Author's personal copy

5.01 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms,

and Meaning
Margaret M McCarthy, University of Maryland School of Medicine, Baltimore, MD, USA
Geert J De Vries and Nancy G Forger, Neuroscience Institute, Georgia State University, Atlanta, GA, USA
Ó 2017 Elsevier Inc. All rights reserved.

5.01.1 Introduction 4
5.01.2 Ten Things We Know 4
5.01.2.1 Hormones Cause Sex Differences by Acting during Development as well as in Adulthood 4
5.01.2.2 There Are Sex Differences in Behavior 5
5.01.2.3 There Are Sex Differences in Physiology 6
5.01.2.4 There Are Sex Differences in Disease Susceptibility 6
5.01.2.5 There Are Sex Differences in Neural Structure, Glial Structure, and Connectivity 7
5.01.2.5.1 Neural Structure 7
5.01.2.5.2 Glial Cells 7
5.01.2.5.3 Synaptic Connectivity 8
5.01.2.6 There Are Sex Differences in Neurochemistry 8
5.01.2.6.1 Sex Differences in g-Aminobutyric Acid Signaling during Development 8
5.01.2.6.2 Sex Differences in Neurotransmitters in Adulthood 8
5.01.2.6.3 Sex Differences in Steroid Receptor Expression 9
5.01.2.7 Androgens as well as Estrogens Play a Role in Sexual Differentiation of the Brain 9
5.01.2.8 Sex Chromosome Complement Contributes to Sexual Differentiation 10
5.01.2.9 Sex Differences Are Context Dependent 11
5.01.2.10 Sexual Differentiation Depends on Four Key Processes 12
5.01.3 Recent Progress in Understanding the Four Key Processes 12
5.01.3.1 Neurogenesis 12
5.01.3.2 Cell Migration 14
5.01.3.3 Cell Death 14
5.01.3.4 Differentiation of Circuits 15
5.01.3.4.1 Axonal Growth 16
5.01.3.4.2 Dendritic Growth and Branching 16
5.01.3.4.3 Synaptogenesis 16
5.01.3.4.4 Differentiation of Neurochemical Phenotype 18
5.01.3.5 The Role of Epigenetic Mechanisms in the Four Key Processes 19
5.01.3.5.1 How Can Effects of Sexual Differentiation Last a Lifetime? 19
5.01.3.5.2 Effects of Manipulating Epigenetic Mechanisms on Sexual Differentiation 20
5.01.3.5.3 Sexual Differentiation of Epigenetic Marks across the Genome 20
5.01.4 Six Unanswered Questions 21
5.01.4.1 What Are the Actual Steroid Levels in the Brain during the Sensitive Period of Sexual Differentiation? 21
5.01.4.2 Do Noncoding RNAs Contribute to Sex Differences in Brain and Behavior? 22
5.01.4.3 Are Sex Differences Necessary? 22
5.01.4.4 Do Sex Differences in Brain Structure Beget Sex Differences in Brain Function? 23
5.01.4.4.1 ‘Canalization’ May Explain the Size and Variability of Sex Differences in Brain Structure and Behavior 24
5.01.4.5 Is Partner Preference Sexually Dimorphic? 24
5.01.4.6 Is the Human Brain Sexual Differentiated? 25
References 26

Abbreviations
AH Anterior hypothalamus GABA g-Aminobutyric acid
AVPV Anteroventral periventricular nucleus GH Growth hormone
BSTp Principal nucleus of the bed nucleus of the stria GnRH Gonadotropin-releasing hormone
terminalis INAH3 Third interstitial nucleus of the anterior
COX-2 Cyclooxygenase-2 hypothalamus
FAK Focal adhesion kinase LH Luteinizing hormone

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4 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
LTP Long-term potentiation
MAP kinase Mitogen-activated protein kinase
MeA Medial amygdala
mPOA Medial preoptic area
PGE2 Prostaglandin E2
PI3 kinase Phosphoinositide 3-kinase
5.01.1 Introduction
It is now over 55 years since publication of the seminal Phoenix,
Goy, Gerall, and Young manuscript (Phoenix et al., 1959), an
influential paper in which four scientists articulated the organi-
zational hypothesis of hormonally controlled sexual differentia-
tion of the brain. For many years the quest to understand how
the brain differed in males and females and led to sex differences
in physiology and behavior was the exclusive domain of scien-
tists self-identifying as ‘neuroendocrinologists’ or ‘behavioral
endocrinologists.’ This is beginning to change as major grant
funding agencies in the United States, Canada, and Europe are
increasingly requiring the inclusion of both male and female
subjects in preclinical or basic science research. The result is
a growing awareness combined with a burgeoning knowledge
base of the pervasive impact of sex on the brain and its myriad
functions. The goal of this chapter is to explore what we have
learned in the years hence. We first list 10 concepts that have
stood the test of time and can safely be considered ‘known.’
The last of these is our view of what we consider the four core
cellular processes that may be influenced by steroids during
sexual differentiation of the brain: (1) cell birth, (2) cell death,
(3) cell migration, and (4) the differentiation of circuits. The
current challenge in the field is to identify the mechanisms by
which steroids alter these four core processes, and a substantial
portion of this chapter is devoted to recent advances in that
domain. Progress is being made, but much remains to be
done. Moreover, there are fundamental questions remaining,
and we conclude this chapter by highlighting several of these.
5.01.2 Ten Things We Know
Making a list is both appealing and daunting. It is appealing
because it highlights what we have accomplished and gives
us confidence in the foundations on which further studies are
built. It is daunting because one might miss something and
inadvertently leave a critical finding off the list. It is also daunt-
ing because it requires that we examine the strength of a partic-
ular finding in the harsh light of new data. Do the initial
conclusions still hold? Were the techniques used appropriate
for the question? Are the findings robust and reliable? Both
the appealing and daunting aspects serve a useful purpose,
and with this in mind, we venture forward.
5.01.2.1 Hormones Cause Sex Differences by Acting during
Development as well as in Adulthood
For the purposes of this chapter, we define sexual differentia-
tion as a process that occurs during a restrictive developmental period
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
POA Preoptic area
SDN Sexually dimorphic nucleus
SDN-POA Sexually dimorphic nucleus of the preoptic area
SNB Spinal nucleus of the bulbocavernosus
VMN Ventromedial nucleus of the hypothalamus
and is irreversible (hence, differentiation). Phoenix and
colleagues called developmental effects of hormones organiza-
tional (Figure 1). In mammals, the focus of this chapter, sexual
differentiation occurs secondarily to sex determination, which
is the process whereby the chromosomal or genetic sex of an
individual determines the fate of the bipotential gonad as
either a testis or an ovary. The Sry gene on the Y chromosome
initiates testicular formation (Koopman et al., 1991), and the
process of sexual differentiation is then directed predominantly
by steroids produced by the gonads. However, the impact of
steroids is largely one sided in that it is the production of testos-
terone that directs the brain (and body) toward a male pheno-
type and the lack of testosterone production by the ovary that
directs the brain (and body) toward a female phenotype. Many
of the morphological and behavioral sex differences described
below are due to organizational effects of hormones directing
the development of neural circuitry that will generate male-
or female-typical functions and behaviors in adulthood.
Not all sex differences in brain function depend on organi-
zational effects, however. As steroid hormones freely cross the
blood–brain barrier, sex differences in levels of circulating
gonadal hormones can be expected to cause sex differences in
neural function in steroid-sensitive areas at the time of
hormone exposure. For example, to engage in male sexual
behavior, animals have to be exposed to testosterone not
only during development, but in adulthood as well. This adult
effect is transient, and Phoenix and colleagues therefore called
such effects activational (Figure 1). There are also activational
effects of steroids that are independent of developmental
effects. For example, male baboons show more yawning than
do females. This sex difference appears to depend entirely on
sex differences in circulating testosterone as castrated male
and ovariectomized female baboons yawn at equally high
levels after testosterone treatment in adulthood (Phoenix and
Chambers, 1982). Similarly, some sex differences in brain
morphology are due to activational effects of steroids (see
below), and there is emerging evidence that sex differences in
cognition may be largely a function of steroids circulating in
adulthood (reviewed in McCarthy and Konkle, 2005). In this
chapter, we will refer to ‘organizational’ and ‘activational’
effects as ‘programming’ and ‘acute,’ respectively, to bring
them in line with terminology used in other areas of develop-
mental biology (cf. Forger et al., 2015a).
It is important to distinguish ‘sexual differentiation’ from
‘sex differences.’ There are many differences between adult
males and females that are a consequence of transient changes
in hormones, reproductive status, or age; these are sex differences,
but are not by our definition sexually differentiated traits. Although
one could argue with our somewhat restricted view of hormon-
ally mediated sexual differentiation of the brain, it serves as
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Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning 5

Figure 1 Sexual differentiation of behavior occurs during a perinatal sensitive period. In rodents the parameters of the sensitive period for sexual
differentiation are operationally defined with the opening of the window being the onset of androgen production by the fetal testis of males late in gesta-
tion and a second surge around birth. The closing of the sensitive period is the developmental time point at which females can no longer be masculin-
ized by treating them with exogenous testosterone. The developmental hormone exposure is akin to early-life programming and determines adult
hormonal effects on sexual and parental behaviors. During the juvenile period, there are not gonadal steroids present but there is a sex difference in
play intensity and frequency which was established by the early-life programming effects of steroids. In rodents, testosterone is aromatized to estradiol
within the brain, and many effects of masculinization are due to the actions of estradiol, while others are directly the result of androgen action.

a useful construct on which to base a discussion of underlying
mechanisms.
5.01.2.2 There Are Sex Differences in Behavior
Sex differences in behavior are ubiquitous among sexually
reproducing species and presumably contribute in some way
to the reproductive success of the organism. Males and females
have evolved different reproductive strategies, necessitating
different morphologies and behaviors. In mammals, sex differ-
ences are commonly seen in sexual behavior, aggression, play
behavior, parental behavior, and performance on cognitive
tasks, to name just a few examples. Since behavior is controlled
by the nervous system, it would follow that the brain must
differ in males and females. However, sex differences in
behavior were recognized long before we considered the brain
to be a sexually dimorphic organ. A scientific approach to the
topic began with observations of effects of hormones on
behavior in domesticated animals such as roosters and
extended to controlled laboratory studies of inbred rodents
in the early 1900s. We now consider it self-evident that
hormones act on the brain to cause sex differences in behavior,
but this was not the initial presumption. A school of thought
prevailed for some time that held that hormonal effects on
peripheral structures, such as the penis, determined behavior
(Beach et al., 1969). In fact, the iconic Phoenix et al. (1959)
paper laying out the organizational/activational hypothesis of
hormone action makes little reference to the brain, but instead
refers to the ‘tissues’ controlling behavior. There is now ample
evidence, discussed below, that these ‘tissues’ include the brain.
Relevant to the understanding of sex differences in behavior
is defining the axis or axes along which the behavior can vary.
Males and females are frequently viewed as differing along
a continuum, with maleness at one end and femaleness on
the other. Individual phenotypes could exist anywhere along
the continuum but on average, males would predominate at
one end and females at the other. For instance, maternal
behavior, as demonstrated by pup retrieval, is much more prev-
alent in female rats (that have recently given birth) than in
males, which almost never spontaneously retrieve pups. Alter-
natively, the sexes may differ on a dependent measure, but only
slightly. In standardized tests of language, for example, girls
score significantly higher than boys, although in this case the
mean sex difference is relatively small, and there is substantial
overlap between the sexes (see Spelke, 2005 for review). None-
theless, both of these behaviors might be said to vary along
a single masculine–feminine axis.
In other cases, multiple axes have been proposed. The best
example of this is the case of sex behavior in the laboratory
rat where sexual differentiation is often viewed as involving
two axes and at least three distinct processes: (1) feminization,
the default developmental program required for a female
behavioral response in adulthood (i.e., the display of lordosis
under the proper hormonal milieu), (2) masculinization,
a hormonally induced process allowing for male sexual
behavior in adulthood (e.g., the display of mounting,
thrusting, and ejaculation), and (3) defeminization, a hormon-
ally induced process to eliminate, mask, or override the femini-
zation process. Emerging evidence suggests that the processes
of masculinization and defeminization occur relatively inde-
pendently and may target distinct neural circuitry. Although
males are normally both defeminized and masculinized, it is
possible to separate these. In mice, for example, estrogen
receptor alpha activation appears required for masculinization,

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6 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
while estrogen receptor beta has a major role in defeminiza-
tion, so that mice with a mutated estrogen receptor beta gene
are masculinized, but not defeminized (Kudwa et al., 2006).
Recent evidence suggests that the cellular mechanisms medi-
ating masculinization and defeminization are also unique
(see below).
One could make the argument that other examples of sexu-
ally dimorphic behavior, such as juvenile play, also vary along
multiple axes. For instance, rough and tumble play by juveniles
is more intense and more frequent in males than females,
across many species (Meaney, 1989; Hines, 2006). Although
males show more rough play, females are not idle while the
males are playing; they are doing something else (in monkeys,
grooming-type ‘play’; in humans, this might be doll play). If
‘play’ is looked at comprehensively, this could suggest two
axes (e.g., rough play and grooming play), which may, like
male and female sexual behavior, be separable. Are males
masculinized on rough play and defeminized on doll play? If
so, this would make play analogous to the multiple axes view
of sex behavior. If not, this raises the question of whether the
differentiation of sexual behavior is really qualitatively
different from the differentiation of other behaviors, such as
play. For example, if female sex behavior and male sex behavior
are considered as two different behaviors entirely, then we are
back to single axes with each individual more feminine or
masculine on each of these two behaviors; the term ‘defemini-
zation’ would not be necessary.
A reader of the sex differences literature will often find
defeminization used interchangeably with masculinization, or
demasculinization used synonymously with feminization. The
term demasculinization is generally not used when discussing
the differentiation of sex behavior, because the masculinization
of behavior has historically been viewed as an active process,
and you cannot deprogram a process that has to be induced
in the first place. However, work on the cellular underpinnings
of sexually dimorphic behaviors suggests otherwise. Neurons
underlying at least some aspects of male sexual behavior
initially form in both sexes and then die in females, analogous
to differentiation of the Wolffian ducts. This suggests that a case
for demasculinization can be made. Thus, while few would argue
that there are lasting and important differences in behavior
between the sexes, there is less agreement about how terms
are applied. One can argue whether this is ‘just semantics,’ or
a significant impediment to clear thinking about the processes
involved.
5.01.2.3 There Are Sex Differences in Physiology
Even before the work of Phoenix et al. (1959), there was
evidence that the brain was sexually differentiated. Female
mammals exhibit profound cyclical variations in the release
of gonadal and pituitary gonadotropic hormones, whereas
males do not. Harris and Jacobsohn (1952) demonstrated
that male pituitary transplants placed directly under the hypo-
thalamus sustained ovarian cycles in female rats. This dis-
proved earlier claims that the pituitary was responsible for
sex differences in gonadotropic hormone release and instead
pointed to the brain as the site of sexual differentiation. Barra-
clough and his collaborators then showed that a single injec-
tion of testosterone propionate given shortly after birth
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
caused a permanent absence of cyclicity in female rats
(Barraclough and Leathem, 1954; Barraclough, 1961). After
identifying the anterior preoptic area (POA) as the site respon-
sible for cyclicity in gonadotropic hormone release, they
concluded: “the anterior preoptic area is undifferentiated at
birth with regard to its subsequent control of gonadotropin
secretion.” They also concluded that in the absence of testos-
terone the anterior POA differentiates to sustain cyclicity,
whereas in the presence of testosterone it “becomes refractory
to both intrinsic and extrinsic activation, and the more tonic
type of male gonadotropin secretion is observed” (Barraclough
and Gorski, 1961). We now know that in response to gonado-
tropin-releasing hormone (GnRH) from the anterior POA, the
pituitary synthesizes and releases the gonadotropins, luteiniz-
ing hormone (LH), and follicle-stimulating hormone. Both
act at a distant target, the gonads, and are essential links in
the hypothalamic–pituitary–gonadal axis.
As with sex behavior, the female pattern, defined as the
ability to respond to an increase in estradiol level with a surge
in LH release (positive feedback), is the default, and the male
pattern (defined as lack of positive feedback to estradiol) is
induced by the actions of testicular steroids during develop-
ment. When systematically compared, however, the sensitive
period for steroid-mediated behavioral defeminization ends
earlier than the sensitive period for defeminization of gonado-
tropin secretion (Diaz et al., 1995).
There are also interesting sex differences in the secretion of
other pituitary hormones, including prolactin and growth
hormone (GH). For example, males have more frequent and
higher amplitude pulses of GH than females (Norstedt and
Palmiter, 1984), and these differences are determined at least
in part by gonadal secretions during perinatal life (Jansson
et al., 1984). The sex difference in GH secretion has important
implications for liver function and body weight: about
75–85% of the genes in the liver exhibit a sex difference in
expression (Rinn and Snyder, 2005), and these differences are
indirectly driven by the sex difference in GH secretion pattern.
Thus sexual differentiation of the secretion pattern of the ante-
rior pituitary has lifelong consequences for the entire body.
5.01.2.4 There Are Sex Differences in Disease Susceptibility
The literature on human neurological and psychiatric disorders
demonstrates some of the most compelling evidence for sex
differences in the brain. Many, if not most, neurological
diseases exhibit sex differences in incidence, severity, or disease
course (see Table 1). For example, men are much more likely
than women to be diagnosed with Parkinson’s disease
(Strickland and Bertoni, 2004; Shulman, 2007), while females
outnumber males in the incidence of Alzheimer’s disease and
multiple sclerosis (Fratiglioni et al., 1997; Barrett, 1999; Swaab
et al., 2003; Schwendimann and Alekseeva, 2007). Among
psychiatric disorders, women exhibit higher rates of anorexia,
bulimia, depression, and essentially all anxiety disorders
(Kessler et al., 1994; Kornstein et al., 2002; Nestler, 2002;
Altemus, 2006; Södersten et al., 2006), whereas men show
a greater incidence of gender identity disorder, early onset
schizophrenia, antisocial personality disorders, and conduct
disorders (Häfner et al., 1993; Kessler et al., 1994; American
Psychiatric Association DSM-IV-TR, 2000).
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Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning 7

Table 1 Many neurological and psychiatric disorders connectivity (e.g., axon projection patterns or synapse
exhibit a sex difference in incidence number), or neurochemistry (e.g., the expression of neuropep-
tides, neurotransmitters, or receptors).
Disorder Relative incidence

Developmental disorders 5.01.2.5.1 Neural Structure

Attention-deficit hyperactivity M>F Several of the earliest recognized and most intensively studied
Asperger’s syndrome M>F neural sex differences were differences in the volume of cell
Autism M>F groups. For example, as first described by Nottebohm and
Dyslexia M>F Arnold (1976) over 40 years ago, there are striking sex differ-
Gilles de la Tourette M>F ences in the volume of several nuclei controlling singing in
Mental retardation M>F
canaries and zebra finches. These volume differences result
Stuttering M>F
from sex differences in cell number, soma size, and dendritic
Adult-onset disorders
Alzheimer’s disease F > Ma complexity (reviewed in Arnold, 1992). Similarly, the sexually
Amyotrophic lateral sclerosis M>F dimorphic nucleus of the medial preoptic area (SDN-POA) of
Anorexia nervosa F>M rats is several times larger in volume in males than in
Antisocial personality M>F females, and this sex difference is also reflected by sex
Bulimia F>M differences in the number and size of neurons in this region
Conduct disorder M>F (Gorski et al., 1978, 1980). Homologous sex differences in
Depression F>M cell groups of the POA are seen in many mammals, including
Gender identity disorder M>F gerbils, guinea pigs, ferrets, sheep, hyenas, monkeys, and
Multiple sclerosis F>M
humans (reviewed in Forger, 2001).
Parkinson’s disease M>F
One of the best-understood sex differences in neural struc-
Schizophrenia (early onset) M>F
Sleep apnea M>F ture involves a sexually dimorphic neuromuscular system.
The spinal nucleus of the bulbocavernosus (SNB) is a cluster
a
Estimates of gender-specific incidence for Alzheimer’s disease of motoneurons in the lumbar spinal cord that innervates stri-
vary. After accounting for sex differences in life span, the risk ated muscles involved in copulation. Although first described
appears to be between 1.5 and 3.0 times higher in women.
in rats (Breedlove and Arnold, 1980), males of many species
have more SNB motoneurons (or the homologous motoneu-
rons in Onuf’s nucleus) than do females. Perhaps owing to
Perhaps most striking, essentially all neurodevelopmental disor-
the relative simplicity of the circuit and the accessibility of
ders are more prevalent in boys than in girls (Gissler et al., 1999;
motoneurons for experimental manipulation, more may be
Zup and Forger, 2002; Swaab et al., 2003; Rutter et al.,
known about cellular mechanisms involved in sexual differen-
2003). This is true for cerebral palsy, mental retardation,
tiation of the SNB than for any other neural sex difference
dyslexia, stuttering, phonological disorder, sleep terrors, atten-
(reviewed in Sengelaub and Forger, 2008). Well-established
tion-deficient disorder, Asperger’s syndrome, autism, and tic
sex differences in volume and/or cell number also exist in the
disorders. In fact, of all the disorders of infancy or childhood
medial amygdala (MeA; reviewed in Cooke, 2006), anteroven-
listed in the Diagnostic and Statistical Manual of Mental Disorders
tral periventricular nucleus of the hypothalamus (AVPV), and
(DSM-IV-TR), published by the American Psychiatric
principal nucleus of the bed nucleus of the stria terminalis
Association, the only exception to the pattern is Rett
(BSTp; reviewed in Forger, 2006). In the AVPV and BSTp, the
syndrome, which affects girls almost exclusively. However,
differences are sexually differentiated, as the sex differences in
this is due to the fact that the disorder is X chromosome-
volume and cell number can be reversed by appropriate
linked; male fetuses inheriting the Rett syndrome mutation
gonadal steroid hormone treatments during development;
are more severely affected than are female fetuses, and rarely
MeA volume, on the other hand, may be an example of a sex
survive to term (Schanen, 1999).
difference due principally to acute effects of hormones (Cooke
The male bias in neurodevelopmental disorders is especially
et al., 1999).
notable since sex differences in childhood disorders are less
likely than are adult-onset disorders to be due to differences
5.01.2.5.2 Glial Cells
in circulating hormones or to experience, and are more likely
Structural sex differences in the nervous system are not limited
to reflect pre- or postnatal brain development. The bias is not
to neurons. For example, astrocytes in the arcuate nucleus and
well understood and strikes us as one of the most understudied
POA of rats are much more complex in males than in females,
areas in psychiatry or medicine.
and this sex difference, too, is dependent on differential expo-
sure to gonadal steroid hormones in males and females
(reviewed in McCarthy, 2008). Another cell type, the microglia,
5.01.2.5 There Are Sex Differences in Neural Structure, Glial
which is actually an immune cell, is also found in different
Structure, and Connectivity
forms in males and females in some brain regions, and this
Many of the sex differences in behavior, physiology, and neuro- difference is determined by early gonadal steroid exposure. In
logical disorders described above can presumably be traced to the POA, microglia are critical contributors to the establish-
sex differences in the nervous system. These neural sex differ- ment of a higher density of dendritic spine synapses on
ences may take many forms, but can be broadly conceptualized neurons of males (Lenz et al., 2013), highlighting the impor-
as differences in structure (e.g., the size or number of neurons), tance of cell-to-cell communication.

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8 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
5.01.2.5.3 Synaptic Connectivity
A persistent problem in the study of sex differences in the brain
is the impact of technical limitations directing the nature of the
research, and this is particularly true in the study of synaptic
connectivity. There are several ways to quantify synapses. The
gold standard is electron microscopy as this is the only way
to see synaptic vesicles and thereby say definitively that
a synapse is truly present. This technique was used very effec-
tively by Arai and Matsumoto in the early 1980s in a series of
reports revealing profoundly sexually dimorphic patterning in
the arcuate nucleus, ventromedial nucleus of the hypothalamus
(VMN), and amygdala (for review, see Matsumoto, 2000). The
down side of quantitative electron microscopy is the small
region sampled and the labor involved, making it difficult to
examine entire brain regions or to compare large numbers of
animals. Golgi impregnation of neurons offers a way to more
quickly quantify the number or density of dendritic
spines and to completely reconstruct dendritic trees. Dynamic
changes in spine density across the life span and a sex difference
in branching were revealed in the VMN using this technique
(Pozzo-Miller and Aoki, 1991; Mong et al., 1999; Todd et al.,
2007). Disadvantages of Golgi impregnation, however, are
that only dendritic spines, and not synapses, can be measured,
and there is also the risk that what is presumed to be a random
selection process (only a subset of neurons take up the Golgi
material), may actually reflect a bias that we do not yet under-
stand. Quantitative Western blot for synaptic proteins can serve
as a proxy measure of synapses, and when used to assess the
protein spinophilin, which is heavily localized to dendritic
spines, has proved useful in cross-group comparisons
(Amateau and McCarthy, 2002a). Again, however, only spine
synapses are detected, leaving completely unexplored the
hormonal regulation of axosomatic synapses. In summary,
no single technique is perfect, and our ability to quantify
specific types of synapses across groups is limited. Nonetheless,
we actually do know a fair amount about sex differences in
synaptic profiles, which are proving to be robust and may be
of profound significance (see Section 5.01.3.4.3).
5.01.2.6 There Are Sex Differences in Neurochemistry
There is a wealth of information on sex differences in neuro-
chemistry. In fact, some of the earliest reports on sex differences
in the brain concerned higher serotonin levels in female versus
male brains (Kato, 1960). Perusal of PubMed suggests that at
least a quarter of the reports on sex differences in the brain
concern sex differences in neurochemistry (e.g., levels and
distribution of neurotransmitter and their receptors, steroid
receptors, enzymes involved in steroid and neurotransmitter
synthesis and catabolism, growth factors, and cytokines).
Several concrete examples are offered below.
5.01.2.6.1 Sex Differences in g-Aminobutyric Acid Signaling
during Development
Contrary to its ubiquitous inhibitory effect in the mature brain,
g-aminobutyric acid (GABA) has emerged as the dominant
source of excitation in the developing brain. This is due to
a reversal of the transmembrane chloride concentration
gradient, resulting in depolarization of the membrane poten-
tial upon opening of the GABAA receptor which is a chloride
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
permeable ionophore. Estrogens enhance the depolarizing
action of GABA by increasing intracellular chloride. As a result,
when the channel opens, the shift in the membrane potential is
greater than it would be without the action of estradiol, and
there is a stronger and more enduring opening of voltage-
gated calcium channels (Nunez and McCarthy, 2008). Since
males have higher estradiol content in the brain during the
developmental period when GABA action is depolarizing,
they also have greater intracellular calcium influx. This can be
both trophic and promote the growth of axons, dendrites,
and synapses, but can also be excitotoxic if in excess (Auger
et al., 2001; Nunez et al., 2003). Moreover, early postnatally,
testosterone and its metabolite estradiol elevate the levels of
GABA and its synthesizing enzymes, GAD 65 and GAD 67, in
several hypothalamic nuclei and the hippocampus of male
rats (Davis et al., 1996c, 1999). So males both make more
GABA and respond to it more strongly, a scenario which
increases excitation broadly in the developing male brain and
may contribute to the greater vulnerability of the male to dele-
terious effects of insult or injury.
5.01.2.6.2 Sex Differences in Neurotransmitters in Adulthood
Numerous sex differences in neurotransmitter and neurotrans-
mitter receptor expression have been reported throughout the
brain. These include modulatory systems not traditionally
linked with reproductive behavior, such as histamine neurons
in the hypothalamus, noradrenaline neurons in the locus
coeruleus, serotonin neurons in the dorsal raphe, and dopa-
mine in the substantia nigra and ventral tegmental area in
animals as well as humans (De Vries, 1990; De Vries and
Simerly, 2002; Cosgrove et al., 2007). The ways in which
neurotransmitter systems can vary are continuing to be discov-
ered. In a manner similar to that seen for depolarizing actions
of GABA during development, a component of the stress axis is
profoundly different in males and females as a result of
a change in sensitivity to corticotropin-releasing factor (CRF),
which acts as a modulatory neuropeptide within the brain.
Adrenergic neurons of the locus coeruleus express receptors
for CRF, and when an animal is stressed, the receptors are inter-
nalized away from the membrane in males but trafficked to the
membrane in females (Bangasser et al., 2010). The impact of
this differential distribution of CRF receptor is further ampli-
fied by ramped-up noradrenergic transmission in females
(Bangasser, 2013; see also Forger et al., 2015b for further
discussion). This example is probably one of many as
researchers become increasingly aware of the importance of
sex as a biological variable in modulating the nervous system.
Because of the widespread influences of these modulatory
systems, the impact of the sex differences could be profound
and may be related to the development of behavioral disorders
that show sex differences in incidence, as described in Section
5.01.2.4 (see also Cosgrove et al., 2007; Becker and Hu, 2008).
Sex differences in neurochemistry may also offer clues as to
the significance of sex differences in brain structure. An
example of this concerns the AVPV. Female mice have more
neurons in the AVPV than males (Forger et al., 2004). A subset
of these neurons express Kiss1 mRNA and its gene product,
kisspeptin, and females mice have 10 times as many kisspeptin
neurons in the AVPV as do males (Clarkson and Herbison,
2006). In rats, the sex difference in kisspeptin expression is
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Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
close to absolute, with males expressing almost no kisspeptin
(Kauffman et al., 2007). In both species, this sex difference
depends on early exposure to gonadal steroids (Kauffman
et al., 2007; Gonzalez-Martinez et al., 2008). Kisspeptin-
containing projections are found in close apposition to
GnRH neurons (Clarkson and Herbison, 2006), and kisspeptin
triggers an LH surge in both rats and mice by stimulating GnRH
neurons (Gottsch et al., 2004; Irwig et al., 2004). Thus, the sex
difference in kisspeptin cell number may contribute to the sex
difference in LH release described in Section 5.01.2.3. Interest-
ingly, estradiol treatment increases Kiss1 mRNA in the AVPV
but decreases it in the arcuate nucleus, which contains a nondi-
morphic group of kisspeptin neurons; it has been suggested
that the former group contributes to the surge and the latter
to the negative feedback of gonadotropin secretion (Dungan
et al., 2006). It is tempting to speculate that the higher number
of kisspeptin neurons in the AVPV gives females the ability to
respond to estradiol treatment with an LH surge, an ability
that males lack. Kisspeptin is probably not the only factor,
however. For example, female rats also have twice as many
neurons in AVPV that coexpress markers of both glutamatergic
and GABAergic signaling (Ottem et al., 2004). These dual
phenotype neurons may synapse on GnRH neurons to control
the switch from negative to positive feedback of estradiol that
occurs around ovulation (see also Section 5.01.3.4.1).
5.01.2.6.3 Sex Differences in Steroid Receptor Expression
Sex differences in gonadal steroid receptor expression have
been reported for several forebrain nuclei of adult mice and
rats. For example, androgen receptor expression is greater in
the BSTp of males than of females (Simerly et al., 1990a;
Roselli, 1991; Shah et al., 2004). In this case, the mechanism
may be related to the same mechanism that causes the differ-
ence in overall cell number in the BSTp (i.e., cell death) because
mice lacking the prodeath gene Bax do not show a sex differ-
ence in overall cell number (Forger et al., 2004; see Section
5.01.3.3.), or in the number of androgen receptor expressing
cells in the BSTp (Holmes et al., 2009a). For other brain areas,
different mechanisms seem to apply. For example, estrogen
receptor immunoreactivity is about 20 times greater in the
BSTp of adult, gonadally intact female mice than of males,
and in this case the difference is due to the acute suppression
of receptor expression by testicular hormones (Kelly et al.,
2013). Estrogen and progesterone receptor binding and/or
mRNA expression in the VMN, preoptic periventricular, and
medial preoptic nuclei also appear to be higher in females
(Simerly et al., 1990; De Vries and Simerly, 2002), but in
many of these cases, it is unclear whether the sex difference is
caused by programming or acute effects of gonadal hormones.
Regardless of the underlying mechanisms, if expression of
hormone receptors causes differences in hormone sensitivity,
they are likely to contribute to sex differences in neural
function.
This may also be the case for sex differences in steroid
receptor expression during development. Gonadal hormones
cause sex differences in the expression of estrogen receptors,
androgen receptors, and aromatase in the developing hypothal-
amus (MacLusky et al., 1985; DonCarlos and Handa, 1994;
McAbee and DonCarlos, 1998), suggesting that the hormones
influence their own subsequent effectiveness. A special case is
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
9
the almost absolute sex difference in the expression of proges-
terone receptors in the medial POA (mPOA) around birth.
Perinatally, male rats express a much higher level of proges-
terone receptors than females (Wagner et al., 1998). This
suggests that exposure of pups to maternal progesterone during
gestation may have different consequences in males than in
females. The role of this sex difference in sexual differentiation
of the mPOA is unclear, but treating males postnatally with the
progesterone receptor blocker, RU486, reduced male sexual
behavior in adulthood (Lonstein et al., 2001). Although other
interpretations are possible, these data are consistent with the
idea that activation of the differently expressed progesterone
receptor is involved in sexual differentiation of the brain.
5.01.2.7 Androgens as well as Estrogens Play a Role
in Sexual Differentiation of the Brain
In early work on sexual differentiation, testosterone was shown
to masculinize and defeminize the behavior of female rats or
guinea pigs (Phoenix et al., 1959; Beach et al., 1969). Testos-
terone is an androgen, which acts primarily by binding to intra-
cellular androgen receptors. Thus, the early literature
understandably often discussed effects of ‘androgenization,’
or the ‘androgenized’ female. However, it soon became evident
that estrogens, in particular 17b estradiol (hereafter, ‘estra-
diol’), mimicked many effects of testosterone, and usually at
lower doses. This was somewhat puzzling because all mamma-
lian fetuses are exposed to elevated estrogens, produced by the
maternal ovaries; how are female fetuses protected from the
effects of estrogen exposure? The apparent paradox was
resolved by two suggestions: first, that a-fetoprotein, an
estrogen-binding protein produced at high levels in developing
animals, binds circulating estrogens and prevents them from
entering the brain. Second, the aromatization hypothesis
proposed that testosterone, released by the perinatal testes,
crosses the blood–brain barrier and is converted to estradiol
in target cells by the aromatase enzyme; estradiol then acts
via estrogen receptors to masculinize the brain and behavior
(for reviews, see Goy and McEwen, 1980; Lephart, 1996).
The importance of a-fetoprotein has recently been
confirmed by examining the brain and behavior of a-fetopro-
tein knockout mice. Females that develop in the absence of
functional a-fetoprotein show very low, male-like levels of
lordosis as adults and high levels of male sexual behavior, as
well as male-like neuroendocrine responses (Bakker et al.,
2006; González-Martínez et al., 2008; Taziaux and Bakker,
2015). In support of the aromatization hypothesis, brain
masculinization can be blocked by treating neonatal male
rats with an aromatase inhibitor or estrogen antagonist (e.g.,
Döhler et al., 1986); the SDN-POA is feminized in male rats
treated with antisense oligonucleotides to the estrogen
receptor (McCarthy et al., 1993); and the behavior of mice is
demasculinized or feminized by a knockout of the estrogen
receptor a or b gene, respectively (Kudwa et al., 2006). These
findings are bolstered by reports that nonaromatizable
androgens, such as dihydrotestosterone, in many cases do not
mimic effects of testosterone on the brain or behavior.
The SNB has long been known as an exception to the rule
that estrogenic metabolites of testosterone are responsible for
masculinization and defeminization of the rodent brain and
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10 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
behavior. SNB cell number in rats was the first neural sex differ-
ence shown to differentiate under the control of androgens,
acting via classical intracellular androgen receptors (Breedlove
and Arnold, 1981; Breedlove et al., 1982). It is now clear that
androgens act at virtually every level of the SNB system, in
development and throughout adult life (reviewed in Sengelaub
and Forger, 2008). Although for some time the SNB was
considered the only morphological sex difference that depends
on androgens and androgen receptors, more recently new roles
of androgens have been identified. For example, male rats lack-
ing functional androgen receptors exhibit partial demasculini-
zation of the volume of the BSTp, VMN, and posterodorsal
MeA (Morris et al., 2005; Durazzo et al., 2007; Dugger et al.,
2007). Androgens may also maintain normal hippocampal
structure in male rats and monkeys (MacLusky et al.,
2006) and play a role in the sexual differentiation of vaso-
pressin innervation in the brain (Han and de Vries, 2003).
This should not come as a surprise, since it has been known
for many years that androgens are required for complete
masculinization of behavior, including juvenile play (Meaney,
1989) and male sexual behavior in some strains of rats (e.g.,
van der Schoot, 1980; the full masculine phenotype can be
achieved with neonatal estradiol treatment in other strains,
however, see Amateau and McCarthy, 2004; Todd et al.,
2005). More recently, androgen receptors have been implicated
in the sexual differentiation of social and olfactory preferences
in mice (Bodo and Rissman, 2007, 2008) and the neuroendo-
crine response to a mild stressor in rats (Zuloaga et al., 2011).
Importantly, although estrogens are critical for differentia-
tion of the rat and mouse brain, androgens may play a more
central role in sexual differentiation of the primate brain (see
Wallen, 2005; Thornton et al., 2009). Human a-fetoprotein
does not have a high affinity for estrogens (Swartz and Soloff,
1974), suggesting that the brains of human fetuses of both
sexes are exposed to estrogens. Experiments of nature also
cast doubt on the importance of estrogens for sexual differenti-
ation in primates. About half a dozen men with inactivating
mutations of the aromatase gene have been reported, and all
appear to be heterosexual with normal sexual function
(reviewed in Jones et al., 2006), although overall libido may
be reduced (Carani et al., 2005). A role for androgen receptors
in sexual differentiation of humans is supported by the obser-
vation that genetic males (XY) with an inactivating mutation of
the androgen receptor gene are not only phenotypically female
with respect to body type, but also female-typical in terms of
gender identity, sexual orientation, and gender role (Hines
et al., 2003; Hines, 2008).
5.01.2.8 Sex Chromosome Complement Contributes
to Sexual Differentiation
Alfred Jost’s proposal in the 1940s (Jost, 1947) that testes are
crucial for the development of the male phenotype, and that
without them the body develops in a female direction, has
been driving research in the field of sexual differentiation for
more than half a century. The more recent discoveries of genes
that direct the differentiation of the primordial gonad into the
testis (Koopman et al., 1991) only require reformulating the
theory on the mechanisms of sexual differentiation into: “sex
chromosomal genes determine the differentiation of the
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
gonads into testes or ovaries; the resulting differences in
gonadal secretions cause all other differences.”
The Jost doctrine, however, may not be as generally appli-
cable as first thought. Several sex differences, for example, in
bird plumage, birdsong, and the development of pouch versus
scrotum in wallabies, are likely to be caused directly by sex
chromosomal genes and not by differences in gonadal
hormones (Arnold, 1996). To test whether this may also apply
to sex differences in the brain, Arnold and colleagues intro-
duced a model system (known as the Four Core Genotypes)
that can distinguish between direct actions of sex chromosomal
complement (XX vs XY) and gonadal hormones on sexually
dimorphic traits (De Vries et al., 2002). Female mice with the
familiar XX genotype were crossed with males with an XY

Sry genotype. These mice lacked the Sry gene on the Y chromo-
some, which normally directs the growth of the testis, but
developed a male phenotype anyway because they had an Sry
transgene inserted on an autosomal chromosome. The
offspring included XX and XY
mice of either gonadal sex
depending on whether they inherited the autosomal Sry trans-
gene or not. Comparing XX and XY
mice within sex (defined
on basis of gonad) revealed a number of differences that appear
to be caused by differences in sex chromosomal complement
independent of gonad type. For example, vasopressin expres-
sion is more masculinized in XY females than in XX females,
and in XY males than in XX males (De Vries et al., 2002). Addi-
tional effects on aggression, habit formation, body weight, and
response to brain injury have since been detected (for reviews,
see Arnold and Chen, 2009; Forger et al., 2015a). Thus we now
know that while hormones act broadly throughout the brain,
every cell in the brain has a genetic sex as a result of chromo-
some complement (Figure 2).
The introduction of the Four Core Genotypes model
provides an important new tool for distinguishing roles of
hormones and genes in establishing or maintaining sex differ-
ences. While first viewed as a means for identifying specific
genes on the X or Y chromosome that direct sex-specific devel-
opment, the model has led to far more nuanced interpretations
of the ways that the sex chromosomes contribute to sex differ-
ences. One example concerns the X chromosome. In females,
one X chromosome in each cell is largely silenced in a process
referred to as random X inactivation, but this process is not
complete, with as many as 15% of X genes in humans escaping
inactivation (Berletch et al., 2010). This means that female cells
have a ‘double dose’ of some X genes. But there are also
multiple other means by which the X can impact an organism.
These include parent of origin allelic variation (also known as
imprinting), cellular mosaicism (a result of random inactiva-
tion of the maternal versus paternal X), and the creation of
an epigenetic sink whereby the inactive X sequesters a dispro-
portionate amount of the proteins that regulate gene expres-
sion (see for review Arnold et al., 2016).
The importance of the number of X chromosomes
becomes apparent in consideration of XO women, referred
to as Turners Syndrome, and XXY men, referred to as Kleinfel-
ter’s Syndrome in humans. It is difficult to determine,
however, whether the phenotypic differences in these
syndromes are due to an altered sex chromosome comple-
ment or due to changes in gonadal hormones, because in
both cases there is a retardation or regression of gonadal
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Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning 11

Figure 2 Both hormones and chromosome complement contribute to sex differences. The mode and means of hormone action have greatly
expanded in the past decade as we now know that steroids can act both via nuclear transcription factor receptors and membrane receptors. Steroids
can also be locally synthesized within the brain, which challenges us to understand what truly local levels of steroid concentrations are. We now also
appreciate that every cell in the brain has a sex due to the chromosome complement being either XX or XY in mammals. Many effects of chromo-
some complement have been identified but precisely how they exert these effects is still being determined. Interactions between steroid hormones
and chromosome complement further add to the complexity.

development. Arnold and colleagues have addressed this
confound with a variant on the Four Core Genotypes model
called the Sex Chromosome Trisomy model which produces
XX, XY, XXY, and XYY mice. By focusing on genes that escape
X inactivation and would therefore be elevated in XXY versus
XYY or XY males they hope to identify which genes explain the
multiple phenotypic effects evident in individuals with sex
chromosome aneuploidies (Arnold et al., 2016).
A different approach is to examine naturally occurring sex
chromosome aneuploidies in humans and ask how they
impact brain parameters compared to normative controls.
Cortical thickness asymmetries are a well-documented pheno-
typic variable in the frontal and occipital cortex of humans
(Lin et al., 2015) and in some regions are found only in males.
The number of sex chromosomes impacted many of the
cortical thickness asymmetries observed in a highly region-
specific and divergent manner for X versus Y, but quite inter-
estingly did not alter those regions in which there was a sex
difference. Moreover, the same aneuploidy (i.e., an extra
X chromosome) had different effects in males versus females,
highlighting the complex interplay of genetic and gonadal sex
(Raznahan et al., 2015).
5.01.2.9 Sex Differences Are Context Dependent
If the pioneers of our field had chosen to work with mice rather
than with rats and guinea pigs, things might have been very
different. Mice do not show consistent sex differences in
male sexual behavior. For example, several reports suggest
that female mice treated with testosterone or estradiol in adult-
hood show mounting and thrusting in response to a receptive
female at levels that are similar to, or even higher than, those
of males (Wersinger et al., 1997; Jyotika et al., 2007), although
other groups find clear a clear sex difference of male sexual
behavior in mice (Vale et al., 1973; Bakker et al., 2006). Strain
differences may account for this variability, but testing condi-
tions are also likely to play a role. For example, sex differences
in response to sleep deprivation depend on whether animals
are stressed or not, with stressed animals showing bigger sex
differences in sleep recovery than unstressed animals (Koehl
et al., 2006). Developmental history may also contribute to
variability. Male prairie voles are spontaneously parental,
whereas virgin females avoid or attack pups (Lonstein and
De Vries, 1999a). This sex difference depends on rearing condi-
tions, however, because if females are raised to adulthood in
the presence of their parents, they too are parental (Lonstein
and De Vries, 2001). Even what appear to be very subtle
changes in developmental history can make a big difference.
In one study, prairie voles showed sex differences in partner
preference formation, or not, depending on whether they
were transported by hand or in a plastic cup during routine
cage changing (Bales et al., 2007).
Context also plays a role in sex differences in human
behavior. For example, one of the most consistent cognitive
sex differences is found in the Mental Rotations Test. In this
famous test, subjects have to mentally rotate a block figure to
match it with a congruent object in a line-up of similarly
shaped but not identical objects. Males outperform females
on this task in a wide range of studies. Interestingly, a much
smaller male advantage is seen if instead of interconnected
cubes, the figures take on a human shape (Alexander and
Evardone, 2008). Similarly, the male advantage in certain
math tests is eliminated or reduced if female subjects are told
in advance that females do as well or better than males on these

Hormones, Brain and Behavior, Third Edition, 2017, 3–32

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12 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning

tests, but sex differences are exacerbated if females are told the
reverse (for review, see Spelke, 2005). In all of these cases
context, whether defined as the conditions during the test or
those leading up to the test, determines the outcome. To
what extent these different behavioral outcomes are reflected
in context-dependent brain structure or neurochemistry is not
known. But when studying sex differences, it is important to
frame any findings as to whether they are persistent or tran-
sient, context dependent or independent, or the result of direct
effects of hormones and chromosome complement or indirect
as a function of somatic sex differences or in the case of
humans, gender differences (Figure 3).
5.01.2.10 Sexual Differentiation Depends on Four Key
Processes
As is evident from the preceding sections, a large number of sex
differences of every stripe have been identified in the mamma-
lian brain. The sheer number and variety of the reported differ-
ences can be bewildering. It may be useful to recognize that
sexual differentiation of any neural trait must, in principle, be
due to effects on one (or a combination) of four basic cellular
processes: cell birth, cell migration, cell death, and the differen-
tiation of neural circuits (Figure 4). This last category includes
the morphological differentiation of cells (i.e., dendritic extent,
axon outgrowth, spine, and synapse formation) as well as
neurochemical differentiation (i.e., which neurotransmitters,
neuropeptides, or hormone receptors are expressed).
To understand at a mechanistic level how hormones differ-
entiate male and female brains, it is important to identify
which of the four key processes are involved. Disentangling
the various possibilities can be surprisingly difficult, however.
For example, assume that a sex difference is found in which
males have more neurons than do females that express neuro-
transmitter ‘x’ in a brain region of interest. If this sex difference
persists even after adult hormone levels are made equivalent,
but can be reversed by developmental exposure to steroids,
we would say that the trait is sexually differentiated, or
programmed, by gonadal steroids. Such a difference could arise
because steroid hormones cause more neurons destined for the
brain region of interest to be born in males (i.e., a hormonal
effect on neurogenesis), or because steroids affect the move-
ment and aggregation of cells, such that more cells overall
come to be associated with the brain region of interest in males.
The difference could also arise because fewer cells die in males
(an effect on cell death), or because gonadal steroids cause
existing cells to express neurotransmitter ‘x’ (i.e., a hormonal
effect on neurochemical differentiation).
Much of the recent progress in understanding sexual differ-
entiation comes from determining which of these basic devel-
opmental processes is controlled by gonadal steroids. At this
point, there is firm evidence that cell death and the morpholog-
ical differentiation of circuits contribute to permanent neural
sex differences (see Section 5.01.3.3), and in at least one system
it now seems clear that hormones direct neurochemical differ-
entiation (see Section 5.01.3.4.4). There are also now estab-
lished incidences of sex differences in neuro- and glial-genesis
in the developing hippocampus and amygdala. Some of this
evidence is presented below.
5.01.3 Recent Progress in Understanding the Four
Key Processes
5.01.3.1 Neurogenesis
For some neural sex differences, the birth of the cells in ques-
tion is complete before the time the gonads differentiate. In
these cases, it is very unlikely that gonadal hormones could
cause the sex difference by controlling neurogenesis, and so
this mechanism can effectively be eliminated. Motoneurons
that populate the SNB, for example, undergo their final mitosis
by the 14th day of gestation in rats (Breedlove et al., 1983), yet
testosterone is not produced by the differentiating testes until
several days later. Thus, it is very unlikely that in this system
gonadal steroid hormones could create a sex difference in cell
number via an alteration of neurogenesis. It is even more

Figure 3 Not all sex differences are made equal. Sex differences come in many varieties. They may be dimorphic, i.e., in distinct forms in each sex,
or they may just vary along a continuum. Some sex differences only emerge under certain conditions and are therefore context dependent, while
others will disappear under certain conditions as the two sexes converge on the same endpoint. Early-life programming of sex differences usually
results in persistent changes, while others appear only transiently in response to the changing hormonal milieu.

Hormones, Brain and Behavior, Third Edition, 2017, 3–32

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Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning 13

Figure 4 Four key principles by which sex differences can be established. There are four major pathways by which neuroanatomical sex differences
can be established. The first is cell genesis, the birth of new cells that will eventually become neurons or glia. To date, there is relatively little
evidence for neurogenesis being an important contributor to the establishment of sex differences in the numbers of neurons or glia in a particular
brain region, but this is beginning to change with new evidence emerging in the telencephalon and in the adult brain. A second potential source for
sex differences in the numbers of neurons in a particular brain region is differential migration into that region. Stuart Tobet and colleagues have
observed hormonally modulation of migratory paths of hypothalamic neurons, but whether this ultimately contributes to sex differences remains to
be determined. The third and one of the best documented sources of sex differences in the number of neurons in a brain region is differential cell
death, with either more cells dying in females, as observed for the sexually dimorphic nucleus, spinal nucleus of the bulbocavernosus, and bed
nucleus of the stria terminalis, or more cells dying in males, as seen in the anteroventral periventricular nucleus. These sex differences in cell death
are attributed to the actions of the gonadal steroids, testosterone, and estradiol, but the precise cellular mediators remain unknown. The fourth key
principle is broadly defined as cellular differentiation and refers to both the neurochemical phenotype of particular neurons and the nature and extent
of synaptic connections made by particular neurons. Sex differences in both neurochemical phenotype and synaptic connectivity are profound, and
advances are being made in identifying the particular cellular mechanisms by which these differences are achieved.

unlikely that postnatal exogenous steroid treatments, which
can masculinize SNB cell number, could affect the birth of
SNB cells.
In other cases, however, gonadal steroid production and the
birth of neurons destined for the area in question may overlap.
This is true for the SDN-POA, for example (Jacobson and
Gorski, 1981; Davis et al., 1996a). Using tritiated thymidine
or bromodeoxyuridine to label dividing cells, one can test for
sex differences in neurogenesis. There are pitfalls, however,
and a sex difference in the number of BrdUþ or thymidine-
labeled cells at sacrifice does not necessarily mean that
neurogenesis was different. For example, if the same number
of cells are born in males and females, but these cells migrate
to different brain areas or die at different rates in males than
in females, a sex difference in labeled cells will result, but
does not reflect a sex difference in neurogenesis. Investigators
have addressed this problem by examining the number of
cells labeled after very short survival times (i.e., presumably
before cell death or migration could occur). Using this type
of analysis, perinatal neurogenesis has been eliminated as
important for sexual differentiation of the SDN-POA or AVPV
(for review, see Forger, 2006).
The hippocampus is one brain region where a sex difference
in early-life neurogenesis has been clearly demonstrated. After
an injection of BrdU at birth, male rats have about twice as
many labeled cells in the hippocampus as do females and,
within in the CA1 region, many of the newly generated cells
become neurons (Zhang et al., 2008; Bowers et al., 2010).
This sex difference in neurogenesis is due primarily to endoge-
nously produced estrogens because the administration of an
aromatase inhibitor or estrogen receptor antagonist at birth
decreases the generation of new cells in males while having
no effect in females (Bowers et al., 2010). Since sex differences
in cell number in the hippocampus of adult rats are small, the
greater cell genesis in males presumably is offset by increased
cell elimination at some point in life.
A sex difference in cell genesis has also been observed in the
developing amygdale; only in this instance the number of
newborn cells is greater in females than males (Krebs-Kraft
et al., 2010). Some of these newborn cells are destined to be
astrocytes while others will become neurons. Evidence suggests
that a sex difference in endocannabinoid tone in the devel-
oping amygdala is the determinant of the greater number of
newborn cells in the females. Males have a higher endocanna-
binoid tone than females in this brain region, and treating
females with mimics of endocannabinoids reduces the number
of newborn cells to that seen in males. Determining if gonadal
steroids modulate endocannabinoid levels is an important next
step in identifying the cellular mechanisms by which this sex
difference occurs.
The genesis of new cells, some of which are neurons, has also
been observed in the AVPV and MeA of adolescent rats; this ‘late’

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14 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
neurogenesis may contribute to the development or mainte-
nance of neuroanatomical sex differences (Ahmed et al., 2008).
5.01.3.2 Cell Migration
Many cells in sexually dimorphic neural regions of mice and
rats (e.g., the POA, BST, AVPV, and SNB) have migrated to their
respective location prior to testis differentiation and the time of
the perinatal sex difference in testosterone synthesis (Bayer,
1987; Bayer and Altman, 1987; Han and De Vries, 1999); the
gross position of these cells is therefore unlikely to be affected
by gonadal hormones. However, cell addition in some brain
regions continues throughout perinatal development (see
discussion in Gotsiridze et al., 2007), and even for early-
arriving cells, gonadal steroids could cause subtle changes in
cell position within a general brain region. For example, adult
male and female rats have similar numbers of estrogen receptor
b-expressing cells in the AVPV, but these cells are more medially
located in females (Orikasa et al., 2002). This sex difference in
the position of cells, however, is not necessarily due to sex
differences in migration. For example, if testosterone increases
the survival of cells in the lateral AVPV, then males will wind up
with more laterally positioned cells. To conclude that cell
migration is sexually dimorphic and/or regulated by gonadal
steroids, therefore, more direct evidence is necessary.
Stuart Tobet and colleagues have addressed this issue using
video microscopy to follow the movements of neurons in orga-
notypic brain slices. In an analysis of the POA/anterior hypo-
thalamus (AH) of embryonic mice, sex differences in the
migratory pathway of labeled cells were found, such that signif-
icantly more medial-to-lateral migration was seen in embry-
onic males than in females (Henderson et al., 1999), which
correlates with a transient sex difference in the location of
phenotypically identified cells in the POA/AH 2 days later
(Wolfe et al., 2005). In addition, the movement of neurons
in slice cultures of the POA/AH is affected by estradiol, but
not by DHT (Knoll et al., 2007), and antagonists to GABA
receptors may also influence cell migration in the hypothal-
amus (McClellan et al., 2008). This latter observation is
intriguing because it suggests that sex differences in neural
activity could influence cell positioning. Although the func-
tional significance of sex differences in cell migration is not
clear, one could imagine that the positioning of cells might
affect afferent input, cell–cell communication on a local scale,
or exposure to diffusible signals. In the case of the estrogen
receptor b-expressing cells in AVPV described above, for
example, the closer proximity of these neurons to the ventricle
in females could increase access to chemosignals in the cerebro-
spinal fluid.
The technical obstacles to obtaining direct evidence for sex
differences in neuronal migration have discouraged all but
a few hearty souls from pursuing this line of investigation.
Therefore, whether this key process is in fact a critical contrib-
utor to programmed sex differences in the brain remains to
be determined.
5.01.3.3 Cell Death
About half of the neurons originally produced during devel-
opment of the vertebrate nervous system are eliminated
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
during a perinatal period of naturally occurring cell death
(Oppenheim, 1991; Mosley et al., 2016). Gonadal steroids
can increase or decrease the rate of this cell death in some
neural regions, and the hormonal control of cell death is
currently the best-established mechanism for creating neural
sex differences in rodents (reviewed in Forger, 2006; Forger
et al., 2015a,b). It is not clear whether cell death actually is
the most important mechanism underlying neural sex differ-
ences, or whether we know a fair amount about the contribu-
tion of cell death to sexual differentiation because it is
relatively easy to find evidence for differential death in males
and females. For example, dying cells can be quantified
based on morphology (pyknosis), or using well-established
techniques such as TUNEL (terminal deoxynucleotidyl nick
end labeling) and immunohistochemistry for activated
caspase-3, which take advantage of biochemical markers of
apoptosis.
Although cell death is a well-established mechanism for
generating neural sex differences, if the evidence is viewed
with a critical eye, the case is clear in only a handful of cases.
These include some of the most ‘famous’ sex differences,
including the SNB, SDN-POA, BSTp, and the AVPV in rats
(Nordeen et al., 1985; Davis et al., 1996a; Chung et al.,
2000; Sumida et al., 1993). There is also some evidence for
a role of cell death in sexual differentiation of rat visual
cortex (Nuñez et al., 2001), the gerbil POA (Holman et al.,
1996), at least one birdsong nucleus (Kirn and DeVoogd,
1989), and frog laryngeal motoneurons (Kay et al., 1999).
In each of these cases, a sex difference in cell number in
adulthood is correlated with a sex difference in the number
of dying cells at some point in development. For example,
adult males have more SNB motoneurons than do females,
and females have more pyknotic cells in the SNB region
around the time of birth (Nordeen et al., 1985). Females
also have more dying cells than do males in the early
postnatal BSTp (Chung et al., 2000; Gotsiridze et al., 2007;
Ahern et al., 2013), and this correlates with greater BSTp cell
number in adult males. In both the SNB and BSTp,
testosterone treatments that eliminate the sex difference in
cell number in adulthood also eliminate the sex difference
in dying cells during early development (Nordeen et al.,
1985; Chung et al., 2000), adding weight to the conclusion
that differential cell death is the mechanism underlying
sexual differentiation.
There are limitations to this type of analysis, however. For
example, counts of the number of pyknotic, TUNEL-positive,
or caspase-3 positive cells are necessarily ‘snapshots’ of the
number of cells dying (or, more specifically, at a particular
stage of apoptosis) at a given moment. In order to determine
whether a sex difference in dying cells can account
quantitatively for a sex difference in neuron number in
adulthood, one would have to know how long a cell appears
apoptotic in the material under study. This variable has not
been determined for any sexually dimorphic region
(although see Nuñez et al., 2000) and estimates of how long
a cell takes to undergo apoptosis vary greatly. Thus, it is
difficult to rule out other mechanisms. For example, in the
rat locus coeruleus, adult females have more neurons than do
males, and cell death is greater in males on the day of birth
(Guillamon et al., 1988). One might be tempted to stop
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Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning 15

there and declare that the mechanism underlying the sex

difference has been demonstrated. However, a more detailed
study of developmental changes in cell number suggests that
the adult sex difference may actually result from
a postpubertal addition of cells in the locus coeruleus of
females (Pinos et al., 2001). Similarly, a greater number of
cells in visual cortex of males correlate with greater cell death
in this region in females postnatally, but a more complete
developmental profile suggests that the mechanism
accounting for the adult sex difference is complex (Nuñez
et al., 2002).
Genetically modified mice have been used to get around
some of these problems and to test more directly the contribu-
tion of cell death to neural sex differences. For example, Bax,
a prodeath gene of the Bcl-2 family is singularly important
for apoptosis in neural development, and deletion of this single
gene nearly eliminates perinatal cell death in many brain
regions (White et al., 1998; Ahern et al., 2013). By examining
Bax knockout mice, it is therefore possible to ask which neural
sex differences depend on differential cell death in males and
females. In mice lacking Bax, the number of cells in the SNB,
AVPV, and BSTp is significantly increased (Forger et al., 2004;
Jacob et al., 2005) and, more important, sex differences in total
cell number in each of these regions are eliminated (Figure 5).
This demonstrates that Bax protein is required for sexually
dimorphic cell death in the mouse forebrain and spinal cord.
Because the animals in these studies were gonadally intact,
Bax gene status overrides endogenous testosterone levels in
determining cell number. Thus, one obligatory gene in the
sexually dimorphic cell death pathway – Bax – has been
identified.
The study of mutant mice has also identified sex differences
Figure 5 The proapoptotic gene, bax, is required for sex differences
not dependent on Bax and demonstrates that the control of cell
in neuron number. (a) Cell death throughout the developing mouse
number may vary not only from region to region, but also
brain is dependent on the prodeath gene, Bax. Dying cells (black dots),
among subtypes of cells within a single region (see below, labeled by immunohistochemistry for activated caspase-3, are much
and Zup et al., 2003; Forger et al., 2004; Semaan et al., 2010; more numerous in newborn wild-type mice (Baxþ/þ) than in Bax
Gilmore et al., 2012). knockout mice (Bax
/
). (b) In adulthood, cell number in the BNSTp
The next question is how testosterone regulates cell death. was greater in wild-type males than in females. Deletion of the Bax
Estrogenic metabolites play the predominant role in sexual gene increased cell number overall and eliminated the sex difference,
differentiation of the AVPV, SDN-POA, and BSTp of rodents suggesting this sex difference depends on developmental cell death.
(McCarthy et al., 1993; Bodo et al., 2006; Hisasue et al., The number of animals per group is indicated at the base of each bar.
2010; Orikasa and Sakuma, 2010; Tsukahara et al., 2011), so n.s., Not significant. (a) Reprinted with permission of Wiley from Ahern,
T.H., Krug, S., Carr, A.V., Murray, E.K., Fitzpatrick, E., Bengston, L.,
the aromatization of testosterone and the activation of
McCutcheon, J., De Vries, G.J., Forger, N.G., 2013. Cell death atlas of
estrogen receptors is a first step. In some cases, effects of
the postnatal mouse ventral forebrain and hypothalamus: effects of age
estrogen receptor activation on Bcl-2 family proteins may be and sex. J. Comp. Neurol. 521, 2551–2569; (b) Reprinted with the
direct, since putative estrogen response elements have been permission of the National Academy of Sciences of the USA from
described in the Bcl-2 and Bcl-xL genes (Pike, 1999; Lin Forger, N.G., Rosen, G.J., Waters, E.M., Jacob, D., Simerly, R.B.,
et al., 2006). Moreover, estradiol increases Bcl-2 and de Vries, G.J., 2004. Deletion of Bax eliminates sex differences in the
decreases Bax expression in the SDN-POA of newborn rats mouse forebrain. Proc. Natl. Acad. Sci. U.S.A. 101, 13666–13671.
(Tsukahara, 2009). Alternatively, the estrogen regulation of
Bcl-2 family members may be parallel to, or downstream of,
5.01.3.4 Differentiation of Circuits
other signaling pathways. In the neonatal rat AVPV, for
example, the use of targeted apoptosis microarrays identified The building of circuits involves at least three morphological
a sex difference in the activity of the tumor necrosis factor components: axonal growth, dendritic growth, and synapto-
(TNF) alpha–TNF receptor 2–NF-kappa B cell survival genesis. In addition to the morphological differentiation of
pathway, as well as a difference in Bax expression (Krishnan circuits, the expression of neurotransmitters and neuropeptides
et al., 2009). Using transcriptome analysis, other novel will determine circuit functionality. There is evidence for
proapoptotic proteins have also been implicated in male- steroids regulating all three morphological processes, although
specific cell death in the neonatal AVPV (Del Pino Sans the preponderance of the data pertains to synaptogenesis and
et al., 2015). particularly to synapses on dendritic spines. This is largely

Hormones, Brain and Behavior, Third Edition, 2017, 3–32

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16 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
due to technical limitations. As noted above, it is simply easier
to quantify dendritic spines than to measure changes in axonal
and dendritic growth. Nonetheless, there is good evidence for
steroid modulation of axonal growth, with one of the best
examples being the building of the functional circuit relevant
to control of the LH surge. There is also good evidence for
effects of steroids on the production of neurotransmitter/
neuropeptides, as described below.
5.01.3.4.1 Axonal Growth
The sexual differentiation of neural circuits was presaged by
landmark studies of Dominique Toran-Allerand on the
profound induction of neurite outgrowth by estradiol from
organotypic explant cultures of the POA, hypothalamus, and
cerebral cortex (for review, see Toran-Allerand, 2005). Neurite
is the appropriate term in these cases, as processes emerging
from neurons grown in culture are not readily distinguished
as dendrites or axons. Explant cultures do, however, allow for
direct application of steroids to the medium and precise quan-
tification of neurite length. In cells cultured from the VMN,
estradiol interacts with neurotropic factor signaling to control
neurite outgrowth, and effects of estradiol appear to be via
a membrane receptor (Carrer et al., 2005). Estradiol can also
have inhibitory effects on neurite growth, for example, on sero-
tonergic neurons derived from the embryonic mesencephalon
(Lu et al., 2004). Unfortunately, studying neurite development
in the actual brain turns out to be quite challenging, and prog-
ress has been relatively slow.
We discussed above one of the most pronounced sex differ-
ences in physiology, the control of gonadotropin secretion
from the anterior pituitary. Male rodents have continuously
pulsatile LH release, whereas females exhibit an LH surge in
response to estradiol. Although, as mentioned above, it is clear
that this sex difference is controlled by the brain, finding out
just where in the brain proved far more complicated than origi-
nally supposed, and may not yet be entirely solved. The obvious
candidates, the GnRH neurons themselves, are largely indistin-
guishable in males and females, suggesting that differences in
afferent input to these neurons is the key variable. One brain
region that may play this role is the AVPV (Wiegand and
Terasawa, 1982; Ronnekleiv and Kelly, 1986; Petersen and
Barraclough, 1989). The results of anterograde labeling experi-
ments indicate that neurons in the AVPV provide direct inputs
to GnRH-containing neurons in the preoptic region (Gu and
Simerly, 1997). A subset of these neurons are probably the
sexually dimorphic kisspeptin and/or glutamatergic/GABAergic
neurons discussed in Section 5.01.2.6.2. In addition,
descending projections from the AVPV to dopaminergic
neurons in the arcuate nucleus are about eight times more
abundant in females than in males (Simerly, 1998). Thus,
axonal output from the AVPV is highly sexually dimorphic,
and this may account for the sex difference in control of
reproductive function.
In addition, AVPV itself receives sexually dimorphic input.
For example, a projection from the BSTp to AVPV is about
10-fold greater in male rats than in females (Gu and Simerly,
1997; Hutton et al., 1998). This is entirely due to a hormonally
mediated sex difference in axon outgrowth during develop-
ment; the projection apparently never forms in females, but
is induced in females treated with testosterone at birth.
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
To explore the basis of this striking sex difference further, Sim-
erly and colleagues cocultured BST and AVPV explants from
neonatal male and female rats. They found a robust outgrowth
from BST to AVPV only when the AVPV came from a male or
a testosterone-treated female (Ibanez et al., 2001); the sex/
hormone status of the BST had no effect on outgrowth. Thus,
a diffusible factor from the AVPV is responsible for controlling
the growth of BST neurons, but the precise identity of this factor
remains to be determined.
5.01.3.4.2 Dendritic Growth and Branching
The first indication that gonadal steroids could influence
dendritic growth came from studies on motoneurons of the
SNB. For example, the castration of adult male rats led to
a 50% reduction in the overall extent of SNB dendritic trees,
which could be prevented by treating castrates with testos-
terone (Kurz et al., 1986). A similar reduction in dendritic
extent was seen in white-footed mice exposed to short day
lengths characteristic of winter (Forger and Breedlove, 1987),
suggesting that hormonally mediated growth and retraction
of SNB dendrites may be a normal feature in seasonally
breeding rodents. Gonadal hormones also control the initial
outgrowth of SNB dendrites in development; in this case,
however, both estrogens and androgens support growth
(Goldstein and Sengelaub, 1994). Interestingly, although
SNB motoneurons abundantly express the androgen receptor,
they do not express estrogen receptors. The effect of estrogens
on SNB dendrites appears to be mediated instead by hormone
action at the target muscles. Estrogen administration at the
muscle supports dendritic growth of SNB motoneurons, and
the local blockade of estrogen receptors at SNB target muscles
results in severely reduced dendritic trees (Nowacek and
Sengelaub, 2006).
5.01.3.4.3 Synaptogenesis
Sexual differentiation of the brain occurs during a perinatal
sensitive period. This is analogous to other developmental
processes that are restricted to sensitive developmental periods,
such as organization of the barrel cortex in response to whisker
activation, or organization of the visual system by light and
color. In both cases, sensory stimuli act to refine an overly
exuberant innervation of a target region by pruning excessive
or superfluous inputs. Similarly, SNB motoneuron dendrites
are pruned from early exuberant growth, and this pruning is
steroid sensitive (Goldstein et al., 1990). However, steroids
also build sexually dimorphic circuits by forming new synapses
‘on demand,’ as will be discussed in this section. Depending on
the brain region, estradiol can either increase or decrease the
density and/or number of dendritic spines and the attendant
synapses. In addition, strikingly different mechanisms are
utilized in different brain regions. We highlight this point by
reviewing effects of estradiol on dendritic spine formation in
three regions: the arcuate nucleus, POA, and VMN.
5.01.3.4.3.1 Arcuate Nucleus
The arcuate nucleus of the hypothalamus represents an impor-
tant site for neuroendocrine integration and contains a variety
of neurons that control hormone secretion from the anterior
pituitary (Everitt et al., 1986; Swanson, 1986). The nucleus is
comprised of numerous cell types, including dopaminergic,
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Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
enkephalinergic, and GABAergic neurons, and many of these
coexpress releasing peptides such as corticotropin-releasing
hormone, GH-releasing hormone, and thyrotropin-releasing
hormone. The arcuate sends its strongest projections to other
parts of the periventricular zone of the hypothalamus (Simerly,
1995; Li et al., 1999) and plays a central role in reproduction,
feeding, and the response to stress. There is a marked and
permanent sexual dimorphism in the arcuate nucleus, in which
females have a two- to threefold higher density of axodendritic
spine synapses than males, while males have two- to threefold
more axosomatic synapses than do females. The pattern is
reversed in males castrated as neonates or females treated
with testosterone at birth (Matsumoto and Arai, 1980). Given
that axosomatic synapses are generally inhibitory while axo-
dendritic spine synapses are excitatory, sex differences in the
relative number of somatic versus spine synapses are likely to
have a profound impact on neuronal excitability, as well as
on the source of afferent input to arcuate neurons. One such
sexually dimorphic projection, from AVPV to the arcuate
nucleus, was discussed above.
The testosterone-induced decrease in arcuate spine synapses
correlates with a marked sex difference in the morphology of
astrocytes in the same brain region (Mong et al., 1996,
1999): males have more complex astrocytes with a stellate
morphology, compared to the relatively simple, bipolar shape
of astrocytes in the arcuate of females. This sex difference is also
determined by estradiol acting within the first few days of life.
In the adult arcuate, this same population of astrocytes is
capable of physically stripping synapses and allowing for rees-
tablishment later. This feature is unique to females and is
a component of the remodeling that occurs across the estrous
cycle (Garcia-Segura et al., 1994). Whether there is an analo-
gous but permanent process in which astrocytes are differenti-
ated by estradiol to suppress the formation of dendritic spine
formation during development in males, remains unknown.
Paramount for establishing a causal link between astrocyte
morphology and the number of dendritic spines on neurons
is determining the primary site of estradiol action. Interestingly,
the estradiol-induced astrocyte stellation was found to require
activation of the GABAA receptor (Mong et al., 2002). And
while astrocytes express GABAA receptors, they do not make
GABA. The rate-limiting enzyme in GABA synthesis, GAD, is
found only in neurons, and estradiol increases the amount
and activity of GAD (see Section 5.01.2.6.1). Therefore, estro-
gens apparently alter the morphology of arcuate astrocytes by
stimulating the synthesis of GABA, which is released from
neurons to act on neighboring astrocytes to induce stellation.
It has yet to be determined whether astrocyte stellation then
results in the permanent suppression of dendritic spine synapse
formation.
5.01.3.4.3.2 The Preoptic Area of the Hypothalamus
The POA has long been established as crucial for the control of
male copulatory behavior (Davidson, 1966; Hansen et al.,
1982). This region also influences other sexually dimorphic
functions, including the control of gonadotropin release,
maternal behavior, and female copulatory behavior, by
virtue of its projections to other sexually dimorphic nuclei
involved in the regulation of these sex-specific functions
(Swanson, 1986; Simerly and Swanson, 1988). Thus it is not
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
17
surprising that the POA is a site of major sex differences in
morphometry. For example, the sex difference in SDN-POA
volume, discussed above, is perhaps the most celebrated sex
difference in the mammalian brain. Close scrutiny of the
POA also reveals additional sex differences at the synaptic level.
As in the arcuate nucleus, the POA of males contains more
complex, stellate astrocytes than that of females (Amateau and
McCarthy, 2002a,b). But the relationship between astrocyte
morphology and dendritic spine patterning is the opposite of
that seen in the arcuate: male rats have a two- to threefold
greater density of dendritic spines in the POA than do females
(Amateau and McCarthy, 2002b). The induction of spines in
the POA of males is permanent, with the pattern established
in the first few days of postnatal life, and persisting until at least
90 days of age (Amateau and McCarthy, 2004).
The sexual differentiation of POA neurons is a complex
process that begins with estradiol upregulation of the enzyme
cyclooxygenase-2 (COX-2), a nodal point in the production
of prostaglandins and the thromboxanes (Hoffman, 2000).
Induction of COX-2 is strongly yoked with an inducible form
of prostaglandin E2 synthase, leading to the preferential
production of prostaglandin E2 (PGE2) over other prosta-
noids. Estradiol treatment of neonatal female rats increases
PGE2 levels in the POA, and this appears to be a direct result
of estradiol induction of COX-2 gene transcription (Amateau
and McCarthy, 2004). There are two effects of PGE2 on astro-
cytes: increased stellation (Amateau and McCarthy, 2002a)
and release of glutamate (Bezzi et al., 1998; Sangrizi et al.,
1999). The glutamate released by the astrocytes in response
to PGE2 is speculated to then activate AMPA receptors on the
neighboring (or originating) neuron to induce the formation
of dendritic spines (Figure 6). The source of the PGE2 produc-
tion is hard to determine precisely but a critical contribution of
the brain’s innate immune cells, the microglia, has been estab-
lished (Lenz et al., 2013). Microglia had previously been
considered only in the context of injury but have recently
received renewed attention as important sculptors of neural
circuits (Schafer et al., 2012). In the POA, males have more
microglia and they both respond to and produce more PGE2
than in females, thereby providing a positive feedback mecha-
nism for maintaining elevated prostaglandin during the sensi-
tive period for sexual differentiation. The magnitude of the
induction of these dendritic spines by prostaglandins is tightly
correlated with the expression of male sexual behavior in adult-
hood. Compared to animals with a low density of POA
dendritic spines, animals with high spine densities exhibit
shorter latencies and higher frequencies of mounting and
thrusting in tests with receptive females (Wright et al., 2008).
5.01.3.4.3.3 Ventromedial Nucleus of the Hypothalamus
As the POA is critical to the control of male reproductive
behavior, the VMN is central to female reproductive behavior
(reviewed in Pfaff et al., 1994). The neural outputs of the
VMN have been examined in detail, and they provide an
anatomical substrate for relaying the acute effects of ovarian
steroid hormones onto neural systems mediating lordosis, as
well as a means of coordinating female copulatory receptivity
with gonadotropin secretion. Despite this, and the intimate
connections between the VMN and other sexually dimorphic
nuclei, the VMN is not dramatically dimorphic in terms of
 Author's personal copy
18 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
Figure 6 Prostaglandins induce masculinization of the preoptic area
neurons and sex behavior. Estradiol in the neonatal preoptic area (POA)
of males binds to the estrogen receptor and induces transcription of the
gene for cyclooxygenase-2 (COX-2), resulting in increased production
of prostaglandin E2 (PGE2). The astrocytes in the male POA are more
complex, with longer and more frequently branching processes than in
females, and this sex difference is mediated in part by PGE2, and in
part by glutamate. More importantly, PGE2 induces the formation of
dendritic spine synapses on POA neurons, and this is also mediated in
part by glutamate. The organization of a greater density of dendritic
spines in the male POA results in masculinization of sex behavior in the
adult animal.
cell number or nuclear volume. It does, however, exhibit a sex
difference in dendritic spines: neurons of the VMN of males
have more excitatory dendritic spine synapses than females,
and VMN dendrites are longer and branch more frequently in
males (Schwarz et al., 2008).
An obvious question is whether prostaglandins mediate
the sex difference in dendritic spines in the VMN, as in the
POA. The answer here is no; nor does GABA appear to play
any significant role in synaptogenesis during development of
this brain region (Todd et al., 2007). There is again, however,
a critical role for glutamate, but it is a very distinct role from
that in the POA. Recent studies indicate that estradiol
promotes the release of glutamate from VMN nerve terminals.
The enhanced release is independent of protein synthesis but
requires activation of phosphoinositide 3-kinase (PI3 kinase).
The impact of the increased glutamate release is activation of
MAP kinase in the postsynaptic neuron, followed by the
induction of spinophilin synthesis and the construction and
maturation of dendritic spine synapses (Schwarz et al.,
2008). There are many novel aspects of this mechanism for
establishing a sexually dimorphic pattern of synaptic connec-
tivity in the VMN, one of the most notable being the apparent
lack of any involvement of astrocytes. Equally notable is the
rapidity of the effects, with estradiol significantly enhancing
glutamate release within 3 h, and the observation that protein
synthesis is not required for the effect of estradiol. Both of
these findings suggest a nongenomic mechanism of action,
which is somewhat surprising given the permanent nature of
the effect. The estrogen receptor is required and appears to acti-
vate PI3 kinase directly, as demonstrated by a marked increase
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
in the phosphorylated form of its substrate, Akt, within 1 h of
estradiol treatment. PI3 kinase has previously been implicated
in neurotransmission, but precisely how it is promoting the
release of glutamate in this instance remains poorly under-
stood. A final piece of evidence makes this series of findings
even more intriguing: there is no requirement for activation
of estrogen receptors in the postsynaptic neuron in which
dendritic spines are being induced by estradiol. The toxin,
latrotoxin, acts presynaptically to stimulate glutamate release
and mimics the effects of estradiol on spine formation in the
VMN. The increase in dendritic spines in latrotoxin-treated
animals was not altered by blocking estrogen action, indi-
cating that there was no contribution of a postsynaptic effect
of estradiol (Schwarz et al., 2008).
From a behavioral perspective, these observations are
equally intriguing. Given that we had previously determined
that masculinization of sex behavior could be achieved inde-
pendently of gonadal steroids by manipulating prostaglandins,
and that males that were treated neonatally with a prosta-
glandin inhibitor were not masculinized but were defeminized
(Todd et al., 2005), we concluded that steroid action in the
VMN must be relevant to the process of defeminization. Thus
there seem to be separate, albeit related, cellular processes
that control masculinization versus defeminization of sexual
behavior.
5.01.3.4.4 Differentiation of Neurochemical Phenotype
In addition to the examples of morphological differentiation
discussed above, there are even more reports on sex differences
in neurotransmitters or neurotransmitter receptor expression
(De Vries, 1990; Fink et al., 1998; Rhodes and Rubin, 1999).
However, two fundamentally different sets of processes could
cause any of these differences: processes that determine the
absolute number of cells capable of expressing a specific attri-
bute (i.e., birth, death, or migration of cells), or processes
that act on preexisting cells to alter their phenotype (Tobet
and Hanna, 1997; De Vries and Simerly, 2002; Forger, 2006).
The former set of processes enriches or depletes areas with cells
that show specific features, e.g., cells that have relatively large
dendritic trees, whereas the second set of processes changes
the features of existing cells, e.g., extend or prune dendritic
trees. As described above, it has been surprisingly difficult to
disentangle these two possibilities, in part because gonadal
hormones often trigger sexual differentiation before the
neurons of interest assume their final phenotype.
A case in point is the sexually dimorphic vasopressin inner-
vation of the brain. This innervation shows one of the most
consistently found neural sex differences among vertebrates
(Figure 7; De Vries and Panzica, 2006), with males having
more vasopressin neurons in the BST and MeA and denser
projections from these areas than do females across many
mammalian species (De Vries and Panzica, 2006). Nonmam-
malian vertebrates show similar sex differences in homologous
vasotocin projections (Moore et al., 2000; Goodson and Bass,
2001; De Vries and Panzica, 2006). This sex difference has
been well studied in rats, where exposure to gonadal steroids
during perinatal life determines the number of vasopressin cells
found in adults (Wang et al., 1993; Han and De Vries, 2003).
Differential cell birth and migration are very unlikely to
contribute to the sex differences, as vasopressin cells are born
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Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
Figure 7 Sex difference in the vasopressin (AVP) innervation of the
lateral septum (LS). The top panels show the AVP innervation in the LS
of a female (left) and male rat (right). Note the higher density of vaso-
pressin fibers in male brains. The graph shows the number of AVP cells
in the bed nucleus of the stria terminalis of male and female wild type
and Bax
/
knockout mice. Note that while the overall number of AVP
cells is higher in bax
/
mice, the sex difference in cell number
remains.
on embryonic days 12 and 13 (al-Shamma and De Vries, 1996;
Han and De Vries, 1999), at least a week before gonadal
hormones trigger their sexual differentiation (Wang et al.,
1993; Han and De Vries, 2003). This leaves differential cell
death or phenotypic differentiation as the two most likely
causes.
Circumstantial evidence favors the latter. Essentially all
vasopressin cells in the BST coexpress the neuropeptide gala-
nin, but not all galanin cells coexpress vasopressin (Planas
et al., 1995a). Because the total number of galanin cells does
not differ between males and females, it was hypothesized
that, during development, higher levels of testosterone act on
existing galaninergic cells to increase the percentage that will
coexpress vasopressin (Planas et al., 1995b). In support, vaso-
pressin and galanin neurons in the BST and MeA of rats show
the same unusual birth profile, with both types of neurons
born days earlier than most surrounding cells (Han and
De Vries, 1999), consistent with the idea that these neurons
belong to a single cohort of pluripotent cells.
The hypothesis that testosterone instructs these cells to
become vasopressinergic, however, is difficult to test directly.
Gonadal steroids determine vasopressin cell number soon after
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
19
birth (Wang et al., 1993; Han and De Vries, 2003), yet the vast
majority of presumptive vasopressin neurons do not begin
expressing vasopressin until several days (in males) to weeks
(in females) later (Szot and Dorsa, 1993). Thus, one cannot
identify the cells of interest during the time the sex difference
in their number is determined. It has also been difficult to
rule out a role for cell death. Mice are especially useful for
differentiating between these two possibilities, as there are
several genetically engineered strains in which cell death is
altered (Lindsten et al., 2005; see Section 5.01.3.3). One can
therefore use such mice as a litmus test for cell death. Mice over-
expressing cell death-reducing factors would probably show
no, or reduced, sexual differentiation if differentiation
depended on cell death. The same would be true for mice lack-
ing proteins required for neuronal cell death, such as the Bax
protein described above. Vasopressin expression was studied
by comparing wild-type mice with two genetically altered
strains: those that overexpress the antiapoptotic factor Bcl-2
specifically in neurons, or mice with a null mutation in the
gene encoding Bax. Neuronal cell death is markedly reduced
throughout the brain, and sex differences in cell number are
reduced or abolished in several brain areas in both mutants
(see Section 5.01.3.3). These mutations do indeed increase
the total number of cells that produce vasopressin. Critically,
however, the sex difference in cell number remains intact
(Figure 7; De Vries et al., 2008). This leaves sexual differentia-
tion of cellular phenotype as the only remaining plausible
mechanism for sexual differentiation of vasopressin expression.
Numerous other neurotransmitters and neuropeptides (e.g.,
dopamine, neurotensin, substance P, and enkephalin) show
sex differences in cell number (reviewed in De Vries, 1990;
Forger et al., 2015a), and for none of these has the cellular
mechanism of sexual differentiation been established. The
search for these mechanisms may be amenable to the same
strategy described here. If, however, one successfully eliminates
cell birth, migration, and death as possible mechanisms, that is
only a modest first step. One then faces the task of identifying
which of a host of possible molecular mechanisms contributes
to the phenotype of differentiating neurons. Understanding the
process underlying phenotypic decisions constitutes one of the
major challenges of neuroscience today, and hormone-
sensitive systems are poised to make a major contribution
because this process can often be manipulated (hormonally)
in sexually dimorphic systems with relative ease. One thing
seems certain: to the extent that sexual differentiation of cell
phenotype involves long-term changes in gene expression,
epigenetic modifications will be involved, as discussed in the
next section.
5.01.3.5 The Role of Epigenetic Mechanisms in the Four Key
Processes
5.01.3.5.1 How Can Effects of Sexual Differentiation Last
a Lifetime?
How can we explain the enduring effects of gonadal hormones
on cell morphology (e.g., changes in dendritic spines) or
neurochemical phenotype (e.g., as seen in the vasopressin
system) described above? In some systems, hormones may
induce permanent changes in the phenotype of neurons by
altering the structures that they innervate. Target-dependent
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20 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
differentiation of neurotransmitter phenotype has been found
in the sympathetic nervous system, for example (Ernsberger
and Rohrer, 1999; Landis, 1990). Gonadal hormones could
also alter neuronal morphology or phenotype by acting directly
on those cells. Either way, epigenetic mechanisms (modifica-
tions of chromatin that do not involve changes to the under-
lying DNA code) are implicated.
The DNA that is common to every cell in the body is epige-
netically modified during early development to generate the
vast array of different cell types in the mature organism, and
many of the cell fate ‘decisions’ are essentially irreversible
(i.e., once a liver cell, always a liver cell). Similarly, to the extent
that gonadal steroid exposure during neonatal life leads to
permanent changes in neuronal cell morphology or neuro-
chemistry, epigenetic mechanisms are likely to be involved.
Even effects of gonadal steroids on cell death may involve
epigenetic mechanisms. For example, testosterone prevents
neurons from dying during the cell death period in the
neonatal SDN-POA and BSTp of mice and rats. However, the
hormone signal that is present on the day of birth does not
cause a sex difference in cell death until 5–7 days later
(Gotsiridze et al., 2007; Davis et al., 1996a) indicating that
a cellular ‘memory’ for the hormone exposure is required.
Indeed, several recent studies confirm the requirement for
epigenetic modifications in sexual differentiation of the brain
(for reviews, see McCarthy et al., 2009; Forger, 2016).
5.01.3.5.2 Effects of Manipulating Epigenetic Mechanisms on
Sexual Differentiation
The fundamental structural unit of chromatin is the nucleo-
some, comprising a length of about 146 base pairs of DNA
wrapped around an octamer of histone proteins. Covalent
modifications of the DNA, or the histone protein tails that
protrude from the nucleosome, influence accessibility to tran-
scriptional machinery and gene expression (for reviews, see
Felsenfeld and Groudine, 2003; Jiang et al., 2008).
The best understood of the histone tail modifications is
acetylation. Histone acetyltransferases add acetyl groups to
histone tails, which generally enhances transcriptional activity,
whereas histone deacetylases (HDACs) remove acetyl groups
and generally reduce transcription (Cosgrove and Wolberger,
2005). Interestingly, steroid hormone receptors, including
androgen and estrogen receptors, recruit coactivators or core-
pressors to target genes that alter histone acetylation
(Kishimoto et al., 2006; Tetel, 2009), and blocking these cofac-
tors can prevent effects of testosterone on morphological and
behavioral sexual differentiation (Auger et al., 2000, 2002).
Given the role of histone acetylation in steroid receptor
action, we hypothesized that this epigenetic mark might be
important for hormonally mediated sexual differentiation.
As described in Section 5.01.3.3, the size of the BSTp
depends on testosterone-regulated cell death in early devel-
opment. To test whether histone acetylation plays a role,
mice were treated with the HDAC inhibitor, valproic acid,
during the first 2 days of life, and the BSTp was examined
at weaning. The valproic acid treatment transiently increased
histone acetylation and prevented masculinization of BSTp
volume and cell number in both males and testosterone-
treated females (Murray et al., 2009). Valproic acid had no
effect on the BSTp of control females, or on cell number in
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
brain regions that are not sexually dimorphic, excluding
a nonspecific effect on cell survival.
Matsuda et al. (2011) took things further by administering
a different HDAC inhibitor, trichostatin A, to newborn rats.
When compared to animals receiving control treatments, tri-
chostatin A-treated rats showed marked impairments in male
sexual behavior in adulthood (Matsuda et al., 2011).
Importantly, the intracerebroventricular administration of
antisense oligonucleotides to block the endogenous
production of HDACs also had the same effect, eliminating
concerns that the inhibition of masculinization was related to
side effects of trichostatin A. These authors also showed that
histones associated with the estrogen receptor alpha and
aromatase genes are differentially acetylated in the mPOA of
perinatal males and females, although whether this is related
to the effects of HDAC blockade on sexual function is not
known. Thus, both the Matsuda et al. (2011) and the Murray
et al. (2009) studies suggest that HDAC activity (and, hence,
the repression of some gene or genes) is required for
masculinization of brain morphology and behavior.
A more recent study by Nugent et al. (2015), however,
found greater DNA methylation in the mPOA of neonatal
female rats. To test the functional significance of this sex differ-
ence, neonatal rats were treated with a DNA methyltransferase
(DNMT) inhibitor during the first 2 days of life, and dendritic
spine density (which is normally higher in the POA of males,
see Section 5.01.3.4.3.2) was examined. DNMT inhibition
masculinized spine density in females and eliminated the sex
difference (Nugent et al., 2015). DNMT-treated females also
displayed male sexual behavior in adulthood (Nugent et al.,
2015). This strongly suggests that active repression of some
gene(s) by DNA methylation is required for normal female
development.
The alert reader may have noticed that the repression of
gene transcription is implicated in normal masculine develop-
ment by the HDAC inhibitor studies, but in normal feminine
development by the DNMT study (Murray et al., 2009;
Matsuda et al., 2011; Nugent et al., 2015). The effect of a given
epigenetic perturbation may depend on species, age, brain
region, and even on the dependent variable measured within
a given brain region. Indeed, the same HDAC inhibitor that
prevents masculinization of total cell number in the mouse
BSTp promotes masculinization of the vasopressin projections
emanating from the BSTp (Murray et al., 2009, 2011). Prelim-
inary findings also suggest that neonatal DNMT inhibition in
mice masculinizes or feminizes neurochemical differentiation
in the mPOA, depending on which neurochemical is examined
(M. Mosley and N. Forger, unpublished; Forger, 2016).
5.01.3.5.3 Sexual Differentiation of Epigenetic Marks across
the Genome
In a complementary approach, a handful of studies have
recently reported sex differences and effects of early hormone
exposure on specific epigenetic marks across the genome. For
example, Gharahmani et al. (2014) examined the effects of
testosterone treatment on the day of birth on the DNA methyl-
ome in the BST/mPOA and striatum of mice. They found
a small number of genes (<70) that were differentially methyl-
ated between control females and testosterone-treated females
on postnatal day 4, and roughly 1000 differentially methylated
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Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
genes in adulthood. A similar pattern – few differences in DNA
methylation on day 4 but tenfold more in adulthood – was
seen when comparing males and females (Gharamani et al.,
2014). Thus, effects of neonatal testosterone exposure were
late-emerging, in what might be termed an ‘epigenetic echo’
of the hormone exposure. Parallel effects were seen in a detailed
study of epigenetic changes to the promoter regions of the
estrogen receptors, alpha and beta, and the progesterone
receptor from birth to adulthood. Sex differences were present
at some times and not others, with new ones appearing long
after the perinatal hormone exposure (Schwarz et al., 2010).
Another interesting finding of the Gharahmani et al. (2014)
study is that those genes exhibiting a sex difference (or an effect
of neonatal testosterone treatment in females) on DNA meth-
ylation were unevenly distributed: 85–90% of them were hypo-
methylated in control females. This was true at both ages
(postnatal day 4 and adulthood) and in both brain regions
(striatum and BST/mPOA). Similarly, a genome-wide analysis
across fetal development in the human brain identified a subset
of genes with a significant sex-by-age interaction on DNA meth-
ylation: among these, the pattern noted was progressive hypo-
methylation in females (Spiers et al., 2015).
Based on a transcriptome analysis, differences in DNA
methylation between groups in the mouse study (Gharahmani
et al., 2014) were not a good predictor of differences in gene
expression. This implies that, for the set of differentially meth-
ylated genes, females use less DNA methylation to achieve the
same rate of transcription. This resonates with findings in
a recent genome-wide study of the distribution of the histone
mark H3K4me3 (three methyl groups on lysine 4 of histone
3): genes with a sex difference in H3K4me3 in adult mice
showed a strong sex bias (75% of them had more of the histone
mark in females), and sex differences in the epigenetic mark did
not predict differences in gene expression (Shen et al., 2015).
Taken together, males and females may use different epigenetic
mechanisms to control gene expression, even when that expres-
sion does not vary (see Forger, 2016).
5.01.4 Six Unanswered Questions
Despite the obvious progress in understanding mechanisms of
sexual differentiation over the past several years, a number of
big questions remain unanswered and, often, unasked. We
discuss several of these below. With the goal of holding up
a magnifying glass to cherished or unexamined beliefs, the
tone here is both more speculative and more provocative
than above.
5.01.4.1 What Are the Actual Steroid Levels in the Brain
during the Sensitive Period of Sexual Differentiation?
Every discipline has its iconic papers, findings so fundamental
that the same manuscript is cited over and over again without
question. As opposed to dogma, these studies do not them-
selves postulate any theory or guiding principle but instead
provide data that can be used to support such. In the field of
sexual differentiation, a small number of studies measuring
serum testosterone and hypothalamic estradiol levels in the
neonatal rat have served such a role (Weisz and Ward, 1980;
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
21
Rhoda et al., 1984). These papers found higher serum testos-
terone and hypothalamic estradiol in males and have been
taken by many as proof that male brains are organized by
steroids derived from the testes. These findings have stood
the test of time and proven enormously useful. The risk of
iconic papers, however, is that findings that are true for one
set of circumstances are often inappropriately generalized to
other circumstances. For 30 years, it has been generally
assumed that the entire male brain is exposed to significantly
higher levels of both androgens and estrogens than the female
brain during the perinatal sensitive period. Moreover, it has
been a foregone conclusion that the source of ‘gonadal’ steroids
found in the brain during the developmental window is the
circulation, which in turn reflects steroidogenesis by the testis.
There are two pieces of information that suggest this
simplistic view should be reevaluated. One is the evidence
that neurons can synthesize steroids de novo from cholesterol
all the way to estradiol (Rune et al., 2002; Prange-Kiel et al.,
2003; Hojo et al., 2004). Thus the brain can serve as its own
gonad. This is best exemplified in the songbird brain where local
estradiol synthesis is critical to masculinization of one of the
song control nuclei (Holloway and Clayton, 2001). This may
also be true in the mammalian brain, but remains to be more
definitively established. The second piece of evidence is that
when steroid levels in the brain are measured in multiple
regions and multiple time points, the concept of uniformly
higher levels in perinatal males is not supported. In fact, in
some brain regions outside the diencephalon, levels of estradiol
can be high in both sexes and higher than in the POA or hypo-
thalamus (Amateau et al., 2004). However, there is a problem
with the methods used to obtain both sets of evidence.
Radioimmunoassay as a way to measure small peptides,
and eventually steroids, was a methodological advance so
major that it garnered a Noble Prize for Roslyn Yallow in
1977. Still in common use today, this technique allows for
reasonably precise quantification of steroids in the circulation
where only the presence of binding globulins is a potential
and easily remedied confound. It is far more difficult, however,
to precisely measure steroids in brain tissue where the dense
concentration of lipids can create false positives, or the strin-
gent extraction protocols required can cause loss of substantial
signal in the small samples generated from individual brain
regions. Quantification techniques that are based on the phys-
ical properties of the steroids themselves, such as liquid or gas
chromatography followed by mass spectrometry, are the most
precise techniques available, but remain prohibitive for most
investigators due to a combination of expense, time, and acces-
sibility. Moreover, the sensitivity of mass spectrometry is at
least 10-fold less than that of radioimmunoassay. As a result,
there is currently a large gap in our knowledge of what are
the precise levels of steroids in particular brain regions during
the dynamic period of sexual differentiation. Not knowing
this information severely handicaps our ability to determine
whether specific parts of the brain are either making steroids
de novo or regulating the rate of metabolism, uptake or binding
to increase or decrease exposure in a meaningful way. More
importantly, not knowing when there are or are not sex differ-
ences in tissue steroid levels at key developmental time
points precludes us from discovering potentially novel mecha-
nisms of sexual differentiation of the brain.
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22 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
5.01.4.2 Do Noncoding RNAs Contribute to Sex Differences in
Brain and Behavior?
In addition to the iconic epigenetic modifications discussed
above (DNA methylation and the covalent modification of
histone tails), other epigenetic mechanisms play important
roles in the control of gene expression. The best-known
example relates to the most pervasive sex difference in mamma-
lian biology: the silencing of one randomly selected X chromo-
some in every cell of females and in no cells of males (Lyon,
1999). The process of random X inactivation is initiated by
the transcription from one X chromosome of a long, noncod-
ing RNA known as XIST (X inactivation-specific transcript;
reviewed in Jeon et al., 2012; Gendrel and Heard, 2014).
XIST coats its chromosome of origin and recruits further epige-
netic marks that cause profound condensation of the inacti-
vated X (Jeon et al., 2012; Gendrel and Heard, 2014). The
widely accepted purpose of random X inactivation is to make
male and female cells more similar by equalizing the func-
tional dosage of X chromosome genes. Thus, somewhat ironi-
cally, an essentially absolute sex difference in an epigenetic
processes (the coating of one X chromosome with XIST and
the countless epigenetic modifications that follow) functions
to make the sexes more alike.
Recently, important roles for other noncoding RNAs in the
control of gene expression have been discovered. For example,
microRNAs are short (22–23 nucleotides), noncoding RNA
molecules that are transcribed from DNA, bind to complemen-
tary sites in the 30 untranslated region of specific messenger
RNAs (mRNAs), and target them for destruction (Bartel,
2009). A single microRNA can silence up to 100 different
mRNAs and thus has the potential for a profound impact on
the posttranscriptional control of gene expression. Whether
microRNAs should be considered an epigenetic mechanism is
up for debate. If your definition of ‘epigenetics’ requires modi-
fications to chromatin, not all microRNAs qualify. However,
some microRNAs also induce DNA or histone changes leading
to chromatin condensation, which clearly fits the bill (Sato
et al., 2011).
Sex differences have recently been described in the expres-
sion of specific microRNAs in whole brain (Morgan and Bale,
2011) or cortex (Murphy et al., 2014) of developing and adult
rats. At least some of the sex differences may be due to
programming effects of gonadal steroids, because the adminis-
tration of an aromatase inhibitor markedly alters the brain
microRNA patterns of neonatal male rats (Morgan and Bale,
2011). Sex differences have also been reported for microRNA
transcription in response to challenges such as endocrine-
disrupting chemicals (Topper et al., 2015) or ischemia (Lusardi
et al., 2014). The profile of circulating microRNAs also differs
in male and female rats after stroke and may be a biomarker
of the severity of stroke outcome (Selvamani et al., 2014).
As far as we are aware, no study has yet shown an effect of
manipulating the expression of a particular microRNA on
a behavioral or brain sex difference. In other words, we are still
awaiting the demonstration that microRNAs contribute to
sexual differentiation of the brain. Some sex differences in
microRNA levels may not cause differences in gene expression
but, as proposed above for sex differences DNA and histone
methylation, may reflect different epigenetic pathways to an
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
equivalent gene expression outcome in males and females.
In the mPOA and VMH of the rat brain, for example, sex differ-
ences in microRNA expression did not correlate with differ-
ences in the expression of mRNAs predicted to be targets of
those microRNAs (Topper et al., 2015).
5.01.4.3 Are Sex Differences Necessary?
Sex differences are seen in essentially all sexually reproduc-
ing species. Given the near universality of differences
between males and females, they seem likely to play a crucial
role. What this role is, however, can be surprisingly unclear.
This is particularly true for neural sex differences, and in
Section 5.01.4.4, we describe some of the difficulties in
ascribing function to known sex differences in the nervous
system.
A related issue is the fact that the degree of sexual dimor-
phism varies enormously. At one extreme are orb-weaving
spiders in which females may be >12 times the size of males
(Hormiga et al., 2000) or, as described above, brain regions
in rats that are several times larger in volume in one sex than
the other. At the other end of the spectrum are species that
appear to exhibit few sex differences or, within a species, sex
differences that are extremely subtle, yet reliable. These obser-
vations raise the question of whether, or when, sex differences
are necessary. Much of what we know about sexual differentia-
tion of the brain relies on observations of rats, mice, and guinea
pigs. These are polygynous species in which, under the favor-
able conditions of the laboratory, essentially all individuals
that achieve adult body size become reproductive and attempt
to mate. In species exhibiting cooperative breeding, in which
some members of a social group suppress reproduction to
contribute to the rearing of the offspring of others, other rules
may apply. Cooperative breeding with reproductive suppres-
sion is seen in a wide variety of mammalian taxa, including
the rodents, canids, veverrids, and many examples among the
primates (Jennions and MacDonald, 1994; Solomon and
French, 1997). The most extreme example is seen in naked
mole-rats (Heterocephalus glaber), which are uniquely social
mammals that exhibit the closest mammalian equivalent to
eusociality. Naked mole-rats live in large subterranean colonies
averaging 70–90 individuals, including a single breeding
female (the queen) and 1–3 breeding males (Jarvis, 1981).
All other members of the colony are reproductively suppressed,
but contribute to overall maintenance and survival of the
colony, including caring for pups (Brett, 1991; Lacey and
Sherman, 1991). Subordinates are not sterile, however, and
can become breeders if removed from the colony and placed
with an opposite-sex partner.
Naked mole-rats are remarkably sexually monomorphic.
Among subordinates, there are no sex differences in overall
body size, anogenital distance, or the expression of a large
variety of behaviors (Lacey et al., 1991; Peroulakis et al.,
2002). In addition, naked mole-rats lack many of the sexual
dimorphisms in the brain and spinal cord that are seen in other
mammals. For example, vasopressin innervation of the septum,
and morphology of the perineal muscles and motoneurons do
not differ between males and females (Peroulakis et al., 2002;
Seney et al., 2006; Rosen et al., 2007; Holmes et al., 2009b).
In addition, in a stereological analysis of several brain regions
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Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
known to be sexually dimorphic in other rodents, male and
female naked mole-rats did not differ on any measure (Holmes
et al., 2007). Instead, brain morphology varied by social status
(i.e,. differences between breeding and nonbreeding animals,
regardless of sex). Even the number of kisspeptin neurons,
which is so dimorphic in other rodents (Section 5.01.2.6.2),
does not differ between subordinate male and female naked
mole-rats, but depends on breeding status (Zhou et al.,
2013). These findings suggest that status, rather than sex, plays
the predominant role in determining neural structure in this
uniquely social mammal.
The reduction in sex differences in naked mole-rats may be
related to their unique reproductive strategy, as in nature, it is
estimated that fewer than 5% of all naked mole-rats ever
achieve reproductive status (Jarvis et al., 1994). Perhaps in
this case, it makes ‘sense’ to rely more on acute than on
programming effects of steroids to control brain morphology
and function. Although naked mole-rats present an extreme
case, cooperative breeding is widespread. It would be inter-
esting to know to what extent programming versus acute effects
of steroids shape the brain in other species with social organi-
zation that differs from that characteristic of common lab
animals.
5.01.4.4 Do Sex Differences in Brain Structure Beget Sex
Differences in Brain Function?
The landmark studies by Phoenix et al. (1959) suggested that
sex differences in neural structure could explain the permanent
effects of hormones on behavior. When such differences were
found, they were typically linked to sex differences in function.
For example, sex differences in the medial POA were related to
sex differences in male sexual behavior and the regulation of
gonadotropic hormone release (Yahr, 1988; Kelley, 1988;
De Vries, 1990; Breedlove, 1992). This made sense at the
time, as the medial POA had indeed been implicated in these
functions. However, there is good evidence that sex differences
in the brain do not only serve to generate sex differences in
physiology and behavior. The opposite may be true as well;
that is, structural sex differences may enable the brain to
generate similar behaviors in males and females in spite of
them being exposed to different hormonal and physiological
conditions (De Vries and Boyle, 1998; De Vries, 2004).
We will illustrate this with the sexually dimorphic vasopressin
innervation.
As mentioned above, most vertebrate species exhibit a sex
difference in vasopressin innervation, such that males have
a greater density of vasopressin fibers projecting from the BST
and MeA to the septum than do females (De Vries and Panzica,
2006). This has been linked to the higher aggression seen in
males (Koolhaas et al., 1990, 1991). Interestingly, in Syrian
hamsters, which lack vasopressin projections of the BST and
MeA altogether (Albers et al., 1991), females are as aggressive
as males and tend to dominate males of similar body weight
(Payne and Swanson, 1970; Huhman et al., 2003). Similarly,
female spotted hyenas are socially dominant and much more
aggressive than males (Hamilton et al., 1986; Glickman et al.,
2006), and the traditionally found sex difference in septal
innervation is absent or reversed in this species (Rosen et al.,
2006). Each of these examples is compatible with the idea
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
23
that sex differences in vasopressin innervation beget sex differ-
ences in function. They also invoke the idea that the size and
direction of sex differences in vasopressin innervation correlate
with the size and direction of sex differences in behavior.
Research in prairie voles, however, suggests that sex differences
in vasopressin innervation cannot be that easily interpreted.
Prairie voles resemble spotted hyenas in that they are highly
social (Getz et al., 1981; Carter et al., 1995). They differ in that
social behaviors, including aggressive and parental behavior,
are remarkably similar between male and female voles
(Villalba et al., 1997; Lonstein and De Vries, 1999a). Paradox-
ically, however, the sex difference in vasopressin expression in
prairie voles is larger than what has been reported for any other
mammal to date (Bamshad et al., 1993, 1994; Wang et al.,
1994b). Evidence suggests that this sex difference in vaso-
pressin innervation contributes to the absence of sex differences
in parental behavior typically seen in other rodents (Lonstein
and De Vries, 2000). Mating appears to release vasopressin in
the septum of male prairie voles (Bamshad et al., 1994;
Wang et al., 1994b). This may stimulate parental behavior,
because injecting exogenous vasopressin into the septum
increases paternal responsiveness and V1a receptor antagonists
reduce it (Wang et al., 1994a). This indicates that the vaso-
pressin innervation of the septum is required for parental
behavior in males. As in other female rodents, hormonal
changes associated with pregnancy and parturition appear
necessary to trigger parental behavior in female prairie voles
(Lonstein and De Vries, 1999b; Hayes and De Vries, 2007).
Because male prairie voles will never get pregnant let alone
give birth, they may need a different strategy to boost their
parental responsiveness. If the sex difference in vasopressin
expression plays this role, then vasopressin can cause as well
as prevent sex differences in social behavior.
This hypothesis is perfectly testable. One would predict in
the former case (vasopressin causes sex differences) that block-
ing vasopressin neurotransmission would blunt or eliminate
sex differences and in the latter case (vasopressin prevents sex
differences) that blocking vasopressin would cause sex differ-
ences in vasopressin-dependent functions that were not there
before. In fact, such tests have already been done. For example,
vasopressin antagonists block social recognition memory in
male but not in female rats, thereby causing a sex difference
that was previously absent (Bluthe and Dantzer, 1990).
Similarly, a knockout in the V1a receptor gene reduces anxiety
in male but not in female mice (Bielsky et al., 2005), exactly
what one would predict if a system is more important for
a function in one sex than in the other. Therefore, the possi-
bility that sex differences can cause as well as prevent sex differ-
ences in function should be considered as two obligate
alternative hypotheses when regarding the function of vaso-
pressin innervation in the brain. It is unlikely that the vaso-
pressin innervation is the only neural system for which this is
true. Of the hundreds of sex differences that have been found
in the central nervous system, only a handful can be clearly
linked to sex differences in behavior. Perhaps the clearest
example is the cluster of motoneurons in the spinal cord that
innervates muscle groups with a clearly sexually dimorphic
function (i.e., the SNB). There is also a strong positive
correlation between the density of dendritic spine synapses
on POA neurons and measures of male sexual behavior
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24 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
(Wright et al., 2008). But of almost all others, we do not know
exactly what advantage the dimorphism gives males and
females in terms of function. Considering the possibility that
these dimorphisms can cause as well as prevent sex differences
in function may help answer this question.
5.01.4.4.1 ‘Canalization’ May Explain the Size and Variability
of Sex Differences in Brain Structure and Behavior
Most neuroanatomical sex differences, be they in synaptic
profile, neurochemical phenotype, astrocyte morphology, or
neurotransmitter levels, are of a relatively modest magnitude.
Typical are one- to twofold differences in an endpoint mean
between males and females. But more importantly, the variance
associated with the mean is often very low, suggesting there is
not a continuum of responses with a great deal of overlap
between males and females but instead a clustering of males
and females at opposite ends of the spectrum. This is suggestive
of a process termed canalization that has been used by evolu-
tionary biologists to explain the robustness of species pheno-
typic traits in the face of constant challenges from a changing
environment (Gursky et al., 2012). Species phenotypic traits
are maintained in a narrow ‘canal’ by various factors that range
from chaperone proteins to miRNAs to even transposons (see
for review McCarthy et al., 2015). It is possible that phenotypic
traits of the brains of males and females are also constrained to
specific ‘canals’ but also not allowed to stray too far apart.
Behavior, on the other hand, is not so tightly constrained as it
is influenced by a multitude of physiological and contextual
factors that must be integrated by the nervous system before
a response is made. Thus it would be predicted that there is
much more overlap in the behavioral phenotypes of males
and females, especially outside the context of reproduction,
and this does indeed seem to be the case.
5.01.4.5 Is Partner Preference Sexually Dimorphic?
Sexual orientation, also referred to as sexual partner preference,
is defined by the sex of the individuals that are arousing or attrac-
tive to the reference individual, whether it be an individual of the
opposite sex (heterosexual), the same sex (homosexual), or both
sexes (bisexual). The estimated frequency of homosexuality in
humans ranges from 2% to 10%, suggesting that the large
majority of males are sexually oriented toward females and the
majority of females are sexually oriented toward males. The over-
whelming prevalence of one sex preferring the other is a constant
across all vertebrate species, as would quite naturally be expected
from the point of view of reproductive success. Nonetheless,
what draws the majority of attention is the much less frequent
phenotype of same-sex preference. Notably, the biological basis
of sexual orientation is a matter of impassioned debate only
when it involves discussion of the etiology of homosexuality.
Few seem to question whether opposite-sex orientation is bio-
logical. But actually we understand little about opposite-sex
attraction, and it can be argued that understanding the biological
basis of same-sex orientation would be greatly advanced by
understanding opposite-sex orientation. Thus, a fundamental
question is whether sexual orientation per se is sexually
differentiated.
The answer to this may depend on how you pose the ques-
tion. If we state that the majority of males prefer females as
Hormones, Brain and Behavior, Third Edition, 2017, 3–32
sexual partners and the majority of females prefer males, then
this sounds like a profoundly sexually dimorphic and presum-
ably differentiated response. Antecedent to this view would be
the assumption that distinct biological processes drive the
neural substrate of partner preference to either a male bias or
a female bias. The existence of distinct processes for a male pref-
erence versus a female preference provides a ready explanation
for why some females prefer other females, some males prefer
males, and why some individuals have no preference.
However, if we take the view that the majority of animals prefer
the opposite sex as partners, then there is no sex difference as
the same drive exists in males and females but it is manifest
differently as a function of one’s own sex. This means that
a component of the neural response is computation of one’s
own sex, which then determines the response to others’ sex.
Given the intensity and early onset of both internal and
external influences of sex on brain development, this is not
outside the realm of possibility. In humans, we are unlikely
to ever be able to definitively separate the impact of nature
from nurture, and our best alternative is the study of naturally
occurring or experimentally manipulated variation in sexual
preference in animals.
The current state of the art of partner preference research is
found on several fronts. These include studies of the program-
ming effects of gonadal steroids and early experience on partner
preference, the neuroanatomical loci controlling partner prefer-
ence, and the study of naturally occurring variation in partner
preference in animal models. Consistent evidence supports
the view that partner preference is organized by gonadal
steroids, such that perinatal androgens, with aromatization to
estrogens in rodents, direct the formation of preference for
a female sexual partner (Brand et al., 1991; Vega Matuszczyk
et al., 1988). In many mammals, odors are the primary signal
indicating sex. Preference can be assessed by determining the
amount of time a test subject prefers to spend with male versus
female stimulus animals or by the amount of time spent inves-
tigating odors generated by stimulus animals. Male- versus
female-specific odors can induce a differential brain response
in the same animal, and likewise, animals of opposite sex
will respond to the same odor differently (Bakker et al.,
1996; Woodley and Baum, 2004). The latter speaks to the
sexual differentiation of partner preference and suggests that
the olfactory system may be the initiation point for subsequent
behavioral responses. In many species, if olfaction is blocked,
there is no partner preference to measure.
Olfaction is important to humans as well, but visual stimuli
are far more potent and the arousal potential of same-sex versus
opposite-sex images depends on the partner preference of the
observer (see for review Baum, 2006). Zebra finches are also
heavily dependent upon vision for expressing partner preference,
and steroids influence partner preference in this species as well
(Adkins-Regan and Leung, 2006). The effect is context depen-
dent, however, because early experience, i.e., being raised in an
environment with a skewed sex ratio, can also strongly influence
adult partner preference in zebra finches.
The neuroanatomical substrate of partner preference begins
with that portion of the brain detecting and decoding the
sex-specific sensory signals originating from the stimulus
animal, be they olfactory, visual, or auditory. But from there,
all signals appear to converge on the POA, and in particular
 Author's personal copy
Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning 25

an SDN within the POA (see Baum, 2006). An SDN is present 5.01.4.6 Is the Human Brain Sexual Differentiated?
in the POA not only in rats, but also in sheep, gerbils, ferrets,
To some an affirmative answer to this question is self-evident:
hamsters, and humans. Lesions of the SDN and its surround
how could the human brain not be subject to the same process
in rats and ferrets either eliminate or reverse sexual preference
that occurs in the majority if not all other mammals as well as
(for review, see Baum, 2006). In humans, the third interstitial
many birds, reptiles, and amphibians? Even invertebrates have
nucleus of the hypothalamus (INAH3) may be homologous
brains that differ in males and females. But others argue, ‘not so
to the SDN-POA of rats and is larger in men than women
fast, humans are exceptional in many ways.’ We are the only
(Allen et al., 1989). Levay (1991) found that INAH3 is
species with complex computational abilities and a sophisti-
smaller in homosexual men than in heterosexual men, and
cated language that includes generation of an historical record.
a second study found a mean difference in the same direction
We also have rich cultural and societal rituals and expectations
that did not, however, reach statistical significance (Byne
that include prescriptions of the appropriate behavior for boys
et al., 2001). Thus, the size of INAH3 may be a marker of
and girls, men and women. These rules and expectations are
partner preference in men, although this conclusion is not
imposed on children even before they are born with the choice
without its detractors.
of a gender-typical name and continue with modes of dress and
Another approach is the use of biomarkers to determine if
even the manner in which adults interact with an infant of one
an individual was exposed to an endocrine environment
sex versus the other. Thus as we have mentioned many times in
in utero that varies from the norm for that sex. These biomarkers
this chapter, it is impossible to parse out the influence of envi-
include long bone length, hand digit ratio, and the detection of
ronment and experience from biology when considering the
small noises made by the inner ear. In general, these studies
brain and behavior of humans. Nonetheless, it is worth a try.
support the conclusion that the prenatal hormonal milieu
One powerful approach is the study of children at a young
contributes to the propensity to show same-sex orientation
age. While the influence of environment and experience cannot
(Balthazart, 2016). But in many human and animal studies,
be eliminated, it is at least lessened compared to that of a full-
a major and unavoidable confound is either the use of surgical
grown adult. Toy choice reflects an interest in different types of
manipulations, such as lesions, or the health status of the
objects, and this varies on average between boys and girls. The
affected individuals, such as the number of HIV-infected
data are messy, with many boys willing to play with girls’ toys
subjects in the homosexual group in human studies. Neither
on occasion, and vice versa. One of the strongest influences is
of these criticisms applies to the study of a naturally occurring
whether a child has a sibling of the opposite sex, demonstrating
variant of homosexuality, the male-preferring domestic ram.
the importance both of exposure and of modeling the behavior
In at least two different study populations, approximately 8%
of other children. Nonetheless, on average, boys spend more
of rams prefer to mount other male rams. The frequency of
time with certain types of toys and girls with others. Melissa
the phenotype is similar to that observed in humans, and there
Hines has spent most of her career studying this phenomenon
are no clear external markers of male-preferring rams. Analyses
and whether prenatal exposure to androgens in girls can influ-
of the brain reveal that the SDN of male-preferring rams is
ence toy choice. She and others have consistently found that
smaller than that of rams that prefer ewes, and it contains fewer
androgen-exposed girls shift their toy preference to that of
aromatase-expressing neurons. This suggests reduced neuronal
boys (reviewed in Hines, 2006). So does this mean there is
exposure to estradiol developmentally and in adulthood may
a ‘toy’ nucleus in the brain that directs boys to like trucks and
be a critical variable in the establishment of same-sex prefer-
girls to like dolls? That seems unlikely. Hines and colleagues
ence in this species (Roselli et al., 2004).
recently added a new dimension to the sexual differentiation
Thus, on balance, we can conclude that partner preference
of play with the observation that girls prefer to mimic the
is sexually differentiated and that there is an important role for
behavior of other girls or women and that it is this aspect of
gonadal steroid exposure in the organization of partner prefer-
their brain development that is influenced by hormones, not
ence, but early experience may also be important. The primary
the desire to play with a particular object or in a particular
detection of the sensory stimulus emanating from a potential
way (Hines et al., 2016). In girls exposed to androgens in utero,
partner is a critical initiating step but the integration and
the desire to mimic other females is lost, and for reasons not
response to the stimulus appear to be encoded within the
well understood, their preference shifts to toys normally
POA. While these are important advances, there remains
preferred by boys.
much to be learned. Work on the genetics of partner prefer-
The work of Hines and others speaks to the behavior of
ence generated a great deal of interest in the early 1990s
humans, but, as noted above, brain and behavior are not
(Hammer et al., 1993), but there has been little progress on
always closely aligned. Many neuroanatomical sex differences
that front. There is a continuing interest in the role of birth
have been reported in the human brain, but the majority of
order and correlations with handedness, particularly for
these relied on postmortem tissue. Because of this, the majority
male homosexuality, and the proposal of the maternal
also involved the brains of adults although one remarkable
immune hypothesis (Blanchard et al., 2006). But again, we
study looked at a nucleus in the hypothalamus from many
know far less than we should. Moreover, the preponderance
different individuals ranging from birth to old age and found
of information is weighted toward understanding male, as
a sex difference that did not appear until late adolescence
opposed to female partner preferences, although this may be
and then waned again in older adults (Swaab et al., 2003).
defensible given the health implications for male versus
More recently, researchers have taken advantage of noninvasive
female homosexuality. Regardless, progress on both is likely
imaging techniques that allow for longitudinal analyses of the
to remain slow given the paucity of researchers and resources
same individual as they mature. The magnitude and direction
currently dedicated to this topic.

Hormones, Brain and Behavior, Third Edition, 2017, 3–32

 Author's personal copy
26 Sexual Differentiation of the Brain: A Fresh Look at Mode, Mechanisms, and Meaning
of sex differences varies with the mode of data acquisition and
analyses (i.e., correcting for total brain volume or not), but
differences in the developmental trajectory of the peak of
cortical gray matter are reliably found and modulated by
androgen receptor allelic variation as well as androgen levels.
White matter increases more rapidly in male brains as develop-
ment proceeds and combined with differences in gray matter
amplify the magnitude of sex differences across the life span
(reviewed in Giedd et al., 2012).
More recently advances in imaging have allowed for measure-
ment of functional connectivity. Images from almost 1000
humans revealed a profound sex difference in the ‘connectome,’
with females showing strong interhemispheric connectivity and
males the opposite, strong intrahemispheric connections
(Ingalhalikar et al., 2014). The authors interpreted their findings
as supportive of the view that females engage multiple tasks at
once and are highly social while males are focused systematizers.
This stereotypical view generated a firestorm of criticism. In
response the authors went on to use a computerized battery of
neurocognitive tasks in combination with imaging and largely
supported their original conclusions (Tunc et al., 2016), but the
controversy here is certainly not resolved.
On the opposite end of the spectrum and equally controver-
sial was a recent report that reexamined several studies
involving imaging and gender-typical activities. Here the
authors concluded that there was no clear predictability of
sex based on the mean responses (Joel et al., 2015). Instead,
they concluded, every brain is a mosaic of male-like, female-
like, and neutral features, and therefore there is no such thing
as a ‘male brain’ or a ‘female brain.’ In some ways this conclu-
sion is intuitively obvious and consistent with the high degree
of regionally specific mechanisms establishing sex differences
in the brain as determined in rodent models. But the finding
was largely misinterpreted by the lay media as demonstrating
there are no sex differences in the brain, which was not the
case even in the Joel et al. study.
This serves as a fitting conclusion to our long treatise on the
modes, mechanisms, and meanings of sex differences in the
brain as it so aptly demonstrates how much we still have to
learn. In some ways, the topic of sex differences in the brain
remains as controversial today as when the first reports were
made in the late 1960s early 1970s. With the changing policies
at major granting agencies, there is likely to be more, not fewer
reports of brain and behavior sex differences. This makes even
more salient the admonishment that scientists bear the burden
of assuring their work is not used or interpreted inappropriately
(Maney, 2016). It is essential that we ‘get it right’ as the impli-
cations of sex differences research reach far beyond the labora-
tory to medical, educational, and public health policies that
impact the daily lives of all members of society.
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