Transgenerational Effects of Prenatal Endocrine Disruption on Reproductive and Sociosexual Behaviors in Sprague Dawley Male and Female Rats

Abstract

Endocrine-disrupting chemicals (EDCs) lead to endocrine and neurobehavioral changes, particularly due to developmental exposures during gestation and early life. Moreover, intergenerational and transgenerational phenotypic changes may be induced by germline exposure (F2) and epigenetic germline transmission (F3) generation, respectively. Here, we assessed reproductive and sociosexual behavioral outcomes of prenatal Aroclor 1221 (A1221), a lightly chlorinated mix of PCBs known to have weakly estrogenic mechanisms of action; estradiol benzoate (EB), a positive control; or vehicle (3% DMSO in sesame oil) in F1-, F2-, and F3-generation male and female rats. Treatment with EDCs was given on embryonic day (E) 16 and 18, and F1 offspring monitored for development and adult behavior. F2 offspring were generated by breeding with untreated rats, phenotyping of F2s was performed in adulthood, and the F3 generation were similarly produced and phenotyped. Although no effects of treatment were found on F1 or F3 development and physiology, in the F2 generation, body weight in males and uterine weight in females were increased by A1221. Mating behavior results in F1 and F2 generations showed that F1 A1221 females had a longer latency to lordosis. In males, the F2 generation showed decreased mount frequency in the EB group. In the F3 generation, numbers of ultrasonic vocalizations were decreased by EB in males, and by EB and A1221 when the sexes were combined. Finally, partner preference tests in the F3 generation revealed that naïve females preferred F3-EB over untreated males, and that naïve males preferred untreated over F3-EB or F3-A1221 males. As a whole, these results show that each generation has a unique, sex-specific behavioral phenotype due to direct or ancestral EDC exposure.

Keywords: Aroclor 1221 (A1221); endocrine-disrupting chemical (EDC); estradiol; mating behavior; paced mating; polychlorinated biphenyl (PCB); social behavior; transgenerational; ultrasonic vocalization (USV).

Figure 1

The transgenerational experimental design. Abbreviations: EDC: endocrine-disrupting chemical, E: embryonic day, DMSO: dimethyl sulfoxide, EB: estradiol benzoate, A1221: Aroclor 1221, and P: postnatal day. Gray shading indicates those generations used in the current study for mating behaviors. The F3 generation was used for sociosexual behaviors.

Figure 2

Boxplots of body weight at euthanasia (~P120) for adult (a) males and (b) females. Data were analyzed by one-way ANOVA or Kruskal–Wallis for effect of F0 treatment, followed by Holm–Sidak or Dunn’s pairwise comparisons. ** p < 0.01. In this and other boxplot graphs, the line represents the median, the lower and upper outline of the boxes the 25th and 75th percentile, respectively, and the lines the 95th percentile.

Figure 3

Boxplots of male mating behavior at P60 for F1 and F2 generations. Sexually naïve males were scored for (a) latency to mount, (b) latency to intromit, (c) latency to ejaculate, (d) postejaculatory interval, (e) mount frequency, (f) intromission frequency, (g) intromission ratio (calculated as number of intromissions divided by number of mounts), and (h) copulatory rate (calculated as the number of mounts and intromissions from the start time until ejaculation). Data were analyzed by one-way ANOVA or Kruskal–Wallis for effect of F0 treatment, followed by Holm–Sidak or Dunn’s pairwise comparisons. F1: n = 14 DMSO, 11 EB, 11 A1221; F2: n = 26 DMSO, 22 EB, 22 A1221. * p < 0.05.

Figure 4

Boxplots of female mating behavior at P60 for F1 and F2 generations. Sexually naïve females were scored for (a) lordosis quotient, (b) lordosis intensity score, (c) latency to first lordosis response, (d) proceptive behavior frequency, (e) proceptive rate, (f) rejection behavior frequency and (g) rejection rate. Data were analyzed by one-way ANOVA or Kruskal–Wallis for effect of F0 treatment, followed by Holm–Sidak or Dunn’s pairwise comparisons. F1: n = 14 DMSO, 11 EB, 12 A1221; F2: n = 23 DMSO, 22 EB, 23 A1221. * p < 0.05.

Figure 5

Ultrasonic vocalizations (USVs) emitted by F3−generation males and females (P60–120) in response to an opposite−sex rat. An untreated control group (UNT) was raised across generations in−house alongside the F3 litters. (a) Timeline of USV experiment; (b) Diagram of experiment on day 3; (c) example spectrogram of recorded USVs; (d) Boxplots of the total number of USV calls (frequency modulated [FM] and non−FM) during the first 5 min of recording by sex; (e) Boxplots of the total number of USV calls with sex combined. Data were analyzed by two−way ANOVA or Aligned Rank Transformation (ART) for effect of F0 treatment and sex, followed by Holm−Sidak or ART−C pairwise comparisons. Males: n = 6 UNT, 10 DMSO, 6 EB, 6 A1221; females: n = 5 UNT, 9 DMSO, 6 EB, 8 A1221. + p < 0.07; ** p < 0.01.

Figure 6

Partner preference (PP) in a mating context by F3-generation males and females (P60–120). The Chooser was a naïve rat purchased from Harlan, given a choice between two opposite-sex rats: an in-lab-generated F3 rat (UNT, DMSO, EB, A1221) and a purchased rat. A preference score was calculated by subtracting time spent with the F3 rat minus time spent with the Harlan rat, in which positive numbers indicate more time spent near the F3-lineage rat and a negative score indicating time towards the Harlan rat. (a) Timeline of PP experiment; (b) diagram of experiment on day 3, with the wire zone shaded in gray; preference scores from the wire zone for (c) naïve female Choosers with F3-lineage males and (d) naïve male Choosers with F3-lineage females. Data were analyzed by linear mixed model (LMM) for effect of F0 treatment within each sex followed by Holm–Sidak pairwise comparisons. LMM estimated marginal means and standard errors are graphed. Males: n = 5 UNT, 9 DMSO, 7 EB, 8 A1221; females: n = 5 UNT, 10 DMSO, 7 EB, 7 A1221. * = p < 0.05 vs. UNT; + = p ≤ 0.083 vs. UNT.