Sex contextualism in laboratory research: Enhancing rigor and precision in the study of sex-related variables.

Understanding sex-related variation in health and illness requires rigorous and precise approaches to revealing underlying mechanisms. A first step is to recognize that sex is not in and of itself a causal mechanism; rather, it is a classification system comprising a set of categories, usually assigned according to a range of varying traits. Moving beyond sex as a system of classification to working with concrete and measurable sex-related variables is necessary for precision. Whether and how these sex-related variables matter-and what patterns of difference they contribute to-will vary in context-specific ways. Second, when researchers incorporate these sex-related variables into research designs, rigorous analytical methods are needed to allow strongly supported conclusions. Third, the interpretation and reporting of sex-related variation require care to ensure that basic and preclinical research advance health equity for all.

Why and How to Account for Sex and Gender in Brain and Behavioral Research.

Long overlooked in neuroscience research, sex and gender are increasingly included as key variables potentially impacting all levels of neurobehavioral analysis. Still, many neuroscientists do not understand the difference between the terms "sex" and "gender," the complexity and nuance of each, or how to best include them as variables in research designs. This TechSights article outlines rationales for considering the influence of sex and gender across taxa, and provides technical guidance for strengthening the rigor and reproducibility of such analyses. This guidance includes the use of appropriate statistical methods for comparing groups as well as controls for key covariates of sex (e.g., total intracranial volume) and gender (e.g., income, caregiver stress, bias). We also recommend approaches for interpreting and communicating sex- and gender-related findings about the brain, which have often been misconstrued by neuroscientists and the lay public alike.

A supergene-linked estrogen receptor drives alternative phenotypes in a polymorphic songbird.

Behavioral evolution relies on genetic changes, yet few behaviors can be traced to specific genetic sequences in vertebrates. Here we provide experimental evidence showing that differentiation of a single gene has contributed to the evolution of divergent behavioral phenotypes in the white-throated sparrow, a common backyard songbird. In this species, a series of chromosomal inversions has formed a supergene that segregates with an aggressive phenotype. The supergene has captured ESR1, the gene that encodes estrogen receptor α (ERα); as a result, this gene is accumulating changes that now distinguish the supergene allele from the standard allele. Our results show that in birds of the more aggressive phenotype, ERα knockdown caused a phenotypic change to that of the less aggressive phenotype. We next showed that in a free-living population, aggression is predicted by allelic imbalance favoring the supergene allele. Finally, we identified cis-regulatory features, both genetic and epigenetic, that explain the allelic imbalance. This work provides a rare illustration of how genotypic divergence has led to behavioral phenotypic divergence in a vertebrate.

Regional epigenetic differentiation of the Z Chromosome between sexes in a female heterogametic system.

In male heterogametic systems, the X Chromosome is epigenetically differentiated between males and females, to facilitate dosage compensation. For example, the X Chromosome in female mammals is largely inactivated. Relative to well-studied male heterogametic systems, the extent of epigenetic differentiation between male and female Z Chromosomes in female heterogametic species, which often lack complete dosage compensation, is poorly understood. Here, we examined the chromosomal DNA methylation landscapes of male and female Z Chromosomes in two distantly related avian species, namely chicken and white-throated sparrow. We show that, in contrast to the pattern in mammals, male and female Z Chromosomes in these species exhibit highly similar patterns of DNA methylation, which is consistent with weak or absent dosage compensation. We further demonstrate that the epigenetic differences between male and female chicken Z Chromosomes are localized to a few regions, including a previously identified male hypermethylated region 1 (MHM1; CGNC: 80601). We discovered a novel region with elevated male-to-female methylation ratios on the chicken Z Chromosome (male hypermethylated region 2 [MHM2]; CGNC: 80602). The MHM1 and MHM2, despite little sequence similarity between them, bear similar molecular features that are likely associated with their functions. We present evidence consistent with female hypomethylation of MHMs and up-regulation of nearby genes. Therefore, despite little methylation differentiation between sexes, extremely localized DNA methylation differences between male and female chicken Z Chromosomes have evolved and affect expression of nearby regions. Our findings offer new insights into epigenetic regulation of gene expression between sexes in female heterogametic systems.

Genome-wide variation in DNA methylation linked to developmental stage and chromosomal suppression of recombination in white-throated sparrows.

Much of our knowledge on regulatory impacts of DNA methylation has come from laboratory-bred model organisms, which may not exhibit the full extent of variation found in wild populations. Here, we investigated naturally-occurring variation in DNA methylation in a wild avian species, the white-throated sparrow (Zonotrichia albicollis). This species offers exceptional opportunities for studying the link between genetic differentiation and phenotypic traits because of a nonrecombining chromosome pair linked to both plumage and behavioural phenotypes. Using novel single-nucleotide resolution methylation maps and gene expression data, we show that DNA methylation and the expression of DNA methyltransferases are significantly higher in adults than in nestlings. Genes for which DNA methylation varied between nestlings and adults were implicated in development and cell differentiation and were located throughout the genome. In contrast, differential methylation between plumage morphs was concentrated in the nonrecombining chromosome pair. Interestingly, a large number of CpGs on the nonrecombining chromosome, localized to transposable elements, have undergone dramatic loss of DNA methylation since the split of the ZAL2 and ZAL2m chromosomes. Changes in methylation predicted changes in gene expression for both chromosomes. In summary, we demonstrate changes in genome-wide DNA methylation that are associated with development and with specific functional categories of genes in white-throated sparrows. Moreover, we observe substantial DNA methylation reprogramming associated with the suppression of recombination, with implications for genome integrity and gene expression divergence. These results offer an unprecedented view of ongoing epigenetic reprogramming in a wild population.

Rapid regulatory evolution of a nonrecombining autosome linked to divergent behavioral phenotypes.

In the white-throated sparrow (Zonotrichia albicollis), the second chromosome bears a striking resemblance to sex chromosomes. First, within each breeding pair of birds, one bird is homozygous for the standard arrangement of the chromosome (ZAL2/ZAL2) and its mate is heterozygous for a different version (ZAL2/ZAL2m). Second, recombination is profoundly suppressed between the two versions, leading to genetic differentiation between them. Third, the ZAL2m version is linked with phenotypic traits, such as bright plumage, high aggression, and low parental behavior, which are usually associated with males. These similarities to sex chromosomes suggest that the evolutionary mechanisms that shape sex chromosomes, in particular genetic degeneration of the heterogametic version due to the suppression of recombination, are likely important in this system as well. Here, we investigated patterns of protein sequence evolution and gene expression evolution between the ZAL2 and ZAL2m chromosomes by whole-genome sequencing and transcriptome analyses. Patterns of protein evolution exhibited only weak signals of genetic degeneration, and few genes harbored signatures of positive selection. We found substantial evidence of transcriptome evolution, such as significant expression divergence between ZAL2 and ZAL2m alleles and signatures of dosage compensation for highly expressed genes. These results suggest that, early in the evolution of heteromorphic chromosomes, gene expression divergence and dosage compensation can prevail before large-scale genetic degeneration. Our results show further that suppression of recombination between heteromorphic chromosomes can lead to the evolution of alternative (sex-like) behavioral phenotypes before substantial genetic degeneration.

Vasoactive intestinal peptide as a mediator of the effects of a supergene on social behaviour.

Supergenes, or linked groups of alleles that are inherited together, present excellent opportunities to understand gene-behaviour relationships. In white-throated sparrows (Zonotrichia albicollis), a supergene on the second chromosome associates with a more aggressive and less parental phenotype. This supergene includes the gene for vasoactive intestinal peptide (VIP), a neuropeptide known to play a causal role in both aggression and parental behaviour. Here, using a free-living population, we compared the levels of VIP mRNA between birds with and without the supergene. We focused on the anterior hypothalamus and infundibular region, two brain regions containing VIP neurons known to play a causal role in aggression and parental behaviour, respectively. First, we show that the supergene enhances VIP expression in the anterior hypothalamus and that expression positively predicts vocal aggression independently of genotype in both sexes. Next, we show that the supergene reduces VIP expression in the infundibular region, which suggests reduced secretion of prolactin, a pro-parental hormone. Thus, the patterns of VIP expression in these two regions are consistent with the enhanced aggression and reduced parental behaviour of birds with the supergene allele. Our results illustrate mechanisms by which elements of genomic architecture, such as supergenes, can contribute to the evolution of alternative behavioural phenotypes.

Inside the supergene of the bird with four sexes.

The white-throated sparrow (Zonotrichia albicollis) offers unique opportunities to understand the adaptive value of supergenes, particularly their role in alternative phenotypes. In this species, alternative plumage morphs segregate with a nonrecombining segment of chromosome 2, which has been called a 'supergene'. The species mates disassortatively with respect to the supergene; that is, each breeding pair consists of one individual with it and one without it. This species has therefore been called the "bird with four sexes". The supergene segregates with a behavioral phenotype; birds with it are more aggressive and less parental than birds without it. Here, we review our efforts to identify the genes inside the supergene that are responsible for the behavioral polymorphism. The gene ESR1, which encodes estrogen receptor α, differs between the morphs and predicts both territorial and parental behavior. Variation in the regulatory regions of ESR1 causes an imbalance in expression of the two alleles, and the degree to which this imbalance favors the supergene allele predicts territorial singing. In heterozygotes, knockdown of ESR1 causes a phenotypic switch, from more aggressive to less aggressive. We recently showed that another gene important for social behavior, vasoactive intestinal peptide (VIP), is differentially expressed between the morphs and predicts territorial singing. We hypothesize that ESR1 and VIP contribute to behavior in a coordinated way and could represent co-adapted alleles. Because the supergene contains more than 1000 individual genes, this species provides rich possibilities for discovering alleles that work together to mediate life-history trade-offs and maximize the fitness of alternative complex phenotypes.

Polymorphisms in sex steroid receptors: From gene sequence to behavior.

Sex steroid receptors have received much interest as potential mediators of human behaviors and mental disorders. Candidate gene association studies have identified about 50 genetic variants of androgen and estrogen receptors that correlate with human behavioral phenotypes. Because most of these polymorphisms lie outside coding regions, discerning their effect on receptor function is not straightforward. Thus, although discoveries of associations improve our ability to predict risk, they have not greatly advanced our understanding of underlying mechanisms. This article is intended to serve as a starting point for psychologists and other behavioral biologists to consider potential mechanisms. Here, I review associations between polymorphisms in sex steroid receptors and human behavioral phenotypes. I then consider ways in which genetic variation can affect processes such as mRNA transcription, splicing, and stability. Finally, I suggest ways that hypotheses about mechanism can be tested, for example using in vitro assays and/or animal models.

Rapid effects of estradiol on aggression depend on genotype in a species with an estrogen receptor polymorphism.

The white-throated sparrow (Zonotrichia albicollis) represents a powerful model in behavioral neuroendocrinology because it occurs in two plumage morphs that differ with respect to steroid-dependent social behaviors. Birds of the white-striped (WS) morph engage in more territorial aggression than do birds of the tan-striped (TS) morph, and the TS birds engage in more parenting behavior. This behavioral polymorphism is caused by a chromosomal inversion that has captured many genes, including estrogen receptor alpha (ERα). In this study, we tested the hypothesis that morph differences in aggression might be explained by differential sensitivity to estradiol (E2). We administered E2 non-invasively to non-breeding white-throated sparrows and quantified aggression toward a conspecific 10 min later. E2 administration rapidly increased aggression in WS birds but not TS birds, consistent with our hypothesis that differential sensitivity to E2 may at least partially explain morph differences in aggression. To query the site of E2 action in the brain, we administered E2 and quantified Egr-1 expression in brain regions in which expression of ERα is known to differ between the morphs. E2 treatment decreased Egr-1 immunoreactivity in nucleus taeniae of the amygdala, but this effect did not depend on morph. Overall, our results support a role for differential effects of E2 on aggression in the two morphs, but more research will be needed to determine the neuroanatomical site of action.

Rapid effects of 17ß-estradiol on aggressive behavior in songbirds: Environmental and genetic influences.

Contribution to Special Issue on Fast effects of steroids. 17ß-estradiol (E2) has numerous rapid effects on the brain and behavior. This review focuses on the rapid effects of E2 on aggression, an important social behavior, in songbirds. First, we highlight the contributions of studies on song sparrows, which reveal that seasonal changes in the environment profoundly influence the capacity of E2 to rapidly alter aggressive behavior. E2 administration to male song sparrows increases aggression within 20 min in the non-breeding season, but not in the breeding season. Furthermore, E2 rapidly modulates several phosphoproteins in the song sparrow brain. In particular, E2 rapidly affects pCREB in the medial preoptic nucleus, in the non-breeding season only. Second, we describe studies of the white-throated sparrow, which reveal how a genetic polymorphism may influence the rapid effects of E2 on aggression. In this species, a chromosomal rearrangement that includes ESR1, which encodes estrogen receptor α (ERα), affects ERα expression in the brain and the ability of E2 to rapidly promote aggression. Third, we summarize studies showing that aggressive interactions rapidly affect levels of E2 and other steroids, both in the blood and in specific brain regions, and the emerging potential for steroid profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS). Such studies of songbirds demonstrate the value of an ethologically informed approach, in order to reveal how steroids act rapidly on the brain to alter naturally-occurring behavior.

Estrogen receptor α polymorphism in a species with alternative behavioral phenotypes.

The evolution of behavior relies on changes at the level of the genome; yet the ability to attribute a behavioral change to a specific, naturally occurring genetic change is rare in vertebrates. In the white-throated sparrow (Zonotrichia albicollis), a chromosomal polymorphism (ZAL2/2(m)) is known to segregate with a behavioral phenotype. Individuals with the ZAL2(m) haplotype engage in more territorial aggression and less parental behavior than individuals without it. These behaviors are thought to be mediated by sensitivity to sex steroids, and the chromosomal rearrangement underlying the polymorphism has captured a prime candidate gene: estrogen receptor 1 (ESR1), which encodes estrogen receptor α (ERα). We therefore hypothesized that the behavioral effects of the ZAL2(m) rearrangement are mediated by polymorphism in ESR1. We report here that (i) the ESR1 promoter region contains fixed polymorphisms distinguishing the ZAL2(m) and ZAL2 alleles; (ii); those polymorphisms regulate transcription efficiency in vitro and therefore potentially do the same in vivo (iii); the local expression of ERα in the brain depends strongly on genotype in a free-living population; and (iv) ERα expression in the medial amygdala and medial preoptic area may fully mediate the effects of genotype on territorial aggression and parenting, respectively. Thus, our study provides a rare glimpse of how a chromosomal polymorphism has affected the brain and social behavior in a vertebrate. Our results suggest that in this species, differentiation of ESR1 has played a causal role in the evolution of phenotypes with alternative life-history strategies.

Evaluation of reference genes for quantitative real-time PCR in the brain, pituitary, and gonads of songbirds.

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbirds: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR in songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology.

Hormonal regulation of vasotocin receptor mRNA in a seasonally breeding songbird.

Behaviors associated with breeding are seasonally modulated in a variety of species. These changes in behavior are mediated by sex steroids, levels of which likewise vary with season. The effects of androgens on behaviors associated with breeding may in turn be partly mediated by the nonapeptides vasopressin (VP) and oxytocin (OT) in mammals, and vasotocin (VT) in birds. The effects of testosterone (T) on production of these neuropeptides have been well-studied; however, the regulation of VT receptors by T is not well understood. In this study, we investigated steroid-dependent regulation of VT receptor (VTR) mRNA in a seasonally breeding songbird, the white-throated sparrow (Zonotrichia albicollis). We focused on VTR subtypes that have been most strongly implicated in social behavior: V1a and oxytocin-like receptor (OTR). Using in situ hybridization, we show that T-treatment of non-breeding males altered V1a and OTR mRNA expression in several regions associated with seasonal reproductive behaviors. For example, T-treatment increased V1a mRNA expression in the medial preoptic area, bed nucleus of the stria terminalis, and ventromedial hypothalamus. T-treatment also affected both V1a and OTR mRNA expression in nuclei of the song system; some of these effects depended on the presence or absence of a chromosomal rearrangement that affects singing behavior, plasma T, and VT immunolabeling in this species. Overall, our results strengthen evidence that VT helps mediate the behavioral effects of T in songbirds, and suggest that the chromosomal rearrangement in this species may affect the sensitivity of the VT system to seasonal changes in T.

Training in the implementation of sex and gender research policies: an evaluation of publicly available online courses.

BACKGROUND: Recently implemented research policies requiring the inclusion of females and males have created an urgent need for effective training in how to account for sex, and in some cases gender, in biomedical studies. METHODS: Here, we evaluated three sets of publicly available online training materials on this topic: (1) Integrating Sex & Gender in Health Research from the Canadian Institutes of Health Research (CIHR); (2) Sex as a Biological Variable: A Primer from the United States National Institutes of Health (NIH); and (3) The Sex and Gender Dimension in Biomedical Research, developed as part of "Leading Innovative measures to reach gender Balance in Research Activities" (LIBRA) from the European Commission. We reviewed each course with respect to their coverage of (1) What is required by the policy; (2) Rationale for the policy; (3) Handling of the concepts "sex" and "gender;" (4) Research design and analysis; and (5) Interpreting and reporting data. RESULTS: All three courses discussed the importance of including males and females to better generalize results, discover potential sex differences, and tailor treatments to men and women. The entangled nature of sex and gender, operationalization of sex, and potential downsides of focusing on sex more than other sources of variation were minimally discussed. Notably, all three courses explicitly endorsed invalid analytical approaches that produce bias toward false positive discoveries of difference. CONCLUSIONS: Our analysis suggests a need for revised or new training materials that incorporate four major topics: precise operationalization of sex, potential risks of over-emphasis on sex as a category, recognition of gender and sex as complex and entangled, and rigorous study design and data analysis.

Recently implemented research policies requiring the inclusion of females and males have created an urgent need for effective training in how to account for sex, and in some cases gender, in biomedical studies. We evaluated three publicly available online trainings on this topic: (1) Integrating Sex & Gender in Health Research from the Canadian Institutes of Health Research; (2) Sex as a Biological Variable: A Primer from the United States National Institutes of Health; and (3) The Sex and Gender Dimension in Biomedical Research, developed as part of "Leading Innovative Measures to Reach Gender Balance in Research Activities (LIBRA)" from the European Commission. We reviewed each course with respect to their coverage of (1) What is required by the policy; (2) Rationale for the policy; (3) Handling of the concepts "sex" and "gender;" (4) Research design and analysis; and (5) Interpreting and reporting data. All three discussed the importance of including males and females to better generalize results, discover potential sex differences, and tailor treatments to men and women. The interconnectedness of sex and gender, how to operationalize sex, and potential downsides of focusing on sex more than other sources of variation were minimally discussed. Notably, all three courses explicitly endorsed invalid analytical approaches that lead to incorrect findings of differences. Our analysis suggests a need for revised or new training materials that cover four major topics: precise operationalization of sex, attention to potential risks of over-emphasizing sex, consideration of gender and sex as complex and intertwined, and rigorous study design and data analysis.

Behavioral characterization of a white-throated sparrow homozygous for the ZAL2(m) chromosomal rearrangement.

The white-throated sparrow is rapidly becoming an important model in the genetics of social behavior because of a chromosomal rearrangement that segregates with a behavioral phenotype. Within a population, 50 % of individuals are heterozygous for a rearranged chromosome 2 (ZAL2(m)). These birds sing more and are more aggressive than the other 50 %, who lack the rearrangement. A disassortative mating system, in which heterozygotes almost never interbreed, ensures that ZAL2(m)/2(m) homozygotes are extremely rare. Here, we provide the first systematic characterization of such a homozygote, a hatch-year female. Her plumage was atypical of her age and sex, resembling that of an adult male. She was extremely vocal and aggressive, dominating her opponents in behavioral tests. Her phenotype was thus an exaggerated version of a typical ZAL2/2(m) heterozygote, supporting the hypothesis that alleles inside the ZAL2(m) rearrangement confer high aggression and further emphasizing this species' value as a model of social behavior.

Considering Sex as a Variable at a Research University: Knowledge, Attitudes, and Practices.

Biomedical research has a history of excluding females as research subjects, which threatens rigor, reproducibility, and inclusivity. In 2016, to redress this bias, the U.S. National Institutes of Health (NIH) implemented a policy requiring the consideration of sex as a biological variable (SABV) in all studies involving vertebrate animals, including humans. Unless strongly justified, females and males must be included in all studies and results reported disaggregated by sex. Recent evidence indicates, however, that misunderstandings of the policy and other significant barriers impede its implementation. To shed light on those barriers at our home institution, we conducted a study funded by the Emory University Specialized Center of Research Excellence on Sex Differences (SCORE). In semistructured interviews of Emory principal investigators in the biological sciences, we noted their knowledge of what the policy entails and why it was implemented, their attitudes toward it, and the extent to which it has or has not changed their research practices. Although attitudes toward SABV were generally positive, most researchers face challenges with respect to its implementation. We suggest interventions that can be mounted at the level of home institutions, such as raising awareness of locally available core facilities, to help address these challenges. More training is needed on what the policy asks of researchers, how sex is defined, the nonhormonal ways that sex differences can manifest, and best practices for statistical analysis of sex-based data. Home institutions may also want to explore ways to lessen the stress associated with rollout of SABV policy.