Sex-specific transgenerational effects on murine thyroid gland imposed by ancestral exposure to neonicotinoid thiacloprid.

Neonicotinoids, a relatively new widely used class of insecticide is used in agriculture to control insect populations. We examined the capacity of ancestral exposure to the neonicotinoid thiacloprid (thia) to induce transgenerational effects on thyroid tissue. Pregnant outbred Swiss female mice were exposed to thia at embryonic days E6.5 to E15.5 using 0, 0.6, and 6 mg/kg/day doses. Thyroid paraffin sections were prepared for morphology analysis. We apply ELISA method to measure T4 and TSH levels, RT-qPCR for gene expression analysis, ChIP-qPCR techniques for sperm histone H3K4me3 analysis, and immunofluorescence microscopy and western blots for protein detection. We observed an alteration in the morphology of thyroids in both males and females in the F3 generation. We observed an increase in T4 hormone in F1 females and a significant T4 level decrease in F3 males. T4 changes in F1 females were associated with a TSH increase. We found that the amount of Iodothyronine Deiodinase 1 (DIO1) (an enzyme converting T4 to T3) was decreased in both F1 and F3 generations in female thyroids. GNAS protein which is important for thyroid function has increased in female thyroids. Gene expression analysis showed that the expression of genes encoding thyroid gland development, chromatin, biosynthesis and transport factors were affected in the thyroid gland in both sexes in F1 and F3. The analysis of sperm histone H3K4me3 showed that H3K4me3 occupancy at the Dio1 locus has decreased while Thyroglobulin (Tg) and Matrix Metallopeptidase 2 (Mmp2) genes have increased H3K4me3 occupancy in the sperm of F3 mice. Besides, DNA methylation analysis of our previously published datasets showed that, in the sperm of F1 and F3 thia-derived mice, several genes related to thyroid function show consistent alterations. Our data suggest that ancestral exposure to thiacloprid affects thyroid function not only in exposed but also in indirectly exposed F3 generation.

Transgenerational epigenetic effects imposed by neonicotinoid thiacloprid exposure.

Neonicotinoids are a widely used class of insecticides that are being applied in agricultural fields. We examined the capacity of a neonicotinoid, thiacloprid (thia), to induce transgenerational effects in male mice. Pregnant outbred Swiss female mice were exposed to thia at embryonic days E6.5-E15.5 using different doses. Testis sections were used for morphology analysis, ELISAs for testosterone level analysis, RT-qPCR and RNA-seq for gene expression analysis, MEDIP-seq and MEDIP-qPCR techniques for DNA methylation analysis, and Western blot for a protein analysis. The number of meiotic double-strand breaks and the number of incomplete synapsed chromosomes were higher in the thia 6-treated group of F3 males. Genome-wide analysis of DNA methylation in spermatozoa revealed that differentially methylated regions were found in all three generations at the promoters of germ cell reprogramming responsive genes and many superenhancers that are normally active in embryonic stem cells, testis, and brain. DNA methylation changes induced by thia exposure during embryonic period are preserved through several generations at important master regulator regions.

Inactivation of Exosc10 in the oocyte impairs oocyte development and maturation, leading to a depletion of the ovarian reserve in mice.

EXOSC10 is a catalytic subunit of the nuclear RNA exosome, and possesses a 3'-5' exoribonuclease activity. The enzyme processes and degrades different classes of RNAs. To delineate the role of EXOSC10 during oocyte growth, specific Exosc10 inactivation was performed in oocytes from the primordial follicle stage onward using the Gdf9-iCre; Exosc10 f/- mouse model (Exosc10 cKO(Gdf9)). Exosc10 cKO(Gdf9) female mice are infertile. The onset of puberty and the estrus cycle in mutants are initially normal and ovaries contain all follicle classes. By the age of eight weeks, vaginal smears reveal irregular estrus cycles and mutant ovaries are completely depleted of follicles. Mutant oocytes retrieved from the oviduct are degenerated, and occasionally show an enlarged polar body, which may reflect a defective first meiotic division. Under fertilization conditions, the mutant oocytes do not enter into an embryonic development process. Furthermore, we conducted a comparative proteome analysis of wild type and Exosc10 knockout mouse ovaries, and identified EXOSC10-dependent proteins involved in many biological processes, such as meiotic cell cycle progression and oocyte maturation. Our results unambiguously demonstrate an essential role for EXOSC10 in oogenesis and may serve as a model for primary ovarian insufficiency in humans. Data are available via ProteomeXchange with identifier PXD039417.

EXOSC10/Rrp6 is essential for the eight-cell embryo/morula transition.

The conserved 3'-5' exoribonuclease EXOSC10/Rrp6 is required for gametogenesis, brain development, erythropoiesis and blood cell enhancer function. The human ortholog is essential for mitosis in cultured cancer cells. Little is known, however, about the role of Exosc10 during embryo development and organogenesis. We generated an Exosc10 knockout model and find that Exosc10-/- mice show an embryonic lethal phenotype. We demonstrate that Exosc10 maternal wild type mRNA is present in mutant oocytes and that the gene is expressed during all stages of early embryogenesis. Furthermore, we observe that EXOSC10 early on localizes to the periphery of nucleolus precursor bodies in blastomeres, which is in keeping with the protein's role in rRNA processing and may indicate a function in the establishment of chromatin domains during initial stages of embryogenesis. Finally, we infer from genotyping data for embryonic days e7.5, e6.5 and e4.5 and embryos cultured in vitro that Exosc10-/- mutants arrest at the eight-cell embryo/morula transition. Our results demonstrate a novel essential role for Exosc10 during early embryogenesis, and they are consistent with earlier work showing that impaired ribosome biogenesis causes a developmental arrest at the morula stage.

Epigenetic Effects Promoted by Neonicotinoid Thiacloprid Exposure.

BACKGROUND: Neonicotinoids, a widely used class of insecticide, have attracted much attention because of their widespread use that has resulted in the decline of the bee population. Accumulating evidence suggests potential animal and human exposure to neonicotinoids, which is a cause of public concern. OBJECTIVES: In this study, we examined the effects of a neonicotinoid, thiacloprid (thia), on the male reproductive system. METHODS: The pregnant outbred Swiss female mice were exposed to thia at embryonic days E6.5 to E15.5 using "0," "0.06," "0.6," and "6" mg/kg/day doses. Adult male progeny was analyzed for morphological and cytological defects in the testes using hematoxylin and eosin (H&E) staining. We also used immunofluorescence, Western blotting, RT-qPCR and RNA-seq techniques for the analyses of the effects of thia on testis. RESULTS: We found that exposure to thia causes a decrease in spermatozoa at doses "0.6" and "6" and leads to telomere defects at all tested doses. At doses "0.6" and "6," thia exposure leads to an increase in meiotic pachytene cells and a decrease in lumen size, these changes were accompanied by increased testis-to-body weight ratios at high dose. By using RNA-seq approach we found that genes encoding translation, ATP production, ATP-dependent proteins and chromatin-modifying enzymes were deregulated in testes. In addition, we found that exposure to thia results in a decrease in H3K9me3 levels in spermatocytes. The changes in H3K9me3 were associated with a dramatic increase in activity of retroelements. CONCLUSION: Our study suggests that gestational exposure to thia affects epigenetic mechanisms controlling meiosis which could lead to deleterious effects on male spermatogenesis.

Developmental exposure to chlordecone induces transgenerational effects in somatic prostate tissue which are associated with epigenetic histone trimethylation changes.

BACKGROUND: Chlordecone (CD), also known as Kepone, is an organochlorine insecticide that has been used in banana crops in the French West Indies. Due to long-term contamination of soils and water, the population is still exposed to CD. Exposure to CD in adulthood is associated with an increased risk of prostate cancer (PCa). OBJECTIVES: We examined the transgenerational effects of CD on murine prostate tissue. METHODS: We exposed pregnant Swiss mice to CD. The prostates from directly exposed (F1) and non-exposed (F3) male progeny were analyzed. We used immunofluorescence, RNA-seq and ChIP-seq techniques for the comprehensive analyses of chromatin states in prostate. RESULTS: We observed an increased prostatic intraepithelial neoplasia phenotype (PIN) in both F1 and F3 generations. Transcriptomic analysis in CD-derived F1 and F3 prostate using RNA-seq revealed that 970 genes in F1 and 218 in F3 genes were differentially expressed. The differentially expressed genes in both datasets could be clustered accordingly to common biological processes, "cell differentiation", "developmental process", "regulating of signaling", suggesting that in both generations similar processes were perturbed. We detected that in both datasets several Hox genes were upregulated; in F1, the expression was detected mainly in Hoxb and Hoxd, and in F3, in Hoxa family genes. Using a larger number of biological replicates and RT-qPCR we showed that genes implicated in testosterone synthesis (Akr1b3, Cyp11a1, Cyp17a1, Srd5a1) were dramatically upregulated in PIN samples; Cyp19a1, converting testosterone to estradiol was elevated as well. We found a dramatic increase in Esr2 expression both in F1 and F3 prostates containing PIN. The PIN-containing samples have a strong increase in expression of self-renewal-related genes (Nanog, Tbx3, Sox2, Sox3, Rb1). We observed changes in liver, F1 CD-exposed males have an increased expression of genes related to DNA repair, matrix collagen and inflammation related pathways in F1 but not in F3 adult CD-derived liver. The changes in RNA transcription were associated with epigenetic changes. Specifically, we found a global increase in H3K4 trimethylation (H3K4me3) and a decrease in H3K27 trimethylation (H3K27me3) in prostate of F1 mice. ChIP-seq analysis showed that 129 regions in F1 and 240 in F3 acquired altered H3K4me3 occupancy in CD-derived prostate, including highest increase at several promoters of Hoxa family genes in both datasets. The alteration in H3K4me3 in both generations overlap 73 genes including genes involved in proliferation regulation, Tbx2, Stat3, Stat5a, Pou2f3 and homeobox genes Hoxa13, Hoxa9. CONCLUSIONS: Our data suggest that developmental exposure to CD leads to epigenetic changes in prostate tissue. The PIN containing samples showed evidence of implication in hormonal pathway and self-renewal gene expression that have the capacity to promote neoplasia in CD-exposed mice.

Ovarian dysfunction following prenatal exposure to an insecticide, chlordecone, associates with altered epigenetic features.

Chlordecone (CD) is an insecticide that was used in the French West Indies for several years to control the banana root borer pest. Given its nonsignificant degradation, it persists in the environment. CD is a carcinogenic compound with reproductive and developmental toxicity and is a recognized endocrine-disrupting chemical. In this study, we examined the effects of CD on female reproductive system of mice with the focus on epigenetic features in ovary. Our data show that gestational exposure to low dose of CD affects meiotic double-strand breaks repair in female embryos. In adult mice derived from CD-treated pregnant females, we observed delayed puberty, decreased number of primordial and increased number of atretic follicles. Gene expression analysis revealed that Rcbtb2 and Rbpms genes were not expressed in embryonic gonads. Estrogen signaling- and oocyte maturation-associated genes were downregulated in adult ovaries. The morphological changes were associated with altered epigenetic features: increased H2Aub and increased H3K27me3 and decreased H4ac and H3K4me3 in embryonic oocytes. The DNA damage-associated, γH2AX marks were detected in the follicles of treated but not control adult ovaries. We also found reduced H3K4me3 and H4ac in fully grown oocytes of the treated ovaries. The ChIP-seq analysis of H3K4me3 in adult ovaries showed that target genes of ZFP57 and TRIM28, which regulate pluripotency and imprinting, were significantly enriched in altered regions. Our study clearly demonstrates that gestational exposure to a low dose of CD impairs the function of female reproductive system and the changes are associated with altered epigenetic features.

Perinatal Exposure to Glyphosate and a Glyphosate-Based Herbicide Affect Spermatogenesis in Mice.

Glyphosate is the most widely used herbicide in the world. Several studies have investigated the effects of glyphosate and glyphosate-based herbicides (GBHs) on male reproduction, but there is still little and conflicting evidence for its toxicity. In this study, we analyzed the effects of glyphosate, alone or in formula, on the male reproductive system. Pregnant mice were treated from E10.5 to 20 days postpartum by adding glyphosate or a GBH (Roundup 3 Plus) to their drinking water at 0.5 (the acceptable daily intake, ADI dose), 5 and 50 mg/kg/day. Male offspring derived from treated mice were sacrificed at 5, 20, and 35 days old (d.o.) and 8 months old (m.o.) for analysis. Our result showed that exposure to glyphosate, but not GBH, affects testis morphology in 20 d.o. and decrease serum testosterone concentrations in 35 d.o. males. We identified that the spermatozoa number decreased by 89% and 84% in 0.5 and 5 mg/kg/day of GBH and glyphosate groups, respectively. Moreover, the undifferentiated spermatogonia numbers were decreased by 60% in 5 mg/kg/day glyphosate group, which could be due to the alterations in the expression of genes involved in germ cell differentiation such as Sall4 and Nano3 and apoptosis as Bax and Bcl2. In 8 m.o. animals, a decreased testosterone level was observed in GBH groups. Our data demonstrate that glyphosate and GBHs could cause endocrine-disrupting effects on male reproduction at low doses. As glyphosate has effects at the ADI level, our data suggest that the current ADI for glyphosate could be overestimated.

Embryonic exposure to the widely-used herbicide atrazine disrupts meiosis and normal follicle formation in female mice.

The widely-used herbicide atrazine (ATZ) is detected in ground and surface water in many countries. Several studies in animals have demonstrated that ATZ has endocrine-disrupting effects on male and female reproduction in many vertebrate species. In this study, we investigated the effects of ATZ exposure on meiosis, a key step in gametogenesis in mammals. The treatment was initiated before oocyte entry into meiosis, which occurs during the embryonic period in females. We found that embryonic exposure to ATZ increases the level of 8-oxo-guanine in the nucleus of meiotic cells, reflecting oxidative stress and affecting meiotic double-strand break repair, chromosome synapsis and crossover numbers. Finally, embryonic exposure to ATZ reduces the number of primordial follicles and increases the incidence of multi-oocyte follicles in adult mice. Our data demonstrate that embryonic exposure to ATZ disrupts prophase I of meiosis and affects normal follicle formation in female mice.