Rodent reproductive behavior among males and females after exposure to endocrine-disrupting chemicals

Abstract

Sexual reproduction—from both physiological and behavioral perspectives—is dependent upon appropriate connections between a diverse, hormone-modulated network of neural regions. Importantly, these substrates are regulated by hormones across the lifespan from early development to adulthood, making them targets of endocrine-disrupting chemicals (EDCs). Rodents, such as mice and rats, are invaluable to the characterization of EDCs because of their sex-specific, stereotyped appetitive and consummatory reproductive behaviors. Phthalates, bisphenol A (BPA), and EDC mixtures pose a salient risk to the health of humans, wildlife, and livestock because these synthetic compounds are ubiquitous due to their widespread use in mass production of consumer and industrial goods. This review outlines how the hypothalamic-pituitary-gonadal axis regulates male and female sexual behaviors, and how phthalates and BPA can perturb appetitive and consummatory behaviors and impact neural substrates that modulate reproductive behavior. We will then discuss how to progress toward a clearer understanding of the reproductive and neurobiological changes that occur due to EDC exposure.

In Brief:

The rodent reproductive behavioral control column (RBCC) of the hypothalamus and the hypothalamic-pituitary-gonadal (HPG) axis are critical systems that regulate copulatory behaviors in both males and females. We review how endocrine disrupting chemicals (EDCs), specifically phthalates, bisphenol-A, and chemical mixtures, dysregulate appetitive and consummatory copulatory behaviors and their neuroendocrine substrates, using mouse and rodent models.

Introduction

Reproduction is critical for species survival as it ensures gene transmission to the next generation. In mammals, successful copulation is regulated by a suite of behaviors that brings opposite-sex conspecifics together so they can combine gametes, leading to fertilization and the reproduction of an organism (Yin & Lin, 2023). Reproductive behaviors are outputs from wide-ranging neurobiological substrates in the central and peripheral nervous systems (CNS, PNS) (Mei et al., 2023). These substrates integrate multi-modal sensory cues—urinary odorants, pheromones, mating calls, and tactile contact, etc.—which represent social conditions necessary for copulation to occur (Wei et al., 2021). These CNS and PNS substrates are important aspects of the wider hypothalamic-pituitary-gonadal axis, the major circuit of sex-hormone signaling (Acevedo‐Rodriguez et al., 2018). During critical periods of sensitivity, such as development and puberty, circulating steroid hormones shape neural structures critical for producing adult sexual behaviors (Been et al., 2022; Phoenix et al., 1959). These organizational effects subsequently modify how neural regions respond to hormones and social cues, which can be described as activational effects. In adult mammals, the action of circulating gonadal hormones is indispensable to both the physiological and ethological components of reproduction (Jennings & De Lecea, 2020; Mei et al., 2023).

This review introduces endocrine-disrupting chemicals (EDCs) and their effect on rodent reproductive behavior. EDCs are potential threats to reproductive health because of these substances can alter hormone signaling systems, such as receptor expression; epigenetic modifications; endocrine metabolism and synthesis; and receptor agonism and antagonism (Lisco et al., 2022; U.S. Environmental Protection Agency, 1998). We focus on phthalates and bisphenol A (BPA), two EDC types that are ubiquitously found in the environment and in humans due to their use in the mass production of plastics and other consumer and industrial goods, including food products and personal care products (Kelly et al., 2020; Koch & Calafat, 2009; Mariana et al., 2023). Phthalates are a category that includes dozens of single compounds, including di(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP). In the real world, humans are exposed to mixtures composed of myriad EDCs, in both environmental and occupational contexts. (Fletcher et al., 2022). Therefore, EDC mixtures are also included in this review.

Here, special focus is also placed on rodents because in vivo rodent assays are integral to U.S. Environmental Protection Agency (EPA) guidelines on characterizing putative EDCs (Vandenberg, 2021). Furthermore, consistent with physiological aspects of EDC definitions, previous rodent studies demonstrate aberrations in sex-steroid synthesis, spermatogenesis, reproductive cyclicity, and birth outcomes due to phthalate and BPA exposure (Barakat et al., 2019; Chiang & Flaws, 2019; Laws et al., 2023; Richter et al., 2007; Rubin, 2011; Siracusa et al., 2018). The underlying regulation of these reproductive events is similar in rodent models and humans. Further, rodents are a consistent and useful model for understanding the behavioral impacts of endocrine-disrupting chemicals as the neural and hormonal regulation of these outputs is known.

Defining and identifying endocrine disrupting chemicals

In 1996, the U.S. Congress passed the Food Quality Protection Act (FQPA), mandating the EPA to test all existing pesticides for endocrine-disrupting properties (Maffini & Vandenberg, 2022). Under the FQPA, the EPA defines EDCs as a broad category of compounds that negatively affect reproductive physiology and behavior, with particular emphasis on the estrogen, androgen, and thyroid hormone (EAT) systems (Vandenberg, 2021; U.S. Environmental Protection Agency, 1998). The EPA utilizes dozens of in vitro and in vivo tests, using rodent, frog, and fish models to identify EDCs and their dose-response curves (Vandenberg, 2021; U.S. Environmental Protection Agency, 1998). It recommends Sprague Dawley and Wistar rats for in vivo assays measuring androgen and estrogen chemical interactions via weight changes of hormone-sensitive organs, such as the uterus, glans penis, liver, and kidney, among others (Endocrine Disruptor Screening Program & U.S. Environmental Protection Agency, 2011a, 2011b). Mating behavior tests, such as latency to copulation among rodents and other mammals, are important criteria to EPA’s definition of EDCs (U.S. Environmental Protection Agency, 1998). One large weakness in the EPA guidelines for identifying EDCs is their coarse evaluation endpoints that lack sensitivity (Vandenberg et al., 2012, 2019; Zoeller et al., 2012). Organ weight changes measure only overt toxicity, and few, if any, human diseases feature these gross metrics as diagnostic criteria (Vandenberg, 2021). Since the FQPA became law 28 years ago, the EPA identified 1,300 high-priority pesticides to evaluate; though fewer than 75 of which have been assessed (Maffini & Vandenberg, 2022). For an in-depth description and analysis of EPA guidelines on EDCs, see Vandenberg, 2021.

Phthalates are alkyl or dialkyl esters of phthalic acid, with at least one dozen unique phthalate chemicals in mass production of various industrial and consumer goods (Dickerson et al., 2012; Oehlmann et al., 2009). Since the 1920s, these synthetic chemicals have been used as additives to polyvinyl chloride (PVC) and other plastics to increase durability and flexibility, and today they are also used as gelling agents, lubricants, and detergents. They are also found in infant toys, plastic food containers, personal care products, and pharmaceuticals (Dickerson et al., 2012). Humans are exposed to a mixture of phthalates every day through ingestion, inhalation, and dermal contact (Heudorf et al., 2007; Mitro et al., 2016; Rowdhwal & Chen, 2018). Infants are perinatally exposed to phthalates through breast milk and amniotic fluid (Högberg et al., 2008; Kim et al., 2018; Renfrew et al., 2008). Importantly, exposure to phthalates during critical developmental windows can permanently alter neuroendocrine signaling pathways resulting in dysregulated gonadal hormone synthesis in adults (Barakat et al., 2019; Hannon & Flaws, 2015; Pell et al., 2017) This is important because it indicates that developmental exposures to phthalates could perturb the endocrine system and alter reproductive behaviors.

BPA is another widely prevalent EDC, with detectable levels observed in over 92 percent of Americans, and is one of the top 50 most-produced chemicals worldwide (Pelch et al., 2011). Around the world, over 2 million metric tons of this synthetic compound are consumed in the production of polymers, resins (epoxy, polysulfone, polyester), rubber chemicals, and other industrial goods. Like phthalates, BPA is found in many consumer-packaged goods including food and beverage products, baby bottles, and medical and dental devices. Although BPA has a short half-life and does not accumulate in human tissue, its ubiquitous environmental presence leads to continuous exposure among humans and animals and has been implicated in maternal-fetal transfer. Animal models show that BPA endocrine disruption includes alterations in hypothalamic and pituitary sex-steroid receptor expression and changes in brain sexual dimorphism (Dickerson et al., 2012). BPA is an estrogenic compound (Dickerson et al., 2012). But it can also dysregulate endocrine systems through interactions with androgen receptor (AR), aryl hydrocarbon receptor (AHR), and estrogen-related receptor-γ, demonstrating the divergent mechanisms that can be impacted by a single EDC. Therefore, it is difficult to anticipate the systemic consequences of EDC exposure, even with artificial, single-compound exposure models used in the laboratory (Pelch et al., 2011).

Reproductive behaviors of rats and mice

Reproductive ethology among rodents is organized into sexually dimorphic appetitive and consummatory phases (Ball & Balthazart, 2008). Appetitive behaviors describe variable searching behaviors, and consummation describes stereotyped behaviors that complete the behavioral goal. Thus, appetitive behaviors increase the likelihood that an organism will perform a consummatory action. With respect to reproductive behavior, many factors, including an animal’s memory of experiences, internal metabolic and hormonal states, and features of the external environment, such as visual and odor cues, influence the transition from an appetitive to consummatory phase (Wei et al., 2021). Because of the diversity of species within the Rodentia order, many salient and nuanced aspects distinguish copulation behaviors and patterns between rodent species, such as extent of partner preference, level of sociality, and duration of copulatory bouts. Here we focus particularly on rats and mice, due to their comparatively similar behaviors, vast research literature describing their reproduction, and common use as laboratory models for both reproductive and toxicology studies.

Appetitive phase of copulatory behavior:

A suite of interactions between adult males and females potentiates reproductive consummation. Both sexes detect, approach, and investigate conspecifics using multisensory domains, such as olfaction and audition (Yin & Lin, 2023). Female rats express a variety of complex behaviors which solicit and encourage male sexual attention, but the expression of these behaviors is dependent on the hormonal state. To attract male attention, females that are in estrus, a period with high circulating estrogens, will approach males, orient towards them, and then quickly dart away. Darting is often accompanied by a hopping gait and ear wiggling (Erskine, 1989; Wei et al., 2021). However, during diestrus, a period when estradiol levels are relatively low, females actively reject male copulatory advances and avoid opposite-sex interactions (Hardy, 1979; Wei et al., 2021). Female mice utilize darting and escape behaviors to pace and to control reproductive stimulation (Johansen et al., 2008). However, it remains unknown whether the darting of female mice has the same solicitory effect as it does in rats (Johansen et al., 2008).

In males, mice will investigate the anogenital region of potential mates, with rats also exploring the female’s face. A male mouse will often push the female with his nose or crawl beneath the female and lift her up (Hull & Dominguez, 2007). Volatile odorants and pheromones in male urine markings and female vaginal secretions contain information such as sex of an individual and reproductive status enabling animals to identify and attract potential mates (Roullet et al., 2011; Wei et al., 2021). Male mice preferentially investigate the urine of females in estrus compared to urine of non-estrus females or other odors (Harmeier et al., 2018). Naïve, sexually inexperienced female mice develop a preference for locations associated with male-soiled bedding (Martínez-García et al., 2009; Wei et al., 2021).

In mice, ultrasonic vocalizations (USVs) are important male social cues that solicit interactions with females. Upon encountering females or the urine of estrus females, males emit USVs (Harmeier et al., 2018). Males will also emit USVs during same-sex social contexts, though male-male interaction calls have simple acoustics compared to more complex harmonic copulatory USVs (Matsumoto & Okanoya, 2018). Physiologically, androgens modulate USV expression, making them vulnerable to potential EDC disruption (Dizinno, 1977). Behaviorally, a subordinate position within a social dominance hierarchy reduces male vocalizations during female interactions (Nyby et al., 1976). Females are attracted to male USVs and are, based on auditory stimulation alone, able to distinguish between males of differing hormonal states. Further, they prefer vocalizations of novel, non-kin males over familiar, male siblings (Egnor & Seagraves, 2016; Musolf et al., 2010; Premoli et al., 2021). Females also preferentially approach prerecorded adult male USVs over isolated pup calls or artificial tones (Hammerschmidt et al., 2009). Less studied are female USVs emitted during copulation; however, female USV production is confirmed to occur during murine copulation (Neunuebel et al., 2015). In rats, both sexes produce 50 kHz vocalizations during sexual interaction and prenatal exposure to EDCs can perturb the pattern of USVs (Hull & Dominguez, 2007; Lenell et al., 2021).

Thus, appetitive behaviors and sexual motivation can be measured by quantifying latency to approach urine, USV calls, or conspecifics, and the duration of time spent investigating these cues/conspecifics. The frequency of hopping and darting are female appetitive actions. Further, USV communication during social and sexual interactions is a hormonally modulated outcome that can provide important insights into both male and female sexual interest in the opposite sex partner (Mhaouty-Kodja, 2020).

Reproductive consummatory behaviors:

The three phases of copulatory consummation among male rodents include mounting, intromission, and ejaculation. During mounting, the male rodent grasps the female with his forepaws, covers her back with his ventral side, and produces several shallow thrusts. During intromission, the male locates her vagina with his penis while producing deeper thrusts. For copulation to result in fertilization, the male ejaculates to introduce his gametes to the female reproductive tract (Wei et al., 2021). Among male rats, the initial shallow pelvic thrusting occurs rapidly, between a frequency of 19 to 23 Hz (Hertz; occurrences per second). During rat intromission, as the male thrusts deeply, the period of physical conjunction between the male and female lasts for 200–300 milliseconds. A series of seven to ten intromissions, spaced apart by 1–2-minute periods of male genital self-grooming, occurs before ejaculation. Ejaculation is defined by a deeper thrust lasting 750 to 2000 milliseconds, a slower dismount from the female, and rhythmic contraction of penile, anal sphincter, and skeletal muscles. A refractory, post-ejaculatory interval (PEI) then occurs for 6 to 10 minutes before the male reengages in copulation. During the PEI, the male again grooms his genitals (Hull & Dominguez, 2007).

Relative to male rats, research literature on male murine copulatory behavior is less robust, and a great diversity of male consummatory reproductive behavior has been observed across mouse strains (Park, 2009). Unlike the relatively few numbers of intromissions occurring before a male rat ejaculates, male mice will intromit between 17 to 142 times before sperm is deposited in the female (Park, 2009). The PEI of a male mouse ranges from hours to days among commonly investigated strains. Typically, after ejaculation, the male mouse maintains a mount-like grasp on the female as the dyad falls to their sides, the male remaining catatonic for 10 to 30 seconds (Park, 2009). Thus, investigators typically measure number of mounts, intromissions, and ejaculations, latency to the first occurrence of these events, and the PEI.

Complementary to male consummatory behaviors is female receptivity, marked by a lordosis posture. Lordosis expression in rats and mice requires the hormonal context of the estrus phase of the reproductive cycle. With this posture, female rodents arch the spine ventrally. Arching exposes the pelvis and external genitalia dorsally to the male, facilitating intromission. Female behavior ultimately gates the success of male copulatory attempts by expressing either lordosis or avoiding his mounts. Similar to males, female rodents also experience a refractory period of low sexual interest and genital self-grooming following copulatory consummation (Yin & Lin, 2023). This has been revealed in both rats and mice when females are allowed to pace the frequency of interactions and mating events with their male partners (Steinberg et al., 2007). Measures of female receptivity among rodents include the lordosis quotient (LQ) and the latency to the first lordosis (Beach, 1976; Young et al., 1937). The LQ is the sum of lordosis events divided by the sum of mounts, multiplied by 100, occurring during the observation period (Beach, 1976). Reproductive efficiency can be measured as the number of intromissions over the number of mounts: A greater ratio indicates more effective male copulation (Jones et al., 2011). A reduced number of intromissions before ejaculation, however, does not equate to more efficient mating because diminished intromissions preceding ejaculation may not provide sufficient penile stimulation to induce the progestational state required for blastocyst implantation (Wilson et al., 1965).

As animals gain sexual experience, the choreography of reproductive behaviors is refined and becomes increasingly efficient, reflecting the animals’ learning and memory (Wei et al., 2021). Dexterous motor skills, such as those required for sex, is hypothesized to be dependent upon the physical repetition of practice (Papale & Hooks, 2018). Therefore, changes in rodent behavior across multiple copulatory encounters should also be considered. For example, hormone primed sexually naïve C57BL/J6 female mice have relatively low receptivity in response to male mating attempts, and females’ maximal sexual receptivity as measured by the LQ does not occur until the fourth encounter, each separated by several days (Bonthuis et al., 2011).

Neurobiological Substrates and the Regulation of Reproductive Behavior

Behavior, whether expressed in solitary or social contexts, is a systemic output reflecting physiological, cognitive, and emotional states of an organism. Considering the neurobiology of rats and mice, two major circuits govern expressions of social behavior: the hypothalamic-pituitary-gonadal (HPG) axis and reproductive behavior control column (RBCC). The HPG axis regulates sex-steroid secretion and function, which gates fertility and reproduction (Figs. 1, 2) (Acevedo‐Rodriguez et al., 2018). This pathway consists of hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei, which integrate signals from gonadal hormones and other brain regions. The hypothalamus receives these inputs, then communicates with the pituitary. The pituitary in turn sends hormonal signals to the gonads. The RBCC enables expression of sexual, parental, and aggressive behaviors and is comprised of connections between the medial preoptic nucleus (MPN), ventrolateral aspect of the ventromedial hypothalamus (VMHvl), and the ventral premammillary nucleus (PMv) (Mei et al., 2023). Excitatory and inhibitory neuronal pathways, utilizing glutamate and GABA (gamma-aminobutyric acid) neurotransmitters respectively, regulate activity of RBCC nuclei (Mei et al., 2023). Many hormonal cues shape RBCC activity, which then regulates social interest and behavior (Mei et al., 2023). Crucially, endocrine signals also modulate the developmental organization of the HPG and RBCC components. Thus, these networks are vulnerable to disruption by phthalates and BPA, among other chemical exposures. It remains to be investigated how EDCs interfere with glutamatergic and GABAergic signals which modulate the hormone-dependent RBCC nuclei that gate reproductive behavior.

Figure 1.

The female hypothalamic-pituitary-gonadal (HPG) axis is a major pathway modulating copulatory receptivity in female rodents. Thus, each HPG component is susceptible to harm from endocrine disrupting chemicals (EDCs). Cyclic feedback across this system establishes the appropriate hormonal context which enables rodent expression of consummatory lordosis. The symbols + and – represent positive and negative feedback actions, respectively. Kisspeptin (kiss) neurons in the anteroventral periventricular nucleus (AVPV) and arcuate (ARC) integrate sex-hormone signals originating from the ovary. The AVPV and ARC transduce these integrated ovarian signals and stimulate neurons which release gonadotropin releasing hormone (GnRH). This release of GnRH can occur in a pulsatile or surge manner. The GnRH signal then stimulates the anterior pituitary to release follicle-stimulating hormone (FSH) and luteinizing hormone (LH). These releasing hormones act on receptors on the ovary which maintains the continuing reproductive cycle.

Figure 2.

The male hypothalamic pituitary gonadal axis (HPG) is a connected system of reproductive structures which regulate reproductive behavior. Each aspect of the HPG axis is a potential target of endocrine disrupting chemical (EDC) action, requiring further study. The – symbols represent negative feedback functions. Functions of the male HPG axis, similar to the female counterpart, are reliant upon hypothalamic GnRH neurons. However, a major dimorphism in the HPG axis is the hypothalamic regulation of GnRH and LH release. Specifically, in rodents, only females demonstrate LH surge signaling and strong positive feedback of gonadal hormones on the hypothalamus. Abbreviations: ARC; Arcuate, AVPV; anteroventral periventricular nucleus, FSH; Follicle-stimulating hormone, GnRH; gonadotropin releasing hormone, LH, Luteinizing hormone.

The HPG Axis and Reproduction

Females:

In both female mice and rats biological fertility and behavioral receptivity are coincident and phase locked to an ovulatory cycle that repeats across four phases (Yin & Lin, 2023). Procession through these phases requires mutual regulation between the central nervous system and the gonads (Fig. 1). Most broadly, kisspeptin-expressing neurons in two hypothalamic nuclei—the arcuate (ARC) and anteroventral periventricular nucleus (AVPV)—detect ovarian estradiol and are hypothesized to act as GnRH pulse and surge generators, respectively (Herbison, 2020). Kisspeptin is released and it stimulates gonadotropin releasing hormone neurons (GnRH). GnRH neurons then release their product which travels to the anterior pituitary. The anterior pituitary releases luteinizing hormone (LH) and follicle stimulating hormone (FSH). These hormones then stimulate ovarian hormone release including estradiol and progesterone (Jennings & De Lecea, 2020; Mei et al., 2023; Yin & Lin, 2023). It is suggested that gradual increases in estradiol during diestrus and proestrus prime the AVPV and cause the GnRH surge in late proestrus; a hypothesized negative estradiol clamp on the ARC regulates GnRH pulse activity, which is subsequently suppressed by post-ovulatory progesterone and results in low-frequency GnRH pulses during estrus (Herbison, 2020).

In diestrus, gonadal estradiol and progesterone have relatively low concentrations (Jennings & De Lecea, 2020). As the cycle transitions from diestrus to proestrus, estradiol rises to a peak in the afternoon, and this rise leads to the positive feedback on the hypothalamus triggering the preovulatory LH surge (Jennings & De Lecea, 2020). Estrus follows during which ovarian follicles reach maturity, rupture, and release an egg or eggs into the oviduct. This is when the female rodent is sexually receptive and will display lordosis. The corpus luteum forms from the ruptured follicles and secretes progesterone (Yin & Lin, 2023). Due to physiological responses of the uterus to progesterone, biological fertility is maximized, with the eggs now accessible to insemination and the uterine walls ready to support implantation of a fertilized egg. Crucially, the sequence of apical estradiol and then an increase in progesterone locks female behavioral fertility—lordosis and receptivity to male insemination—to the time of her maximal biological fertility (Jennings & De Lecea, 2020; Yin & Lin, 2023).

In adult males, kisspeptin neurons in the ARC and AVPV activate GnRH neurons in the hypothalamus, which then release their product and cause the release of FSH and LH from the anterior pituitary (Fig. 2) (Nakamura et al., 2022). However, unlike females, there is no GnRH surge at the level of the hypothalamus or anterior pituitary. In both rats and mice, male kisspeptin neurons in the AVPV do not respond to elevate levels of estradiol, suggesting that sexual dimorphism in some aspect of the AVPV-kisspeptin neurons is a key factor underlying the absence of the LH surge in male mice and rats (Biehl et al., 2018; Clarkson & Herbison, 2006; De Vries & Södersten, 2009; Kauffman, 2022; Kauffman et al., 2007; Simonneaux, 2020; Smith et al., 2006; Williams III & Kriegsfeld, 2012). At the level of the testes, LH primarily acts by binding to receptors on Leydig cells, which produce testosterone. FSH acts by binding to its receptor found on Sertoli cells, which produce inhibin. Testosterone has negative feedback and inhibits the release of GnRH from the hypothalamus and the release of FSH and LH from the pituitary. Inhibin has negative feedback at the level of the anterior pituitary, which leads to the suppression of FSH (Tilbrook & Clarke, 2001). Estradiol, aromatized from testosterone, exerts negative feedback on the male HPG axis at the level of pituitary and hypothalamus. Within the brain, estradiol binds to receptors on kisspeptin-neurons which then inhibits kisspeptin and GnRH release (L. Hu et al., 2008; Kaprara & Huhtaniemi, 2018).

Male sexual behavior and endocrine-disrupting chemicals

Among male rodents, the most consistent behavioral consequence of exposure to EDCs is decreased appetitive and consummatory sexual behaviors. This is indicated by increased latencies to consummatory copulatory behaviors and reduced USVs produced during mating. There are exceptions to this as some EDC exposed males actually have shortened latencies to copulatory events and reduced frequencies of these events. Despite these disrupted reproductive outcomes, male preference for female over male conspecifics remains intact. Ultimately, studies examining various EDCs exposures across gestational, lactational, pubertal, and adult periods still find that male rodents can successfully copulate and are fertile, regardless of exposure paradigm.

Altered Male Sexual behaviors but Preserved Preference for Females

Experiments confirming EDC-induced reductions in sexual motivation and performance have been conducted with C57BL/6J mice and Wistar rats. Published studies (Supplemental Table 1) have tested the impacts of DEHP, BPA, DBP, a phthalate mixture, and a “NeuroMix” which contains phthalates and BPA in addition to other EDCs on reproductive characteristics. In mice, following chronic DEHP exposure (5 and 50 ug/kg/d) during adulthood, the latency to first intromission and ejaculation increased by about four times when compared to control animals (60 minutes compared to 15 minutes) (Dombret et al., 2017). Adult exposure to a mixture of phthalates (Supplemental Table 1) decreased mount and intromission frequency and increased the latency to the first ejaculation (Ducroq et al., 2023). Pubertal and developmental DEHP exposures at relatively low doses (tolerable daily intake; TDI or No Observed Adverse Effect Level; NOAEL) also increased the latency to mounting, intromission, and ejaculation events but increased the number of mounts, thrusts and intromissions (Capela & Mhaouty-Kodja, 2021). Among pubertally exposed mice, these delays occurred during both naïve and experienced copulatory encounters. For developmental exposure, only sexually experienced males had delayed consummatory latencies (Dombret et al., 2017). Wistar rats dosed with DEHP at 500 mg/kg/d from gestation day 1 (GD1) to postnatal day 21 (PND21) had an increased latency to intromission (38.3±2.3 to 64.0±6.4 seconds control vs. DEHP exposure, relatively) (Dalsenter et al., 2006). In male C57BL/6J mice, adult exposure to DEHP alone or as part of a phthalate mixture also altered male USVs during copulations. Specifically, males had fewer total vocalizations and a reduced cumulative duration of vocalizations emitted during copulation (Ducroq et al., 2023).

BPA exposure also affects sexual behaviors of male animals. BPA exposure from PND26–202 (176 days) at TDI and NOAEL doses caused subtle delays in male consummatory copulatory behavior (Picot et al., 2014; Wu et al., 2020). Within a ten-hour observation window, chronically exposed (NOAEL) male rats failed to ejaculate while paired with unexposed female partners. However, during a separate ten-day copulation period, exposed males successfully mated, albeit with significantly delayed detection of the first sperm plug compared to control mating pairs (5 vs. 3 days) (Wu et al., 2020). Male rats prenatally exposed to BPA throughout gestation had an increased intromission latency when tested with a sexually receptive female, although they also demonstrated an increase in the number of intromissions (Farabollini et al., 2002).

While increased latencies to reproductive consummation likely describe a reduction in male sociosexual motivation after EDC exposure, their preference for female over male conspecifics remains intact. Neither chronic-adult nor chronic-developmental exposure to BPA altered male C67BL/6J opposite-sex preference (Picot et al., 2014). Further, males exposed to single phthalates or in mixture maintained a bias for investigating estrus or hormone primed females over non-receptive females. (Capela & Mhaouty-Kodja, 2021).

Among sexually naive male C57BL6/J mice exposed to BPA at TDI levels, numbers of intromissions (15.20±1.99) and thrusts (390.40±57.29) were significantly reduced compared to vehicle controls (29.92±5.67; 588.69±54.35) (Picot et al., 2014). Sexually experienced TDI exposed males also had significantly fewer intromissions (8.23±1.38 vs 14.00±2.34) and thrusts (284.85±33.21 vs. 365.33±51.73) compared to control males, however the number of mounts was unchanged.

Interestingly, chemical exposures can also shorten latencies and increase frequencies of male sexual behaviors. Adult male, sexually naive Wistar rate exposed in utero to DBP at 100 mg/kg/d from GD15 through GD21 had decreased latencies to copulatory events. During a 15-minute assay, the latency to the initial mounting occurred about 7 times earlier in DBP-exposed compared to control animals (15.2 sec vs. 109.4 seconds, respectively). The first intromission occurred at 125.2 seconds in controls compared to 21.2 seconds in exposed males (Reznikov et al., 2021). In addition to this pattern of shorter latencies, DBP exposure also increased intromissions and ejaculations. In fact, male controls did not reach ejaculation during the testing period (15 minutes), but males prenatally exposed to DBP had an average of 1.2 ejaculations during that same period. Prenatal DBP exposed males with sexual experience maintained this pattern of increased intromission and ejaculation frequency compared to control males. DBP-exposed rats more readily achieved ejaculation as well, with the PEI decreasing by a factor of 3 (129. vs. 416.2 seconds) (Reznikov et al., 2021). Male attractiveness to opposite sex animals remains relatively unexplored across environmental toxicology and reproductive biology studies. This can be assessed by having female investigate between an EDC-exposed or control male, or their cues. Females choosing between urine from a control male compared to either a DEHP exposed male (50 ug/kg/d) or a phthalate mixture exposed male preferentially spent more time investigating control urine (Ducroq et al., 2023).

Thus, the expression of sexual behaviors in EDC exposed males are aberrant and males can exhibit an increase or a decrease in latencies to initial mounts, intromissions, or ejaculations. Potential explanations, which are not mutually exclusive, are that the salience or processing of sensory cues such as odor or USVs is altered, there are defects in endocrine signaling that modulate consummatory sexual behavior, neural regions that regulate these behavioral outputs have been altered in development, or there are perturbations in responsiveness to the activational effects of hormones in adulthood. We will address these possibilities in the next section.

Potential mechanisms of EDC alteration of sexual behavior in males

Male sexual behaviors and functions such as ejaculation are mediated by the HPG axis (see above), hypothalamic nuclei (medial preoptic area [MPOA], VMHvl) of the RBCC, the medial amygdala (MeA), and the bed nucleus of the striata terminalis (BNST) (Fig. 3). Activity of both the MPOA and VMHvl promote male copulatory behavior, unlike the PMv (Mei et al., 2023; Wei et al., 2021). Thus, these neural substrates are a major interest regarding EDC-induced perturbations. Motivation to vocalize and mate is under modulation of the medial preoptic nucleus (MPN), which integrates olfactory signals (incoming from the olfactory bulb, MeA, and bed nucleus of the striata terminalis [BNST]) into behavioral outputs (Dombret et al., 2017; Newman, 1999). The MPN and sexually dimorphic nucleus (SDN), are subregions within the MPOA (Dominguez and Hull, 2005; He et al., 2013) which are also broadly important to sexual behavior (Mei et al., 2023; Tsuneoka & Funato, 2021). These regions regulate copulatory behaviors, express the steroid hormone receptors AR, estrogen receptor type 1 (ESR1; also known as ER-alpha) and type 2 (ESR2; also known as ER-beta) and are also sexually dimorphic in size, cell number, or expression of specific peptides such as Calbindin or tyrosine hydroxylase (TH), depending on the region (Mei et al., 2023; Morishita et al., 2021; Tsukahara & Morishita, 2020; Wei et al., 2021) Changes in the sexual dimorphic properties of brain regions can demonstrate perturbations in neural development (Dominguez & Hull, 2005; He et al., 2013).

Figure 3.

Male reproductive consummation occurs when the male (brown mouse) grasps the flanks of the female (grey mouse), thrusts his pelvis, and intromits his penis into her vagina. Male copulation depends on the MPOA and VMHvl of the RBCC. In contrast to females the PMv does not regulate reproductive behavior (Mei et al., 2023; Wei et al., 2021). The excitatory (glutamatergic) and inhibitory (GABAergic) connections in this circuit connect hormone-sensitive nuclei and require further investigation with respect to EDCs. Most interesting, Bayless et al., 2023 report that GABAergic projections from the BNST to the MPOA are initially inhibitory but become excitatory. Abbreviations: BNSTp; Bed nucleus of the stria terminalis, posterior part; GABA; Gamma-aminobutyric acid; MeA; Medial amygdala; MPOA; Medial preoptic area; RBCC; Reproductive behavioral control column; VMHvl; Ventromedial hypothalamus, ventrolateral part.

Excitatory and inhibitory neurons connect a number of hormone-sensitive brain nuclei which regulate behavioral responses to specific external (social) and internal (endocrine) contexts. The social environment regulates both male and female rodent sexual motivation and copulation via the transduction of multimodal sensory cues including opposite-sex pheromones, volatile odors, and vocalizations. Chemosensory stimuli travel through the olfactory tract to stimulate the BNST and MeA, which then engage the MPOA and VMHvl of the RBCC (Wei et al., 2021). In male rodents, the specific nuclei and the circuitry between nuclei is still being investigated (Bayless et al., 2023). However, both the BNST and MeA synapse on the MPOA and VMHvl via GABAergic neurons (Bayless et al., 2023; Li & Dulac, 2018). Interestingly, these GABAergic neurons are initially inhibitory, but then induce an excitatory signal that is received by the MPOA (Bayless et al., 2023). Activation of the MPOA is necessary to elicit male mounting behavior. The MPOA and VMHvl mutually regulate each other: the MPOA inhibits the VMHvl, and the VMHvl excites the MPOA (Wei et al., 2021) (Fig. 3). While both the MPOA and (to a lesser extent) the VMHvl promote male sexual behavior, in vivo experiments demonstrate their temporally specific roles: During the onset of mounting, the VMHvl becomes activated, but this activity is suppressed during intromission (Guo et al., 2023; Lin et al., 2011; Mei et al., 2023). In contrast, male MPOA firing activity increases at the time a female is first encountered until ejaculation, when firing is suppressed (Shimura et al., 1994). The exact mechanism through which BNST and MeA inhibition balances the ratio of activity between the MPOA and the VMHvl, and their respective behavioral outputs in males, remains to be elucidated. In contrast to females, PMv activity in the RBCC is not known to affect male reproductive behavior (Fig. 3). Further, relatively little is known how auditory stimuli impacts the RBCC regulation of social behavior; however, recent work has investigated how endocrine (oxytocin) signaling affects parental behavior and cortical activity (Marlin et al., 2015; Wei et al., 2021).

Phthalates and the male rodent brain:

Exposure to relatively low doses of DEHP (5, 50 ug/kg/d) during male mouse adulthood does not change AR or ESR1 protein expression in the MPA, as measured by Western blot (Supplemental Table 2). However, pubertal exposure to 50 ug/kg/d causes a decrease in AR protein. Similar to adult DEHP exposure, pubertal male mouse exposure does not alter ESR1 protein (Capela & Mhaouty-Kodja, 2021).

There are additional changes observed in the hypothalamus after relatively low DEHP exposure during adulthood, specifically in the MPN. These include increased relative protein expression of GFAP and actin; and decreased number of AR-immunoreactive neurons and mRNA expression. Increased GFAP (glial fibrillary acidic protein) is a key indicator of astrocyte activation and diminished androgen. Therefore, it is unsurprising that adult DEHP exposure also increases the expression of the early-phase astrocyte activation marker NDRG2. Further, in the MPN, proteomic analysis identified 22 differentially expressed proteins implicated in the functioning of mitochondria, protein folding/chaperones, neuroprotection, cellular metabolism, nuclear chromatin, and the cytoskeleton. Androgen modulation of cytoskeletal proteins GFAP and beta-actin underlie organizational changes in glia and neurons. These organizational changes are important to many neural functions, including reproductive behavior (Dombret et al., 2017).

BPA and the male rodent brain:

Changes in hypothalamic cell number and expression of TH have been observed in male Sprague Dawley rats after BPA exposure. Specifically, acute injections of BPA on PND 1 and 2 significantly increased the number of TH-immunoreactive cells in the AVPV in males (PND19) (Patisaul et al., 2006), increased the size of the AVPV, and increase the number of Calbindin positive cells in the SDN (PND85) (Patisaul et al., 2007). In contrast, a longer gestational and lactational exposure to BPA in rats did not alter TH cell number (Rubin et al., 2006). In male C57BL6/J mice exposed to BPA in utero, there was no change the number of kisspeptin or calbindin cells in the preoptic area of the hypothalamus (Picot et al., 2014). The NeuroMix EDC formulation which Gore et al., 2022 utilizes includes BPA, DEHP, and DBP, alongside other chemicals that dysregulate the endocrine system, such as vinclozolin and PCB. Male rats exposed in utero to this mix had a decrease in ESR2 expression in the MeA, but there was no change in AR expression.

EDC mixtures and the male rodent brain:

A phthalate-only mixture, as well as NeuroMix, have been investigated with chronic adult and chronic embryonic exposures, using mouse and rat, respectively (Supplemental Table 2). In mice, in the MPOA there were pan-metabolic changes after exposure to a phthalate-only mixture. These changes coincided with increased AHR expression and diminished AR protein expression. ESR1 protein expression was unchanged (Ducroq et al., 2023). NeuroMix (which includes phthalates, BPA, and other EDCs) did not disrupt Ar mRNA expression in the MeA or paraventricular nucleus (PVN). Similarly, in the PVN, Esr2 mRNA expression was unchanged, however, Esr2 mRNA expression decreased in the MeA (Gore et al., 2022).

Female sexual behavior and endocrine-disrupting chemicals

In females, reduced sexual motivation results from DEHP exposure, both as a single chemical and as a component of a phthalate mixture (Supplemental Table 3). Female mice typically prefer a male over a female conspecific; however, this preference is lost as measured by a sex-discrimination index. Specifically, females exposed to DEHP or a phthalate mixture in adulthood did not prefer an opposite sex male compared to a female (Adam et al., 2021). Female rats exposed to NeuroMix from GD8- GD18 had no significant preference for a testosterone treated male stimulus compared to a male not treated with hormone. This contrasts with control females who did prefer to investigate a hormone treated male (Gore et al., 2022). Reduced female reproductive interest was also observed as an increased rate of events where females actively rejected male mounting (Adam et al., 2021). However, despite these indicators of reduced motivation, exposed females maintain lordosis capacity (Adam et al., 2021).

Female consummatory behavior, specifically lordosis number or lordosis quotient, decreases in response to EDC exposure. Female mice exposed to DEHP or a phthalate mixture had a reduced LQ (Adam et al., 2021). A similar result was seen in Wistar-Omamichi rats exposed to the individual phthalates DBP, diisononyl phthalate (DiNP), or Di-2-ethylhexyl adipate (DEHA) during gestation and lactation (Lee et al., 2006). In comparison to phthalate exposures, effects of BPA on lordosis behaviors are mixed. Early postnatal exposure to BPA in Long-Evans and Wistar rats did not alter the LQ when females were hormone primed with estradiol and progesterone and tested as adults; however, a similar developmental exposure to BPA reduced hopping and darting proceptive behaviors (Adewale et al., 2009; Monje et al., 2009; Ryan et al., 2010). In paced mating experiments, where chambers are designed to allow females to regulate their interaction with male partners, female Wistar rats administered BPA—either before or after birth—had increased lordosis frequency and approached the males faster when compared to controls (Farabollini et al., 2002). The expected opposite-sex preference and investigatory bias in females was maintained after BPA TDI and NOAEL exposure (GD15-weaning) in C57BL/6 mice (Naulé et al., 2014). Similar to the increased lordosis behavior among the BPA Wistar rats, in utero BPA administration in C57BL/6 mice caused increased female consummatory behavior, as measured by the LQ however, this increased consummation occurred only during naïve, not experienced, copulatory encounters (Naulé et al., 2014). Prenatal exposure to NeuroMix resulted in a decreased investigation of male stimuli or nose touching with male stimuli (Gore et al., 2022).

Female attractiveness to males is impaired following exposure to environmental chemicals. Multiple assays utilizing female urinary cues, anesthetized females, or active female subjects as investigatory targets presented to sexually experienced males, confirm that DEHP exposure and phthalate mix exposure reduces female attractiveness (Adam et al., 2021). Specifically, males spent more time investigating a control female or her urine compared to exposed females. Reductions in male copulatory vocalizations while paired with DEHP exposed females further confirmed that exposed females were less attractive compared to control animals.

Potential mechanisms of EDC alteration of sexual behavior and lordosis in females

As described above for male rats and mice, female sexual behaviors and responses depend upon appropriate development and hormone signaling within the HPG axis and specific hypothalamic brain regions including the AVPV, ARC, MPOA, VMH, and BNST (Yin & Lin, 2023). Similar to males, the MeA and BNST transduce chemosensory cues representing male conspecifics to the RBCC nuclei (MPOA, VMHvl, PMv) (Yin & Lin, 2023). Lordosis expression is dependent upon chemosensation, with disruption to the olfactory organs causing a reduction in female receptivity (Yin & Lin, 2023). Behavioral outputs from activation of RBCC nuclei differ between the sexes. The MPOA inhibits lordosis, while activation of the lateral subdivision of the ventrolateral part of the VMH (VMHvll) and the PMv promote expression of female reproductive consummation (Mei et al., 2023; Yin & Lin, 2023). As in males, excitatory glutamatergic and inhibitory GABAergic neuronal connections across the RBCC and associated nuclei regulate hormone-sensitive brain activity and behavioral expressions (Fig. 4). Demonstrating the disinhibitory effect of the MeA on the MPOA, reduction in MeA activity (GABAergic cells in particular) reduces lordosis (DiBenedictis et al., 2012; Johnson et al., 2021; McCarthy et al., 2017; Rajendren & Moss, 1993). Similar to the MeA, the BNSTpr region may influence the expression of lordosis stimulated by the VMHvll through inhibition and disinhibition mechanisms (Yamamoto et al., 2018). Specifically, repression of female receptivity during the post-consummatory refractory period is a function of the BNSTpr, which is hypothesized to be mediated through this region’s concentrated GABAergic efferents to the VMHvl and PMv (Dong & Swanson, 2004; Ortiz-Juza et al., 2021). These circuits still require additional investigation to determine how they modulate female sexual motivation and consummatory behaviors.

Figure 4.

The female rodent (grey) expresses lordosis, i.e., sexual receptivity, characterized by a back in dorsiflexion that facilitates intromission from the male (brown). Lordosis is regulated via glutamatergic (excitatory) and GABAergic (inhibitory) connections across the MPOA, VHMvll, and PMv of the reproductive behavioral control column (Yin & Lin, 2023). Chemosensory social cues, such as male pheromones and volatile odors, transduced via the BNSTpr and MeA, are upstream regulators of the RBCC. The MPOA suppresses lordosis, while the VMHvll promotes lordosis (Mei et al., 2023). The PMv also promotes lordosis, though less so than the VMHvll (Mei et al., 2023). Changes in the balance of activation of these nuclei modulates female sexual receptivity. Each of these nuclei and their connections is an area requiring further exploration, regarding EDCs. Abbreviations: BNSTpr; Bed nucleus of the stria terminalis, principal nucleus, GABA; Gamma-aminobutyric acid, MeA; Medial amygdala, MPOA; Medial preoptic area, PAG; Periaqueductal gray, PMv; Ventral premammillary nucleus, VMHvll; Ventromedial hypothalamus, ventrolateral part, lateral subdivision.

Phthalates and the female rodent brain:

DBP, known to have both estrogenic and anti-androgenic mechanisms, causes increased mRNA expression of kisspeptin and kisspeptin receptor (GPR54) in the AVPV following early postnatal (P1–5) subcutaneous administration in Sprague Dawley rats. No changes in ESR1 mRNA in this region were seen; however, an increase in ESR2 occurred (J. Hu et al., 2013). With the same early postnatal DBP exposure, relative mRNA expression of GPR54, Esr1, and Esr2 in the ARC decreased. Though, as in the AVPV, relative kisspeptin mRNA levels were increased (J. Hu et al., 2013) (Supplemental Table 4).

Hu et al., 2013 also applied short-term DBP administration during female rat puberty (PND26–30) to understand the impact on the expression of kisspeptin and kisspeptin receptor, ESR1, and ESR2. In the AVPV, acute pubertal exposure increased kisspeptin and GPR54 mRNA expression at low dosages (0.5 mg/kg) and decreased expression at 5 mg/kg and 50 mg/kg. Esr1 relative expression increased at all three dosages in the AVPV, though no changes in Esr2 were noted. Similar to acute, postnatal DBP exposure, pubertal exposure causes inconsistent changes between the AVPV and ARC. Decreases in ARC kisspeptin relative mRNA expression occurred at 5 and 50 mg/kg, in contrast to changes observed in the AVPV. Non-monotonic alterations in relative GPR54 expression resulted from pubertal administration, with the low (0.5 mg/kg) and high doses (50 mg/kg) causing increases, while 5 mg/kg was unchanged. However, in the ARC, relative expression of Esr1 and Esr2 mRNA were unchanged, regardless of dose of exposure (J. Hu et al., 2013).

Chronic adult exposure to DEHP via oral gavage at relatively high dosages (1000 and 3000 mg/kg/d) increased hypothalamic GnRH levels in female Wistar rat (Liu et al., 2014). This pattern was replicated in pubertal Wistar rat exposed chronically at lower dosages, with GnRH protein levels increased at 250 mg/kg/d, 500 mg/kg/d, and 1000 mg/kg/d. Surprisingly, no differences in relative GnRH mRNA expression were observed after exposure, at any concentration of DEHP (Liu et al., 2016).

BPA and the female rodent brain:

Gestational and early postnatal rodent BPA exposure changes neuroendocrine expression and sexual differentiation of the brain. Decreases in AVPV TH immunoreactivity were observed after embryonic exposure, but no changes in the ARC or SDN-POA volume occurred (Dickerson et al., 2012). Similarly, a decrease in AVPV TH-immunoreactive cell number occurred in CD-1 female mice, following exposure from gestation through lactation (Rubin et al. 2006, Wolstenholme et al., 2011). Postnatal BPA exposure increased ESR1 protein and mRNA in the female POA, while only increased Esr1 mRNA has been reported in the medial basal hypothalamus (Dickerson et al., 2012).

EDC mixtures and the female rodent brain:

Across the hypothalamus (BNST, VMH, MPOA, ARC) and MeA, no changes in ESR1 cell number or density have been observed after adult murine exposure to a phthalate mixture. However, progesterone-receptor (PR) cells decreased in number and density within the VMH. Mixture exposure also diminished PR cell number in the MeA, but PR-cell density remained unchanged. Opposite from the MeA, PR-cell density was reduced in the BNST, but BNST PR-cell numbers unchanged due to adult murine phthalate mixture exposure. Reduced PR-cell numbers were also observed in the MPOA after exposure to a phthalate mixture, but the results of PR density analysis in the MPOA are unreported (Adam et al., 2021).

The NeuroMix EDC formulation did not change adult Esr2 expression within the PVN, an area important to HPG regulation and social behavior. Similarly, no difference in Ar or Esr2 expression was observed in the MeA (Gore et al., 2022).

Conclusions and Future Directions

In this review we discussed the effects of BPA, phthalates, and mixtures on the reproductive behaviors of mice and rats. We examined the impacts of exposures that occurred during adulthood, puberty, and prenatal and postnatal periods and we have described a mosaic of altered neural and behavioral changes. Males exposed to a variety of EDCs demonstrate robust retention of investigatory preference for opposite-sex conspecifics and hormone-primed females (Capela & Mhaouty-Kodja, 2021; Dombret et al., 2017; Ducroq et al., 2023; Gore et al., 2022; Picot et al., 2014). Males exposed to EDCs can successfully copulate but the timing and frequency of consummatory events (intromission, ejaculation) is altered. Among male rodents, changes in AR and ESR1 and ESR2 expression are highly specific to brain region, dosage, and exposure period. AR is often, but not reliably, reduced in the MPOA after DEHP exposure. However, ESR1 protein expression appears normal relative to controls across different ages of EDC exposure (Capela & Mhaouty-Kodja, 2021; Ducroq et al., 2023). Combined BPA and phthalate exposure via NeuroMix decreases Esr2 mRNA in the MeA, but Ar mRNA expression remains unchanged (Gore et al., 2022). Furthermore, no changes in AR or ESR1 protein have been observed in the BNST or MeA following TDI BPA exposure during adulthood (Picot et al., 2014).

In females, EDC exposure typically resulted in reduced mate-preference and sex-discrimination (Adam et al., 2021; Gore et al., 2022). Further, in studies where females were exposed to phthalates during critical windows of development there was reduced female lordosis in response to male mounting (LQ) (Adam et al., 2021; Lee et al., 2006). BPA exposures produced a more varied effect on lordosis behavior with developmental exposures leading to no change, or an increase in lordosis (Adewale et al., 2009; Farabollini et al., 2002; Naulé et al., 2014; Ryan et al., 2010). Thus, one pattern that emerges from these studies in females is that there is a need for stronger behavioral characterization of female appetitive sexual behaviors in rats and mice. For example, it is unknown if ethograms of female rats apply to their murine counterparts. More broadly, studies that examine reproductive behaviors as an outcome should also include a comprehensive analysis that examines neural markers underlying these behaviors.

Humans, animals, and the environment are exposed to a mixture of EDCs every day, thus there is an urgent need to understand how they can impact physiology. The complexity of environmentally relevant EDC dosages and mixtures, alongside the widespread influence of EDCs throughout the neuroendocrine system, presents many challenges to a full characterization of these chemicals. Real-world effects of EDCs are necessarily intricate because organisms encounter mixtures that include many different chemical classes, such as phthalates and BPA, with outcomes emerging from potentially additive effects. Even when simplistic models of single-EDC exposure are studied, non-linear dose-response curves create an additional dimension of difficulty to toxicological research (Vandenberg et al., 2012). Despite these challenges, to move forward, studies on both environmentally relevant EDC mixtures and the single-chemical models of endocrine disruption are needed to identify the dose-dependent effects. Studying single chemicals also remains integral to understanding mechanism and providing context for appreciating non-monotonic dose-response curves. Further, environmentally relevant doses should be used to represent human exposures more accurately. Further, there is need for work that comprehensively analyzes how EDC effects are dependent upon on the age, duration (acute, chronic) and window of exposure, since activational and organizational neuroendocrine effects are dynamic across the lifespan. Studies examining the impact of adult exposures on sexual behaviors are relatively sparse.

Quantifying copulatory behaviors is a powerful method that can be used to characterize the deleterious impact of EDCs on physiology. Reproductive functions are dependent on appropriate gonadal sex-steroid hormone signaling during development to organize the neural circuitry that modulates behavior. Further, in adulthood, multifaceted CNS involvement is required for the transduction and integration of external sensory input into appropriate behavioral outputs. Because rodent sexual behaviors and their associated neurobiological circuitry are characterized, stereotyped and sex-specific, investigating the behavioral consequences of EDC exposure gives us hints into the neuroendocrine mechanisms by which chemicals can have an impact. Further, the conserved neural circuits involved in the expression of reproductive behaviors in mammals can provide insights into how EDCs affect human physiology. Identifying how EDCs such as phthalates, BPA, and chemical mixtures affect physiology is a vital public health concern due to the ubiquitous and unavoidable environmental exposure that humans, wildlife, and livestock encounter.