Mehmet Uzumcu

Epigenetic Transgenerational Actions of Endocrine Disruptors and Male Fertility

Transgenerational effects of environmental toxins require either a chromosomal or epigenetic alteration in the germ line. Transient exposure of a gestating female rat during the period of gonadal sex determination to the endocrine disruptors vinclozolin (an antiandrogenic compound) or methoxychlor (an estrogenic compound) induced an adult phenotype in the F1 generation of decreased spermatogenic capacity (cell number and viability) and increased incidence of male infertility. These effects were transferred through the male germ line to nearly all males of all subsequent generations examined (that is, F1 to F4). The effects on reproduction correlate with altered DNA methylation patterns in the germ line. The ability of an environmental factor (for example, endocrine disruptor) to reprogram the germ line and to promote a transgenerational disease state has significant implications for evolutionary biology and disease etiology.

Developmental Methoxychlor Exposure Affects Multiple Reproductive Parameters and Ovarian: Folliculogenesis and Gene Expression in Adult Rats

Methoxychlor (MXC) is an organochlorine pesticide with estrogenic, anti-estrogenic, and anti-androgenic properties. To investigate whether transient developmental exposure to MXC could cause adult ovarian dysfunction, we exposed Fischer rats to 20 microg/kg/day (low dose; environmentally relevant dose) or 100 mg/kg/day (high dose) MXC between 19 days post coitum and postnatal day 7. Multiple reproductive parameters, serum hormone levels, and ovarian morphology and molecular markers were examined from prepubertal through adult stages. High dose MXC accelerated pubertal onset and first estrus, reduced litter size, and increased irregular cyclicity (P<0.05). MXC reduced superovulatory response to exogenous gonadotropins in prepubertal females (P<0.05). Rats exposed to high dose MXC had increasing irregular estrous cyclicity beginning at 4 months of age, with all animals showing abnormal cycles by 6 months. High dose MXC reduced serum progesterone, but increased luteinizing hormone (LH). Follicular composition analysis revealed an increase in the percentage of preantral and early antral follicles and a reduction in the percentage of corpora lutea in high dose MXC-treated ovaries (P<0.05). Immunohistochemical staining and quantification of the staining intensity showed that estrogen receptor beta was reduced by high dose MXC while anti-Mullerian hormone was upregulated by both low- and high dose MXC in preantral and early antral follicles (P<0.05). High dose MXC significantly reduced LH receptor expression in large antral follicles (P<0.01), and down-regulated cytochrome P450 side-chain cleavage. These results demonstrated that developmental MXC exposure results in reduced ovulation and fertility and premature aging, possibly by altering ovarian gene expression and folliculogenesis.

Early Life Exposure to Endocrine-Disrupting Chemicals Causes Lifelong Molecular Reprogramming of the Hypothalamus and Premature Reproductive Aging

Gestational exposure to the estrogenic endocrine disruptor methoxychlor (MXC) disrupts the female reproductive system at the molecular, physiological, and behavioral levels in adulthood. The current study addressed whether perinatal exposure to endocrine disruptors re-programs expression of a suite of genes expressed in the hypothalamus that control reproductive function and related these molecular changes to premature reproductive aging. Fischer rats were exposed daily for 12 consecutive days to vehicle (dimethylsulfoxide), estradiol benzoate (EB) (1 mg/kg), and MXC (low dose, 20 μg/kg or high dose, 100 mg/kg), beginning on embryonic d 19 through postnatal d 7. The perinatally exposed females were aged to 16-17 months and monitored for reproductive senescence. After euthanasia, hypothalamic regions [preoptic area (POA) and medial basal hypothalamus] were dissected for real-time PCR of gene expression or pyrosequencing to assess DNA methylation of the Esr1 gene. Using a 48-gene PCR platform, two genes (Kiss1 and Esr1) were significantly different in the POA of endocrine-disrupting chemical-exposed rats compared with vehicle-exposed rats after Bonferroni correction. Fifteen POA genes were up-regulated by at least 50% in EB or high-dose MXC compared with vehicle. To understand the epigenetic basis of the increased Esr1 gene expression, we performed bisulfite conversion and pyrosequencing of the Esr1 promoter. EB-treated rats had significantly higher percentage of methylation at three CpG sites in the Esr1 promoter compared with control rats. Together with these molecular effects, perinatal MXC and EB altered estrous cyclicity and advanced reproductive senescence. Thus, early life exposure to endocrine disruptors has lifelong effects on neuroendocrine gene expression and DNA methylation, together with causing the advancement of reproductive senescence.

Epigenetic effects of endocrine-disrupting chemicals on female reproduction: An ovarian perspective

The link between in utero and neonatal exposure to environmental toxicants, such as endocrine-disrupting chemicals (EDCs) and adult female reproductive disorders is well established in both epidemiological and animal studies. Recent studies examining the epigenetic mechanisms involved in mediating the effects of EDCs on female reproduction are gathering momentum. In this review, we describe the developmental processes that are susceptible to EDC exposures in female reproductive system, with a special emphasis on the ovary. We discuss studies with select EDCs that have been shown to have physiological and correlated epigenetic effects in the ovary, neuroendocrine system, and uterus. Importantly, EDCs that can directly target the ovary can alter epigenetic mechanisms in the oocyte, leading to transgenerational epigenetic effects. The potential mechanisms involved in such effects are also discussed.

Fetal and neonatal exposure to the endocrine disruptor methoxychlor causes epigenetic alterations in adult ovarian genes.

Exposure to endocrine-disrupting chemicals during development could alter the epigenetic programming of the genome and result in adult-onset disease. Methoxychlor (MXC) and its metabolites possess estrogenic, antiestrogenic, and antiandrogenic activities. Previous studies showed that fetal/neonatal exposure to MXC caused adult ovarian dysfunction due to altered expression of key ovarian genes including estrogen receptor (ER)-beta, which was down-regulated, whereas ERalpha was unaffected. The objective of the current study was to evaluate changes in global and gene-specific methylation patterns in adult ovaries associated with the observed defects. Rats were exposed to MXC (20 microg/kgxd or 100 mg/kg.d) between embryonic d 19 and postnatal d 7. We performed DNA methylation analysis of the known promoters of ERalpha and ERbeta genes in postnatal d 50-60 ovaries using bisulfite sequencing and methylation-specific PCRs. Developmental exposure to MXC led to significant hypermethylation in the ERbeta promoter regions (P < 0.05), whereas the ERalpha promoter was unaffected. We assessed global DNA methylation changes using methylation-sensitive arbitrarily primed PCR and identified 10 genes that were hypermethylated in ovaries from exposed rats. To determine whether the MXC-induced methylation changes were associated with increased DNA methyltransferase (DNMT) levels, we measured the expression levels of Dnmt3a, Dnmt3b, and Dnmt3l using semiquantitative RT-PCR. Whereas Dnmt3a and Dnmt3l were unchanged, Dnmt3b expression was stimulated in ovaries of the 100 mg/kg MXC group (P < 0.05), suggesting that increased DNMT3B may cause DNA hypermethylation in the ovary. Overall, these data suggest that transient exposure to MXC during fetal and neonatal development affects adult ovarian function via altered methylation patterns.

Effect of the methoxychlor metabolite HPTE on the rat ovarian granulosa cell transcriptome in vitro.

Ovarian granulosa cells play a central role in steroidogenesis, which is critical for female reproduction. Follicle-stimulating hormone (FSH) promotes cyclic adenosine monophosphate (cAMP)-mediated signaling to regulate granulosa cell steroidogenesis. We have shown previously that 2,2-bis-(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) inhibits FSH- and dibutyryl cAMP-stimulated steroidogenesis and affects the messenger RNA levels of steroidogenic pathway enzymes in rat granulosa cells. However, HPTE showed a differential effect in FSH- and cAMP-stimulated cells in that HPTE more completely blocked FSH- when compared to cAMP-driven steroidogenesis. The objective of this study was to analyze the effects of HPTE on global gene expression profiles in untreated granulosa cells and those challenged with FSH or cAMP. Granulosa cells from immature rats were cultured with 0, 1, 5, or 10 microM HPTE in the presence or absence of either 3 ng FSH/ml or 1mM cAMP for 48 h. Total RNA was isolated for real-time quantitative PCR and microarray analysis using the GeneChip Rat Genome 230 2.0 and ArrayAssist Microarray Suite. An investigation of changes in gene expression across all HPTE treatments showed that HPTE altered more genes in FSH- (approximately 670 genes) than in cAMP-stimulated cells (approximately 366 genes). Analysis confirmed that HPTE more effectively inhibited FSH- than cAMP-induced steroid pathway gene expression and steroidogenesis. Furthermore, expression patterns of novel genes regulating signal transduction, transport, cell cycle, adhesion, differentiation, motility and growth, apoptosis, development, and metabolism were all altered by HPTE. This study further established that HPTE exerts differential effects within the granulosa cell steroidogenic pathway and revealed that these effects include broader changes in gene expression.

The hepatocyte growth factor system as a regulator of female and male gonadal function

The hepatocyte growth factor (HGF) system comprises HGF, its receptor (the c-met tyrosine kinase), HGF activator (HGFA) protein, and HGFA inhibitor (HAI). The components of the HGF system have been identified in a plethora of tissues to include the ovary and testis. In its traditional context, the HGF system works via paracrine- and autocrine-mediated feedback in which HGF (of mesenchymal origin) binds and activates c-met (within epithelial cells); target cells then respond to HGF via any number of morphogenic and functional changes. The concomitant presence of HGFA and HAI suggests that HGF bioactivity can be locally modulated. A number of studies have collectively shown that the mammalian ovary and testis contain HGF, c-met, and HGFA; very little is currently known regarding HAI within the gonad. Within the ovary, HGF controls numerous key functions which collectively regulate the growth and differentiation of ovarian follicles; these include cell growth, steroidogenesis, and apoptosis within theca cells and/or granulosa cells. Comparatively, less is known about the function of HGF within the testicular Leydig and Sertoli cells, but evidence is emerging that HGF may regulate somatic cell function, including Leydig cell steroidogenesis. Changes in the cellular origin of HGF and c-met during fetal and postnatal testicular development suggest that HGF, in collaboration with other growth factors, may regulate important aspects of testicular cell morphogenesis and differentiation which enable male sexual viability. Likewise, experimental evidence showing that HGF can modulate many vital processes which enable ovarian follicle growth, differentiation, and function indicate the importance of HGF in female reproduction. This review presents what is currently known regarding the expression of the HGF system and its function within the ovary and testis.

Epigenetic mechanisms in the actions of endocrine-disrupting chemicals: gonadal effects and role in female reproduction.

There is a heightened interest and concern among scientists, clinicians and regulatory agencies as well as the general public, regarding the effects of environmental endocrine-disrupting chemicals (EDCs). In this review, we identify the main epigenetic mechanisms and describe key ovarian processes that are vulnerable to the epigenetic actions of EDCs. We also provide an overview of the human epidemiological evidence documenting the detrimental effects of several common environmental EDCs on female reproduction. We then focus on experimental evidence demonstrating the epigenetic effects of these EDCs in the ovary and female reproductive system, with an emphasis on methoxychlor, an organochlorine pesticide. We conclude the review by describing several critical issues in studying epigenetic effects of EDCs in the ovary, including transgenerational epigenetic effects.

Targeted Genome-Wide Methylation and Gene Expression Analyses Reveal Signaling Pathways Involved in Ovarian Dysfunction after Developmental EDC Exposure in Rats

ABSTRACT Transient exposure to methoxychlor (MXC), an environmental endocrine-disrupting chemical, during fetal and neonatal stages causes ovarian dysfunction in pubertal, adult, and aging animals. Adult animals have reduced number of ovulations and abnormal follicular composition associated with altered gene expression and DNA methylation patterns. To test the hypothesis that the ovarian epigenomic changes induced by MXC are detectable following the exposure period, leading to altered gene expression by adulthood, we conducted a targeted genome-wide methylation study using Nimblegen 3x720K CpG Island Plus RefSeq Promoter Arrays. Control (vehicle), low-dose MXC (20 μg/kg/day), or high-dose MXC (100 mg/kg/day) treatments were administered between Embryonic Day 19 and Postnatal Day (PND) 7. Ovaries were collected at PND 7 immediately after exposure or at adulthood, PND 60. Array hybridizations were conducted with genomic DNA after methylated DNA immunoprecipitation and the array data were analyzed. DNA methylation events were functionally annotated, and candidate loci common to all the treatments or unique to some treatments were identified. Specific loci encoding signaling molecules such as the regulatory subunit p85 of phosphoinositide-3-kinase, insulin-like growth factor-1 receptor, Harvey rat sarcoma viral oncogene, insulin receptor, and forkhead box protein O3 were identified to be hypermethylated in MXC-treated ovaries at PND 7 and/or PND 60. Examination of gene expression changes with TaqMan low-density arrays revealed that nearly 25% of the genes that were assayed were downregulated. These data demonstrate that key molecules in specific signaling pathways such as PTEN signaling, IGF-1 signaling, or rapid estrogen signaling are epigenetically altered in MXC-exposed ovaries, which is associated with ovarian dysfunction and female infertility.

Long-term effects of early-life exposure to environmental oestrogens on ovarian function: role of epigenetics.

Oestrogens play an important role in development and function of the brain and reproductive tract. Accordingly, it is considered that developmental exposure to environmental oestrogens can disrupt neural and reproductive tract development, potentially resulting in long‐term alterations in neurobehaviour and reproductive function. Many chemicals have been shown to have oestrogenic activity, whereas others affect oestrogen production and turnover, resulting in the disruption of oestrogen signalling pathways. However, these mechanisms and the concentrations required to induce these effects cannot account for the myriad adverse effects of environmental toxicants on oestrogen‐sensitive target tissues. Hence, alternative mechanisms are assumed to underlie the adverse effects documented in experimental animal models and thus could be important to human health. In this review, the epigenetic regulation of gene expression is explored as a potential target of environmental toxicants including oestrogenic chemicals. We suggest that toxicant‐induced changes in epigenetic signatures are important mechanisms underlying the disruption of ovarian follicular development. In addition, we discuss how exposure to environmental oestrogens during early life can alter gene expression through effects on epigenetic control potentially leading to permanent changes in ovarian physiology.