Ancestral environmental exposures such as toxicants, abnormal nutrition or stress can promote the epigenetic transgenerational inheritance of disease and phenotypic variation. These environmental factors induce the epigenetic reprogramming of the germline (sperm and egg). The germline epimutations can in turn increase disease susceptibility of subsequent generations of the exposed ancestors. A variety of environmental factors, species and exposure specificity of this induced epigenetic transgenerational inheritance of disease is discussed with a consideration of generational toxicology. The molecular mechanisms and processes involved in the ability of these inherited epimutations to increase disease susceptibility are discussed. In addition to altered disease susceptibility, the potential impact of the epigenetic inheritance on phenotypic variation and evolution is considered. Observations suggest environmentally induced epigenetic transgenerational inheritance of disease is a critical aspect of disease etiology, toxicology and evolution that needs to be considered.

Environmentally induced epigenetic transgenerational inheritance of disease.

An organism’s reproductive fitness is sensitive to the environment, integrating cues of resource availability, ecological factors, and hazards within its habitat. Events that challenge the environment of an organism activate the central stress response system, which is primarily mediated by the hypothalamic–pituitary–adrenal (HPA) axis. The regulatory functions of the HPA axis govern the cardiovascular and metabolic system, immune functions, behavior, and reproduction. Activation of the HPA axis by various stressors primarily inhibits reproductive function and is able to alter fetal development, imparting a biological record of stress experienced in utero. Clinical studies and experimental data indicate that stress signaling can mediate these effects through direct actions in the brain, gonads, and embryonic tissues. This review focuses on the mechanisms by which stress activation of the HPA axis impacts fertility and fetal development.

Stress and the HPA Axis: Balancing Homeostasis and Fertility.

Parents’ stressful experiences can influence the offspring’s vulnerability to many pathological conditions, including psychopathologies, and their effects may even endure for several generations. Nevertheless, the cause of this phenomenon has not been determined, and only recently have scientists turned to epigenetics to answer this question. There is extensive literature on epigenetics, but no consensus exists with regard to how and what can (and must) be considered to study and define epigenetics processes and their inheritance. In this work, we aimed to clarify and systematize these concepts. To this end, we analyzed the dynamics of epigenetic changes over time in detail and defined 3 types of epigenetics: a direct form of epigenetics (DE) and 2 indirect epigenetic processes: within (WIE) and across (AIE). DE refers to changes that occur in the lifespan of an individual, due to direct experiences with his environment. WIE concerns changes that occur inside of the womb, due to events during gestation. Finally, AIE defines changes that affect individual predecessors (parents, grandparents, etc.), due to events that occur even long before conception and that are somehow (e.g., through gametes, the intrauterine environment setting) transmitted across generations. This distinction allows us to organize the main body of epigenetic evidence according to these categories and then focus on the latter (AIE), referring to it as a faster route of informational transmission across generations—compared with genetic inheritance—that guides human evolution in a Lamarckian (i.e., experience-dependent) manner. Of the molecular processes that are implicated in this phenomenon, well-known (methylation) and novel (non-coding RNA) regulatory mechanisms are converging. Our discussion of the chief methods that are used to study epigenetic inheritance highlights the most compelling technical and theoretical problems of this discipline. Experimental suggestions to expand this field are provided, and their practical and ethical implications are discussed extensively.

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Epigenetic Inheritance: Concepts, Mechanisms and Perspectives.

Exposure of male mice to early life stress alters the levels of specific sperm miRNAs that promote stress-associated behaviors in their offspring. To begin to evaluate whether similar phenomena occur in men, we searched for sperm miRNA changes that occur in both mice and men exposed to early life stressors that have long-lasting effects. For men, we used the Adverse Childhood Experience (ACE) questionnaire. It reveals the degree of abusive and/or dysfunctional family experiences when young, which increases risks of developing future psychological and physical disorders. For male mice, we used adolescent chronic social instability (CSI) stress, which not only enhances sociability defects for >1 year, but also anxiety and defective sociability in female offspring for multiple generations through the male lineage. Here we found a statistically significant inverse correlation between levels of multiple miRNAs of the miR-449/34 family and ACE scores of Caucasian males. Remarkably, we found members of the same sperm miRNA family are also reduced in mice exposed to CSI stress. Thus, future studies should be designed to directly test whether reduced levels of these miRNAs could be used as unbiased indicators of current and/or early life exposure to severe stress. Moreover, after mating stressed male mice, these sperm miRNA reductions persist in both early embryos through at least the morula stage and in sperm of males derived from them, suggesting these miRNA changes contribute to transmission of stress phenotypes across generations. Since offspring of men exposed to early life trauma have elevated risks for psychological disorders, these findings raise the possibility that a portion of this risk may be derived from epigenetic regulation of these sperm miRNAs.

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Reduced levels of miRNAs 449 and 34 in sperm of mice and men exposed to early life stress

Transgenerational plasticity (TGP) occurs when the environment experienced by a parent influences the development of their offspring. In this article, we develop a framework for understanding the m...

An Integrative Framework for Understanding the Mechanisms and Multigenerational Consequences of Transgenerational Plasticity

Although previous studies found that I -time acute stress applied during follicle maturation impaired oocyte competence, it is unknown whether repeated chronic stress, which is known to cause animal behavioral adaptation, would damage oocytes when applied during follicle growth. In this study, female mice were exposed to repeated restraint stress (RRS) or unpredictable stress (UPS) for different days before equine chorionic gonadotropin injection to initiate oocyte prematuration development and to observe effects of different Stressors on oocytes in the growing follicles. The results showed that although oocyte pre- and postimplantation development was unaffected when mice were exposed to RRS or UPS once a day for 4 days, development was impaired when mice were exposed to RRS for 8 or more days or to UPS twice a day for 4 days (4 x 2). The 4 x 2 UPS caused more oxidative stress in oocytes and severer apoptosis in antral follicles than did the 4-day RRS. The RRS mice were stressed consistently from days I to 23 of restraint, and the stress that a mouse had 4x2 UPS was severer than that from 4-day RRS. The results suggest that (I) the degree that a stress damages oocytes is the product of duration x severity of the stress; (2) RRS impaired oocyte developmental potential through cumulative effects on growing follicles; and (3) preantral follicles were not as sensitive to stress as antral follicles were.

Stresses on Female Mice Impair Oocyte Developmental Potential:Effects of Stress Severity and Duration on Oocytes at the Growing Follicle Stage

While effects of gestational, neonatal or adolescent stress on psychological alterations in progeny have been extensively studied, much less is known regarding the effects of adult pre-gestational life events on offspring behavior. Although full siblings often display behavioral differences, whether the different parental life events prior to different pregnancies contribute to these behavioral differences among siblings is worth studying. In this study, male and female adult mice were restrained for 60 days before mating with unstressed or stressed partners. F1 offspring were examined for anxiety or mated to generate F2. Both F1 females and males from restrained mothers and/or fathers showed significantly reduced anxiety and serum cortisol and increased mRNA levels of glucocorticoid receptor and brain-derived neurotrophic factor compared to control offspring from unstressed parents. Similar behavioral and molecular changes were also observed in F2 females and males. Although restraint of adolescent mice reduced anxiety in F1 of both sexes, social instability of them increased anxiety predominantly in F1 females. Thus, adult pre-gestational restraint reduced offspring's anxiety across generations; different stressors on parents may cause different phenotypes in offspring; individual behaviors can depend on adult life experiences of parents.

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Parental life events cause behavioral difference among offspring: Adult pre-gestational restraint stress reduces anxiety across generations.

Although previous studies indicated that cumulus cells (CCs) accelerate oocyte aging by releasing soluble factors, the factors have yet to be characterized. While demonstrating that CCs promoted oocyte aging by releasing soluble Fas ligand (sFasL), our recent study suggested that CCs might secrete other factors to mediate oocyte aging as well. This study tested whether CCs accelerate oocyte aging by secreting tumor necrosis factor (TNF)-α. The results showed that mouse CCs undergoing apoptosis released soluble TNF-α (sTNF-α) during in vitro aging. While ethanol activation rates were higher, the maturation-promoting factor (MPF) activity was lower significantly after culture of cumulus-denuded oocytes (DOs) in medium conditioned with CCs for 36 h than in medium conditioned for 24 h. Aging mouse oocytes expressed TNF-receptor 1. The CCs released equal amounts of sTNF-α and sFasL during aging in vitro, and the TNF-α-knockdown CCs secreted less sFasL than the control CCs did. Treatment of DOs in vitro with sTNF-α significantly accelerated their aging. The aging-promoting effect of sTNF-α was significantly reduced in TNF-α-knocked-down CCs and in CCs from the TNF-α-knockout mice. It is concluded that mouse CCs accelerate oocyte aging by secreting sTNF-α as well as sFasL.

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Cumulus cell-released tumor necrosis factor (TNF)-α promotes post-ovulatory aging of mouse oocytes

Mechanisms by which psychological stress damages oocytes are largely undetermined. Although a previous study showed that the stress-induced corticotrophin-releasing hormone (CRH) elevation impaired oocyte competence by triggering apoptosis of ovarian cells, how CRH causes apoptosis in ovarian cells and oocytes is unknown. In this study, we have examined the hypothesis that restraint stress (RS)-induced CRH elevation triggers apoptosis of ovarian cells and impairs oocyte competence through activating the Fas/FasL system. The results showed that RS of female mice impaired oocyte competence, enhanced expression of CRH and CRH receptor (CRH-R) in the ovary, and induced apoptosis while activating the Fas/FasL system in mural granulosa cells (MGCs) and oocytes. Injecting mice with CRH-R1 antagonist antalarmin significantly alleviated the adverse effect of RS on oocyte developmental potential. Treatment of cultured MGCs recapitulated the effects of CRH and antalarmin on apoptosis and Fas/FasL expression in MGCs. Silencing FasL gene by RNA interference in cultured MGCs further confirmed the involvement of the Fas/FasL system in the CRH triggered apoptosis of ovarian cells. It is concluded that the RS-induced CRH elevation triggers apoptosis of ovarian cells and impairs oocyte competence via activation of the Fas/FasL system.

Restraint-induced corticotrophin-releasing hormone elevation triggers apoptosis of ovarian cells and impairs oocyte competence via activation of the Fas/FasL system.

Studies have indicated that psychological stress impairs human fertility and that various stressors can induce apoptosis of testicular cells. However, the mechanisms by which psychological stress on males reduces semen quality and stressors induce apoptosis in testicular cells are largely unclear. Using a psychological (restraint) stress mouse model, we tested whether male psychological stress triggers apoptosis of spermatozoa and spermatogenic cells through activating tumour necrosis factor (TNF)-α signalling. Wild-type or TNF-α-/- male mice were restrained for 48 h before examination for apoptosis and expression of TNF-α and TNF receptor 1 (TNFR1) in spermatozoa, epididymis, seminiferous tubules and spermatogenic cells. The results showed that male restraint significantly decreased fertilization rate and mitochondrial membrane potential, while increasing levels of malondialdehyde, active caspase-3, TNF-α and TNFR1 in spermatozoa. Male restraint also increased apoptosis and expression of TNF-α and TNFR1 in caudae epididymides, seminiferous tubules and spermatogenic cells. Sperm quality was also significantly impaired when spermatozoa were recovered 35 days after male restraint. The restraint-induced damage to spermatozoa, epididymis and seminiferous tubules was significantly ameliorated in TNF-α-/- mice. Furthermore, incubation with soluble TNF-α significantly reduced sperm motility and fertilizing potential. Taken together, the results demonstrated that male psychological stress induces apoptosis in spermatozoa and spermatogenic cells through activating the TNF-α system and that the stress-induced apoptosis in spermatogenic cells can be translated into impaired quality in future spermatozoa.

Qiao-Qiao Kong

Papers

Stresses on Female Mice Impair Oocyte Developmental Potential:Effects of Stress Severity and Duration on Oocytes at the Growing Follicle Stage

Parental life events cause behavioral difference among offspring: Adult pre-gestational restraint stress reduces anxiety across generations.

Cumulus cell-released tumor necrosis factor (TNF)-α promotes post-ovulatory aging of mouse oocytes

Restraint-induced corticotrophin-releasing hormone elevation triggers apoptosis of ovarian cells and impairs oocyte competence via activation of the Fas/FasL system.

Restraint stress of male mice triggers apoptosis in spermatozoa and spermatogenic cells via activating the TNF-α system.