Bisphenol-A analogs induce lower urinary tract dysfunction in male mice.

Bisphenol-A (BPA), an estrogenic endocrine disrupting chemical, significantly impacts numerous diseases and abnormalities in mammals. Estrogens are known to play an important role in the biology of the prostate; however, little is known about the role of bisphenols in the etiology of prostate pathologies, including benign prostate hyperplasia (BPH) and associated lower urinary tract dysfunction (LUTD). Bisphenol-F (BPF) and bisphenol-S (BPS) are analogs often used as substitutes for BPA; they are both reported to have in vitro and in vivo estrogenic effects similar to or more potent than BPA. The objective of this study was to assess the role of these bisphenols in the development of LUTD in adult male mice. In adult mice exposed to BPA, BPS or BPF, we examined urinary tract histopathology and physiological events associated with urinary dysfunction. Mice treated with bisphenols displayed increased bladder (p < 0.005) and prostate (p < 0.0001) mass, and there was an increased number of prostatic ducts in the prostatic urethra (p < 0.05) and decreased size of the urethra lumen (p < 0.05) compared to negative controls. After two months of bisphenol exposure, mice displayed notable differences in cystometric tracings compared to controls, consistent with LUTD. Treatment of male mice with all bisphenols also induced voiding dysfunction manifested by detrusor instability and histologic changes in the prostatic urethra of male rodents, consistent with LUTD. Our results implicate BPA and its replacements in the development and progression LUTD in mice and provide insights into the development and progression of BPH/LUTS in men.

Sexual characteristics of adult female mice are correlated with their blood testosterone levels during prenatal development.

Mice produce litters containing many pups, and the female fetuses that develop between male fetuses have significantly higher concentrations of the male sex steroid testosterone in both their blood and amniotic fluid than do females that develop between other female fetuses. These two types of females differ during later life in many sexually related characteristics. Thus, individual variation in sexual characteristics of adult female mice may be traceable to differential exposure to testosterone during prenatal development because of intrauterine proximity to male fetuses.

The regulation of infanticide and parental behavior: implications for reproductive success in male mice.

Infanticide has been proposed to be a pathological response to overcrowding or other forms of environmental stress and thus a maladaptive behavior. However, in male house mice this behavior is predictable and is modulated by learning. Committing infanticide can increase a male's reproductive success and in some situations may therefore be an adaptive behavior.

High fetal estrogen concentrations: correlation with increased adult sexual activity and decreased aggression in male mice.

In the house mouse (Mus musculus), fetuses may develop in utero next to siblings of the same or opposite sex. The amniotic fluid of the female fetuses contains higher concentrations of estradiol than that of male fetuses. Male fetuses that developed in utero between female fetuses had higher concentrations of estradiol in their amniotic fluid than males that were located between other male fetuses during intrauterine development. They were also more sexually active as adults, less aggressive, and had smaller seminal vesicles than males that had developed between other male fetuses in utero. These findings raise the possibility that during fetal life circulating estrogens may interact with circulating androgens both in regulating the development of sex differences between males and females and in producing variation in phenotype among males and among females.

TRIENNIAL REPRODUCTION SYMPOSIUM: Environmental programming of reproduction during fetal life: Effects of intrauterine position and the endocrine disrupting chemical bisphenol A.

During critical periods in fetal life, there is an increased vulnerability to perturbations in endocrine function due to environmental factors. Small shifts in concentrations of hormones that regulate the differentiation of organs, such as estradiol and testosterone, can have permanent effects on morphology, enzymatic activity, and hormone receptors in tissues as well as neurobehavioral effects. These changes can lead to effects throughout life, including impacting the risk for various diseases (referred to as the Developmental Origins of Adult Health and Disease hypothesis). The intrauterine position phenomenon concerns the consequence for fetuses of randomly implanting next to embryos of the same or opposite sex. An intrauterine position next to males vs. females results in small differences in serum testosterone and estradiol during fetal life that are associated with marked effects on life history (such as lifetime fecundity) in both males and females born in litters (mice, rats, gerbils, rabbits, and swine) as well as human twins. Research with mice subsequently demonstrated that a very small experimental change in fetal serum estradiol levels altered organogenesis and caused permanent changes in organ function. Taken together, these findings led to the hypothesis that environmental chemicals that mimic or antagonize hormone action (e.g., endocrine disrupting chemicals) could also be causing harm at very low exposures (the "low dose" hypothesis) within the range of exposure of humans, domesticated animals, and wildlife. There is now extensive evidence from experimental laboratory animals, sheep, and humans that fetal exposure to very low (presumably safe) doses of the endocrine disrupting chemical bisphenol A (BPA), which exhibits estrogenic activity, can cause permanent changes that can increase the risk of a wide array of diseases. The reasons that federal regulatory agencies are ignoring the massive literature showing adverse effects of BPA and other endocrine disrupting chemicals are discussed.

Prenatal exposure to endocrine disrupting chemicals: effects on behavioral development.

Numerous chemicals released into the environment by man are able to disrupt the functioning of the endocrine system by binding to hormonal receptors. Exposure to estrogenic endocrine disruptors during critical periods in fetal life can alter the development of reproductive organs, the neuroendocrine system and subsequent behavior. We present a series of studies on the effects of exposure during fetal life to low, environmentally relevant doses of two pesticides, o,p'DDT and methoxychlor, and of low doses of the synthetic estrogen, diethylstilbestrol on subsequent neuro-behavioral development in house mice. The main findings can be summarized as follows: (1) Mice prenatally exposed to methoxychlor showed changes in reflex development. Exposure to a very low dose of methoxychlor appeared to produce an increased reactivity during early postnatal life. (2) Methoxychlor exposed periadolescent mice showed a decreased reaction time exploring both a novel environment and a novel object. (3) The onset of male intrasex aggression appeared to be delayed in males prenatally exposed to low doses of methoxychlor, since exposed males showed low levels of aggressive interactions during early adolescence but not after they reached adulthood. (4) The rate of depositing urine marks in a novel environment was increased in males prenatally exposed to DES, and also to o,p'DDT and methoxychlor. (5) The proportion of both males and females attacking a same-sex conspecific was increased in mice prenatally exposed to low doses of DES and, marginally, to o,p'DDT. This effect appeared to be related to a decreased latency to attack. However, males prenatally exposed to o,p'DDT displayed a decreased intensity of aggression. The possible implications of perturbing the hormonal milieu during fetal development on the modulation of developmental turnpoints and future behavioral responses are discussed.

Control of the preovulatory release of luteinizing hormone by steroids in the mouse.

The regulation of the preovulatory release of LH by steroids was examined in the mouse, a species in which ovulation is strongly influenced by priming pheromones. Ovariectomized mice were implanted with estradiol in Silastic capsules to involve negative feedback. Preovulatory-like LH surges then were induced by injections of either estradiol benzoate (EB) or progesterone. LH surges were not observed in the absence of steroid injection. LH surges always occurred at lights out on a 14-h light, 10-h dark cycle on the day after EB injection but occurred on the same day as progesterone injection. The amount of EB or progesterone injected seemed unimportant but, in either case, had to be given within a limited diurnal period of sensitivity. LH surges comparable to those of intact proestrous females were produced either by injecting both EB and progesterone or by manipulating the background dose of encapsulated estradiol. In the latter regard, when ovariectomized females were implanted with a wide range of doses of estradiol (0.1--1000 microgram/capsule), a decided window phenomenon became apparent. That is, LH surging could be induced by steroid injections only within a limited range of background doses of encapsulated estradiol. The relationship of the above findings to the pheromonal control of LH secretion and ovulation in mice is as yet unclear.

Developmental effects of endocrine-disrupting chemicals in wildlife and humans.

Large numbers and large quantities of endocrine-disrupting chemicals have been released into the environment since World War II. Many of these chemicals can disturb development of the endocrine system and of the organs that respond to endocrine signals in organisms indirectly exposed during prenatal and/or early postnatal life; effects of exposure during development are permanent and irreversible. The risk to the developing organism can also stem from direct exposure of the offspring after birth or hatching. In addition, transgenerational exposure can result from the exposure of the mother to a chemical at any time throughout her life before producing offspring due to persistence of endocrine-disrupting chemicals in body fat, which is mobilized during egg laying or pregnancy and lactation. Mechanisms underlying the disruption of the development of vital systems, such as the endocrine, reproductive, and immune systems, are discussed with reference to wildlife, laboratory animals, and humans.

Effects of altered prenatal hormonal environment on expression of autoimmune disease in NZB/NZW mice.

F1 hybrid New Zealand Black (NZB) x New Zealand White (NZM) (NZB/NZW) mice spontaneously develop an autoimmune disease analogous to systemic lupus erythematosus (SLE). Testosterone experts a powerful suppressive effect on this disorder in adult NZB/NZW mice. A series of experiments was designed to determine if disease would also be suppressed by exposing fetal NZB/NZW mice to increased testosterone. A model was developed in which NZB dams carrying NZB/NZW fetuses were treated with testosterone in a dose adequate to masculinize the external genitalia in female fetuses. NZB/NZW mice that were derived from testosterone-treated dams and control NZB/NZW offspring were followed in a longevity study and had serial assays to assess development of SLE. Additional experiments were carried out to measure lymphocyte subsets and responses to mitogens. Results were compared with F1 hybrid offspring of C57BL/6 dams crossed with DBA/2 males, which are not autoimmune and do not develop SLE. Spleen cells from these groups were tested for Thy 1.2, CD4, CD8, and IgM receptors, and for responses to the mitogens Concanavalin A (ConA) and lipopolysaccharide. Control male NZB/NZW fetuses had unexpectedly high serum estradiol, which decreased significantly with maternal testosterone treatment. The testosterone-exposed male NZB/NZW fetuses developed into adults that lived longer than male NZB/NZW controls. Testosterone treatment of the dam was associated with elevated terminal anti-DNA levels but did not alter markers of renal diseases in adult NZB/NZW mice of either sex. Testosterone-exposed NZB/NZW females had altered T-lymphocyte subsets and testosterone-exposed males had increased response to ConA compared to controls. In male NZB/NZW fetuses whose mothers were administered testosterone, the naturally high level of circulating estradiol observed in untreated male fetuses was decreased significantly. This decrease was associated with an increase in longevity. This unique observation has important implications for fetal exposure to endocrine disruptors in the environment.

Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol.

We have developed a relative binding affinity-serum modified access (RBA-SMA) assay to determine the effect of serum on the access of xenoestrogens to estrogen receptors within intact cultured MCF-7 human breast cancer cells. We used this assay to predict low dose activity of two xenoestrogens in mice. In serum-free medium, bisphenol A, a component of polycarbonates and of resins used to line metal food cans, showed a lower relative binding affinity (RBA; 0.006%) than octylphenol (0.072%) and nonylphenol (0.026%), which are used as surfactants in many commercial products (all RBAs are relative to estradiol, which is equal to 100%). In 100% serum from adult men, bisphenol A showed a higher RBA (0.01%) than in serum-free medium and thus enhanced access to estrogen receptors relative to estradiol. In contrast, octylphenol showed a 22-fold decrease in RBA (0.0029%) and nonylphenol showed a 5-fold decrease in RBA (0.0039%) when measured in adult serum. This indicates that, relative to estradiol, serum had less of an inhibitory effect on the cell uptake and binding in MCF-7 cells of bisphenol A, while serum had a greater inhibitory effect on octylphenol and nonylphenol relative to estradiol. Extrapolation of these relative activities in adult serum predicted that the estrogenic bioactivity of bisphenol A would be over 500-fold greater than that of octylphenol in fetal mouse serum. Bisphenol A and octylphenol were fed to pregnant mice at 2 and 20 micrograms/kg/day. Exposure of male mouse fetuses to either dose of bisphenol A, but to neither dose of octylphenol, significantly increased their adult prostate weight relative to control males, which is consistent with the higher predicted bioactivity of bisphenol A than octylphenol in the RBA-SMA assay. In addition, our findings show for the first time that fetal exposure to environmentally relevant parts-per-billion (ppb) doses of bisphenol A, in the range currently being consumed by people, can alter the adult reproductive system in mice.

Evaluating the effects of endocrine disruptors on endocrine function during development.

The major concerns with endocrine disruptors in the environment are based mostly on effects that have been observed on the developing embryo and fetus. The focus of the present manuscript is on disruption of three hormonal systems: estrogens, androgens, and thyroid hormones. These three hormonal systems have been well characterized with regard to their roles in normal development, and their actions during development are known to be perturbed by endocrine-disrupting chemicals. During development, organs are especially sensitive to low concentrations of the sex steroids and thyroid hormones. Changes induced by exposure to these hormones during development are often irreversible, in contrast with the reversible changes induced by transient hormone exposure in the adult. Although it is known that there are differences in embryonic/fetal/neonatal versus adult endocrine responses, minimal experimental information is available to aid in characterizing the risk of endocrine disruptors with regard to a number of issues. Issues discussed here include the hypothesis of greater sensitivity of embryos/fetuses to endocrine disruptors, irreversible consequences of exposure before maturation of homeostatic systems and during periods of genetic imprinting, and quantitative information related to the shape of the dose-response curve for specific developmental phenomena.

Developmental effects of estrogenic chemicals are predicted by an in vitro assay incorporating modification of cell uptake by serum.

Many estrogenic chemicals found in the environment (xenoestrogens) show a lower affinity for plasma estrogen binding proteins relative to the natural estrogens such as estradiol. These binding proteins, which include alphafetoprotein in rats and mice, sex hormone binding globulin in humans, and albumin in all species, regulate estrogen uptake into tissues. Therefore, the in vivo estrogenic potency relative to estradiol of xenoestrogens that show lower binding to these serum proteins will thus be underestimated in assays that compare the potency of xenoestrogens to estradiol and do not take serum binding into account. We have examined the effects of the binding components in serum on the uptake of a number of xenoestrogens into intact MCF-7 human breast cancer cells. Since most estrogenic chemicals are not available in radiolabeled form, their uptake is determined by competition with [3H]estradiol for binding to estrogen receptors (ER) in an 18-h assay. Serum modified access (SMA) of cell uptake of xenoestrogens is calculated as the RBA in serum-free-medium divided by the RBA in serum, and the bioactive free fraction of xenoestrogen in serum is then also calculated. We predicted the concentration of two xenoestrogens, bisphenol A and octylphenol, required to alter development of the prostate in male mouse fetuses. Whereas octylphenol was predicted to be a more potent estrogen than bisphenol A when tested in serum-free medium, our assay predicted that bisphenol A would be over 500-times more potent than octylphenol in fetal mice. The finding that administration of bisphenol A at a physiologically relevant dose predicted from our in vitro assay to pregnant mice from gestation day 11 to 17 increased adult prostate weight in male offspring relative to controls (similar to the effect of estradiol), while the same doses of octylphenol did not alter prostate development, provided support for our hypothesis.

Variation in infanticide and parental behavior in male mice due to prior intrauterine proximity to female fetuses: elimination by prenatal stress.

When sexually-naive male mice are placed together with newborn young, some males will commit infanticide (kill the young) while others will behave parentally (retrieve the young to a nest and keep them warm). The intrauterine position of male mouse fetuses, which is recorded at Cesarean delivery, has previously been found to influence the titers of estradiol that male fetuses are exposed to in utero. In adulthood, most male mice that developed in utero between male fetuses (2 M males) behaved parentally toward young, while most males that developed in utero between female fetuses (0 M males) committed infanticide. When 0 M and 2 M males were castrated at birth and tested with young in adulthood, few males committed infanticide. But, when these same males were tested with young after 25 days of treatment with testosterone, more 0 M than 2 M males committed infanticide and more 2 M than 0 M males behaved parentally. In contrast to the above findings, more 2 M than 0 M males that were castrated when 28 days old behaved parentally without treatment with testosterone; few of the non-parental males committed infanticide, but, instead, ignored the young. Finally, stressing pregnant mice by placing them under bright lights during the last third of pregnancy eliminated the effect of developing next to female fetuses in the male offspring, and all prenatally-stressed males resembled 2 M males in their behavior toward young: most prenatally-stressed males were parental rather than infanticidal when tested with young. The significance of these findings to models of hormonal effects on sexual differentiation is discussed.

Time-contingent change in infanticide and parental behavior induced by ejaculation in male mice.

About 50% of sexually-naive male CF-1 mice (Mus musculus) commit infanticide (kill young). But, 80-90% of male mice commit infanticide between 1-4 days after mating. Between 12 and 50 days after mating infanticide is inhibited and most males (80-100%) behave parentally toward young (they build a nest, retrieve and groom the young, and hover over the young to keep them warm). Beginning at 60 days after mating, infanticide is again facilitated and parental behavior is inhibited (70-80% of males commit infanticide). The facilitation and inhibition of infanticide as a function of time after mating is mediated by the act of ejaculating rather than by mounting and intromitting during mating or by cohabiting with a female either before or after mating. The experience of committing infanticide either prior to mating or within the first 4 days after mating does not influence the facilitation of parental behavior that occurs 12 days after mating. But, repeated contact with young after males become parental serves to maintain parental behavior for an extended period of time. The age of the young (newborn-20 days old) utilized as stimulus animals does not influence these time-dependent changes in the behavior of male mice after ejaculation. This phenomenon is unique in that a response (infanticide) to a novel stimulus (young) is facilitated, then inhibited, and then facilitated again as a function of time between an event (ejaculation) and exposure to the novel stimulus.(ABSTRACT TRUNCATED AT 250 WORDS)

The influence of reproductive state on infanticide by wild female house mice (Mus musculus).

The majority of female mice (Mus musculus) from laboratory stocks are spontaneously parental. In contrast, the majority of adult wild female house mice exhibit infanticide (the killing of preweanling young), but the frequency with which infanticide is observed varies as a function of age and reproductive state. Prepubertal females were less likely to exhibit infanticide (39%) than were adult virgin females (61%). The frequency of infanticide increased during pregnancy, with over 90% of females exhibiting infanticide just before parturition. But, after parturition, previously infanticidal females cared for their own litters. When lactating female mice were tested after two days of separation from their own nursing young for their behavior toward a novel newborn pup on either the tenth or twenty-fifth day after parturition, the proportion of the females that exhibited infanticide was not significantly different from that of adult virgin females (about 60%). After only two hours of separation from their own nursing young on the tenth day after parturition, however, all females continued to exhibit parental behavior toward a novel pup.

Blood flow in the uterine loop artery and loop vein is bidirectional in the mouse: implications for transport of steroids between fetuses.

In rodents, steroids are able to pass between fetuses. Although not previously examined in mice, we have provided evidence that blood flow in the uterine loop artery in rats is bidirectional and that steroids are transported between fetuses by diffusion via the uterine lumen, not via the uterine blood vessels of the mother. The direction of blood flow in the uterine loop artery and vein feeding each uterine horn in house mice was examined on day 17 of pregnancy. Dye was injected into the heart to determine the direction of blood flow in the uterine artery while injection of dye into individual placentae was used to determine the direction of blood flow in the uterine vein. Blood entered the loop artery from both the dorsal and caudal ends and was thus bidirectional. Venus blood flow from placentae was in a rostral direction from placentae in the rostral portion of a uterine horn and in a caudal direction from placentae in the caudal portion of a uterine horn. Comparison of anogenital distance and body weight at birth using a variety of classification schemes, based on different assumptions about the mechanism and direction of transport of steroids between fetuses, showed that the only scheme which accounted for variation in anogenital distance at birth in female mice was that which was based on the number of directly adjacent male siblings in utero. Taken together with recent findings in rats reported elsewhere, we conclude that blood flow in the uterine blood vessels is bidirectional, and steroids are transported between fetuses by diffusing through the amniotic fluid and across the fetal membranes of adjacent fetuses.(ABSTRACT TRUNCATED AT 250 WORDS)

Individual variation in the neural timing of infanticide and parental behavior in male house mice.

In male house mice (Mus domesticus and M. musculus), the act of coital ejaculation provides a fail-safe neural signal for timing the birth of their offspring. A unique aspect of this phenomenon is the extraordinary latency that can occur between the stimulus (ejaculation) and its adaptive neural response (male mice cease killing pups and behave parentally toward them). Thus the inhibition of infanticide is routinely time-delayed for many days after mating. In the absence of mating, cohabitation with a female will not inhibit infanticide in CF-1 stock males (M. domesticus), whereas the birth of pups in the male's home cage will inhibit infanticide. But with regard to the ejaculatory phenomenon, which also includes the spontaneous reemergence of infanticide 50-60 days after mating, this entire behavioral cycle toward pups can occur in the total absence of regular time cues from a light/dark cycle following ejaculation. However, exposure to photoperiodic (L:D 12:12) or constant light (LL) accelerated the transition time from infanticide to parenting after ejaculation, while in constant dark (DD), the transition time to parenting was significantly prolonged. The time interval between ejaculation and the inhibition of infanticide, which varied among individuals first mated at 6 months of age, was repeatable when the same males were remated at 9 months of age; however, when males were again mated at 18 months of age, the time interval between ejaculation and parenting was dramatically prolonged. In general, coital ejaculation triggers a neural timing system that cannot be explained by any presently known physiological mechanism. Our results do suggest, however, that the neural timing variation observed among individuals is influenced by sex steroid exposure during late fetal development.

Urine marking and maternal aggression of wild female mice in relation to anogenital distance at birth.

A series of experiments were conducted with wild house mice to verify the effect of intrauterine position on females' anogenital distance at birth (AGD) and to examine the relationships between a female's AGD, used as a bioassay of androgen exposure during fetal life, and her social behavior and reproductive success in adulthood. Experiment 1 showed that cesarean-delivered females that developed in utero between two males (2 M females) have significantly longer AGD's than females positioned between two females (0 M females). We then categorized naturally delivered females shortly after birth as having a long, medium or short AGD. In adulthood, these females were tested for their behavior towards unfamiliar pups, their rate of urine-marking in response to a variety of social stimuli, postpartum aggression and success in protecting their litters in response to male and female intruders. Adult females with different AGD's at birth did not differ either in their behavior toward pups or in their rate of urine marking. Conversely, males housed across a wire mesh partition from a long-AGD female deposited a higher number of urine marks than those exposed to a short-AGD female. When tested after delivering a litter, long-AGD females displayed more tail-rattling (a component of agonistic behavior) towards intruders of both sexes in comparison to short-AGD females. These results are consistent with the hypothesis that females with a long AGD are exposed to higher levels of Testosterone during fetal life than females with a short AGD. Although not related to AGD, other measures of maternal aggression were affected by postpartum day, sex of intruders and a female's infanticidal potential while a virgin.

Seminal vesicle and preputial gland response to steroids in adult male mice is influenced by prior intrauterine position.

There are differences in serum steroid concentrations during fetal life between male mice that develop between two male fetuses (2M males, with elevated testosterone) and between two female fetuses (0M males, with elevated estradiol). The present studies were undertaken to determine whether prior intrauterine position would influence the weight of seminal vesicles and preputial glands in adult male mice. To eliminate any potential differences between 2M and 0M males in circulating gonadal steroids, all males were castrated in adulthood and implanted with silastic capsules containing testosterone (T), dihydrotestosterone (DHT) or a combination of T and estradiol-17 beta (E2) or DHT and E2. Three weeks later, preputial glands were significantly heavier in 2M than 0M males after treatment with T but not DHT. Seminal vesicles were also significantly heavier (blotted wet weight) in 2M than 0M males after treatment with T. For 2M males, seminal vesicles weighed the same in response to treatment with T or DHT. However, relative to the effect of T, DHT significantly increased seminal vesicle weight in 0M males such that they were equivalent to weights in 2M males treated with T or DHT. This finding suggests that seminal vesicles in 0M males have lower concentrations of 5 alpha-reductase and, thus, a lower capacity to metabolize T to DHT which is required for normal seminal vesicle function. There were no significant effects of E2 (in combination with T or DHT) on seminal vesicle or preputial gland weight.