Early Life Exposure to Endocrine-Disrupting Chemicals Causes ...

ORIGINAL

RESEARCH

Early Life Exposure to Endocrine-Disrupting Chemicals Causes Lifelong Molecular Reprogramming of the Hypothalamus and Premature Reproductive Aging Andrea C. Gore, Deena M. Walker, Aparna M. Zama, AnnMarie E. Armenti, and Mehmet Uzumcu Institute for Neuroscience (A.C.G., D.M.W.), Division of Pharmacology and Toxicology and Center for Molecular Toxicology (A.C.G.), and Institute for Cellular and Molecular Biology (A.C.G.), The University of Texas at Austin, Austin, Texas 78712; and Department of Animal Sciences (A.M.Z., A.E.A., M.U.), Rutgers University, New Brunswick, New Jersey 08901

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 reprograms 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 ␮g/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 chemicalexposed 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. (Molecular Endocrinology 25: 2157–2168, 2011)

T

he control of reproductive aging in female mammals involves a complex interplay of the hypothalamus, pituitary, and ovary. The hypothalamus plays an important role in this life transition, with hypothalamic GnRH neurons changing the synthesis and release of the decapeptide with aging in a species-specific manner (1, 2). Throughout the life cycle, regulatory inputs to GnRH neu-

rons from neuropeptides (e.g. kisspeptin), neurotransmitters [e.g. glutamate and ␥-aminobutyric acid (GABA)], and neurotrophic factors (e.g. TGF and IGF) control GnRH gene expression and release, and the balance of these inputs determine the final output of GnRH release from nerve terminals into the portal capillary system that vascularizes the anterior pituitary gland (3– 6). In addition, feed-

ISSN Print 0888-8809 ISSN Online 1944-9917 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/me.2011-1210 Received August 1, 2011. Accepted September 20, 2011. First Published Online October 27, 2011

Abbreviations: DMSO, Dimethylsulfoxide; EB, estradiol benzoate; EDC, endocrine-disrupting chemical; ER, estrogen receptor; GABA, ␥-aminobutyric acid; HPG, hypothalamicpituitary-gonadal; MBH, medial basal hypothalamus; MXC, methoxychlor; NRF1, nuclear respiratory factor 1; POA, preoptic area; STAT, signal transducer and activator of transcription.

Mol Endocrinol, December 2011, 25(12):2157–2168

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Endocrine Disruption of Reproductive Aging

back from peripheral hormones, such as sex steroids (7, 8), regulates hypothalamic output via actions of estradiol, progesterone, and androgens on their respective steroid hormone receptors that are abundant in the hypothalamus. This complex network of neurons, glia, and steroid regulation changes substantially during reproductive aging (9 –12). Nevertheless, relatively little is known about how the hypothalamus changes with age, and its causeand-effect role on reproductive senescence. The timing and progression of reproductive senescence is determined by a combination of genetic and environmental factors. To date, most research has focused on genetic predispositions (13). Nevertheless, the environment is likely to play a role, and there is speculation that endocrine-disrupting chemicals (EDC) may hasten reproductive aging, with the potential outcome of shortening an individual’s reproductive lifespan. EDC are compounds in the environment that act upon the body’s hormonal systems and include industrial contaminants, plastics/plasticizers, pesticides, and other compounds (14). Recent studies show that exposures to EDC during key developmental periods, especially prenatal/early postnatal life, can cause molecular/cellular changes that affect the function of the affected tissues later in life, a concept referred to as the fetal/developmental basis of adult disease (15). The mechanisms for these effects are diverse and probably involve epigenetic molecular changes, including DNA methylation and histone modifications, the consequences of which are manifested later in life. Intriguingly, the latency between exposure and adult dysfunction extends into the realm of reproductive aging. Animal studies show that reproductive aging is accelerated by EDC [methoxychlor (MXC) (16), bisphenol A (17), and dioxins (18)], and recent epidemiological evidence links developmental EDC exposures to accelerated menopause [diethylstilbestrol (19) and perfluorocarbons (20)]. The current study sought to draw mechanistic connections between early life developmental exposure to an estrogenic endocrine disruptor, MXC, its actions on the


hypothalamus, and the consequences on reproductive senescence. MXC is an organochlorine pesticide with estrogenic and antiestrogenic properties (16, 21, 22). Some studies have investigated effects of MXC on the female hypothalamus (23), but few have taken a mechanistic approach to understanding how MXC may affect hypothalamic gene expression (24, 25), and none has linked early life exposures to reproductive aging. Amid concerns that infertility is on the rise (26, 27), understanding the links between EDC exposures and reproductive aging are critical. Furthermore, such work extends observations on the fetal basis of adult disease beyond the young adult and into the realm of aging. Finally, this study focused on the hypothalamic mechanisms by which EDC may reprogram the neuroendocrine circuit regulating reproduction. Therefore, as a whole, this project’s goal was to determine early life programming of the entire life cycle’s reproductive capacity and the underlying molecular mechanisms.

Results Loss of estrous cyclicity is accelerated in perinatal endocrine-disrupted rats Rats were monitored by daily vaginal smears for a minimum of 12 consecutive days per month, or longer (up to 20 d) if more time was required to establish cyclicity status, during the 5-month period leading up to euthanasia. Table 1 shows that most dimethylsulfoxide (DMSO)treated rats were still exhibiting regular cycles at 16 –17 months of age. The low-dose MXC group had some diminution in regular cycles with aging, but this was only significantly different from the vehicle group at 14 months. By contrast, the high-dose MXC and the estradiol benzoate (EB)-treated groups had few (if any) rats still exhibiting cycles by 13 months of age. Fisher’s exact test revealed that these latter groups were significantly different from the control group from 13 to 17 months of age (P ⬍ 0.001– 0.0001). Thus, perinatal EDC treatment was associated with premature reproductive failure.

TABLE 1. Estrous cycle classification for aging rats treated between E19 and P7 DMSO 20 ␮g of MXC 100 mg of MXC 1 mg of EB

13 Months 83% Reg n⫽6 100% Reg n⫽9 0% Reg** n⫽9 0% Reg** n⫽5

14 Months 100% Reg n⫽6 78% Reg* n⫽9 22% Reg** n⫽9 0% Reg** n⫽5




Fisher’s exact test was conducted with significance at *, P ⬍ 0.001; **, P ⬍ 0.0001 vs. control (DMSO) at the same age. Reg, Regular estrous cycles of 4 –5 d; n, sample size at each age.

A

35

Estradiol

30

30 #

#

20 15 10

Progesterone

10

MXC high

MXC low

EB

0

DMSO

MXC high

MXC low

EB

DMSO

20 15

5

*

5 0

B


25

25

P4 (ng/ml)

E2 (pg/ml)


FIG. 1. Serum concentrations of estradiol (A) and progesterone (B) in aging female rats at 16 –17 months of age. Levels of estradiol were significantly lower in the EB group compared with all others. Levels were also lower in the two MXC groups compared with DMSO. Serum progesterone concentrations were unaffected by perinatal exposure to EDC. #, P ⬍ 0.05 vs. control; *, P ⬍ 0.005 vs. all other groups.

Serum estradiol and progesterone concentrations in endocrine-disrupted rats Serum hormone levels were measured in terminal blood samples. Figure 1 shows that serum estradiol concentrations in the aged females were affected by perinatal treatment (P ⬍ 0.005, Kruskal-Wallis). Levels of estradiol were significantly lower in the EB group than all the other groups (Mann-Whitney post hoc, P ⬍ 0.005). Both the low-dose and high-dose MXC groups also differed from the control (P ⬍ 0.05). For progesterone, no differences were determined among the groups (P ⫽ 0.375). Hypothalamic gene expression is reprogrammed in aging, endocrine-disrupted rats The low-density PCR array was used to measure expression of 48 genes in the preoptic area (POA) and medial basal hypothalamus (MBH) of perinatally exposed rats euthanized at aging endpoints. Figure 2 shows expression of those genes in the POA that differed in the aging rats by 50% or more between the perinatal vehicleand EDC-treated groups and based on raw P values of less than 0.05 before Bonferroni correction. Seventeen genes were differentially regulated by perinatal EDC treatments according to these criteria, including steroid hormone receptors/coregulatory factors/binding partners (Esr1, AR, Pgr, Srd5a1, Sts, and Arnt); glutamate/GABA receptor subunits (Gria3, Grin2b, Grik2, and Gabbr1); neurotrophic factors/receptors (Tgfa, Tgfb1, and Igf1r); neuropeptides/receptors (Kiss1, Kiss1r, and Gnrhr); and the transcription factor, Stat5b. Of these 17 genes, 15 showed a similar pattern of expression in the aging rats, with mRNA levels higher in the EB and/or MXC high-dose groups compared with vehicle. Two genes had unique expression patterns. Kiss1 mRNA levels were significantly lower in the EB group compared with all other

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groups (P ⬍ 0.001). Gnrhr mRNA levels were higher in the high-dose MXC group compared with all others (P ⬍ 0.05, nonsignificant after Bonferroni correction). After Bonferroni correction, the two POA genes significantly affected by perinatal EDC treatment were: Esr1 (P ⱕ 0.001) and Kiss1 (P ⬍ 0.001) (Fig. 2). In the MBH, gene expression of only one transcript (solute carrier 17a6, vesicular glutamate transporter 2) was higher in the MXC high-dose group compared with all others (P ⬍ 0.05, nonsignificant after Bonferroni correction). Other MBH genes were not altered by perinatal EDC treatments. Supplemental Tables 1 and 2, published on The Endocrine Society’s Journals Online web site at http://mend.endojournals.org, show relative gene expression of those genes that were not affected in either POA (Supplemental Table 1) and all genes in MBH (Supplemental Table 2). DNA methylation of estrogen receptor (ER)␣ (Esr1) gene by perinatal EDC Because the Esr1 gene was significantly up-regulated in the brain of aging females, we performed bisulfite conversion and pyrosequencing of two sites of the Esr1 regulatory regions in exon 1b and intron 1 to determine whether long-term epigenetic programming of the Esr1 gene had occurred. These regions were chosen because DNA methylation of CpG sites in these two gene regions is regulated by maternal behavior or perinatal estrogen when assessed in young adults (28 –30). Fourteen CpG sites in exon 1b, and 11 sites in intron 1, were analyzed by pyrosequencing for percentage of methylation. The total percentage of methylation of all the CpG sites within the two regions of analysis was unaffected by perinatal EDC treatment (Fig. 3). The sequences and location of CpG sites of the regions of analysis are also shown in Fig. 3. When individual CpG sites were examined for percentage of methylation, one site in exon 1b (⫹46 from the transcription start site) and two sites in intron 1 (⫹1733 and ⫹1786 from the transcription start site) were up-regulated in the perinatally EB-treated rats compared with controls (Fig. 4). Putative transcription factors in exon 1b and intron 1 of Esr1 MatInspector analysis of the exon 1b and intron 1 sequences of the ER␣ gene revealed numerous putative transcription factor sites on the forward (⫹) and reverse (⫺) strands. Table 2 shows the list of transcription factors with more than 80% similarity to the prototypical sequences in the 221-nucleotide sequence assayed for exon 1b. Forty putative transcription factors with homology to the rat prototypical sequences were identified, with similar distribution on the forward (⫹) and reverse (⫺) DNA strand. There were seven potential binding sites for six

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A

Steroid receptors, co-regulators and binding partners Esr1 (ERα) AR Pgr

3

p = 0.001

2

(p = 0.004)

2 (p = 0.05)

DMSO

2

EB 1

1

0

0

MXC low

1

MXC high 0

2 Srd5a1 (5α reductase) 2 Sts (steroid sulfatase) (p = 0.006)

Fold-change in gene expression

(p = 0.006)

1

1

0

0

Arnt

(p = 0.002)

2 1 0

B

Neurotransmitter receptors Grin2b (NR2b) Gria3 (GluR3)

2

(p = 0.035)

1 0

2

(p = 0.034)

1

Grik2 (KA2) (p = 0.021)

2

1.6 1.2 0.8

1

0

tivator/cell cycle regulator, two nuclear respiratory factor 1 (NRF1), one cell cycle-dependent element, and one signal transducer and activator of transcription (STAT) sites were identified. The intron 1 sequence also contained a total of five STAT sites in the region of analysis.

0.4 0

0

Gabbr1 (GABAB-R1) (p = 0.012)

Body weight is affected by perinatal EDC Rats were weighed on the day of euthanasia. As shown in Fig. 5, the EB group had a significantly higher body weight than rats in all of the other treatment groups (P ⬍ 0.005). Post hoc analysis (Fisher’s least significant difference) showed that the EB group was significantly heavier than all other groups (P ⬍ 0.01 vs. all).

C

Cytokines & Receptors Tgfa (p = 0.038) 1.6 Tgfb1 2 1.2 1

0.8

Igf1r

(p = 0.027)

2

(p = 0.011)

Discussion

The current data show that early life exposures to environmental endocrine disrup0 0 0 tors hasten premature reproductive aging, D Neuropeptides & Receptors E Transcription Factor thereby diminishing an individual’s lifetime Gnrhr (p = 0.028) Kiss1r (p = 0.035) Stat5b 1.6 10 Kiss1 p < 0.001 2 reproductive capacity. Whether this is the (p = 0.019) 6 8 1.2 case in women remains to be proven, al6 4 0.8 though epidemiological data suggest that 1 4 2 0.4 certain EDCs are associated with earlier age 2 at menopause. Women who had been ex0 0 0 0 posed to high levels of dioxin due to a FIG. 2. Low-density PCR analysis identified 48 transcripts in the POA, and data shown for those transcripts with raw P ⬍ 0.05 grouped into 5 classes: A) steroid hormone chemical plant explosion in Seveso, Italy, signaling; B) neurotransmitter signaling; C) cytokine signaline; D) neuropeptide showed increased risk for early menopause signaling; and E) transcription factors. 17 genes met these criteria, and of those, 15 had (31). Body burden of DDE (dichlorodipheexpression patterns with levels higher in the EB and/or MXC high-dose group compared with DMSO. For this group, Esr1 gene expression met significance at P ⬍ 0.001 (DMSO nyldichloroethylene), a metabolite of the and MXC low dose were both significantly lower than EB and MXC high dose). Two organochlorine pesticide DDT (dichlorodigenes had unique expression patterns. Kiss1 gene expression was significant at P ⬍ phenyltrichloroethane), was associated with 0.001, with levels significantly lower in the EB group compared with all others. Gnrhr earlier age at menopause in a population of expression had highest levels in the MXC high-dose group. Raw P values are shown for each gene. Gene expression data for the remaining POA genes (P ⬎ 0.05) are presented women studied in North Carolina (32). A in Supplemental Table 1. cross-sectional study conducted on Hispanic women relating their body burdens of ortranscription factors (one transcription factor had two ganochlorine pesticides to age at menopause showed that sites) in the hypermethylated ⫹46 region. In the 174 nucleotide sequence of intron 1 (Table 3), 33 menopause occurred at earlier ages in women exposed to DDT, transcription factors were found that met the 80% ho- DDE, ␤-hexachlorocyclohexane, and trans-nonachlor (33). mology cut-off. A cluster of 13 overlapping transcription These results are consistent with the current study’s results. By contrast, other human studies show no relationship befactors distributed across the (⫹) and (⫺) DNA strand was found in the 20-nucleotide region from 1827 to 1846 tween serum EDC and age at menopause (polychlorinated binucleotides downstream from the transcription start site phenyl, Refs. 32, 34), and one study indicated that on average, in exon 1b, a region characterized by four adjacent re- menopause occurred 3 months later in women handling pestipeating CpG sites (see Fig. 3). Within that region, five cides than in controls (35). To our knowledge, links between putative ZF5 POZ domain zinc finger, four E2F-myc ac- MXC and menopause have not been studied in humans. 0.4

1



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TABLE 2. Transcription factors in the exon 1b sequence with more than 80% homology to the rat prototypical sequences Family YBXF ETSF NF1F SORY MYOD SORY HOXF YY1F WHNF AP4R MYOD NEUR CTCF EGRF SP1F MAXF KLFS HESF EBOX EBOX LTFM CP2F NOLF PRDM NFKB FAST FKHD EGRF SP1F CDEF E2FF TF2B HOMF ZFO3 HEAT HEAT INRE SORY HOXF TEAF

Description Y-box binding transcription factor Human/murine ETS factors Nuclear factor 1 SOX/SRY-sex/testis-determining factors and related HMG box factors Myoblast determining factors SOX/SRY-sex/testis-determining factors and related HMG box factors Paralog hox genes 1– 8 from the four hox clusters A, B, C, D Activator/repressor binding to transcription initiation site Winged helix binding sites AP4 and related proteins Myoblast determining factors NeuroD, Beta2, HLH domain pancreas transcription factor 1 CTCF and BORIS gene family transcrip-tional regulators EGR/NGF induced protein C and related factors GC-box factors SP1/GC Myc associated zinc fingers Krueppel like transcription factors Vertebrate homologs of enhancer of split complex E-box binding factors E-box binding factors Lactotransferrin motif CP2-erythrocyte factor related to Drosophila Elf1 Neuron-specific olfactory factor PRD1-BF1 and RIZ homologous (PR) domain proteins (PRDM) Nuclear factor ␬ B/c-rel FAST-1 SMAD interacting proteins Forkhead domain factors EGR/NGF induced protein C and related factors GC-box factors SP1/GC Cell cycle regulators: Cell cycle dependent element E2F-myc activator/cell cycle regulator RNA polymerase II transcription factor IIB Homeodomain transcription factors C2H2 zinc finger transcription factors 3 Heat shock factors Heat shock factors Core promotor initiator elements SOX/SRY-sex/testis-determining factors and related HMG box factors Paralog hox genes 1– 8 from the four hox clusters A, B, C, D TEA/ATTS DNA binding domain factors

Position Position Matrix (from) (to) Anchor Strand similarity ⫺143 ⫺131 ⫺137 (⫺) 0.889 ⫺134 ⫺114 ⫺124 (⫺) 0.855 ⫺130 ⫺110 ⫺120 (⫹) 0.929 ⫺128 ⫺104 ⫺116 (⫺) 0.925 ⫺127 ⫺111 ⫺119 (⫺) 0.911 ⫺125 ⫺101 ⫺113 (⫺) 0.984 ⫺123 ⫺105 ⫺114 (⫺) 0.966 ⫺121 ⫺101 ⫺111 (⫹) 0.877 ⫺109 ⫺99 ⫺104 (⫺) 0.974 ⫺94 ⫺78 ⫺86 (⫹) 0.851 ⫺94 ⫺78 ⫺86 (⫺) 0.928 ⫺93 ⫺81 ⫺87 (⫹) 0.964 ⫺82 ⫺56 ⫺69 (⫹) 0.843 ⫺78 ⫺62 ⫺70 (⫹) 0.888 ⫺77 ⫺61 ⫺69 (⫹) 0.910 ⫺74 ⫺62 ⫺68 (⫹) 0.913 ⫺74 ⫺58 ⫺66 (⫹) 0.824 ⫺65 ⫺51 ⫺58 (⫺) 0.870 ⫺64 ⫺52 ⫺58 (⫹) 0.912 ⫺63 ⫺51 ⫺57 (⫺) 0.928 ⫺61 ⫺53 ⫺57 (⫺) 0.984 ⫺47 ⫺29 ⫺38 (⫹) 0.881 ⫺42 ⫺20 ⫺31 (⫹) 0.888 ⫺42 ⫺13 ⫺27 (⫺) 0.802 ⫺36 ⫺22 ⫺29 (⫺) 0.824 ⫺23 ⫺7 ⫺15 (⫺) 0.851 ⫺15 2 ⫺7 (⫹) 0.926 ⫺3 14 6 (⫺) 0.808 19 35 27 (⫺) 0.879 24 36 30 (⫺) 0.949 25 41 33 (⫹) 0.864 28 34 31 (⫹) 1.000 31 49 40 (⫹) 0.899 34 46 40 (⫺) 0.916 35 59 47 (⫺) 0.953 36 60 48 (⫹) 0.886 39 49 44 (⫹) 0.945 39 63 51 (⫺) 0.872 44 62 53 (⫺) 0.942 49 61 55 (⫹) 0.967

Positions are shown relative to the transcription start site in exon 1b. (⫹), Forward; (⫺), reverse.

There are plausible explanations for these different outcomes across the human data, not the least of which are differences in the nature of the EDC studied. Humans (and wildlife) are exposed to complex mixtures of environmental contaminants, and exposures occur throughout the life cycles. Although epidemiological studies are valuable, the ability to draw causal connections between exposure and outcome is not possible. Notably, authors on both sides of this literature make the point that there is not a linear dose-response relationship between body burden and biological outcome. Thus, differences among studies may also result from making inferences across different exposure levels that may not be comparable from one population to another. Last, it is becoming more

widely accepted that exposures to EDCs during critical developmental windows may have latent effects (14). However, to draw this kind of connection in humans, it is necessary to relate secular trends in the timing of menopause to exposures received 40 –50 yr ago. Thus, animal studies are necessary to understand causal relationships and to discern the underlying mechanisms. Developmental EDC exposures hasten reproductive senescence Our current results and others show that the reproductive life cycle, which includes puberty, adult reproductive function, and aging, is significantly altered by perinatal EDC exposures. Estrogenic EDCs, such as MXC, cause

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A

% Methylation

13


Exon 1b

B

18

12

17

11

16


However, these animals still have a follicular pool, making it likely that the hypothalamus is not only a target of perinatal MXC but that its reprogramming hastens reproductive aging.

Intron 1

Developmental reprogramming of the hypothalamus 9 14 Hypothalamic function is vulnerable to perinatal EDC actions (reviewed in Ref. 39), 8 13 and our current data add novel information about the molecular mechanisms by which this occurs. We show lifelong changes in hypothalamic gene expression associated with premaC Exon 1b sequence ture reproductive aging, in a region-specific -156 -100 manner. In the POA of rats, a complex regu-45 latory neural/glial network controlling HPG +12 reproductive function was up-regulated by D Intron 1 sequence 50% or more by high-dose MXC or EB treat+1733 ments, relative to control. The three major ste+1789 +1843 roid hormone receptors (ER␣, androgen re+1898 ceptor, and progesterone receptor), glutamate/ FIG. 3. Methylation of CpG sites in the Esr1 gene was assayed by bisulfite reaction GABA receptors, growth factors (TGF␣ and and pyrosequencing. For exon 1b (A), total methylation across the 14 sites analyzed TGF ␤), the Kiss1 receptor, and the transcripwas not significantly altered by the perinatal EDC exposure. Similarly, total methylation of 11 sites in intron 1 (B) was unaffected by treatment. The regions of tion factor Stat5b showed this pattern of exanalysis are shown in C (exon 1b) and D (intron 1). In C and D, CpG sites are pression. In addition, a key neuropeptide reghighlighted in gray. The transcription start site in exon 1b is indicated by bold ulating GnRH release, kisspeptin (Kiss1 gene), underlined text. Numbers to the left of the sequence (B and C) are nucleotide was down-regulated in the EB group, and the positions relative to the transcription start site in exon 1b. GnRH receptor was up-regulated in the MXC high-dose group. The sum of these changes advancements in the timing of puberty in females (16; reviewed in Ref. 36). This earlier timing of puberty, how- would be integrated at the level of GnRH output from the ever, is accompanied by a diminution of fertility in peri- hypothalamus to pituitary and subsequently to the gonad. natally treated animals when those rats were examined at Although GnRH gene expression itself was not altered, d 50 – 60 (16). Together with results showing earlier re- this is not surprising in light of evidence that it is probably productive senescence, these results as a whole indicate GnRH release and not gene expression that is most highly that the reproductive life cycle is substantially shorter in regulated, including during reproductive aging (reviewed EDC-exposed animals. This finding is consistent with re- in Ref. 4). sults for bisphenol A (17), dioxins (18), and others (reThe two genes significantly affected in the POA by viewed in Ref. 35) but not heretofore studied from the perinatal EDC exposure were Kiss1 (down-regulated in hypothalamic molecular approach. the EB group) and Esr1 (ER␣; up-regulated in the EB and The control of natural reproductive aging, and disruption MXC high-dose groups). The importance of these two of these processes by EDC resulting in premature reproducgenes on female reproduction, both independently as well tive senescence, involves coordinated physiological proas in combination, cannot be understated. Kiss1 neurons cesses regulated by the hypothalamic-pituitary-gonadal (HPG) axis. At least some of the reproductive aging process in the POA play a crucial role in the capacity for rodents caused by MXC is due to actions on the ovary. Perinatal and other species to undergo ovulatory processes (40; exposure disrupted folliculogenesis, leading to an increased reviewed in Ref. 41). This role extends to reproductive number of preantral and early antral follicles that failed to aging, which is accompanied by changes in the Kiss1 popachieve maturation and ovulation, and fewer/no corpora ulation as shown in rats, monkeys, and humans, with a lutea were detectable (16). This was accompanied by cel- concurrent decline in ovulatory capacity (9, 10, 42). lular/molecular changes to the ovary. Other EDCs have Furthermore, the Kiss1 system is vulnerable to EDC been shown to have ovarian follicular effects, including exposures (43– 45). Together with the current work, polychlorinated biphenyls (37) and bisphenol A (17, 38). the hypothalamic Kiss1 system is an important target MXC high

MXC low

EB

DMSO

MXC high

MXC low

EB

15

DMSO

10



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current literature suggesting that expression of Esr1 is epigenetically regu35 lated in the hypothalamus (28, 30, 48, 49), we found that methylation at three 30 sites (⫹46 site of exon 1b, ⫹1733 and ⫹1786 sites of intron 1) was increased 25 p = 0.046 in the EB group. The MXC rats were 20 not significantly different from control in their DNA methylation pattern, pos15 sibly because their reproductive func10 tion is still maintained (albeit diminished) with aging. In the EB rats, by 5 contrast, the high dosage given perina0 tally had profound consequences on re-155 -129 -122 -104 -82 -80 -73 -33 +8 +29 +31 +38 +46 +65 productive aging, gene expression, and DNA methylation pattern. EB rats also B Intron 1 25 showed higher body weight at death, DMSO suggesting that adipogenesis and/or the p = 0.006 EB 20 hypothalamic control of energy balMXC low p = 0.033 MXC high ance was dysregulated by the perinatal treatment. 15 It was surprising to us that both Esr1 mRNA levels and CpG methyl10 ation of the Esr1 promoter were increased in the EB group, because the relationship between gene expression 5 and CpG methylation is predicted to be inverse. This has been shown in the 0 brain for Esr1 and for other genes, such +1733 +1755 +1773 +1786 +1820 +1834 +1836 +1838 +1840 +1885 +1905 as Bdnf (28, 30, 48, 49). Several factors FIG. 4. Methylation of individual CpG sites in exon 1b (A) and intron 1 (B) of Esr1 is shown for the four perinatal treatment groups. In exon 1b, one site located at ⫹46 relative to the may explain the lack of such a relationtranscription start site in exon 1 differed by perinatal treatment, with levels significantly higher ship for the EB-treated rats in the curin the EB group compared with DMSO. In intron 1, two sites located at ⫹1733 and ⫹1786 rent study. First, there are numerous were also affected, again with the EB group having significantly higher percentage of other CpG sites on the Esr1 gene whose methylation compared with DMSO. methylation pattern was not measured for future mechanistic studies on how developmental herein but which may play different regulatory roles in EDC exposures alter hypothalamic function. Esr1 gene expression. Second, other epigenetic and nonThe Esr1 gene was significantly up-regulated by EB and epigenetic regulatory factors (e.g. coregulatory factors high-dose MXC, a finding consistent with previous work showing that estrogenic EDCs can affect hypothalamic Esr1 and transcription factors) may come into play in the final gene or ER␣ protein expression (45; reviewed in Ref. 36). measure of Esr1 mRNA levels. Third, histone modificaThe same hypothalamic cells may coexpress Kiss 1 and ER␣ tions, not measured in this study, can contribute substan(46). Recent work has shown that positive feedback regula- tially to the epigenetic regulation of gene expression. tion of the HPG axis requires coexpression of ER␣ within Fourth, the POA is a highly heterogeneous tissue, with Kiss1 neurons (40, 47). These estrogen-sensitive kisspeptin- different cell types that may be differentially affected at ergic neurons are a logical target for further research on the gene and protein level by perinatal endocrine disrupendocrine disruptors and are potentially vulnerable to long- tors. Fifth, the timing of exposure and the long lag until gene expression measurements may come into play. Interm regulation by perinatal exposure. deed, DNA methylation is not fixed throughout the life cycle but may undergo dynamic change postnatally. This Lifelong epigenetic changes in Esr1 induced by has even been shown for Esr1, whose DNA methylation perinatal EDC Based on our observation of long-lasting effects of profile undergoes shifts from postnatal d 1 to 20 to 60 in MXC and EB on Esr1 gene expression, together with rats (29). Similarly, this concept applies to developmental % Methylation

% Methylation

40

A

Exon 1b

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TABLE 3. Transcription factors in the intron 1 sequence with more than 80% homology to the rat prototypical sequences Family E2FF PLAG KLFS INSM STAT HNF1 NFAT MYT1 MYT1 STAT STAT EVI1 GATA OVOL ZF5F E2FF NRF1 ZF5F E2FF ZF5F NRF1 E2FF ZF5F E2FF ZF5F CDEF STAT VTBP NKXH RUSH STAT LTFM KLFS

Description E2F-myc activator/cell cycle regulator Pleomorphic adenoma gene Krueppel like transcription factors Insulinoma associated factors Signal transducer and activator of transcription Hepatic nuclear factor 1 Nuclear factor of activated T-cells MYT1 C2HC zinc finger protein MYT1 C2HC zinc finger protein Signal transducer and activator of transcription Signal transducer and activator of transcription EVI1-myleoid transforming protein GATA binding factors OVO homolog-like transcription factors ZF5 POZ domain zinc finger E2F-myc activator/cell cycle regulator Nuclear respiratory factor 1 ZF5 POZ domain zinc finger E2F-myc activator/cell cycle regulator ZF5 POZ domain zinc finger Nuclear respiratory factor 1 E2F-myc activator/cell cycle regulator ZF5 POZ domain zinc finger E2F-myc activator/cell cycle regulator ZF5 POZ domain zinc finger Cell cycle dependent element Signal transducer and activator of Transcription Vertebrate TATA binding protein factor NKX homeodomain factors SWI/SNF related nucleophosphoproteins with a RING finger DNA binding motif Signal transducer and activator of transcription Lactotransferrin motif Krueppel like transcription factors

Position (from) 1746 1747 1757 1759 1784 1792 1792 1795 1798 1800 1802 1808 1811 1812 1827 1827 1827 1828 1828 1829 1829 1829 1830 1830 1831 1834 1844 1851 1851 1854

Position (to) 1762 1769 1773 1771 1802 1808 1810 1807 1810 1818 1820 1824 1823 1826 1841 1843 1843 1845 1845 1844 1845 1845 1844 1846 1845 1846 1862 1867 1869 1864

Anchor 1754 1758 1765 1765 1793 1800 1801 1801 1804 1809 1811 1816 1817 1819 1834 1835 1835 1835 1836 1836 1837 1837 1837 1838 1838 1840 1853 1859 1860 1859

Strand (⫺) (⫺) (⫺) (⫺) (⫺) (⫹) (⫹) (⫹) (⫺) (⫺) (⫹) (⫹) (⫹) (⫺) (⫹) (⫺) (⫺) (⫺) (⫹) (⫹) (⫺) (⫺) (⫺) (⫹) (⫹) (⫹) (⫺) (⫹) (⫹) (⫺)

Matrix similarity 0.815 1.000 0.928 0.917 0.952 0.852 0.872 0.967 0.817 0.910 0.961 0.821 0.982 0.831 0.966 0.897 0.830 0.952 0.894 1.000 0.802 0.946 0.919 0.908 0.967 0.906 0.934 0.850 0.964 0.993

1854 1855 1869

1872 1863 1885

1863 1859 1877

(⫺) (⫺) (⫺)

0.980 0.910 0.925

Positions are shown relative to the transcription start site in exon 1b. (⫹), Forward; (⫺), reverse.

Body Weight

340

*

320 300 280 260 240

MXC high

MXC low

EB

220

DMSO

Terminal body weight (g)

exposures to EDCs affecting DNA methylation with profiles that undergo dynamic change well into aging in the prostate gland (50). In our rats, this concept could be tested by comparing hypothalamic Esr1 methylation pat-

FIG. 5. Body weight (g) is shown for rats on the day of euthanasia at 16 –17 months of age. The EB group had significantly heavier body weights than all other groups. *, P ⬍ 0.01 vs. all other treatments.

terns of neonatally endocrine-disrupted rats throughout postnatal life into aging, an area that we are currently investigating. We scanned the Esr1 sequence for the presence of putative transcription factors to guide future analyses of gene regulatory regions that may be affected by developmental estrogenic EDC exposures. In exon 1b, the hypermethylated CpG at ⫹46 is within the binding sites for several transcription factors, such as SORY, HEAT, and ZFO3, that were shown to play roles as transcriptional repressors (51–55), which may relate to the lack of expected inverse correlation between the methylation and gene expression. In intron 1, there was a cluster of transcription factors (ZF5 POZ domain zinc finger, E2F-myc activator/cell cycle regulator, NRF1, cell cycle-dependent element, and STAT) overlying the GC-rich region from ⫹1827 to 1846. The ZF5 transcription factor is best studied for its role as a transcriptional repressor (56). E2F is better associated with transcriptional activation and,


more recently, with histone H3K56 acetylation (57). NRF1 is well known for its role in mitochondrial function, but recently has been associated with the mediation of estrogen effects on ER␣ gene transcription (58), something that may be relevant to the current finding for specific effects of EB on DNA methylation at that site in intron 1. Finally, the intron 1 sequence also contained five STAT sites in our region of analysis, a finding that highlights the potential importance of this transcription factor in Esr1 regulation.

Conclusions The current findings extend the ever-growing body of research collectively termed the fetal/developmental basis of adult disease to the end of the life spectrum. Our results show that not only do perinatal EDC reprogram hypothalamic gene expression but that they cause long-term changes in epigenetic properties of the Esr1 gene and possibly others not studied herein. These findings have substantial implications for humans. By hastening senescence, EDC may eliminate the possibility of biological children for women who may postpone childbirth for personal or professional reasons. Considering the important roles of estrogens on targets in body and brain, early reproductive senescence may accelerate some disease-related states associated with menopause and affect quality of life in the aging population of women.

Materials and Methods Animals and husbandry Fischer (CDF) inbred rats were obtained from Charles River Laboratories (Wilmington, MA) to generate timed-pregnant females. Animals in this study were siblings of those described in Ref. 16 and had identical husbandry in the labs at Rutgers University. Rats were fed a soy-free scientific diet 5V01 rat chow (Lab Diet manufactured by PMI Nutrition International LLC, Brentwood, MO) and tap water ad libitum. They were allowed to develop to 16 –17 months of age. During this time, they were subjected to periodic monitoring of estrous cycles by daily vaginal smears (see Ref. 16 for details). The cycles were categorized as regular (4 –5 d), prolonged (6⫹ d), persistent estrus, or persistent diestrus (59). All procedures in the present study were carried out in accordance with the guidelines of the Rutgers University Animal Care and Facilities Committee.

Treatments Experimental subjects were exposed to one of four treatments (below) for a total of 12 consecutive days. Daily injections began with the pregnant dam (ip) on gestational d 19 through parturition on d 22 (four prenatal days). Then, injections continued with the pups (sc) from postnatal d 0 (birth) to d 7 (eight


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postnatal days). Treatments were: 1) vehicle control [DMSO sesame oil at 1:2 (three litters, n ⫽ 5 individuals)]; 2) EB (1 mg/kg 䡠 d) as a positive estrogenic control (three litters, n ⫽ 5 individuals); 3) MXC (Sigma, St. Louis, MO), 20 ␮g/kg 䡠 d (lowdose MXC; five litters, n ⫽ 9 individuals); and 4) 100 mg/kg 䡠 d (high-dose MXC; five litters, n ⫽ 9 individuals) in 1 ml of vehicle per kilogram of body weight. The choice of doses of MXC and EB was based on the companion study, in which all animals were treated to cause ovarian disruption (16). The 20 ␮g/kg 䡠 d was selected as an environmentally relevant, low dose of MXC (60, 61). The 100 mg/kg 䡠 d MXC is considered a midrange dose, chosen for comparison with published studies (23, 62, 63). The EB dose was selected to be high so as to ensure disruption of both ovary and hypothalamus by an estrogenic compound.

Euthanasia and tissue/blood collection At 16 –17 months of age, female rats were euthanized by decapitation between 1400 and 1600 h. Rats with regular estrous cycles were euthanized on the day of proestrus; on this light cycle, rats are in the preovulatory period of the cycle, when hypothalamic activity is increasing and serum LH and estradiol concentrations are rising. Noncycling rats were euthanized on persistent diestrus or persistent estrus. Trunk blood was collected and serum separated by centrifugation and stored at ⫺80 C. Brains were rapidly removed, and the POA and the MBH were blocked as described previously (64), snap frozen in liquid nitrogen, and stored at ⫺80 C.

RNA extraction and real-time PCR After shipment to the University of Texas, RNA was extracted and purified, treated with deoxyribonuclease (TURBO DNA kit; Applied Biosystems, Inc., Foster City, CA), and purity and integrity were assessed on the bioanalyzer 2100 (Agilent, Cedar Creek, TX). cDNA conversion was carried out on 2 ␮g of cytoplasmic RNA (High-capacity cDNA reverse transcription kit; Applied Biosystems, Inc.), aliquoted, and processed for realtime PCR as published (45, 65). A 48-gene TaqMan PCR-based array (Applied Biosystems, Inc.) was designed and used for realtime PCR reactions, which were run on the ABI 7900 real-time PCR machine using parameters described elsewhere (45, 65, 66). Data were analyzed using the normalized Ct (␦-Ct) for each sample before transformation to fold change.

Hormone RIA Serum 17␤-estradiol and progesterone were measured in duplicate 100 ␮l of serum samples using commercially available RIA kits, according to the manufacturer’s instructions (Coat-ACount RIA kits; Siemens Medical Solutions, Malvern, PA). The assay sensitivity, intra- and interassay coefficients of variance were 8 pg/ml, 5.5 and 6.4% (respectively) for estradiol, and 0.2 ng/ml, 4.6 and 5.9% for progesterone.

DNA methylation analysis of the ER␣ promoter During the extraction of RNA described above, the genomic DNA fraction was isolated from the nuclear fraction of the POA and MBH samples used for cytoplasmic RNA isolation. Briefly, nuclear pellets were resuspended in high salt buffer and treated with proteinase K followed by a short (10 min) ribonuclease A treatment. Samples were then extracted with phenol/chloroform and precipitated in 100% isopropanol at ⫺20 C overnight. Pel-

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leted DNA was resuspended in nuclease-free H2O and aliquoted at 20 ng/␮l. A 50-␮l aliquot for each DNA sample was sent to EpigenDX (Worcester, MA) for bisulfite reaction and pyrosequencing of the ER␣ regulatory regions in exon 1b and intron 1. Sequences of these regions are shown in Fig. 3.

Transcription factor analysis Using MatInspector software (Genomatix, Inc., Munich, Germany), the intron 1 and exon 1b sequences used by EpigenDX for DNA methylation analysis were scanned to determine which putative rat transcription factor binding sites were localized in these regions.


3.

4. 5. 6.

7.

Analysis and statistics Experimental subjects derived from three to five litters per treatment and a total of five to nine rats were used per group. No significant effects were found when birth litter was used as a covariate. Furthermore, intra- and interlitter variability were comparable. Therefore, individual rats were used as the unit of analysis for statistical purposes. ANOVA was used to compare differences among treatment groups. Post hoc analysis was performed (Fisher’s projected least significant difference) when a main treatment effect at P ⬍ 0.05 was identified. When appropriate, Bonferroni corrections were made for multiple analyses and data reported using Tukey post hoc tests. In a few cases, data did not meet assumptions of homogeneity of variance based on Levene’s test and were instead analyzed by the nonparametric Kruskal-Wallis test. For estrous cyclicity, Fisher’s exact test was used to compare differences between treated animals with controls.

8.

9.

10.

11.

12.

13.

Acknowledgments We thank David Crews, Ph.D. (University of Texas at Austin) and David Sweatt, Ph.D. (University of Alabama at Birmingham, AL) for helpful discussions about this article.

14.

15.

Address all correspondence and requests for reprints to: Andrea C. Gore, Ph.D., The University of Texas at Austin, Division of Pharmacology and Toxicology, C0875, Austin, Texas 78712. E-mail: [email protected]. Present address for A.E.A.: Stony Brook University, Department of Neurosurgery, Stony Brook, New York 11794. This work was supported by National Institutes of Health Grants ES018139 and ES07784 (to A.C.G.), F31 AG034813 (to D.M.W.), and ES013854 (to M.U.) and by the National Institute of Environmental Health Sciences Center Grant ES005022 (to M.U.). Disclosure Summary: The authors have nothing to disclose.

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