Pre- and postnatal bisphenol A treatment results in persistent deficits in the sexual behavior of male rats, but not female rats, in adulthood

Perinatal administration of the endocrine disruptor bisphenol A (BPA) reportedly inhibits the sexual behavior of sexually naïve adult male rats. In order to evaluate the effects of BPA administration during early development on later reproductive behavior, we administered one of five doses of bisphenol A daily to pregnant female rats throughout gestation and lactation, and quantified the appetitive and consummatory sexual behaviors of the resultant male and female offspring over multiple sexual encounters in adulthood. Males receiving low dose perinatal BPA (50 μg/kg bw/day) showed persistent deficits in sexual behavior in adulthood. Males receiving the highest dose (5 mg/kg bw/day), however, were indistinguishable from controls with respect to consummatory sexual behaviors but showed decreased latencies to engage in those behaviors when sexually naïve, with significant non-linear, or U-shaped, dose-response relationships observed on the first and last day of testing. Adult female sexual behavior was not affected by early BPA administration at any dose tested. These results are consistent with previous reports that BPA exerts behavioral effects especially at low doses, and further indicates that BPA can cause lasting impairment of sexual behavior in males, but does not alter the normal development of female appetitive or consummatory sexual behaviors. To our knowledge, this is the first report indicating that adult sexual performance is impaired in sexually experienced animals following perinatal exposure to bisphenol A.     

Developmental exposure to environmental estrogens alters anxiety and spatial memory in female mice

Humans and wildlife are exposed to numerous anthropogenic drugs and pollutants. Many of these compounds are hormonally active, and recent evidence suggests that the presence of these endocrine disruptors permanently alters normal development and physiology in a variety of vertebrate species. Here, we report on the effects of developmental exposure to two common estrogenic pollutants, bisphenol A and ethinyl estradiol on sexually dimorphic, non-reproductive behavior. Mice (Mus musculus domesticus) were exposed to environmentally relevant levels of these chemicals (2 and 200 microg/kg/day for bisphenol A and 5 microg/kg/day for ethinyl estradiol) throughout prenatal and early postnatal development. As adults, the animals were observed in a variety of tests measuring sexually dimorphic behaviors including short-term spatial memory (in a radial-arm maze and a Barnes maze) and anxiety (in an elevated-plus maze and a light/dark preference chamber). Developmental exposure to ethinyl estradiol was found to masculinize behavior in all of the assays used. Bisphenol A increased anxious behavior in a dose-dependent fashion but had no effect on spatial memory. These results indicate that non-reproductive, sexually dimorphic behavior is sensitive to endocrine disruption. In addition, these experiments suggest that both humans and wildlife are being exposed to levels of these endocrine disrupting compounds that are sufficient to disrupt the development of the nervous system and that may have permanent consequences on sexually dimorphic behaviors.     

Inhibition of male chick phenotypes and spermatogenesis by Bisphenol-A

Bisphenol-A (BPA) has been reported to bind to the estrogen receptor (ER) and also to act as a xenoestrogen on the reproductive system of many species. In our previous study, a high dose of BPA disturbed the growth of the comb and testes of male chickens. In this study, the exposure of relatively low doses of BPA on the growth of the male chicken phenotypes was investigated. White Leghorn male chicks were orally administered various doses of BPA (2 microg to 200 mg/kg) from 2 weeks of age, and thereafter the comb, wattle and testes were examined at 5, 10, 15, 20 and 25 weeks of age. Although the body weight showed no significant difference among the birds of all ages, the growth of above organs was significantly affected in the chicks even with a minimal dose of 2-microg BPA. These inhibitory effects appeared in a dose-dependent manner. Histologically, the growth of the testes was negatively affected by exposure to over 20-microg/kg BPA: namely, the development of seminiferous tubuli and spermatogenesis were severely inhibited. The mRNA expressions of ERalpha and the aromatase gene (p450arom) increased in the testes in a dose-dependent manner after BPA administration. Accordingly, even low doses of BPA delayed the growth of the male chicken phenotype either by a direct effect or by an indirect response resulting in an increase in both of the endogenous estrogen levels and hyper-sensitivity to estrogen.     

Relationship between male-male and male-female sexual behavior in 5-6-month-old male lambs

To characterize male–male sexual behavior during lamb development, to relate it with lamb body and testicular growth, and with sexual behavior toward estrual ewes, 40 Milchschaf male lambs, weaned at 45 days of age, were kept with ewes that were nursing younger lambs. Experimental lambs were weighed and scrotal circumference was measured every 2 weeks. Male–male sexual behavior was observed during 1–2 h every 2 weeks after birth until 7 months of age. Observations were recorded more intensively (3–4 h on five different days) for 2 weeks (5–6 months of age) as male–male sexual behavior increased during that period. Both mounting and mounted lambs were identified. An individual mounting index (MI) was calculated. To study male–female sexual behavior, lambs were individually located with two estrual ewes, and during 5 min the number of ano-genital sniffing, lateral approaches, mounts, and mounts with ejaculation were recorded. From those data, a libido index was also calculated. Male–male mounts (n = 308) were observed. Courtship behavior was displayed in 25% of interactions; mounts were accepted in 72.1% of attempted mounts. Mounts without previous courtship were accepted more frequently than mounts with previous courtship (P = 0.002). Lamb weight and scrotal circumference were not different according to MI groups. Lambs that mounted more times estrual ewes (first tertile) had greater (P = 0.04) MI (0.61 ± 0.10) than lambs with medium (0.27 ± 0.09) and less (0.30 ± 0.10) MI. The regression between MI and heterosexual libido index was r = 0.33 (P < 0.05). In summary, intensive male–male sexual activity during a short period of male lamb development was observed. There was a positive relationship between sexual behavior of male lambs towards other male lambs and towards estrual ewes.     

Effects of folic acid on testicular toxicity induced by bisphenol-A in male Wistar rats

We investigated the protective effect of the folic acid (FA) against bisphenol-A (BPA) induced toxicity in rat testis. We used four groups of seven adult male Wistar albino rats. The control group was fed corn oil, the BPA group was given BPA, the FA group was given FA and the FA + BPA group was given FA initially followed by BPA 1 h later. The BPA, FA and corn oil were administered by oral gavage for 14 days. At the end of the experiment, testis sections were examined for histological and histomorphometric characteristics. The TUNEL method was used to detect apoptosis and immunohistochemistry was used to examine the distribution of spermatogonial stem cells. Levels of serum testosterone were measured, and sperm viability and morphology were determined. The histological structure of the testis was normal in the control and FA groups. Although the number of TUNEL positive cells/tubule increased, the seminiferous epithelium height (SEH) at stages VII?VIII decreased in the BPA group compared to the control, FA and FA + BPA groups. The number of TUNEL positive cells/tubule decreased and the SEH at stages VII?VIII increased in the FA + BPA group compared to the BPA group. No significant difference in spermatogonial stem cells was found among groups. The level of serum testosterone and percentage of viable sperm was significantly lower, while the head, midpiece and total sperm abnormalities were significantly higher in the BPA treated group compared to control, FA, FA + BPA groups. It appears that the toxic effects of BPA on testis might be minimized by FA treatment.     

Ginkgo biloba extract enhances male copulatory behavior and reduces serum prolactin levels in rats

The aim of this study was to investigate the effects of Ginkgo biloba extract (EGb 761) on male copulatory behavior in rats. EGb 761 (1 mg/ml) induced significant production of testosterone (T) in rat Leydig cells in vitro. Its effects on sexual behavior were then tested in Long-Evans male rats after 7, 14, 21, or 28 days of oral gavage of vehicle (distilled water) or EGb 761 at doses of 10, 50, or 100 mg/kg. Administration of 50 mg/kg of EGb 761 for 28 days and of 100 mg/kg for 14 or 21 days significantly increased intromission frequency compared to controls on the same day. An increase in ejaculation frequency was seen after treatment with 50 mg/kg of EGb 761 for 14, 21, or 28 days when compared to either the control group on the same day or the same group on day 0. A reduction in ejaculation latency was only seen after administration of 50 mg/kg of EGb 761 for 14 days compared to the vehicle-treated group. After treatment for 28 days, no significant difference was seen in mount latency, intromission latency, serum T levels, reproductive organ weight, sperm number, or levels of the metabolite of dopamine, 3,4-dihydroxyphenylacetic acid in the brain with any dose of EGb 761, but significantly reduced serum prolactin levels and increased dopamine levels in the medial preoptic area and arcuate nucleus were seen at the dose of 50 mg/kg. These findings show that EGb 761 (especially at the dose of 50 mg/kg) enhances the copulatory behavior of male rats and suggest that the dopaminergic system, which regulates prolactin secretion, may be involved in the facilitatory effect of EGb 761.     

Long-term effects of pubertal anabolic-androgenic steroid exposure on reproductive and aggressive behaviors in male rats

The current study examined acute and long-term effects of anabolic-androgenic steroid (AAS) exposure during puberty on copulation, vocalizations, scent marking, and intermale aggression, both with and without tail pinch, in intact male rats. Animals received 5 mg/kg of testosterone, nandrolone, stanozolol, or vehicle, beginning at puberty. After 5 weeks, behavior tests were performed while continuing AAS injections. AAS treatment was then discontinued. Behaviors were tested during 3-5 weeks, 9-11 weeks, and 15-17 weeks of withdrawal. During AAS administration, stanozolol males showed significant reductions in all behaviors compared with controls, except aggression with tail pinch. Nandrolone treatment significantly reduced vocalizations and scent marking, and testosterone had no significant effect on behavior. During withdrawal, behaviors in stanozolol males recovered to control levels at variable rates: aggression at 4 weeks; mounts, vocalizations, and scent marking at 9 weeks; and ejaculations at 15 weeks of withdrawal. Stanozolol males showed significantly higher levels of tail pinch-induced aggression during every withdrawal test. Nandrolone-treated males scent-marked at control levels by 9 weeks withdrawal but displayed significantly fewer vocalizations and significantly more tail pinch-induced aggression than controls for the entire study. Testosterone-treated males scent-marked significantly below controls at 3 weeks withdrawal and showed significantly more tail pinch-induced aggression at 5 weeks withdrawal. All three AAS significantly increased tail pinch-induced aggression compared with corresponding nontail pinch tests, even at study endpoint. These results suggest that alterations in androgen-dependent behaviors by pubertal AAS exposure can persist long after drug exposure, and some effects may even be permanent.     

Hormonal responses to different sexually related conditions in male rats

Plasma levels of corticosterone (C) and testosterone (T) increase after sexual activity in males of several species. However, the physiological significance of these increases has not been elucidated. In the present study, hormonal response to different conditions linked to sexual activity was assessed. In the first experiment, plasma levels of C and T were assessed both in sexually experienced and naive male rats after the following conditions: (A) control group, without sexual stimulation; (B) males exposed to ovariectomized females; (C) males exposed to intact, non-receptive females; (D) males exposed to receptive females with the vagina obstructed, to avoid intromission; (E) males exposed to receptive females: but separated by a grid that prevents physical contact; (F) males exposed to receptive females during 30 min. In a second experiment, experienced male rats were allowed to repeatedly copulate until reaching the criteria for sexual exhaustion, and 24 h later, they were allowed to copulate. Once sexually related conditions ended, males were killed and their blood was obtained. C and T plasma levels were assessed by HPLC with ultraviolet (UV) detection. Results indicate that T did not increase significantly in naive male in any sexual condition, while in the experienced males, significant increases were observed with the mere presence of a receptive female and also after ejaculation. These increases were significantly larger in experienced males. On the other hand, C also increased in all sexual conditions, both in experienced and naive rats; however, the increase observed was larger in experienced males. Regarding sexual satiety, both C and T increased after copulating ad libitum to satiety. T increased almost three-fold compared to control, while C increased two-fold. No significant changes were observed in either one of the steroids 24 h after sexual exhaustion, even though males remained with a receptive female during an hour. These results show that sexual experience has an important influence on the hormonal response to sexual activity. C rises could be directly related to sexual arousal involved in the different sexual conditions, while T rises seem to have a direct relationship with both the motivation and execution aspects of masculine sexual behavior.     

Assessment of human contamination of estrogenic endocrine-disrupting chemicals and their risk for human reproduction

There is broad human exposure to estrogenic endocrine-disrupting chemicals (EDCs), but the data sets that exist are primarily for various environmental media such as food and water rather than the most relevant internal exposure. We have detected various kind of EDC contamination in humans including dioxin and bisphenol A (BPA) widely used for the production of plastic products. BPA was present in serum and follicular fluid at approximately 1–2 ng/ml, as well as in fetal serum and full-term amniotic fluid, confirming passage through the placenta. An approximately five-fold higher concentration, 8.3 ± 8.7 ng/ml, was revealed in amniotic fluid at 15–18 weeks of gestation, compared to other fluids showing increased exposure at the critical developmental period in humans. Interestingly, serum BPA concentrations were significantly higher in normal men and in women with polycystic ovary syndrome (PCOS) compared with normal women possibly due to differences in the androgen-related metabolism of BPA. These findings may provide some insight into the metabolism of EDCs in human and the pathophysiology of endocrine disorders such as PCOS. Dioxin contamination in relationship to development of endometriosis is also discussed.     

Peripubertal exposure to male odors influences female puberty and adult expression of male-directed odor preference in mice

Testosterone-dependent olfactory signals emitted by male are well known to accelerate female puberty in mice (Vandenbergh effect). However, it remains unclear whether these chemosignals also influence adult expression of male-directed odor preference. Therefore, we exposed female mice to intact or castrated male bedding (vs clean bedding as control) during the peripubertal period (postnatal day (PD) 21–38) and measured male-directed odor preference in adulthood. At PD45 or PD60, females exposed to intact male odors, and thus showing puberty acceleration, preferred to investigate odors from intact males over females or castrated males. Females exposed to castrated male odors did not show puberty acceleration but preferred male (intact or castrated) over female odors. Finally, control females did not show any odor preference when tested at PD45, although a preference for male odors emerged later (PD60). In a second experiment, females that were exposed to intact male odors after pubertal transition (PD36–53) also preferred intact male over castrated male odors. In conclusion, our results indicate that peripubertal exposure to male odors induced early expression of male-directed odor preference regardless of puberty-accelerating effect and that induction of male-directed odor preference is not specific to the peripubertal period.     

Neonatal exposure to endocrine active compounds or an ERbeta agonist increases adult anxiety and aggression in gonadally intact male rats

Endocrine active compounds (EACs) have been shown to influence a number of reproductive endpoints but less is known about how they might affect other hormone dependent behaviors including anxiety and aggression. Recent evidence suggests that these effects may be mediated through the beta form of the estrogen receptor (ERbeta). Using male Long Evans rats, we sought to determine how neonatal exposure to EACs affects anxiety and aggression in adulthood. Anxiety was assessed using the elevated plus maze and aggression was assessed 8 weeks later using the resident intruder test. To gain insight into which ER subtype (ERalpha vs ERbeta) might be mediating these effects we used agonists specific for ERalpha (1,3,5-tris(4-Hydroxyphenyl)-4-propyl-1H-pyrazole (PPT)) or ERbeta (Diarylpropionitrile (DPN)) as additional treatment groups. For these experiments the synthetic EAC bisphenol-A (BPA) and the phytoestrogen metabolite equol (EQ) were used. Male neonates were injected with either 0.05 ml sesame oil (control), 50 microg estradiol benzoate (EB), 1 mg/kg DPN, 1 mg/kg PPT, 50 microg/kg BPA, or 10 mg/kg EQ daily for 4 days beginning on the day of birth (PND 0). Compared to the oil treated controls, significantly fewer open arm entries were made by the males neonatally treated with DPN, EQ, or BPA. The DPN and EQ treated males were also more aggressive compared to the controls. These findings suggest that neonatal exposure to EACs with agonistic activity on ERbeta may influence affective behavior in adulthood, including anxiety and aggression.     

Metabolism of bisphenol A in zebrafish (Danio rerio) and rainbow trout (Oncorhynchus mykiss) in relation to estrogenic response

The kinetics of bisphenol A (BPA) were investigated in zebrafish (Danio rerio) exposed to 100 microg BPA/l. BPA uptake was measured during a 7-day period followed by an elimination phase of similar duration. After 2, 6, 12, 24, 48, 72, 120 and 168 h of uptake/elimination, fish were analysed for their content of BPA, bisphenol A glucuronic acid (BPAGA) and bisphenol A sulfate (BPAS). Within the first 24 h steady state levels of BPA, BPAGA and BPAS were reached and the total body concentrations were calculated to be 569, 12,600 and 39.9 ng/g fish, respectively. Elimination rates of the three compounds in zebrafish were estimated by fitting the data to a compartment model. An initial rapid elimination phase was observed for BPA and BPAS with total body half lives (T(1/2)) of <1.1 h and 30 min, followed by a slower second elimination phase with T(1/2) values of 139 and 71 h, respectively. Excretion of BPAGA occurred from a single compartment with a T(1/2) of 35 h. The steady state concentration of BPA and its metabolites were investigated in rainbow trout (Oncorhynchus mykiss) exposed to 100 microg BPA/l. The toxicokinetic parameters from zebrafish and rainbow trout were compared; including previously published data on the rainbow trout. The data indicate that the smaller estrogenic sensitivity observed for the zebrafish may be caused by a more rapid metabolism of BPA in the zebrafish liver.     

Repeated exposure to social stress alters the development of agonistic behavior in male golden hamsters

In male golden hamsters, exposure to social stress during puberty alters aggressive behavior. Interestingly, agonistic behavior undergoes two major transitions during puberty: a decline in attack frequency and a shift from play fighting to adult-like aggression. Based on previous observations, we developed an approach for characterizing offensive responses as play fighting or adult-like. The present studies had two aims. First, we validated our approach by looking at the development of attack types during puberty. Second, we looked at the effects of repeated social stress on the development of agonistic behavior by repeatedly exposing individuals to aggressive adults during puberty. In the first phase of the study, our results point to three different developmental periods. Initially, animals engage in agonistic behavior though attacks targeted at the face and cheeks. This period lasts from Postnatal Day 20 (P-20) to P-40 (early puberty). This phase corresponding to play fighting is followed by a transitional period characterized by attacks focused on the flanks (from P-40 to P-50, mid-puberty). Afterward, animals perform adult-like aggression characterized by attacks focused on the belly and rear. Our data also show that repeated exposure to aggressive adults has two separate effects on the development of agonistic behavior. Repeated social stress accelerated the onset of adult-like agonistic responses. Furthermore, attack frequency, while decreasing during puberty, remained at a higher level in early adulthood in stressed animals. These results show that repeated exposure to social stress during puberty alters the development of agonistic behavior both qualitatively and quantitatively.     

Long-term exposure to a continuous 900 MHz electromagnetic field disrupts cerebellar morphology in young adult male rats

The pathological effects of exposure to an electromagnetic field (EMF) during childhood and adolescence may be greater than those from exposure during adulthood. We investigated possible pathological changes in the cerebellum of adolescent rats exposed to 900 MHz EMF daily for 25 days. We used three groups of six 21-day-old male rats as follows: unexposed control group (Non-EG), sham-exposed group (Sham-EG) and an EMF-exposed group (EMF-EG). EMF-EG rats were exposed to EMF in an EMF cage for 1 h daily from postnatal days 21 through 46. Sham-EG rats were placed in the EMF cage for 1 h daily, but were not subjected to EMF. No procedures were performed on the Non-EG rats. The cerebellums of all animals were removed on postnatal day 47, sectioned and stained with cresyl violet for histopathological and stereological analyses. We found significantly fewer Purkinje cells in the EMF-EG group than in the Non-EG and Sham-EG groups. Histopathological evaluation revealed alteration of normal Purkinje cell arrangement and pathological changes including intense staining of neuron cytoplasm in the EMF-EG group. We found that exposure to continuous 900 MHz EMF for 1 h/day during adolescence can disrupt cerebellar morphology and reduce the number of Purkinje cells in adolescent rats.     

Aluminum affects glial system and behavior of rats

Aluminum (Al) has been associated with neuronal dysfunction. These neuronal changes may involve glial alterations. We intend to evaluate the consequence of Al on the glial system and the behavior of rats exposed chronically to 0.3% of aluminum chloride in drinking water during 4 months in adulthood (A) or since intra-uterine age (IU); animals from this latter group were sacrificed at four months of age. Our data show an intense glial fibrillary acidic protein (GFAP)-immunoreactivity with a high density of astrocytes in both treated groups compared with controls. However, in IU rats, astrocytes display prominent glial cell bodies and processes. A and IU rat groups perform a significantly reduced locomotor activity. However, using the dark/light box test, the IU rats prefer to spend more time in the enlightened compartment compared to other groups. Behavioral and glial changes caused by Al exposure bring support for the role of Al in brain dysfunction involving glial alterations.     

Decreased apoptosis in the forebrain of adult male medaka (Oryzias latipes) after aqueous exposure to ethinylestradiol

Endocrine disrupting compounds (EDCs), especially those that are estrogenic, are an issue of growing concern because they may ultimately adversely affect wildlife survival. 17-beta-Estradiol and its synthetic counterpart, 17-alpha-ethinylestradiol, two common EDCs, are associated with intersex conditions and impaired male reproductive behavior in fish. Male and female Japanese medaka (Oryzias latipes) were exposed to 10 ng/l ethinylestradiol for 6 months. Using terminal dideoxynucleotidyl-mediated dUTP nick end-labeling (TUNEL) to quantitate cell death, we found that ethinylestradiol-exposed males had significantly fewer apoptotic cells in the forebrain compared to untreated males and exposed females. Our results show that the effects of ethinylestradiol exposure are highly variable among individuals of the same species and even within tissues of the same individual. Thus, when examining the effects of EDCs on natural populations, data from a variety of tissues should be examined and the interpretation of any effects should include consideration of tissue-specific processes.     

Static magnetic field exposure affects behavior and learning in rats

The present work investigated the behavioral effects of a moderate exposure (1 h per day for 5 consecutive days) to a static magnetic field (SMF, 128 mT) in male rats. SMF effects were evaluated in two sets of control and SMF-exposed rats. One set of animals was used for evaluation of SMF potential effects on emotional behaviors in the elevated plus maze and in the open field. The other set of animals was tested for learning and memory abilities in different procedures of the Morris water maze task. We found no significant difference between control and SMF-exposed rats in anxiety tests. However, the ratio of open arms time in the plus maze was reduced by half in SMF-exposed rats. In the Morris water maze, SMF-exposed rats were partially impaired during the initial learning task as well as in the retention task at one week. We conclude that static magnetic field exposure altered emotional behaviors in the plus maze and led to cognitive impairments, or at least to substantial attention disorders, in the Morris water maze.     

The influence of androgenic and estrogenic hormones on sexual behavior in castrated adult male pigs

The objectives of these studies were to evaluate the influence of testosterone propionate (TP), estradiol cypionate (EC), dihydrotestosterone propionate (DHTP), EC + TP, EC + DHTP, and TP + DHTP on traits of masculine sexual behavior in castrated adult male pigs of different breeds. Masculine sexual behavior was restored and maintained by TP, whereas EC initially activated sexual behavior, including copulation and ejaculation, but was unable to sustain copulatory behavior for the 8- to 18-week periods that were evaluated. Treatment with DHTP was ineffective for stimulation of sexual behavior; thus, it is suggested that testosterone promotes some aspects of masculine sexual behavior in male pigs via aromatization to estrogen, but both androgen and estrogen are required for maintenance of the full complement of masculine sexual behavior traits.