Long-term effects of early life exposure to environmental estrogens on ovarian function: Role of epigenetics

Abstract

Estrogens play an important role in development and function of the brain and reproductive tract. Accordingly, it is thought that developmental exposure to environmental estrogens can disrupt neural and reproductive tract development potentially resulting in long-term alterations in neurobehavior and reproductive function. Many chemicals have been shown to have estrogenic activity whereas others affect estrogen production and turnover resulting in disruption of estrogen signaling pathways. However, these mechanisms and the concentrations required to induce these effects cannot account for the myriad adverse effects of environmental toxicants on estrogen sensitive target tissues. Hence, alternative mechanisms are thought 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 estrogenic chemicals. We suggest that toxicant-induced changes in epigenetic signatures are important mechanisms underlying disruption of ovarian follicular development. In addition, we discuss how exposure to environmental estrogens during early life can alter gene expression through effects on epigenetic control potentially leading to permanent changes in ovarian physiology.

1) Introduction

The gonadal steroid estradiol (E₂) plays a central role in neurogenesis, synaptogenesis, regulation of neurotrophin expression, and sexual differentiation of the brain. Recent evidence suggests that the estrogenic activity of environmental contaminants is also associated with adverse effects on neurodevelopment and neurobehavior (1–6). The developing brain undergoes phases of cell neurogenesis, proliferation, differentiation, density, migration, and death; the brain also expresses high levels of estrogen receptors. While it has been shown that sexual dimorphism exists in brain development, it is clear that regardless of gender, the actions of estrogens on the developing brain are typically permanent and range from establishing sex differences to trophic and neuroprotective effects. Gonadal estrogens affect target organs throughout the body including the brain. Estrogens can also act as local autocrine or paracrine signals that impact only the microenvironment, including various regions of the brain. The fetal environment is replete with estrogens originating in the maternal circulation and placenta as well as from direct synthesis in fetal neurons. All of these parameters occur to varying degrees, but depending on the response to estrogenic (or androgenic) signals, the cells are either protected from naturally occurring apoptosis or induced to undergo cell death (7).

Estrogens both promote and prevent synaptogenesis in the developing brain. Estrogens are involved in establishment of synaptic patterns that endure into adulthood and subserve the neural networks of steroid-regulated physiology and behavior. Estrogens can suppress the formation of dendritic spine synapse in the arcuate nucleus. Estrogens increase levels of the amino acid transmitter GABA by up-regulating the rate-limiting enzyme glutamic acid decarboxylase. GABA acts on GABA-A receptors of astrocytes and induces process growth and branching, resulting in an increased stellate morphology (8) compared to those not exposed to estrogens. On the other hand, estrogen exposure in the preoptic and ventromedial hypothalamus promotes the development and stabilization of dendritic spine synapses which involve glutamate and ionotropic glutamate receptors (9). Because estrogens are an important aspect of neurodevelopment with multifaceted signaling mechanisms that not only influence sex differentiation but a myriad of unique and opposing effects in different regions of the brain at different times during development, there is a potential for both direct but also indirect (via ovarian function) effects of environmental estrogens.

Exposure to endocrine disruptors during fetal and early postnatal life produces different long-term alterations on various brain areas. For example, neonatal exposure to bisphenol A (BPA) decreased the inhibition of GnRH activity by RFamide-related peptide-3 (RFRP3) neurons. This effect may be related with the advanced puberty observed in these animals (10). On the other hand, the perinatal exposure to estradiol benzoate (EB) or methoxychlor (MXC) produced an early reproductive senescence in rats which is related with developmental reprogramming of hypothalamic genes. Specifically, expression of Esr1 and Kiss1 genes were altered in this animal model (11). Other brain areas not related to reproduction could be affected by early exposure to endocrine disruptors. For example, exposure to estradiol valerate (EV) at postnatal day (PND) 1 in rats produced an increase in dopamine content in striatum, substantia nigra, and ventral tegmental area when rats were adult. In addition, these rats lacked of amphetamine induced locomotor activity in the adulthood, possibly because of a decrease of dopamine transporter (DAT), the molecular target of amphetamine (12).

As in the brain, exposure to environmental estrogens produces adverse effects in other estrogen-sensitive tissues including the uterus (13) and the ovaries (14–17). Human exposure to environmental contaminants has been widely documented through large scale biomonitoring (18–24) and epidemiological studies (25–29). While several different environmental contaminants have been measured in human ovarian follicular fluid (28–31), the impact on circulating E₂ concentrations and associated health consequences are largely unknown. However, evidence from studies with wildlife suggests that chemicals in the environment can adversely affect ovarian biology (32, 33) across the lifespan.

The ovary is composed of follicles at different stages of development and stroma. Follicles at their advance stages are composed of the theca externa and interna (androgen production), a basement membrane, granulosa cells (sites of E₂ and anti-Müllerian hormone (AMH) synthesis) and an oocyte. Folliculogenesis (follicle recruitment and growth to ovulation) is regulated in a stage-dependent manner by oocyte derived factors (e.g. bone morphogenetic protein-15 (BMP-15), growth differentiation factor-9 (GDF-9)), gonadotropins (follicle stimulating hormone (FSH) and luteinizing hormone (LH) in early antral and Graafian follicles) and several growth regulators (e.g. transforming growth factor-β (TGF-β), AMH, inhibin-B, activin, vascular endothelial growth factor, FOXL2, and insulin like growth factors and binding proteins (reviewed in (34)). Follicles are recruited into the growing pool of follicles where their initial stages of development are gonadotropin independent. From the time follicles are formed in utero (or early postnatal in case of rodents), follicles begin to develop but in the absence of FSH stimulation they undergo atresia with much of the follicle population lost before regular menstrual/estrous cycles commence with the onset of puberty. Once regular reproductive cycles are established follicles reach the secondary stage where FSH support from the pituitary provides the trigger to begin steroidogenesis. Steroid production in the ovary involves a two-cell process in which androgens produced in theca cells are transported to the granulosa where they are converted by aromatase to E₂ or estrone. Tissue culture studies demonstrate that environmental contaminants can both increase the expression of steroidogenic enzymes such as steroidogenic acute regulatory protein (StAR) and aromatase (35, 36), the rate limiting enzyme in the conversion of androgens to estrogens (estrone and E₂), as well as increase the expression of enzymes involved in the metabolism of gonadal steroids (37, 38). Furthermore, animal studies have demonstrated that exposure to chemicals with hormone-like activity change functional characteristics of steroid-dependent target tissues (reviewed in (39)).

Studies performed in animal models illustrate potential effects of early exposure to estrogenic compounds on ovarian development and function. The specific processes that are most vulnerable to estrogenic compounds are (i) follicular formation (also known as follicular assembly), (ii) follicular growth, and (iii) follicular maturation and ovulation.

(i) Follicular formation/follicular assembly

In mice, primordial germ cells (PGCs) arrive in the ovary and divide by mitosis between PND10.5 and PND13.5. These cells named oogonia enter meiosis I by PND 13.5 and form the oocytes. Then oocytes progress until prophase I and arrest in the diplotene stage. During maturation, oocytes are disposed in clusters and are surrounded by somatic cells forming germ cell cysts (also named oocyte nests). From gestational day 17.5 until PND 5 this germ cell nest breakdown, many of the oocytes undergo programmed cell death and remaining oocytes are surrounded by pre-granulosa cells. This process, known as follicular formation or follicular assembly, sets the primordial pool of follicles (40, 41). A large list of factors, including neurotrophins, growth factors and transcription factors participate in the follicular assembly (40). Exposure to E₂ or estrogenic compounds in newborn rodents inhibits and delays the follicular assembly (42, 43) and is associated with abnormally assembled follicles, containing more than one oocyte per follicle (multi-oocyte follicles; MOF) (44–49). Compounds with estrogenic activity have been demonstrated to decrease the number of primordial follicles (14, 15, 46, 50) potentially shortening reproductive lifespan.

(ii) Follicular growth

Once formed most primordial follicles remain quiescent to be recruited throughout the reproductive life although some immediately start growing prior to puberty. After female reach puberty, group of follicles are recruited in cohort to grow up until early antral stage, independently of central control as mentioned earlier. Neonatal exposure to estrogenic chemicals, such as BPA (51), MXC (17, 52), and EV (53) has been linked to reduced growth and development of primary, secondary and antral follicles. Since, early stages of follicular development do not depend upon gonadotropin influence; the ovary could be a direct target of the disruption by estrogenic compounds.

(iii) Follicular maturation and ovulation

Developmental exposure to estrogenic compounds could interfere with the final maturation of follicles. Thus, follicles, instead of completing their final maturation to ovulate, they form follicular cysts in the ovaries (16, 51, 54, 55). Therefore, environmental estrogens are thought to be potentially important in the pathogenesis of polycystic ovary syndrome (56, 57). This could result from a direct effect in the ovary as well as indirectly at the level of brain under the influence of estrogenic chemicals. The evidence for the disruptive effects of estrogenic compounds on the differentiation of brain in female has long been known (58–61). Evidence for direct effects of environmental estrogens in the ovary exists. For example, prepubertal rats that were exposed to estrogenic chemicals (e.g., MXC) during fetal and neonatal developmental periods ovulate fewer eggs in response to exogenous gonadotropins (52), suggesting reduced gonadotropin responsiveness in the ovary.

Although exposure to environmental estrogenic compounds and their effects on human reproductive function remains uncertain, there is a growing literature demonstrating adverse effects of environmental chemicals in animal models as well as wildlife species (reviewed in (62)). However, owing to inherent limitations of animal models and the assumption that human exposure to environmental chemicals are frequently thought to be too low to induce adverse health effects in humans, analogous to those documented in animal models, alternative mechanisms are sought to explore the links between exposure and adverse effects documented in epidemiological studies. There is a growing concern that exposure to environmental estrogens during early development can produce irreversible long-term programming of physiology including adverse effects on reproductive physiology and development (Summarized in Table 1). Recent research has advanced the knowledge of mechanisms that could explain how exposure to estrogens during fetal and neonatal development produces long-term alterations in the function of different organs. In this context, we review the epigenetic mechanisms of gene expression that regulate ovarian development and function and provide examples of epigenetic reprogramming that could explain long-term alterations in ovarian function produced by exposure to estrogenic compounds during development.

Table 1.

Endocrine Disrupting Chemicals (EDCs): Mechanism, Source and Effects on Ovarian Function.

Compound/Potential Targets/Source	Does of exposure/species	Main Effects	References
Bisphenol-A (BPA) ESR Plastics industry	1, 10 and 100 μg/ml in vitro, mice cultured antral follicles	Inhibits follicular growth, induces atresia, and inhibits steroidogenesis and expression of steroidogenic enzymes in antral follicles from adult FVB, C57BL/6, and CD-1mice.	127
	500 μg per pup PND1-10 daily rat sc injection	Alters the hypothalamic-pituitary-gonadal axis in female Sprague-Dawley rats and induces development of follicular cysts in the ovary, leading to infertility. Increases testosterone and estradiol levels and decreases progesterone.	51
	20 μg/kg PND7-14 daily mice hypodermic injection	Inhibits methylation of imprinted genes during oogenesis via the ER signaling pathway, affecting the development of oocytes and folliculogenesis.	106
	50 mg or 50 μg/kg PC14-E9.5 or PC14-E12.5 daily mice diet	Disturbances in tissue-specific DNA methylation and gene expression in imprinted loci, leading to abnormalities in placental development.	78
	0.5 mg/kg E30-90 daily ewe sc injection	Decreases miRNAs targeting SOX family genes (sex determination and embryonic development), kit ligand (follicular assembly establishment of reserve pool of primordial follicles) and insulin related genes (follicular development and steroid production).	121
	150 μg/pup PND 1-5 daily mice sc injection	Induces polyovular follicles or multi-oocyte follicles (having more than one oocyte in a follicle).	47
	0.05 and 20 mg/kg PND 1, 3, 5, and 7 rats sc injection	Reduces primordial follicles and increased growing follicles. Primordial and recruited showed increased ERβ and proliferation in recruited follicles.	15
	50 ug/kg and 50 mg/kg PND 1 rats sc injection	Produces early pubertal onset. Large antral-like follicles and follicular cysts, lower numbers of corpora lutea and acyclicity.	54
	2, 20, and 200 μg/kg E1-19 daily mice orally	Dose-dependent, brain region-specific and sex-specific effects of BPA on gene expression of ERs, ERα, ERβ and estrogen-related receptor-γ that varied across brain regions, and changes DNA methyltransferase (DNMT) 1 and DNMT3A in the juvenile cortex and hypothalamus. Changes in behavior: hyperactive phenotype in males and hypoactive phenotype in females.	104
	4.4, 44, and 440 uM in vitro cultured antral follicles PND 32 mice	BPA (440 μM) inhibits follicle growth. BPA (44 and 440 uM) inhibit progesterone, dehydroepiandrosterone, androstenedione, estrone, testosterone, and estradiol production.	115
	3 nM and 300 nM in vitro cultured Metaphase II oocytes mice	Exposure to 3 nM BPA was associated with slightly accelerated follicle development, increased allele methylation errors in differentially methylated regions of maternally imprinted genes, and decreased histone H3K9 trimethylation and interkinetochore distance.	124
	10 and 100 ug per pup PND1-5 daily mice sc injection	Blocks cyst breakdown and rise polyovular follicles and primordial follicles.	46
Methoxychlor (MXC) ESR1 ESR2 AR Pesticide	50, 100, or 500 mg/kg PND3-10 daily rats sc injection	Inhibits early ovarian development and stimulates AMH production directly in the ovary.	17
	100 mg/kg E19 to PND7 daily rats ip or sc injection	Induces hypermethylation of the promoter region of Esr2 in the adult ovary with a decrease in the ESR2 protein and induces Dnmt3b expression in the ovary.	101
	0.5 or 1mg per pup PND 1-14 daily mice ip injection	Produces ovarian atrophy, relative ovarian weight loss, depletion of corpora lutea and follicular cysts in 0.5 mg doses	55
	20 μg/kg and 100 mg/kg E19 to PND7 daily rats ip or sc injection	Hypermethylation and/or downregulation in: regulatory subunit p85 of phosphoinositide-3-kinase, insulin-like growth factor-1 receptor, Harvey rat sarcoma viral oncogene, insulin receptor, and FOXO3 in the ovary.	102
	20 μg/kg and 100 mg/kg E19 to PND7 daily rats ip or sc injection	Reduces ovulation and induces early puberty, irregular cyclicity, sub-fertility, and premature reproductive aging.	52
Di(2-ethylhexyl) phthalate (DEHP) ESR Androgen production Plastic Industry medical devices building products containing polyvinyl chloride	20 and 40 μg/kg PND7-14 daily mice hypodermic injection	Decreases number of the primordial follicles at pubertal and adult age. Decreases methylation of maternal imprinted genes Igf2 and Peg3 in oocytes, which leads to abnormalities in oocyte and embryo development.	50
Genistein ESR Phytoestrogen (soy)	1 nM to 100 uM in vitro cultured ovaries for 7 days PND1 mice	Impairs formation of primordial follicular pool in the ovary.	42
	10 and 100 μg/pup PND1-5 daily mice sc injection	Increases number of multi-oocyte follicles.	48
Estradiol valerate (EV) and Estradiol Benzoate (EB) ESR Synthetic estradiol	10 mg/kg PND1, 7 and 14 rats sc injection	Reduces follicular growth and maturation in the ovary, decreases steroidogenesis, disrupts estrous cyclicity, and impairs reproductive performance.	53
	5 mg/kg PND 1-14 daily mice ip injection	Induces multi-oocyte follicles in adult ovaries.	42
	10 μg/kg PN1-34 day rats sc injection	Advances vaginal opening, and induces multi-oocyte follicles in the ovary.	49
	0,1 mg per pup PND1 sc injection	Increased ovarian expression of nerve growth factor (Ngfb) and p75 low-affinity neurotrophic receptor (Ngfr) mRNAs. Early vaginal opening, disrupted cyclicity, appearance of follicular cyst, absence of corpus luteum, infertility and reduced number of primordial follicles	16
	25 ug per pup PND 1 rats sc injection	Early pubertal onset. Ovaries undersized and absence of folliculogenesis. Absense of sexual receptivity after ovariectomy and hormone replacement.	54
Diethylstilbestrol (DES) ESR Synthetic estrogen	0, 0.75, 1.25, 2.5, or 25ug per pup PND1-5 daily Rats sc injection	Dose-dependent increase in miR-29a, miR-29b, and miR-29c expression, this resulted in a decrease in DNMT1, DNMT3a, and DNMT3b expression in testes	105
	5 μg/kg PND 1-14 daily lambs sc injection	Decline in the stock of primordial follicles, increase in number of multi-oocyte follicles and increase in the number of antral atretic follicles	14
	0.2 and 20 ug/kg PND 1,3,5 and 7 rats sc injection	Produce a decrease in primordial follicles and increased growing follicles. Increase the incidences of multi-oocyte follicles.	15
	1 ug per pup PND1-5 daily mice sc injection	Produces a higher incidence in polyovular follicles and the oocytes from polyovular follicles have a smaller capacity for fertilization.	44
	3 ug per pup PND1 and 2 mice sc injection	Induces appearance of polyovular follicles.	45
	10 and 100 ug per pup PND1-5 daily mice sc injection	Blocks the cyst breakdown and rises polyovular follicles and primordial follicles.	46

AMH, anti-Mullerian hormone; E, embryonic day; PND, postnatal day; PC, pre-coital day.

2) Epigenetic mechanisms and their roles in ovarian development and function

The term epigenetics was first coined by Conrad Waddington in 1942 to describe “the branch of biology which studies the causal interactions between genes and their products which bring the phenotype into being.” (63). Today, epigenetics is commonly defined as the study of long-lived but reversible DNA modifications that can change gene expression without changing the DNA sequence (64). There are three major epigenetic mechanisms: (i) DNA methylation, which refers to methylation of cytosine in CpG dinucleotides and is generally associated with a down regulation of gene expression; (ii) histone modifications, which refers to post-translational modifications of various amino acid moieties in histone core proteins and include acetylation and methylation of lysines. Some of these modifications (permissive histone marks) render the regulatory regions of genes more accessible to binding of transcription factors and increase gene expression while some (suppressive histone marks) renders it less accessible and decrease gene expression, and (iii) non-coding RNA (ncRNAs), refer to functional RNAs that do not encode proteins, but generally inhibit the expression of other genes. Non-coding RNAs are classified according to their sizes: microRNAs (miRNAs), which are comprised of 21–25 nucleotides; small RNAs, 100–200 nucleotides; and long ncRNAs > 200–10,000 nucleotides. All three mechanisms collectively and cooperatively modify chromatin structures and regulate gene expression (for reviews (64–69)). The present discussion will focus on the specific mechanisms that regulate ovarian development and function, including germ cell differentiation and epigenetic reprogramming (Figure 2). The role of epigenetic mechanisms in the development and function of the ovary can be viewed from two perspectives: (a) germ cell epigenetic reprogramming; and (b) follicular formation and development.

Figure 2. Developmental EDC exposure and potential epigenetic mechanisms in altered brain and ovarian function.

The normal ovarian development and function are strictly regulated by precise epigenetic mechanisms (DNA methylation, histone modifications, and miRNA) that coordinate the spatio-temporal expression of key genes. These genes are transcription factors, cell cycle proteins, growth factors, hormone synthesis enzymes and receptors, which participate in different stages of development of the follicles. Exposure to environmental estrogens alters these mechanisms of control, affecting gene expression in the ovary and leading to dysfunction and disease (See section 3).

2.1. Germ cell epigenetic reprogramming

During mammalian development, two major epigenetic reprogramming events occur: one in the preimplantation embryo and the other is in PGCs. Epigenetic programming in preimplantation embryo refers to the global erasure of DNA methylation patterns in the maternal and paternal genomes in all loci except imprinted genes and tandem repeats such as intracisternal A particles (IAPs) (70), and the reestablishment of new DNA methylation patterns according to the cell lineage fate (71). Although epigenetic modifications continue as the cells and tissues further differentiate, these modifications are limited and remain in the trajectory that is initially established.

However, PGCs is an exception, and they undergo a global epigenetic reprogramming at later stage of embryonic development. Specifically, around embryonic (E) day 7.5 in mice, PGCs begin to differentiate within the embryo proper (72), and their initial DNA methylation profile resembles that of the embryonal cells from which they differentiate (71). The PGCs gain germ cell-specific expression patterns between E8.5 and E11.5, during which they migrate and colonize the bipotential gonad, an embryonic structure that differentiates into the testis or ovary depending on the genetic sex. Gonadal sex determines the fate of PGCs, which differentiate into either female or male germ cells. Germ cells in both sexes proliferate rapidly until E13–14, when male germ cells enter mitotic arrest and female germ cells enter meiosis and arrest at meiosis I (73, 74). Between E8.5 and E12.5, the genome of PGCs, including the imprinted loci, undergoes a major demethylation process. While the timing of the demethylation process in male and female germs cells is the same, the remethylation timelines are sex-specific. Remethylation in male PGCs takes place in utero several days after erasure between E14.5 and E16.5 and is mostly complete by birth (75, 76). In contrast, female germ cell remethylation begins postnatally (PND 1 – 5) and continues throughout the growth of the oocyte. In addition, imprinting patterns are established based on the size of the oocyte rather than the age of the animal (77). Thus the evidence suggests that as each follicle is recruited into the growing follicular pool, the imprinting patterns of the associated oocyte are established. Any exposure to environmental chemicals such as BPA during imprinting establishment can disrupt the imprinting patterns and the resultant embryo suffers from altered gene expression at these loci and developmental abnormalities, especially in the placenta (78).

DNA methylation and the enzymatic machinery, namely de novo DNA methyltransferases (DNMTs) that establish DNA methylation patterns play major roles in genomic imprinting (65). Therefore, gonadal expression levels of DNMTs closely follow the timing of the establishment of the imprinting marks (79). Some imprinted loci also involve histone marks (80) and non-coding RNAs (81). During germ cell epigenetic reprogramming, besides imprinted loci, some repeat sequences (e.g., LINE1) lose their methylation marks and are de novo remethylated along with imprinted sequences (82). However, sequences with high mutagenic potential such as IAPs are protected from demethylation, which are believed to mediate transgenerational epigenetic inheritance (83).

2.2. Follicle formation and development

The main somatic cell types that are associated directly with follicles (granulosa and thecal cells) are regulated by various epigenetic mechanisms, including DNA methylation (84), histone modifications (85), and non-coding RNA (86) throughout different follicular stages. Steroidogenic factor 1 (Sf1), which is expressed in granulosa cells in the embryonic gonad (87), is regulated by promoter DNA methylation at the earliest stage of gonadal differentiation. While the Sf1 promoter in the embryonic ovary had no or minimal DNA methylation, it is highly methylated in embryonic liver or kidney, which reflects the expression patterns of the gene in these organs (88). It is likely that theca cell differentiation also involves epigenetic mechanisms, but relatively little is known regarding the initial cell specification of thecal cell precursors (89).

In postnatal and adult ovary, epigenetic mechanisms continue to play roles in differentiation of these two somatic cells. In granulosa cell, down-regulation of inhibin α during luteinization is closely regulated by histone modifications as well as DNA methylation in the promoter region of this gene (90). In contrast, up-regulation of steroidogenic acute regulatory protein (StAR) and down-regulation of aromatase (Cyp19a1) following gonadotropin treatment do not involve DNA methylation but are regulated primarily by histone modifications (91). In thecal, cells, when the levels of permissive histone marks are pharmacologically elevated at the promoter region in cytochrome P450 17A1 (CYP17A1), gene expression increases leading to an increase in androgen production (92). In contrast, in bovine thecal cells, the LH surge reduces chromatin accessibility of the CYP17A1 gene and its mRNA levels (93). In addition, patterns of epigenetic marks are determined by the cell types within the follicle as well as the follicular stage. While DNA methylation is not involved in preovulatory down-regulation of CYP19A1 in the follicles, it plays a major role in the permanent silencing of CYP19A1 in the corpus luteum (94). The roles of miRNAs in various aspects of ovarian development and function, including, gonadal differentiation (95), follicle formation (96), selection, maturation (97), ovulation (98), and luteinization (99) as well as atresia (100), have also been shown.

In summary, the main epigenetic events as relate to the ovary that are likely to be vulnerable to adverse effects of environmental factors, including endocrine disruptors are (i) the epigenetic reprogramming of female germ cells and establishment of maternal imprinting patterns, and (ii) initial differentiation and continuous adaptation of somatic components of ovarian follicles to the various stages of folliculogenesis and corpus luteum function. Experimental evidence supporting these potential epigenetic events is provided in Section 3. In addition, it is important to note that, the epigenetic reprogramming of the early embryo is also vulnerable to influence of environmental factors including endocrine disruptors. The consequence of this influence not only would be on the ovary and female germ cells, but it would also be on the entire developing organism ((78) and references within).

3) Effects of environmental estrogenic compounds on epigenetic regulation of gene expression in the ovary and its relation with ovarian function and development

Emerging evidence suggests changes in epigenetic control of gene expression as a potential mechanism underlying long-term and irreversible alterations in ovarian physiology arising from developmental exposure to endocrine disruptors (62, 101, 102). Owing to the central role of estrogen receptors (ESR) in the ovary during development (103), cells of ovarian follicles are thought to be potential targets of disruption by endogenous or environmental estrogens. Exposure to environmental chemicals with hormone-like activity in early life, such as MXC, BPA, EB and di(2-ethylhexyl) phthalate (DEHP) produces changes in the expression of DNMT in different tissues (50, 101, 104–106). DNMT can affect the patterns of methylation of promoter regions of genes usually causing a repression of gene expression. Methylation of CpG island or promoter regions decreases the accessibility of transcription factors to the binding sites in the DNA. If the binding of a stimulating transcription factor to the DNA is blocked the gene down-regulates. Conversely, if the binding of a transcription repressor is blocked the gene up-regulates. Ovarian exposure to MXC, BPA, and DEHP has been shown to alter the expression of DNMT resulting in potential alterations to the epigenome (50, 101, 106). The ovary is regulated by a myriad of transcription factors (107) which in turn regulate the expression of genes related to hormone action, extrinsic innervation, and paracrine growth factors. All these regulators act coordinately to promote follicular development, ovulation and ovarian hormone production. As may be predicted, changes in DNMT can deregulate these highly coordinated inputs and alter ovarian physiology. Despite this, the mechanism which explains how an exposure in early life produce a change in the expression of genes that can persist until adulthood, in the absence of the stimuli, remains unknown.

Long-term ovarian alterations produced by different environmental signals during fetal and early postnatal development could result from an adaptive or maladaptive response of the body to the environmental signal. In this context, it was shown that exposure to chemicals with hormone-like activity in early life leads to long-term alterations in expression of critical genes, such as ESR2, in the ovary. One such chemical is chlorinated organic pesticide MXC. Although it is currently de-registered by the EPA in the U.S., MXC is an excellent model endocrine disruptor since its major metabolites (i.e., HPTE and mono-OH-MXC) act as ESR1 agonists as well as ESR2 and androgen receptor antagonists (108). These activities represent many hormone-like chemicals in the environment. Exposure to MXC during fetal and neonatal development produces hypermethylation of the promoter region of Esr2 in the adult ovary with a decrease in the ESR2 protein (101). This decrease in ESR2 could produce a lower response to endogenous E₂ in adulthood and as a consequence, an alteration in follicular development. Direct effects of E₂ which favor cell proliferation and follicular development have been demonstrated (109); however, much of the paracrine regulators of folliculogenesis and its receptors have estrogen response elements (EREs). The expression of these paracrine factors can be affected by this permanently altered response to E₂ through the estrous cycle. For example, AMH which is produced by granulosa cells of growing pre-antral follicles has EREs and is increased in adulthood after exposure to MXC or BPA in early life (17, 110). E₂ may have a repressive effect on Amh expression and the high expression of Amh could be the result of lower response to E₂ during the estrous cycle in adulthood. Alternatively, exogenous estrogens may stimulate Amh expression through the ERE leading to an increase in AMH (111). AMH has a repressive effect on early follicular development and primordial follicle recruitment as well as FSH-induced follicular maturation (112–114) which is consistent with the findings of a lower number of developing follicles in different models of estrogenic disruption (16, 17, 53, 115).

Other genes subject to epigenetic control are important in regulation of ovarian function and are permanently modified by developmental exposure to estrogenic compounds (102) including insulin growth factor-1 receptor (IGF-1R). IGF-1R agonism leads to activation of PI3K/AKT and MAPK pathways which is related to a protection of cells from Fas-L induced apoptosis and stimulation of granulosa cell proliferation (116, 117). IGF-1R is down-regulated in adult rats exposed to MXC in the neonatal period. This is consistent with the phenotype of MXC-treated animals, which showed reduced follicular development (17). Interestingly, IGF-1 secretion is influenced by FSH (118) and hence some of the effects of FSH on follicular development are mediated by IGF1. In this context, the lower expression of IGF1-R in MXC treated rats could produce a lower response of the ovary to the effects of FSH. Sympathetic innervation of the ovary is also thought to be important in ovarian regulation (119). Norepinephrine activates the β-2 adrenoreceptor and increase expression of FSHR favoring follicular development (120). β-2 adrenoreceptor gene is hypermethylated after early exposure to MXC (102). Other genes of intracellular proteins important for cell signaling pathways such as Pik3r1, Prkar2a, Pak3, and Foxo3a, are hypermethylated in the adult rat ovary after early exposure to MXC (102). On the other hand, the exposure to DEHP decreases methylation of maternal imprinted genes Igf2 and Peg3 in oocytes, which leads to abnormalities in oocyte and embryo development (50). In the same way, BPA is also known to alter methylation pattern of Igf2 and Peg3 in oocytes affecting the development of oocytes and folliculogenesis (106). In addition, daily exposure to 10 mg BPA per kg bw from 2 weeks prior mating to E9.5 or E12.5, which corresponds to germ cell and early embryonic epigenetic reprogramming, produces a tissue-specific DNA methylation and gene expression disturbances in imprinted loci (Snrpn, Ube3a, Igf2, Kcnq1ot1), leading to placental abnormalities at E12.5 in mice. Daily exposure to 10 μg BPA per kg bw, a dose considered to be safe for humans, was also effective in causing alteration at some of these loci. Importantly, effects of both doses were stage-dependent: A daily exposure between E5.5 and E12.5 had no observable effect (78).

Exposure to environmental estrogens during pregnancy can also modulate ovarian miRNA expression in the fetus (121). Exposure to BPA in sheep produces a decrease in miRNAs targeting Sry-related high-mobility-group box (SOX) family genes, kit ligand, and insulin-related genes (121). SOX genes are implicated in sex determination and embryonic development (122), while Kit ligand signaling is important for follicular assembly and the correct establishment of the reserve pool of primordial follicles (123). A silencing of Kit ligand by miRNA may result in an abnormal follicular assembly and could explain defects observed in animals exposed to environmental estrogens. On the other hand silencing of insulin related genes as IGF1 and IGF2 could explain the phenotype of reduced follicular growth in animals exposed to environmental estrogens during fetal and neonatal development. With regard to histone modifications, BPA produces a significant decrease in histone H3K9 trimethylation, a repressive histone mark, in cultured follicles (124); however, in vivo effects of developmental exposure to estrogenic compounds on histone modifications in the ovary are not known. Regardless, recent studies have shown that developmental exposure to phytoestrogen, genistein, affects histone marks in the uterus leading to alteration in gene expression and abnormal growth during adulthood (125).

In addition to the epigenetic reprogramming of ovarian somatic cells after the exposure to environmental estrogens, the PGC can also be a target of epigenetic disruption (50, 106). These epigenetic alterations could potentially damage the function of oocytes affecting both oogenesis and embryogenesis. Another consideration of epigenetic modifications of PGCs by environmental estrogens is whether these modifications of the epigenome can be hereditable to future generations. There is little information about the transgenerational inheritance of epigenetic modifications in ovarian follicular development after exposure to estrogenic compounds (126). The possibility that estrogens can produce a transgenerational inheritance of ovarian disease through the permanent alteration of germ cell epigenome needs to be considered.

4) Conclusions

Exposure to environmental estrogens has been of concern due to the potential impact in the development and function of the ovaries, reproductive tract and other estrogen sensitive tissues such as the brain. Because fetal and neonatal developmental stages are sensitive periods, exposure to estrogenic compounds could produce irreversible epigenetic reprogramming of ovarian genes that control ovarian follicular development that could, in turn, lead to abnormalities in adulthood. Studies in animal models have shown that developmental exposure to environmentally relevant levels of these chemicals can produce changes in epigenetic mechanisms and gene expression changes (50, 101, 102, 106, 121, 124). In some of these studies, these changes are also associated with ovarian dysfunction (11, 50, 106). In the future it will be important (i) to establish causal links among epigenetic alterations and gene expression and functional changes in the ovary, (ii) to determine whether these chemicals can induce additional epigenetic alterations, gene expression changes and ovarian dysfunction or whether additional environmental chemicals can cause similar changes in the ovary, and (iii) to determine whether human females with reproductive diseases/dysfunction display similar patterns of epigenetic alterations that have been demonstrated in animal models. Finally, the possibility that the effects of the estrogenic chemicals can be transmitted through epigenetic reprogramming of germ cells affecting ovarian follicular development should be assessed in the future.

Figure 1. Developmental EDC exposure and possible mechanisms to alter brain and ovarian functions.

Endocrine-disrupting chemicals (EDC) have different mechanism of actions which mediate the final effects on ovarian function. Among them, the most recognized are through activating estrogenic receptors either by binding to them or by modifying estradiol (E2) availability [e.g., E2, bisphenol A, methoxychlor (MXC), di(2-ethylhexyl) phthalate (DEHP), diethylstilbestrol]. Other important mechanisms are the antiandrogenic proprieties of some compounds (e.g., MXC and DEHP) and the thyroid modulating effects of others (not reviewed). EDC can directly target the brain or the ovaries due the presence estrogen and androgen receptors in both tissues. However, the mechanisms of regulations between both tissues lead to the possibility that disruption of ovarian physiology affect the brain and vice versa.