Developmental Exposure to Ethinylestradiol Affects Reproductive Physiology, the GnRH Neuroendocrine Network and Behaviors in Female Mouse

Abstract

During development, environmental estrogens are able to induce an estrogen mimetic action that may interfere with endocrine and neuroendocrine systems. The present study investigated the effects on the reproductive function in female mice following developmental exposure to pharmaceutical ethinylestradiol (EE2), the most widespread and potent synthetic steroid present in aquatic environments. EE2 was administrated in drinking water at environmentally relevant (ENVIR) or pharmacological (PHARMACO) doses [0.1 and 1 μg/kg (body weight)/day respectively], from embryonic day 10 until postnatal day 40. Our results show that both groups of EE2-exposed females had advanced vaginal opening and shorter estrus cycles, but a normal fertility rate compared to CONTROL females. The hypothalamic population of GnRH neurons was affected by EE2 exposure with a significant increase in the number of perikarya in the preoptic area of the PHARMACO group and a modification in their distribution in the ENVIR group, both associated with a marked decrease in GnRH fibers immunoreactivity in the median eminence. In EE2-exposed females, behavioral tests highlighted a disturbed maternal behavior, a higher lordosis response, a lack of discrimination between gonad-intact and castrated males in sexually experienced females, and an increased anxiety-related behavior. Altogether, these results put emphasis on the high sensitivity of sexually dimorphic behaviors and neuroendocrine circuits to disruptive effects of EDCs.

Keywords: GnRH; endocrine disruption; ethinylestradiol; neuroendocrinology; reproduction; sexual behavior.

Figure 1

Experimental design. F1 females were exposed to 0.1 and 1 μg of ethinylestradiol (EE2)/kg (body weight)/day, corresponding respectively to environmentally-relevant (ENVIR) and pharmacological (PHARMACO) doses. Exposure began at embryonic day (E) 10 by exposing F0 pregnant and then lactating dams through their drinking water until postnatal day (PND) 21. After weaning, F1 females continued to be exposed through drinking water until PND40. Vaginal opening was assessed during the peripubertal period (PND23 to PND32). Neuroanatomical studies were performed on 8-week-old females. Estrus cyclicity was assessed between 14 and 17 weeks of age. Maternal behavior was assessed on nulliparous females. Social-sexual preference tests were performed on sexually-naïve females, lordosis behavior was then assessed in 7 trials which were followed by a second test of social-sexual preference. Anxiety-like behavior was assessed in an elevated plus maze device. Finally, females were mated with fertile males to assess fecundity and fertility.

Figure 2

Effects of developmental EE2 exposure on reproductive physiology. (A) Body weight of female mice exposed to the vehicle (Control; n = 20) or to EE2 [ENVIR (n = 13) and PHARMACO (n = 20)] was evaluated from PND22 up to PND92. Data are expressed as means ± SEM. (B) Vaginal opening pattern for each experimental group. Cumulative percentages of female mice showing vaginal opening according to age and experimental condition are presented from PND23 to PND32. Dissimilar letters indicate significant differences at each PND, p < 0.05 using the Chi² test of Pearson (a = ENVIR vs. Control. b = PHARMACO vs. Control. (C,D) The estrus cycle was evaluated daily for 14- to 18-week-old females during three cycles. (C) Estrus cycle length given as a percentage of females showing a complete cycle over four consecutive days (4 days) and females that did not complete a cycle over 5 days of vaginal cytology analyses (>4 days). Chi² test comparing Control vs. ENVIR or Control vs. PHARMACO groups, ^***p < 0.001. (D) Average occurrence of proestrus/estrus (follicular phase) vs. metestrus/diestrus (luteal phase) stages and per experimental group during 3 weeks of vaginal cytology analyses in adult females. One-way ANOVA (F = 4.32). ^*Tukey's multiple comparison test; ^*p < 0.05. Paired t-test: ^#p < 0.05; ^###p < 0.001.

Figure 3

EE2 altered the establishment of GnRH neuron network. (A)Total number of GnRH immunoreactive neurons in the POA for each experimental group. (B) Curve fit of the distribution of GnRH neurons centered on the OVLT through a rostro-caudal axis from the median septum (-400 μm) up to the medial preoptic area mPOA (+400 μm). Extra sum-of-square-test F_{(6, 103)} = 5.75, P < 0.0001. (C) Quantification of the labeled area of GnRH in three parts of the median eminence (posterior, middle and anterior). Tukey's box-plots of GnRH labeling according to the experimental group. The band is the median and (+) is the mean. Kruskal-Wallis test; ^*p < 0.05. Dunn's Multiple Comparison test (statistically different from Control). Numbers in brackets are effectives of animals per group. (D) Representative photographs of immunohistochemical labeling with DAB-Ni staining of GnRH perikarya in the Organum Vasculum of the Lamina Terminalis (OVLT) (upper panel) and immunohistochemical staining with fluorescence of GnRH fibers in the median eminence (ME) (lower panel). 3V: third ventricle. Scale bar = 100 μm.

Figure 4

The number of kisspeptin neurons was not disturbed by EE2 exposure. (A) Tukey's boxplots of the numbers of kisspeptin neurons counted in the periventricular preoptic nucleus (PVpo). (+) is the mean, numbers in brackets are effective of animals per group. No statistical differences were detected. (B) Photomicrographs of coronal brain sections counterstained with DAPI (blue) and immunostained for kisspeptin (red). Arrowheads indicate kisspeptin perikarya along the third ventricle (3V). Scale bar = 20 μm.

Figure 5

EE2 impaired social and sexual behaviors of females. (A) Sexual preference tested in naïve (without any contact with an adult male) and experienced (after mating behavior tests) females at the estrus stage. Histograms present the time spent sniffing the gonad-intact or the castrated male over 10 min. ^*** Unpaired t-test comparing the average time spent by females with the gonad-intact or the castrated male. (B,C) Female sexual behavior (lordosis behavior): females were tested on 7 trials at the day of estrus: for each female, a trial was carried out once per cycle. (B) Lordosis quotient (LQ): number of lordosis postures/total number of successful mounts × 100. Two-way ANOVA, p = 0.02. (C) Percentages of females showing lordosis posture during behavioral tests. Chi² test comparing each exposed group to the Control group for each trial. ^ap = 0.06; ^**p < 0.01; ^***p < 0.001.

Figure 6

EE2-exposure increased anxiety of females. (A) Percentages of time spent in the open arms of an elevated plus maze (EPM) (Kruskal Wallis test; p = 0.001). (B) Numbers of entries into the open arms of the EPM (Kruskal Wallis test; p = 0.01). Bonferroni multiple comparison test: ^*p < 0.05; ^*p < 0.001; ^***p < 0.001.