This story is a common refrain I hear when discussing my research on sex differences in the brain. There is no single correct answer when it comes to human behavior. Some researchers would insist that there is nothing parents can do to suppress the innate tendencies of boys...

“We have raised our children in a gender-neutral household since the day they were born, and we never allowed any sort of weapons, not even a water pistol,” a young mother told me emphatically from the microphone in the lecture hall where I’d just given a talk on the differences between male and female brains. “But the other day my seven-year-old son bit his peanut butter and jelly sandwich into the shape of a gun and started shooting his little sister with it!” The audience laughed appreciatively; everyone had a similar story. “What did we do wrong?” she pleaded.

Just how large those differences are, however, is the crux of an ongoing debate in science. And how much a brain’s function can be attributed to biology versus cultural expectations is a challenging question to answer. Confounding the issue is the concept of gender, a purely human construct that can itself influence brain development. Gender refers to both personal and societal perceptions of one’s sex, and embodies all the complexities of cultural expectations, inherent biases, and predetermined norms of behavior, each of which differs for boys and girls and can affect the young brain. Debates are most heated on questions of sex differences in cognitive abilities and emotionality, and for good reason: biological evidence of superior cognitive ability in one sex could have devastating consequences for equality.

To justify this male bias in laboratory experiments, most research­ers maintain that there are no sex differ­ences in brain function outside of the con­text of reproduction.

Studies of laboratory animal models—for which social biases and constructs such as gender are absent—have revealed significant anatomical differences between the brains of males and females that arise in fetal and early postnatal development, as well as a role for hormones, which differ greatly between the sexes, in the functioning of the adult brain. For these reasons, researchers assiduously avoid experimenting with female animals. A recent comparison of the representation of male and female animals in preclinical research found the discipline of neuroscience to be one of the most strongly skewed toward the exclusive study of males, with five times more studies conducted solely with male animals than with females or a mixture of the sexes.1 To justify this male bias in laboratory experiments, most researchers maintain that there are no sex differences in brain function outside of the context of reproduction, and that the so-called masculinization of the male brain occurs only in those areas that govern reproductive behaviors.

But there is now increasing evidence that differences in brain function are prevalent across the sex divide, and that these differences manifest in surprising ways in animal models of both health and disease. (See “Gender bias in neuropsychiatric disorders.”) Many sex differences in adult brain structure and behaviors are the result of in utero organizational effects of gonadal steroid hormones, in particular androgens and their aromatized derivatives, estrogens, both of which are present in substantially higher concentrations in male fetuses due to testicular steroidogenesis. Brain differences between the sexes can also arise from diverse factors, including the expression of genes carried on the sex chromosomes and discrepancies in maternal treatment of male and female progeny. Together, these factors mediate differences in neurogenesis, myelination, synaptic pruning, dendritic branching, axonal growth, apoptosis, and other neuronal parameters.

This is not to say that everything is different. Indeed, much of the brain and its functions are indistinguishable between the two sexes. But when it is different, the question is, how did the differences come about? By what cellular mechanisms did the course of development change in a particular region that differs between males and females?

Early studies have focused on the usual suspects: neurotransmitters, neurotrophins, and transcription factors, for example. But we are now in the midst of a major rethinking of the origins of sex differences in the mammalian brain with a shift in emphasis away from traditional agents and a new understanding of steroid hormone action.

Female by default

© EVAN OTO/SCIENCE SOURCEThe gonads of the developing fetus are the epicenters of sex determination. All other primary and secondary sex characteristics depend on hormones emanating from the testes or ovaries at specific points later in development. By default, the gonadal precursor will differentiate into an ovary; formation of a testis requires a transcription factor coded for by the Sry gene on the Y chromosome. Likewise, the brain will develop as a female brain by default and be directed towards masculinization only if exposed to the steroids produced by the testis.

Developmental masculinization of the brain leads to significant structural differences in the brains of the two sexes. (See illustration.) Some brain regions are larger in males; others are smaller. Collections of cells that constitute nuclei or subnuclei of the brain differ in overall size due to differences in cell number and/or density, as well as in the number of neurons expressing a particular neurotransmitter. The length and branching patterns of dendrites and the frequency of synapses also vary between males and females—in specific ways in specific regions—as does the number of axons that form projections between nuclei and across the cerebral hemispheres. Even nonneuronal cells are masculinized. Astrocytes in parts of the male brain are more “bushy,” with longer and more frequent processes than those in the same regions of the female brain. And microglia, modified macrophages that serve as the brain’s innate immune system, are more activated in parts of the male brain and contribute to the changes seen in the neurons.

Steroid hormones induce such changes by binding to transcription factors that then translocate to the cell nucleus to initiate gene transcription. For example, estradiol binds to its receptor to induce expression of the gene for cyclooxygenase, which mediates the rate-limiting step in the production of a short-lived signaling molecule called prostaglandin E2 (PGE2). A little more than 10 years ago, my colleagues and I made the surprising discovery that PGE2 is both necessary and sufficient for the fetal masculinization of the preoptic area, a brain region that is essential for sexual behavior in male mice.2 In males, levels of PGE2 are upregulated selectively in this brain region by estradiol-induced synthesis of the cyclooxygenase enzyme. PGE2 then initiates a signal transduction cascade that leads to activation of AMPA glutamate receptors and the formation and stabilization of synapses on the dendrites of neurons in this brain region. As a result, male mice have twice the density of excitatory synapses in the preoptic area as females, and this positively correlates with expression of male copulatory behavior in adulthood.3

We subsequently discovered that microglia, which have recently begun to be appreciated for their role in sculpting neuronal circuits,6 are the predominant source of PGE2.4 Not only are there more of these innate immune cells in young male brains, their morphology reflects a more activated state, and they produce more PGE2 than do the microglia in female brains. Pharmacological treatments given early in development to shift microglia away from an activated state resulted in lower PGE2 production and prevented masculinization induced by estradiol.5 Thus, a nonneuronal cell, microglia, and an inflammatory mediator, PGE2, are essential for the normal masculinization of the preoptic area in mice.

Another region of the brain that is masculinized during development is the amygdala, which in addition to its roles in the processing of emotions is a key region regulating social play behavior by juveniles, sometimes called rough-and-tumble play, which differs markedly in males and females across a wide range of species. The dimorphism in the frequency and intensity of play is particularly interesting in that it is expressed during a time of life when there are minimal to no circulating steroids, and thus any differences in males and females are either genetic or the result of earlier organizational effects of steroids on the brain.7 Sex differences in the synaptic patterning of the amygdala are not as readily apparent as in the preoptic area, but there is a notable difference in cell genesis during the neonatal sensitive period—at least the first four days of life in mice and up to a week in rats—with the amygdala of females making more new neurons and astrocytes than the same region in males.8

This particular sex difference appears to be mediated by endocannabinoids, natural ligands for the receptors that are activated by the psychoactive components of marijuana. Specifically, higher endocannabinoid levels in the male amygdala act to suppress cell genesis. Increasing endocannabinoid levels or administering endocannabinoid mimetics to females during the first week of life reduces the level of cell genesis in their amygdalas to that of males. And, quite interestingly, this correlates with an increase in rough-and-tumble play by these females as juveniles.

Although it is unknown how endocannabinoids reduce cell genesis in the amygdala, emerging evidence suggests the resident microglia of this brain region may be critical mediators of cell number, just as they are elsewhere in the brain. Microglia can regulate cell number in two ways: by phagocytosing dead or dying cells, or by engulfing and actually killing live cells, a process recently termed phagoptosis.9 Appropriate control of cell number is critical to a healthy brain. If dying cells are not efficiently removed, toxic cell contents are spilled into the extracellular space, leading to additional cell death. Conversely, if cells proliferate excessively, the ability to form and maintain organized connections is lost. Microglia are essential guardians of both these processes, and ongoing work suggests that this is likely also true in the control of sex differences in cell number in specific subnuclei.

Epigenetics and the brain

The hormonally mediated masculinization of the brain is referred to as an “organizational” event in recognition of its relative permanency, but how this state endures has been unknown. In the preoptic area, an area closely associated with the hypothalamus and which controls male sexual behavior, we find consistent sex differences in synaptic density across rodent life stages. Males have about twice as many synapses for a given length of a neuronal dendrite as females have, and this is true in newborn rats, adolescents, and adults.3 Something is maintaining the spacing of the synapses.

One likely suspect is epigenetic modifications to the genome, which we now know can store such cellular memory. By interfering with DNA methyltransferases (DNMTs) to cause widespread demethylation of the genome, my group found evidence of greater DNMT activity in female rats that correlated perfectly with an increase in DNA methylation for the brain region controlling masculinization of sexual behavior.10 Inhibiting DNMTs in females during the first week of life resulted in rats that were more male-like in both brain structure and behavior, presumably as a consequence of reduced DNA methylation and increased expression of a suite of genes critical for masculinization. Surprisingly, if we treated females with a DNMT inhibitor outside of the sensitive period, they were still masculinized, suggesting that DNA methylation is critical to the maintenance of feminization by actively repressing masculinization genes. The same was found to be true for mice in which the enzyme DNMT3a was genetically deleted in the preoptic area. Identification of what genes are emancipated by the loss of methylation is ongoing, but early analysis implicates genes associated with microglia and with mast cells, another component of the innate immune system of the brain.

Changes in the epigenome are a component of sexual differentiation of the brain, but we are only beginning

to crack this complex code.

The role of DNA methylation in brain sex differences is not cut-and-dried, however. The canonical view is that epigenetic marks are established early and then endure. But studies have found there can also be a delayed epigenetic response to early hormonal treatment, a sort of epigenetic echo. For example, geneticist Eric Vilain of the University of California, Los Angeles, and colleagues observed many more sex differences in DNA methylation in adult mice than in newborns, both in the striatum and the preoptic area, and that treatment of newborn female mice with testosterone shifted their DNA methylation profile to that of males, but not until they were adults.11 In a similar study, researchers at the University of Maryland in Baltimore found sex differences in methylation of the promoter regions of the estrogen and progesterone receptors in the hippocampus, preoptic area, and hypothalamus, but the pattern of methylation changed over the course of the animals’ lives, from neonate to adolescent to adult.12 There is clearly an organizational effect of hormones in the brain, but the appearance of those effects in the epigenome is not tied closely to the time of exposure. How this is occurring at the cellular level is currently a mystery.

Histone modifications also appear to be important in the differentiation of male and female brains. One particular histone modification, called H3K4me3, clusters at transcription start sites and is generally, but not exclusively, associated with increased gene expression. A genome-wide analysis in the murine preoptic area found some 250 genes with a sex difference in the amount of associated H3K4me3, more than 70 percent of which were higher in females. Many of these genes were involved in synaptic transmission, neuronal growth, and differentiation.14

Not surprisingly, there are also sex differences in the levels of histone deacetylases (HDACs), which mediate such epigenetic marks. There are higher levels of HDACs in the preoptic area of neonatal male mice, and these enzymes tend to be associated with the promoter regions of the estrogen receptor and the aromatase enzyme, which makes estradiol. Deacetylation is associated with decreased gene expression, and both the estrogen receptor and aromatase are more highly expressed in males prenatally, but decline after birth when testosterone levels drop and masculinization is finalized. Blocking HDAC activity during the first week of life impairs male sexual performance in adulthood, confirming the importance of deacetylation for normal masculinization.13

Thus, just as with DNA methylation, changes in the epigenome of the histones are a component of sexual differentiation of the brain, but we are only beginning to crack this complex code.

The mosaic brain

© ISTOCK.COM/RUDALL30/MARINAZAKHAROVASo to what extent do these brain sex differences identified in rodents also exist in humans? While we can’t experiment on humans for obvious reasons, we can rely on “natural experiments” in which a hormonal profile or sensitivity has been altered due to genetic anomalies. Two well-studied examples are congenital adrenal hyperplasia (CAH), in which the adrenal glands produce excessive androgens during fetal development, and complete androgen insensitivity syndrome (CAIS), in which a mutation in the androgen receptor makes it incapable of binding testosterone and other androgens. In both cases, gonadal development occurs according to the chromosomally dictated sex—i.e., XX embryos will develop ovaries and XY embryos, testes—but the secondary sex characteristics often align with the opposite sex. CAH girls are born with masculinized genitalia, for example, due to their in utero androgen exposure, while CAIS boys appear as normal girls when born due to the lack of differentiation of the external male sex organs.

These conditions provide the opportunity to ask whether brain sex matches gonadal sex. In the case of CAIS, the answer is emphatically no, as these XY individuals consistently identify as females. This finding is in line with the notion that early life exposure to androgens is necessary for development of a male identity. For the CAH girls, the shift in hormonal profile is not as dramatic as that for CAIS individuals, and thus the changes in brain and behavior are also less dramatic. Still, there is typically evidence for a degree of “masculinization” of their brains when assessed for behavioral traits such as toy choice. Thus, despite some differences between humans and animal models, the preponderance of evidence supports the notion that humans undergo a hormonally mediated process of sexual differentiation of the brain just like animals.

The brain is a mix of relative degrees of masculinization in some areas and feminization in others.

However, while both the popular and scientific presses make reference to “male” and “female” brains, the brain is in reality not a unitary organ like the liver or the kidney. It is a compilation of multiple independent yet interacting groups of cells that are subject to both external and internal factors. This is abundantly true for hormonal modulation, with many and varied signal transduction pathways invoked. As a result, it is quite literally impossible for the brain to take on a uniform “maleness” or “femaleness.” Instead, the brain is a mix of relative degrees of masculinization in some areas and feminization in others. On average, there are likely to be some areas that are more strongly feminized in a female and others that are more strongly masculinized in a male, but averages are never predictive of an individual’s profile. Moreover, a mosaic is not a blend—there is not a continuum of maleness to femaleness—and there are many parameters that are neutral in regard to sex, with no consistent differences between males and females.

Evolutionarily, the creation of a maleness-femaleness mosaic within one brain makes sense, providing organisms with greater variability and therefore adaptability to changing environments. But another striking aspect of brain sexual differentiation that my colleagues and I have noted is that for each endpoint we examine, we find the magnitude of the sex difference to be constrained within the relatively low range of just one- to twofold. While this is still significantly greater than the extremely small variance within each sex, it is by no means colossal, as one might describe the difference between a male peacock’s tail and that of a peahen. It is as if something is both pushing the brains of the sexes apart and keeping them together at the same time.

This interpretation is consistent with the concept of canalization, originally proposed by British biologist Conrad Waddington in the late 1940s and now embraced by evolutionary biologists as a means by which species maintain robustness in the face of ever-present internal and external challenges. Chaperone proteins and other agents act to buffer an organism against changes in pH or salinity and other environmental threats by assisting in proper protein folding or maintaining order in intracellular traffic, for example. We propose that, during embryonic development or during the first week of life, many sexually differentiated endpoints are subject to canalization, assuring that males will stay in one canal and females in another, and that the two canals will never merge or grow too far apart.

In humans, an additional canalization factor could be parental, societal, and cultural influences early in life. Gender-specific behaviors may be rewarded, for example, or punished if considered not in line with a child’s sex. While these factors remain difficult to tease apart, it is clear that the brains of males and females diverge as they develop, and it should be self-evident that using only male animals to probe mammalian brain function does not reveal the whole picture.

© ISTOCK.COM/FRIKOTA GENDER BIAS IN NEUROPSYCHIATRIC DISORDERS Some neuropsychiatric disorders are thought to originate during fetal development, even if patients are not typically diagnosed until adolescence or young adulthood. Of these, most are much more common in males. Other disorders begin to manifest at puberty or later in life, and these occur more frequently in females. The biological reasons for these sex biases in disease prevalence are currently under investigation. Major depressive disorder: One of the most common neuropsychiatric disorders, MDD is considered strongly gender biased, with women twice as likely as men to be diagnosed. This bias is seen worldwide, suggesting a biological as opposed to cultural origin. Dysregulation of the stress axis and its convergence with the dynamic nature of reproductive hormones in women are implicated as root causes of greater risk in women, although more recent evidence suggests this dysregulation may have its origins in very early childhood. However, the importance of other variables contributing to the gender bias, such as the willingness of women to seek help while men tend to self-medicate with drugs and alcohol, cannot be discounted. Anorexia nervosa: Strictly postpubertal in onset, anorexia nervosa is predominantly a young woman’s disease, with a gender bias greater than 10:1 that is almost assuredly driven by perceived societal pressures. Interestingly, bulimia nervosa, a disorder of binge eating but in which normal body weight is maintained, is much less gender biased, with women only three times as likely as men to suffer the disorder. Autism spectrum disorder: While ASD was originally considered only twice as prevalent in boys, recent estimates put the ratio closer to 5:1. A currently popular but unproven theory postulates that elevated testosterone in utero leads to ASD-like behaviors by placing boys on the extreme end of the male spectrum. A counter-theory is that girls are underdiagnosed for ASD due to physician bias and a different presentation, with fewer social and cognitive defects. Others argue that girls are more resilient and require a greater load of genetic insult before the disorder manifests, and empirical evidence supports this view for those limited instances in which a genetic origin of ASD is clear. Attention deficit hyperactivity disorder: Reports of the degree to which ADHD occurs more frequently in boys than girls vary widely and are likely influenced as much by cultural factors as biological ones. Additionally, males tend to show greater impairments, making them at least four times more likely to be diagnosed. Schizophrenia: When considered for the population overall, there is no clear gender bias in the frequency of schizophrenia. However, diagnosis is much more common in boys and young men than in girls, whereas diagnosis in middle age or older is substantially more frequent in women. Differential responses to stress, with distinct brain regions being over- or underactivated in men versus women, further contribute to divergence in the disease. Biplolar disorder: Rates of bipolar disorder do not vary between men and women, yet a genetic polymorphism strongly associated with the disorder is relevant to risk in women but not men. This highlights how much we have to learn about the nature of sex differences in neuropsychiatric disorders and the multiple ways in which some differences can manifest.

Margaret M. McCarthy is chair of the Department of Pharmacology and a member of the Program in Neuroscience at the University of Maryland School of Medicine in Baltimore.

References

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Correction (October 7): This story has been updated to correctly reflect that deacetylation is associated with decreased, not increased, gene expression. The Scientist regrets the error.