Exposure of Adult Female Mice to Low Doses of di(2-ethylhexyl) Phthalate Alone or in an Environmental Phthalate Mixture: Evaluation of Reproductive Behavior and Underlying Neural Mechanisms

Abstract

Background: We have previously shown that adult male mice exposure to low doses of an ubiquitous endocrine disruptor, di(2-ethylhexyl) phthalate (DEHP), alters courtship behavior.

Objective: The effects of adult exposure to low doses of DEHP alone or in an environmental phthalate mixture on estrous cyclicity, reproductive behavior, and underlying neural structures were analyzed in female mice.

Methods: Two-month-old C57BL/6J females were exposed orally for 6 wk to DEHP alone (0, 5 or $50\mu g/\mathrm{kg}/d$) or to DEHP ($5\mu g/\mathrm{kg}/d$) in a phthalate mixture. Estrous cyclicity was analyzed in intact mice, and behavior [lordosis, olfactory preference, partner preference, ability to stimulate male ultrasonic vocalizations (USVs)] was measured in ovariectomized mice primed with estradiol and progesterone. Immunohistochemical studies were conducted in the neural structures involved in behavior for estrogen receptor (ER) $\alpha$ and progesterone receptor (PR).

Results: Exposure to DEHP alone or in mixture lengthened the estrous cycle duration, with a shorter proestrus and longer estrus and metestrus stages. Under normalized hormonal levels, females exposed to DEHP alone or in mixture exhibited altered olfactory preference. A lower lordosis behavior and ability to attract and stimulate male emission of courtship USVs was observed, probably due to modifications of pheromonal emission in exposed females. The behavioral alterations were associated with a lower number of PR-expressing neurons, without changes in $\text{ER}\alpha$, in the neural circuitry underlying sexual behavior. The majority of effects observed was comparable between the two DEHP doses and were driven by DEHP in the mixture.

Conclusions: Exposure to environmental doses of DEHP alone or in mixture altered several components of female sexual behavior in mice, probably through selective disruption of neural PR signaling. Together with the previously reported vulnerability of male mice, this finding suggests a major impact of exposure to phthalates on sexual reproduction, including in other species with similar neural regulatory processes. https://doi.org/10.1289/EHP7662.

Figure 1.

Effects of adult female mice exposure to DEHP alone or in mixture on estrous cyclicity. A. Representation of five consecutive estrous cycles (a–e) in 4 females exposed either to the vehicle (control), DEHP at 5 (DEHP-5) or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$ (DEHP-50), or to a phthalate mixture (Mix). The duration of the estrous cycles (in days) are indicated. (B–C) Mean duration of the estrous cycle (B) and mean duration of each stage of the estrous cycle (C) in female mice. Data expressed as $\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}$ for 15 females per treatment group. Kruskal-Wallis analysis showed a treatment effect on the duration of the estrous cycle ($p=0.0001$), proestrus, estrus, and metestrus ($p=0.0001$). Post hoc analyses (*$p<0.05$, **$p<0.01$, ***$p<0.001$ vs. the control group) are indicated. (D–E). Body (D) and uterine weights (E) are indicated as $\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}$. Treatment effect on uterine weight shown by one-way ANOVA ($\#p<0.05$). Summary data for panels B, C, D, and E can be found in Table S3. Note: ANOVA, analysis of variance; DEHP, di(2-ethylhexyl) phthalate; SEM, standard error of the mean.

Figure 2.

Effects of adult female mice exposure to DEHP alone or in mixture on lordosis behavior and olfactory preference. (A) Lordosis behavior was tested in naive (Test 1) and experienced (Test 2) females in the presence of a sexually experienced male with a two-week interval duration. (B) Percentage of female mice ($n=11–13\text{\hspace{0.17em}per\hspace{0.17em}treatment\hspace{0.17em}group}$) showing lordosis behavior in the four treatment groups exposed to the vehicle (Veh, control), DEHP at 5 or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$ or to a phthalate mixture (Mix). (C) Lordosis quotient, number of female lordosis posture/number of male mounts, was calculated in Tests 1 and 2 for the four treatment groups ($\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}$). Kruskal-Wallis analysis showed a treatment effect of treatment for Test 2 ($p=0.001$); post hoc analyses (*$p<0.05$, **$p<0.01$, ***$p<0.001$ vs. the control group) are indicated. (D) Rejection quotient, number of female rejection behavior/number of male mounts, was calculated in Tests 1 and 2 for the four treatment groups. Kruskal-Wallis analysis showed a treatment effect for Test 2 ($p=0.0201$); post hoc analyses (*$p<0.05$ vs. the control group) are indicated. (E–H). Olfactory preference of females toward an anesthetized male and female was measured in a Y-maze paradigm (E). Total time spent in chemo-investigation by female mice (F) and number of entries into the male and female stimulus arms (G) are presented as $\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}$. The discrimination index (H), time spent by exposed females in male investigation minus the time spent in female investigation divided by the total time of investigation, is expressed as $\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}$. One-way ANOVA showed a treatment effect on the discrimination index ($p=0.0289$); positive index for the control and DEHP-5 groups (*$p<0.05$ and **$p<0.01$) are indicated. Summary data for panels B, C, D, F, G and H can be found in Table S4. Note: ANOVA, analysis of variance; DEHP, di(2-ethylhexyl) phthalate; SEM, standard error of the mean.

Figure 3.

Effects of adult female mice exposure to DEHP alone or in mixture on partner preference. (A–C) In a three-chamber test, a sexually experienced male had choice between a control female exposed to the Veh and a female exposed to DEHP alone or in mixture (A). The number of entries of males into the chamber of control female vs. the chamber of female exposed to DEHP at 5 (DEHP-5) or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$ (DEHP-50), or to a phthalate mixture (Mix) (B), and the percentage of time spent investigating each female (C) are represented as $\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}$. Males were used for 11–13 females per treatment group. Paired $t$-test or Wilcoxon test are indicated (*$p<0.05$, **$p<0.01$, ***$p<0.001$ vs. the control group). (D–F) In a Y-maze test, a sexually experienced male had the choice between an anesthetized female exposed to the Veh and a female exposed to DEHP alone or in mixture (D). The number of entries of males into the arm of Veh vs. the arm of DEHP- or Mix-exposed female (E) and the percentage of time spent investigating each female (F) are presented as $\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}$. Paired t-test or Wilcoxon test (*$p<0.05$, **$p<0.01$, ***$p<0.001$ vs. the control group). (G–I) In a Y-maze paradigm, a sexually experienced male had the choice between urine from vehicle-exposed females and urine from females exposed to DEHP alone or in mixture (G). The number of entries of males into the arm of Veh vs. the arm of DEHP or Mix-exposed urine (H) and the percentage of time spent investigating the Veh vs. DEHP or Mix-exposed female urine (I) are presented as $\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}$. Paired t-test or Wilcoxon test (*$p<0.05$, **$p<0.01$, ***$p<0.001$ vs. the control group) are indicated. Summary data for panels B, C, E, F, H, and I can be found in Table S5. Note: DEHP, di(2-ethylhexyl) phthalate; SEM, standard error of the mean; Veh, vehicle.

Figure 4.

Effects of adult female mice exposure to DEHP alone or in mixture on the emission of male USVs. (A) Total number of USV emitted by sexually experienced males in the presence of females exposed to the Veh, DEHP at 5 or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$, or to a phthalate mixture (Mix) during the 4-min recording. Data are expressed as $\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}$. Males were used for 11–13 females per treatment group. (B–D) Total number ($\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}$) of syllables of the simple (B), complex (C), and frequency jump (D) category. A significant treatment effect was found by one-way ANOVA on the number of total USVs ($p=0.0023$), downward ($p=0.022$), complex ($p=0.041$), mixed ($p=0.023$), and frequency-jump syllables ($p=0.005$), and by Kruskal-Wallis on the number of short syllables ($p=0.039$). Post hoc analyses (*$p<0.05$, **$p<0.01$ vs. the control group) are indicated. (E) Total duration ($\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}$) of ultrasonic vocalizations emitted by males. (F–H) Total duration ($\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}$) of syllables of the simple (F), complex (G), and frequency jump (H) categories. A significant treatment effect was found by one-way ANOVA on the duration of total USVs ($p=0.031$) and complex syllables ($p=0.022$), by Kruskal-Wallis for the duration of downward ($p=0.028$) and mixed syllables ($\#p=0.034$); post hoc analyses (*$p<0.05$ vs. the control group) are indicated. Summary data for panels A, B, C, D, E, F, G, and H can be found in Table S6. Note: ANOVA, analysis of variance; DEHP, di(2-ethylhexyl) phthalate; SEM, standard error of the mean; USV, ultrasonic vocalization; Veh, vehicle.

Figure 5.

Effects of adult exposure to DEHP alone or in mixture on neural $\mathrm{ER\alpha }\text{-immunoreactivity}$ in the facilitatory system of female mice. (A–C) Representative nuclear $\text{ER}\mathrm{\alpha }$-immunolabeling in the MeA (A), BNST (B), and VMH (C) of females exposed to the Veh, DEHP at 5 (DEHP-5) or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$ (DEHP-50), or to a phthalate mixture (Mix). Scale bar: $100\mathrm{\mu }\mathrm{m}$; (D–I). Quantitative analyses of the number of $\text{ER}\mathrm{\alpha }\text{-immunoreactive}$ (ir) cells (D–F) and mean fluorescence density (G–I) in the indicated brain areas. Data are $\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}.$ of 6 females per treatment group. Summary data for panels D, E, F, G, H, and I can be found in Table S7. Note: AC, anterior commissure; BNST, bed nucleus of stria terminalis; DEHP, di(2-ethylhexyl) phthalate; MeA, medial amygdala; OC, optic chiasma; PR, progesterone receptor; SEM, standard error of the mean; Veh, vehicle control; VMH, ventromedial hypothalamus.

Figure 6.

Effects of adult female mice exposure to DEHP alone or in mixture on neural PR-immunoreactivity in the facilitatory system. (A–C) Representative nuclear PR-immunolabeling in the MeA (A), BNST (B), and VMH (C) of females exposed to the Veh, DEHP at 5 (DEHP-5) or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$ (DEHP-50), or to a phthalate mixture (Mix). Scale bar: $100\mathrm{\mu }\mathrm{m}$; (D–I) Quantitative analyses of the number of PR-immunoreactive (ir) cells (D–F) and mean fluorescence density (G–I) in the indicated brain areas. Data are $\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}.$ of 6 females per treatment group. A significant treatment effect was found by one-way ANOVA on cell number in the MeA ($p<0.0001$) and VMH ($p=0.0010$), and on density in the BNST ($p=0.0003$) and VMH ($p=0.0022$), whereas Kruskal-Wallis analysis showed a treatment effect on cell number in the BNST ($p=0.0096$) and density in the MeA ($p=0.0107$). Post hoc analyses (*$p<0.05$, **$p<0.01$, ***$p<0.001$ vs. the control group) are indicated. Summary data for panels D, E, F, G, H, and I can be found in Table S8. Note: AC, anterior commissure; ANOVA, analysis of variance; BNST, bed nucleus of stria terminalis; DEHP, di(2-ethylhexyl) phthalate; MeA, medial amygdala; OC, optic chiasma; PR, progesterone receptor; SEM, standard error of the mean; Veh, vehicle control; VMH, ventromedial hypothalamus.

Figure 7.

Effects of adult female mice exposure to DEHP alone or in mixture on neural $\text{ER}\mathrm{\alpha }\text{-}$ and PR-immunoreactivity in the inhibitory system. (A–B) Representative nuclear $\text{ER}\mathrm{\alpha }\text{-}$ (A) and PR-immunolabeling (B) in the MPOA and ARC of female mice exposed to the Veh, DEHP at 5 (DEHP-5) or $50\mathrm{\mu }\mathrm{g}/\mathrm{kg}/\mathrm{d}$ (DEHP-50), or to a phthalate mixture (Mix). Scale bar: $100\mathrm{\mu }\mathrm{m}$; $3\mathrm{V}$: third ventricle. (C–D) Quantitative analyses of the number of $\text{ER}\mathrm{\alpha }\text{-}$ (C) and PR-immunoreactive (ir) cells (D) in the indicated brain areas. Data are $\text{means}\pm \mathrm{S}.\mathrm{E}.\mathrm{M}.$ of 6 females per treatment group. A significant treatment effect was found by one-way ANOVA on the number of PR-immunoreactive cells in the MPOA ($p=0.0003$) and ARC ($p=0.0428$). Post hoc analyses (*$p<0.05$, ***$p<0.001$ vs. the control group) are indicated. Summary data for panels C and D can be found in Table S9. Note: ANOVA, analysis of variance; ARC, arcuate nucleus; DEHP, di(2-ethylhexyl) phthalate; ER, estrogen receptor; MPOA, medial preoptic area; PR, progesterone receptor; SEM, standard error of the mean; Veh, vehicle control.

Figure 8.

Proposed mechanisms for behavioral effects of adult exposure to DEHP in sexual partners. Chronic oral exposure of male and female mice to DEHP affected the physiological balance between the key neural sex steroid signaling pathways in the neural circuitry underlying sexual behavior. A lower expression of neural AR in males and neural PR in females occurred without effects on neural $\text{ER}\mathrm{\alpha }$ expression. The resulting lower neural $\text{AR}/\text{ER}\mathrm{\alpha }$ ratio in males might be related to a lower emission of USVs and the ability to attract female partners. In females, the lower neural $\text{PR}/\text{ER}\mathrm{\alpha }$ ratio was associated with a lower olfactory discrimination and expression of lordosis behavior. In addition, modifications in female pheromonal cues probably due to peripheral effects of phthalates were related to a lower ability of exposed females to stimulate male courtship behavior such as USV emission. This suggests that combined modifications induced by DEHP exposure in both sexual partners may greatly impact mating and sexual reproduction in rodents. The red arrows represent the lowered sex steroid receptor ratio and male and female behaviors induced by adult DEHP exposure. Note: AR, androgen receptor; DEHP, di(2-ethylhexyl) phthalate; ER, estrogen receptor; PR, progesterone receptor; USVs, ultrasonic vocalizations.