division , and cell death  pathways.
gene-chip analysis revealed that atz treatment affects many cellular processes, including steroid-hormone metabolism, oxidative phosphorylation and xenobiotic metabolism.
genome-wide sequencing reveals global changes in h3k4me <dig> marks following atz treatment
h3k4me <dig> is a pivotal mark of active chromatin because it acts as a platform for the binding of multiple histone acetyltransferases, histone demethylases and nucleosome-remodeling complexes  <cit> .
as h3k4me <dig> marks are essential for meiosis, we asked whether treatment with atz affects these marks.
to this end, we analyzed the genome-wide distribution of h3k4me <dig> marks in testis tissue.
we found that  <dig> peaks were changed ; of these,  <dig> peaks were increased in atz-treated mice, and only  <dig> were reduced.
we confirmed the randomly chosen differential peaks by chip-qpcr  .fig.
<dig> atrazine globally affects the distributions of h3k4me <dig> peaks.
a the distribution of differential h3k4me <dig> peaks within genic and intergenic regions.
the differential peaks were identified as described in the material and methods section; the genic regions are sequences located within genes , while the intergenic regions are places that do not overlap the genic regions.
the majority of differential peaks are located in the genic regions.
b the genome-wide distribution of differential h3k4me <dig> peaks in treated samples: the x-axis shows the chromosome coordinates; the y-axis shows the chromosome number, and the red bars show the positions of the differential peaks on the chromosomes.
the number of peaks per chromosome correlates with chromosome length; the longest, chromosome  <dig>  has the greatest number of differential peaks
among the differential h3k4me <dig> marks,  <dig> were located within coding genes ; this comprised only 54 % of total altered peaks, almost half of which were located in intergenic regions .
<dig>  % of differential peaks are located within tsss.
large numbers of peaks were located far from transcription start sites, suggesting that distally located elements, such as enhancers and ncrna, are affected and may contribute to gene regulation.
we compared the differential peaks to known ncrna maps and found  <dig> peaks overlapping ncrna .
the functions of most ncrnas have yet to be elucidated.
because endocrine function is affected in atz-treated animals, we asked whether h3k4me <dig> peaks changed in the vicinity of steroidogenesis genes or genes regulating steroid-hormone function.
both increased and decreased numbers of h3k4me <dig> sites were found in different parts of the ncoa <dig> gene : h3k4me <dig> marks were reduced at exon  <dig> and increased near the tss region in treated animals ; expression of the latter gene was downregulated.
we found increased h3k4me <dig> marks in cyp19a <dig>  also known as aromatase .
the number of h3k4m <dig> marks was also decreased within the star gene , a finding in agreement with the microarray data .
because star activity is essential for steroid-hormone biosynthesis, decreased h3k4me <dig> marks and star transcription suggest that the function of star in steroid-hormone metabolism is modified in atz-treated animals.fig.
<dig> atz affects the number of h3k4me <dig> marks and mrna levels of genes involved in androgen signaling and testosterone metabolism.
a the differential h3k4me <dig> peaks in the ncoa <dig> gene , b
cyp19a <dig> gene and c
star gene.
the y-axis presents the plotted values of tags at each position after normalizing to all reads in the same dataset ; the x-axis shows the schema of the gene; rectangles are exons, and lines are introns.
note that the differential regions are located within genes.
the h3k4me <dig> profiles from control biological replicates are shown in red, and those from atz-treated mice are in blue.
for illustration, the “integrative genomic viewer”  software version  <dig> .
<dig> was used.
d qpcr analysis of the ncoa <dig>  cyp19a <dig>  and star genes.
qpcr analysis of rna samples was performed in three independent experiments.
primer sequences for each gene are shown in the materials and methods section.
the data are presented as fold change in treated samples compared to controls, *p <  <dig> , **p <  <dig>
subsequently, we asked whether differential h3k4me <dig> regions were located near genes that share common biological functions.
we chose the subset of differential peaks within +/−1 kb genes and performed functional annotation using david software .
we identified seven significantly enriched gene ontology  clusters .table  <dig> the gene ontology  clusters identified by david
the most significant go cluster contained gtpase regulatory activity genes.
most of these genes are related to small ras gtpase and arf gtpase signal transduction regulators.
arf <dig> is required for vesicle transport  <cit>  and mitochondrial homoeostasis  <cit> .
other enriched clusters include protein transport, maintenance of protein localization, cell junctions, and metal-ion binding .
enriched cluster  <dig> contains genes regulating apoptosis and the dna-damage response.
we performed functional annotation of genes located upstream and downstream of ncrna .
we found that differential peaks overlapping ncrna were surrounded by genes encoding proteins involved in adherens junctions and phagocytosis.
sertoli cells are responsible for the phagocytic elimination of apoptotic spermatogenic cells during normal and pathological conditions  <cit>  suggesting that atz may increase the function of phagocytosis.
by using fold change cut-off value ≥ <dig> we found that  <dig> genomic loci have differential h3k4me <dig> marks .
<dig> peaks are located within tss of some genes ,  <dig> peaks are far from tss of any genes, suggesting the marks at distally located promoters, enhancers and non-coding rnas are deregulated.
the differential peaks are located within genomic regions nearby genes of spermatogenesis function ; cell adhesion and motility ; transcription regulation and signaling ; oxidoreductase , cell cycle and cell death .
the analysis confirms that atz damage spermatogenesis function most likely due to metabolism of atz.
our data show that atz changes the number of h3k4me <dig> marks in genes with a wide range of important cellular functions.
comparison of chip-seq and gene-chip data reveal common genes related to gtpase regulation, mitochondrial function and steroid-hormone levels
chip-seq and gene-chip have different sensitivities.
the chip-seq method is very sensitive; it involves integrating a large number of short sequencing reads.
compared to rna sequencing, the gene-chip assay is less sensitive in detecting genes with very low expression and is less accurate in detecting highly expressed genes  <cit> .
to compare the two datasets, we calculated the averaged signal values of chip-seq peaks at the tss of differentially expressed genes.
we found that  <dig> genes were present  in both datasets, while six differential genes identified by gene-chip had no h3k4me <dig> peaks either because they were encoded by mitochondrial dna or because no peaks were identified near the tss.
all differentially expressed gtpase-regulating genes had altered h3k4me <dig> marks, suggesting that the transcription of genes was mediated via epigenetic modifications of histones.
however,  <dig> differential genes had no changes in h3k4me <dig> marks at their tss; the expression of mrna from these genes could be modulated by other mechanisms, such as distally located enhancers, epigenetic mechanisms and ncrna.
thus, atrazine induces epigenetic modifications at the tss of the majority of differentially expressed genes.table  <dig> the gene-chip and chip-seq agreement
acsm2
cox18
dhrs1
gbf1
mrpl27
arfgef1
xpo7
entpd4
g3bp2
gm16677
gm17019
sar1a
ldlr
star
nmt1
psma1
psph
rrn3
gm9758
speer4b
speer4c
speer4e
speer7-ps1
speer8-ps1
tbx18
kif18b
trim45
1700020n15rik
ccdc103
cpsf7
eftud2
eif5
fryl
hdac5
huwe1
mid1
pkd1
psap
smim7
tmem165
9230104l09rik
ccl28
gm5859
klk1b22
lilrb4
nd6
regions showing altered epigenetic marks are enriched at the nr5a2-binding site
to address whether regulatory dna motifs are present within regions containing altered epigenetic marks, we used meme-chip  <cit> , which is designed to discover motifs in large sets of dna sequences.
we analyzed the summits of 100-bp sequences within differential peaks.
parameters and details are described in the materials and methods.
we found a significantly enriched motif among the differential peaks.
we compared this motif with known motifs using tomtom  <cit>  and found that part of the discovered motif sequence was significantly similar to the nr5a2-binding site .
recent work showed that atz activates nr5a receptors via phosphorylation and increases camp production and pi3k signaling  <cit> .
to further understand the involvement of nr5a <dig> in the transcriptional regulation of atz-treated mice, we performed a functional annotation of genes located near presumed nr5a2-binding sites in atz-treated mice.
we found enrichment of genes with enzyme inhibitor activity  and genes encoding proteins involved in the immune response , locomotory behavior  and lipoprotein metabolism  .fig.
<dig> the motif discovered by meme-chip.
the top image is a statistically over-represented sequence within the differential peaks discovered by meme-chip  <cit> .
the bottom image is the consensus sequence within the motif discovered by meme-chip.
this consensus sequence significantly resembles an nr5a2-binding site
our data show that nr5a2-binding sites are significantly enriched in differential peaks in atz-treated testes tissue, suggesting an important role for nr5a <dig> in the transcriptional network of atz- treated mice.
chip-seq reveals deregulation of h3k4me <dig> marks associated with dsbs
we compared the altered distribution of h3k4me <dig> peaks observed in our study to a published dsb-hotspot maps of the c57bl/6 j mouse strain  <cit> .
h3k4me <dig> marks within  <dig> regions were elevated in genomic regions overlapping dsb hotspots .
fifty-seven of these h3k4me <dig> peaks were located within genes.
most of the differential h3k4me <dig> peaks were associated with the strongest dsb hotspots .fig.
<dig> differential peaks are associated with dsb regions.
a the association of differential h3k4me <dig> peaks with the hottest dsb hotspots.
the dmc <dig> tags associated with differential h3k4me <dig> peaks were extracted from published datasets  <cit> , and intensity values were calculated and presented as log on the left plot.
the right plot is the intensity of all dsb hotspots signal from the whole dataset  <cit> .
the median signal of dsb peaks associated with differential h3k4me <dig> peaks was higher than the median signal of all dsbs hotspots, suggesting that differential trimethylation peaks in atz-treated mice are associated with the strongest hotspots.
b the par region in atz-treated mice has a decreased number of h3k4me <dig> marks.
the dsb map was downloaded from geo from a previously published work  <cit> .
plots from controls are in red, plots from treated samples are in blue, and the cpg island is shown in yellow
it is remarkable that h3k4me <dig> marks at the par region of x chromosomes were decreased in atz-treated mice .
this region of par contains a cpg island with  <dig> cpgs.
interesting, altered h3k4me <dig> peaks associated with dsbs were located within very large mouse genes .
erbb <dig> and nrxn <dig> are known to contain common fragile sites   <cit> , which are regions of profound genomic instability  <cit> .
many breakpoints are enriched in at stretches, which affect replication efficiency by forming secondary structures that stall progression of the replication fork  <cit> .
we examined the at content in genes and found increased at content and at stretches .table  <dig> the altered h3k4me <dig> peaks are overlapping with dsb sites in very large genes
erbb4
nrxn1
hs6st3
cntnap5b
hpse2
gmds
dennd1a
our data show that atz treatment affects h3k4me <dig> marks at genomic regions overlapping dsbs regions, including par and very large genes.
discussion
the objective of this study was to investigate the effects of atz in meiosis, which is essential for the production of normal gametes.
we used atrazine for our study because it is widely distributed; it is the most common pollutant in the environment and is still in use in many countries.
mice were treated with doses lower than those used in many other studies but exceeding the authorized limits.
we chose this dose to determine whether it can alter molecular events underlying gametogenesis, although it does not trigger apoptosis or cause obvious changes in testicular morphology.
we treated mice for three weeks to cover the period from entry into meiosis until the diplotene stage of prophase i. specifically, we analyzed meiotic progression, rna expression and the distribution of genome-wide histone h3k4me <dig> marks.
the effects of atz on mitochondria and steroid-hormone metabolism
one of the reasons why atz is toxic to the testis is its effect on mitochondrial function.
our data suggest that the testicular cells of exposed mice suffer from impaired mitochondrial function, a result consistent with the findings of previous studies  <cit> .
rats chronically exposed to atz have swollen mitochondria with disrupted cristae  <cit> .
many studies have demonstrated that the oxidative stress induced by atrazine causes increased activity of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione-s-transferase, increasing the production of ros  <cit> .
we found that exposure of mice to a relatively low dose of atz led to diminished testosterone levels.
decreased testosterone can be explained by impaired cholesterol metabolism and increased activity of aromatase cyp19a <dig>  which converts testosterone to estradiol.
we found an increase in the level of cyp19a <dig> mrna and increased numbers of h3k4me <dig> marks in this gene.
the increase in cyp19a <dig> activity after atz exposure has been demonstrated in many studies  <cit> .
it was suggested that atz activates cyp19a <dig> via nr5a-receptor binding  <cit> .
there are nr5a1-binding sites in the cyp19a <dig> promoter.
however, pezzi et al.
proposed that nr5a <dig> plays an important role in the regulation of cyp19a <dig> expression in leydig cells  <cit> .
we also found a putative binding site for nr5a <dig> within altered h3k4me <dig> peaks in cyp19a <dig>  our data support the hypothesis that nr5a <dig> plays an essential role in activating cyp19a <dig>  it has been suggested that the stimulatory effects of atz on the nr5a receptor subfamily  are mediated by receptor phosphorylation, amplification of camp and pi3k signaling  <cit> .
these authors proposed that hormone networks are altered in many species exposed to atz via the convergence of nr5a activity and camp signaling  <cit> .
we found enrichment of several putative nr5a sites near genes encoding proteins involved in the immune response and lipoprotein metabolism.
recent work has shown that the silencing of nr5a <dig> in colon cancer cell lines affects signal transduction, gtpase activity, cholesterol transport, and apolipoprotein expression  <cit> .
we propose that nr5a <dig> regulates a number of cellular pathways and promotes transcriptional network activation under pathological conditions.
effects of atz exposure on meiosis and recombination
specifically, we found increased numbers of cells in the early stages  of meiosis in atz-treated mice.
we also detected increased numbers of cells with dmc <dig> foci on the sex chromosomes.
chromatin at dsb sites contains a special environment near prdm9-binding motifs, which are nucleosome-depleted  <cit> .
prdm9-modified nucleosomes create an environment for dna repair machinery  <cit> .
we suggest that dsb hotspots serve as sensitive targets of dna-damaging reagents because of their open chromatin structure and enrichment with repeated elements.
we found an increase in h3k4me <dig> at dsb sites associated with strong hotspots, probably reflecting inefficient dsb repair at strong dsb hotspots or repression of hr.
if recombination goes wrong way it will be suppressed by cellular mechanisms.
recently, it was found that the helicase srs <dig> in yeast  <cit> , antirecombinase rtel- <dig> in c. elegans  <cit> , and human pari  <cit>  suppress inappropriate recombination, which is highly toxic to cells as a source of genomic rearrangements.
the disrupting effect of atz is most striking at the sex chromosomes.
recombination at sex chromosomes occurs at 40-mb-long pseudoautosomal regions of x and y-chromosomes.
recombination at the par region differs significantly between sexes: in males, this region has the highest recombination rate  <cit> .
par has a different chromatin structure; par dna occupies unusually long chromosomal axes  <cit> , which may play a role in accessibility to dna damaging factors.
dsb formation and repair at par is a limiting step in meiosis  <cit> .
atz can modulate dsb machinery at par.
the toxic effect of atz on sex chromosomes was previously shown in drosophila, in which it significantly increased x-linked recessive lethality and x or y loss after treatment by larval feeding  <cit> .
it is also important that the h3k4me <dig> peak at par includes a cpg island, a region where dsb repair could be affected due to chromatin silencing and reduced access to dna repair machinery  <cit> .
altered h3k4me <dig> peaks are associated with dsbs in large genes, such as erbb <dig> and nrxn <dig>  within human analogues of these genes, there are sites of breakage.
common fragile sites  are highly conserved between species, and these regions are often enriched in structural variants, including duplications and retrotransposon elements  <cit> .
dna damage at cfs arises due to exogenous factors, including uv and ionizing radiation, chemotherapy and reactive oxygen species  <cit> .
exposure to pesticides results in increased susceptibility of fragile sites to breakage in workers exposed to pesticides  <cit> .
it is possible that we failed to detect a set of dsbs not associated with recombination because a set of dsbs induced by atz is absent from published datasets.
future work will address this possibility.
in summary, our data elucidates the molecular mechanisms underlying atz’s negative effect on meiosis.
importantly, we found a number of effects at the molecular level at a stage in which no morphological changes are detectable.
CONCLUSIONS
in this study, we demonstrated that atz affects meiosis in adult and young prepubertal animals.
atz affects recombination during meiosis and many cellular processes, leading to a decrease in the number of spermatozoa in mice.
in addition, the technical approaches used in this research will facilitate the design of studies aimed at understanding the effects of environmental toxicants on reproduction.

 environmental factors can alter dna methylation and epigenetic modifications in the germline. we studied the effects of the widely used herbicide atrazine on meiosis. in many countries, atz is the most common contaminant detected in rivers. atz residues are even detected in soil and aqueous environments in countries where it has been banned. low levels of atz metabolites in pregnant women are associated with low birth weight. in mammals, atz is metabolized by several groups of xenobiotic-metabolizing enzymes. the metabolism of atz interferes with oxidative phosphorylation and cytochrome p function, resulting in decreased oxygen consumption. atz is known to alter reproductive processes in rodents, reptiles, birds, goats, amphibians and fish. atz targets many tissues, including the testes, ovaries, brain, liver. the effects of atz on reproduction have been extensively studied; however, the effect of atz on meiosis has not been elucidated. meiosis is the division of germ cells and is essential for the production of haploid gametes. in most organisms, homologous recombination is important for the proper segregation of chromosomes during meiosis. hr predominantly occurs in discrete areas of the genome known as recombination hotspots. histone h trimethylation at lysine by prdm methyltransferase is indispensable for the proper formation of double-strand breaks throughout the genome, except in the pseudoautosomal region of x chromosome. germ cells lacking prdm are unable to repair dsbs at the pachytene stage of gametogenesis, and knock-out mice are infertile in both sexes. it has also been shown that environmental pollutants affect hr both in male and female. the first study illustrating the effects of environmental exposure on meiosis was the bisphenol a treatment study, in which the authors showed that embryonic exposure to bpa causes meiotic anomalies, which lead to aneuploidy in adult female mice. importantly, meiotic effects in animals were induced by environmentally relevant doses of bpa. bpa exposure of adult rat delays meiosis initiation and induces accumulation of meiotic dna dsbs in the late meiotic stage. in nematodes bpa exposure also results in impaired chromosome synapsis and disruption of meiotic double-strand break repair progression of c. elegans. we hypothesized that atz may also alter meiosis because it reduces sperm number and changes testis morphology in animals. here, we studied the mechanisms by which atz affects meiosis in mice. we showed that exposure to atz increases testosterone levels and reduces the number of spermatozoa in epididymis, causing a delay of meiosis. using gene-chip and chip-seq analysis of h3k4me marks, we found that a wide range of cellular functions, including gtpase activity, mitochondrial function and steroid-hormone metabolism, are affected by atz. furthermore, histone h3k4me marks are enriched in treated mice in regions of strong recombination within very large genes and reduced at the par region of the x chromosome. we therefore propose that atz disrupts normal meiosis.  experimental design we treated adult male mice with atz at a concentration of 100 mg/l in water for three weeks and followed with two weeks without treatment to cover one full round of spermatogenesis. given that mice drink approximately 5 ml of water per day, we estimate that the average intake of atz was approximately 25 mg/kg/day. this dose is relatively low compared to that used previously. we chose this dose of atz because doses higher than 50 mg/kg/day are known to change the weight of internal organs and are considered as toxic. to study the effect of atz on the first wave of spermatogenesis, we treated newborn animals from postnatal day through day dpp by administering atz suspended in oil to the lactating dams at a dose of 25 mg/kg/day. low levels of atrazine do not affect testicular morphology and cell viability after treatment, the mice were euthanized, and reproductive organs were dissected. we measured the organs and body weights of treated and control animals. we found that the body weight and reproductive organs were not affected at this dose. to evaluate morphological changes in seminiferous tubules caused by atz, we analyzed paraffin-embedded sections stained with hematoxylin and eosin and compared matching stages from treated and control animals. the morphological analysis did not reveal any significant changes in either the seminiferous tubules or the epididymis ). however, we could not exclude small changes in the proportion of cells, which are difficult to distinguish by this method. we verified cell populations by staining with propidium iodide and sorting by facs and we comcluded that cell populations were not significantly affected by this treatment ). to examine whether the number of germ or sertoli cells had changed, we immunostained testicular tissue with antibodies specific for undifferentiated spermatogonial stem cells and sertoli cells ). we found that treatment did not significantly alter the number or proportion of those cell types. to evaluate the rate of apoptosis, we performed tunel assays using the in situ cell-death detection kit. we did not detect significant changes in the rate of apoptosis in adult atz-treated mice compared to control. thus, treatment with a relatively low dose of atrazine does not affect organ weight or the cell populations within seminiferous tubules. decreased sperm count and serum testosterone levels in atz-treated mice one essential metric of reproductive state is the number of spermatozoa per epididymis. to evaluate the effect of atz, we counted spermatozoa in the epididymis of treated and control mice. we found that atz-treated mice had a significantly lower sperm count than untreated animals. to assess endocrine function after treatment, we measured serum levels of testosterone and follicle-stimulating hormone. we found that serum testosterone was times lower in treated animals, while the level of fsh was not significantly affected. our data show that a relatively low dose of atrazine decreases sperm counts and serum testosterone levels.fig. low-dose atz treatment decreases spermatozoa numbers and testosterone levels. a the number of spermatozoa in the epididymis decreased. b the amount of testosterone in the serum decreased; n = 10/group; values are means +/−sd, while fsh was not significantly affected analysis of the synaptonemal complex reveals delayed meiosis we hypothesized that a decrease in sperm count might reflect defects in meiotic progression or spermiogenesis. we thus asked whether atz negatively affects the meiotic stages of spermatogenesis. to analyze meiotic progression in treated mice, we performed surface spreads from testis tissue and analyzed synaptonemal-complex formation. the sc is formed exclusively during prophase of meiosis, and its major role is to bring the chromosomes together. prophase i is subdivided into several sub-stages. we immunostained the surface cell spreads with antibodies against sycp and γh2ax and then classified cells into meiotic substages as previously described,. during the leptotene phase, there is an increase in the number and intensity of γh2ax-positive domains throughout the nucleus. in the zygotene stages, the number of γh2ax domains and the intensity of γh2ax staining decreased, as the staining was concentrated over the chromatin. at the early pachytene stage, γh2ax had almost disappeared from the chromatin of the autosomes, but there were still some visible traces of marks on autosomes, and marks appeared to cover the sex chromosomes. at the late pachytene stage, γh2ax was detected only at the sex chromosomes. the proportions of prophase i substages detected in control mice were similar to those detected by other authors. however, we found that the number of early stage spermatocytes increased in atz-treated animals, while the late stages decreased. in control mice, the largest fraction of spermatocytes was in the late pachytene stage, where γh2ax markers were detectable only in sex chromosomes. in atz-treated animals, the majority of spermatocytes showed patterns of γh2ax staining typical of early stages. we performed quantitative analysis of early pachytene cell fractions in control and treated mice, and we detected three major defects in atz-treated mice: incomplete synapsing of one or two chromosomes, which was often detected together with the formation of branched chromosome structures; sex-body defects. we found that the number of cells with incomplete synapses increased times in treated cells compared to untreated cells, the number of cells with sex-chromosome defects increased times.fig. atrazine causes delayed meiosis in mice. a γh2ax staining changes during meiotic prophase i substages. b the distribution of meiotic substages in control and atz-treated mice; cells from control and treated mice were classified according to γh2ax and sycp staining. note that the number of spermatocytes in the zygotene and early pachytene stages is increased in atz-treated animals, while the number of spermatocytes in the late stages decreased. c surface spreads from control and atz-treated testis stained with anti-γh2ax and anti-sycp antibodies. in control mice, the majority of meiotic cells have γh2ax marks at the sex bodies only. in atz-treated mice, the majority of meiotic cells have γh2ax marks at many chromosomes the performed analysis of immunostaining pattern of γh2ax marks helped us to evaluate the meiotic progression, however, we do not exclude the fact that our observation is not perfect due to the possibility that meiosis in some cells is deregulated and that the stages could not be defined exactly. our analysis is designed to observe the general view of meiotic progress. we complete this finding by analyzing meiotic progress during the first wave of spermatogenesis. taken together, our data show that atz has an effect on meiotic progression and increases the number of synapsing defects. atz treatment delays meiotic progression due to persistence of dsbs we next asked whether the increase in early stage meiotic cells occurred due to the persistence of dsbs breaks. chromosomes undergo dsb formation at the leptotene-zygotene stage. at this stage, a maximal number of dsbs can be detected by staining surface spreads with an antibody against dmc which is a dna-binding protein of dsbs. we found that the number of cells with dmc foci increased with atz treatment. most of the cells positive for dmc in atz-treated animals had foci at the sex chromosomes. the increase in the number of dmc1-positive cells with treatment could be the result of inefficient repair of dsbs in sex chromosomes.fig. the proportions of dmc1- and atr-positive cells increase upon treatment. a spreads from control and atz-treated mice were stained with anti-dmc and anti-sycp. in control mice, the dsbs are repaired at this stage, such that no dmc foci are detectable. in atz-treated samples, several dmc foci are detectable at the pachytene stage. b spreads stained with anti-atr and anti-sycp. in control mice, the atr signal is detected in sex bodies only, while atr staining is detected at several chromosomes in atz-treated mice. c quantitative analysis of atr staining. the spreads were costained with atr and sycp antibodies and classified according to pattern: sex body only and sex body with staining of autosomal chromosomes to further examine synapsing efficiency in atz-treated mice, we stained chromosomes with an antibody against atr, a marker of asynapsed chromosomes and silenced chromatin. atr acts stage-specifically to regulate multiple aspects of mammalian meiotic silencing. if asynapsis persists at the pachytene stage, atr leads to the induction of repressive post-translational modifications and irreversible gene silencing over many megabases. to analyze the role of atr in atz-treated mice, we counted the number of cells that showed staining of atr at the sex body only or that showed simultaneous atr staining of both xy bivalents and autosomes. in atz-treated mice, there was times more atr staining of autosomes. consistent with previous data on the role of atr in chromatin silencing, our experiments showed that atz treatment increases the number of chromosomes with incomplete synapsing. delayed meiotic progression and increased apoptosis in young prepubertal animals to analyze the effect of the atz on meiotic progression during the first wave of spermatogenesis, we treated newborn males with atz. young prepubertal animals have a higher proportion of cells at any given meiotic stage because the first wave of spermatogenesis is generally synchronous. the analysis of the testicular sections of treated animals revealed that the number of diplotene cells decreased times compared to untreated animals, while the numbers of early pachytene and zygotene cells increased times. these data are consistent with the results obtained from treated adult mice showing that atz has a profound effect on meiosis. as the first wave of spermatogenesis involves germ-cell apoptosis to regulate the number of germ cells under normal conditions, we assayed this process following atz treatment. atz treatment of young mice increased the number of apoptotic cells compared to controls. the increased apoptosis rate in young mice is the result of an elevated number of germ cells with meiotic defects, which are eliminated during the first wave of spermatogenesis. thus, atz exposure of young prepubertal mice causes a delay in meiosis similar to that observed in adult males.fig. atrazine delays meiosis in prepubertal mice. h&e straining of histological sections of a control and atz-treated mice. the atz-treated mice have reduced numbers of cells in the seminiferous tubules. c the percentages of seminiferous tubule cross-sections in which preleptotene and leptotene cells, zygotene and early pachytene cells, pachytene cells, diplotene d spermatocytes and round spermatids represent the most advanced types of germ cells in control and atz-treated testes at postnatal day the percentage of empty tubules is also indicated. d tunel assays on testis histological sections from 20-day-old control and atz-treated mice. the results are presented as a percent of tubules containing apoptotic cells, and the data of at least five mice are plotted atz treatment affects the expression of genes involved in mitochondrial function, steroid-hormone function and gtpase activity to assess how atz affects spermatogenesis at the molecular level, we performed a comparative analysis of gene expression using gene-chip analysis. using a fold-change cutoff value of and a p-value cutoff of <, we identified genes that were differentially expressed between atz-treated and control mice. the cellular heterogeneity of seminiferous tubules limits the sensitivity of this approach, as patterns of gene expression may vary by cell type. we confirmed differential expression by qpcr. some differentially expressed genes were transcription factors, and their roles in spermatogenesis have not yet been elucidated. the expression of oxidative phosphorylation-related genes was increased. these genes might be activated due to oxidative stress. oxidative stress is the result of increased production of oxidizing species caused by the metabolism of atz. we confirmed that oxidative phosphorylation gene expression was associated with increased ros by measuring hydrogen peroxide in the serum.fig. atrazine affects mrna transcription and increases production of hydrogen peroxide. a qpcr analysis of rna samples was performed in three independent experiments. qpcr primer sequences for each gene are shown in the materials and methods. the data are presented as fold change in treated samples compared to the controls, *p < , **p <. b hydrogen peroxide levels increase in the serum of treated animals. the measurement of hydrogen peroxide in the serum of at least three independent experiments is shown. the data are presented as average luminescence values, *p <  the transport of cholesterol across the mitochondrial membrane is a key rate-limiting step in the regulation of steroid-hormone biosynthesis. we found decreased levels of mrnas encoding star, a major protein responsible for cholesterol transport. the level of ldlr also decreased, which affects cholesterol metabolism. other target genes included small gtpase regulatory proteins, such as g3bp nmt sar1a, xpo, gbf and arfgef. arf gtpase is also required for the maintenance of mitochondrial functionality and dynamics. we performed the analysis by using fold change cut-off value ≥ the short list includes differentially expressed genes. these are genes, which play role in anti-oxidative stress response, xenobiotic metabolism, cell division, and cell death pathways. gene-chip analysis revealed that atz treatment affects many cellular processes, including steroid-hormone metabolism, oxidative phosphorylation and xenobiotic metabolism. genome-wide sequencing reveals global changes in h3k4me marks following atz treatment h3k4me is a pivotal mark of active chromatin because it acts as a platform for the binding of multiple histone acetyltransferases, histone demethylases and nucleosome-remodeling complexes. as h3k4me marks are essential for meiosis, we asked whether treatment with atz affects these marks. to this end, we analyzed the genome-wide distribution of h3k4me marks in testis tissue. we found that peaks were changed; of these, peaks were increased in atz-treated mice, and only were reduced. we confirmed the randomly chosen differential peaks by chip-qpcr.fig. atrazine globally affects the distributions of h3k4me peaks. a the distribution of differential h3k4me peaks within genic and intergenic regions. the differential peaks were identified as described in the material and methods section; the genic regions are sequences located within genes, while the intergenic regions are places that do not overlap the genic regions. the majority of differential peaks are located in the genic regions. b the genome-wide distribution of differential h3k4me peaks in treated samples: the x-axis shows the chromosome coordinates; the y-axis shows the chromosome number, and the red bars show the positions of the differential peaks on the chromosomes. the number of peaks per chromosome correlates with chromosome length; the longest, chromosome has the greatest number of differential peaks among the differential h3k4me marks, were located within coding genes; this comprised only 54 % of total altered peaks, almost half of which were located in intergenic regions.  % of differential peaks are located within tsss. large numbers of peaks were located far from transcription start sites, suggesting that distally located elements, such as enhancers and ncrna, are affected and may contribute to gene regulation. we compared the differential peaks to known ncrna maps and found peaks overlapping ncrna. the functions of most ncrnas have yet to be elucidated. because endocrine function is affected in atz-treated animals, we asked whether h3k4me peaks changed in the vicinity of steroidogenesis genes or genes regulating steroid-hormone function. both increased and decreased numbers of h3k4me sites were found in different parts of the ncoa gene: h3k4me marks were reduced at exon and increased near the tss region in treated animals; expression of the latter gene was downregulated. we found increased h3k4me marks in cyp19a also known as aromatase. the number of h3k4m marks was also decreased within the star gene, a finding in agreement with the microarray data. because star activity is essential for steroid-hormone biosynthesis, decreased h3k4me marks and star transcription suggest that the function of star in steroid-hormone metabolism is modified in atz-treated animals.fig. atz affects the number of h3k4me marks and mrna levels of genes involved in androgen signaling and testosterone metabolism. a the differential h3k4me peaks in the ncoa gene, b cyp19a gene and c star gene. the y-axis presents the plotted values of tags at each position after normalizing to all reads in the same dataset; the x-axis shows the schema of the gene; rectangles are exons, and lines are introns. note that the differential regions are located within genes. the h3k4me profiles from control biological replicates are shown in red, and those from atz-treated mice are in blue. for illustration, the “integrative genomic viewer” software version. was used. d qpcr analysis of the ncoa cyp19a and star genes. qpcr analysis of rna samples was performed in three independent experiments. primer sequences for each gene are shown in the materials and methods section. the data are presented as fold change in treated samples compared to controls, *p < , **p <  subsequently, we asked whether differential h3k4me regions were located near genes that share common biological functions. we chose the subset of differential peaks within +/−1 kb genes and performed functional annotation using david software. we identified seven significantly enriched gene ontology clusters.table the gene ontology clusters identified by david the most significant go cluster contained gtpase regulatory activity genes. most of these genes are related to small ras gtpase and arf gtpase signal transduction regulators. arf is required for vesicle transport and mitochondrial homoeostasis. other enriched clusters include protein transport, maintenance of protein localization, cell junctions, and metal-ion binding. enriched cluster contains genes regulating apoptosis and the dna-damage response. we performed functional annotation of genes located upstream and downstream of ncrna. we found that differential peaks overlapping ncrna were surrounded by genes encoding proteins involved in adherens junctions and phagocytosis. sertoli cells are responsible for the phagocytic elimination of apoptotic spermatogenic cells during normal and pathological conditions suggesting that atz may increase the function of phagocytosis. by using fold change cut-off value ≥ we found that genomic loci have differential h3k4me marks. peaks are located within tss of some genes, peaks are far from tss of any genes, suggesting the marks at distally located promoters, enhancers and non-coding rnas are deregulated. the differential peaks are located within genomic regions nearby genes of spermatogenesis function; cell adhesion and motility; transcription regulation and signaling; oxidoreductase, cell cycle and cell death. the analysis confirms that atz damage spermatogenesis function most likely due to metabolism of atz. our data show that atz changes the number of h3k4me marks in genes with a wide range of important cellular functions. comparison of chip-seq and gene-chip data reveal common genes related to gtpase regulation, mitochondrial function and steroid-hormone levels chip-seq and gene-chip have different sensitivities. the chip-seq method is very sensitive; it involves integrating a large number of short sequencing reads. compared to rna sequencing, the gene-chip assay is less sensitive in detecting genes with very low expression and is less accurate in detecting highly expressed genes. to compare the two datasets, we calculated the averaged signal values of chip-seq peaks at the tss of differentially expressed genes. we found that genes were present in both datasets, while six differential genes identified by gene-chip had no h3k4me peaks either because they were encoded by mitochondrial dna or because no peaks were identified near the tss. all differentially expressed gtpase-regulating genes had altered h3k4me marks, suggesting that the transcription of genes was mediated via epigenetic modifications of histones. however, differential genes had no changes in h3k4me marks at their tss; the expression of mrna from these genes could be modulated by other mechanisms, such as distally located enhancers, epigenetic mechanisms and ncrna. thus, atrazine induces epigenetic modifications at the tss of the majority of differentially expressed genes.table the gene-chip and chip-seq agreement acsm2 cox18 dhrs1 gbf1 mrpl27 arfgef1 xpo7 entpd4 g3bp2 gm16677 gm17019 sar1a ldlr star nmt1 psma1 psph rrn3 gm9758 speer4b speer4c speer4e speer7-ps1 speer8-ps1 tbx18 kif18b trim45 1700020n15rik ccdc103 cpsf7 eftud2 eif5 fryl hdac5 huwe1 mid1 pkd1 psap smim7 tmem165 9230104l09rik ccl28 gm5859 klk1b22 lilrb4 nd6 regions showing altered epigenetic marks are enriched at the nr5a2-binding site to address whether regulatory dna motifs are present within regions containing altered epigenetic marks, we used meme-chip, which is designed to discover motifs in large sets of dna sequences. we analyzed the summits of 100-bp sequences within differential peaks. parameters and details are described in the materials and methods. we found a significantly enriched motif among the differential peaks. we compared this motif with known motifs using tomtom and found that part of the discovered motif sequence was significantly similar to the nr5a2-binding site. recent work showed that atz activates nr5a receptors via phosphorylation and increases camp production and pi3k signaling. to further understand the involvement of nr5a in the transcriptional regulation of atz-treated mice, we performed a functional annotation of genes located near presumed nr5a2-binding sites in atz-treated mice. we found enrichment of genes with enzyme inhibitor activity and genes encoding proteins involved in the immune response, locomotory behavior and lipoprotein metabolism.fig. the motif discovered by meme-chip. the top image is a statistically over-represented sequence within the differential peaks discovered by meme-chip. the bottom image is the consensus sequence within the motif discovered by meme-chip. this consensus sequence significantly resembles an nr5a2-binding site our data show that nr5a2-binding sites are significantly enriched in differential peaks in atz-treated testes tissue, suggesting an important role for nr5a in the transcriptional network of atz- treated mice. chip-seq reveals deregulation of h3k4me marks associated with dsbs we compared the altered distribution of h3k4me peaks observed in our study to a published dsb-hotspot maps of the c57bl/6 j mouse strain. h3k4me marks within regions were elevated in genomic regions overlapping dsb hotspots. fifty-seven of these h3k4me peaks were located within genes. most of the differential h3k4me peaks were associated with the strongest dsb hotspots.fig. differential peaks are associated with dsb regions. a the association of differential h3k4me peaks with the hottest dsb hotspots. the dmc tags associated with differential h3k4me peaks were extracted from published datasets, and intensity values were calculated and presented as log on the left plot. the right plot is the intensity of all dsb hotspots signal from the whole dataset. the median signal of dsb peaks associated with differential h3k4me peaks was higher than the median signal of all dsbs hotspots, suggesting that differential trimethylation peaks in atz-treated mice are associated with the strongest hotspots. b the par region in atz-treated mice has a decreased number of h3k4me marks. the dsb map was downloaded from geo from a previously published work. plots from controls are in red, plots from treated samples are in blue, and the cpg island is shown in yellow it is remarkable that h3k4me marks at the par region of x chromosomes were decreased in atz-treated mice. this region of par contains a cpg island with cpgs. interesting, altered h3k4me peaks associated with dsbs were located within very large mouse genes. erbb and nrxn are known to contain common fragile sites, which are regions of profound genomic instability. many breakpoints are enriched in at stretches, which affect replication efficiency by forming secondary structures that stall progression of the replication fork. we examined the at content in genes and found increased at content and at stretches.table the altered h3k4me peaks are overlapping with dsb sites in very large genes erbb4 nrxn1 hs6st3 cntnap5b hpse2 gmds dennd1a our data show that atz treatment affects h3k4me marks at genomic regions overlapping dsbs regions, including par and very large genes. discussion the objective of this study was to investigate the effects of atz in meiosis, which is essential for the production of normal gametes. we used atrazine for our study because it is widely distributed; it is the most common pollutant in the environment and is still in use in many countries. mice were treated with doses lower than those used in many other studies but exceeding the authorized limits. we chose this dose to determine whether it can alter molecular events underlying gametogenesis, although it does not trigger apoptosis or cause obvious changes in testicular morphology. we treated mice for three weeks to cover the period from entry into meiosis until the diplotene stage of prophase i. specifically, we analyzed meiotic progression, rna expression and the distribution of genome-wide histone h3k4me marks. the effects of atz on mitochondria and steroid-hormone metabolism one of the reasons why atz is toxic to the testis is its effect on mitochondrial function. our data suggest that the testicular cells of exposed mice suffer from impaired mitochondrial function, a result consistent with the findings of previous studies. rats chronically exposed to atz have swollen mitochondria with disrupted cristae. many studies have demonstrated that the oxidative stress induced by atrazine causes increased activity of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, and glutathione-s-transferase, increasing the production of ros. we found that exposure of mice to a relatively low dose of atz led to diminished testosterone levels. decreased testosterone can be explained by impaired cholesterol metabolism and increased activity of aromatase cyp19a which converts testosterone to estradiol. we found an increase in the level of cyp19a mrna and increased numbers of h3k4me marks in this gene. the increase in cyp19a activity after atz exposure has been demonstrated in many studies. it was suggested that atz activates cyp19a via nr5a-receptor binding. there are nr5a1-binding sites in the cyp19a promoter. however, pezzi et al. proposed that nr5a plays an important role in the regulation of cyp19a expression in leydig cells. we also found a putative binding site for nr5a within altered h3k4me peaks in cyp19a our data support the hypothesis that nr5a plays an essential role in activating cyp19a it has been suggested that the stimulatory effects of atz on the nr5a receptor subfamily are mediated by receptor phosphorylation, amplification of camp and pi3k signaling. these authors proposed that hormone networks are altered in many species exposed to atz via the convergence of nr5a activity and camp signaling. we found enrichment of several putative nr5a sites near genes encoding proteins involved in the immune response and lipoprotein metabolism. recent work has shown that the silencing of nr5a in colon cancer cell lines affects signal transduction, gtpase activity, cholesterol transport, and apolipoprotein expression. we propose that nr5a regulates a number of cellular pathways and promotes transcriptional network activation under pathological conditions. effects of atz exposure on meiosis and recombination specifically, we found increased numbers of cells in the early stages of meiosis in atz-treated mice. we also detected increased numbers of cells with dmc foci on the sex chromosomes. chromatin at dsb sites contains a special environment near prdm9-binding motifs, which are nucleosome-depleted. prdm9-modified nucleosomes create an environment for dna repair machinery. we suggest that dsb hotspots serve as sensitive targets of dna-damaging reagents because of their open chromatin structure and enrichment with repeated elements. we found an increase in h3k4me at dsb sites associated with strong hotspots, probably reflecting inefficient dsb repair at strong dsb hotspots or repression of hr. if recombination goes wrong way it will be suppressed by cellular mechanisms. recently, it was found that the helicase srs in yeast, antirecombinase rtel- in c. elegans, and human pari suppress inappropriate recombination, which is highly toxic to cells as a source of genomic rearrangements. the disrupting effect of atz is most striking at the sex chromosomes. recombination at sex chromosomes occurs at 40-mb-long pseudoautosomal regions of x and y-chromosomes. recombination at the par region differs significantly between sexes: in males, this region has the highest recombination rate. par has a different chromatin structure; par dna occupies unusually long chromosomal axes, which may play a role in accessibility to dna damaging factors. dsb formation and repair at par is a limiting step in meiosis. atz can modulate dsb machinery at par. the toxic effect of atz on sex chromosomes was previously shown in drosophila, in which it significantly increased x-linked recessive lethality and x or y loss after treatment by larval feeding. it is also important that the h3k4me peak at par includes a cpg island, a region where dsb repair could be affected due to chromatin silencing and reduced access to dna repair machinery. altered h3k4me peaks are associated with dsbs in large genes, such as erbb and nrxn within human analogues of these genes, there are sites of breakage. common fragile sites  are highly conserved between species, and these regions are often enriched in structural variants, including duplications and retrotransposon elements. dna damage at cfs arises due to exogenous factors, including uv and ionizing radiation, chemotherapy and reactive oxygen species. exposure to pesticides results in increased susceptibility of fragile sites to breakage in workers exposed to pesticides. it is possible that we failed to detect a set of dsbs not associated with recombination because a set of dsbs induced by atz is absent from published datasets. future work will address this possibility. in summary, our data elucidates the molecular mechanisms underlying atz’s negative effect on meiosis. importantly, we found a number of effects at the molecular level at a stage in which no morphological changes are detectable.  in this study, we demonstrated that atz affects meiosis in adult and young prepubertal animals. atz affects recombination during meiosis and many cellular processes, leading to a decrease in the number of spermatozoa in mice. in addition, the technical approaches used in this research will facilitate the design of studies aimed at understanding the effects of environmental toxicants on reproduction. 
furthermore, treated mice display enriched histone h3k4me marks in regions of strong recombination, within very large genes and reduced marks in the pseudoautosomal region of x chromosome. rt-qpcr was used to validate differentially expressed genes or differential peaks. environmental factors such as pesticides can cause phenotypic changes in various organisms, including mammals. genome-wide mapping of h3k4me marks distribution was done by chip-sequencing of testis tissue using illumina technologies. using gene-chip and chip-seq analysis of h3k4me marks, we found that a broad range of cellular functions, including gtpase activity, mitochondrial function and steroid-hormone metabolism, are affected by atz. we studied the effects of the widely used herbicide atrazine on meiosis, a key step of gametogenesis, in male mice. our data demonstrate that atrazine exposure interferes with normal meiosis, which affects spermatozoa production.
 environmental factors such as pesticides can cause phenotypic changes in various organisms, including mammals. we studied the effects of the widely used herbicide atrazine on meiosis, a key step of gametogenesis, in male mice. methods gene expression pattern was analysed by gene–chip array. genome-wide mapping of h3k4me marks distribution was done by chip-sequencing of testis tissue using illumina technologies. rt-qpcr was used to validate differentially expressed genes or differential peaks.  we demonstrate that exposure to atz reduces testosterone levels and the number of spermatozoa in the epididymis and delays meiosis. using gene-chip and chip-seq analysis of h3k4me marks, we found that a broad range of cellular functions, including gtpase activity, mitochondrial function and steroid-hormone metabolism, are affected by atz. furthermore, treated mice display enriched histone h3k4me marks in regions of strong recombination, within very large genes and reduced marks in the pseudoautosomal region of x chromosome.  our data demonstrate that atrazine exposure interferes with normal meiosis, which affects spermatozoa production. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords meiosisdouble-strand breaksgene-chipchip-seqh3k4me3atrazineatip-avenir 2013r13139nssmagulova fatima http://dx.doi.org/ /501100006459université européenne de bretagne irset chaire d'excellencesmagulova fatima http://dx.doi.org/ /501100004543china scholarship council issue-copyright-statement© the author 2015 