Mate preference behavior is an essential first step in sexual selection and is a critical determinant in evolutionary biology. Previously an environmental compound (the fungicide vinclozolin) was found to promote the epigenetic transgenerational inheritance of an altered sperm epigenome and modified mate preference characteristics for three generations after exposure of a gestating female.
The current study investigated gene networks involved in various regions of the brain that correlated with the altered mate preference behavior in the male and female. Statistically significant correlations of gene clusters and modules were identified to associate with specific mate preference behaviors. This novel systems biology approach identified gene networks (bionetworks) involved in sex-specific mate preference behavior. Observations demonstrate the ability of environmental factors to promote the epigenetic transgenerational inheritance of this altered evolutionary biology determinant.
Combined observations elucidate the potential molecular control of mate preference behavior and suggests environmental epigenetics can have a role in evolutionary biology.
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Epigenetics; Brain; Networks; Evolution; Behavior
Sex steroid hormones coordinate neurotransmitter systems in the male brain to facilitate sexual behavior. Although neurotransmitter release in the male brain has been well documented, little is known about how androgens orchestrate changes in gene expression of neurotransmitter receptors. We used male whiptail lizards (Cnemidophorus inornatus) to investigate how androgens alter neurotransmitter-related gene expression in brain regions involved in social decision-making. We focused on three neurotransmitter systems involved in male-typical sexual behavior, including the NMDA glutamate receptor, nitric oxide, and dopamine receptors. Here we show that in androgen-treated males, there are coordinated changes in neurotransmitter-related gene expression. In androgen-implanted castrates compared to blank-implanted castrates (control group), we found associated increases in neuronal nitric oxide synthase (nNOS) gene expression in the nucleus accumbens, preoptic area and ventromedial hypothalamus, a decrease of NR1 gene expression (obligate subunit of NMDA receptors) in the medial amygdaloid area and nucleus accumbens, and a decrease in D1 and D2 dopamine receptor gene expression in the nucleus accumbens. Our results support and expand the current model of androgen-mediated gene expression changes of neurotransmitter-related systems that facilitate sexual behavior in males. This also suggests that the proposed evolutionarily ancient reward system that reinforces sexual behavior in amniote vertebrates extends to reptiles.
androgens; neuronal nitric oxide synthase; glutamate; sexual behavior; dopamine receptors
In the red-eared slider turtle (Trachemys scripta), a species with temperature-dependent sex determination (TSD), the expression of the aromatase gene during gonad development is strictly limited to the female-producing temperature. The underlying mechanism remains unknown. In this study, we identified the upstream 5′-flanking region of the aromatase gene, gonad-specific promoter, and the temperature-dependent DNA methylation signatures during gonad development in the red-eared slider turtle. The 5′-flanking region of the slider aromatase exhibited sequence similarities to the aromatase genes of the American alligator, chicken, quail, and zebra finch. A putative TATA box was located 31 bp upstream of the gonad-specific transcription start site. DNA methylation at the CpG sites between the putative binding sites of the fork head domain factor (FOX) and vertebrate steroidogenic factor 1 (SF1) and adjacent TATA box in the promoter region were significantly lower in embryonic gonads at the female-producing temperature compared the male-producing temperature. A shift from male- to female-, but not from female- to male-, producing temperature changed the level of DNA methylation in gonads. Taken together these results indicate that the temperature, particularly female-producing temperature, allows demethylation at the specific CpG sites of the promoter region which leads the temperature-specific expression of aromatase during gonad development.
Epigenetics is a perspective, not a set of techniques. Molecular biology and genetics are the dominant disciplines in biology today, but practitioners of these fields have only recently ‘rediscovered’ the importance of the environment. This has led to increasing research into molecular epigenetics and the interface between the environment and gene regulation. Beyond the study of epigenetic mechanisms at the level of the gene, more investigation of epigenetic outcomes at the level of both the individual organism and the evolution of the population is needed.
Evolutionary change is a product of selection. Selection operates on the phenotype, and its consequences are manifest in representation of the genotype in successive generations. Of particular interest to both evolutionary and behavioral biologists is the newly emerging field of epigenetics and behavior. Two broad categories of epigenetic modifications must be distinguished. Context-Dependent epigenetic change can be observed if the environmental factors that bring about the epigenetic modification persists (e.g., the frequency and quality of maternal care modifying the brain and future behavior of the offspring each generation). Because the environment induces epiallelic change, removing the causative factor can reverse a Context-Dependent epigenetic state. Germline-Dependent epigenetic change occurs when the epigenetic imprint is mediated through the germline. Such effects are independent of the causative agent and there is no evidence at present that a Germline-Dependent epigenetic state can be reversed. Finally, only Germline-Dependent epigenetic modifications can be truly transgenerational. Although an individual’s life history is progressive and continuous, it might usefully be viewed as the cumulation of divisions; each period emerging from what has gone before and, at the same time, setting the stage for what follows. These life history stages are somewhat arbitrary, with many traits spanning conventional divisions, but each period tends to have its own characteristic ethologies and particular contribution to neural and behavioral phenotypes. To understand how theses episodes ‘fit’ together, it is necessary to deconstruct early life events and study each period both in its’ own right and how it interacts with the preceding and subsequent stages. Lastly, it seems intuitive that Germline-Dependent and Context-Dependent epigenetic modifications interact, resulting in the individual variation observed in behaviors, but until now this hypothesis has never been tested experimentally.
transgenerational; individual variation; life history; prenatal; postnatal; adolescence; litter
Progesterone and its nuclear receptor are critical in modulating reproductive physiology and behavior in female and male vertebrates. Whiptail lizards (genus Cnemidophorus) are an excellent model system in which to study the evolution of sexual behavior, as both the ancestral and descendent species exist. Male-typical sexual behavior is mediated by progesterone in both the ancestral species and the descendant all-female species, although the molecular characterization and distribution of the progesterone receptor protein throughout the reptilian brain is not well understood. To better understand the gene targets and ligand binding properties of the progesterone receptor in whiptails, we cloned the promoter and coding sequence of the progesterone receptor and analyzed the predicted protein structure. We next determined the distribution of the progesterone receptor protein and mRNA throughout the brain of C. inornatus and C. uniparens by immunohistochemistry and in situ hybridization. We found the progesterone receptor to be present in many brain regions known to regulate social behavior and processing of stimulus salience across many vertebrates, including the ventral tegmental area, amygdala, nucleus accumbens and several hypothalamic nuclei. Additionally, we quantified immunoreactive cells in the preoptic area and ventromedial hypothalamus in females of both species and males of the ancestral species. We found differences between both species and across ovarian states. Our results significantly extend our understanding of progesterone modulation in the reptilian brain and support the important role of the nuclear progesterone receptor in modulating sexual behavior in reptiles and across vertebrates.
progesterone receptor; reptile; sexual behavior; social behavior; parthenogenesis
Temperature-dependent sex determination (TSD) was first reported in 1966 in an African lizard. It has since been shown that TSD occurs in some fish, several lizards, tuataras, numerous turtles, and all crocodilians. Extreme temperatures can also cause sex reversal in several amphibians and lizards with genotypic sex determination. Research in TSD species indicates that estrogen signaling is important for ovary development and that orthologs of mammalian genes play a role in gonad differentiation. Nevertheless, the mechanism that actually transduces temperature into a biological signal for ovary versus testis development is not known in any species. Classical genetics could be used to identify the loci underlying TSD, but only if there is segregating variation for TSD. Here, we employ the “animal model” to analyze inheritance of sexual phenotype in a 13-generation pedigree of captive leopard geckos, Eublepharis macularius, a TSD reptile. We directly demonstrate genetic variance and genotype-by-temperature interactions for sex determination. Additive genetic variation was significant at a temperature that produces a female-biased sex ratio (30°C) but not at a temperature that produces a male-biased sex ratio (32.5°C). Conversely, dominance variance was significant at the male-biased temperature (32.5°C), but not at the female-biased temperature (30°C). Non-genetic maternal effects on sex determination were negligible in comparison to additive genetic variance, dominance variance, and the primary effect of temperature. These data show for the first time that there is segregating variation for TSD in a reptile and consequently that a QTL analysis would be practicable for identifying the genes underlying TSD.
maternal effects; quantitative genetics; reptile; sex ratio; temperature-dependent sex determination
Background: The links between nature and nurture need to be redefined to accommodate anthropogenic chemical contamination. Although some local remediation of contamination has occurred, at the global level this is simply not possible. Contaminants are spread by population migration, by introduction via the food chain, and through air and water currents, even to regions that were never exposed directly to these environmental insults. In recognizing and accepting this worldwide change, we must move on and consider the types of adaptations that could occur as a consequence.
Objectives: We propose a paradigm shift in the field that integrates various disciplines involved in the study of environmental contamination to recognize that contamination is widespread and cannot be remedied at the global level.
Discussion: Greater effort must be placed on integrative and interdisciplinary studies that explicitly illuminate how the causal mechanisms and functional outcomes of related processes operate at each level of biological organization while at the same time revealing the relations among the levels.
Conclusions: To anticipate and understand the future, we must devote more study to what is likely to happen and less to what has happened. Only then will we begin to understand how ancestral environmental exposures act at both the level of the individual and the level of their descendants to influence all aspects of life history.
endocrine-disrupting chemicals; epigenetics; evolutionary biology; heritable; proximate effect; ultimate effect
The developmental processes underlying gonadal differentiation are conserved across vertebrates, but the triggers initiating these trajectories are extremely variable. The red-eared slider turtle (Trachemys scripta elegans) exhibits temperature-dependent sex determination (TSD), a system where incubation temperature during a temperature-sensitive period of development determines offspring sex. However, gonadal sex is sensitive to both temperature and hormones during this period – particularly estrogen. We present a model for temperature-based differences in aromatase expression as a critical step in ovarian determination. Localized estrogen production facilitates ovarian development while inhibiting male-specific gene expression. At male-producing temperatures aromatase is not upregulated, thereby allowing testis development.
temperature-dependent sex determination; Trachemys scripta; estrogen; aromatase; ovary
Although gonadogenesis has been extensively studied in vertebrates with genetic sex determination, investigations at the molecular level in nontraditional model organisms with temperature-dependent sex determination are a relatively new area of research. Results show that while the key players of the molecular network underlying gonad development appear to be retained, their functions range from conserved to novel roles. In this review, we summarize experiments investigating candidate molecular players underlying temperature-dependent sex determination. We discuss some of the problems encountered unraveling this network, pose potential solutions, and suggest rewarding future directions of research.
temperature; sex determination; gonad; reptile; development
The steroidogenic enzyme CYP17 is responsible for catalyzing the production of androgenic precursors, while CYP19 converts testosterone to estradiol. De novo neurosteroidogenesis in specific brain regions influences steroid hormone dependent behaviors. In the all-female lizard species Cnemidophorus uniparens, individuals alternately display both male-like mounting and female-like receptivity. Mounting is associated with high circulating concentrations of progesterone following ovulation (PostOv), while receptivity is correlated with estrogen preceding it (PreOv). At a neuroanatomical level, the preoptic area (POA) and ventromedial nucleus of the hypothalamus (VMN) are the foci of the male-typical mounting and female-typical receptivity, respectively. In this study, we indirectly test the hypothesis that the whiptail lizard brain is capable of de novo neurosteroidogenesis by cloning fragments of the genes encoding two steroidogenic enzymes, CYP17 and CYP19, and examining their expression patterns in the C. uniparens brain. Our data indicate that these genes are expressed in the C. uniparens brain, and more importantly in the POA and VMN. Using radioactive in situ hybridization, we measured higher CYP17 mRNA levels in the POA of PostOv lizards compared to receptive PreOv animals; CYP19 mRNA levels in the VMN did not change across the ovarian cycle. To our knowledge, these are the first data suggesting that the reptilian brain is capable of de novo steroidogenesis. This study also supports the idea that non-gonadal sources of steroid hormones locally produced in behaviorally relevant brain loci are central to the mediation of behavioral output.
sexual behavior; androgen; estrogen; sexual differentiation
Individuals vary in their sociosexual behaviors and reactivity. How the organism interacts with the environment to produce this variation has been a focus in psychology since its inception as a scientific discipline. There is now no question that cumulative experiences throughout life history interact with genetic predispositions to shape the individual’s behavior. Recent evidence suggests that events in past generations may also influence how an individual responds to events in their own life history. Epigenetics is the study of how the environment can affect the genome of the individual during its development as well as the development of its descendants, all without changing the DNA sequence. Several distinctions must be made if this research is to become a staple in behavioral neuroendocrinology. The first distinction concerns perspective, and the need to distinguish and appreciate, the differences between Molecular versus Molar epigenetics. Each has its own lineage of investigation, yet both appear to be unaware of one another. Second, it is important to distinguish the difference between Context-Dependent versus Germline-Dependent epigenetic modifications. In essence the difference is one of the mechanism of heritability or transmission within, as apposed to across, generations. This review illustrates these distinctions while describing several rodent models that have shown particular promise for unraveling the contribution of genetics and the environment on sociosexual behavior. The first focuses on genetically-modified mice and makes the point that the early litter environment alters subsequent brain activity and behavior. This work emphasizes the need to understand behavioral development when doing research with such animals. The second focuses on a new rat model in which the epigenome is permanently imprinted, an effect that crosses generations to impact the descendants without further exposure to the precipitating agent. This work raises the question of how events in generations past can have consequences at both the mechanistic, behavioral, and ultimately evolutionary levels.
Development; Genetically-modified mice; Knockout; Imprinting; Molar epigenetics; Context-Dependent epigenetic modification; Germline-Dependent epigenetic modification; Neural network; Cytochrome oxidase
In mammals, the formative environment for social and anxiety-related behaviors is the family unit; in the case of rodents, this is the litter and the mother-young bond. A deciding factor in this environment is the sex ratio of the litter and, in the case of mice lacking functional copies of gene(s), the ratio of the various genotypes in the litter. Both Sex and Genotype ratios of the litter affect the nature and quality of the individual's behavior later in adulthood, as well as metabolic activity in brain nuclei that underlie these behaviors. Mice were raised in litters reconstituted shortly after to birth to control for sex ratio and genotype ratio (wild type pups versus pups lacking a functional estrogen receptor α). In both males and females, the Sex and Genotype of siblings in the litter affected aggressive behaviors as well as patterns of metabolic activity in limbic nuclei in the social behavior network later in adulthood. Further, this pattern in males varied depending upon the Genotype of their brothers and sisters. Principal Components Analysis revealed two components comprised of several amygdalar and hypothalamic nuclei; the VMH showed strong correlations in both clusters, suggesting its pivotal nature in the organization of two neural networks.
life history; sex differences; genotype differences; sibling; aggression
Embryonic exposure to the endocrine disruptor vinclozolin during gonadal sex determination promotes an epigenetic reprogramming of the male germ-line that is associated with transgenerational adult onset disease states. Further analysis of this transgenerational phenotype on the brain demonstrated reproducible changes in the brain transcriptome three generations (F3) removed from the exposure. The transgenerational alterations in the male and female brain transcriptomes were distinct. In the males, the expression of 92 genes in the hippocampus and 276 genes in the amygdala were transgenerationally altered. In the females, the expression of 1,301 genes in the hippocampus and 172 genes in the amygdala were transgenerationally altered. Analysis of specific gene sets demonstrated that several brain signaling pathways were influenced including those involved in axon guidance and long-term potentiation. An investigation of behavior demonstrated that the vinclozolin F3 generation males had a decrease in anxiety-like behavior, while the females had an increase in anxiety-like behavior. These observations demonstrate that an embryonic exposure to an environmental compound appears to promote a reprogramming of brain development that correlates with transgenerational sex-specific alterations in the brain transcriptomes and behavior. Observations are discussed in regards to environmental and transgenerational influences on the etiology of brain disease.
Females alter their mate choices as they transition through different reproductive stages; however, the proximal mechanisms for such behavioral fluctuation are unclear. In many taxa, as females transition through different reproductive stages, there is an associated change in hormone levels; therefore, we examined whether fluctuation in hormone levels serves as a proximal mechanism for within-individual variation in mate choice in female túngara frogs (Physalaemus pustulosus). We manipulated hormone levels of females by administering 0, 10, 100, 500 or 1000 IU of human chorionic gonadotropin (HCG), which is a ligand for luteinizing hormone (LH) receptors and will therefore cause increased gonadal hormone production. Phonotaxis assays were conducted to measure three aspects of mate choice behavior before and after HCG administration; receptivity (response to a conspecific mate signal), permissiveness (response to a signal that is less attractive than conspecific signals) and discrimination (ability to discern signal differences). The probability of response to a conspecific and an artificial hybrid signal significantly increased at the highest HCG doses. The difference in mean response time between pre- and post-HCG tests was significantly different for both the receptivity and permissiveness tests among the five doses. Increased permissiveness, however, was not due to decreased discrimination because females could discriminate between calls even at the highest HCG doses. These hormonal manipulations caused the same behavioral pattern we reported in females as they transitioned through different reproductive stages (Lynch, K.S., Rand, A.S., Ryan, M.J., Wilczynski, W., 2005. Plasticity in female mate choice associated with changing reproductive states. Anim. Behav. 69, 689–699), suggesting that changes in hormone levels can influence the female’s mate choice behavior.
Male choice; Receptivity; Reproductive hormones; Anuran
R-Spondin1 (Rspo1) is a novel regulator of the Wnt/β-catenin signalling pathway. Loss-of-function mutations in human RSPO1 cause testicular differentiation in 46, XX females, pointing to a role in ovarian development. Here we report the cloning and comparative expression analysis of R-SPONDIN1 orthologues in the mouse, chicken and red-eared slider turtle, three species with different sex-determining mechanisms. Evidence is presented that this gene is an ancient component of the vertebrate ovary-determining pathway.
Gonadal RSPO1 gene expression is female up-regulated in the embryonic gonads in each species at the onset of sexual differentiation. In the mouse gonad, Rspo1 mRNA is expressed in the somatic cell lineage at the time of ovarian differentiation (E12.5–E15.5), with little expression in germ cells. However, the protein is localised in the cytoplasm and at the cell surface of both somatic (pre-follicular) and germ cells. In the chicken embryo, RSPO1 expression becomes elevated in females at the time of ovarian differentiation, coinciding with female-specific activation of the FOXL2 gene and estrogen synthesis. RSPO1 protein in chicken is localised in the outer cortical zone of the developing ovary, the site of primordial follicle formation and germ cell differentiation. Inhibition of estrogen synthesis with a specific aromatase inhibitor results in a decline in chicken RSPO1 expression, indicating that RSPO1 is influenced by estrogen. In the red-eared slider turtle, which exhibits temperature-dependent sex determination, up-regulation of RSPO1 occurs during the temperature-sensitive period, when gonadal development is responsive to temperature. Accordingly, RSPO1 expression is temperature-responsive, and is down-regulated in embryos shifted from female- to male-producing incubation temperatures.
These results indicate that RSPO1 is up-regulated in the embryonic gonads of female vertebrates with different sex-determining mechanisms. In all instances, RSPO1 is expressed in the incipient ovary. These findings suggest that R-SPONDIN1 is an ancient, conserved part of the vertebrate ovary-determining pathway.
Copulatory behaviors are generally dependent on testicular androgens in male vertebrates, being eliminated by castration and re-instated by testosterone administration. It is postulated that a critical factor in this hormonal gating is up-regulation of neuronal nitric oxide synthase (nNOS) in the preoptic area, and consequent enhanced nitric oxide synthesis in response to stimuli associated with a receptive female. Previous studies have suggested that nNOS protein is more abundant in behaviorally relevant preoptic regions of testosterone-exposed animals than in hormone-deprived controls. This study sought to elucidate the molecular events underlying this apparent up-regulation by examining preoptic nNOS mRNA abundance at several time points following testosterone administration in a castration and replacement paradigm. Castrated male whiptails (Cnemidophorus inornatus) were implanted with testosterone, and at four time points over the subsequent 18 days their sexual behavior was tested. A rostral periventricular area previously implicated in hormonal gating of male-typical copulatory behavior was then excised by laser microdissection, and nNOS transcript abundance was assessed by quantitative PCR. As neither this technique nor nNOS mRNA measurements have previously been performed in this area of the brain, expression was concommitantly assayed on adjacent sections by in situ hybridization or NADPH diaphorase histochemistry. Results are consistent with transcriptional up-regulation of nNOS by testosterone and a central role for the enzyme in mediating hormonal gating of copulatory behavior.
anteroventral periventricular preoptic area; Cnemidophorus inornatus; in situ hybridization; laser microdissection; NADPH diaphorase histochemistry
Genes are not expressed in isolation any more than social behavior has meaning outside of society. Both are in dynamic flux with the immediate environment that the gene/individual finds itself, which in turn establishes the timing, pattern, and conditions of expression. This means that complex behaviors and their genetic underpinnings should be viewed as a cumulative process, or as the result of experiences up to that point in time and, at the same time, as setting the stage for what will follow. The evidence indicates that as experiences accumulate throughout life, early experiences shape how genes/individuals will respond to later experiences, whereas later experiences modify the effects of these earlier experiences. A method of graphically representing and analyzing change in gene and neural networks is presented. Results from several animal model systems will be described to illustrate these methods. First, we will consider the phenomenon of temperature-dependent sex determination in reptiles. We will illustrate how the experience of a particular temperature during a sensitive period of embryogenesis sculpts not only the patterns of expression of genes involved in sex determination and gonadal differentiation but also the morphological, physiological, neuroendocrine, and behavioral traits of the adult phenotype. The second model system concerns the effects of the sex ratio in the litter in rats, and the genotype ratio in the litter of transgenic mice, on the nature and frequency of maternal care and how this in turn influences the patterns of activation of identified neural circuits subserving the offspring's sociosexual behavior when it is an adult.
Reptile; Knockout mouse; Rat; Epigenetic; Cytochrome oxidase; Analysis
Hormone–neurotransmitter interactions form an important link through which hormones influence a variety of behavioral processes. Typically, sexual behavior is dimorphic with males mounting receptive females. In the all-female lizard species Cnemidophorus uniparens, individuals display both male-like pseudocopulation and female-like receptivity. These respective behavioral states are correlated with high circulating concentrations of progesterone following ovulation and of estrogen preceding it. In sexual species, serotonin is involved in male-typical mounting, and, as reported here, in male-like pseudosexual behavior in this unisexual species. In the first study, C. uniparens were ovariectomized and treated systemically with exogenous androgen, a hormonal regimen that results in individuals displaying only male-like pseudosexual behavior. An increase in serotonin levels in the preoptic area coupled with the suppression of male-like pseudocopulation was observed in androgen-treated lizards injected with 5-hydroxytryptophan (the precursor of serotonin) and clorgyline (a monoamine oxidase inhibitor) compared to vehicle-treated controls. Our second experiment involved ovariectomizing lizards and either injecting them with estradiol or implanting them with either an empty (Blank) or a progesterone- or testosterone-containing Silastic capsule. Treatment with para-chlorophenylalanine (an inhibitor of tryptophan hydroxylase) facilitated male-like pseudosexual behavior depending on the circulating hormonal milieu and decreased serotonin levels in the preoptic area. Our data suggest that serotonin is inhibitory to male-like pseudosexual behavior in C. uniparens but more importantly that the hormonal environment modulates the serotonin system at the level of the preoptic area, with the serotonergic system then establishing behavioral thresholds that allow for this behavior to be “gated”.
Serotonin; Reptile; Sexual behavior; Thresholds; Neurotransmitters; Hormones; Preoptic area
Based on Tinbergen’s view of the study of behavioural development we describe some recent advances and their importance in this field. We argue that the study of behavioural development should combine both proximate and ultimate approaches, and can help to understand how early subtle environmental factors shape consistent individual variation both between and within sexes. This is illustrated by reviewing the profound effects of incubation temperature on the development of brain and social behaviour in the leopard gecko, a species with temperature-dependent sex determination, and the effects of early exposure to steroid hormones on social behaviour in rodents and especially birds. Both are maternal effects: incubation temperature can be partly determined by the nest site where the mother deposited her eggs, while in both oviparous and viviparous vertebrates maternal hormones reach and influence the embryo. In the gecko, incubation temperature affects sexual and aggressive behaviour, growth, the hypothalamus-pituitary-gonadal axis, as well as the size, connectivity and metabolic capacity of certain brain areas. In this way not only is the gonad type determined, but so too is the morphological, physiological, neural, and behavioural phenotype established that explains much of within-sex variation. In rodents, maternal hormones affect similar aspects. In avian species, maternal hormones, deposited in the eggs, vary systematically between and within clutches and have both short- and long-lasting effects on competitive behaviour. Evidence suggests that mothers adaptively adjust hormone allocation to the environmental context. In addition, we discuss some effects of postnatal experience on behavioural development in geckos, mice and bird species. Our results also illustrate how the study of animal models other than rodents can help in understanding important developmental processes.
behavioural development; bird; cytochrome oxidase; individual differentiation; lizard; maternal androgens; temperature-dependent sex determination
In this article we consider the importance of assessing endocrine disruption in a large new cohort that has been proposed, the National Children's Study (NCS). We briefly review evidence that endocrine disruption is a potentially important hypothesis for human studies and weigh the need to assess endocrine disruption in the NCS. We note the salient features of earlier, similar cohort studies that serve as reference points for the design of the NCS. Finally, we discuss features of the NCS that would allow or enhance assessment of endocrine disruption, even if endocrine disruption were not a primary hypothesis motivating the study. At this time, the evidence supporting endocrine disruption in humans with background-level exposures is not strong. Thus, a compelling rationale for the NCS will probably need to be based on core hypotheses that focus on other issues. Nonetheless, if properly designed, the NCS could serve as an excellent resource for investigating future hypotheses regarding endocrine disruption.