C H A P T E R
11
Sex Differences in the Brain: Focus
on Developmental Mechanisms
Margaret M. McCarthy
University of Maryland School of Medicine, Baltimore, MD, United States
O U T L I N E
11.1 Introduction
129
11.2 Historical Perspective and Current Status
130
11.3 Conceptualizing Sex Differences
131
11.4 Steroid Hormones Program the Developing
Brain
11.4.1 Female Development is the Default
11.4.2 Critical Periods for Sexual Differentiation
of the Brain
11.5 Mechanisms of Steroid-Induced
Masculinization
11.5.1 Steroids and Steroid Receptors
11.5.2 Sex Differences in the Brain are
Programmed by Steroids
11.5.3 Cell Death is Developmentally Regulated
by Steroids in Brain Regions Controlling
Reproduction
11.5.4 Cell Birth is also Developmentally
Regulated but in Brain Regions not
Directly Associated with Reproduction
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11.6 Elucidating Mechanisms of Normal Brain
Development in Males and Females Provides
Clues to Sources of Vulnerability and
Resilience
145
References
145
137
11.1 INTRODUCTION
Males and females have different physiologies and
they behave differently, that is an absolute and raises
little concern when the subjects under discussion are
animals. But when it comes to translating that absolute
to humans there is both emotional and scientifically rigorous debate.1,2 And that is as it should be. If health
and policy decisions are to be made based on sex as a
Principles of Gender-Specific Medicine.
DOI: http://dx.doi.org/10.1016/B978-0-12-803506-1.00033-4
11.5.5 Nonneuronal Cells can be Key
Contributors to Sexual Differentiation
11.5.6 Steroids Induce Epigenetic Changes to
the DNA to Induce and Maintain Sex
Differences
11.5.7 Steroids can also Act Rapidly to Induce
Enduring Change in Neural Circuits
11.5.8 Steroids Indirectly Modulate Neuronal
Excitability in the Developing Brain
11.5.9 Sex Chromosome Complement also Matters
to Brain Development
variable, the science should be substantial in amount,
broad in scope, and high in caliber. When the question is
sex differences in the brain, that goal cannot be achieved
without a strong foundational base of research in
animal models. But can rats and mice or even nonhuman
primates really inform us as to the myriad of ways that
boys and girls and men and women differ? Probably
not. Humans are singular in their use of complex language and computational skills and our social societies
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© 2017 Elsevier Inc. All rights reserved.
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11. MECHANISMS ESTABLISHING SEX DIFFERENCES IN BRAIN
and attendant cultural norms are far more variable and
complicated than even the most sophisticated animal
groups.
Humans are also singular for possessing gender,
defined as a combination of self and societal perception of an individual’s sex. We can’t ask animals if they
know what sex they are and how they feel about it, so
this parameter remains unique to humans. The power of
gender to impact the developing brain begins at the earliest stages of life, with parents speaking to and physically interacting with newborns in a sex-biased way. The
influence only grows from there as children are both
actively and passively driven to conform to gender
norms.3 Thus it is impossible to truly separate out the
impact of environment and experience from biology in
sculpting the brains of humans in ways that may differ
in males and females.
Given the enormity of the gap between humans and
other species, some say there is little to no value in the
study of animal models. But this denies the value of
understanding mechanism, i.e., understanding the precise way in which molecular, biochemical, and cellular
processes are impacted by the biological variable of sex.
These discoveries can only be made when exploiting the
experimental power of animal models, in particular the
many advantages offered by rats and mice.
Initially the dose of androgen was sufficiently high that
the females had male-like genitalia and so their malelike behavior could be either brain- or body-derived. But
subsequent experiments lowered the dose sufficiently
that the females were feminized in appearance but still
behaved as males, leading to the speculation that it was
indeed the brain that regulated their male-like behavior.
However, there was one important caveat: the females
required male-like hormone levels in order to behave as
males in adulthood. Moreover, they were immune to the
impact of female hormones and could not be induced to
exhibit female sexual receptivity. This was subsequently
codified as the Organizational/Activational Hypothesis
meaning that early developmental hormone effects
organize the brain which then must be activated by the
appropriate hormonal profile in adulthood (Fig. 11.1).
Shortly after the studies of Phoenix and colleagues, a
separate group of researchers at UCLA were making the
parallel discovery that developing females exposed to
androgens were either frankly sterile or became progressively less fertile as adulthood progressed.6 Again the
initial source was not thought to be the brain but instead
a direct effect of hormones on the developing ovary or
possibly the pituitary. Both target organs were eventually
rejected as the origins of the hormone-induced fertility
and the brain identified as the culprit via its regulation
11.2 HISTORICAL PERSPECTIVE
AND CURRENT STATUS
Attribution of the first recorded endocrinology experiment is given to Arnold Berthold in 1849 for his observations of the impact of removing the testis in roosters
which not only changed their physical characteristics,
but also impacted their behavior. That the gonads were
the source of a masculinizing substance was known
long before then, dating to at least Aristotle. Yet it
wasn’t entirely clear that the hormones of the gonads
were the driving force of sex differences in behavior.
Instead, one of the grandfathers of the field of neuroendocrinology, Frank Beach, postulated that it was the
hormonal effects on the body that mattered and then
the body determined behavior. Put simply, if an individual possessed the genitals and body type of a male
they would attempt to mate with females and behave
aggressively toward other males. The brain was considered secondary and essentially in service to the demands
of the body phenotype (see for review Ref. 4). This is
not an entirely unreasonable view. It is also a testable
hypothesis, a task performed in the now iconic 1959
publication of Phoenix, Goy, Gerall, and Young,5 contemporaries of Beach. These scientists treated pregnant
Guinea pigs with high doses of androgens and then
observed the behavior of the female offspring as adults.
FIGURE 11.1
Organizational/activational hypothesis. Sex chromosome complement directs differentiation of the bipotential gonad.
The Sry gene of the Y chromosome codes for a testis determining factor. In the absence of Sry the gonad will become an ovary. The fetal
and neonatal testis produces high levels of androgens, which serve as
precursors to estrogens and together these steroid hormones direct the
parallel but distinct processes of brain masculinization and defeminization. As a result, when testicular steroids again rise in adulthood
male-typic behaviors are activated. In females the developing ovary
remains quiescent and the brain undergoes the default process of feminization. In adulthood a feminized brain will respond to the cyclical
production of ovarian hormones with changes in sexual receptivity
and fertility.
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
11.3 Conceptualizing Sex Differences
of LH secretion from the pituitary.7 Specifically it is the
GnRH neurons of the diencephalon which control LH
release. In males there is relatively continuous pulsatile release of LH, resulting in relatively continuous
steroidogenesis and sperm production by the testis.
In females, however, the pattern of LH release varies
across the reproductive cycle with the culmination being
a massive surge of release that induces ovulation by the
ovary. In females exposed to high levels of androgen
developmentally, the LH surge doesn’t happen and so
instead the ovary develops large preovulatory follicles
that remain essentially stuck, never releasing their ova,
resulting in infertility.
The discoveries that adult physiology and behavior
were programmed developmentally and differently in
males and females were major advances in our understanding of reproduction and spawned the subdisciplines
of behavioral endocrinology and neuroendocrinology.
But in essence the impact stopped there, with the topic
of sex differences being relegated to a boutique area
focused almost entirely on reproduction. The nascent
field of neuroscience, which dates back only to the early
1970s, did not embrace the notion of either the value or
relevance of sex differences in the brain, further walling off the topic. Ironically, the isolation became worse
with the path breaking work of the Bruce McEwen laboratory in the 1990s demonstrating a nonreproductive
endpoint that was profoundly influenced by hormonal
changes across the estrus cycle. They reported that the
synaptic profile of hippocampal neurons, a brain region
central to learning and memory, shifted up to 30% in
females at different stages of reproductive readiness.8
First met with incredulity but eventually accepted in
the face of overwhelming data, this finding had the perhaps unexpected effect of further marginalizing sex differences research by alarming researchers not studying
reproduction. The reasoning went that including female
subjects in studies of fundamental aspects of nervous
system function would only introduce variability and
noise, and therefore it was best to use exclusively male
rats and mice, gradually leading to the elimination of
female subjects from preclinical research that was not
directly relevant to reproduction.9 Ironically, in 1993 an
act of the US congress, the National Institutes of Health
Revitalization Act, mandated equal representation of
women, children, and minorities in preclinical research
and was thus at the same time having the opposite effect,
significantly increasing the representation of underrepresented groups in clinical trials.10
Today we are in the midst of a paradigm shift that has
forced a reckoning in our views of the brain and neuroscience research. This can be attributed to a convergence
of the following factors. First, what was long known
to be true, that most scientists only study males, was
established to be true with actual data.9,11 Second, failed
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clinical trials based on preclinical data overwhelmingly
were shut down due to adverse events in women.12
Third, the gender bias in neuropsychiatric disorders
across the life span and greater vulnerability of males to
neurological disorders and birth injuries demanded that
attention be paid to the binary variable of sex.1 Lastly,
accumulating evidence of novel mechanisms by which
sex differences are established highlighted how little
we know about the fundamental rules governing brain
development in both males and females and illuminated
the potential benefits of learning such rules for all.
11.3 CONCEPTUALIZING SEX
DIFFERENCES
As the conversation about sex differences in the brain
has grown more sophisticated there have been increased
attempts to establish exactly what is meant when an
endpoint is deemed “different” in males versus females.
The term sexually dimorphic is evoked frequently and
inappropriately. Widespread generalizations across species and endpoints further obscure the picture. Getting
the semantics right is essential as it is important to know
when a sex difference should be attended to, when it
should be acknowledged but not necessarily incorporated into any conclusions and when it should just be
ignored. It is equally important to know when there is
no sex difference in an endpoint and this takes equal
rigor to that of establishing that a sex difference exists.13
A primary source of confusion as well as rancor in
the discussion of sex differences is the bundling together
of endpoints that should not be considered in the same
framework. The major dividing line is those endpoints
that are physical markers, meaning neuroanatomy and
neurophysiology, and those that are the external manifestation of neural functioning, meaning behavior and
physiology (Table 11.1). The two sets of endpoints are
connected, and indeed the goal of studying neuroanatomy and neurophysiology is to understand the control
of physiology and behavior, but the connection is often
much looser than we care to acknowledge.
Both behavior and physiology are outputs from multifactorial inputs that must be integrated and weighted
before a response is made. For instance, all of the neural
circuits for mating behavior may be fully activated but
if a predator is nearby the circuits controlling freezing
will override those for mating. Likewise for foraging
and feeding, a predator will certainly trump the urge to
forage, but an attractive and available mating partner
will trump the urge to forage. There is a hierarchy of
control that assures survival in the short term but also
motivates for successful reproduction in the long term.
When we assess the neural circuits controlling these
complex behaviors and find a sex difference, which is
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
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11. MECHANISMS ESTABLISHING SEX DIFFERENCES IN BRAIN
TABLE 11.1 Categories of Sex Differences Quantified in Animal Models
Neuroanatomy
Neural function
Physiology
Behavior
Cell number and density (neurons,
astrocytes and microglia)
Firing threshold and rate
Stress axis
activation—HPAA
Courtship, mating, and parenting
Cell size and shape
Neurotransmitter synthesis and
release
Reproductive axis
activation—HPGA
Foraging and feeding
Synaptic patterning
Neurotransmitter or modulator
receptor number
Metabolism
Learning and memory
Connectivity
Signal transduction coupling
Habit formation (i.e., addiction)
Myelination
Membrane trafficking
Aggression (male:male and maternal)
Neurochemical phenotype
Steroid and steroid receptor levels
Anxiety and emotionality
Peptide synthesis and binding
Audible communication (song in birds
or ultrasonic vocalizations in rodents)
Trophic factor synthesis and binding
Chemical communication
Sexual preference
well established for the circuit controlling mating behavior, the relative importance of that difference only matters in the context of when the behavior is expressed, it
provides a valence for external stimuli to be integrated
and a calculation made as to whether the behavior is
executed or not. This is perhaps best illustrated with
mating behavior in which a male’s motivation to mate
will override the motivation to eat relatively easily. But
females must maintain a critical body mass of fat in order
to reproduce and so the motivation to mate is properly
tempered against that set point. A sex difference in the
valences of the various nodes in both the mating and
feeding circuits presumably underlies the sex difference
in behavior, but precisely how this is achieved is beyond
our current abilities in neuroscience to assess. We also
know the neural circuits for mating are fully differentiated neonatally but the behavior will not be expressed
until adulthood. Thus the neuroanatomical changes
endure across the life span but the behavioral changes
are both transient and context dependent (Fig. 11.2).
11.4 STEROID HORMONES PROGRAM
THE DEVELOPING BRAIN
As briefly reviewed above, steroids from the gonads
are the determining factor for many sex differences in
brain and behavior. To recap, in males the fetal testis
produces high levels of androgens that will masculinize the brain so that in adulthood the animal responds
to testosterone with execution of male-typic behavior and male physiology. Conversely, females are not
exposed to high levels of androgens developmentally
and in adulthood respond to ovarian hormones with
female-typic behavior and physiology. The processes of
masculinization and feminization are separate but not
exclusive as evidenced by a third process of defeminization which occurs in males and removes the capacity for
the male to respond to female hormones as an adult (see
for review Ref. 14).
11.4.1 Female Development is the Default
It is important to examine what this statement actually means. That female development is the default does
not mean its not active, it is very much an active process,
it just means that it is the developmental program that
will proceed in the absence of an additional stimulus.
In the case of the gonad, which begins as undefined,
the additional stimulus is expression of the Sry gene
located on the Y chromosome and which codes for a
testis differentiating factor.15 This factor is now known
to be a transcription factor that suppresses expression of
aromatase and promotes expression of Sox-9, initiating
a cascade of cell signaling pathways that execute the
complex process of testis formation. If Sry is mutated
or missing, the developmental progression of the undifferentiated gonad will proceed towards an ovary. And if
Sry is accidentally or purposely translocated to the X or
an autosome, testis differentiation will proceed.
Similar to the undifferentiated gonad, the brain also
proceeds by default as female, but in this case the differentiation stimulus originates in the gonads with the
surge of androgen production prenatally. In this way
nature assures that gonadal sex and brain sex are concordant, an essential feature for successful reproduction.
Experimentally the default differentiation of the female
provides the advantage that treatment with exogenous
androgens can initiate the process of masculinization,
thereby providing an experimental tool for interrogating
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
11.4 Steroid Hormones Program the Developing Brain
133
FIGURE 11.2
Sex differences in context. When behavioral or neuroanatomical endpoints are found to differ in males and females it is important to interpret them in the appropriate context. Neuroanatomical substrates that are organized early in development may be enduring but the
behaviors they control are only expressed in response to the appropriate hormonal milieu or circumstances. Neuroanatomically differentiated
substrates sometimes converge back to baseline during the juvenile hiatus and then reappear in adulthood. Alternatively, sometimes males and
females have similar endpoints under basal conditions but diverge in response to nutritional or psychological challenges. Conversely, males
and females may converge from different endpoints in order to compensate for unanticipated costs of the distinct reproductive costs inherent
to each sex.
the cellular mechanisms. On the other hand, that feminization is the default also makes it very difficult to dissect, other than as a process that is not masculinization.
The study of defeminization provides some insight, but
not all sexually differentiated endpoints include defeminization as a component, limiting the generalizability.
11.4.2 Critical Periods for Sexual
Differentiation of the Brain
Critical periods are defined as opportunistic windows
during which essential developmental milestones must
be met or are forever foreclosed. One of the best characterized critical periods is that for development of the
visual system. Spontaneous waves of excitation from
the retina to the brain prior to eye opening (in cats and
rodents) initiates a critical period for synaptic innervation which is then refined by both intrinsic and extrinsic
stimuli. If one eye is delayed from opening during a
defined period of development, the visual field of the
contralateral cortex will be invaded by the ipsilateral
projections of the open eye and the first eye will be forever “blinded” even if perfectly functional. Even more
dramatically, if animals, in this case kittens, are reared in
an environment in which there are no vertical lines, only
horizontal, they will be forever blind to horizontal lines.
This critical period involves a matching of the external
environment to the construction of an internal neural
circuit and is based on a use-it-or-lose-it principle. In
other words there is an initial overly exuberant innervation of the brain by the optic nerve and then this is
pruned back and refined in response to specific visual
stimuli (see for review Ref. 16).
For sexual differentiation of the brain the initial trigger is the endogenous production of androgens by the
male fetal testis, which begins in late gestation in rodents
and at the beginning of the second trimester in humans.
What initiates the sudden and dramatic upswing in fetal
testis androgen production wasn’t known until a recent
study which discovered a small set of kisspeptin neurons which appear only transiently and only in males to
stimulate fetal GnRH neurons which in turn stimulate
fetal pituitary LH release and the induction of steroidogenesis by the testis.17 Kisspeptin neurons were already
known for their essential role in puberty onset, again
via the regulation of GnRH neurons to establish the LH
pulse generator in males, and ultimately the capacity for
an LH surge in females (see for review Ref. 18).
In humans fetal steroid levels are high mid-gestation
but begin to drop by term in response to negative feedback
from the high steroid levels associated with pregnancy.
At birth this feedback inhibition is lost and the newborn
hypothalamic-pituitary-gonadal axis rebounds with prodigious gonadal steroidogenesis. Levels of androgens in
infant males reach that of puberty, leading to the term
minipuberty. This period of elevated steroids contributes
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11. MECHANISMS ESTABLISHING SEX DIFFERENCES IN BRAIN
to masculinization of the genitalia, spermagenesis, and
somatic features. A contribution to masculinization of
the brain is speculated but not clearly established (see
for review Ref. 19). To date there is no clear role for minipuberty in feminization. In contrast to the transition to a
new state of reproductive readiness that follows puberty,
the induction of steroidogenesis by the fetal testis is temporary. In humans levels drop by 1–3 months and are
almost absent by 6 months. In rodents levels drop within
hours of birth. In both cases precisely how this occurs is
unknown but is likely due to maturation of the HPG axis
and establishment of central inhibition.
Given the tightly constrained period of steroidogenesis,
it is at first reasonable to assume that the decline in steroid
levels is also the end of the critical period, but studies in
rodents demonstrate this is not the case. Because females
are not exposed to high levels of steroid they remain
sensitive to exogenous treatment with a masculinizing
dose. In rodents this sensitivity endures into the postnatal
period but wanes after about the first week, with some
variation depending on the endpoint. In humans it is
often stated that the end of the critical period is prenatal,
but as noted above there is some evidence for an impact
of the minipuberty that occurs in the first few months of
life. There also are no experiments administering male
levels of hormone to females, for obvious reasons, and
so we don’t really know when the critical period ends in
humans. Also unknown has been the mechanism of how
the sensitive period ends, but recent studies suggest an
essential role for epigenetics (see below Section 11.5.6).
11.5 MECHANISMS OF STEROIDINDUCED MASCULINIZATION
Identifying the mechanisms by which steroids exert
an enduring effect on the developing brain requires first
determining which steroid, and second what receptor
the steroid is acting through. This might seem straightforward given that the testis produces testosterone and
there is only one androgen receptor (AR). But studies in
rodents have again demonstrated that the answer is not
simple at all.
11.5.1 Steroids and Steroid Receptors
Shortly after the establishment of the Organizational/
Activational hypothesis the animal model of choice
switched from the Guinea pig to the laboratory rat for the
simple reasons of larger litter size and shorter gestation.
Fortuitously the critical period for masculinization also
extends postnatally in the rat—not so true in the Guinea
Pig. In the course of these early studies using postnatal
androgen treatment of females, estradiol was included
as a presumptive control steroid as it was considered a
“female” hormone. But to the researchers’ surprise estradiol was not only capable of masculinizing the female
brain; it was even more potent than testosterone. The
discovery that the brain has high levels of the enzyme
aromatase, which converts androgens into estrogens,
was a key puzzle piece for solving the mystery and
is codified as the Aromatization Hypothesis.20 But this
raised another question, given the high levels of circulating estrogens in the bloodstream of the dam as well as
the amniotic fluid, how do female pups avoid becoming
masculinized? This mystery was solved with the discovery that alpha-fetoprotein, a steroid binding globulin
with a high affinity for estradiol, is found at high levels
in the fetal circulation during the critical period. Both
male and female fetuses make alpha-fetoprotein and
so both sequester the maternal estrogens in the bloodstream, depriving the steroid access to the brain. But the
circulating testosterone in the fetal male has unfettered
access to the brain, easily crossing the blood–brain barrier and then converted into estrogens once inside neurons. Thus only males experience elevated estrogens in
the brain during the critical period. If alpha-fetoprotein
is deleted, say by a genetic null mutation, then indeed
all of the female offspring are masculinized.21
Estrogens, a class of compounds that includes estradiol, estriol, and estrone, bind to and activate multiple
receptors. Two of these are canonical nuclear transcription factor receptors, ER-alpha and ER-beta, coded for
by the Esr1 and Esr2 genes, respectively. Most of the
signature endpoints subject to sexual differentiation
(i.e., sexual and aggressive behavior, LH release) are
mediated by ER-alpha but ER-beta has been specifically
implicated in the defeminization process,22 although this
process itself remains poorly understood. There are also
rapidly mediated membrane effects of estrogens either
through separate G-protein coupled receptors or the
classic nuclear receptors. Even the ability to respond to
estrogen with rapid effects is subject to sexual differentiation in the hippocampus.23
In primates the hormonal control of masculinization is slightly different. First, androgens rather than
estrogens are the dominant masculinizing hormones.24
While there is ample aromatase in the brain of primates,
including humans,25 all the evidence points to the AR
and androgen levels as the principal directors of the
masculinization process. This comes in part from experiments in nonhuman primates, but also from so-called
naturally occurring experiments in humans. Most compelling are women with complete androgen insensitivity
due to a mutated AR. These women are XY but phenotypically female with the exception of undescended
testis, no uterus and a shortened vagina. They are also
psychosexually differentiated as women with little to
no evidence of gender identity dysphoria or homosexuality (reviewed in Ref. 26). This doesn’t mean there
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
11.5 Mechanisms of Steroid-Induced Masculinization
is no role for estrogens in primate brain masculinization, and indeed there is some indirect evidence for that
coming again from “natural experiments,” as in the
case in men with mutated ER or aromatase genes. But
these individuals are exceedingly rare in comparison to
androgen-insensitive women and so the role of estrogen
in masculinization of primate, including human, brain
remains an open question.
Because androgens are predominant in primates this
causes some to question the utility of research on rodents
for understanding the human condition in regards to
sex differences. But steroids as a class are a different
type of signaling molecule than most others in the brain.
Steroids themselves do not alter neuronal activity or
directly impact cell proliferation, survival, or differentiation. Instead steroids induce or repress other signaling
molecules such as growth factors, receptors, neurochemicals, etc., and these are the agents that actually mediate
change. So, the critical question is what are these signaling molecules and are they common to the process
of masculinization across species but induced by a different steroid? i.e., estrogens in rodents and androgens
in primates. Until we know those signaling molecules
this question cannot be answered, but major advances
on this front of late suggests that progress is beginning
to be made.
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11.5.2 Sex Differences in the Brain
are Programmed by Steroids
The proper formation of the brain involves a complex
interplay of temporally orchestrated interdependent
events. Cell proliferation, migration, differentiation, and
integration are all regulated steps, as is neurochemical
phenotype determination, dendritic and axonal growth
and branching, and synaptogenesis. Cell death and synaptic pruning are essential for final maturation of neural
circuits. Layered on these neuronal responses are the
equally important and developmentally regulated astrocytes, oligodendrocytes, and the brain’s innate immune
system cells, the microglia. Steroid hormones regulate
every one of these processes and cell types at some point
in time in some region of the developing brain, resulting in enduring sex differences in the neural landscape
(Fig. 11.3). Bringing order to such a wide ranging panorama of events is found in unifying principles that act
across multiple brain regions, but also by in depth analyses of tightly defined endpoints.
Before discussing both some unifying principles and
in depth analyses, it is worth the time to set the historical context. The earliest attempts to rigorously identify
sex differences in the brain began with high resolution
electron microscopy in rodents in the mid-1960s. The
FIGURE 11.3 Multiple endpoints are modulated by steroids during development. The first mechanism identified as determining hormonally-mediated sexual differentiation was differential cell death in which males and females produce the same number of neurons in a particular
subnucleus or brain region but more of them die in one sex versus the other. It is now known that many cellular endpoints are modulated by
steroids developmentally, via unique mechanisms and in distinct brain regions at varying times but more often perinatally. Many of these changes
involve epigenetic modification of the genome to both establish and maintain the sex difference.
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
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11. MECHANISMS ESTABLISHING SEX DIFFERENCES IN BRAIN
thinking at the time was that we had moved past the
notion of a difference in the intelligence of men and
women based on brain size or some other metric, and
that if sex differences did exist they would be small
and nuanced. And indeed two groups of researchers did
find small and nuanced sex differences, some that only
appeared in response to a lesion (reviewed in Ref. 14).
Even this was big news at the time, resulting in publication in the highest caliber scientific journals. About 10
years later, a graduate student at Rockefeller University
named Art Arnold contemplated the brains of canaries and zebra finches. Like most song birds, only the
males of these species sing a complex and learned song.
Arnold and his mentor, Fernando Nottebohm reasoned
that there must be a brain region that controlled this
singing ability and they predicted it would either be
much smaller or not exist in female song birds. They
were indeed correct and reported the first major sex difference in a vertebrate brain, so major in fact that it was
visible to the naked eye once the brains were sectioned
and stained for visualization. Legend has it that this
report prompted another graduate student on the other
side of the United States, Roger Gorski at UCLA, to step
back and take a more distant view of the rodent brain,
leading him to discover the sexually dimorphic nucleus
of the preoptic area (SDN-POA), or sexually dimorphic
nucleus (SDN) for short (reviewed in Ref. 14).
11.5.3 Cell Death is Developmentally
Regulated by Steroids in Brain Regions
Controlling Reproduction
The SDN is a small collection of nissl-dense cells and
is 3–5 times larger in male compared to female rats.
Because of the ease of quantification of the size of this
nucleus (it’s really a subnucleus), the opportunity was
presented to ask simple questions. Is it larger in males
because more neurons are born there? Or because more
neurons migrate to that spot? Perhaps more neurons
differentiate into the phenotype in the male or they are
larger or less densely packed? Or lastly, what seemed
the least likely, is it because more neurons die in the
female? As is so often the case, it was the least expected
answer that turned out to be true. Males and females
start life with the same rate of neuronal proliferation in
the SDN, but in response to the elevated estradiol of the
male more neurons survive. In the absence of the trophic
actions of estradiol in females, most of the neurons die.
The cell death occurs during the critical period and treatment of females with exogenous estradiol will save the
neurons from death, but only if the treatment is during
that period.27
This same principle was subsequently found to apply
to a set of motor neurons in the spinal cord, a subdivision
of the bed nucleus of the stria terminalis (pBNST) and
another small subnucleus called the anteroventral
periventricular region, or AVPV (reviewed in Ref. 28).
Interestingly, however, the AVPV is reversed from the
others in that rather than estrogens being prosurvival,
there is an active initiation of cell death in the AVPV of
males so that in the end the region is smaller than that
of females.29
Each of these brain regions is essential to reproduction in some way. The SDN is embedded in the preoptic
area (POA), which is the central node of the neural circuitry controlling male sexual behavior as well as maternal behavior. The pBNST is closely aligned with the
POA and helps to integrate olfactory and social stimuli.
The motor neurons of the spinal cord are in the spinal
nucleus of the bulbocavernosus, or SNB, and control the
penis. Lastly, the AVPV is critical to the generating of the
LH surge in females that is requisite for ovulation. But
a role in reproduction and a role for steroid-modulated
cell death is where the commonality ends. For all of the
nuclei but the SNB the principal hormone mediating
cell death is estradiol, having a neuroprotective effect
in the SDN and pBNST but a neurotoxic effect in the
AVPV. The cellular pathways of estradiol’s effects are at
times overlapping and at times unique per region and
not completely understood. The BAX gene is critical to
cell death in the AVPV, BNST, and SNB even though the
direction of death is different in the two sexes as is the
hormonal control.30 But within the AVPV there are two
types of neurons that die in response to estradiol stimulation, GABA neurons and dopamine neurons, and here
the cellular mechanism is different for each neuronal
phenotype (Fig. 11.4). In the SNB, androgens mediate
cell survival and a feedback from the muscles innervated
by these motor neurons is a critical component (see for
review Refs. 28,31,32).
The end result of steroid hormone controlled cell
death in each region is the creation of a volumetric
sex difference, meaning the subnucleus is smaller in
one sex versus the other due to more cells dying in
that sex during the critical period. Because death is
permanent, it was naturally assumed this established a
permanent structural sex difference in the brain with no
further modification. But this notion requires revision
in light of the surprising observation that ongoing cell
genesis, including neurogenesis, maintains the larger
AVPV in females and SDN in males.33 This challenges
both the contention that the only place in the brain
adult neurogenesis occurs is in the hippocampus and
subventricular zone, and the concept of rigid structural
sex differences in the brain. Because the volumetric
sex difference must be actively maintained it is also
subject to modification if those factors maintaining it
are disrupted.
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
11.5 Mechanisms of Steroid-Induced Masculinization
FIGURE 11.4 One hormone, many effects. In rodents, estradiol
(E2) derived from testicular androgens is the dominant steroid mediating masculinization. In one region, the sexually dimorphic nucleus
of the preoptic area (SDN-POA), E2 promotes cell survival but in the
nearby anteroventral periventricular nucleus (AVPV), the same steroid
induces cell death. At the same time, E2 stimulates neurogenesis in the
male hippocampus and there is a lower rate of cell genesis in the amygdala of the same animals, resulting in females having a higher rate of
cell proliferation. In each region the mechanism by which E2 exerts
these divergent effects is distinct and in some cases not yet known.
11.5.4 Cell Birth is also Developmentally
Regulated but in Brain Regions not Directly
Associated with Reproduction
For many years the control of cell death by steroids
stood as the only clear mechanism by which a brain
region was differentiated. This notion too, however, has
required revising in light of observations of sex differences in cell genesis in the hippocampus and amygdala
during the critical period (Fig. 11.4). Interestingly, both
of these brain regions are more involved in cognitive and
emotional responses than pure reproductive functions.
In the neonatal rat hippocampus males have twice the
rate of cell genesis as females as determined by injection
of the cell birth marker BrdU, a thymidine analog that is
incorporated into the DNA during cell division. When
the fate of these cells is determined, the majority of the
newly born cells that survive in males become neurons,
up to 80%, whereas less than half do so in females.34
This is a rather remarkable sex difference and raises
two interesting questions: (1) how is this sex difference
137
regulated and (2) what is the functional significance?
The answer to the first question begins with exploration
of the hormonal basis of the sex difference, an obvious
place to start. Administering estradiol or testosterone
to females increases their rate of cell genesis to that of
males, and blocking estradiol synthesis or antagonizing
the estrogen receptor reduces cell genesis in males to
even below that of females.34 Thus it would seem this
is another straightforward example of a hormonallymediated sex difference during the critical period. But
there is one critical caveat, when the amount of estradiol
in the hippocampus is measured in males and females
at the time of the sex difference in cell genesis, there is
no sex difference.35 And not only is there no sex difference, the amount of estradiol in the hippocampus is
extremely low relative to other brain regions, including
even the cortex. The same is true for testosterone and
dihydrotestosterone.35 There also are no sex differences
in the amount of estrogen or AR in the developing hippocampus. Thus the developing hippocampus appears
to be exquisitely sensitive to steroids, yet steroids do not
appear to be the driver of the sex difference.
If steroid levels are not the primary source of the sex
difference in cell genesis, there must be additional factors that either differentially regulate the sensitivity to
the small amount of steroid that is present in male and
female hippocampus, or a nonhormonal source that originates with the sex chromosomes. Candidates include
genes on the X chromosome that escape inactivation and
that could suppress neurogenesis in females, or genes on
the Y chromosome that promote neurogenesis in males.
Neither have been identified to-date. Alternatively,
microRNAs that originate on the X chromosome could
broadly modulate autosomal gene expression to modulate neurogenesis.36 Currently it is not known precisely
how the sex difference in hippocampal neurogenesis
during the critical period is regulated, and the potential
for a combination of hormonal and genetic regulation,
including epigenetic, is also possible. Understanding at
the cellular and molecular level how this fundamental
process is being regulated so differently in males and
females has broad ranging implications for normal neural development and potentially for neuropsychiatric
disorders in which the dysregulation of the hippocampus is strongly implicated.
In the adult the hippocampus is key to two broad functional categories: learning and memory, and stress and
anxiety. Anatomically the hippocampus is also broadly
divided into ventral and dorsal subdivisions which are
preferentially involved in learning versus stress responding.37 Both of these functional responses differ broadly
in males and females and the sex difference in neurogenesis exists in both the dorsal and ventral regions.
In rodent animal models the hippocampus is strongly
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
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11. MECHANISMS ESTABLISHING SEX DIFFERENCES IN BRAIN
associated with spatial learning as assessed by the
Morris water maze or other food reward-based mazes.
What was originally considered a superior performance
by male animals is now understood to be an impact of
context that differs for the two sexes. Specifically, if the
conditions under which the animal is tested are stressful, females perform poorly, but if there is ample familiarization with the task before hand, there is either no
sex difference or in some cases females perform better
than males.38,39 Additionally, males and females attend
to different contextual cues when navigating the maze.40
Males rely on geographic cues such as directionality (i.e.,
north vs south, right vs left), whereas females use local
cues (swim in the direction of the object on the wall), an
effect that has been generalized to humans.41 By manipulating the presence of geographic versus local cues,
the relative performance of males and females changes.
Why this is the case remains a mystery, but origins in the
sex difference in neurogenesis during the critical period
is one possibility.
The hippocampus is also central to the negative feedback arm of the hypothalamic-pituitary-adrenal (HPA)
axis. Glucocorticoid receptors are heavily expressed
by hippocampal neurons which transmit an inhibitory
message to the paraventricular nucleus (PVN) to shut
down the activation of the HPA axis following a stressful event.42 Many factors can modulate or impair the
negative feedback loop, including sex and reproductive
status. Just as with spatial learning, stress responding is
highly contextualized and sex differences in the activation of the HPA axis depend on past experience and the
type of stressor. Stage of life also seems to impact the
response of each sex, with males being more susceptible to prenatal stress and females more susceptible to
adolescent stressors (see for review Ref. 43). Again, why
these differences exist is not known but it is an active
area of investigation by laboratories around the world.
Little consideration has been given to the idea that neonatal neurogenesis may be a contributing variable to sex
differences in stress responding.
The complex and context dependent nature of learning and stress response, and how they impact each
other, makes understanding the role of developmental
sex differences in those systems even more challenging. In brain regions controlling reproduction it is clear
that developmental events are setting the stage for adult
functions. But HPA axis modulation and hippocampaldependent spatial learning begin well before reproductive capacity is reached. Indeed about two weeks of age
in the rodent is when both these functions come online.44 Cells that are born in the neonatal hippocampus
require about two weeks to differentiate into neurons
and begin to integrate into the neural network. Thus in
males, in which substantially more neurons are born in
the first few days, there would be many more immature
FIGURE 11.5 Juvenile social play behavior is sexually differentiated. In all species which exhibit play behavior, characterized in
rodents by chases, pins, pounces, and boxing, males engage with a
higher frequency and intensity than females. The sex difference is
robust and stable over many days. The medial amygdala is a key
brain region controlling this sex difference in play which is organized
by gonadal steroids perinatally but is expressed at a time in life when
there are no circulating steroids, therefore the behavior is not activated
as is the case of sexual, maternal, and aggressive behaviors.
neurons integrating into the hippocampal network at
two weeks of age compared to females in which the
overall maturation would be greater. As a result, the
way new information is processed and encoded into
the developing brain may be quite different in males
and females and have enduring consequences for adult
functioning.
The amygdala is a collection of nuclei with associated
functions relevant to social behavior, emotionality, and
fear-based learning.45–47 One particular social behavior
that is controlled by the amygdala is displayed only during a restricted life period, juvenile play.48 In all species
that play, which is the majority in mammals, males are
observed to play with a higher physical intensity and
frequency, leading to the term rough-and-tumble play.49
This robust and pan-species sex difference is particularly
of interest because it occurs at a time in life when there
are no circulating sex steroids (Fig. 11.5). However the
propensity to play is programmed by steroids during
the same critical period as all of the other organized sex
differences identified to-date.50,51
The amygdala is often referred to as a sexually dimorphic brain region with reports of a larger size in males
for specific subnuclei and the dendritic profile of neurons therein.52–55 There is also a sex difference in cell
genesis in the neonatal amygdala only in this case more
new cells are born in the female, and many of them will
differentiate into astrocytes as well as neurons.56 Indirect
evidence is consistent with this sex difference being
hormonally determined, meaning higher androgens
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
11.5 Mechanisms of Steroid-Induced Masculinization
in males suppresses cell proliferation in the amygdala. Intriguingly, the cell signaling pathway involves
endocannabinoids,56 what some call the brain’s own
marijuana.57 Endocannabinoids are membrane-derived
locally acting modulators best known for their ability
to inhibit GABA or glutamate release from presynaptic terminals.58 The two dominant endocannabinods are
anandamide and 2-aceylglycerol, or 2AG and their concentration in the brain is tightly regulated by associated
degradative enzymes. There are two receptors for endocannabinoids, CB1 and CB2, both G-protein coupled and
widely distributed throughout the brain.59 There are no
sex differences reported for CB1 or CB2 levels but developing males have a higher endocannabinoid tone in the
amygdala during the first few days of life because of
lower levels of the associated degradative enzymes.56
Administration of exogenous endocannabinoid agonists
to females raises the tone and, surprisingly, reduces cell
genesis to the level of males. How this occurs at the cellular level is not known but there is a correlation with the
later appearance of juvenile play. Thus females in which
neonatal cell genesis is suppressed by endocannabinoid
treatment play like males as juveniles, meaning they
exhibit more frequent physical encounters, such as pinning, boxing, pouncing, and chasing. At this time there
is only a correlation between the sex difference in cell
genesis and the sex difference in play, but both are modulated by endocannabinoids in a consistent direction.56
11.5.5 Nonneuronal Cells can be Key
Contributors to Sexual Differentiation
The POA is a small region just rostral to the hypothalamus and above the optic chiasm (hence its name
as it is “pre” to the optic nerve). As already discussed,
the SDN resides here and is one of the largest mammalian neuroanatomical sex differences (Fig. 11.6). In rats
there is also a robust sex difference in the morphology of
neurons in this region in the density of synapses on the
dendrites, specifically spine synapses which are known
to be excitatory glutamate synapses. On a given length
of dendrite males have 2–3 times more spine synapses as
females.60 This brain region is essential for expression of
male sexual behavior in adulthood and the higher density of synapses endures across the life span. On the individual level, the density of spines is correlated with the
intensity of male sexual behavior, further corroborating
the importance of this developmentally organized and
then hormonally activated synaptic profile to behavior.61
The sex difference in synaptic profile of the POA falls
into the category of a classic hormonally mediated sexually differentiated endpoint in that treating newborn
females with a masculinizing dose of estradiol during
the critical period fully recapitulates the masculinization process. But if estradiol treatment occurs outside
139
FIGURE 11.6 The innate immune system mediates sexual differentiation. The preoptic area (POA) is the most important brain region
for appropriate expression of adult male sexual behavior. The SDN is
found here and on neurons found outside the SDN but in the POA
the density of excitatory synapses is 2–3 times greater in males than
females. The sex difference in synapses is determined by the inflammatory signaling molecule prostaglandin E2 (PGE2), which is found
at higher levels in the male POA compared to the female. Microglia
are the brains innate immune cells and derived from modified macrophages. Microglia display a range of morphologies and their shape is
representative of their activational state. Ameboid-like microglia are
more activated and both respond to and produce PGE2. This morphology of microglia is also more prevalent in the POA of newborn males
and is the source of the higher PGE2 in their brains. Source: Modified
from McCarthy MM, Pickett LA, VanRyzin JW, Kight KE. Surprising origins of sex differences in the brain. Horm Behav. 2015;76:3–10.
the critical window, there is no change in the synaptic
profile of the POA nor will the females behave as males
in adulthood.
None of this is particularly surprising but where the
surprises come is in the mechanism of hormone action.
Given the endpoint in question is the density of synapses a likely candidate for the target of hormone action
would be a neurotransmitter or its associated receptors.
But instead, the critical signaling molecule is prostaglandin E2 (PGE2), a membrane-derived signaling molecule
that binds to G-protein coupled receptors broadly distributed throughout the brain. This is remarkably similar to the endocannabinoid system and they are indeed
actually synthesized from the same lipid precursor.62
New born male rat pups have higher levels of PGE2,
and higher levels of the synthesizing enzymes COX-1
and COX-2.63 If males are treated with the COX inhibitor,
indomethacin, shortly after birth, they display little to no
male sexual behavior as adults. Conversely, if females
are treated with a masculinizing dose of estradiol both
the level of PGE2 and the COX enzymes rise to the level
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
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11. MECHANISMS ESTABLISHING SEX DIFFERENCES IN BRAIN
of males and, more importantly, injecting PGE2 directly
in the POA of newborn female rats is equally effective
at masculinizing this brain region as peripheral estradiol
injection.63 In fact a single injection of PGE2 is sufficient
to permanently alter the synaptic profile and behavior
in females.64 This raises the question of the source of the
PGE2 and whether the single injection is actually initiating further prostaglandin production.
The brain possesses an innate immune system consisting of modified macrophages called microglia, an
unfortunate name given they have no real relation
to glia and they aren’t all that micro. Microglia originate in the embryonic yolk sac and migrate into the
CNS prior to the closing of the blood–brain barrier.65
They tile throughout the brain and lay down stem
cells from which they continuously replenish.66 Until
recently the primary function attributed to microglia
was the removal of cellular debris following an injury
that involved necrotic cell death.67,68 The primary signaling molecule for phagocytosis is ATP but microglia
also respond to and make prostaglandins.69 Recently a
more nuanced view of microglia has emerged, particularly regarding their role in development as sculptures
of neural networks by regulating the pruning of synapses and the size of proliferative neuronal precursor
pools.70–72 The morphology of microglia is an indicator
of their activational state, which changes in response to
local conditions. Ramified microglia have long branching processes with which they check on neighboring
neurons. These are considered nonactivated surveying
microglia.73,74 On the opposite end of the spectrum are
ameboid shaped microglia which have a blebby appearance representative of their activated state following an
insult (Fig. 11.6). Visualization of microglia is achieved
by immunohistochemical detection of the cell specific
protein Iba1 and the activational state categorized by the
relative shape of the cell. This was done for the microglia
of the developing POA in male and female rat pups and
a striking sex difference discovered. In males the microglia were in an activated state while in the females they
were overwhelmingly in the ramified surveying state. If
females are masculinized by estradiol injection, so are
their microglia. And, if females are treated with a single
injection of PGE2 their microglia become activated and
generate still more PGE2, thereby initiating the masculinization synaptogenicprocess.75,76 Thus microglia are
intimate partners with neurons for building the neural
circuit driving male sexual behavior.
Connecting PGE2 to increased dendritic spine density
requires first identifying the GPRC by which the PGE2
signal is transduced. There are four principle receptors
for PGE2, EP1–4, but they are a promiscuous and facile
group with much cross-talk.77 A combination of pharmacological and gene expression manipulation approaches
converged on EP2 and EP4 as the key receptors,61 both
of which are adenylate cyclase linked and lead to activation of protein kinase A.78 Specifically a form of PKA
that is localized to the head and neck of dendritic spines
where it regulates the phosphorylation of select subunits of the AMPA form of the glutamate receptor.64
Phosphorylation increases the trafficking of the AMPA
receptor to the membranes of both neurons and astrocytes,79 maximizing responsiveness to glutamate release,
which can be induced by PGE2.80 Thus the following
sequence of events appears to occur in the POA of neonatal males. First, estradiol stimulates production of
PGE2 by upregulating COX-1 and COX-2. This PGE2
then activates microglia which make still more PGE2,
the combination of which leads to activation of PKA,
phosphorylation of AMPA receptors which move to
the membrane, and induction of glutamate release by
neighboring astrocytes. The released glutamate activates
the membrane clustered AMPA receptors leading to the
formation and stabilization of dendritic spine synapses
(reviewed in Ref. 36). So three cell types, one of which
is nonneuronal, the immune microglia, are required for
the masculinization of the developing brain.
11.5.6 Steroids Induce Epigenetic Changes
to the DNA to Induce and Maintain Sex
Differences
Epigenetics literally means above the genome and
refers to modifications to the DNA and associated histones that will change gene expression but not be heritable in the classic sense. Through this mechanism early
life experiences, including in the womb, can imprint
on the genome characteristics that will be beneficial to
the predicted future environment.81 The two canonical
forms of epigenetic change are direct modification of the
DNA via methylation of cytosines proximal to guanines
on the DNA strand, and decoration of the histones surrounding DNA with methyl, acetyl, or other modifying
groups. In either case the access of transcription factors
to gene promoters and enhancer sites is either facilitated
or sterically hindered. The two forms of modification
interact in complex and still poorly understood ways
(reviewed in Refs. 82,83).
The original invocation of epigenetics was as a means
to explain how every cell in the body could contain the
entire genome yet differentiate into and maintain a differentiated phenotype as a hepatocyte versus an islet cell
and so on. Because the maintenance of cell fate is vitally
important the epigenetic suppression of gene expression
is strong and enduring. Consistent with this need, the
covalent bond between the methyl group and 5’ carbon on cytosine is irreversible. This is proving not to be
the case in the brain, however, where epigenetic regulation is far more dynamic than expected,84 including an
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
11.5 Mechanisms of Steroid-Induced Masculinization
FIGURE 11.7 Epigenetic modifications mediate sexual differentiation. There are two canonical mechanisms for epigenetic modification of gene expression. Chromatin remodeling involves addition and
subtraction of acetyl, methyl, and other groups to the histone tails associated with nucleosomes. These modifications are achieved by specific
enzymes. Sex differences in the amount of changes to the histones suggest that enzymatic activity differs in some regions of male and female
brains during development. Pharmacology inhibition of the enzymes
confirms a functional role for these modifications by disrupting the
normal sexual differentiation process. Epigenetic changes to the DNA
involve the addition of a methyl group to cytosines proximal to guanines. This too is achieved enzymatically and once the methyl group is
added it recruits methyl binding proteins (MBDs) which further suppress gene expression by sterically hindering the binding of transcription factors. Methylation patterns of various genes differ in specific
brain regions of males and females. DNA methylation also recruits
enzymes that modulate histones (HDACs) and is one way in which
the two forms of epigenetic modifications interact. Source: Modified from
Nugent BM, McCarthy MM. Epigenetic underpinnings of developmental sex
differences in the brain. Neuroendocrinology 2011;93(3):150–158.
enzymatic pathway for demethylation that was originally discovered as a component of DNA repair.85 The
acetylation and methylation of histones are also actively
regulated following novel experiences.86,87
Steroid receptors by their very nature as transcription
factors interact directly with the DNA and some associated cofactors exert histone modifying activity,88 making
them an obvious candidate as epigenetic modifiers. Both
DNA methylation and histone modifications have been
identified as contributors to the sexual differentiation
process (Fig. 11.7). Sex differences in the amount of specific histone marks are found in specific brain regions,89,90
and inhibition of the enzymes that place acetyl groups
on the lysine residues of histone tails during the critical
period impairs masculinization of sexual behavior.91 The
naturally occurring progression of cell death that leads
to a sex difference in the size of the BNST is also modulated by histone acetylation, with the larger size in males
141
being prevented if histone deacetylating enzymes are
blocked, which increases acetylation and suppression of
gene expression during the critical period.92 This implies
that in the normal course of events estrogen receptor and
its associated cofactors directly deactylate genes critical
for neuronal survival, thereby increasing expression of
those genes.
Direct changes to the DNA via methylation are also
implicated in sexual differentiation of brain and behavior. Both isoforms of the estrogen receptor as well as
the progesterone receptor show dynamic and hormonally modulated patterns of DNA methylation in their
promoter regions.93 Dynamic in that sex differences and
hormonal effects seen in newborns are no longer apparent but replaced by additional changes in juveniles.
A still different pattern emerges in adults. Moreover, these
changes are region-specific. In general, however, these
changes do not seem directly related to expression levels
of the receptors and may instead reflect past expression.
Similarly, an in depth analysis of the methylation of the
genome following neonatal hormone treatment found
very few changes on the short term but dramatic changes
in the adult,94 suggesting an epigenetic “echo.”
The most causal connection between DNA methylation and the sexual differentiation process is found in
the study of neonatal rats treated with inhibitors of
the methyl transferase enzymes directly into the brain
and the impact on adult neuranatomy and behavior
assessed.2 Under normal conditions, females were found
to have higher levels of overall DNA methylation in the
POA and when this was reduced by pharmacological
inhibition of the key enzymes, not only was the sex difference in DNA methylation eliminated, these females
had a male-like synaptic pattern in the POA and exhibited male sexual behavior as adults. Analyses of gene
expression revealed that many genes are epigenetically
repressed in females during development and that a
function of higher steroid levels in the neonatal male
is to inhibit methylation of DNA and emancipate those
genes to achieve masculinization of brain and behavior
(Fig. 11.8). Moreover, the higher DNA methylation in
females also determines the closing of the critical period
as pharmacological inhibition of the key enzymes outside this time also induced masculinization, whereas
administration of steroid did not. These observations
demand a rethinking of the female developmental program as the default as there is clearly an active repression of the male developmental program. The process
of masculinization also requires rethinking as one of
the primary impacts of steroids is not to directly induce
gene transcription but to instead broadly modify the
genome via DNA methylation. An important consequence of this strategy is that DNA methylation can be
reversed, either chemically or perhaps by physiology
and experience.
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
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11. MECHANISMS ESTABLISHING SEX DIFFERENCES IN BRAIN
FIGURE 11.8
Sex differences in DNA methylation differentiate the POA and adult male sexual behavior. The enzymes that confer methyl
groups to cytosine residues are more active in the newborn female POA and are suppressed by the higher estradiol found in the males. As a
result there is more overall DNA methylation in females, leading to suppression of genes required for masculinization. In males, the DNA is
demethylated following inhibition of the methylation enzymes (DNMT), allowing key masculinization genes to be expressed, leading to masculinization of the brain and adult male sexual behavior.
11.5.7 Steroids can also Act Rapidly to Induce
Enduring Change in Neural Circuits
Given the enduring nature of sexually differentiated traits and the canonical slow action of steroids it
was naturally assumed that all hormonal effects in the
developing brain required gene transcription and protein translation. But this assumption was overthrown
by the observation that estradiol activates a signal transduction pathway in the hypothalamus within minutes.
PI3 kinase in presynaptic terminals phosphorylates its
dominant substrate, Akt, 30 min after estradiol administration. The activation of PI3 kinase requires the classic
estrogen receptor be present in the presynaptic neuron,
but presumably action is restricted to the membrane.
Via a mechanism not yet known, activated PI3 kinase
induces glutamate release which binds to postsynaptic
glutamate receptors, activating MAP kinase and induction of dendritic branching.95 As a result the dendritic
tree of neurons in the male hypothalamus are larger and
more complex than that of females. Functionally, this
neuroanatomical network change appears to underlie the
process of defeminization in which the male brain loses
the capacity to express female sexual behavior.96 Why
the default feminization pathway needs to be removed
in males remains a mystery but it clearly involves a separate mechanism from that of masculinization.
11.5.8 Steroids Indirectly Modulate Neuronal
Excitability in the Developing Brain
Neurotransmission as a target of steroid hormone
modulation during development seemed obvious but
largely failed to deliver as a mechanism for establishing sex differences. But steroids do modulate excitability during the critical period, and interestingly this
is achieved in a manner similar to that noted above,
through the modulation of kinases.
The amino acid GABA is the dominant inhibitory
neurotransmitter of the mature brain, so vitally important that even modest antagonism of its receptors can
lead to seizures and ultimately death. But surprisingly,
early in development GABA is the dominant source of
excitation and is vitally important to the maturation of
healthy neuronal networks.97 This chameleon change is
achieved by reversing the concentration gradient of a
single molecule, chloride.98 GABA-A receptors are chloride permeable ion channels which when open allow
chloride to flux down its concentration gradient. In
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
11.5 Mechanisms of Steroid-Induced Masculinization
143
FIGURE 11.9
Steroids modulate excitation in developing neurons via depolarizing GABA. (A) GABA is an excitatory amino acid neurotransmitter. (B) The GABA-A receptor is a chloride permeable ionophore that allows chloride to flow into or out of the cell depending on
driving force (a combination of concentration gradient and electric charge). (C) In immature neurons the intracellular chloride concentration is
high because it is actively pumped into the cell by NKCC1. The activity of the pump is regulated by phosphorylation. Estradiol increases the
activity of the pump by increasing the production of specific kinases (SPAK and OSR1) which phosphorylate the pump. (D) As a result, GABA
is more excitatory in males than females and leads to a larger and longer lasting influx of calcium into the cell via L-type voltage-gated calcium
channels. Over the course of development GABA gradually reverts to its mature inhibitory action but this progression takes longer in males than
females. (E) The calcium influx induced by excitatory GABA activates many cellular processes, including the phosphorylation of the transcription
factor CREB (pCREB). When the amount of cells expressing pCREB is quantified following administration of a GABA-A agonist, there is more
in several regions of the male brain compared to the female.
mature neurons chloride is maintained at low intracellular levels by an electroneutral pump called KCC2 for its
ability to pump both potassium and chloride. When the
channel opens chloride flows into the cell and hyperpolarizes the membrane, pushing it further away from the
threshold for an action potential. In immature neurons,
however, the opposite occurs. Intracellular chloride is
maintained at a high concentration by a different pump
called NKCC1 because it pumps sodium, potassium and
chloride.99 In this case when the GABA receptor channel
opens chloride rushes out of the cell, depolarizing the
membrane which is also studded with voltage-gated calcium channels (VGCC). The L-type VGCC is particularly
sensitive to low voltage changes and opens in response
to the GABA-induced depolarization, allowing calcium
into the cell and activation of a myriad of signal transduction and cellular processes that promote growth and
maturation.100 Ultimately the neuron matures and the
chloride potential is reversed to maintain hyperpolarization and inhibition.101
Steroids potently enhance the excitatory actions of
GABA but not by a direct effect on neurotransmitter
synthesis or receptor number or even predominantly
by directly affecting the chloride pumps, although there
are some sex differences in expression.102 Instead, estradiol upregulates two highly specific kinases, SPAK and
OSR1, both of which phosphorylate NKCC1 to increase
its activity.103 As a result, higher intracellular chloride
is achieved and when the GABA-A receptor opens the
magnitude of the transmembrane chloride concentration gradient is higher, leading to a stronger depolarization and an increased frequency and duration of L-type
VGCC opening.104 Ultimately this means more calcium
enters the cell and all the attendant processes that go
with that are more intense (Fig. 11.9).105 Quantification
of calcium influx in cultured hippocampal neurons finds
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11. MECHANISMS ESTABLISHING SEX DIFFERENCES IN BRAIN
more neurons that respond to GABA as depolarizing if
they are pretreated with estradiol, and the peak of the
calcium influx is greater and for a longer duration.106 In
primates (Papio anubis), in utero estradiol is confirmed to
enhance the excitatory actions of GABA,107 while in the
rat model continuous treatment with estradiol maintains
the depolarizing action of GABA, artificially extending
the developmental period.108 This could have important
consequences if neonates are exposed to estrogenic compounds in the environment.
11.5.9 Sex Chromosome Complement also
Matters to Brain Development
The emphasis on hormones as the sole source of sex
differences in the brain held sway for over 30 years.
Chromosome complement was considered only important as the determinant of gonadal differentiation and all
else flowed from there. But it is undeniable that every
cell in the brain has a sex, meaning it is either XX or XY,
and that means there are some genes that are potentially
expressed differently in males and females.109 In females
one X chromosome is subject to inactivation in order to
provide dosage compensation compared to males with a
single X. Therefore only the Y would be at issue and there
are overwhelmingly few Y chromosome specific genes
and they are largely concerned with spermatogenesis.
Thus it was easy to dismiss chromosome complement as
a source of sex differences in the brain. But there were
persistent instances in which hormones could not quite
explain everything regarding sexual differentiation of
brain and behavior, especially in birds (which have WW
and WZ for male and female sex chromosomes) and this
prompted Art Arnold, who made the first discovery of
a major neuroanatomical sex difference in the brain in
birds, to turn his attention to the role of genetic sex.110
The challenge was how to separate the impact of chromosome complement from gonadal phenotype and this
is where the modifiability of mouse genetics provided
a powerful tool. The Sry gene on the Y chromosome is
essential for testis determination. By deleting the gene
from the Y chromosome and reinserting it on an autosome, scientists were able to generate a mouse that had
an XX chromosome compliment but developed testes.
Likewise, the same manipulation led to the creation of
XY mice that developed ovaries. By comparing XX and
XY animals that both have ovaries or both have testis the
impact of chromosome complement could be assessed
independently of gonadal phenotype (Fig. 11.10). This is
referred to as the 4-core genotype model.111 Early studies
focused predominantly on reproductive endpoints such
as mating and parenting behavior and the associated
neural underpinnings but found little to no effect of
chromosome complement. This was reassuring in that it
confirmed 50 years of research about the importance of
FIGURE 11.10
The 4-core genotype allows for separation of hormonal versus sex chromosome effects. Every cell in the brain of mammals is either XX or XY. In order to isolate the impact of that genetic
difference from gonadal differentiation, a mouse line was generated
in which the testis determining gene Sry was transferred to an autosome. This allows for the generation of XX animals with testis and XY
animals with ovaries. By comparing XX to XX-sry the effects of hormones can be assessed independent of the sex chromosomes whereas
comparison of XX-sry to XY or XX to XY-no Sry allows for assessing the
impact of chromosome complement in animals with the same gonadal
phenotype. Many effects of sex chromosome complement have been
detected using this approach.
hormones to sexual differentiation of brain and behavior. However, when the analyses extended outside the
realm of reproduction, effects of chromosome complement were found on aggression, habit formation, feeding
behavior, and so on.112 This is not to say that hormones
have no effect on those endpoints and how they are
organized developmentally, but that there is a contribution to the variability in responses between males and
females that comes from the sex chromosomes.
Once a role for sex chromosomes in sex differences
in behavior was confirmed the race was on to identify
the key genes. But this has turned out to be harder than
expected. There are several ways in which chromosome
compliment could impact the developing brain differently in males and females. First would be a gene on
the Y chromosome outside the pseudoautosomal region
which would only be expressed in males brains, but
as noted above, other than Sry most of these genes are
related to spermatogenesis. Second would be a gene
on the X chromosome that escapes inactivation and
therefore predicted to be at twice the level in females
as males. The number of genes that escape inactivation
has recently been found to be much more than originally thought: up to 15% in humans,113 and so this is a
possibility. Moreover, the X chromosome is particularly
enriched in genes associated with neural development
and cognitive functioning. To-date no mammalian genes
on the X chromosome that contribute to sex differences
in brain development have been identified. The third
possibility is that the presence of an inactivated X chromosome is not neutral. X inactivation is an energetically
PRINCIPLES OF GENDER-SPECIFIC MEDICINE
REFERENCES
expensive epigenetic process that involves heavy methylation of the DNA and associated changes to the
chromatin in order to suppress expression.114 There is
speculation that this process sequesters many of the key
enzymes and other factors needed for epigenetic modifications of autosomes, thereby exerting wide ranging
effects throughout the genome. This effect is referred
to as a heterochromatin sink (reviewed in Ref. 115).
Consistent with this interpretation is the increasingly
deleterious consequence of each additional X chromosome in cases of sex chromosome aneuploidy such as
XXY versus XXXY. However, multiple Y chromosomes
also has deleterious consequences for brain development.116,117 Sex chromosome aneuploidies always have
associated gonadal dysfunction, making it difficult to
cleanly separate hormonal and genetic effects. Ongoing
studies involving animal models in which these variables can be separated,118 as done for the 4-core genotype
model, will advance our knowledge on this front.
11.6 ELUCIDATING MECHANISMS OF
NORMAL BRAIN DEVELOPMENT IN
MALES AND FEMALES PROVIDES CLUES
TO SOURCES OF VULNERABILITY AND
RESILIENCE
Early life is not kind to males. Intrauterine mortality
is so much greater than for females that a profoundly
higher rate of conception of male fetuses converges to
parity by birth.119 When male fetuses do survive they
are more likely to have prenatal strokes, be born prematurely, suffer a birth injury, and fare far worse than
females if one occurs. Neuropsychiatric disorders with
origins in development exhibit a profound gender bias
in prevalence and/or presentation. Boys are diagnosed
with autism spectrum disorder (ASD), attention deficit
and hyperactivity disorders, and early onset schizophrenia at markedly higher rates than girls. Neurological
disorders such as dyslexia, stuttering, and Tourette’s
syndrome are 2–3 times more frequent in boys (reviewed
in Refs. 81,120–125). Yet the biological basis for the fragility of the developing male is entirely unknown.
It is tempting to speculate that the higher testosterone
levels experienced by the developing and adult male
are the source of the vulnerability, and indeed there is
evidence in experimental models that androgens can
increase neurotoxicity following an insult, but the same
androgens can also be neuroprotective.126 The toxic versus protective effects are obviously mediated by different
mechanisms but this highlights the importance of looking beyond the steroid itself to understand the actions it
is exerting. Only by identifying the mechanism can we
hope to understand how steroids might be contributing
to sex differences in either vulnerability or resilience.
145
The potential of this approach is just beginning to be
realized. The four- to five fold higher rates of diagnosis of
ASDs in boys has proven maddeningly difficult to unravel.
A recent, extensive transcriptomic analysis of postmortem tissue from autistic and nonaffected individuals was
used to distinguish between two nonexclusive hypotheses: (1) risk genes for autism are expressed at higher
levels in males, versus (2) genes involved in normal
male brain development are more highly expressed in
males with autism.127 The authors concluded the latter,
that rather than autism risk genes being higher in males
it is the genes normally involved in male brain development that were being overexpressed. More importantly,
many of these genes were disproportionately associated
with neuroinflammation. The authors were comfortable
reaching that conclusion because of extensive corroboration from the basic science research on neuroinflammatory signaling molecules such as prostaglandins, and
the key role played by microglia and astrocytes in the
masculinization process in rodents, as reviewed above.
Hopefully this is the first of many examples in which
basic science in animal models will inform us as to the
origins and potential treatments of human disorders that
vary profoundly in frequency, severity, and presentation
in boys versus girls and men versus women.
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