David Pafford Crews - US grants

The specific objective of this proposal is to examine the causal mechanisms and functional outcomes of the two major annual reproductive tactics -- associated and dissociated -- exhibited by higher vertebrates. In many seasonally breeding vertebrates, gamete maturation and maximum secretion of sex steroid hormones immediately precede or coincide with courtship and copulatory (mating) behavior. This annual pattern may be termed the associated reproductive tactic, or prenuptial gametogenesis (Figure 1). A markedly different annual pattern is exhibited in many vertebrates, including some mammals, in which the gametes are produced only after the breeding season has ended; the gametes are then stored until the next breeding period. In these species, mating occurs when the gonads are not producing gametes and blood concentrations of sex steroid hormones are basal. This pattern may be referred to as a dissociated reproductive tactic, or postnuptial gametogenesis (Figure 1). I will fucus on one representative species of each reproductive tactic. The green anole lizard is similar to many laboratory and domesticated mammals and birds showing the associated tactic. In contrast, the red-sided garter snake shows the dissociated patterns. In many instances a direct comparison of these two species will be made, whereas in other instances gaps in our knowledge must be filled before conceptually valid comparisons can be made. Thus, some of the proposed experiments deal with only one species or tactic. Ultimately, however, my goal is to compare the two tactics at as many levels of organization as are feasible and resonable. Such a broad approach is crucial if important generalities underlying vertebrate reproductive processes are to emerge. My proposed studies will also contribute directly to our understanding of related areas of reproductive biology, including gamete storage and animal husbandry.

DESCRIPTION (provided by applicant): The goal of the proposed research is to elucidate the role of steroid hormones in controlling sexually dimorphic behaviors. Male-typical copulatory behaviors such as mounting and intromission are dependent on androgens in all vertebrates studied, but the mechanisms by which these hormones influence the brain and behavior are not well understood. Whiptail lizards of the genus Cnemidophorus enable a powerful comparative approach to understanding these mechanisms because some species are sexual, exhibiting the typical vertebrate pattern of sexually dimorphic behavior (estrogen-dependent receptivity in females and androgen-dependent mounting in males), and other species are all-female, reproducing clonally and exhibiting both male-like and female-like behavior, according to ovarian hormone levels. The experiments proposed involve C. inornatus, a typical sexual species, and C. uniparens, which is all-female. C. uniparens individuals, just like C. inornatus females, are receptive when their circulating estrogen levels are high, but unlike C. inornatus, they also exhibit the complete repertoire of male-typical copulatory behavior when presented with a receptive female during the periovulatory phase of the ovarian cycle when progesterone levels are high. This "pseudocopulatory" behavior can be evoked in the laboratory by administration of exogenous progesterone, and the operational goal of this research is to determine how progesterone is able to mimic the normal function of androgen in this way. Specific experiments will investigate how the progesterone receptor comes to be expressed in the preoptic area of the brain that is thought to mediate male-typical copulatory behavior, and in the process will yield information about the normal function of this area, as well as the developmental regulation of steroid hormone receptors. Other studies will elucidate the actions of androgens and progesterone on the neurotransmitters dopamine and nitric oxide, which are important components in the neural control of sexual behavior. This functional interchangeability of androgens and progestins has obvious fundamental implications for human health. All women normally experience fluctuating levels of progesterone, and millions of women use exogenous progestins. There is also a marked diurnal rhythm [in] progesterone levels in men, and progestins (usually in conjunction with testosterone) are beginning to be studied as a male contraceptive. The interaction of these progestins with the functions of other steroid hormones therefore warrants thorough investigation. From a scientific point of view, the hormonal control of sexual behavior is a particularly tractable model for elucidating the way in which particular genes, expressed in particular neural circuits, can affect specific behaviors, because the stimuli and behaviors involved are simple, the brain areas involved are well characterized, and the relationship between hormones and genes is comparatively well understood.

Mammals, birds, and reptiles constitute the amniote vertebrates. In all mammals and birds, and in many reptiles, sex is determined at fertilization by genotype (genotypic sex determination or GSD). In some reptiles, however, the temperature at which the egg incubates determines whether the embryo hatches as a male or a female (temperature-dependent sex determination or TSD) Thus, in these species, embryos are initially bipotential and only later does channelization toward the male or female phenotype occur. The initial bipotentiality and later determination of sex by species exhibiting TSD offer a unique model system for studies of the mechanisms of sex determination and sexual differentiation in all amniote vertebrates, including man. We have discovered recently that the administration of estrogen to embryos incubating at male-producing temperatures causes ovarian development, over-riding any temperature effect. The proposed research will investigate (i) how this estrogen effect occurs, (ii) whether, under normal conditions, steroid hormones are present before the gonad is committed to testicular/ovarian development, and (iii) whether embryonic steroids have a similar role in the differentiation of non-gonadal phenotypes as in mammals and birds. We also have discovered that the temperature that an embryo experiences profoundly affects its adult morphology, physiology, and behavior. The mechanisms by which incubation temperature organizes adult sexuality will be studied through hormonal manipulations of embryos and neonates by castration and/or administration of steroid hormones, synthetic hormone agonists and antagonists, enzyme inhibitors, and steroid antisera. Together, these experiments will determine the extent to which GSD and TSD have similar physiological or biochemical bases in the control of gonad determination and in the biopsychology of sexual differentiation. The proposed studies are of fundamental importance to understanding, the development of sexuality in vertebrates, including the relation of different levels of sexuality within an individual. The studies will use multiple techniques, including behavioral testing, histology, -immunocytochemistry, monoclonal antibodies for hormone receptors, synthetic steroid agonists and antagonists, high-performance liquid chromatography, gas chromatography/mass. spectrometry, and radioimmunoassay.

This proposal requests continued support of a broadly based research program that seeks to understand (i) the evolution of hormone-brain-behavior mechanisms underlying sexual behaviors, and (ii) the dual neural circuits that subserve these behaviors. This goal is relevant to mental health since information on the brain mechanisms that control normal behavior provides the context in which to determine and evaluate psychopathology. The approach used is both comparative and multidiscipline. 'Me comparative method emphasizes the need for different model systems if we are to elucidate both the general rules which govern behaviors as when as their historical roots. Multidisciplinary studies spanning the molecular to the population levels of biological organization provide a multifaceted, yet integrated, perspective of individual behavior. The animal model systems to be used are parthenogenetic whiptail lizards and their sexual ancestors. The unisexual species is known to have descended directly from extant sexual species, thereby allowing direct ancestor-descendant comparisons. Because all individuals have ovaries, yet exhibit both male-like and female-like pseudosexual behaviors, the complication of having two gonadal sexes, each with their own particular hormonal milieu, is removed. This makes it possible to study the neural circuits that underlie mounting and receptive behavior in a manner not possible with more common laboratory animals. The studies proposed center on a comparison of the sexual ancestral species and their parthenogenetic descendants. They employ behavioral observation in both the field and the laboratory, functional neurology of specific behaviors as assessed by hormonal manipulation including intracranial implantation, hormone-receptor analysis, radioimmunoassay, immunocytochemistry, and autoradiography. Some experiments will exploit the fact that progesterone stimulates, rather than inhibits, the sexual behavior of males of the parental species and that progesterone activates male-like pseudosexual behavior in the unisexual descendant species. These studies will examine the mechanism of hormone action at the level of the receptor. Other studies will localize the sites of hormone action in the brain, show how different sexual behaviors are reflected in dimorphisms in brain nuclei, and identify and describe the dual neural circuits controlling mounting and receptive behavior.

ABSTRACT Proposal: #96-23546 PI: Crews & Rhen Title: Evolutionary origin of behavioral organization Organization of reproductive and aggressive behaviors by estradiol (via aromatization of testosterone in mammals) during early development is well documented in birds and mammals. There is a correlation between the organized and heterogametic sex in these groups; males are organized (lose the ability to display female-typical behavior) in mammals, whereas females are organized (lose the ability to display male-typical behavior) in birds. The opposite sex retains the capacity to display both female- and male-typical behavior. A similar correlation is observed in other vertebrates with genotypic sex determination. Alternate explanations are (1) that a developmental constraint results in organization of the heterogametic sex, or (2) that there is a functional relationship between organization and gonochorism. Reptiles with environmental sex determination do not fit into the constraint paradigm because they lack a heterogametic sex. In contrast, these species may be organized if organization is adaptive. In this study, manipulation of embryonic temperature (and thereby gonadal sex) and adult hormones will be performed to distinguish among hypotheses about organization. Log-linear, multivariate, and path analyses will be used to analyze reproductive and aggressive behaviors. Results will (1) illuminate the proximate cause of behavioral variation in an ESD species, and (2) for the first time delineate and test alternate hypotheses about the evolutionary causes of behavioral organization in the context of sex determining mechanisms.

DESCRIPTION (Adapted from applicant's abstract): This research addresses the fundamental question of what determines behavioral variation. Many environmental variables can produce predictable effects on phenotype. One such variable is the incubation temperature of eggs in many reptiles. In the leopard gecko, embryos become male or female depending upon their temperature during development. In addition, between-sex as well as within-sex differences attributable to incubation temperature have been found in morphology, secretion of and sensitivity to steroid hormones, sociosexual behavior, reproductive success, and in the neuroanatomy and metabolic activity of brain areas that mediate sociosexual behaviors. These environmental effects are analogous to the effect of intrauterine environment in mammals, including humans. Given the homology of the endocrine and nervous systems across vertebrates, it is important to determine if homologous mechanisms underlie these analogous effects on behavior. If the mechanism underlying environmental effects on behavior in the leopard gecko is conserved (i.e., via sex steroids), this research will lend new insight into the evolution of sexual differentiation because temperature-dependent sex determination is thought to be the evolutionary precursor to genotypic sex determination (present in birds and mammals) and because reptiles are the ancestors of both birds and mammals. If the mechanism is different (i.e., direct temperature effects), this research would elucidate a novel process of sexual differentiation that may also be present in birds and mammals but, because of homeothermy, is masked. This latter possibility is especially important because young birds and mammals cannot regulate their body temperature as do adults. The proposed research is broadly categorized into three groups: thermoregulation and its relation to sociosexual behaviors (i.e., the degree to which the neural substrates mediating thermoregulatory and sociosexual behavior overlap), the development of the neural phenotypes of these brain regions and hormone milieus, and the effects of hormonal manipulations during development and neural manipulations in adulthood on thermoregulatory and sociosexual behaviors. The final category of experiments is particularly important as it will discern whether the incubation temperature effects are direct or indirect.

Many egg-laying reptiles lack sex chromosomes, depending instead upon the incubation temperature of the egg to determine the sex of their offspring, a process known as temperature-dependent sex determination (TSD). How temperature both stimulates and inhibits genetic cascades to determine the type of gonad and direct sexual differentiation is the focus of this application. This investigator and his colleagues have developed the red-eared slider turtle (Trachemys scripta elegans) as an animal model system to study TSD. These researchers have demonstrated that male and female gonadal development are the result of separate genetic cascades influenced by steroid hormones, and that specific enzymes activate or inhibit steroid hormone effects on sex determination. In the red-eared slider system, female (or ovary) determination involves estrogens whereas male (or testis) determination involves nonaromatizable androgens. Mammals and turtles share a common evolutionary history and recent findings indicate that despite a difference in the trigger (environmental temperature vs. sex chromosomes), the red-eared slider shares many characteristics of sexual development with the mammalian system, including the same genes involved along the sex determination pathway. Experiments are designed to identify the genes involved in testis formation and then to determine how temperature and sex hormones accomplish this feat. For example, relative levels of gene expression will be measured using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis. Localization of gene expression will be analyzed with in situ hybridization. The ability to manipulate sex in a primitive vertebrate species by incubation temperature, exogenous hormones, and other agents provides unparalleled experimental control, thereby enabling more detailed analysis of the normal pattern of gene expression during sex determination than is possible with other amniotes (reptiles, birds and mammals are known as the amniote or higher vertebrates) having sex chromosomes. This work is important for several reasons. First, it has long been assumed that steroid hormones of maternal or embryonic origin are not involved in gonad formation in mammals and birds. The work with TSD reptiles indicates that this conclusion may be premature. Second, since TSD may represent the evolutionary precursor to sex chromosomes, potential temperature and steroid effects in sex determination may be present, but partly or wholly masked, in warm-blooded vertebrates. Third, temperature has not been adequately investigated as a factor in steroid hormone action at the genetic level in warm-blooded animals despite numerous studies documenting how both hormone responsiveness and hormone action are markedly dependent on temperature.

Our understanding of the process by which individuality emerges is still rudimentary, but it is clear that the environment in which the individual develops, and its sociosexual interactions as an adult, are central to this process. This project addresses how the experiences passively acquired as an embryo interact with those of the adult, when the individual has a degree of behavioral regulation of its own environment. Behavioral and neural plasticity is at the root of these individual differences. Thus, the overall goal of the proposed research is to determine how embryonic experience and sexual experience later in life interact to affect adult male sexual behavior, and to reveal how this in turn effects the neural circuitry underlying behavior. Specifically, using the leopard gecko (Eublepharis macularius) as a model system, experiments will investigate the mechanisms involved in mediating the interaction between embryonic and adult experience. Obvious candidate mechanisms have two properties: they are influenced by the embryonic environment and they are capable of affecting sexual behavior and learning during adulthood. The mesolimbic dopamine system satisfies both these criteria and it is hypothesized that varying extent of embryonic hormone exposure organizes this system and the resulting differences mediate how the two morphs respond to experience. These studies are long-term and will involve the participation of undergraduate and graduate students for their execution. Students will be expected to present their findings at national meetings and be co-authors on papers published in peer-reviewed journals.

DESCRIPTION (provided by applicant): Endocrine-disrupting chemicals (EDCs) are compounds in the environment that perturb endocrine systems. EDCs are ubiquitous in the modern world, and detectable in virtually all humans and wildlife. Many effects of EDCs are mediated by hormone receptors such as estrogen receptors (ERs) that are widely distributed in the brain, and thereby EDCs perturb neurobiological functions. The hypothalamus, hippocampus and amygdala are brain regions with high expression of ERs, and are proven targets for endocrine disruption. However, the cellular, molecular and physiological processes for these effects, and the functional behavioral implications for exposures of these brain regions to EDCs, are not well explored. Furthermore, the brain has a number of structural and functional sexual dimorphisms that develop early in life, a process that is sculpted by actions of estrogens on ERs, and disrupted by EDCs. Therefore, it is the overarching goal of this research to determine the molecular and cellular mechanisms by which exposure of developing fetuses to ecologically-relevant levels of EDCs perturb the sexual differentiation of specific brain areas, and the consequences on behaviors regulated by these regions. The proposed studies will fill a gap in knowledge by testing effects of perinatal exposure to a class of endocrine-disrupting chemicals, specifically polychlorinated biphenyls (PCBs), on sexually dimorphic neurobiological processes. PCBs are a ubiquitous and persistent environmental contaminant, and while banned for decades, they are still prevalent in soil and groundwater, leach into food and water, and are detectable in tissues of virtually all humans. The proposed studies will use a well-established rat model already in use in the PIs'labs to test effects of exposure to PCBs during a critical hormone-sensitive developmental window of late gestation, a critical period for brain sexual differentiation during which the developing nervous system is exquisitely sensitive to both endogenous and exogenous hormones, particularly estrogens. The experiments will quantify expression of a network of estrogen-regulated genes (Aim 1);elucidate some potential epigenetic mechanisms for regulation of identified targets (Aim 2);determine the manifestation of gene expression changes through protein immunohistochemistry (Aim 3);and ascertain the final outcome as a behavioral phenotype (Aim 4). These experiments have broad implications for humans as exposure to PCBs is universal, persistent, and has wide-ranging effects on health and disease. Therefore, understanding the latent effects of PCBs on neural development, and their underlying mechanisms, can inform public policy, medical interventions, and prevention. In addition, results on PCBs, used as a "model" EDC for decades, can help us better understand effects of other estrogenic EDCs still in common use such as those in plastics, pesticides, and beyond. PUBLIC HEALTH RELEVANCE: Exposures of humans to PCBs and other estrogenic EDCs are universal, and these compounds have wide- ranging effects on health and disease. Therefore, understanding the latent effects of PCBs on neural development, and their underlying mechanisms, can inform medical interventions and prevention, and guide public health policy. The rat model is highly conserved with humans, and it enables us to test the cause-and- effect relationship between prenatal PCBs, the development of adult disease, and the neural mechanisms underlying these biomedical processes.

DESCRIPTION (provided by applicant): Endocrine-disrupting chemicals (EDCs) are compounds in the environment that perturb endocrine systems. EDCs are ubiquitous in the modern world, and detectable in virtually all humans and wildlife. Many effects of EDCs are mediated by hormone receptors such as estrogen receptors (ERs) that are widely distributed in the brain, and thereby EDCs perturb neurobiological functions. The hypothalamus, hippocampus and amygdala are brain regions with high expression of ERs, and are proven targets for endocrine disruption. However, the cellular, molecular and physiological processes for these effects, and the functional behavioral implications for exposures of these brain regions to EDCs, are not well explored. Furthermore, the brain has a number of structural and functional sexual dimorphisms that develop early in life, a process that is sculpted by actions of estrogens on ERs, and disrupted by EDCs. Therefore, it is the overarching goal of this research to determine the molecular and cellular mechanisms by which exposure of developing fetuses to ecologically-relevant levels of EDCs perturb the sexual differentiation of specific brain areas, and the consequences on behaviors regulated by these regions. The proposed studies will fill a gap in knowledge by testing effects of perinatal exposure to a class of endocrine-disrupting chemicals, specifically polychlorinated biphenyls (PCBs), on sexually dimorphic neurobiological processes. PCBs are a ubiquitous and persistent environmental contaminant, and while banned for decades, they are still prevalent in soil and groundwater, leach into food and water, and are detectable in tissues of virtually all humans. The proposed studies will use a well-established rat model already in use in the PIs' labs to test effects of exposure to PCBs during a critical hormone-sensitive developmental window of late gestation, a critical period for brain sexual differentiation during which the developing nervous system is exquisitely sensitive to both endogenous and exogenous hormones, particularly estrogens. The experiments will quantify expression of a network of estrogen-regulated genes (Aim 1); elucidate some potential epigenetic mechanisms for regulation of identified targets (Aim 2); determine the manifestation of gene expression changes through protein immunohistochemistry (Aim 3); and ascertain the final outcome as a behavioral phenotype (Aim 4). These experiments have broad implications for humans as exposure to PCBs is universal, persistent, and has wide-ranging effects on health and disease. Therefore, understanding the latent effects of PCBs on neural development, and their underlying mechanisms, can inform public policy, medical interventions, and prevention. In addition, results on PCBs, used as a model EDC for decades, can help us better understand effects of other estrogenic EDCs still in common use such as those in plastics, pesticides, and beyond.

ABSTRACT Every human has a body burden of endocrine-disrupting chemicals (EDCs), and levels of EDCs correlate with reproductive, endocrine, and neurobehavioral deficiencies. As environmental stressors, EDCs interact with other types of stressors to increase chronic disease and impair the quality of life. This proposal seeks to understand how the developmental trajectory of an individual is shaped by the interaction of behavioral stress during two life stages in the context of contamination. We focus on the social behavioral phenotype, as shifts in the reaction norms of social behavior can profoundly change an individual?s relationship to its community, his/her reproductive success, and mental health. We postulate that neurodevelopment in a contaminated world changes an individual?s baseline social phenotype, and that further life stressors overlaid upon the EDC phenotype create greater deflections from the behavioral norm. We will approach this question in several novel ways. First, we will model constant low-level exposure to a mixture of common-use EDCs throughout life, and assess emotional reactivity and sociality. Second, we will examine the effects of a typical life challenge (mild stress) during two critical life periods: to the mother (during pregnancy), to the individual during adolescence, or both. Third, we will compare males and females; this enables us to identify susceptibilities that may relate to well-established gender differences in disease and neurobehavioral dysfunction. Finally, by measuring changes in neuromolecular activity and neuroanatomical organization in a defined network of interconnected limbic and forebrain nuclei regulating the social phenotype, we can gain mechanistic insights into these processes. Thus, our overarching hypothesis is that each life stressor (lifelong EDC exposures, mild prenatal stress, and mild stress during adolescence) concatenates to shift the reaction norms for neurodevelopment and social behavior, and that the combination of stressors exacerbates adverse outcomes for neurobiological health. Mechanistically, we further propose that EDCs and stressors modulate this phenotype through perturbing the normal complementarity of estrogen and androgen signaling in the social decision-making network of the brain. There are 3 Specific Aims. Aim I will establish the sexually dimorphic behavioral phenotype of a lifetime of exposure to low levels of a mixture of common-use EDCs, upon which is superimposed mild stress during critical life stages (gestational, adolescent, or both). Aim II will determine underlying neuromolecular mechanisms for the changes caused by lifelong EDC exposures and gestational/adolescent stressors, focusing on estrogen/androgen-sensitive circuits. Aim III will identify neuroanatomical and cytoarchitectural substrates for the changes caused by EDCs and stressors, prioritizing estrogen-androgen signaling pathways. Proposed work has the potential to have a broad impact, ranging from societal and government policies, the health crisis caused by increasing chronic disease, and understanding fundamental biological principles at the molecular, cellular and organismal levels.