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

**Bailey A. Kermath 1 , Lindsay M. Thompson 2 , Justin R. Jefferson 2 , Mary H. B. Ward <sup>2</sup> and Andrea C. Gore 1,2, \***


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

#### **1. Introduction**

Endocrine-disrupting chemicals (EDCs) interfere with hormone action within an organism [1,2]. These chemicals, or mixture of chemicals, act upon the neuroendocrine systems that govern physiological processes such as reproduction, immune function, metabolism, and sex-typical behaviors in adulthood. Exposure to environmental EDCs during critical periods of development such as gestation can alter the organization of these neuroendocrine systems and predispose organisms towards disease and maladaptive traits. Known as the Developmental Origins of Health and Disease or DOHaD [3], this phenomenon has been well studied for a variety of health outcomes in individuals who experienced direct exposure early in life (F1 generation). Regarding neuroendocrine functions and hormonedependent behaviors, the focus of this study, exposures to EDCs including bisphenol A

**Citation:** Kermath, B.A.; Thompson, L.M.; Jefferson, J.R.; Ward, M.H.B.; Gore, A.C. Transgenerational Effects of Prenatal Endocrine Disruption on Reproductive and Sociosexual Behaviors in Sprague Dawley Male and Female Rats. *Toxics* **2022**, *10*, 47. https://doi.org/10.3390/ toxics10020047

Academic Editors: Tracie Baker and Jessica Plavicki

Received: 7 December 2021 Accepted: 14 January 2022 Published: 20 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

(BPA), phthalates, and persistent organic pollutants such as polychlorinated biphenyls (PCBs) induce adverse phenotypic outcomes in animal studies [4–17], and are associated with increased prevalence of neurobehavioral disorders in epidemiological studies in humans [18–22].

EDCs also exert actions on the F2 generation, exposed as germ cells within the F1 embryo. The F3 generations and beyond can exhibit phenotypic changes in the absence of direct exposure, presumably through germline epigenetic inheritance [23,24]. Although few in number, studies on inter- and transgenerational effects of EDCs have reported sexually dimorphic effects on behaviors, especially those influenced by early life endogenous hormones ([16,25–32]; reviewed in [33]). More research comparing generational effects is needed to better understand how legacy chemicals that are no longer actively manufactured but are still persistent in the environment, such as PCBs, may lead to heritable effects generations later.

The current study aims to build upon previous studies in the lab that identified transgenerational effects of PCBs on physiology, behavior, and hypothalamic gene expression throughout development [29,30,34,35]. Here, we extend these findings by examining mating behavior and sociosexual ultrasonic vocalization and partner preference activity in the F1, F2 and F3 generations to show sex- and generation-specific disruption in adult female and male rats.

#### **2. Materials and Methods**

#### *2.1. Experimental Design and Animal Husbandry*

All animal protocols were conducted in accordance with NIH and USDA guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Texas at Austin. Sprague Dawley rats were obtained from Harlan Laboratories (Houston, TX, USA), switched to the low-phytoestrogen Harlan-Teklad 2019 Global Diet ad libitum, and housed in same-sex groups (2–3 per cage) under constant humidity and temperature (21–22 ◦C) and a partially reversed 12:12 L:D cycle (lights on at 2400 h). Virgin females were impregnated in house. The morning after a sperm-positive vaginal smear was termed embryonic day (E) 1. On E16 and E18, during the period of sexual differentiation of the brain, F0 dams were weighed and randomly injected with one of three treatment groups: 1 mg/kg Aroclor 1221 (A1221, an estrogenic PCB mixture, administered intraperitoneally [i.p.]), 50 µg/kg estradiol benzoate (EB; administered subcutaneously [s.c.]), or a negative vehicle control (3% DMSO in sesame oil, injected i.p. or s.c., and combined into one DMSO group). Dosages and routes were selected to be identical to other studies in our lab and to be human relevant [35–39]. F0 litters were spread over 6 cohorts for a total of: DMSO, *n* = 14; EB, *n* = 11; A1221, *n* = 12.

Behavioral and physiological reproductive endpoints were examined after rats reached sexual maturity, using 1 male and female from each litter (Figure 1). F1 males and females were examined for sexual behaviors as young adults (P60) while mated to naïve rats (purchased from Harlan). After behavioral testing, F1 females carried litters to term. F2 offspring were also observed for sexual behavior during mating at P60 and the pregnant F2 dams carried the F3 generation to term. Finally, F3 maternal-maternal lineage females and paternal-paternal lineage males were examined for adult sociosexual behaviors (P60–120). A set of untreated rats (UNT, *n* = 6) were raised in the lab alongside the F3 offspring as an additional negative control group, in which dams were restrained and finger-poked to simulate an injection. Harlan-raised males and females used for F3 sociosexual experiments were received at 2 months of age and allowed to acclimate to the lab for 3–4 weeks before experimentation.

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 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.

#### 2.2. Tissue Collection *2.2. Tissue Collection*

Males and female rats were euthanized between P113–127. For all rats, adrenals and gonads were removed, weighed, and normalized to body weight. Trunk blood was collected from F1 and F2 rats, allowed to clot and spun at 1500× g for 5 min. Serum was separated and stored at −80 °C until further analysis. Males and female rats were euthanized between P113–127. For all rats, adrenals and gonads were removed, weighed, and normalized to body weight. Trunk blood was collected from F1 and F2 rats, allowed to clot and spun at 1500× *g* for 5 min. Serum was separated and stored at −80 ◦C until further analysis.

#### 2.3. Serum Hormone Assays *2.3. Serum Hormone Assays*

F1- and F2-generation serum samples were used to investigate the concentrations of circulating testosterone (males) and estradiol (females). Concentrations of serum testosterone were detected in duplicate using an RIA kit, as recommended by the manufacturer (Cat. No. 07189102, MP Biomedicals, Santa Ana, CA, USA). The assay range was 0.1–10 ng/mL, assay sensitivity 0.03 ng/mL and intra-assay variability 1.8%. Serum estradiol samples were run in duplicate using the estradiol RIA kit (Cat. No. DSL-4800, Beckman Coulter, Brea, CA, USA). The assay range was 5–720 pg/mL, assay sensitivity 2.2 pg/mL and intra-assay variability 3.0%. F1- and F2-generation serum samples were used to investigate the concentrations of circulating testosterone (males) and estradiol (females). Concentrations of serum testosterone were detected in duplicate using an RIA kit, as recommended by the manufacturer (Cat. No. 07189102, MP Biomedicals, Santa Ana, CA, USA). The assay range was 0.1–10 ng/mL, assay sensitivity 0.03 ng/mL and intra-assay variability 1.8%. Serum estradiol samples were run in duplicate using the estradiol RIA kit (Cat. No. DSL-4800, Beckman Coulter, Brea, CA, USA). The assay range was 5–720 pg/mL, assay sensitivity 2.2 pg/mL and intra-assay variability 3.0%.

#### *2.4. Ovariectomy and Hormone Priming for Sociosexual Experiments*

2.4. Ovariectomy and Hormone Priming for Sociosexual Experiments Stimulus females used in the ultrasonic vocalization testing were ovariectomized. During surgery, an estradiol Silastic capsule was placed s.c. between the shoulder blades. After recovery, these rats received a s.c. dose of 590 μg progesterone 4 h prior to use to induce receptivity. For the other behaviors, females remained ovarian-intact but were hormone-primed to ensure receptivity during experiments. Ovarian-intact females were given 50 μg estradiol s.c. 52 h, and 590 μg progesterone 4 h, prior to behavioral testing [32]. In all cases, receptivity was confirmed with a sexually experienced male that was Stimulus females used in the ultrasonic vocalization testing were ovariectomized. During surgery, an estradiol Silastic capsule was placed s.c. between the shoulder blades. After recovery, these rats received a s.c. dose of 590 µg progesterone 4 h prior to use to induce receptivity. For the other behaviors, females remained ovarian-intact but were hormone-primed to ensure receptivity during experiments. Ovarian-intact females were given 50 µg estradiol s.c. 52 h, and 590 µg progesterone 4 h, prior to behavioral testing [32]. In all cases, receptivity was confirmed with a sexually experienced male that was otherwise not used in the experiment.

#### otherwise not used in the experiment. *2.5. Reproductive Behavior and Fertility in F1 and F2 Rats*

2.5. Reproductive Behavior and Fertility in F1 and F2 Rats To determine whether prenatal endocrine disruption adversely affects adult reproductive behavior in the F1 and F2 generations, mating trials were conducted at P60 in a non-paced setting. F1 and F2 females were tested on the day of behavioral estrus with a To determine whether prenatal endocrine disruption adversely affects adult reproductive behavior in the F1 and F2 generations, mating trials were conducted at P60 in a non-paced setting. F1 and F2 females were tested on the day of behavioral estrus with a sexually experienced, Harlan-purchased male. F1 and F2 males were tested with sexually

naïve, Harlan females in behavioral estrus. Mating trials were performed under dim red light and videotaped for subsequent scoring. Males were acclimated for 10 min to the mating chamber (30 × 38 cm) 5 h before the trial and then returned to the same chamber for 5 min immediately before the trial start at 1600 h. The start time was recorded when the female was placed into the mating chamber. Trials only proceeded if the female was receptive and the male displayed mounting behavior within the first 20 min.

Videos were scored by an experimenter blind to treatment for the following male sexual behaviors: mount frequency, intromission frequency, latencies to mount, intromit, and ejaculate, and the postejaculatory interval (PEI). Because the experimental males were sexually inexperienced and thus slow to display mating behavior, their ejaculation latencies and PEI scores were capped at 30 min after the first mount and 15 min after ejaculation, respectively. Intromission rate was calculated as number of intromissions over the number of mounts with or without penetration. Copulatory rate was calculated as the number of mounts and intromissions from the start time until ejaculation. Female sexual behaviors scored were proceptive (hops and darts only, as ear wiggling could not be scored from the videotape), receptive (lordosis quotient, or the percentage of lordosis responses for the first 10 male mounts, and lordosis intensity score, rating the magnitude of each spinal dorsiflexion from 0 to 3, with 0 representing no spinal dorsiflexion and 3 an exaggerated dorsiflexion and head and rump elevation) and rejection (kicking, boxing, biting, escape, rolling) behaviors for the first 10 male copulatory acts. We further calculated the proceptive rate and rejection rate as the number of acts over the time scored and the latency to display the first lordotic response.

#### *2.6. USV Recording in Sociosexual Context in F3 Rats*

USVs were elicited in a sociosexual context for the F3 generation and recorded in a glass chamber (30 × 76 × 45 cm) equipped with an ultrasonic microphone (CM16, Avisoft Bioacoustics, Glienicke/Nordbahn, Germany), as published [6,29]. USVs were sampled at a 250 kHz sampling rate with 16-bit resolution through an A/D card (National Instruments, Austin, TX, USA) using RECORDER NA-DAQ software (v4.2.16, Avisoft Bioacoustics, Glienicke/Nordbahn, Germany). All trials were performed 1–3 h after lights off under dim red light. Experimental rats were sexually naïve, F3 EDC- and control-lineage males and females, aged P60–P120. F3 females were ovarian-intact and hormone-primed to be receptive on the final day of testing. Each experimental rat underwent three separate days of trials, following a previously validated protocol [40]. Days 1 and 2 consisted of a 10-min trial in the recording chamber to habituate the animals and obtain baseline USV recordings. On the final day, a sexually experienced stimulus rat of the opposite sex was placed into the chamber with the experimental rat, separated by a wire mesh partition. They were allowed to interact through the mesh wire for 5 min at which point the stimulus rat was removed from the room and 10 min of USVs were recorded from the experimental rat. Recorded USVs were analyzed with SASlab Pro software (v5.2.07, Avisoft Bioacoustics, Glienicke/Nordbahn, Germany), which automatically measures the number and acoustic parameters of USVs. Sonograms were generated under a 512 FFT-length and 75% overlap frame setup. As flat 50 kHz USVs may have unique communicative properties compared to calls with frequency modulation, USVs were separated into flat and frequency-modulated (FM) calls using an unbiased and replicable technique that categorizes USVs based on their bandwidth, or the maximum peak frequency minus the minimum peak frequency. Calls with a bandwidth of 5 kHz or more were classified as FM and a bandwidth of less than 5 kHz as flats (non-FM) [41,42]. The total number of 50 kHz USVs, number of FM and non-FM calls for the first 5 min of each recording session were analyzed.

#### *2.7. Partner Preference in F3 Rats*

F3 EDC- and control-lineage males and females were used after USV testing, approximately 4–7 h after lights off under dim red light. Partner preference trials were conducted as previously described [32]. All rats were sexually naïve and all females remained go-

nadally intact but were hormone primed to be receptive on the final day of testing. Trials were conducted in a glass arena (122 × 46 cm) and recorded by a video camera connected to ANY-maze software (v4, Stoelting Co., Wood Dale, IL, USA). In order to determine whether F3 EDC- or control-lineage rats could be distinguished from untreated animals in a mating-induced partner preference paradigm, we placed an F3 experimental rat (A1221-, EB-, DMSO-lineage or UNT) opposite a Harlan rat as the Stimulus rats. After Stimulus rats were placed on opposing sides of the arena, a Chooser rat of the opposite sex was allowed to explore and interact with the stimulus rats through wire mesh dividers [32]. Harlan-raised males and females were used as the Choosers and were a separate set from those used as stimulus animals.

Chooser rats were habituated to the empty arena in a 10-min trial on days 1 and 2. On day 3, Stimulus rats were placed behind opposite wire mesh dividers and allowed to acclimate for 5 min. Next, the Chooser rat was placed in the center of the arena and given 10 min to explore and interact with the Stimulus rats across the wire mesh. Trials were repeated up to three times, in which the location and identity of the stimulus rats were exchanged to avoid confounding biases. Behaviors (grooming, rearing, facial investigation, contact with Plexiglas dividers, speed) and total time and total time active (the combination of all scored behaviors) spent in each zone were scored by an experimenter blind to treatment and analyzed by ANY-maze software (v4, Stoelting Co., Wood Dale, IL, USA). Behavior from the area immediately surrounding the wire divider of the stimulus rat (the wire zone) was used for analysis. Data from the Harlan stimulus rat were subtracted from the F3-lineage rat to calculate a preference score in which positive numbers indicate more time spent near the F3-lineage rat.

#### *2.8. Statistical Analysis*

Data were analyzed with R 4.1.0 [43], the rstatix [44], the emmeans [45], the lme4 [46], the lmerTest [47], and the ARTool [48–50] packages. Scores over 2.5 standard deviations were considered outliers and removed from the analysis. When outliers were present, only one outlier was detected and removed per group with the one exception of the number of proceptive behaviors in the female F2-DMSO group, in which two outliers were removed. Outliers were distributed evenly across groups. Maternal and paternal lines in the F2 generation were combined for statistical analysis as parental lineage did not significantly impact the endpoints examined. For all somatic (F1, F2 and F3 generations) and mating behavior (F1 and F2 only) outcomes, a one-way analysis of variance (ANOVA) was run for Treatment. Kruskal–Wallis tests were used when data did not meet the Levene's homogeneity of variance or Shapiro–Wilk normality tests. Holm–Sidak or Dunn pairwise post hoc comparisons were run when a significant main effect was found. USV parameters were analyzed with a two-way ANOVA for Sex and Treatment. If data did not meet ANOVA assumptions, even after attempts of data transformation techniques, we used the Aligned Rank Transform (ART) for non-parametric factorial ANOVA [49,50] and the corresponding ART-C pairwise post hoc comparisons [48]. Finally, wire zone preference scores from the partner preference test were run separately for males and females using a linear mixed model with F0 treatment as a Fixed Variable, Animal ID as a Random Variable, and Trial Number as a Repeated Variable. For all data, alpha was set to 0.05.

#### **3. Results**

A summary of statistically significant results is provided in Table 1.


**Table 1.** Summary of significant results. Somatic (F1, F2, F3) Table 1. Summary of significant results. Table 1. Summary of significant results.

EB A1221 EB A1221

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Table 1. Summary of significant results.

DMSO (or UNT for Partner Preference). FM: Frequency modulated. n.s. and -: No significant effects. #: Number. Decreased compared to DMSO (or UNT for Partner Preference). Increased compared to DMSO (or UNT for Partner Preference). FM: Frequency modulated. n.s. and -: No significant ef-# Plexiglas bouts - - - Ultrasonic Vocalizations (F3) DMSO (or UNT for Partner Preference). FM: Frequency modulated. n.s. and -: No significant ef-DMSO (or UNT for Partner Preference). FM: Frequency modulated. n.s. and -: No significant effects. #: Number. Decreased compared to DMSO (or UNT for Partner Preference). Increased compared to Decreased compared to DMSO (or UNT for Partner Preference). Increased compared to Time rearing (trend) - # Rearing bouts (trend) - - - (combined for the sexes) EB A1221 (combined for the sexes) EB A1221 Partner Preference Behavior (F3) Total time active (trend) - - Ultrasonic Vocalizations (F3) (combined for the sexes) EB A1221 # Rearing bouts (trend) - - - Time at Plexiglas (trend) - # Rearing bouts (trend) - - - Time at Plexiglas (trend) - Time rearing (trend) - # Rearing bouts (trend) - - - Total time active (trend) - - Time rearing (trend) - Decreased compared to DMSO (or UNT for Partner Preference). Adrenal weight (normalized) - - (trend, F1 and F3) - Increased compared to DMSO (or UNT for Partner Preference). FM: Frequency modulated. n.s. and -: No significant effects. #: Number.

DMSO (or UNT for Partner Preference). FM: Frequency modulated. n.s. and -: No significant ef-

3.1.1. Males

3.1.2. Females

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Table 1. Summary of significant results.

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#### 3.1. Transgenerational Somatic Changes fects. #: Number. A trend was observed for an F0 treatment effect (EB slightly larger than DMSO) in nor-(combined for the sexes) EB A1221 3.1. Transgenerational Somatic Changes 3.1. Transgenerational Somatic Changes fects. #: Number. Time at Plexiglas (trend) - Decreased compared to DMSO (or UNT for Partner Preference). Increased compared to DMSO (or UNT for Partner Preference). FM: Frequency modulated. n.s. and -: No significant ef-Time rearing (trend) - # Plexiglas bouts - - - # Plexiglas bouts - - - Ultrasonic Vocalizations (F3) Time at Plexiglas (trend) - # Rearing bouts (trend) - - - Uterine weight (normalized) - A1221 > EB (F2) - - Hormones (F1, F2)—Estradiol (females), Testosterone (males)—n.s. *3.1. Transgenerational Somatic Changes*

Few somatic changes were detected in the measured outcomes for EDC-lineage rats.

DMSO (or UNT for Partner Preference). FM: Frequency modulated. n.s. and -: No significant ef-

#### 3.1. Transgenerational Somatic Changes malized adrenal weights of F1 (F(2,33) = 2.997, p = 0.064) and F3 (F(2,25) = 2.856, p = 0.076) 3.1.1. Males # Plexiglas bouts - - - Decreased compared to DMSO (or UNT for Partner Preference). Increased compared to Decreased compared to DMSO (or UNT for Partner Preference). Increased compared to Ultrasonic Vocalizations (F3) # Plexiglas bouts - - - Time at Plexiglas (trend) - 3.1.1. Males

3.1.1. Males

fects. #: Number.
