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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

2021; National Institute of Environmental Health Sciences; Volume: 129; Issue: 1 Linguagem: Inglês

10.1289/ehp7662

1552-9924

Nolwenn Adam, Linda Brusamonti, Sakina Mhaouty‐Kodja,

Effects and risks of endocrine disrupting chemicals

Vol. 129, No. 1 ResearchOpen AccessExposure 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 Nolwenn Adam, Linda Brusamonti, and Sakina Mhaouty-Kodja Nolwenn Adam Sorbonne Université, CNRS, Institut national de la santé et de la recherche médicale (Inserm); Neuroscience Paris Seine — Institut de Biologie Paris Seine, Paris, France , Linda Brusamonti Sorbonne Université, CNRS, Institut national de la santé et de la recherche médicale (Inserm); Neuroscience Paris Seine — Institut de Biologie Paris Seine, Paris, France , and Sakina Mhaouty-Kodja Address correspondence to Sakina Mhaouty-Kodja, Sorbonne Université, CNRS UMR 8246, INSERM U1130, 7 quai St Bernard, Bât A 3ème étage 75005, Paris, France. Telephone: +331 44 27 91 38. Email: E-mail Address: [email protected] Sorbonne Université, CNRS, Institut national de la santé et de la recherche médicale (Inserm); Neuroscience Paris Seine — Institut de Biologie Paris Seine, Paris, France Published:27 January 2021CID: 017008https://doi.org/10.1289/EHP7662Cited by:1AboutSectionsPDF Supplemental Materials ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail AbstractBackground: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 50μg/kg/d) or to DEHP (5μg/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) α 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 ERα, 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/EHP7662IntroductionPhthalates are among the most frequently detected organic pollutants in the environment (Gao and Wen 2016), due to their extensive use as plasticizers in several commonly used products. Di(2-ethylhexyl) phthalate (DEHP) is the most abundant molecule of this family (Gao and Wen 2016). Previous studies in humans reported associations between phthalate metabolites in urine and reduced anogenital distance in boys (Bornehag et al. 2015) and interest in sexual activity in women (Barrett et al. 2014) or altered age of pubertal onset in girls (Berger et al. 2018). Experimental studies using rodents described adverse effects of developmental exposure to DEHP on sexual differentiation of the urogenital tract in males, age of puberty and testicular and ovarian functions (for review: Hannon and Flaws 2015; Howdeshell et al. 2008; Rowdhwal and Chen 2018). However, the potential effects of adult exposure to DEHP on the neural regulation of reproductive behavior have received less attention. In this context, we previously showed that exposure of adult male mice to DEHP at the tolerable daily intake dose (TDI) of 50μg/kg/d (EFSA 2005, 2019) or at lower doses close to the environmental exposure altered courtship behavior (Dombret et al. 2017). In particular, DEHP exposure lowered the emission of ultrasonic vocalizations (USVs) and the ability to attract females and delayed the initiation of mating. In female rodents, previous studies reported that acute exposure to DEHP during adulthood alters estrous cyclicity and ovarian function (Chiang et al. 2020; Davis et al. 1994; Hannon et al. 2014; Herreros et al. 2013; Li et al. 2012). Whether and how adult exposure to low environmental doses of DEHP affects female behavior and underlying neural structures remains to be investigated. Indeed, the effects of exposure to phthalates on female sexual behavior were analyzed only for perinatal exposure (Guerra et al. 2010; Lee et al. 2006).In female rodents, the expression of sexual behavior is limited to the estrus phase of the cycle, coinciding with ovulation (for review: Mhaouty-Kodja et al. 2018). This behavior is induced by a hormonal sequence involving the preovulatory surge of estradiol, which triggers both the ovulatory surge of pituitary luteinizing hormone (LH) and, via an estrogen receptor (ER) α-mediated action, the up-regulation of progesterone receptor (PR). Progesterone liberated following ovarian stimulation by LH induces female receptivity. Female sexual behavior includes an attractivity phase during which the female stimulates male behavior by emitting pheromones and a copulatory phase with the female adopting a receptive posture called lordosis when being approached from behind for insemination by the courting male. All these behavioral patterns are controlled by a neural circuitry involving the olfactory bulb, which transmits chemo-signals to the medial and posteromedial cortical amygdala and then to the bed nucleus of the stria terminalis and the ventromedial hypothalamus. This principal facilitatory system for lordosis behavior is activated by estradiol and progesterone during the estrus phase, as mentioned above. Inversely, the constraints exerted by the inhibitory system involving the hypothalamic preoptic and arcuate nuclei are lifted during this period.In the present study, we characterized the effects of chronic exposure of adult female mice to DEHP alone or in an environmental phthalate mixture on reproductive behavior and underlying neural structures. For this purpose, adult C57BL/6J female mice were assigned to one of four exposure groups. The first three groups included females exposed orally for 6 wk to the vehicle (control), DEHP at the TDI dose of 50μg/kg/d, or DEHP at 5μg/kg/d. The DEHP dose of 5μg/kg/d is within the environmental exposure range; this dose induced behavioral alterations in male mice following adult or pubertal exposure (Capela and Mhaouty-Kodja 2021; Dombret et al. 2017). To mimic the environmental coexposure to phthalates (Martine et al. 2013; Anses 2015), the fourth group of females was exposed to a phthalate mixture containing DEHP at 5μg/kg/d, dibutyl phthalate (DBP) at 0.5μg/kg/d, butylbenzyl phthalate (BBP) at 0.5μg/kg/d, diisobutyl phthalate (DiBP) at 0.5μg/kg/d and diethyl phthalate (DEP) at 0.25μg/kg/d. A first cohort of female mice including the four treated groups was analyzed for estrous cyclicity and body and uterine weights. A second cohort of females was ovariectomized and primed with estradiol and progesterone to induce their receptivity (acceptance of male mounting and display of lordosis behavior in response to mounts) under similar hormonal conditions. These females were analyzed for their lordosis and rejection behaviors as well as for olfactory preference in the presence of sexually experienced males. The ability of females and their pheromonal cues to attract male partners and induce the male emission of courtship USVs was also investigated. In this second cohort, locomotor activity was measured, and body weight (BW) was monitored during the whole period of treatment and behavioral analyses. The neural structures involved in the expression of sexual behavior and belonging to the facilitatory and inhibitory systems of lordosis behavior were studied for the number of ERα- and PR-immunoreactive neurons and mean fluorescence density.MethodsAnimals and TreatmentsStudies were performed in accordance with the French and European legal requirements (Decree 2010/63/UE) and were approved by the "Charles Darwin" Ethical committee (project number 01490-01). The experiments were reported following the Animal Research: Reporting In Vivo Experiments (ARRIVE) guidelines.Animals were obtained from breeding of male and female C57BL/6J mice (Janvier Labs) and housed in nest-enriched polysulfone cages with polysulfone bottles. Mice were kept at 22°C under an inverted light schedule, i.e., the dark time began at 1330 hours (1:30 P.M.) with a 12:12 h light–dark cycle, and fed a standard diet (A03–10; Safe-diets) with free access to food and water. Offspring were mixed at weaning to avoid potential litter effects (no more than one female per litter per cage, and one to two females per litter in each treatment group) and allowed to grow until 8 wk of age.Oral exposure was performed as previously described (Dombret et al. 2017). Phthalates (Sigma-Aldrich) were dissolved in ethanol and water (1% and 40% of prepared food, respectively) and incorporated by the experimenter into powdered food (A03–10; Safe-diets) that was then reconstituted into pellets. Eight-week-old females were fed ad libitum with chow containing the vehicle [i.e., ethanol and water (1% and 40% of prepared food, respectively; control group)], DEHP (CAS 117-81-7) at 50 or 5μg/kg/d (DEHP-50 and DEHP-5 groups, respectively), or a phthalate mixture (Mix group) containing DEHP at 5μg/kg/d, DBP (CAS 84-74-2) at 0.5μg/kg/d, BBP (CAS 85-68-7) at 0.5μg/kg/d, DiBP (CAS 84-69-5) at 0.5μg/kg/d, and DEP (CAS 84-66-2) at 0.25μg/kg/d. The composition of the phthalate mixture was based on French and European studies showing an external coexposure to these molecules (Martine et al. 2013) and the presence of their metabolites in urinary samples (Anses 2015; Dewalque et al. 2014). The ratio of DEHP over the other phthalates was determined on the basis of the estimated daily intake in France and Europe (Dewalque et al. 2014; Martine et al. 2013). Mice were weighed weekly, and phthalate doses were adjusted to their BWs and calculated for a daily food intake of 5g per animal. Reconstituted pellets were prepared every week immediately after animal weighing, stored at 4°C, and changed twice a week. Analyses started after 6 wk of exposure, and treatments were maintained during the whole period of the study.Experiments were conducted on two cohorts of female mice, each including 4 treated groups. The first cohort including 15 intact females per treatment group was subjected to analyses of estrous cyclicity; body and uterine weights were collected from all these females at necropsy (Figure S1). Behavioral analyses were conducted on a second cohort including 11 females from the control group, 12 from the DEHP-5 group, 13 from the DEHP-50 group, and 13 females from the Mix group, which were all ovariectomized and hormonally primed (Figure S1). At the end of behavioral analyses, females were sacrificed, and body and uterine weights were measured; the brains collected from 6 females per treatment group were processed for immunohistochemical analyses. All analyses were performed by blind observation, because females were identified by numbers attributed at weaning without any information concerning their treatment details.Estrous CyclicitySix weeks after the beginning of exposure, analyses of the estrous cycle were started while maintaining the treatment. Vaginal smears flushed with physiological saline were taken daily from females for 7 wk. The estrous cycle phases were identified by microscopy after hematoxylin/eosin coloration of the vaginal smears. The cycle duration was calculated as the average mean of days spent in seven complete cycles. The number of days spent in each stage of the estrous cycle was also determined. BW was examined during the whole period of treatment and estrous cycle analyses. The estrous cycle was monitored until sacrifice by pentobarbital injection (120mg/kg) to measure body and uterine weights of females at the metestrus stage.Behavioral TestsFour weeks after the beginning of exposure, female mice were ovariectomized under general anesthesia (xylazine 10mg/kg and ketamine 100mg/kg). At the time of ovariectomy, all females received 1cm subcutaneous SILASTIC™ implants (3.18mm outer diameter ×1.98mm inner diameter; Dow Corning) filled with 50μg of estradiol benzoate (Sigma-Aldrich) in 30μL sesame oil and sealed at each end with SILASTIC™ adhesive as previously described (Dombret et al. 2017; Naulé et al. 2014, 2015; Raskin et al. 2012). Two weeks later, behavioral analyses were started, and each female was given a subcutaneous injection of 1mg/100μL progesterone (Sigma-Aldrich) dissolved in sesame oil 4–5 h before each test to induce female receptivity. Tests were conducted under red-light illumination, 2 h after lights off and were videotaped for later analyses. They were conducted following the order indicated below and in Figure S1, starting with lordosis, then olfactory preference, partner preference, USV analysis, and ending with locomotor activity. All females of the second cohort were analyzed in the behavioral tests. Control untreated males were sexually experienced before the beginning of tests, which were performed with a different male per female (lordosis and USV tests) or per a pair of females or urine (partner preference tests). The devices used in the tests, with the exception of animal home cages, were cleaned with 10% ethanol between trials.Lordosis.Females were tested twice: in a first test (naive) and 2 wk later after this first sexual experience in a second test (sexually experienced). Each female was introduced into the cage of a sexually experienced male used as a partner. Tests ended after 20 min. The percentage of females exhibiting lordosis behavior, lordosis quotient (lordosis number/number of mounts) and rejection quotient (rejection number/number of mounts) were calculated for each subject in response to male mounting (Naulé et al. 2014, 2015).Olfactory preference.Olfactory preference was assessed in an enclosed plexiglass Y-maze as previously described (Capela et al. 2018; Dombret et al. 2017; Picot et al. 2014). Female mice were allowed to become familiar with the maze, where two empty perforated goal boxes were placed at each end, for 10 min over two consecutive days. On the day of the test, females were offered the choice between a sexually receptive female and a gonadally intact male, which were placed in the goal boxes. Stimuli were anesthetized to avoid any social interaction. Exposed females did not have direct access to these stimuli, but the perforated walls of goal boxes allowed air to flow from the boxes into the maze. The total time spent in chemo-investigation and the number of entries into each arm of the maze were scored during the 10-min test. The discrimination index was calculated as the time spent by exposed females in male investigation (M) minus the time spent in female investigation (F) divided by the total time of investigation (M-F)/(M+F).Partner Preference TestsThree-chamber test.Sexually experienced males were allowed to become familiar, for 10 min over 2 consecutive days, with the testing arena where two perforated goal boxes were placed in the side chambers as previously described (Dombret et al. 2017). On the day of the test, each male was placed in the neutral chamber and allowed to freely explore each chamber of the testing arena for 10 min. A female treated with DEHP alone or in mixture was placed inside a goal box and randomly assigned to the left or right chamber, while a vehicle-treated female was placed inside a goal box in the other chamber. The number of entries into each compartment and the time spent sniffing each female by the male over the 10-min test were scored.Y-maze test.Sexually experienced males were allowed to become familiar with the maze for 10 min over 2 consecutive days. On the day of the test, male mice were offered the choice between an anesthetized female from the vehicle group and an anesthetized female from the groups exposed to DEHP alone or in mixture, placed at each end of the maze inside perforated goal boxes.In the second version of this paradigm, female mice were replaced by their urine collected 1 h before the test. For this purpose, an equivalent volume of urine collected from all females of each treatment group was mixed, and 10μL of this mix was applied on a piece of filter paper. On the day of the test, male mice were offered the choice between a filter paper containing the urine from the vehicle group and a filter paper containing the urine from the groups exposed to DEHP alone or in mixture, placed at each end of the maze inside perforated goal boxes.For both tests, the time spent by males in chemo-investigation of each stimulus and the number of entries into each arm were scored during the 10-min test.Ultrasonic vocalizations.Each male was tested in its home cage in the presence of a female from one of the four treatment groups as previously described (Capela et al. 2018, 2019; Dombret et al. 2017). After the introduction of a female, vocalizations were recorded for 4 min with an UltraSoundGate microphone (Avisoft Bioacoustics), which was connected to an ultrasound recording interface plugged into a computer equipped with the Avisoft-SASLab Pro (version 5.2.09; Avisoft Bioacoustics) recording software. Vocalizations were analyzed using Avisoft-SASLab Pro (Avisoft Bioacoustics). Spectrograms were generated for each detected call (frequency resolution: FFT-length: 512; frame size: 100%; overlap: 50%). The parameters used for the automatic quantification of the vocalizations were: cutoff frequency of 30 kHz, element separation based on an automatic single threshold with a hold time of 15 ms. Syllables were identified and grouped into three main categories (simple, complex, frequency-jump). The total number and duration of USVs were analyzed, as well as the number and duration of each syllable.Locomotor activity.The computed circular corridor used to measure activity was made of two concentric cylinders crossed by four diametrically opposite infrared beams (Dombret et al. 2017; Raskin et al. 2009). The locomotor activity was counted when animals interrupted two successive beams and had thus traveled a quarter of the circular corridor. Activity was recorder for 120 min and was expressed as cumulative activity over the whole 120-min test.Body and uterine weight measurements.BW of female mice of the second cohort was monitored weekly during the whole period of treatment and behavioral analyses. At the end of behavioral experiments, animals were sacrificed by pentobarbital injection (120mg/kg), and the uterus was collected and weighed. The results were expressed as absolute body and uterine weights, and as relative uterine weight (percentage of BW).ImmunohistochemistryBrains from perfused animals were post-fixed overnight in 4% paraformaldehyde in phosphate buffered saline (PBS). Brains were then sliced into coronal sections of 30μm in a vibratome and processed for immunolabeling. Sections were blocked for 2 h with 2% normal donkey serum (Sigma-Aldrich) in PBS that contained 0.3% Triton-X100, then incubated with polyclonal anti-ERα antibody diluted at 1:400 (Santa Cruz Biotechnology) or polyclonal anti-PR antibody diluted at 1:400 (Dako-Agilent) for 72 h at 4°C. Immunofluorescence was performed with an Alexa Fluor 488-conjugated chicken antirabbit secondary antibody diluted at 1:500 (Life Technologies-Invitrogen) for 2 h at room temperature in the dark. After several rinses with PBS, sections were rinsed in water, mounted in Mowiol® and stored at 4°C in the dark. Sections were scanned using a high-resolution NanoZoomer Hamamatsu scanner (Hamamatsu Corporation). The number of labeled cells and mean fluorescence per section were counted by NDP.view (NDP.view2, Hamamatsu, Hamamatsu Corporation) and ImageJ software (ImageJ 1.53, NIH; Abràmoff et al. 2004), respectively, in anatomically matched sections identified using the Mouse Brain Atlas (Paxinos and Franklin 2001). ERα- and PR-immunoreactive cells were analyzed in the medial amygdala within an area of 0.56 mm2 (plate 46), in the bed nucleus of stria terminalis within an area of 0.70 mm2 (plate 30), in the ventromedial hypothalamus within an area of 0.12 mm2 (plate 46), in the medial preoptic area within an area of 0.80 mm2 (plate 30), in the arcuate nucleus within an area of 0.20 mm2 (plate 46) and in the posteromedial cortical amygdala within an area of 0.56 mm2 (plate 46).StatisticsData were expressed as means±S.E.M., except for those reporting the percentage of females exhibiting lordosis behavior, and analyzed by GraphPad Prism 6 (GraphPad Software).Normality tests (Kolmogorov-Smirnov and Shapiro-Wilks tests) were performed. Two-way analysis of variance (ANOVA) was used to analyze the main effect of exposure and stimulus on the number of entries for olfactory preference. The Kruskal-Wallis test was used to analyze the effect of exposure on the estrous cycle and stage durations, BW, lordosis and rejection quotients, the number of syllables (short, upward, one-jump), the duration of syllables (short, upward, downward, modulated, mixed, one-jump, and frequency-jump), the number of ERα-immunoreactive cells in the medial amygdala and arcuate nucleus, and the number of PR-immunoreactive cells in the medial amygdala and bed nucleus of stria terminalis. Dunn's post hoc tests were used to determine group differences. Student's one-sample t-test with 0 as the theoretical value was used to analyze the discrimination index for olfactory preference. Partner preference was analyzed by the Student's paired t-test or the Wilcoxon test. One-way ANOVA was used to analyze the effect of exposure on the remaining data. Bonferroni's post hoc tests were used to determine group differences. p-Values of <0.05 were considered to be significant.ResultsEffects of Adult Female Mice Exposure to DEHP Alone or in Mixture on the Estrous CycleAdult female mice were exposed orally for 6 wk, and analyses of the estrous cycle were started while maintaining the treatment for a further 7 wk. Figure 1A shows five consecutive estrous cycles represented for one female from each treatment group. Females exposed to DEHP alone or in phthalate mixture had cycles with longer durations compared with the control female. Quantitative analyses showed an effect of treatment on the mean cycle duration (p=0.0001), with significant longer durations for DEHP-5 (+33%; p=0.0003), DEHP-50 (+35%; p=0.0001) and Mix groups (+20%; p=0.027) than control females (Figure 1B). Detailed analyses of the duration of each stage showed different effects depending on the stage of the estrous cycle. An effect of treatment was observed on the proestrus stage (p=0.0001), with a shorter duration in the DEHP-5 (−12%; p=0.0021), DEHP-50 (−11%; p=0.0016), and Mix groups (−16%; p=0.0001) compared with control females (Figure 1C). There was also an effect of treatment on the estrus (p=0.0001) and metestrus durations (p=0.0001), whereas the diestrus stage remained unaffected (p=0.77). In particular, a longer duration of the estrus stage was noticed for the three treatment groups (+64% for the DEHP-5 group, +38% for the DEHP-50 group, and +52% for the Mix group compared with the control group). Similarly, the metestrus stage was longer for the DEHP-5 (+87%) and DEHP-50 groups (+97%) in comparison with control females.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 50μg/kg/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 means±S.E.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 means±S.E.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.Monitoring of BW during the whole period of treatment and estrous cycle analyses showed no significant differences between the treatment groups (Figure S2A). After estrous cycle analyses, body and uterine weights were measured at the metestrus stage. There was no effect of treatment on BW (p=0.48; Figure 1D), but an effect on uterine weight was detected (p=0.04), with a mean increase of 25% in comparison with the control group (Figure 1E). Post hoc analyses did not show significant differences between the treatment groups.Effects of Adult Mice Exposure to DEHP Alone or in Mixture on Lordosis Behavior and Olfactory PreferenceTo determine the behavioral effects of DEHP alone or in a phthalate mixture at comparable hormonal levels, all the following analyses were performed on ovariectomized females, which received implants containing similar estradiol levels. Females were administered progesterone 4–5 h before each behavioral test to induce their receptivity.Lordosis behavior was first analyzed in naive (Test 1) and sexually experienced females (Test 2), in response to mounts of sexually experienced males as presented in Figure 2A. The percentage of females showing at least a lordosis posture was not statistically different between the four treatment groups for Tests 1 and 2, although a tendency toward lower percentages was observed in females exposed to DEHP alone or in phthalate mixture (Figure 2B). The quantification of the lordosis quotient showed no effect of treatment on Test 1 (p=0.21), but a significant effect on Test 2 (p=0.001) (Figure 2C). Post hoc analyses showed a lower quotient of the DEHP-5, DEHP-50 and Mix groups in females, in comparison with the control group (−55%, p=0.0045; −56%, p=0.0049; −54%, p=0.0062, respectively). This was mainly because behavior in Test 2 was improved in control females (+147% vs. Test 1), whereas it remained low in the three other treated groups. The rejection quotient in Test 2 was also affected by treatment (p=0.0201), but not in Test 1 (p=0.0931), with a significant higher quotient in the DEHP-50 group (+271%, p=0.0281 vs. the control group) (Figure 2D).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 per treatment group) showing lordosis behavior in the four treatment groups exposed to the vehicle (Veh, control), DEHP at 5 or 50μg/kg/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 (means±S.E.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 means±S.E.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 means±S.E.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.Female sexual behavior is activated by olfactory cues emitted by the male partner. We tested the ability of females to discriminate between male and female odors in preference tests using gonadally intact males vs. sexually receptive females (Figure 2E). In this Y-maze paradigm, the total time spent sniffing the stimuli was equivalent for females from the four exposure groups (p=0.53) (Figure 2F). Two-way ANOVA of the number of entries into each arm showed no e