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Anxiety disorders are a major public health concern and current treatments are inadequate for many individuals. Anxiety is more common in women than men and this difference arises during puberty. Sex differences in physiological stress responses may contribute to this variability. During puberty, gonadal hormones shape brain structure and function, but the extent to which these changes affect stress sensitivity is unknown. We examined how pubertal androgens shape behavioral and neural responses to social stress in California mice (Peromyscus californicus), a model species for studying sex differences in stress responses. In adults, social defeat reduces social approach and increases social vigilance in females but not males. We show this sex difference is absent in juveniles, and that prepubertal castration sensitizes adult males to social defeat. Adult gonadectomy does not alter behavioral responses to defeat, indicating that gonadal hormones act during puberty to program behavioral responses to stress in adulthood. Calcium imaging in the medioventral bed nucleus of the stria terminalis (BNST) showed that social threats increased neural activity and that prepubertal castration generalized these responses to less threatening social contexts. These results support recent hypotheses that the BNST responds to immediate threats. Prepubertal treatment with the nonaromatizable androgen dihydrotestosterone acts in males and females to reduce the effects of defeat on social approach and vigilance in adults. These data indicate that activation of androgen receptors during puberty is critical for programming behavioral responses to stress in adulthood.

Understanding sex differences in physiology and disease requires the identification of the molecular agents that cause phenotypic sex differences. Two groups of such agents are genes located on the sex chromosomes, and gonadal hormones. The former have coherent linkage to chromosomes that form differently in the two sexes under the influence of genomic forces that are not related to reproductive function, whereas the latter have a direct or indirect relationship to reproduction. Evidence published in the past 5 years supports the identification of several agents of sexual differentiation encoded by the X chromosome in mice, including Kdm5c, Kdm6a, Ogt and Xist. These X chromosome agents have wide pleiotropic effects, potentially influencing sex differences in many different tissues, a characteristic shared with the gonadal hormones. The identification of X chromosome agents of sexual differentiation will facilitate understanding of complex intersecting gene pathways underlying sex differences in disease.

Sex differences in physiology and disease in mammals result from the effects of three classes of factors that are inherently unequal in males and females: reversible (activational) effects of gonadal hormones, permanent (organizational) effects of gonadal hormones, and cell-autonomous effects of sex chromosomes, as well as genes driven by these classes of factors. Often, these factors act together to cause sex differences in specific phenotypes, but the relative contribution of each and the interactions among them remain unclear. Here, we used the four core genotypes (FCG) mouse model with or without hormone replacement to distinguish the effects of each class of sex-biasing factors on transcriptome regulation in liver and adipose tissues. We found that the activational hormone levels have the strongest influence on gene expression, followed by the organizational gonadal sex effect, and last, sex chromosomal effect, along with interactions among the three factors. Tissue specificity was prominent, with a major impact of estradiol on adipose tissue gene regulation and of testosterone on the liver transcriptome. The networks affected by the three sex-biasing factors include development, immunity and metabolism, and tissue-specific regulators were identified for these networks. Furthermore, the genes affected by individual sex-biasing factors and interactions among factors are associated with human disease traits such as coronary artery disease, diabetes, and inflammatory bowel disease. Our study offers a tissue-specific account of the individual and interactive contributions of major sex-biasing factors to gene regulation that have broad impact on systemic metabolic, endocrine, and immune functions.

Extensive practice and knowledge of the methods and effects of castration of male livestock and even humans has been widespread since ancient times, but only a few decades ago did neutering (including spaying) become a routine part of canine husbandry. In the US, the 6-month neuter became standard practice. Only recently, however, have some of the consequences of this major physiological alteration become evident. As the data-based study on 35 breeds reveals, there are major breed differences associated with effects of neutering, especially with early neutering, including increased risks of joint disorders and some cancers. The study of mixed-breed dogs reveals that the risk of joint disorders is increased in the large dogs. Implications of breed-specific and sex-specific effects for age of neutering have prompted the consideration of a new paradigm with regard to this practice. This involves focusing on each individual dog when deciding upon the appropriate age of neutering to avoid increasing the risk of a joint disorder or cancer above that inherent for the breed. For many breeds, particularly the smaller dogs, no effects were found for the age of neutering on joint disorders and the cancers followed. In these cases, the caregiver has a wide range of choice for neutering without increasing the dog's risk for these diseases. In the future, additional research may reveal more about other increased risks for age-related cognitive dysfunction or elevated levels of luteinizing hormone caused by gonad removal, and lead to revised guidelines.

Extensive practice and knowledge of the methods and effects of castration of male livestock and even humans has been widespread since ancient times, but only a few decades ago did neutering (including spaying) become a routine part of canine husbandry. In the US, the 6-month neuter became standard practice. Only recently, however, have some of the consequences of this major physiological alteration become evident. As the data-based study on 35 breeds reveals, there are major breed differences associated with effects of neutering, especially with early neutering, including increased risks of joint disorders and some cancers. The study of mixed-breed dogs reveals that the risk of joint disorders is increased in the large dogs. Implications of breed-specific and sex-specific effects for age of neutering have prompted the consideration of a new paradigm with regard to this practice. This involves focusing on each individual dog when deciding upon the appropriate age of neutering to avoid increasing the risk of a joint disorder or cancer above that inherent for the breed. For many breeds, particularly the smaller dogs, no effects were found for the age of neutering on joint disorders and the cancers followed. In these cases, the caregiver has a wide range of choice for neutering without increasing the dog's risk for these diseases. In the future, additional research may reveal more about other increased risks for age-related cognitive dysfunction or elevated levels of luteinizing hormone caused by gonad removal, and lead to revised guidelines.

Adolescence is characterized by rapid brain development in white matter (WM) that is attributed in part to surges in gonadal hormones. To date, however, there have been few longitudinal investigations relating changes in gonadal hormones and WM development in adolescents. We acquired diffusion-weighted MRI to estimate mean fractional anisotropy (FA) from 10 WM tracts and salivary testosterone from 51 females and 29 males (ages 9-14 years) who were matched on pubertal stage and followed, on average, for 2 years. We tested whether interactions between sex and changes in testosterone levels significantly explained changes in FA. We found positive associations between changes in testosterone and changes in FA within the corpus callosum, cingulum cingulate, and corticospinal tract in females (all ps

Evolution of genetic mechanisms of sex determination led to two processes causing sex differences in somatic phenotypes: gonadal differentiation and sex chromosome dosage inequality. In species with heteromorphic sex chromosomes, the sex of the individual is established at the time of formation of the zygote, leading to inherent sex differences in expression of sex chromosome genes beginning as soon as the embryonic transcriptome is activated. The inequality of sex chromosome gene expression causes sexual differentiation of the gonads and of non-gonadal tissues. The difference in gonad type in turn causes lifelong differences in gonadal hormones, which interact with unequal effects of X and Y genes acting within cells. Separating the effects of gonadal hormones and sex chromosomes has been possible using mouse models in which gonadal determination is separated from the sex chromosomes, allowing comparison of XX and XY mice with the same type of gonad. Sex differences caused by gonadal hormones and sex chromosomes affect basic physiology and disease mechanisms in most or all tissues.

BackgroundSex differences in obesity and related diseases are well established. Gonadal hormones are a major determinant of these sex differences. However, sex differences in body size and composition are evident prior to exposure to gonadal hormones, providing evidence for gonadal-independent contributions attributable to the XX or XY sex chromosome complement. Large-scale genetic studies have revealed male/female differences in the genetic architecture of adipose tissue amount and anatomical distribution. However, these studies have typically neglected the X and Y chromosomes.Scope of the reviewHere we discuss how the sex chromosome complement may influence obesity, lipid levels, and inflammation. Human sex chromosome anomalies such as Klinefelter syndrome (XXY), as well as mouse models with engineered alterations in sex chromosome complement, support an important role for sex chromosomes in obesity and metabolism. In particular, the Four Core Genotypes mouse model-consisting of XX mice with either ovaries or testes, and XY mice with either ovaries or testes-has revealed an effect of X chromosome dosage on adiposity, hyperlipidemia, and inflammation irrespective of male or female gonads. Mechanisms may include enhanced expression of genes that escape X chromosome inactivation.Major conclusionsAlthough less well studied than effects of gonadal hormones, sex chromosomes exert independent and interactive effects on adiposity, lipid metabolism, and inflammation. In particular, the presence of two X chromosomes has been associated with increased adiposity and dyslipidemia in mouse models and in XXY men. The enhanced expression of genes that escape X chromosome inactivation may contribute, but more work is required.

BackgroundSex differences in obesity and related diseases are well established. Gonadal hormones are a major determinant of these sex differences. However, sex differences in body size and composition are evident prior to exposure to gonadal hormones, providing evidence for gonadal-independent contributions attributable to the XX or XY sex chromosome complement. Large-scale genetic studies have revealed male/female differences in the genetic architecture of adipose tissue amount and anatomical distribution. However, these studies have typically neglected the X and Y chromosomes.Scope of the reviewHere we discuss how the sex chromosome complement may influence obesity, lipid levels, and inflammation. Human sex chromosome anomalies such as Klinefelter syndrome (XXY), as well as mouse models with engineered alterations in sex chromosome complement, support an important role for sex chromosomes in obesity and metabolism. In particular, the Four Core Genotypes mouse model-consisting of XX mice with either ovaries or testes, and XY mice with either ovaries or testes-has revealed an effect of X chromosome dosage on adiposity, hyperlipidemia, and inflammation irrespective of male or female gonads. Mechanisms may include enhanced expression of genes that escape X chromosome inactivation.Major conclusionsAlthough less well studied than effects of gonadal hormones, sex chromosomes exert independent and interactive effects on adiposity, lipid metabolism, and inflammation. In particular, the presence of two X chromosomes has been associated with increased adiposity and dyslipidemia in mouse models and in XXY men. The enhanced expression of genes that escape X chromosome inactivation may contribute, but more work is required.

Biospecimen collection in the Adolescent Brain Cognitive Development (ABCD) study - of hair samples, shed deciduous (baby) teeth, and body fluids - will serve dual functions of screening for study eligibility, and providing measures of biological processes thought to predict or correlate with key study outcomes on brain and cognitive development. Biosamples are being collected annually to screen for recency of drug use prior to the neuroimaging or cognitive testing visit, and to store for the following future studies: (1) on the effects of exposure to illicit and recreational drugs (including alcohol and nicotine); (2) of pubertal hormones on brain and cognitive developmental trajectories; (3) on the contribution of genomics and epigenomics to child and adolescent development and behavioral outcomes; and (4) with pre- and post-natal exposure to environmental neurotoxicants and drugs of abuse measured from novel tooth analyses. The present manuscript describes the rationales for inclusion and selection of the specific biospecimens, methodological considerations for each measure, future plans for assessment of biospecimens during follow-up visits, and preliminary ABCD data to illustrate methodological considerations.

Biospecimen collection in the Adolescent Brain Cognitive Development (ABCD) study - of hair samples, shed deciduous (baby) teeth, and body fluids - will serve dual functions of screening for study eligibility, and providing measures of biological processes thought to predict or correlate with key study outcomes on brain and cognitive development. Biosamples are being collected annually to screen for recency of drug use prior to the neuroimaging or cognitive testing visit, and to store for the following future studies: (1) on the effects of exposure to illicit and recreational drugs (including alcohol and nicotine); (2) of pubertal hormones on brain and cognitive developmental trajectories; (3) on the contribution of genomics and epigenomics to child and adolescent development and behavioral outcomes; and (4) with pre- and post-natal exposure to environmental neurotoxicants and drugs of abuse measured from novel tooth analyses. The present manuscript describes the rationales for inclusion and selection of the specific biospecimens, methodological considerations for each measure, future plans for assessment of biospecimens during follow-up visits, and preliminary ABCD data to illustrate methodological considerations.

Pyramidal neurons in the neocortex receive a majority of their synapses on dendritic spines, whose growth, gain, and loss regulate the strength and identity of neural connections. Juvenile brains typically show higher spine density and turnover compared to adult brains, potentially enabling greater capacity for experience-dependent circuit 'rewiring'. Although spine pruning and stabilization in frontal cortex overlap with pubertal milestones, it is unclear if gonadal hormones drive these processes. To address this question, we used hormone manipulations and in vivo 2-photon microscopy to test for a causal relationship between pubertal hormones and spine pruning and stabilization in layer 5 neurons in the frontal cortex of female mice. We found that spine density, gains, and losses decreased from P27 to P60 and that these measures were not affected by pre-pubertal hormone injections or ovariectomy. Further analyses of spine morphology after manipulation of gonadal hormones suggest that gonadal hormones may play a role in morphological maturation and dynamics. Our data help to segregate hormone-sensitive and hormone-insensitive maturational processes that occur simultaneously in dorsomedial frontal cortex. These data provide more specific insight into adolescent development and may have implications for understanding the neurodevelopmental effects of changes in pubertal timing in humans.

Pyramidal neurons in the neocortex receive a majority of their synapses on dendritic spines, whose growth, gain, and loss regulate the strength and identity of neural connections. Juvenile brains typically show higher spine density and turnover compared to adult brains, potentially enabling greater capacity for experience-dependent circuit 'rewiring'. Although spine pruning and stabilization in frontal cortex overlap with pubertal milestones, it is unclear if gonadal hormones drive these processes. To address this question, we used hormone manipulations and in vivo 2-photon microscopy to test for a causal relationship between pubertal hormones and spine pruning and stabilization in layer 5 neurons in the frontal cortex of female mice. We found that spine density, gains, and losses decreased from P27 to P60 and that these measures were not affected by pre-pubertal hormone injections or ovariectomy. Further analyses of spine morphology after manipulation of gonadal hormones suggest that gonadal hormones may play a role in morphological maturation and dynamics. Our data help to segregate hormone-sensitive and hormone-insensitive maturational processes that occur simultaneously in dorsomedial frontal cortex. These data provide more specific insight into adolescent development and may have implications for understanding the neurodevelopmental effects of changes in pubertal timing in humans.

BackgroundMicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression by targeting specific mRNA species for degradation or interfering with translation. Specific miRNAs are key regulators of adipogenesis, and are expressed at different levels in adipose tissue from lean and obese mice. The degree of lipid accumulation and distribution of white adipose tissue differs between males and females, and it is unknown whether sex differences in adipose tissue-specific miRNA expression may contribute to this dimorphism. Typically, sex differences are attributed to hormones secreted from ovaries or testes. However, the sex chromosome complement (XX versus XY) is also a determinant of sex differences and may regulate miRNA expression in adipocytes.ResultsTo identify sex differences in adipose tissue miRNA expression and to understand the underlying mechanisms, we performed high-throughput miRNA sequencing in gonadal fat depots of the Four Core Genotypes mouse model. This model, which consists of XX female, XX male, XY female, and XY male mice, allowed us to assess independent effects of gonadal type (male vs. female) and sex chromosome complement (XX vs. XY) on miRNA expression profiles. We have also assessed the effects of a high fat diet on sex differences in adipose tissue miRNA profiles. We identified a male-female effect on the overall miRNA expression profile in mice fed a chow diet, with a bias toward higher expression in male compared to female gonadal adipose tissue. This sex bias disappeared after gonadectomy, suggesting that circulating levels of gonadal secretions modulate the miRNA expression profile. After 16 weeks of high fat diet, the miRNA expression distribution was shifted toward higher expression in XY vs. XX adipose tissue. Principal component analysis revealed that high fat diet has a substantial effect on miRNA profile variance, while gonadal secretions and sex chromosome complement each have milder effects.ConclusionsOur results demonstrate that the overall miRNA expression profile in adipose tissue is influenced by gonadal hormones and the sex chromosome complement, and that expression profiles change in response to gonadectomy and high fat diet. Differential miRNA expression profiles may contribute to sex differences in adipose tissue gene expression, adipose tissue development, and diet-induced obesity.

Anxiety and depression symptoms increase dramatically during adolescence, with girls showing a steeper increase than boys after puberty onset. The timing of the onset of this sex bias led us to hypothesize that ovarian hormones contribute to depression and anxiety during puberty. In humans, it is difficult to disentangle direct effects of gonadal hormones from social and environmental factors that interact with pubertal development to influence mental health. To test the role of gonadal hormones in anxiety- and depression-related behavior during puberty, we manipulated gonadal hormones in mice while controlling social and environmental factors. Similar to humans, we find that mice show an increase in depression-related behavior from pre-pubertal to late-pubertal ages, but this increase is not dependent on gonadal hormones and does not differ between sexes. Anxiety-related behavior, however, is more complex during puberty, with differences that depend on sex, age, behavioral test, and hormonal status. Briefly, males castrated before puberty show greater anxiety-related behavior during late puberty compared to intact males, while pubertal females are unaffected by ovariectomy or hormone injections in all assays except the marble burying test. Despite this sex-specific effect of pubertal hormones on anxiety-related behavior, we find no sex differences in intact young adults, suggesting that males and females use separate mechanisms to converge on a similar behavioral phenotype. Our results are consistent with anxiolytic effects of testicular hormones during puberty in males but are not consistent with a causal role for ovarian hormones in increasing anxiety- and depression-related behavior during puberty in females.

Despite accumulating evidence from animal models demonstrating that prenatal alcohol exposure (PAE) results in life-long neuroendocrine dysregulation, very little is known on this topic among humans with fetal alcohol spectrum disorders (FASD). We expected that alterations in gonadal hormones might interfere with the typical development of white matter (WM) myelination, and in a sex-dependent manner, in human adolescents with FASD. In order to investigate this hypothesis, we used diffusion tensor imaging (DTI) to assess: 1) whether or not sex moderates the impact of PAE on WM microstructure; and 2) how gonadal hormones relate to alterations in WM microstructure in children and adolescents affected by PAE.Methods61 youth (9 to 16 yrs.; 49% girls; 50% PAE) participated as part of the Collaborative Initiative on Fetal Alcohol Spectrum Disorders (CIFASD). DTI scans and passive drool samples were obtained to examine neurodevelopmental associations with testosterone (T) and dehydroepiandrosterone (DHEA) levels in boys and girls, and estradiol (E2) and progesterone (P) levels in girls. Tract-based spatial statistics were utilized to generate fractional anisotropy (FA) and mean diffusivity (MD) for 9 a priori WM regions of interest (ROIs).ResultsAs predicted, alterations in FA were observed in adolescents with PAE relative to controls, and these differences varied by sex. Girls with PAE exhibited lower FA (Inferior fronto-occipital and Uncinate fasciculi) while boys with PAE exhibited higher FA (Callosal body, Cingulum, Corticospinal tract, Optic radiation, Superior longitudinal fasciculus) relative to age-matched controls. When gonadal hormone levels were examined in relation to DTI measures, additional group differences in FA were revealed, demonstrating that neuroendocrine factors are associated with PAE-related brain alterations.ConclusionsThese findings provide human evidence that PAE relates to sex-specific differences in WM microstructure, and underlying alterations in gonadal hormone function may, in part, contribute to these effects. Determining PAE-effects on neuroendocrine function among humans is an essential first step towards developing novel clinical (e.g., assessment or intervention) tools that target hormone systems to improve on-going brain development among children and adolescents with FASD.

Men and women exhibit significant differences in obesity, cardiovascular disease, and diabetes. To provide better diagnosis and treatment for both sexes, it is important to identify factors that underlie the observed sex differences. Traditionally, sex differences have been attributed to the differential effects of male and female gonadal secretions (commonly referred to as sex hormones), which substantially influence many aspects of metabolism and related diseases. Less appreciated as a contributor to sex differences are the fundamental genetic differences between males and females, which are ultimately determined by the presence of an XX or XY sex chromosome complement. Here, we review the mechanisms by which gonadal hormones and sex chromosome complement each contribute to lipid metabolism and associated diseases, and the current approaches that are used to study them. We focus particularly on genetic approaches including genome-wide association studies in humans and mice, -omics and systems genetics approaches, and unique experimental mouse models that allow distinction between gonadal and sex chromosome effects.

The frontal cortex matures late in development, showing dramatic changes after puberty onset, yet few experiments have directly tested the role of pubertal hormones in cortical maturation. One mechanism thought to play a primary role in regulating the maturation of the neocortex is an increase in inhibitory neurotransmission, which alters the balance of excitation and inhibition. We hypothesized that pubertal hormones could regulate maturation of the frontal cortex by this mechanism. Here, we report that manipulations of gonadal hormones do significantly alter the maturation of inhibitory neurotransmission in the cingulate region of the mouse medial frontal cortex, an associative region that matures during the pubertal transition and is implicated in decision making, learning, and psychopathology. We find that inhibitory neurotransmission, but not excitatory neurotransmission, increases onto cingulate pyramidal neurons during peri-pubertal development and that this increase can be blocked by pre-pubertal, but not post-pubertal, gonadectomy. We next used pre-pubertal hormone treatment to model early puberty onset, a phenomenon increasingly observed in girls living in developed nations. We find that pre-pubertal hormone treatment drives an early increase in inhibitory neurotransmission in the frontal cortex, but not the somatosensory cortex, suggesting that earlier puberty can advance cortical maturation in a regionally specific manner. Pre-pubertal hormone treatment also accelerates maturation of tonic inhibition and performance in a frontal-cortex-dependent reversal-learning task. These data provide rare evidence of enduring, organizational effects of ovarian hormones at puberty and provide a potential mechanism by which gonadal hormones could regulate the maturation of the associative neocortex.

BackgroundMicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression by targeting specific mRNA species for degradation or interfering with translation. Specific miRNAs are key regulators of adipogenesis, and are expressed at different levels in adipose tissue from lean and obese mice. The degree of lipid accumulation and distribution of white adipose tissue differs between males and females, and it is unknown whether sex differences in adipose tissue-specific miRNA expression may contribute to this dimorphism. Typically, sex differences are attributed to hormones secreted from ovaries or testes. However, the sex chromosome complement (XX versus XY) is also a determinant of sex differences and may regulate miRNA expression in adipocytes.ResultsTo identify sex differences in adipose tissue miRNA expression and to understand the underlying mechanisms, we performed high-throughput miRNA sequencing in gonadal fat depots of the Four Core Genotypes mouse model. This model, which consists of XX female, XX male, XY female, and XY male mice, allowed us to assess independent effects of gonadal type (male vs. female) and sex chromosome complement (XX vs. XY) on miRNA expression profiles. We have also assessed the effects of a high fat diet on sex differences in adipose tissue miRNA profiles. We identified a male-female effect on the overall miRNA expression profile in mice fed a chow diet, with a bias toward higher expression in male compared to female gonadal adipose tissue. This sex bias disappeared after gonadectomy, suggesting that circulating levels of gonadal secretions modulate the miRNA expression profile. After 16 weeks of high fat diet, the miRNA expression distribution was shifted toward higher expression in XY vs. XX adipose tissue. Principal component analysis revealed that high fat diet has a substantial effect on miRNA profile variance, while gonadal secretions and sex chromosome complement each have milder effects.ConclusionsOur results demonstrate that the overall miRNA expression profile in adipose tissue is influenced by gonadal hormones and the sex chromosome complement, and that expression profiles change in response to gonadectomy and high fat diet. Differential miRNA expression profiles may contribute to sex differences in adipose tissue gene expression, adipose tissue development, and diet-induced obesity.

Adaptation virtually defines survival. For mammals, arguably, no other developmental milestone is exemplified by--nor more reliant on--the sudden and dramatic behavioral alterations observed in the maternal female, which rapidly must undergo change in order to express a large suite of proper and effective maternal behaviors. As pregnancy progresses, as well as during lactation, when pup cues are rich and rampant, the female is literally transformed from an organism that actively avoided offspring-related signals, to one highly motivated by those same cues to build nests, be attracted to pups and to retrieve, group, groom, crouch-over, care for, and protect, the young. Ancillary responses such as reference memory, spatial learning, foraging (including predation), and boldness improve in mothers compared to virgins. Such modifications arise early and are persistent, with neural benefits that last well into senescence. Evolutionarily, such enhancements have likely reduced the maternal burdens associated with sheltering and feeding the vulnerable young; collectively, this strengthens the mother's/parent's reproductive fitness and that of the pups in which all this effort is invested. Of the many behaviors that change as a function of pending or concurrent maternity, therefore, what is the role of modifications to resilience, the ability to withstand the numerous, unpredictable, and threatening environmental events that the mother/parent must daily, indeed, multiply daily, face and thwart in order to bring the offspring from pups to fully functioning adults. We explore these questions, and their connections, here in a multi-disciplinary manner focused on the constellation of change that summates to fundamentally alter the female for the rest of her life. Behavior, brain, neurochemistry and genes are fundamentally changed as the substrate for reproduction unfolds and expresses its inherent plasticity.

Adaptation virtually defines survival. For mammals, arguably, no other developmental milestone is exemplified by--nor more reliant on--the sudden and dramatic behavioral alterations observed in the maternal female, which rapidly must undergo change in order to express a large suite of proper and effective maternal behaviors. As pregnancy progresses, as well as during lactation, when pup cues are rich and rampant, the female is literally transformed from an organism that actively avoided offspring-related signals, to one highly motivated by those same cues to build nests, be attracted to pups and to retrieve, group, groom, crouch-over, care for, and protect, the young. Ancillary responses such as reference memory, spatial learning, foraging (including predation), and boldness improve in mothers compared to virgins. Such modifications arise early and are persistent, with neural benefits that last well into senescence. Evolutionarily, such enhancements have likely reduced the maternal burdens associated with sheltering and feeding the vulnerable young; collectively, this strengthens the mother's/parent's reproductive fitness and that of the pups in which all this effort is invested. Of the many behaviors that change as a function of pending or concurrent maternity, therefore, what is the role of modifications to resilience, the ability to withstand the numerous, unpredictable, and threatening environmental events that the mother/parent must daily, indeed, multiply daily, face and thwart in order to bring the offspring from pups to fully functioning adults. We explore these questions, and their connections, here in a multi-disciplinary manner focused on the constellation of change that summates to fundamentally alter the female for the rest of her life. Behavior, brain, neurochemistry and genes are fundamentally changed as the substrate for reproduction unfolds and expresses its inherent plasticity.

We measured diurnal rhythms of food intake, as well as body weight and composition, while varying three major classes of sex-biasing factors: activational and organizational effects of gonadal hormones, and sex chromosome complement (SCC). Four Core Genotypes (FCG) mice, comprising XX and XY gonadal males and XX and XY gonadal females, were either gonad-intact or gonadectomized (GDX) as adults (2.5months); food intake was measured second-by-second for 7days starting 5weeks later, and body weight and composition were measured for 22weeks thereafter. Gonadal males weighed more than females. GDX increased body weight/fat of gonadal females, but increased body fat and reduced body weight of males. After GDX, XX mice had greater body weight and more fat than XY mice. In gonad-intact mice, males had greater total food intake and more meals than females during the dark phase, but females had more food intake and meals and larger meals than males during the light phase. GDX reduced overall food intake irrespective of gonad type or SCC, and eliminated differences in feeding between groups with different gonads. Diurnal phase of feeding was influenced by all three sex-biasing variables. Gonad-intact females had earlier onset and acrophase (peak) of feeding relative to males. GDX caused a phase-advance of feeding, especially in XX mice, leading to an earlier onset of feeding in GDX XX vs. XY mice, but earlier acrophase in GDX males relative to females. Gonadal hormones and SCC interact in the control of diurnal rhythms of food intake.

Epidemiologic studies have shown sex differences in ischemic stroke. The four core genotype (FCG) mouse model, in which the testes determining gene, Sry, has been moved from Y chromosome to an autosome, was used to dissociate the effects of sex hormones from sex chromosome in ischemic stroke outcome. Middle cerebral artery occlusion (MCAO) in gonad intact FCG mice revealed that gonadal males (XXM and XYM) had significantly higher infarct volumes as compared with gonadal females (XXF and XYF). Serum testosterone levels were equivalent in adult XXM and XYM, as was serum estrogen in XXF and XYF mice. To remove the effects of gonadal hormones, gonadectomized FCG mice were subjected to MCAO. Gonadectomy significantly increased infarct volumes in females, while no change was seen in gonadectomized males, indicating that estrogen loss increases ischemic sensitivity. Estradiol supplementation in gonadectomized FCG mice rescued this phenotype. Interestingly, FCG male mice were less sensitive to effects of hormones. This may be due to enhanced expression of the transgene Sry in brains of FCG male mice. Sex differences in ischemic stroke sensitivity appear to be shaped by organizational and activational effects of sex hormones, rather than sex chromosomal complement.

Mounting evidence suggests that environmental factors-in particular, those that we are exposed to during perinatal life-can dramatically shape the organism's risk for later diseases, including neurobehavioral disorders. However, depending on the environmental insult, one sex may demonstrate greater vulnerability than the other sex. Herein, we focus on two well-defined extrinsic environmental factors that lead to sexually dimorphic behavioral differences in animal models and linkage in human epidemiological studies. These include maternal or psychosocial stress (such as social stress) and exposure to endocrine-disrupting compounds (such as one of the most prevalent, bisphenol A [BPA]). In general, the evidence suggests that early environmental exposures, such as BPA and stress, lead to more pronounced behavioral deficits in males than in females, whereas female neurobehavioral patterns are more vulnerable to later in life stress. These findings highlight the importance of considering sex differences and developmental timing when examining the effects of environmental factors on later neurobehavioral outcomes.

Mounting evidence suggests that environmental factors-in particular, those that we are exposed to during perinatal life-can dramatically shape the organism's risk for later diseases, including neurobehavioral disorders. However, depending on the environmental insult, one sex may demonstrate greater vulnerability than the other sex. Herein, we focus on two well-defined extrinsic environmental factors that lead to sexually dimorphic behavioral differences in animal models and linkage in human epidemiological studies. These include maternal or psychosocial stress (such as social stress) and exposure to endocrine-disrupting compounds (such as one of the most prevalent, bisphenol A [BPA]). In general, the evidence suggests that early environmental exposures, such as BPA and stress, lead to more pronounced behavioral deficits in males than in females, whereas female neurobehavioral patterns are more vulnerable to later in life stress. These findings highlight the importance of considering sex differences and developmental timing when examining the effects of environmental factors on later neurobehavioral outcomes.

Sexual dimorphism in body weight, fat distribution, and metabolic disease has been attributed largely to differential effects of male and female gonadal hormones. Here, we report that the number of X chromosomes within cells also contributes to these sex differences. We employed a unique mouse model, known as the "four core genotypes," to distinguish between effects of gonadal sex (testes or ovaries) and sex chromosomes (XX or XY). With this model, we produced gonadal male and female mice carrying XX or XY sex chromosome complements. Mice were gonadectomized to remove the acute effects of gonadal hormones and to uncover effects of sex chromosome complement on obesity. Mice with XX sex chromosomes (relative to XY), regardless of their type of gonad, had up to 2-fold increased adiposity and greater food intake during daylight hours, when mice are normally inactive. Mice with two X chromosomes also had accelerated weight gain on a high fat diet and developed fatty liver and elevated lipid and insulin levels. Further genetic studies with mice carrying XO and XXY chromosome complements revealed that the differences between XX and XY mice are attributable to dosage of the X chromosome, rather than effects of the Y chromosome. A subset of genes that escape X chromosome inactivation exhibited higher expression levels in adipose tissue and liver of XX compared to XY mice, and may contribute to the sex differences in obesity. Overall, our study is the first to identify sex chromosome complement, a factor distinguishing all male and female cells, as a cause of sex differences in obesity and metabolism.

Sexual dimorphism in body weight, fat distribution, and metabolic disease has been attributed largely to differential effects of male and female gonadal hormones. Here, we report that the number of X chromosomes within cells also contributes to these sex differences. We employed a unique mouse model, known as the "four core genotypes," to distinguish between effects of gonadal sex (testes or ovaries) and sex chromosomes (XX or XY). With this model, we produced gonadal male and female mice carrying XX or XY sex chromosome complements. Mice were gonadectomized to remove the acute effects of gonadal hormones and to uncover effects of sex chromosome complement on obesity. Mice with XX sex chromosomes (relative to XY), regardless of their type of gonad, had up to 2-fold increased adiposity and greater food intake during daylight hours, when mice are normally inactive. Mice with two X chromosomes also had accelerated weight gain on a high fat diet and developed fatty liver and elevated lipid and insulin levels. Further genetic studies with mice carrying XO and XXY chromosome complements revealed that the differences between XX and XY mice are attributable to dosage of the X chromosome, rather than effects of the Y chromosome. A subset of genes that escape X chromosome inactivation exhibited higher expression levels in adipose tissue and liver of XX compared to XY mice, and may contribute to the sex differences in obesity. Overall, our study is the first to identify sex chromosome complement, a factor distinguishing all male and female cells, as a cause of sex differences in obesity and metabolism.

BackgroundHuman breast cancer is a heterogeneous disease, histopathologically, molecularly and phenotypically. The molecular basis of this heterogeneity is not well understood. We have used a mouse model of DCIS that consists of unique lines of mammary intraepithelial neoplasia (MIN) outgrowths, the premalignant lesion in the mouse that progress to invasive carcinoma, to understand the molecular changes that are characteristic to certain phenotypes. Each MIN-O line has distinguishable morphologies, metastatic potentials and estrogen dependencies.MethodsWe utilized oligonucleotide expression arrays and high resolution array comparative genomic hybridization (aCGH) to investigate whole genome expression patterns and whole genome aberrations in both the MIN-O and tumor from four different MIN-O lines that each have different phenotypes. From the whole genome analysis at 35 kb resolution, we found that chromosome 1, 2, 10, and 11 were frequently associated with whole chromosome gains in the MIN-Os. In particular, two MIN-O lines had the majority of the chromosome gains. Although we did not find any whole chromosome loss, we identified 3 recurring chromosome losses (2F1-2, 3E4, 17E2) and two chromosome copy number gains on chromosome 11. These interstitial deletions and duplications were verified with a custom made array designed to interrogate the specific regions at approximately 550 bp resolution.ResultsWe demonstrated that expression and genomic changes are present in the early premalignant lesions and that these molecular profiles can be correlated to phenotype (metastasis and estrogen responsiveness). We also identified expression changes associated with genomic instability. Progression to invasive carcinoma was associated with few additional changes in gene expression and genomic organization. Therefore, in the MIN-O mice, early premalignant lesions have the major molecular and genetic changes required and these changes have important phenotypic significance. In contrast, the changes that occur in the transition to invasive carcinoma are subtle, with few consistent changes and no association with phenotype.ConclusionWe propose that the early lesions carry the important genetic changes that reflect the major phenotypic information, while additional genetic changes that accumulate in the invasive carcinoma are less associated with the overall phenotype.

BackgroundHuman breast cancer is a heterogeneous disease, histopathologically, molecularly and phenotypically. The molecular basis of this heterogeneity is not well understood. We have used a mouse model of DCIS that consists of unique lines of mammary intraepithelial neoplasia (MIN) outgrowths, the premalignant lesion in the mouse that progress to invasive carcinoma, to understand the molecular changes that are characteristic to certain phenotypes. Each MIN-O line has distinguishable morphologies, metastatic potentials and estrogen dependencies.MethodsWe utilized oligonucleotide expression arrays and high resolution array comparative genomic hybridization (aCGH) to investigate whole genome expression patterns and whole genome aberrations in both the MIN-O and tumor from four different MIN-O lines that each have different phenotypes. From the whole genome analysis at 35 kb resolution, we found that chromosome 1, 2, 10, and 11 were frequently associated with whole chromosome gains in the MIN-Os. In particular, two MIN-O lines had the majority of the chromosome gains. Although we did not find any whole chromosome loss, we identified 3 recurring chromosome losses (2F1-2, 3E4, 17E2) and two chromosome copy number gains on chromosome 11. These interstitial deletions and duplications were verified with a custom made array designed to interrogate the specific regions at approximately 550 bp resolution.ResultsWe demonstrated that expression and genomic changes are present in the early premalignant lesions and that these molecular profiles can be correlated to phenotype (metastasis and estrogen responsiveness). We also identified expression changes associated with genomic instability. Progression to invasive carcinoma was associated with few additional changes in gene expression and genomic organization. Therefore, in the MIN-O mice, early premalignant lesions have the major molecular and genetic changes required and these changes have important phenotypic significance. In contrast, the changes that occur in the transition to invasive carcinoma are subtle, with few consistent changes and no association with phenotype.ConclusionWe propose that the early lesions carry the important genetic changes that reflect the major phenotypic information, while additional genetic changes that accumulate in the invasive carcinoma are less associated with the overall phenotype.

BackgroundLittle is known about the effects of organic solvents on male reproductive health. To assess fertility and reproductive endocrine function in solvent-exposed men, we investigated time-to-pregnancy using a retrospective cohort design and cross-sectionally measured reproductive hormone concentrations in painters and millwrights compared to a reference group of carpenters.MethodsDetailed occupational, exposure, medical, and time-to-pregnancy histories were obtained by telephone interview. Plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone concentrations were determined by immunoassay. Exposure indices, which summarized working life exposure to total solvents, chlorinated solvents, aromatic solvents, and thinners, degreasers, varnishes, and adhesives as a category were calculated from exposure histories.ResultsFSH concentrations increased significantly with increasing exposure indices for all solvents and for chlorinated solvents. There were no significant associations of solvent exposure indices with LH or testosterone levels. LH, FSH, and testosterone concentrations also did not differ by job title. Using Cox regression, time-to-pregnancy was non-significantly longer in the painters and millwrights than the carpenters. There was no significant association between time-to-pregnancy and any of the solvent exposure indices; however, it should be noted that some of the pregnancies occurred more than 20 years previously, potentially reducing the reliability of the retrospectively collected pregnancy and exposure data.ConclusionsThe significant associations between FSH levels and solvent exposure indices suggest the potential for adverse effects of solvent exposures on reproductive function in men.

BackgroundLittle is known about the effects of organic solvents on male reproductive health. To assess fertility and reproductive endocrine function in solvent-exposed men, we investigated time-to-pregnancy using a retrospective cohort design and cross-sectionally measured reproductive hormone concentrations in painters and millwrights compared to a reference group of carpenters.MethodsDetailed occupational, exposure, medical, and time-to-pregnancy histories were obtained by telephone interview. Plasma luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone concentrations were determined by immunoassay. Exposure indices, which summarized working life exposure to total solvents, chlorinated solvents, aromatic solvents, and thinners, degreasers, varnishes, and adhesives as a category were calculated from exposure histories.ResultsFSH concentrations increased significantly with increasing exposure indices for all solvents and for chlorinated solvents. There were no significant associations of solvent exposure indices with LH or testosterone levels. LH, FSH, and testosterone concentrations also did not differ by job title. Using Cox regression, time-to-pregnancy was non-significantly longer in the painters and millwrights than the carpenters. There was no significant association between time-to-pregnancy and any of the solvent exposure indices; however, it should be noted that some of the pregnancies occurred more than 20 years previously, potentially reducing the reliability of the retrospectively collected pregnancy and exposure data.ConclusionsThe significant associations between FSH levels and solvent exposure indices suggest the potential for adverse effects of solvent exposures on reproductive function in men.