Using RAD‐seq to recognize sex‐specific markers and sex chromosome systems

Next‐generation sequencing methods have initiated a revolution in molecular ecology and evolution (Tautz et al. 2010 ). Among the most impressive of these sequencing innovations is restriction site‐associated DNA sequencing or RAD‐seq (Baird et al. 2008 ; Andrews et al. 2016 ). RAD‐seq uses the Illumina sequencing platform to sequence fragments of DNA cut by a specific restriction enzyme and can generate tens of thousands of molecular genetic markers for analysis. One of the many uses of RAD‐seq data has been to identify sex‐specific genetic markers, markers found in one sex but not the other (Baxter et al. 2011 ; Gamble & Zarkower 2014 ). Sex‐specific markers are a powerful tool for biologists. At their most basic, they can be used to identify the sex of an individual via PCR. This is useful in cases where a species lacks obvious sexual dimorphism at some or all life history stages. For example, such tests have been important for studying sex differences in life history (Sheldon 1998 ; Mossman & Waser 1999 ), the management and breeding of endangered species (Taberlet et al. 1993 ; Griffiths & Tiwari 1995 ; Robertson et al. 2006 ) and sexing embryonic material (Hacker et al. 1995 ; Smith et al. 1999 ). Furthermore, sex‐specific markers allow recognition of the sex chromosome system in cases where standard cytogenetic methods fail (Charlesworth & Mank 2010 ; Gamble & Zarkower 2014 ). Thus, species with male‐specific markers have male heterogamety (XY) while species with female‐specific markers have female heterogamety (ZW). In this issue, Fowler & Buonaccorsi ( 2016 ) illustrate the ease by which RAD‐seq data can generate sex‐specific genetic markers in rockfish (Sebastes). Moreover, by examining RAD‐seq data from two closely related rockfish species, Sebastes chrysomelas and Sebastes carnatus (Fig.  1 ), Fowler & Buonaccorsi ( 2016 ) uncover shared sex‐specific markers and a conserved sex chromosome system.     

Post-translational histone modifications and their interaction with sex influence normal brain development and elaboration of neuropsychiatric disorders

Sex differences in the risk for and expression of various brain disorders have been known for some time. Yet, the molecular underpinnings of these sex differences as well as how sex modifies normal brain development are still poorly understood. It has recently become known that epigenetic mechanisms play an essential role in establishing and maintaining sex differences in neurodevelopment and disease susceptibility. Epigenetic mechanisms such as post-translational modifications of histones (histone PTMs) integrate various hormonal and external environmental influences to affect genomic output, and this appears to occur in a sex-dependent manner. The present review aims to highlight current understanding of the role of histone PTMs in the sexual differentiation of the brain under normal conditions and how sex-specific modulation of histone PTMs may be involved in psychiatric conditions including autism spectrum disorder (ASD), schizophrenia, and major depressive disorder (MDD). The role of sex chromosome genes as sex-specific histone modifiers and their importance in sexually differentiating the brain will be discussed. Further, the contribution of sex-specific histone PTM marks in the placenta in programming the sexually dimorphic developmental course of the brain and susceptibility to diseases/disorders will be reviewed. Prenatal programming may have a long-lasting effect on the adult brain and behavior but due to the interaction of histone PTMs and its modifiers with fluctuating hormone levels and external influences over the lifespan, the process remains dynamic. Although a few studies indicate an association between sex and histone PTM-related mechanisms in ASD, schizophrenia, and MDD, more research is needed to fully appreciate the interactive effects of histone PTMs and sex in the development and manifestation of these disorders. Understanding the interactions between sex and histone PTMs will advance our understanding of psychiatric disorders and potentially guide development of future treatments tailored specifically to each sex.     

Let's talk about sex: A rigorous statistical framework to assign the sex of individuals from reduced‐representation sequencing data

Molecular markers have been used to identify the sex of sampled individuals for several decades, but the time‐consuming development phase prevented their application in many systems. Recently, a growing number of papers have applied reduced‐representation sequencing (RRS) protocols to the identification of sex‐specific markers without the use of test crosses or prior genomic information. While such an approach has great advantages in terms of versatility and ease of use, the “shotgun sequencing” nature of RRS data sets leads to a high amount of missing data, which results in statistical challenges to the confident assignment of sex to individuals. In this issue of Molecular Ecology Resources, Stovall et al. (Molecular Ecology Resources, 18, 2018) provide a statistical framework to answer two questions: (1) how many individuals of one sex only must possess a genotype for this locus to be considered significantly sex‐specific? and (2) How many sex‐specific loci must an individual of unknown sex possess (in a given data set) to be confidently assigned a sex? The statistical pipeline introduced, and applied to samples of New Zealand fur seal (Arctocephalus forsteri) to identify 90 sex‐specific loci, should be broadly applicable to a large number of species and constitutes a nice addition to the molecular ecology toolkit in the genomics era.     

Why boys will be boys and girls will be girls: Human sex development and its defects

Among the most defining events of an individual's life, is the development of a human embryo into male or a female. The phenotypic sex of an individual depends on the type of gonad that develops in the embryo, a process which itself is determined by the genetic setting of the individual. The development of the gonads is different from any other organ, as they possess the potential to differentiate into two functionally distinct organs, testes, or ovaries. Sex development can be divided into two distinctive processes, “sex determination,” which is the commitment of the undifferentiated gonad into either a testis or an ovary, a process that is genetically programmed in a critically timed manner and “sex differentiation,” which takes place through hormones produced by the gonads, once the developmental sex determination decision has been made. Disruption of any of the genes involved in either the testicular or ovarian development pathway could lead to disorders of sex development. In this review, we provide an insight into the factors important for sex determination, their antagonistic actions and whenever possible, references on the “prismatic” clinical cases are given. Birth Defects Research (Part C) 108:365–379, 2016. © 2016 Wiley Periodicals, Inc.     

Biological factors underlying sex differences in neurological disorders

The prevalence, age of onset, pathophysiology, and symptomatology of many neurological and neuropsychiatric conditions differ significantly between males and females. Females suffer more from mood disorders such as depression and anxiety, whereas males are more susceptible to deficits in the dopamine system including Parkinson's disease (PD), attention-deficit hyperactivity disorder (ADHD), schizophrenia, and autism spectrum disorders (ASD). Until recently, these sex differences have been explained solely by the neuroprotective actions of sex hormones in females. Emerging evidence however indicates that the sex chromosome genes (i.e. X- and Y-linked genes) also contribute to brain sex differences. In particular, the Y-chromosome gene, SRY (Sex-determining Region on the Y chromosome) is an interesting candidate as it is expressed in dopamine-abundant brain regions, where it regulates dopamine biosynthesis and dopamine-mediated functions such as voluntary movement in males. Furthermore, SRY expression is dysregulated in a toxin-induced model of PD, suggesting a role for SRY in the pathogenesis of dopamine cells. Taken together, these studies highlight the importance of understanding the interplay between sex-specific hormones and sex-specific genes in healthy and diseased brain. In particular, better understanding of regulation and function of SRY in the male brain could provide entirely novel and important insights into genetic factors involved in the susceptibility of men to neurological disorders, as well as development of novel sex-specific therapies.     

Sex,stress, and epigenetics: regulation of behavior in animal models of mood disorders

Stress-related psychiatric disorders, such as unipolar depression and post-traumatic stress disorder (PTSD), occur more frequently in women than in men. Emerging research suggests that sex differences in receptors for the stress hormones, corticotropin releasing factor (CRF) and glucocorticoids, contribute to this disparity. For example, sex differences in CRF receptor binding in the amygdala of rats may predispose females to greater anxiety following stressful events. Additionally, sex differences in CRF receptor signaling and trafficking in the locus coeruleus arousal center combine to make females more sensitive to low levels of CRF, and less adaptable to high levels. These receptor differences in females could lead to hyperarousal, a dysregulated state associated with symptoms of depression and PTSD. Similar to the sex differences observed in CRF receptors, sex differences in glucocorticoid receptor (GR) function also appear to make females more susceptible to dysregulation after a stressful event. Following hypothalamic pituitary adrenal axis activation, GRs are critical to the negative feedback process that inhibits additional glucocorticoid release. Compared to males, female rats have fewer GRs and impaired GR translocation following chronic adolescent stress, effects linked to slower glucocorticoid negative feedback. Thus, under conditions of chronic stress, attenuated negative feedback in females would result in hypercortisolemia, an endocrine state thought to cause depression. Together, these studies suggest that sex differences in stress-related receptors shift females more easily into a dysregulated state of stress reactivity, linked to the development of mood and anxiety disorders. The implications of these receptor sex differences for the development of novel pharmacotherapies are also discussed.     

Differential TNF-signaling in chronic inflammatory disorders

TNF-alpha is a pleiotropic cytokine with strong proinflammatory and immunomodulatory properties. TNF-alpha plays a critical role in many acute or chronic inflammatory diseases and anti-TNF-strategies have proven to be clinically effective. Two TNF-specific cell surface receptors TNF-R1 and TNF-R2 have been identified and the function of these receptors and the downstream intracellular signal transduction pathways have been extensively studied in vitro. For a long time TNF-R1 was considered to be the predominant mediator of TNF-signaling, whereas TNF-R2 was ascribed only auxilliary function. However, there is increasing clinical and experimental evidence for an important independent role of p80 signaling in chronic inflammatory conditions. It is conceivable that the multiple TNF-mediated chronic inflammatory disorders differ in terms of the ligand form (soluble TNF-alpha versus membrane bound TNF-alpha), the receptor (TNF-R1 versus TNF-R2) and the downstream signaling cascades utilized. The elucidation of the specific characteristics of TNF-signaling in distinct inflammatory disorders will lead to a better understanding ot the pathogenesis of these diseases and will be the basis for the development of more specific and more efficient therapeutic approaches.     

Sex differences in molecular and cellular substrates of stress

Women are twice as likely as men to suffer from stress-related psychiatric disorders, like unipolar depression and post-traumatic stress disorder. Although the underlying neural mechanisms are not well characterized, the pivotal role of stress in the onset and severity of these diseases has led to the idea that sex differences in stress responses account for this sex bias. Corticotropin-releasing factor (CRF) orchestrates stress responses by acting both as a neurohormone to initiate the hypothalamic-pituitary-adrenal (HPA) axis and as a neuromodulator in the brain. One target of CRF modulation is the locus coeruleus (LC)-norepinephrine system, which coordinates arousal components of the stress response. Hypersecretion of CRF and dysregulation of targets downstream from CRF, such as the HPA axis and LC-norepinephrine system, are characteristic features of many stress-related psychiatric diseases, suggesting a causal role for CRF and its targets in the development of these disorders. This review will describe sex differences in CRF and the LC-norepinephrine system that can increase stress sensitivity in females, making them vulnerable to stress-related disorders. Evidence for gonadal hormone regulation of hypothalamic CRF is discussed as an effect that can lead to increased HPA axis activity in females. Sex differences in the structure of LC neurons that create the potential for hyperarousal in response to emotional stimuli are described. Finally, sex differences at the molecular level of the CRF(1) receptor that make the LC-norepinephrine system more reactive in females are reviewed. The implications of these sex differences for the treatment of stress-related psychiatric disorders also will be discussed.     

The impact of culture on sex assignment and gender development in intersex patients.

The appearance of the external genitalia is the major determinant of the social sex, which is announced at or shortly after birth. In the absence of normal development of the external genitalia, definitive gender assignment and its announcement have to be postponed. While over the past 20 years the pathogenesis of most disorders causing abnormal development of the genitalia have been elucidated, our knowledge regarding the impact of these defects upon the psychosexual development is rather rudimentary. This information, however, is needed not only to establish criteria for correct sex assignment but also to design relevant outcome studies. Culture is an important part of the context in which decisions are made on sex assignment of patients with abnormalities of the external genitalia. Cultural differences in dealing with intersexuality and intersex individuals not only influences the patient's own psychosexual development but also medical decisions regarding sex assignment and consecutive management. There is evidence that attitudes concerning gender and sexuality, including the acceptance of intersexuality, differ significantly between various cultures. Thus cross-cultural studies might allow a new approach in dealing with intersexed persons, their families, and their social background, a most important aspect considering the recent discussions and criticisms of patients and individuals affected with intersex disorders.     

Divergence or disorder?: the politics of naming intersex

The conditions once known under the umbrella terms intersex and hermaphroditism are now generally being called disorders of sex development in medical settings. The terms might seem synonymous, but in fact there are significant differences with controversial consequences. Hermaphroditism, an older term that can still be found in many medical writings, is vague, demeaning, and sensationalistic, conjuring mythic images of monsters and freaks. In the 1990s, activists advocated intersex to describe discordance between the multiple components of sex anatomy, but that word alienated many parents of affected children, as it suggests a self-conscious alternative gender identity and sexuality. Disorders of sex development also refers to intersex, but it deemphasizes the identity politics and sexual connotations associated with intersex, avoids the degradation associated with hermaphrodite, and instead highlights the underlying genetic or endocrine factors that cause prenatal sex development to take an unusual path. I argue that using disorder is problematic, because it implies medical conditions in need of repair, when some intersex anatomies, though atypical, do not necessarily need surgical or hormonal correction. I advocate a less pathologizing new term, divergence of sex development, that might reduce some of the conflict over nomenclature and satisfy intersex people, their parents, and their doctors.     

Environmental sex reversal,Trojan sex genes,and sex ratio adjustment: conditions and population consequences

The great diversity of sex determination mechanisms in animals and plants ranges from genetic sex determination (GSD, e.g. mammals, birds, and most dioecious plants) to environmental sex determination (ESD, e.g. many reptiles) and includes a mixture of both, for example when an individual’s genetically determined sex is environmentally reversed during ontogeny (ESR, environmental sex reversal, e.g. many fish and amphibia). ESD and ESR can lead to widely varying and unstable population sex ratios. Populations exposed to conditions such as endocrine‐active substances or temperature shifts may decline over time due to skewed sex ratios, a scenario that may become increasingly relevant with greater anthropogenic interference on watercourses. Continuous exposure of populations to factors causing ESR could lead to the extinction of genetic sex factors and may render a population dependent on the environmental factors that induce the sex change. However, ESR also presents opportunities for population management, especially if the Y or W chromosome is not, or not severely, degenerated. This seems to be the case in many amphibians and fish. Population growth or decline in such species can potentially be controlled through the introduction of so‐called Trojan sex genes carriers, individuals that possess sex chromosomes or genes opposite from what their phenotype predicts. Here, we review the conditions for ESR, its prevalence in natural populations, the resulting physiological and reproductive consequences, and how these may become instrumental for population management.     

Inheritance of intersex disorders.

Intersex disorders result from abnormalities of the sex chromosomes, gonads, internal and external genitalia, sex hormones and gender role. This article reviews the literature on intersex disorders, outlining the characteristics and mode of inheritance, if known, of each. For appropriate and effective management and counselling of patients and their families, physicians must have a good knowledge of the development of the genital tract and of the interaction between genetic sex and environmental influences.     

Genetic and epigenetic effects in sex determination

Sex determination is a complex and dynamic process with multiple genetic and environmental causes, in which germ and somatic cells receive various sex‐specific features. During the fifth week of fetal life, the bipotential embryonic gonad starts to develop in humans. In the bipotential gonadal tissue, certain cell groups start to differentiate to form the ovaries or testes. Despite considerable efforts and advances in identifying the mechanisms playing a role in sex determination and differentiation, the underlying mechanisms of the exact functions of many genes, gene–gene interactions, and epigenetic modifications that are involved in different stages of this cascade are not completely understood. This review aims at discussing current data on the genetic effects via genes and epigenetic mechanisms that affect the regulation of sex determination. Birth Defects Research (Part C) 108:321–336, 2016. © 2016 Wiley Periodicals, Inc.     

Review disorders of sex development: The evolving role of genomics in diagnosis and gene discovery

Disorders of Sex Development (DSDs) are a major paediatric concern and are estimated to occur in around 1.7% of all live births (Fausto‐Sterling, Sexing the Body: Gender Politics and the Construction of Sexuality, Basic Books, New York, 2000). They are often caused by the breakdown in the complex genetic mechanisms that underlie gonadal development and differentiation. Having a genetic diagnosis can be important for patients with a DSD: it can increase acceptance of a disorder often surrounded by stigma, alter clinical management and it can assist in reproductive planning. While Massively Parallel Sequencing (MPS) is advancing the genetic diagnosis of rare Mendelian disorders, it is not yet clear which MPS assay is best suited for the clinical diagnosis of DSD patients and to what extent other established methods are still relevant. To complicate matters, DSDs represent a wide spectrum of disorders caused by an array of different genetic changes, many of which are yet unknown. Here we discuss the different genetic lesions that are known to contribute to different DSDs, and review the utility of a range of MPS approaches for diagnosing DSD patients. Birth Defects Research (Part C) 108:337–350, 2016. © 2016 Wiley Periodicals, Inc.     

Loss of Wnt5a disrupts primordial germ cell migration and male sexual development in mice

Disruptions in the regulatory pathways controlling sex determination and differentiation can cause disorders of sex development, often compromising reproductive function. Although extensive efforts have been channeled into elucidating the regulatory mechanisms controlling the many aspects of sexual differentiation, the majority of disorders of sex development phenotypes are still unexplained at the molecular level. In this study, we have analyzed the potential involvement of Wnt5a in sexual development and show in mice that Wnt5a is male-specifically upregulated within testicular interstitial cells at the onset of gonad differentiation. Homozygous deletion of Wnt5a affected sexual development in male mice, causing testicular hypoplasia and bilateral cryptorchidism despite the Leydig cells producing factors such as Hsd3b1 and Insl3. Additionally, Wnt5a-null embryos of both sexes showed a significant reduction in gonadal germ cell numbers, which was caused by aberrant primordial germ cell migration along the hindgut endoderm prior to gonadal colonization. Our results indicate multiple roles for Wnt5a during mammalian reproductive development and help to clarify further the etiology of Robinow syndrome (OMIM 268310), a disease previously linked to the WNT5A pathway.     

Mesenchymal stem cells: A new diagnostic tool?

Mesenchymal stem cells (MSCs) are progenitor cells capable of self-renewal that can differentiate in multiple tissues and, under specific and standardized culture conditions, expand in vitro with little phenotypic alterations. In recent years, preclinical and clinical studies have focused on MSC analysis and understanding the potential use of these cells as a therapy in a wide range of pathologies, and many applications have been tested. Clinical trials using MSCs have been performed (e.g., for cardiac events, stroke, multiple sclerosis, blood diseases, auto-immune disorders, ischemia, and articular cartilage and bone pathologies), and for many genetic diseases, these cells are considered an important resource. Considering of the biology of MSCs, these cells may also be useful tools for understanding the physiopathology of different diseases, and they can be used to develop specific biomarkers for a broad range of diseases. In this editorial, we discuss the literature related to the use of MSCs for diagnostic applications and we suggest new technologies to improve their employment.     

Adolescent maturation of the prefrontal cortex: Role of stress and sex in shaping adult risk for compromise

Adolescence is a highly dynamic period of development, which includes the final organizational phases of neural maturation within the prefrontal cortex (PFC). The organizational events of neural pruning and myelination occur in a sex‐specific manner, potentially giving rise to the disparities in mood disorders in adulthood. Because of the extended developmental time period of the PFC, environmental insults, including psychosocial stressors, may play a major role in steering the maturation of this region. In this review, the literature surrounding the sex specific alterations that occur in the PFC in rodent models following adolescent stress will be discussed. This will be complimented by a brief review on the state of human research in PFC sex differences in the development of white matter and cytoarchitecture across the lifespan. Taken together, the impact of developmental psychosocial stress on the circuitry of the PFC and resulting adult phenotypes will be summarized with a focus on the importance of considering sex differences in order to build a better understanding of developmental influences on adult disorders.     

Omega-3 fatty acids and neuropsychiatric disorders

Epidemiological evidence suggests that dietary consumption of the long chain omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), commonly found in fish or fish oil, may modify the risk for certain neuropsychiatric disorders. As evidence, decreased blood levels of omega-3 fatty acids have been associated with several neuropsychiatric conditions, including Attention Deficit (Hyperactivity) Disorder, Alzheimer's Disease, Schizophrenia and Depression. Supplementation studies, using individual or combination omega-3 fatty acids, suggest the possibility for decreased symptoms associated with some of these conditions. Thus far, however, the benefits of supplementation, in terms of decreasing disease risk and/or aiding in symptom management, are not clear and more research is needed. The reasons for blood fatty acid alterations in these disorders are not known, nor are the potential mechanisms by which omega-3 fatty acids may function in normal neuronal activity and neuropsychiatric disease prevention and/or treatment. It is clear, however, that DHA is the predominant n-3 fatty acid found in the brain and that EPA plays an important role as an anti-inflammatory precursor. Both DHA and EPA can be linked with many aspects of neural function, including neurotransmission, membrane fluidity, ion channel and enzyme regulation and gene expression. This review summarizes the knowledge in terms of dietary omega-3 fatty acid intake and metabolism, as well as evidence pointing to potential mechanisms of omega-3 fatty acids in normal brain functioning, development of neuropsychiatric disorders and efficacy of omega-3 fatty acid supplementation in terms of symptom management.     

Mouse models for disorders of mitochondrial fatty acid beta-oxidation

Mitochondrial beta-oxidation of fatty acids is vital for energy production in periods of fasting and other metabolic stress. Human patients have been identified with inherited disorders of mitochondrial beta-oxidation of fatty acids with enzyme deficiencies identified at many of the steps in this pathway. Although these patients exhibit a range of disease processes, Reye-like illness (hypoketotic-hypoglycemia, hyperammonemia and fatty liver) and cardiomyopathy are common findings. There have been several mouse models developed to aid in the study of these disease conditions. The characterized mouse models include inherited deficiencies of very long-chain acyl-CoA dehydrogenase, long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, mitochondrial trifunctional protein-alpha, and medium-/short-chain hydroxyacyl-CoA dehydrogenase. Mouse mutants developed, but presently incompletely characterized as models, include carnitine palmitoyltransferase-1a and medium-chain acyl-CoA dehydrogenase deficiencies. In general, the mouse models of disorders of mitochondrial fatty acid beta-oxidation have shown clinical signs that include Reye-like syndrome and cardiomyopathy, and many are cold intolerant. It is expected that these mouse models will provide vital contributions in understanding the mechanisms of disease pathogenesis of fatty acid oxidation disorders and the development of appropriate treatments and supportive care.