Differential Responses to Same and Opposite Sex Odors by Adult House Mice Are Associated with Anogenital Distance

Intrauterine position (IUP) of female and male fetuses in litter-bearing mammals can affect their physiology, morphology and behavior. The relationship between anogenital distance (AGD) and IUP was used as a bioassay for the degree of exposure of female and male fetuses to hormones in utero . Based on laboratory work in several rodent species, the following predictions were made for house mice ( Mus musculus domesticus ): (1) female mice should prefer odors from males with larger AGDs because such males are more aggressive, could protect more resources, and are better parents than males with smaller AGDs; (2) male mice should prefer odors from females with smaller AGDs because these females produce more offspring and are better parents than females with larger AGDs. We also tested the prediction that within sexes, mice should avoid odors from mice with larger AGDs because such mice are more aggressive. Responses to odors in traps were used to test these predictions for house mice living in outdoor enclosures using odor-baited traps. Both predictions were confirmed. Furthermore, mice of both sexes tended to avoid odor cues from individuals of the same sex that had larger AGDs, probably to decrease chances of an aggressive encounter that could result in injury.     

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 ÷ 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.     

Sexual dimorphism in esterified steroid levels in the gastropod Marisa cornuarietis: the effect of xenoandrogenic compounds

Molluscs can conjugate a variety of steroids to form fatty acid esters. In this work, the freshwater ramshorn snail Marisa cornuarietis was used to investigate sex differences in endogenous levels of esterified steroids. Testosterone and estradiol were mainly found in the esterified form in the digestive gland/gonad complex of M. cornuarietis, and males had higher levels of esterified steroids than females (4-10-fold). Additionally, the ability of several xenobiotics, namely tributyltin (TBT), methyltestosterone (MT) and fenarimol (FEN) to interfere with the esterification of testosterone and estradiol was investigated. All three compounds induced imposex - appearance of male sexual characteristics in females. Exposure to TBT led to a decrease in both esterified testosterone (60-85%) and estradiol (16-53%) in females after 100 days exposure, but had no effect on males. Exposure to FEN and MT did not alter levels of esterified steroids in males or in females, although exposed females developed imposex after 150 days exposure. The decrease in esterified steroids by TBT could not be directly linked with a decrease in microsomal acyl-CoA:testosterone acyltransferase (ATAT) activity, which catalyzes the esterification of steroids. In fact, ATAT activity was marginally induced in organisms exposed to TBT for 50 days (1.3-fold), and significantly induced in males and females exposed to MT for 50 days (1.8- and 1.5-fold, respectively), whereas no effect on ATAT activity was observed after 150 days exposure.     

Steroid levels in crinoid echinoderms are altered by exposure to model endocrine disruptors

Sexual steroids (testosterone and estradiol) were measured in the whole body of wild specimens of the crinoid Antedon mediterranea collected from the Tyrrhenian Sea (Italy). Testosterone levels (274-1,488 pg/g wet weight (w.w.)) were higher than those of estradiol (60-442 pg/g w.w.) and no significant differences between males and females were observed. No clear seasonal trend was either detected - individuals from February, June and October 2004 analyzed - apart from a peak of estradiol in males in autumn. Nonetheless, dramatic changes on tissue steroid levels were observed when individuals were exposed to model androgenic and anti-androgenic compounds for 2 and 4 weeks. The selected compounds were 17 alpha-methyltestosterone (17 alpha-MT), triphenyltin (TPT), fenarimol (FEN), cyproterone acetate (CPA), and p,p'-DDE. Endogenous testosterone levels were significantly increased after exposure to 17 alpha-MT, TPT and FEN, while different responses were observed for estradiol; 17 alpha-MT and FEN increased endogenous estradiol (up to seven-fold), and TPT lead to a significant decrease. Concerning the anti-androgenic compounds, CPA significantly reduced testosterone in a dose-dependent manner without altering estradiol levels, whereas specimens exposed to p,p'-DDE at a low dose (24 ng/L) for 4 weeks showed a four-fold increase in T levels. Overall, the data show the ability of the selected compounds to alter endogenous steroid concentrations in A. mediterranea, and suggest the existence in this echinoderm species of vertebrate-like mechanisms that can be affected by exposure to androgenic and anti-androgenic chemicals.     

Paradoxical effects of maternal stress on fetal steroids and postnatal reproductive traits in female mice from different intrauterine positions

We examined effects of maternal stress on prenatal serum concentrations of testosterone and estradiol and on postnatal reproductive traits in female mice from different intrauterine positions. On Day 18 of fetal life, control females positioned in utero between two male fetuses (2M females) had higher concentrations of testosterone and lower concentrations of estradiol in serum than control female fetuses located between two females (0M females). Control females positioned between a male and a female fetus (1M females) had intermediate levels of both hormones. Prior intrauterine position in control females accounted for differences in genital morphology (length of the anogenital separation) at birth and length of estrous cycles during adulthood. Maternal stress eliminated these postnatal differences due to prior intrauterine position: all 0M, 1M, and 2M female offspring of stressed mothers exhibited postnatal traits that were indistinguishable from those of control 2M females. Maternal stress resulted in an increase of over 1 ng/ml in serum testosterone in all female fetuses; the magnitude of the increase was similar for 0M, 1M, and 2M females. There was no effect of maternal stress on serum concentrations of estradiol in 0M and 2M female fetuses. Maternal stress resulted in a dramatic change in the postnatal traits of 0M females, whereas 2M females showed no change. Since the effect of maternal stress on sex steroids was similar among fetuses from different intrauterine positions but postnatal response to maternal stress varied by intrauterine position, other components of the endocrine system may mediate effects of maternal stress on these postnatal characteristics.     

Negative feedback regulation of gonadotropin secretion by androgens in fetal rhesus macaques

Previously we described sex differences in circulating gonadotropin concentrations (greater in females) in fetal rhesus macaques, and demonstrated that these sex differences relate, at least in part, to the negative feedback actions of testicular secretions. A fully functional gonadal-hypothalamic-pituitary feedback relationship is present as early as Day 100 of gestation in fetal males because castration at this time results in a dramatic increase (greater than 10-fold) in fetal luteinizing hormone (LH) concentrations. Although short-term (6-h) treatment of fetuses with testosterone (T) 3 wk after gonadectomy (GX) does not lower LH levels in males, it is completely effective in females. These data suggest that either T is not the primary testicular factor responsible for feedback suppression of LH in fetal males, or the hypothalamic-pituitary axis becomes insensitive to T after GX. To determine if immediate treatment with T after GX is effective in maintaining LH levels, we gonadectomized five fetal rhesus males on Days 98-104 of gestation and immediately implanted crystalline-T-containing intraabdominal Silastic capsules. An additional five fetuses were treated with the nonaromatizable androgen dihydrotestosterone (DHT). Umbilical arterial samples for hormone analysis were obtained prior to GX and again approximately 3 wk later. Serum from control males (n = 11) castrated in utero on Day 100 of gestation contained significantly greater concentrations of LH and follicle-stimulating hormone (FSH) 3 wk after the operation than before GX. Five sham-operated male fetuses did not have elevated levels of either LH or FSH in their serum on Day 120 of gestation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic aspects of the reactions of humoral immunity to collagen in man and animals. II. Modifications of the autoimmune status to collagen type I in NZB x NZW (F1) mice by sex hormones during ontogeny

The sex hormones, estradiol and testosterone, are able to modulate the status of spontaneous reactions of humoral immunity to type I collagen in ontogenesis of NZB x NZW (F1) females. Administration of estradiol to puber and unpuber females leads to a significant increase in the reactivity levels. The autoimmune status to type I collagen in NZB x NZW (F1) males is nonreactive to sex hormones influence. The results obtained corroborate the suggestion of the important role of sex hormones in formation of sex dimorphism and age variability to autoimmunity to type I collagen.     

Dysregulation of neonatal hippocampal cell genesis in the androgen insensitive Tfm rat

The first two weeks of life are a critical period for hippocampal development. At this time gonadal steroid exposure organizes sex differences in hippocampal sensitivity to activational effects of steroids, hippocampal cell morphology and hippocampus dependent behaviors. Our laboratory has characterized a robust sex difference in neonatal neurogenesis in the hippocampus that is mediated by estradiol. Here, we extend our knowledge of this sex difference by comparing the male and female hippocampus to the androgen insensitive testicular feminized mutant (Tfm) rat. In the neonatal Tfm rat hippocampus, fewer newly generated cells survive compared to males or females. This deficit in cell genesis is partially recovered with the potent androgen DHT, but is more completely recovered following estradiol administration. Tfm rats do not differ from males or females in the level of endogenous estradiol in the neonatal hippocampus, suggesting other mechanisms mediate a differential sensitivity to estradiol in male, female and Tfm hippocampus. We also demonstrate disrupted performance on a hippocampal-dependent contextual fear discrimination task. Tfm rats generalize fear across contexts, and do not exhibit significant loss of fear during extinction exposure. These results extend prior reports of exaggerated response to stress in Tfm rats, and following gonadectomy in normal male rats.     

Social regulation of plasma estradiol concentration in a female anuran

The behavior of an individual within a social aggregation profoundly influences behavior and physiology of other animals within the aggregation in such a way that these social interactions can enhance reproductive success, survival and fitness. This phenomenon is particularly important during the breeding season when males and female must synchronize their reproductive efforts. We examined whether exposure to conspecific social cues can elevate sex steroid levels, specifically estradiol and androgens, in female túngara frogs (Physalaemus pustulosus). We compared plasma estradiol and androgen concentrations in wild-caught females before and after exposure to either natural mate choruses or random tones. After exposure to mate choruses for 10 consecutive nights, estradiol concentrations were significantly elevated whereas there was no significant elevation in estradiol concentrations in the group of females exposed to random tones for 10 nights. Plasma androgen concentrations were not significantly changed after exposure to either natural mate choruses or random tones for 10 consecutive nights. Social modulation of estradiol concentrations may be important in maintaining a female's reproductive state while males are chorusing. To our knowledge, this is the first study to demonstrate social regulation of estradiol concentration in female anurans.     

Sex Steroids and the Differentiation of Avian Reproductive Behavior

Experiments in which avian embryos are treated with sex steroidsor steroid antagonists suggest that sexual differentiation ofreproductive behavior (and thus differentiation of the brainmechanisms for such behavior) is controlled by steroids producedby the embryonic gonads. In chickens and Japanese quail, maleshatched from eggs treated with estradiol or testosterone duringincubation are feminized (demasculinized); they fail to exhibitmasculine sexual behavior as adults, and no longer are behaviorallydistinguishable from females. Some evidence suggests that testosteronemay mimic the feminizing action of estradiol by being convertedto an estrogen in the embryonic brain. Genetic female quailexposed to an antiestrogen during embryonic development aremasculinized; they exhibit an increased ability to display themasculine copulatory pattern. Thus the behavior of these speciesis feminized by embryonic exposure to sex steroids, the anhormonal(neutral) sex for behavioral differentiation appears to be themale, and females appear to result from estrogen produced bythe embryonic ovaries. In contrast, sex steroid treatment ofmammals early in development masculinizes behavior, the femaleis the neutral sex, and males result from fetal androgen secretion.These opposite patterns of psychosexual differentiation in birdsand mammals are correlated with a major difference between theavian and mammalian sex-determining mechanism. Implicationsfor other vertebrates are discussed.     

Disruption of Parenting Behaviors in California Mice,a Monogamous Rodent Species,by Endocrine Disrupting Chemicals

The nature and extent of care received by an infant can affect social, emotional and cognitive development, features that endure into adulthood. Here we employed the monogamous, California mouse (Peromyscus californicus), a species, like the human, where both parents invest in offspring care, to determine whether early exposure to endocrine disrupting chemicals (EDC: bisphenol A, BPA; ethinyl estradiol, EE) of one or both parents altered their behaviors towards their pups. Females exposed to either compound spent less time nursing, grooming and being associated with their pups than controls, although there was little consequence on their weight gain. Care of pups by males was less affected by exposure to BPA and EE, but control, non-exposed females appeared able to “sense” a male partner previously exposed to either compound and, as a consequence, reduced their own parental investment in offspring from such pairings. The data emphasize the potential vulnerability of pups born to parents that had been exposed during their own early development to EDC, and that effects on the male, although subtle, also have consequences on overall parental care due to lack of full acceptance of the male by the female partner.     

Perinatal exposure to progesterone, estradiol, or mifepristone affects sexual differentiation of behavior in opossums (Monodelphis domestica)

The effects of perinatal exposure to progesterone (P) and estradiol (E) on sexual differentiation of behavior and morphology were examined by treating male and female gray short-tailed opossums on postnatal day 8 with progesterone alone (P), P plus estradiol (E) (PE), the P receptor antagonist mifepristone/RU486 (MIF), or corn oil control (C) and gonadectomizing them before puberty. When given female hormone replacement therapy in adulthood and tested with intact stimulus males, MIF animals showed less female-typical aggressive threat behavior than animals in other treatment groups. Stimulus males scent marked in more tests involving females than males and in more tests involving MIF animals than animals in other treatment groups. Body weight was lower in females than in males and was lower in MIF animals than in animals in other treatment groups, and P females failed to show female-typical genital locks after copulation. Sexual receptivity was similar in males and females and, while not decreased by any perinatal hormone treatment, was higher in PE males than in animals of either sex in any treatment group. These findings suggest that perinatal exposure to P is associated with the organization of feminine threat behavior and the defeminization of attractivity, body weight and genital anatomy in this marsupial. Reasons for these findings and for why female sexual receptivity is enhanced by perinatal exposure to exogenous E only in an endogenous masculine environment are discussed.     

Structural teratogenicity evaluation of methyl chloride in rats and mice after inhalation exposure

One hundred bred Fischer-344 female rats were exposed daily for 6 hours to atmospheres containing 0, 100, 500, or 1,500 ppm methyl chloride, 25 females per exposure concentration, from gestation day (gd) 7 through gd 19. On gd 20, the females were sacrificed for evaluation of maternal reproductive and fetal parameters. Maternal and fetal toxicity was apparent at the highest exposure concentration. There were no methyl chloride-induced external, skeletal, or visceral abnormalities seen in the fetuses. One hundred thirty-two C57BL/6 female mice bred to C3H males to produce B6C3F1 offspring were exposed daily for 6 hours to atmospheres containing 0, 100, 500, or 1,500 ppm methyl chloride, 33 females per exposure concentration, from gd 6 through gd 17. Exposure to the entire 1,500-ppm group was terminated on gd 10-14, with the animals killed in extremis. Selective necrosis of neurons in the internal granular layer of the cerebellum, ranging from individual cell involvement to focal areas comprising large numbers of neurons, was found in all females. On gd 18, the females from the other treatment groups, all of which survived, were killed for evaluation of maternal reproductive and fetal parameters. No evidence was seen of maternal or fetal toxicity in these exposure groups. There were no significant alterations in external appearance in fetuses from any of the exposure groups. Visceral examination of mouse fetuses revealed a small, but statistically significant, incidence of heart defects in litters of the 500-ppm group. The anomaly, a reduction or absence of the atrioventricular valve, chordae tendineae, and papillary muscle, was observed on the left side (bicuspid valve) in three fetuses and the right side (tricuspid valve) in six fetuses: three males and six females. It is concluded that methyl chloride inhalation exposure in pregnant rats, during critical periods of embryo and fetal development, is not teratogenic at concentrations which elicit maternal and fetal toxicity. In pregnant mice, methyl chloride was severely toxic to dams following 4 days or more of exposure to 1,500 ppm in air. Methyl chloride, at 500, but not 100 ppm, was teratogenic in mice, leading to a malformation in the heart. No embryo-fetal toxicity or teratogenicity was associated with exposure of mice, during critical periods of embryo and fetal development, to 100 ppm of ethyl chloride.     

Urinary sex steroids during sexual development in female mice and in proximate novel males.

The experiments described here were designed to determine whether males' capacity to accelerate female pubertal development is reflected in females' urinary steroid levels in mice, and whether steroids in males' urine are influenced by exposure to developing females. In the first experiment, measures from urine collected daily from female mice aged 31-59 days showed a gradual rise in 17beta-estradiol levels and a distinct linear rise in progesterone levels. In a second experiment, daily steroids were measured in females aged 30-42 days while they were either housed alone or underneath two novel outbred males. Females exposed to males showed accelerated development at day 43 in uterine weight, and to a lesser extent in ovarian and whole-body weights. Average steroid levels did not significantly differ between conditions, but intra-individual variance in estradiol measures was greater in male-exposed than in isolated females. Creatinine levels were higher in isolated females. Males exposed to developing females excreted higher levels of estradiol in their urine compared to isolated males. These data suggest that excreted steroids can reflect general pubertal development, but may not fully reflect substantial morphological impacts of exposure to novel males. Elevations of estrogen levels in males exposed to developing females could help to account for precocious puberty in such females.     

Interaction of bisphenol A (BPA) and soy phytoestrogens on sexually dimorphic sociosexual behaviors in male and female rats

Concerns have been raised regarding the potential for endocrine disrupting compounds (EDCs) to alter brain development and behavior. Developmental exposure to bisphenol A (BPA), a ubiquitous EDC, has been linked to altered sociosexual and mood-related behaviors in various animal models and children but effects are inconsistent across laboratories and animal models creating confusion about potential risk in humans. Exposure to endocrine active diets, such as soy, which is rich in phytoestrogens, may contribute to this variability. Here, we tested the individual and combined effects of low dose oral BPA and soy diet or the individual isoflavone genistein (GEN; administered as the aglycone genistin (GIN)) on rat sociosexual behaviors with the hypothesis that soy would obfuscate any BPA-related effects. Social and activity levels were unchanged by developmental exposure to BPA but soy diet had sex specific effects including suppressed novelty preference, and open field exploration in females. The data presented here reinforce that environmental factors, including anthropogenic chemical exposure and hormone active diets, can shape complex behaviors and even reverse expected sex differences.     

Prenatal testosterone exposure leads to hypertension that is gonadal hormone-dependent in adult rat male and female offspring

Prenatal testosterone exposure impacts postnatal reproductive and endocrine function, leading to alterations in sex steroid levels. Because gonadal steroids are key regulators of cardiovascular function, it is possible that alteration in sex steroid hormones may contribute to development of hypertension in prenatally testosterone-exposed adults. The objectives of this study were to evaluate whether prenatal testosterone exposure leads to development of hypertension in adult males and females and to assess the influence of gonadal hormones on arterial pressure in these animals. Offspring of pregnant rats treated with testosterone propionate or its vehicle (controls) were examined. Subsets of male and female offspring were gonadectomized at 7 wk of age, and some offspring from age 7 to 24 wk received hormone replacement, while others did not. Testosterone exposure during prenatal life significantly increased arterial pressure in both male and female adult offspring; however, the effect was greater in males. Prenatal androgen-exposed males and females had more circulating testosterone during adult life, with no change in estradiol levels. Gonadectomy prevented hyperandrogenism and also reversed hypertension in these rats. Testosterone replacement in orchiectomized males restored hypertension, while estradiol replacement in ovariectomized females was without effect. Steroidal changes were associated with defective expression of gonadal steroidogenic genes, with Star, Sf1, and Hsd17b1 upregulation in testes. In ovaries, Star and Cyp11a1 genes were upregulated, while Cyp19 was downregulated. This study showed that prenatal testosterone exposure led to development of gonad-dependent hypertension during adult life. Defective steroidogenesis may contribute in part to the observed steroidal changes.     

Sex of incubation neighbours influences hatchling sexual phenotypes in an oviparous lizard

In many litter-bearing mammals and in a few viviparous reptiles the sex ratio of the entire brood or the sex of the adjacent fetuses induces sex-specific differences in the hatchling’s phenotype. This study examines whether the sex of incubation neighbours affects hatchling characteristics in oviparous common lizards (Lacerta vivipara). Oviparous common lizards lay eggs with thin eggshells and, therefore, are an optimal model organism for studying the effects of hormone leakage among developing embryos since the strongest evidence for prenatal sex ratio effects on offspring development comes from viviparous populations of the same species. Groups of three eggs were incubated together and were categorised according to the sex of the resulting hatchlings as either homosex (three hatchlings of the same sex) or heterosex (one male or one female hatchling plus two siblings of the opposite sex). Hatchlings incubated adjacent to siblings of the same sex had larger body mass and body condition. Males tended to have lower ventral scale counts when incubated with other males. Conversely, females tended to have more ventral scales when incubated with other females, indicative of a more feminised phenotype. There was also a significant interaction between hatchling sex and incubation environment with respect to the length of the fourth digit of the hindlimb, likely indicative of masculinisation in heterosex females. This study suggests steroid diffusion between adjacent eggs in a minimally manipulative experiment and provides the first evidence for developmental effects of the exogenous hormonal environment in near natural conditions in an oviparous amniote. Implications of these results for the evolution of within-clutch sex ratio are discussed.