The Conflict between Regulatory Agencies over the 20,000-Fold Lowering of the Tolerable Daily Intake (TDI) for Bisphenol A (BPA) by the European Food Safety Authority (EFSA).

BACKGROUND: The European Food Safety Authority (EFSA) recommended lowering their estimated tolerable daily intake (TDI) for bisphenol A (BPA) 20,000-fold to 0.2 ng/kg body weight (BW)/day. BPA is an extensively studied high production volume endocrine disrupting chemical (EDC) associated with a vast array of diseases. Prior risk assessments of BPA by EFSA as well as the US Food and Drug Administration (FDA) have relied on industry-funded studies conducted under good laboratory practice protocols (GLP) requiring guideline end points and detailed record keeping, while also claiming to examine (but rejecting) thousands of published findings by academic scientists. Guideline protocols initially formalized in the mid-twentieth century are still used by many regulatory agencies. EFSA used a 21st century approach in its reassessment of BPA and conducted a transparent, but time-limited, systematic review that included both guideline and academic research. The German Federal Institute for Risk Assessment (BfR) opposed EFSA's revision of the TDI for BPA. OBJECTIVES: We identify the flaws in the assumptions that the German BfR, as well as the FDA, have used to justify maintaining the TDI for BPA at levels above what a vast amount of academic research shows to cause harm. We argue that regulatory agencies need to incorporate 21st century science into chemical hazard identifications using the CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity) nonguideline academic studies in a collaborative government-academic program model. DISCUSSION: We strongly endorse EFSA's revised TDI for BPA and support the European Commission's (EC) apparent acceptance of this updated BPA risk assessment. We discuss challenges to current chemical risk assessment assumptions about EDCs that need to be addressed by regulatory agencies to, in our opinion, become truly protective of public health. Addressing these challenges will hopefully result in BPA, and eventually other structurally similar bisphenols (called regrettable substitutions) for which there are known adverse effects, being eliminated from all food-related and many other uses in the EU and elsewhere. https://doi.org/10.1289/EHP13812.

Prenatal and postnatal exposure to polychlorinated biphenyls alter follicle numbers, gene expression, and a proliferation marker in the rat ovary.

Polychlorinated biphenyls (PCBs) were used in industrial applications until they were banned in the 1970s, but they still persist in the environment. Little is known about the long-term effects of exposure to PCB mixtures on the rat ovary during critical developmental periods. Thus, this study tested whether prenatal and postnatal exposures to PCBs affect follicle numbers and gene expression in the ovaries of F1 offspring. Sprague-Dawley rats were treated with vehicle or Aroclor 1221 (A1221) at 1 mg/kg/day during embryonic days 8-18 and/or postnatal days (PND) 1-21. Ovaries from F1 rats were collected for assessment of follicle numbers and differential expression of estrogen receptor 1 (Esr1), estrogen receptor 2 (Esr2), androgen receptor (Ar), progesterone receptor (Pgr), and Ki-67 (Ki67) at PNDs 8, 32, and 60. Sera were collected for measurement of estradiol concentrations. Prenatal exposure to A1221 significantly decreased the number of primordial follicles and the total number of follicles at PND 32 compared to control. Postnatal PCB exposure borderline increased Ki67 gene expression and significantly increased Ki67 protein levels (PND 60) compared to control. Combined prenatal and postnatal PCB exposure borderline decreased Ar expression (PND 8) compared to control. However, PCB exposure did not significantly affect the expression of Pgr, Esr1, and Esr2 or serum estradiol concentrations compared to control at any time point. In conclusion, these data suggest that PCB exposure affects follicle numbers and levels of the proliferation marker Ki67, but it does not affect expression of some sex steroid hormone receptors in the rat ovary.

Pre- and postnatal developmental exposure to the polychlorinated biphenyl mixture aroclor 1221 alters female rat pituitary gonadotropins and estrogen receptor alpha levels.

Polychlorinated-biphenyls (PCBs) are industrial compounds, which were widely used in manufacturing of electrical parts and transformers. Despite being banned in 1979 due to human health concerns, they persist in the environment. In humans and experimental model systems, PCBs elicit toxicity in part by acting as endocrine-disrupting chemicals (EDCs). Aroclor 1221 (A1221) is a weakly estrogenic PCB mixture known to alter reproductive function in rodents. EDCs can impact hormone signaling at any level of the hypothalamic-pituitary-gonadal (HPG) axis, and we investigated the effects of A1221 exposure during the prenatal and postnatal developmental periods on pituitary hormone and steroid receptor expression in female rats. Examining offspring at 3 ages, postnatal day 8 (P8), P32 and P60, we found that prenatal exposure to A1221 increased P8 neonate pituitary luteinizing hormone beta (Lhb) mRNA and LHß gonadotrope cell number while decreasing LH serum hormone concentration. No changes in pituitary hormone or hormone receptor gene expression were observed peri-puberty at P32. In reproductively mature rats at P60, we found pituitary follicle stimulating hormone beta (Fshb) mRNA levels increased by prenatal A1221 exposure with no corresponding alterations in FSH hormone or FSHß expressing cell number. Estrogen receptor alpha (ERα) mRNA and protein levels were also increased at P60, but only following postnatal A1221 dosing. Together, these data illustrate that exposure to the PCB A1221, during critical developmental windows, alters pituitary gonadotropin hormone subunits and ERα levels in offspring at different phases of maturation, potentially impacting reproductive function in concert with other components of the HPG axis.

Chronic periadolescent leuprolide exposure affects the development of reproductive physiology and behavior of female and male rats differently, but both mature after treatment termination.

BACKGROUND: GnRH agonists have been used to halt the development of puberty in children with precocious puberty since the 1980s. Recently, drugs like Lupron Depot® (leuprolide acetate), have been used to suppress pubertal progression in adolescents who are questioning their gender identity. However, few preclinical studies have been conducted to investigate potential effects of using GnRH agonists in this context. METHODS: The present study tested the effects of daily leuprolide treatment (50 µg/kg, postnatal day (PD) 25-50) on pubertal onset in female (i.e., vaginal opening) and male (i.e., preputial separation) Long-Evans rats. The first estrous cycle immediately after vaginal opening was also measured. Sexual behavior and sexual motivation were tested using the partner-preference paradigm. Female rats were tested during the first behavioral estrus after treatment ended (between PD 51-64). Male rats were tested weekly for four consecutive weeks starting three days after treatment ended (PD 53). RESULTS: Consistent with previous findings, leuprolide significantly delayed pubertal onset in both female and male rats. In addition, the first estrous cycle during the treatment period was disrupted by leuprolide, as indicated by a failure to cycle into estrus after vaginal opening until treatment ended. However, leuprolide affected neither sexual motivation nor fertility when female rats were tested within 14 days of leuprolide treatment ending. In contrast, the development of copulatory behavior and sexual motivation was significantly delayed by leuprolide in male rats; however, mature reproductive behavior was observed by the fourth week post-treatment. CONCLUSIONS: Taken together with previous findings, the present results indicate that male rats may be more sensitive to periadolescent leuprolide administration, taking longer to overcome the effects of leuprolide than female rats. Nevertheless, not long after leuprolide treatment is discontinued, sex-typical reproductive physiology and behavior emerge fully in female and male rats, indicating that the drug's effects are not permanent. If translatable to humans, leuprolide may be a reversible option to give adolescents more time to consider their gender identity with minimal long-term effects on sexual development.

The medial preoptic area and acute cocaine's stimulant effects in rats: Potential influences of estradiol and biological sex.

The medial preoptic area (mPOA) in the hypothalamus is an important integrator of neuroendocrine signaling and a key regulator of both natural and drug-induced reward. Although the mPOA modulates sex differences in other behaviors, whether it also modulates sex differences in cocaine response remains unclear. To help us better understand the mPOA's role in sex differences associated with cocaine response, we examined cocaine-induced changes in locomotion and neural activity in the mPOA of male and female rats. In addition, neural activity in the striatum, a brain area known to be involved in cocaine response, was examined for comparison purposes. Fos, the protein product of the immediate early gene c-fos, was used as the marker of neural activity. Locomotion chambers were used to measure behavior, radioimmunoassays and vaginal lavages were used to determine hormonal status, and immunohistochemical assays were used to quantify Fos. To account for the effects of gonadal hormones, rats were left gonadally intact and categorized as either 'low-estradiol' or 'high-estradiol' based on their hormonal status on test day. Results indicate that high-estradiol females experienced greater cocaine-induced mPOA Fos-immunoreactivity (Fos-ir) and displayed greater cocaine-induced locomotion than low estradiol females. Conversely, high-estradiol males experienced less cocaine-induced mPOA Fos-ir and displayed less cocaine-induced locomotion than low-estradiol males. Cocaine-induced Fos-ir in the mPOA also correlated with cocaine-induced Fos-ir in areas of the striatum already associated with cocaine response. These findings further support the mPOA's role in the endocrine-mediated response to cocaine. It also identifies the mPOA as a contributor to sex differences in cocaine response and potential differences in vulnerability to developing cocaine use disorders.

Sex-specific Effects of Endocrine-disrupting Chemicals on Brain Monoamines and Cognitive Behavior.

The period of brain sexual differentiation is characterized by the development of hormone-sensitive neural circuits that govern the subsequent presentation of sexually dimorphic behavior in adulthood. Perturbations of hormones by endocrine-disrupting chemicals (EDCs) during this developmental period interfere with an organism's endocrine function and can disrupt the normative organization of male- or female-typical neural circuitry. This is well characterized for reproductive and social behaviors and their underlying circuitry in the hypothalamus and other limbic regions of the brain; however, cognitive behaviors are also sexually dimorphic, with their underlying neural circuitry potentially vulnerable to EDC exposure during critical periods of brain development. This review provides recent evidence for sex-specific changes to the brain's monoaminergic systems (dopamine, serotonin, norepinephrine) after developmental EDC exposure and relates these outcomes to sex differences in cognition such as affective, attentional, and learning/memory behaviors.

Endocrine-disrupting chemicals.

In this Quick guide, Bertram et al. discuss the environmental sources of endocrine-disrupting chemicals and their effects on biological systems.

Hormonal contraceptives alter amphetamine place preference and responsivity in the intact female rat.

Hormonal contraceptives (HCs) containing synthetic ovarian hormones are commonly used among reproductive aged women; HCs alter the physiological state of the user by interfering with endogenous hormone concentrations and their actions on the reproductive tract. As ovarian hormones modulate the incidence of substance abuse disorders in women, this experiment explores how modulating female rat ovarian hormonal states with an HC containing the synthetic progestin levonorgestrel influences measures of drug preference and responsivity. First, rats underwent food-light Pavlovian conditioning to measure conditioned orienting, a known predictor of amphetamine (AMP) place preference. Then, rats were conditioned and tested for AMP place preference with either an HC implant or during estrous cycle stages associated with opposing ovarian hormone levels, that is, proestrus (P) or metestrus/diestrus (M/D), while recording ultrasonic vocalizations (USVs) as an index of he donic drug responsivity. Because of dopamine's (DA's) role in reward learning and memory, DA cell number and activity were examined using tyrosine hydroxylase and FOS immunohistochemistry after a final AMP challenge. Conditioned orienting did not differ between cycling and HC-implanted rats. HC rats emitted fewer USVs in response to AMP, showed marginally less AMP place preference, and had lower DA cell activity in the substantia nigra after AMP compared to P rats. M/D rats showed a similar behavioral profile and neural response as HC rats. This experiment suggests ovarian hormones affect drug preference and responsivity, while providing novel insight into how hormone-altering contraceptives may reduce these measures. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Daily GnRH agonist treatment effectively delayed puberty in female rats without long-term effects on sexual behavior or estrous cyclicity.

The present study examined the long-term effects of suppressing puberty with a GnRH agonist on reproductive physiology and behavior in female rats. We have recently reported that administration of the GnRH agonist leuprolide acetate (25 µg/kg) daily between postnatal day (PD) 25-50 delayed puberty and disrupted the development of copulatory behavior and sexual motivation in male rats. However, pilot data from our lab suggest that this low dose of leuprolide acetate (25 µg/kg) was not high enough to significantly delay puberty in female rats. Therefore, we injected female Long-Evans rats with leuprolide acetate at a higher dose (50 µg/kg) or 0.9% sterile saline, daily , starting on PD 25 and ending on PD 50. Vaginal opening was monitored daily starting on PD 30 for signs of pubertal onset and first estrous cycle. In addition, we measured estrous cyclicity starting approximately 2 weeks after the last injection of leuprolide (∼PD 64). Immediately after monitoring estrous cyclicity, the female rats were mated on their first day in behavioral estrus using the partner-preference paradigm, with and without physical contact (PD 95-110). We found that this dose of leuprolide (50 µg/kg) significantly delayed puberty; however, neither estrous cyclicity nor sexual motivation was significantly affected by periadolescent exposure to leuprolide. Together with our findings in male rats, these results add to our understanding of the developmental effects of chemically suppressing puberty in rats.

Effects of sugar cane extract on steroidogenesis in testicular interstitial cells of male Japanese quail (Coturnix japonica).

Sugar cane extract (SCE) is the end product of glucose, fructose, and sucrose elimination in molasses. SCE has various biological effects, such as anti-inflammation and antioxidation, and it is commonly found in animal feed. The present research is aimed at investigating the reproductive endocrine influence of SCE in male Japanese quails (Coturnix japonica) by feeding SCE containing food. In addition, in vitro Leydig cell culture was conducted to clarify the mechanism of SCE's influence. Our results showed that SCE feed extended the latency to the first neck grab, decreased male quail testis and epididymis weights, cloaca gland size, and reduced serum testosterone concentrations. Steroidogenic enzymes 3ßHSD, 17ßHSD, P450c17, and P450scc gene expression in the testis were decreased in the SCE groups. Western blot analysis showed decreased 3ßHSD in the testis after feeding SCE. Isolated testicular interstitial cells cultured with SCE and ovine-LH suppressed testosterone secretion and 3ßHSD gene expression. In conclusion, SCE as a feed additive has an impact on the sexual behavior and reproductive function of male Japanese quail, with the suppression of steroidogenesis in the Leydig cell. Our results may provide beneficial information to the livestock management and the poultry industry.

Prenatal Exposure to an EDC Mixture, NeuroMix: Effects on Brain, Behavior, and Stress Responsiveness in Rats.

Humans and wildlife are exposed to endocrine-disrupting chemicals (EDCs) throughout their lives. Environmental EDCs are implicated in a range of diseases/disorders with developmental origins, including neurodevelopment and behavior. EDCs are most often studied one by one; here, we assessed outcomes induced by a mixture designed to represent the real-world situation of multiple simultaneous exposures. The choice of EDCs, which we refer to as "NeuroMix," was informed by evidence for neurobiological effects in single-compound studies and included bisphenols, phthalates, vinclozolin, and perfluorinated, polybrominated, and polychlorinated compounds. Pregnant Sprague Dawley rats were fed the NeuroMix or vehicle, and then offspring of both sexes were assessed for effects on postnatal development and behaviors and gene expression in the brain in adulthood. In order to determine whether early-life EDCs predisposed to subsequent vulnerability to postnatal life challenges, a subset of rats were also given a stress challenge in adolescence. Prenatal NeuroMix exposure decreased body weight and delayed puberty in males but not females. In adulthood, NeuroMix caused changes in anxiety-like, social, and mate preference behaviors only in females. Effects of stress were predominantly observed in males. Several interactions of NeuroMix and stress were found, especially for the mate preference behavior and gene expression in the brain. These findings provide novel insights into how two realistic environmental challenges lead to developmental and neurobehavioral deficits, both alone and in combination, in a sex-specific manner.

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

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.

Two Hits of EDCs Three Generations Apart: Effects on Social Behaviors in Rats, and Analysis by Machine Learning.

All individuals are directly exposed to extant environmental endocrine-disrupting chemicals (EDCs), and indirectly exposed through transgenerational inheritance from our ancestors. Although direct and ancestral exposures can each lead to deficits in behaviors, their interactions are not known. Here we focused on social behaviors based on evidence of their vulnerability to direct or ancestral exposures, together with their importance in reproduction and survival of a species. Using a novel "two hits, three generations apart" experimental rat model, we investigated interactions of two classes of EDCs across six generations. PCBs (a weakly estrogenic mixture Aroclor 1221, 1 mg/kg), Vinclozolin (antiandrogenic, 1 mg/kg) or vehicle (6% DMSO in sesame oil) were administered to pregnant rat dams (F0) to directly expose the F1 generation, with subsequent breeding through paternal or maternal lines. A second EDC hit was given to F3 dams, thereby exposing the F4 generation, with breeding through the F6 generation. Approximately 1200 male and female rats from F1, F3, F4 and F6 generations were run through tests of sociability and social novelty as indices of social preference. We leveraged machine learning using DeepLabCut to analyze nuanced social behaviors such as nose touching with accuracy similar to a human scorer. Surprisingly, social behaviors were affected in ancestrally exposed but not directly exposed individuals, particularly females from a paternally exposed breeding lineage. Effects varied by EDC: Vinclozolin affected aspects of behavior in the F3 generation while PCBs affected both the F3 and F6 generations. Taken together, our data suggest that specific aspects of behavior are particularly vulnerable to heritable ancestral exposure of EDC contamination, that there are sex differences, and that lineage is a key factor in transgenerational outcomes.

Exposure to environmental chemicals and perinatal psychopathology.

Women are nearly twice as likely to develop mood disorders compared with men, and incidence is greatest during reproductive transitions, including pregnancy and postpartum. Because these periods are characterized by dramatic hormonal and physiologic changes, there is heightened susceptibility to external factors, such as exposure to environmental toxicants, which may play a role in maternal psychopathology. The purpose of this scoping review was to provide an overview of studies conducted in humans and animal models on the effects of nonoccupational exposure to environmental chemicals on maternal psychopathology during the perinatal period. The largest number of studies examined exposure to environmental tobacco smoke and antenatal depression and showed consistently positive findings, although more prospective studies using biomarkers for exposure assessment are needed. The few studies examining persistent organic pollutants such as polybrominated diphenyl ethers and perinatal depression were consistent in showing associations with increased depressive symptoms. Results were mixed for exposure to heavy metals and non-persistent chemicals, but a strong literature in animal models supported an association between bisphenols and phthalates and reduced maternal behavior and care of pups after parturition. Biological mechanisms may include endocrine disruption, neurotransmitter system impairment, alterations in gene expression, and immune activation and inflammation. Additional longitudinal studies that include biospecimen collection are essential to furthering the understanding of how environmental toxicants during pregnancy may affect perinatal psychopathology and the underlying mechanisms of action. Future work should also leverage the parallels between animal and human maternal behavior, thereby highlighting the opportunity for multidisciplinary work in this avenue.

Effects of endocrine-disrupting chemicals on hypothalamic oxytocin and vasopressin systems.

Exposures to endocrine disrupting chemicals (EDCs) perturb hormonal systems. EDCs are particularly problematic when exposure happens in the fetus and infant due to the high sensitivity of developing organisms to hormone actions. Previous work has shown that prenatal polychlorinated biphenyl (PCB) exposure disrupts hypothalamic development, reproductive physiology, mate preference behavior, and social behaviors in a sexually dimorphic manner. Based on evidence that EDCs perturb social behaviors in rodents, we examined effects of PCBs on the neuropeptides oxytocin (OXT) and vasopressin (AVP) that are involved in regulating these behaviors. Rats were exposed prenatally (gestational days 16 and 18) to the weakly estrogenic PCB mixture Aroclor 1221 (0.5 or 1 mg/kg), to estradiol benzoate (EB, a positive control), or to the vehicle (3% dimethyl sulfoxide). In adult (~P90) brains, we counted immunolabeled oxytocin and vasopressin cell numbers in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. EDCs did not change absolute numbers of oxytocin or vasopressin cells in either region, although there were some modest shifts in the rostral-caudal distribution. Second, expression of genes for these nonapeptides (Oxt, Avp), their receptors (Oxtr, Avpr1a), and the estrogen receptor beta (Esr2), was determined by qPCR. In the PVN, there were dose-dependent effects of PCBs in males (Oxt, Oxtr), and effects of EB in females (Avp, Esr2). In the SON, Oxt, and Esr2 were affected by treatments in males. These changes to protein and gene expression caused by prenatal treatments suggest that transcriptional and posttranscriptional mechanisms play roles in mediating how EDCs reprogram hypothalamic development.

Sex differences in conditioned orienting and the role of estradiol in addiction-related behaviors.

Conditioned orienting response (OR) is a form of cue-directed behavior thought to indicate increased attentional and/or motivational processing of reward-associated stimuli. OR as a phenotype has been shown to predict both direct drug proclivity in female rats and behaviors indirectly related to drug proclivity in male rats, but no extant research has compared males and females in terms of their OR behavior or its notable substrates. As females are at increased risk for substance abuse, and the ovarian hormone estradiol is often cited as a driving factor for this predilection, it is important to characterize sex differences between males and females and explore what, if any, contribution estradiol has in behaviors which predict substance abuse. In these experiments, male and female rats [intact or ovariectomized (OVX) with/without estradiol replacement] were compared on a battery of behavioral tasks, including OR, novelty-seeking, attentional set-shifting, and ultrasonic vocalizations (USVs) to amphetamine treatment. Female rats, regardless of estradiol replacement, had higher OR scores than males. OR score was a predictor of attention impairments, and estradiol availability contributed to this relationship in females. Sex differences were not observed in novelty-seeking, attentional set-shifting, or USV response to amphetamine; however, estradiol replacement did alter the presentation of these behaviors. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Epigenetics, estrogenic endocrine-disrupting chemicals (EDCs), and the brain.

Endocrine-disrupting chemicals (EDCs) are a major public health concern, yet there are gaps in knowledge about the mechanisms for their effects. The brain is incredibly sensitive to small changes in the hormonal environment during early development, with natural sex differences in gonadal hormones shaping the organization of sex-specific neural circuits early in life. EDC exposure during these periods can lead to lifelong impacts on neurobiological health outcomes. Recently, epigenetic mechanisms have been proposed as a potential molecular mechanism for effects of endogenous hormones on the organization of developing brain circuits, leading to speculation that these mechanisms may underlie the long-term impacts of EDC exposure. Of these mechanisms, DNA methylation, associated with gene suppression, and histone marks, associated with gene suppression or activation, will be discussed. We review the evidence for hormones and their role in brain sexual differentiation; underlying epigenetic mechanisms; and how two classes of estrogenic EDCs, BPA and PCBs, may interfere with these processes to change brain structure and function.

Daily GnRH agonist treatment delays the development of reproductive physiology and behavior in male rats.

The present study was designed to examine the effects of suppressing pubertal onset with leuprolide acetate, a gonadotropin releasing hormone (GnRH) agonist. Starting on postnatal day (PD) 25, male Long-Evans rats were injected daily with either leuprolide acetate (25 µg/kg dissolved in 0.9% sterile physiological saline; n = 13) or sterile physiological saline (1.0 ml/kg 0.9% NaCl; n = 14) for a total of 25 days. Males were monitored daily for signs of puberty (i.e., preputial separation). On the last day of leuprolide treatment (PD 50), half of each treatment group was injected with 10.0 µg of estradiol benzoate (EB) daily for three consecutive days (PD 50-52) and 1.0 mg of progesterone (P) on the 4th day (PD 53), whereas the other half of each treatment group received oil injections. Four hours after P injections, all subjects were given the opportunity to interact with a gonadally-intact male and a sexually receptive female rat (i.e., a partner-preference test with and without physical contact). Copulatory behavior and sexual motivation were measured. Hormone injections and mating tests were repeated weekly for a total of 3 consecutive weeks. Results showed that leuprolide delayed puberty as well as the development of copulatory behavior and the expression of sexual motivation. By the last test, the leuprolide-treated subjects showed signs of catching up, however, many continued to be delayed. Estradiol and progesterone mildly feminized male physiology (e.g., decreased testes weight and serum testosterone) and behavior (e.g., increased lordosis), but did not interact with leuprolide treatment.