Tracy L. Bale

Sex-Specific Programming of Offspring Emotionality after Stress Early in Pregnancy

Prenatal stress is associated with an increased vulnerability to neurodevelopmental disorders, including autism and schizophrenia. To determine the critical time window when fetal antecedents may induce a disease predisposition, we examined behavioral responses in offspring exposed to stress during early, mid, and late gestation. We found that male offspring exposed to stress early in gestation displayed maladaptive behavioral stress responsivity, anhedonia, and an increased sensitivity to selective serotonin reuptake inhibitor treatment. Long-term alterations in central corticotropin-releasing factor (CRF) and glucocorticoid receptor (GR) expression, as well as increased hypothalamic–pituitary–adrenal (HPA) axis responsivity, were present in these mice and likely contributed to an elevated stress sensitivity. Changes in CRF and GR gene methylation correlated with altered gene expression, providing important evidence of epigenetic programming during early prenatal stress. In addition, we found the core mechanism underlying male vulnerability may involve sex-specific placenta responsivity, where stress early in pregnancy significantly increased expression of PPARα (peroxisome proliferator-activated receptor α), IGFBP-1 (insulin-like growth factor binding protein 1), HIF3α (hypoxia-inducible factor 3a), and GLUT4 (glucose transporter 4) in male placentas but not females. Examination of placental epigenetic machinery revealed basal sex differences, providing further evidence that sex-specific programming begins very early in pregnancy, and may contribute to the timing and vulnerability of the developing fetus to maternal perturbations. Overall, these results indicate that stress experience early in pregnancy may contribute to male neurodevelopmental disorders through impacts on placental function and fetal development.

Early Life Programming and Neurodevelopmental Disorders

For more than a century, clinical investigators have focused on early life as a source of adult psychopathology. Early theories about psychic conflict and toxic parenting have been replaced by more recent formulations of complex interactions of genes and environment. Although the hypothesized mechanisms have evolved, a central notion remains: early life is a period of unique sensitivity during which experience confers enduring effects. The mechanisms for these effects remain almost as much a mystery today as they were a century ago. Recent studies suggest that maternal diet can program offspring growth and metabolic pathways, altering lifelong susceptibility to diabetes and obesity. If maternal psychosocial experience has similar programming effects on the developing offspring, one might expect a comparable contribution to neurodevelopmental disorders, including affective disorders, schizophrenia, autism, and eating disorders. Due to their early onset, prevalence, and chronicity, some of these disorders, such as depression and schizophrenia, are among the highest causes of disability worldwide according to the World Health Organization 2002 report. Consideration of the early life programming and transcriptional regulation in adult exposures supports a critical need to understand epigenetic mechanisms as a critical determinant in disease predisposition. Incorporating the latest insight gained from clinical and epidemiological studies with potential epigenetic mechanisms from basic research, the following review summarizes findings from a workshop on Early Life Programming and Neurodevelopmental Disorders held at the University of Pennsylvania in 2009.

Paternal Stress Exposure Alters Sperm MicroRNA Content and Reprograms Offspring HPA Stress Axis Regulation

Neuropsychiatric disease frequently presents with an underlying hyporeactivity or hyperreactivity of the HPA stress axis, suggesting an exceptional vulnerability of this circuitry to external perturbations. Parental lifetime exposures to environmental challenges are associated with increased offspring neuropsychiatric disease risk, and likely contribute to stress dysregulation. While maternal influences have been extensively examined, much less is known regarding the specific role of paternal factors. To investigate the potential mechanisms by which paternal stress may contribute to offspring hypothalamic–pituitary–adrenal (HPA) axis dysregulation, we exposed mice to 6 weeks of chronic stress before breeding. As epidemiological studies support variation in paternal germ cell susceptibility to reprogramming across the lifespan, male stress exposure occurred either throughout puberty or in adulthood. Remarkably, offspring of sires from both paternal stress groups displayed significantly reduced HPA stress axis responsivity. Gene set enrichment analyses in offspring stress regulating brain regions, the paraventricular nucleus (PVN) and the bed nucleus of stria terminalis, revealed global pattern changes in transcription suggestive of epigenetic reprogramming and consistent with altered offspring stress responsivity, including increased expression of glucocorticoid-responsive genes in the PVN. In examining potential epigenetic mechanisms of germ cell transmission, we found robust changes in sperm microRNA (miR) content, where nine specific miRs were significantly increased in both paternal stress groups. Overall, these results demonstrate that paternal experience across the lifespan can induce germ cell epigenetic reprogramming and impact offspring HPA stress axis regulation, and may therefore offer novel insight into factors influencing neuropsychiatric disease risk.

The Epigenetics of Sex Differences in the Brain

Epigenetic changes in the nervous system are emerging as a critical component of enduring effects induced by early life experience, hormonal exposure, trauma and injury, or learning and memory. Sex differences in the brain are largely determined by steroid hormone exposure during a perinatal sensitive period that alters subsequent hormonal and nonhormonal responses throughout the lifespan. Steroid receptors are members of a nuclear receptor transcription factor superfamily and recruit multiple proteins that possess enzymatic activity relevant to epigenetic changes such as acetylation and methylation. Thus steroid hormones are uniquely poised to exert epigenetic effects on the developing nervous system to dictate adult sex differences in brain and behavior. Sex differences in the methylation pattern in the promoter of estrogen and progesterone receptor genes are evident in newborns and persist in adults but with a different pattern. Changes in response to injury and in methyl-binding proteins and steroid receptor coregulatory proteins are also reported. Many steroid-induced epigenetic changes are opportunistic and restricted to a single lifespan, but new evidence suggests endocrine-disrupting compounds can exert multigenerational effects. Similarly, maternal diet also induces transgenerational effects, but the impact is sex specific. The study of epigenetics of sex differences is in its earliest stages, with needed advances in understanding of the hormonal regulation of enzymes controlling acetylation and methylation, coregulatory proteins, transient versus stable DNA methylation patterns, and sex differences across the epigenome to fully understand sex differences in brain and behavior.

Maternal High-Fat Diet Effects on Third-Generation Female Body Size via the Paternal Lineage

The health consequences of in utero exposure to maternal obesity on future generations are concerning because they contribute to increased rates of diabetes, cardiovascular disease, and metabolic syndrome. We previously reported that maternal high-fat diet exposure in mice resulted in an increase in body size and reduced insulin sensitivity that persisted across two generations via both maternal and paternal lineages. However, because the first generations primordial germ cells may be affected by gestational exposure, analysis of phenotype transmission into a third generation (F3) is necessary to determine whether stable epigenetic programming has occurred. Therefore, we have examined the body size and insulin sensitivity of male and female F3 offspring. We found that only females displayed the increased body size phenotype, and this effect was only passed on via the paternal lineage. The finding of a paternally transmitted phenotype to F3 female offspring supports a stable germline-based transgenerational mode of inheritance; thus we hypothesized that imprinted genes may be involved in this epigenetic programming. Using a quantitative TaqMan Array for imprinted genes to examine paternally or maternally expressed loci in F3 female livers, we detected a potential dynamic pattern of paternally expressed genes from the paternal lineage that was not noted in the maternal lineage. These findings suggest that the environmental influence on developmental regulation of growth and body size may be the result of broad programming events at imprinted loci, thereby providing sex specificity to both the transmission and inheritance of traits related to disease predisposition.

Maternal high-fat diet promotes body length increases and insulin insensitivity in second-generation mice.

Maternal obesity and diet consumption during pregnancy have been linked to offspring adiposity, cardiovascular disease, and impaired glucose metabolism. Furthermore, nutrition during development is clearly linked to somatic growth. However, few studies have examined whether phenotypes derived from maternal high-fat diet exposure can be passed to subsequent generations and by what mechanisms this may occur. Here we report the novel finding of a significant body length increase that persisted across at least two generations of offspring in response to maternal high-fat diet exposure. This phenotype is not attributable to altered intrauterine conditions or maternal feeding behavior because maternal and paternal lineages were able to transmit the effect, supporting a true epigenetic manner of inheritance. We also detected a heritable feature of reduced insulin sensitivity across two generations. Alterations in the GH secretagogue receptor (GHSR), the GHSR transcriptional repressor AF5q31, plasma IGF-I concentrations, and IGF-binding protein-3 (IGFBP3) suggest a contribution of the GH axis. These studies provide evidence that the heritability of body length and glucose homeostasis are modulated by maternal diet across multiple generations, providing a mechanism where length can increase rapidly in concert with caloric availability.

Early Prenatal Stress Epigenetically Programs Dysmasculinization in Second-Generation Offspring via the Paternal Lineage

Studies have linked sex-biased neurodevelopmental disorders, including autism and schizophrenia, with fetal antecedents such as prenatal stress. Further, these outcomes can persist into subsequent generations, raising the possibility that aspects of heritability in these diseases involve epigenetic mechanisms. Utilizing a mouse model in which we previously identified a period in early gestation when stress results in dysmasculinized and stress-sensitive male offspring, we have examined programming effects in second-generation offspring of prenatally stressed (F2-S) or control (F2-C) sires. Examination of gene expression patterns during the perinatal sensitive period, when organizational gonadal hormones establish the sexually dimorphic brain, confirmed dysmasculinization in F2-S males, where genes important in neurodevelopment showed a female-like pattern. Analyses of the epigenomic miRNA environment detected significant reductions in miR-322, miR-574, and miR-873 in the F2-S male brain, levels that were again more similar to those of control females. Increased expression of a common gene target for these three miRNAs, β-glycan, was confirmed in these males. These developmental effects were associated with the transmission of a stress-sensitive phenotype and shortened anogenital distance in adult F2-S males. As confirmation that the miRNA environment is responsive to organizational testosterone, neonatal males administered the aromatase inhibitor formestane exhibited dramatic changes in brain miRNA patterns, suggesting that miRNAs may serve a previously unappreciated role in organizing the sexually dimorphic brain. Overall, these data support the existence of a sensitive period of early gestation when epigenetic programming of the male germline can occur, permitting transmission of specific phenotypes into subsequent generations.

Transgenerational epigenetic programming via sperm microRNA recapitulates effects of paternal stress

Significance Studies examining paternal exposure to diverse environmental stimuli propose that epigenetic marks in germ cells, including small noncoding RNAs such as microRNA (miR), transmit experience-dependent information from parent to offspring. However, these nongenetic mechanisms of transgenerational inheritance are poorly understood, specifically how these germ-cell marks may act postfertilization to enact long-term changes in offspring behavior or physiology. In this study, through zygote microinjection of nine specific sperm miRs previously identified in our paternal stress mouse model, we demonstrate that sperm miRs function to reduce maternal mRNA stores in early zygotes, ultimately reprogramming gene expression in the offspring hypothalamus and recapitulating the offspring stress dysregulation phenotype. Epigenetic signatures in germ cells, capable of both responding to the parental environment and shaping offspring neurodevelopment, are uniquely positioned to mediate transgenerational outcomes. However, molecular mechanisms by which these marks may communicate experience-dependent information across generations are currently unknown. In our model of chronic paternal stress, we previously identified nine microRNAs (miRs) that were increased in the sperm of stressed sires and associated with reduced hypothalamic–pituitary–adrenal (HPA) stress axis reactivity in offspring. In the current study, we rigorously examine the hypothesis that these sperm miRs function postfertilization to alter offspring stress responsivity and, using zygote microinjection of the nine specific miRs, demonstrated a remarkable recapitulation of the offspring stress dysregulation phenotype. Further, we associated long-term reprogramming of the hypothalamic transcriptome with HPA axis dysfunction, noting a marked decreased in the expression of extracellular matrix and collagen gene sets that may reflect an underlying change in blood–brain barrier permeability. We conclude by investigating the developmental impact of sperm miRs in early zygotes with single-cell amplification technology, identifying the targeted degradation of stored maternal mRNA transcripts including sirtuin 1 and ubiquitin protein ligase E3a, two genes with established function in chromatin remodeling, and this potent regulatory function of miRs postfertilization likely initiates a cascade of molecular events that eventually alters stress reactivity. Overall, these findings demonstrate a clear mechanistic role for sperm miRs in the transgenerational transmission of paternal lifetime experiences.

Stress sensitivity and the development of affective disorders

Depressive disorders are the most common form of mental illness in America, affecting females twice as often as males. The great variability of symptoms and responses to therapeutic treatment emphasize the complex underlying neurobiology of disease onset and progression. Evidence from human and animal studies reveals a vital link between individual stress sensitivity and the predisposition toward mood disorders. While the stress response is essential for maintenance of homeostasis and survival, chronic stress and maladaptive responses to stress insults can lead to depression or other affective disorders. A key factor in the mediation of stress responsivity is the neuropeptide corticotropin-releasing factor (CRF). Studies in animal models of heightened stress sensitivity have illustrated the involvement of CRF downstream neurotransmitter targets, including serotonin and norepinephrine, in the profound neurocircuitry failure that may underlie maladaptive coping strategies. Stress sensitivity may also be a risk factor in affective disorder development susceptibility. As females show an increased stress response and recovery time compared to males, they may be at an increased vulnerability for disease. Therefore, examination of sex differences in CRF and downstream targets may aid in the elucidation of the underlying causes of the increased disease presentation in females. While we continue to make progress in our understanding of mood disorder etiology, we still have miles to go before we sleep. As an encouraging number of new animal models of altered stress sensitivity and negative stress coping strategies have been developed, the future looks extremely promising for the possibility of a new generation of drug targets to be developed.

Sex differences and stress across the lifespan

Sex differences in stress responses can be found at all stages of life and are related to both the organizational and activational effects of gonadal hormones and to genes on the sex chromosomes. As stress dysregulation is the most common feature across neuropsychiatric diseases, sex differences in how these pathways develop and mature may predict sex-specific periods of vulnerability to disruption and increased disease risk or resilience across the lifespan. The aging brain is also at risk to the effects of stress, where the rapid decline of gonadal hormones in women combined with cellular aging processes promote sex biases in stress dysregulation. In this Review, we discuss potential underlying mechanisms driving sex differences in stress responses and their relevance to disease. Although stress is involved in a much broader range of diseases than neuropsychiatric ones, we highlight here this area and its examples across the lifespan.