A mixture of the "antiandrogens" linuron and butyl benzyl phthalate alters sexual differentiation of the male rat in a cumulative fashion.

Prenatal exposure to environmental chemicals that interfere with the androgen signaling pathway can cause permanent adverse effects on reproductive development in male rats. The objectives of this study were to 1) determine whether a documented antiandrogen butyl benzyl phthalate (BBP) and/or linuron (an androgen receptor antagonist) would decrease fetal testosterone (T) production, 2) describe reproductive developmental effects of linuron and BBP in the male, 3) examine the potential cumulative effects of linuron and BBP, and 4) investigate whether treatment-induced changes to neonatal anogenital distance (AGD) and juvenile areola number were predictive of adult reproductive alterations. Pregnant rats were treated with either corn oil, 75 mg/kg/day of linuron, 500 mg/kg/day of BBP, or a combination of 75 mg/kg/day linuron and 500 mg/kg/day BBP from gestational Day 14 to 18. A cohort of fetuses was removed to assess male testicular T and progesterone production, testicular T concentrations, and whole-body T concentrations. Male offspring from the remaining litters were assessed for AGD and number of areolae and then examined for alterations as young adults. Prenatal exposure to either linuron or BBP or BBP + linuron decreased T production and caused alterations to androgen-organized tissues in a dose-additive manner. Furthermore, treatment-related changes to neonatal AGD and infant areolae significantly correlated with adult AGD, nipple retention, reproductive malformations, and reproductive organ and tissue weights. In general, consideration of the dose-response curves for the antiandrogenic effects suggests that these responses were dose additive rather than synergistic responses. Taken together, these data provide additional evidence of cumulative effects of antiandrogen mixtures on male reproductive development.

An environmental antiandrogen, vinclozolin, alters the organization of play behavior.

During mammalian sexual differentiation, the androgens, testosterone and dihydrotestosterone are critical for the organization of the male phenotype. In rats, play behavior is sexually dimorphic. Administration of exogenous androgens during the perinatal period results in masculine-like play behavior of juveniles. Recently, there has been increasing concern about the potential for environmental endocrine-disrupting chemicals (EDCs) to alter sexual differentiation in mammals. One such EDC is the fungicide and androgen receptor (AR) antagonist, vinclozolin. We tested whether developmental exposure to an EDC could alter androgen-dependent behaviors such as play. To examine this possibility, neonatal male rats were injected from Postnatal Days (PND) 2 to 3 with corn oil, pharmacological antiandrogen flutamide (50 mg/kg/day) or vinclozolin (200 mg/kg/day); whereas neonatal females were treated either with corn oil or testosterone propionate (TP, 250 microg/kg/day). At PNDs 36-37, animals were observed for social play. Behaviors associated with general social activity, such as sniffing and dorsal contact, were unaffected by treatment or sex. However, play behavior in males treated with flutamide or vinclozolin was significantly reduced to near-female levels when compared to control males. Play behavior in females exposed to TP during the neonatal period was significantly increased when compared with control females. Hence, this study suggests that perinatal exposure to vinclozolin, an environmental antiandrogen, can alter androgen-dependent behavior, such as play, in the male rat.

Spontaneous mutation in the db gene results in obesity and diabetes in CD-1 outbred mice.

Five allelic mutants of the diabetes (db) gene have been previously described in mice and rats causing obesity, infertility, and varying degrees of diabetes. We have identified a new, spontaneous mutation resulting in obesity and diabetes in a colony of CD-1 outbred mice, Mus musculus domesticus. Genetic complementation studies indicated that the new mutation was an allele of the diabetes locus. Sequence analysis of cDNA fragments showed a deletion of one G residue located in exon 12 of the leptin receptor gene. The mutation, Lepr(db-NCSU), results in a frameshift and reduces Lepr transcript levels 10-fold. Mutant mice drank up to four times more water and were up to two times heavier than wild-type mice. Blood glucose and plasma insulin and leptin concentrations were sexually dimorphic among affected mice, suggesting an effect of sex steroids. Mortality of affected males was 100% by 5 mo, whereas affected females survived up to 10 mo of age.

Species, interindividual, and tissue specificity in endocrine signaling.

The activity of endocrine-active agents exhibits specificity at many levels. Differential responsiveness to these agents has been observed between different species and extends to interindividual differences within a species and between different tissues as well. In cases where they have been identified, the biologic and molecular mechanisms underlying this specificity are quite diverse. Determinants of species specificity include differences that exist in receptor binding, gene transcription, and cellular responses to endocrine-active compounds between species. Interindividual differences in responsiveness may be determined at the level of genetic polymorphisms in hormone-metabolizing enzymes, hormone receptors, and in those genes that are transactivated by these receptors, as well as during changing windows of susceptibility that occur as a function of age, such as prenatal and postmenopausal exposures. Extrinsic factors such as diet can also impact individual susceptibility to endocrine-active agents. Tissue-specific determinants of susceptibility are well documented, but little is known regarding the mechanisms underlying these different responses. Differences in the expression of accessory proteins for steroid hormone receptors and different patterns of receptor expression, estrogen receptor alpha and estrogen receptor beta; for example, may contribute to tissue specificity, as may differences in the pattern of expression of other genes such as hormone-metabolizing enzymes. The use of animal model systems and development of appropriate mathematical models has the potential to yield additional valuable information for elucidating the role of these determinants of specificity at low-dose exposures and for improved risk assessments for the adverse health effects of endocrine-active compounds.

Chronic cocaine exposure affects stimulus-induced but not spontaneous behavior of the near-term mouse fetus.

Pregnant female mice were injected subcutaneously with a 40-mg/kg dose of cocaine-HCl or physiological saline from day 1 through day 17 of gestation. On day 18 of gestation, dams were surgically prepared to allow the behavior of their fetuses to be observed. Spontaneous motor behavior was unaffected by cocaine exposure. Cocaine exposure potentiated motor responses of the fetuses to ammonia and to control injections of saline into the amniotic sac. Restriction of umbilical blood flow caused a specific stereotyped response in saline-injected fetuses, which is in agreement with studies of other species. This response was markedly potentiated in fetuses exposed to cocaine. The results suggest that the mouse may be a viable model for studies of the neurodevelopmental effects of gestational cocaine exposure and are discussed in relation to current models of the effects of long-term cocaine exposure on brain neurochemistry.

Androgen exposure and reproductive behavior of an induced ovulator, the pine vole (Microtus pinetorum).

The actions of steroid hormones on brain and behavior are classically divided into organizational effects that are permanent and occur early in development and activational effects that are temporary and occur throughout life. Here, we test the hypothesis that in an induced ovulator, testosterone defeminizes only those neural tissues that rely on synergistic interactions of estrogen and progesterone for normal function in adulthood. Female voles, Microtus pinetorum, injected with testosterone (T) or oil neonatally were paired with males for an 8-week period. During the pairing, androgenized and oil-treated females spent a similar amount of time investigating the caudal and rostral regions of the males. Males spent significantly less time investigating the caudal and rostral regions of androgenized females. Androgenized females mounted males, did not exhibit lordosis, and were less likely to be mounted by males. Moreover, none of the 10 androgenized females gave birth, whereas 8 of 9 control females gave birth. Androgenized females were also not capable of being stimulated into reproductive condition by males. Injection of 0.5 microg of estradiol benzoate for 4 consecutive days resulted in reduced uterine hypertrophy in androgenized females. These results support the original organizational-activational hypothesis by showing that neonatal androgenization defeminizes and masculinizes female pine voles.

Chemical cues are necessary but insufficient for reproductive activation of female pine vole (Microtus pinetorum).

Among various arvicoline rodents, reproduction is influenced to varying degrees by social factors, including behavioral or chemical cues. Since previous research suggested that chemosignals from adult males reproductively activate female pine voles (Microtus pine-torum), we sought to determine specifically what types of stimuli promote the activation response. In these experiments, female were exposed to unfamiliar adult males, or to some combination of cues from males, or were housed alone. Using uterine mass as a measure of reproductive activation, we found that females were not activated by exposure either to male urine by itself or to male-soiled bedding by itself, but full contact with a male clearly resulted in heavier uteri. Females whose vomeronasal organs were surgically excised failed to undergo reproductive activation when housed with males. Finally, females allowed physical contact by being housed directly underneath males had heavier uteri than did females whose housing allowed contact only with the chemical cues from males. Among female arvicoline rodents, it appears that there exists a physiological continuum between absolute dependence on both contact and chemical cues from males vs. absolute independence for reproductive activation. The present results place female pine voles closer to the former extreme than to the latter.

Serological evidence for zoonotic hantaviruses in North Carolina rodents.

In a survey of seven species of wild rodents (n = 423) collected between October 1993 and March 1994 from the three principal ecological biomes of North Carolina (USA), we found hantavirus antibodies in seven (2%) of 301 Peromyscus spp. Hantavirus antibodies were detected in P. leucopus and P. maniculatus captured from mountain and coastal island biomes. Three mice were positive for Sin Nombre virus, while four others had antibodies to Seoul virus or a related agent. Two mice serologically positive for Sin Nombre virus were collected from inside a private mountain domicile. We conclude that the risk of human exposure to hantaviruses in North Carolina resembles that for most other areas of the continental United States.

The anogenital distance index, a predictor of the intrauterine position effects on reproduction in female house mice.

The anogenital distances (AGD) of newborn female house mice vary as a function of prior intrauterine position. Females with long AGDs are more likely to be derived from intrauterine positions adjacent to males (2M) than females not adjacent to males (0M). Females with a male on one side (1M) show intermediate AGDs. Hence the AGD reflects the degree of androgenization experienced by the female in utero and correlates with a number of androgen-dependent anatomical, physiological, and behavioral events in adulthood. This experiment tested the usefulness of AGD measurements of female house mice taken at weaning rather than at birth as an index of prior androgenization. The AGD was normalized for body weight at weaning to yield an anogenital distance index (AGDI). Intrauterine position (IUP) was determined by caesarian section. Pups were marked and reared by foster mothers. Comparison of AGDI showed that 0M females had a significantly lower mean AGDI score than 2M females and 1M females were intermediate. This confirmed that AGDI reflects prior IUP and can be used as an index of prenatal androgenization. While testing for responsiveness to male urine, a stimulus known to accelerate puberty, only females from the 1M and 2M positions differed from controls indicating that 0M females had already attained puberty. Choosing females from unknown IUPs with short-AGDIs, mid-AGDIs, and long AGDIs and treating them with either urine or saline on the nose for the 4 days after weaning yielded much the same response indicating that AGDI can also be used to preselect females for sensitivity to factors influencing puberty. These results demonstrate that some of the variability known to be related to intrauterine position can be predicted by AGDI, a relatively easy measure to acquire at weaning in commonly used laboratory rodents. Such preselection could reduce variability of experimental results in the conduct of studies related to rodent reproduction and may reduce the number of animals needed without loss of predictive ability.

Management, breeding, and reproductive performance of pine voles.

Pine voles (Microtus pinetorum) serve as an interesting rodent model for studies of fossorial adaptation and mammalian mating systems. They are unusual among rodents in displaying a cooperative system of breeding. Here we describe the management of a breeding colony of pine voles. This species is relatively easy to maintain in the laboratory, although care must be taken to provide sufficient water in their diet. Details of reproduction, as well as growth and development of the young, are included. Pine voles have a smaller litter size (2.3 +/- 0.9 pups at birth) than do other arvicoline rodents but are still prolific breeders, producing, on average, a litter per month for more than 12 months. These rodents are useful for comparison with other arvicoline rodents, as well as providing an opportunity to investigate the behavior, physiology, and reproduction of an easily managed cooperatively breeding mammal.

Mother's prior intrauterine position affects the sex ratio of her offspring in house mice.

Sex ratio alterations related to environmental factors occur in several mammals, but no mechanism has been identified to explain the adjustment. Intrauterine position (IUP) may provide the context in which such alterations occur. Previous studies on house mice and gerbils reveal that the position of a fetus in the uterus in relation to the sex of its neighbors influences its later anatomy, physiology, and behavior. The anogenital distance (AGD) of females located between two males (2M) is longer than that of females not between two males (OM). We have found that the IUP, as determined by cesarean section and by an index of the AGD, correlates with the sex ratio of the litters produced by female mice. The sex ratio of the first litter born to 2M females was 58% males, for 1M females was 51% males and for OM females was 42% males. The effect on sex ratio continues into the second litter. The number of pups produced by mothers of different IUPs in her first two litters did not differ, suggesting that the sex ratio adjustment occurs prior to parturition. These results provide a basis for the natural variability observed in sex ratios of litter-bearing mammals and suggest that one or more intrauterine mechanisms may be responsible for environmentally related sex ratio alterations.

Effect of chronic cocaine on reproduction in female house mice.

The effect of chronic cocaine exposure on the reproductive success of juvenile female house mice was studied. We followed two generations of female mice to examine the consequence of cocaine treatment on developmental and reproductive parameters such as weight gain, first estrus, impregnation, fertility, and maternal success. Twenty-two-day-old female mice were given cocaine at a daily total of 40 mg/kg body weight, delivered by two SC injections of 20 mg/kg each, until they were mated and inseminated by experienced males. The treatment attenuated weight gain and delayed puberty in the females but had no discernible effect on their pups. Administration of cocaine to lactating mothers decreased the weaning weight of their pups. Juvenile females previously nursed by mothers receiving cocaine and receiving 40 mg/kg cocaine daily themselves were impregnated at older ages than controls. Nevertheless, once these juveniles reached puberty, they mated successfully and their reproductive parameters did not differ from those of control mice. Thus, chronic cocaine treatment of juvenile female mice slows body growth and development but has little effect on the offspring produced later when they reached adulthood.

Effect of cocaine on the production of puberty-accelerating pheromone by male mice.

This study examined whether chronic cocaine exposure could reduce reproductive fitness of adult male mice by interfering with their production of the puberty-accelerating pheromone, an androgen-dependent urinary pheromone that accelerates puberty in juvenile female mice. Administered at a high dose of 60 mg/kg body weight/day, cocaine caused mortality, body weight loss, and suppression of circulating testosterone during the first week of treatment. However, at 40 mg/kg/day, it resulted in little adverse effect on these parameters. Animals showed habituation to repeated cocaine exposure by regaining part of the lost weight and reelevating suppressed testosterone level at later stages of treatment. Urine samples collected from animals receiving 60 mg/kg cocaine daly for 2 weeks lost the puberty-accelerating effect. However, neither a 3-day treatment of the same dose nor a lower dose of 40 mg/kg reduced the effectiveness. The diminished effect of cocaine-treated male mouse urine might reflect lowered testosterone levels with a lag of 10 to 15 days, similar to that of castrated male mouse urine. These results indicate that cocaine has no direct effect on the production of priming pheromone, and its metabolites in the urine did not affect the response of juvenile females to the pheromone.

Cocaine attenuates puberty acceleration in female house mice.

The onset of puberty in female house mice is advanced by exposure to a male urinary pheromone. This study tested whether cocaine could modify the juvenile female mouse's response to this pheromone. Puberty acceleration, as measured by uterine weight change, is inhibited by daily SC administration of 30 or 40 mg/kg body weight cocaine HCl between 20 and 26 days of age. Two daily injections of 20 mg/kg cocaine reduced both uterine development and body weight gain. Thus, cocaine may reduce an animals' reproductive fitness by isolating it from its social environment. At higher doses, cocaine can delay body growth, as well as the onset of puberty.

Analysis of puberty-accelerating pheromones.

Two experiments were performed to test putative puberty-accelerating pheromones. In the first experiment, 37 weanling female house mice of the ICR strain were exposed to 1 of the following 3 treatments: an airborne mixture of 0.05 M isoamylamine and 0.05 M isobutylamine, fresh male mouse urine, or distilled water, as the control. Neither the amine mixture nor the male urine accelerated first estrus in the mice following airborne exposure to these compounds. In the second experiment, 37 weanling female mice of the same strain were exposed to the same chemicals as in the first experiment by direct contact to the oro-nasal groove. The mixture of isoamylamine and isobutylamine did not accelerate puberty, but direct contact with the male urine accelerated puberty as evidenced by uterine weights.

Puberty acceleration in female mice induced with a partially purified male urine extract: effects on catecholamine release from the olfactory bulbs and hypothalamus.

In the present experiment peri-pubertal female mice were treated with a partially purified puberty accelerating urine extract (PAUE). Mice treated with the PAUE showed an advance in the onset of puberty as indicated by significantly increased uterine weights. Treatment with the PAUE did not alter basal or potassium- (K+, 30 mM) stimulated release of catecholamines (dopamine or norepinephrine) from either anterior or posterior superfused olfactory bulb tissue fragments. There was, however, an overall significantly greater amount of basal and K(+)-stimulated release of NE from the posterior vs. the anterior olfactory bulb. Potassium-stimulated-, but not basal, release of catecholamines from the medial basal hypothalamus of PAUE-treated female mice were increased, with dopamine showing a statistically significant difference compared to water-treated females. These data demonstrate that treatment with the PAUE is a very effective means to accelerate the onset of puberty and results in accompanying increases in catecholaminergic activity, in particular dopamine, within the medial basal hypothalamus.

Neuroendocrine responses to social regulation of puberty in the female house mouse.

First estrus is advanced in female house mice exposed to an adult male and delayed in those housed in groups. Experiments were conducted to explore possible mechanisms by which the hypothalamus integrates these puberty-regulating social signals. Female mice weaned at 21 days of age were placed in groups of 8 (G8JF), a juvenile female with a juvenile male (JFJM) or juvenile female with an adult male (JFAM). All females were ovariectomized on day 28 and sacrificed on day 29. There was no significant difference between treatments in the postovariectomy rise in LH. Next, female mice were weaned, assigned to G8JF, JFJM or JFAM treatments and ovariectomized on day 22. Females were sacrificed on day 29, 3 h after injection with either 1.0 micrograms of estradiol, or vehicle. Estradiol significantly suppressed LH in all three treatments, with no differences between treatments. Two-way ANOVA (social treatment x estradiol treatment) revealed no differences or interactions in brain catecholamines as a result of estradiol injection. The G8JF treatment significantly increased norepinephrine, (NE), dopamine (DA) and its metabolite 3,4-dihydroxy-phenylacetic acid in the mediobasal hypothalamus (MBH), and the 3-methoxy-4-hydroxy-phenylglycol/NE ratio in the preoptic area (POA). In the final experiments, isolate prepubertal female mice were treated with either water or male urine (MU) on the oronasal groove. Eight days of MU treatment resulted in significant uterine growth, however there were no differences in serum LH, POA or MBH catecholamines or POA and median eminence LHRH. One hour after application of MU, serum LH was significantly elevated, however, there were no differences in accessory olfactory bulb catecholamines. These results suggest that the mechanism by which male and grouped female exposure alters first estrus may not involve changes in sensitivity to estradiol negative feedback. Grouping may delay first estrus through the negative effects of DA on the LHRH system in the MBH. We observed no differences that might help to explain how male exposure causes LH release.

Effect of intrauterine position and social density on age of first reproduction in wild-type female house mice (Mus musculus).

In laboratory strains of mice, reproductive maturation is influenced by both the fetal and the peripubertal social environments experienced by females. Intrauterine position (IUP) for female fetuses is identified by the number of adjacent males; where 0M have zero, 1M have one, and 2M have two male neighbors. We sought to confirm, using wild-type female house mice (Mus musculus), the previous finding in the CF-1 strain of laboratory mouse that males prefer 0M over 2M females as mates. We recorded the age at first reproduction for females of known IUP housed either individually with a male or in a group of 6 females and a male. In neither housing condition were there significant differences in age of first reproduction among females of different IUPs, but 0M females that were group housed had perforate vaginas at an earlier age than other group-housed females. Mean (+/- SE) age at first reproduction was 58.1 (+/- 3.3) for paired and 126.6 (+/- 5.1) for group-housed females. The reproductive suppression observed in the grouped females was probably due to the interaction of inhibitory urinary chemosignals, low body weights, and female dominance hierarchies.