Multi- and Transgenerational Outcomes of an Exposure to a Mixture of Endocrine-Disrupting Chemicals (EDCs) on Puberty and Maternal Behavior in the Female Rat

Abstract

Background: The effects of endocrine-disrupting chemicals (EDCs) on fertility and reproductive development represent a rising concern in modern societies. Although the neuroendocrine control of sexual maturation is a major target of EDCs, little is known about the potential role of the hypothalamus in puberty and ovulation disruption transmitted across generations.

Objectives: We hypothesized that developmental exposure to an environmentally relevant dose of EDC mixture could induce multi- and/or transgenerational alterations of sexual maturation and maternal care in female rats through epigenetic reprograming of the hypothalamus. We investigated the transmission of a disrupted reproductive phenotype via the maternal germline or via nongenomic mechanisms involving maternal care.

Methods: Adult female Wistar rats were exposed prior to and during gestation and until the end of lactation to a mixture of the following 13 EDCs: di-n-butyl phthalate (DnBP), di(2-ethylhexyl) phthalate (DEHP), bisphenol A (BPA), vinclozolin, prochloraz, procymidone, linuron, epoxynaxole, dichlorodiphenyldichloroethylene, octyl methoxynimmate, 4-methylbenzylidene camphor (4-MBC), butylparaben, and acetaminophen. Perinatally exposed offspring (F1) were mated with unexposed males to generate germ cell (F2) and transgenerationally exposed (F3 and F4) females. Sexual maturation, maternal behavior, and hypothalamic targets of exposure were studied across generations.

Results: Germ cell (F2) and transgenerationally (F3) EDC-exposed females, but not F1, displayed delayed pubertal onset and altered folliculogenesis. We reported a transgenerational alteration of key hypothalamic genes controlling puberty and ovulation (Kiss1, Esr1, and Oxt), and we identified the hypothalamic polycomb group of epigenetic repressors as actors of this mechanism. Furthermore, we found a multigenerational reduction of maternal behavior (F1-F3) induced by a loss in hypothalamic dopaminergic signaling. Using a cross-fostering paradigm, we identified that the reduction in maternal phenotype was normalized in EDC-exposed pups raised by unexposed dams, but no reversal of the pubertal phenotype was achieved.

Discussion: Rats developmentally exposed to an EDC mixture exhibited multi- and transgenerational disruption of sexual maturation and maternal care via hypothalamic epigenetic reprogramming. These results raise concerns about the impact of EDC mixtures on future generations. https://doi.org/10.1289/EHP8795.

Figure 1.

Multi- and transgenerational design of an EDC mixture exposure on maternal behavior and sexual maturation via the female lineage. Adult female rats (F0) were exposed to a mixture of 13 EDCs at relevant exposure concentrations starting 2 wk before mating and until the end of lactation. Mating was carried out for period of 2 wk. Depending on the day of pregnancy during mating, adult females were exposed for a total of 49–56 d, which spanned the period before, during, and after gestation. The four subsequent generations were evaluated for sexual maturation (VO, GnRH interval interpulse, estrous cycle, and folliculogenesis) and maternal behavior (from P2 to P8). Massive parallel RNA sequencing was carried out using hypothalamic (MBH-PoA) explants from the F1 and F3 generation to decipher direct (F1) vs. transgenerational (F3) target genes of the EDC mixture exposure, followed by qPCR validation at three time points, P6, 21, and 60. Target genes were studied for histone posttranslational modifications and DNA methylation using chromatin immunoprecipitation (ChIP) and bisulfite sequencing (BS), respectively. Note: EC, estrus cycle; EDC, endocrine-disrupting chemical; VO, vaginal opening.

Figure 2.

Pubertal timing (VO) and GnRH interpulse interval across generations (F1–F4 generation) after exposure to EDC mixture or vehicle (F0 generation). (A–D, left) Cumulative percentage of female rats reaching VO in F1 ($n=51–56/\text{group}$), F2 ($n=50–52/\text{group}$), F3 ($n=15–24/\text{group}$) or F4 ($n=47–64/\text{group}$). Samples originate from 15 litters per group per generation. Data were analyzed with Student’s $t$-test using the average of the day at vaginal opening as variable. (A and C, right). GnRH interpulse interval was measured in F1 and F3 females at P20 ex vivo. Hypothalamic explants were incubated individually, and sequential sampling was carried out every 7.5 min for 4 h followed by radioimmunoassay for GnRH ($n=4/\text{group}$). Sample originates from four different litters per group per generation. Data were analyzed using Student’s $t$-test. Bars represent $\text{mean}\pm \text{SEM}$ (*$p<0.05$, ***$p<0.001$ vs. CTL). Summary data are reported in Table S3. Note: CTL, control; EDC, endocrine-disrupting chemical; SEM, standard error of the mean; VO, vaginal opening.

Figure 3.

Estrous cycle, ovarian follicle development, and ovarian weight across generation (F1–F3 generation) of female rats exposed to EDC mixture or vehicle (F0 generation). (A–C, left) Percentage of females showing regular cycle and average time spent in each stage of the estrous cycle in F1 ($n=20/\text{group}$), F2 ($n=15/\text{group}$), or F3 ($n=14–15/\text{group}$) animals. Samples originate from eight different litters per group per generation. Data were analyzed using a two-way ANOVA (A–C, middle). Average number of ovarian follicles, corporal lutea, and cysts per cubic millimeter in F1, F2, and F3 females at P60 ($n=9–10/\text{group}$). Samples originate from eight different litters per group per generation. Data were analyzed using a two-way ANOVA (A–C, right). Normalized ovarian weight (ovarian weight per body weight in grams) measured at P60 in F1 ($n=14/\text{group}$), F2 ($n=16/\text{group}$), and F3 ($n=9–10/\text{group}$) females. Samples originate from eight different litters per group per generation. Data was analyzed using Student’s $t$-test. Bars represent $\text{mean}\pm \text{SEM}$ (*$p<0.05$, **$p<0.01$, ***$p<0.001$ vs. CTL). Summary data are reported in Table S3. Note: ANOVA, analysis of variance; CTL, control; D, diestrus; E, estrus; EDC, endocrine-disrupting chemical; P, proestrus; Reg. cycle, regular cycle; SEM, standard error of the mean.

Figure 4.

Kiss1, Esr1, Oxt, Cart, and Pomc mRNA expression and promoter chromatin state in F3 females ancestrally exposed to EDC mixture or vehicle. (A) Expression of Kiss1 Esr1, Oxt, Cart, and Pomc mRNA in the MBH-PoA of infant (P6), prepubertal (P21), and adult (P60) female rats as determined by qPCR ($n=6/\text{group}$). Samples originated from six different litters per group. RNA expression data were normalized by dividing each individual value by the average of the control group at every time point. (B) Abundance of the TrxG-dependent activating marks H3K4me3 and H3K9ac and the PcG-dependent repressive mark H3K27me3 at the Kiss1, Esr1, Oxt, and Pomc promoter in the prepubertal MBH-PoA of females ancestrally exposed to a mixture of EDC (F3 generation), as measured by ChIP ($n=6/\text{group}$). EDC data was normalized to control. Samples originated from six different litters per group. Dotted red lines represent repressive histone modifications (H3K27me3). Green solid lines represent activatory histone modifications (H3K27me3 and H3K9ac). Bars represent $\text{mean}\pm \text{SEM}$ (*$p<0.05$, **$p<0.01$, ***$p<0.001$ vs. CTL, Student’s $t$-test). Summary data are reported in Table S3. Note: AU, arbitrary units; ChIP, chromatin immunoprecipitation; EDC, endocrine-disrupting chemicals; Oxt, oxytocin; SEM, standard error of the mean; Th, tyrosine hydroxylase.

Figure 5.

Maternal behavior displayed by female rats exposed to a mixture of EDC throughout four generations. (A–D, left) Time spent by dams displaying licking and grooming behavior toward pups after direct (F0 generation, $n=15/\text{group}$), in utero, and through lactation (F1 generation, $n=10-11/\text{group}$, obtained from 5 different litters per group), germ-cell (F2 generation, $n=11/\text{group}$ obtained from 5 different litters per group) or ancestral (F3 generation, $n=11/\text{group}$, obtained from 5 different litters per group) exposure to an EDC mixture or vehicle from P2 to 8. Bar graphs show pooled time spent licking and grooming from P2–8. (A–D, right). Time spent by dams resting alone outside the nest not being involved in maternal care in F0–F3 females. Bar graphs show pooled time spent resting alone from P2–8. Plotted lines represent average $\text{time}\pm \text{SEM}$ (*$p<0.05$ vs. CTL, two-way ANOVA followed by Sidak’s multiple comparisons test). Summary data are reported in Table S3. Note: ANOVA, analysis of variance; CTL, control; EDC, endocrine-disrupting chemical; SEM, standard error of the mean.

Figure 6.

Dopaminergic signaling proteins, mRNA expression and chromatin state in the female rat in utero (F1 generation) and ancestrally (F3 generation) exposed to EDC mixture or vehicle. (A) Expression of Th, Dnm1, Drd1, and Darpp32 mRNA in the MBH-PoA of infant (P6), prepubertal (P21) and adult (P60) F1 female as determined by qPCR ($n=6/\text{group}$). Samples originates from six different litters per group. RNA expression data were normalized by dividing each individual value by the control group average value at every time point. (B) Methylation state at 13 CpG sites of the Th gene promoter from MBH-PoA explants of F1 females at P21 ($n=6/\text{group}$). Samples originates from six different litters per group. (C) Abundance of the TrxG-dependent activating marks H3K4me3 and H3K9ac and the PcG-dependent repressive mark H3K27me3 and H3K9me3 at the Th promoter in the prepubertal MBH-PoA of F1 females, as measured by ChIP ($n=6/\text{group}$). Samples originate from 6 different litters per group. (D) Abundance of Th-immunoreactive cells (green) within the mPoA of F1 prepubertal female rats. (E) Quantification of Th immunoreactivity in the SN, VTA, and mPoA of F1 females. Bars represent mean number of cells per $\text{cubic millimeter}\pm \text{SEM}$; (F) Expression of Th and Drd1 mRNA in the MBH-PoA of infant (P6), prepubertal (P21), and adult (P60) F3 females ($n=6/\text{group}$). Samples originates from 6 different litters per group. (G) Methylation state at 13 CpG sites of the Th gene promoter from MBH-PoA explants of F3 females at P2 ($n=6/\text{group}$) Samples originates from six different litters per group. (H) Abundance of H3K4me3, H3K9ac, H3K27me3, and H3K9me3 histone posttranslational modifications at the Th promoter in the prepubertal MBH-PoA of F3 females, as measured by ChIP ($n=6/\text{group}$). Dotted red lines represent repressive histone modifications (H3K27me3 and H3K9me3). Green solid lines represent activatory histone modifications (H3K27me3 and H3K9ac). EDC data from panels A, C, F, and H were normalized to control data. Samples originates from six different litters per group. Bars represent $\text{mean}\pm \text{SEM}$ (*$p<0.05$, **$p<0.01$, ***$p<0.001$ vs. CTL). Data was analyzed using either two-way ANOVA (6A), followed by Sidak’s multiple comparisons test or Student’s $t$-test (6B–H). Summary data are reported in Table S3. Note: 3V, third ventricle; ANOVA, analysis of variance; AU, arbitrary units; ChIP, chromatin immunoprecipitation; CTL, control; EDC, endocrine-disrupting chemical; SN, substantia nigra; Th, tyrosine hydroxylase; VTA, ventral tegmental area.

Figure 7.

Sexual maturation and mRNA expression data in cross-fostered F2 offspring. (A) Average age at vaginal opening of cross-fostered germ cell (F2) EDC-exposed pups or controls raised by either in utero EDC-exposed dams or controls ($n=12–19/\text{group}$). Samples originates from 6–12 F1 generation litters per group. (B) Percentage of cross-fostered females having regular cycle and time spent in each stage of the estrous cycle ($n=10/\text{group}$). (C) Time spent by cross-fostered F2 dams displaying licking and grooming ($n=8/\text{group}$). (D) Average age at VO in F3 females ($n=10/\text{group}$). Samples originates from $6–10\text{\hspace{0.17em}litters}$. (E–H and M–P) Expression of Kiss1, Esr1, Oxt, and Th mRNA in the MBH-PoA of cross-fostered F2 and F3 pups at P21, as determined by qPCR ($n=6/\text{group}$). Samples originates from six litters. Data from the CE, EC, and EE groups were normalized to the CC group. (I–L and Q–T) Abundance of the TrxG-dependent activating marks H3K4me3, H3K9ac, or the PcG-dependent repressive mark H3K27me3 at the Kiss1, Esr1, Oxt, and Th promoter in the prepubertal MBH-PoA of cross-fostered F2 and F3 females, as measured by ChIP ($n=6/\text{group}$). Samples originates from six litters. Summary data are reported in Table S4. Bars represent $\text{mean}\pm \text{SEM}$ (*$p<0.05$, **$p<0.01$, ***$p<0.001$ vs. CC; †$p<0.05$, ††$p<0.01$, †††$p<0.001$ vs. CE, one-way ANOVA). Note: ANOVA, analysis of variance; CC, control pup raised by control dam; CE, germ-cell EDC-exposed pup raised by control dam; ChIP, chromatin immunoprecipitation; D, diestrus; E, estrus; EC, control pup raised by in utero EDC-exposed dam; EDC, endocrine-disrupting chemical; EE, germ-cell EDC-exposed pup raised by in utero EDC-exposed dam; Oxt, oxytocin; P, proestrus; Reg. cycle, regular cycle; SEM, standard error of the mean; Th, tyrosine hydroxylase; VO, vaginal opening.