ChapterPDF Available

Sexual Differentiation and Sex Differences in Neural Development

July 2018
Current Topics in Behavioral Neurosciences 43

July 2018
43

DOI:10.1007/7854_2018_56

In book: Neuroendocrine Regulation of Behavior

Authors:

Alexandra Turano

Utah State University

Brittany Osborne

University of Delaware

Sex determination occurs at the moment of conception, as a result of XX or XY chromosome pairing. From that point, the body undergoes the process of sexual differentiation, inducing the development of physical characteristics that are easily distinguishable between the sexes and are often reflected in one’s physical appearance and gender identity. Although less apparent, the brain also undergoes sexual differentiation. Sex differences in the brain are organized during a critical period of neural development and have an instrumental role in determining the physiology and behavior of an individual throughout the lifespan. Understanding the extent of sex differences in neurodevelopment also influences our understanding of the potential risk for a number of neurodevelopmental, neurological, and mental health disorders that exhibit strong sex biases. Advances made in our understanding of sexually dimorphic brain nuclei, sex differences in neural cell communication, and sex differences in the communication between the brain and peripheral organs are all research fields that have provided valuable information related to the physiological and behavioral outcomes of sex differences in brain development. More recently, investigations into the impact of epigenetic mechanisms on sexual differentiation of the brain have indicated that changes in gene expression, via epigenetic modifications, also contribute to sexual differentiation of the developing brain. Still, there are a number of important questions and ideas that have arisen from our current understanding of sex differences in neurodevelopmental processes that necessitate more time and attention in this field.

Sex determination and sexual differentiation of the brain. Sex determination occurs at the moment of conception, when the embryo receives either two X chromosomes (female embryo) or an X and a Y chromosome (male embryo) from the mother and the father. If the developing embryo has a Y chromosome, this chromosome contains the sex-determining region of the Y chromosome (SRY gene) that is expressed early in development within specific tissues, in particular the undifferentiated gonads. The SRY gene encodes for a protein known as the testes-determining factor, which differentiates the gonads into a testis early in development. In the absence of the Y chromosome, in females, the gonads differentiate into ovaries. Later, the testes begin to secrete testosterone and dihydrotestosterone (DHT), which differentiate the body and the brain into a male phenotype. In particular, the external genitalia are masculinized; the Wolffian duct develops into the ducts, epididymis, vas deferens, and seminal vesicles; and, importantly for this chapter, the brain is masculinized. In the absence of any testosterone secretion, the external genitalia develops as female, the M?llerian duct develops as the uterus and fallopian tubes, and the brain is feminized

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The basic processes of neural development that occur in a sex-specific manner. (a) Proliferation of cells or neurogenesis (the generation of new neurons)

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Epigenetic mechanisms by which sex differences in the brain are established during neural development. (1)

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Sexual Differentiation and Sex Differences

in Neural Development

Alexandra Turano, Brittany F. Osborne, and Jaclyn M. Schwarz

Contents

1 Introduction

2 Sex Determination and Sexual Differentiation

2.1 Biological Sex is Determined at the Time of Conception

2.2 Sex Hormones Induce Sexual Differentiation of the Body

2.3 Sex Hormones Induce Sexual Differentiation of the Brain

2.4 Genetic Sex Differences Impact the Developing Brain

3 Sex Differences in the Size of Brain Nuclei

3.1 The Spinal Nucleus of the Bulbocavernosus

3.2 The Anteroventral Periventricular Nucleus of the Hypothalamus

3.3 The Bed Nucleus of the Stria Terminalis

4 Sexual Differentiation of the Brain Requires Communication Across and Involvement

of Multiple Cell Types in the Brain

4.1 An Immune Molecule and a Neurotransmitter Coordinate Masculinization

of the Preoptic Area

4.2 Sexual Differentiation Across the Synapse in the Ventromedial Nucleus

of the Hypothalamus

4.3 Development of Neuropeptides Involved in Sex-Speciﬁc Behaviors

4.4 Microglia Induce Sex-Speciﬁc Neuronal Development

4.5 Sex Differences in Cell Genesis and Fate During the Development of Cognitive

Circuits

5 A Whole Body Perspective on Sex Differences in Neural Development

5.1 The Gut Microbiome

5.2 The Liver

5.3 The Placenta

6 Epigenetics and the Sexual Differentiation of the Brain

7 Conclusions

References

Abstract Sex determination occurs at the moment of conception, as a result of XX

or XY chromosome pairing. From that point, the body undergoes the process of

A. Turano, B. F. Osborne, and J. M. Schwarz (*)

Department of Psychological and Brain Sciences, University of Delaware, Newark, DE, USA

e-mail: jschwarz@psych.udel.edu

©Springer International Publishing AG, part of Springer Nature 2018

Curr Topics Behav Neurosci

DOI 10.1007/7854_2018_56

sexual differentiation, inducing the development of physical characteristics that are

easily distinguishable between the sexes and are often reﬂected in one’s physical

appearance and gender identity. Although less apparent, the brain also undergoes

sexual differentiation. Sex differences in the brain are organized during a critical

period of neural development and have an instrumental role in determining the

physiology and behavior of an individual throughout the lifespan. Understanding

the extent of sex differences in neurodevelopment also inﬂuences our understanding

of the potential risk for a number of neurodevelopmental, neurological, and mental

health disorders that exhibit strong sex biases. Advances made in our understanding

of sexually dimorphic brain nuclei, sex differences in neural cell communication,

and sex differences in the communication between the brain and peripheral organs

are all research ﬁelds that have provided valuable information related to the physio-

logical and behavioral outcomes of sex differences in brain development. More

recently, investigations into the impact of epigenetic mechanisms on sexual differ-

entiation of the brain have indicated that changes in gene expression, via epigenetic

modiﬁcations, also contribute to sexual differentiation of the developing brain. Still,

there are a number of important questions and ideas that have arisen from our current

understanding of sex differences in neurodevelopmental processes that necessitate

more time and attention in this ﬁeld.

Keywords Hormones · Neural development · Sexual determination ·

Sexual differentiation

1 Introduction

The brain develops very early in prenatal life via a number of important processes

that produce the cells, shape the structures, and reﬁne the connections within the

central nervous system. The goal of these neurodevelopmental processes is to create

a brain that controls our physiology and behavior for the rest of our lives; but also,

these processes produce the identifying characteristics and personality traits that we

associate with each and every one of us. One of the most notable and arguably more

important deﬁning characteristics of ourselves is our sex. The sex of many mam-

mals, including humans, is determined at the moment of conception, via the simple

process of sex determination that almost belies its importance; however, that single

moment of sex determination subsequently shapes the physiology, brain, behavior,

and health outcomes of the individual for a lifetime.

The physical characteristics that deﬁne our sex are often quite obvious and are

usually enhanced in our appearance by our gender identity, which is reﬂected in our

personality, our mannerisms, and our personal style. What may be less obvious, but

equally fascinating, are the sex differences that are established in our brains during

the process of neural development. The primary goal of these sex differences in the

brain is to control our physiology and certain behaviors in a sex-speciﬁc manner, and

in humans these sex differences also deﬁne our gender identity and inﬂuence our

sexuality. Just as important, sex differences in the brain can inﬂuence the risk for

certain neurodevelopmental disorders, neurological disorders, and mental health

A. Turano et al.

disorders; thus, it is important that we understand how sex differences in the

brain are established during early brain development and maintained throughout

the lifespan in order to better understand how these differences inﬂuence our

mental health and well-being.

The goal of this chapter is to summarize our understanding of how sex differences

in the brain are established during neural development in the context of how these

sex differences regulate sex-speciﬁc behaviors later in life. We will identify histor-

ical and recent ﬁndings that highlight the various mechanisms by which sex differ-

ences in the brain are established during development. Another goal throughout this

chapter will be to identify important future questions or areas of research in the ﬁeld

of sexual differentiation of the brain and sex differences in neural development.

2 Sex Determination and Sexual Differentiation

2.1 Biological Sex is Determined at the Time of Conception

Sex determination occurs at the moment of conception, when a female’s egg is

fertilized by a male’s sperm. The egg and sperm each contribute one half of the

genetic material to the resulting zygote. Each human zygote contains 23 chromo-

somes, including 22 pairs of autosomes and 1 pair of sex chromosomes. The egg

contributes one X chromosome to the zygote, whereas the sperm may contribute

either one X or one Y chromosome. If the sperm contributes an X chromosome, the

resulting zygote will become a female. She will possess two of the same allosomal

(sex) chromosomes, one X chromosome from the egg and one X chromosome from

the sperm. Conversely, if the sperm contributes a Y chromosome, the zygote will

become a male (Fig. 1). He will have two different allosomes, an X chromosome

from the egg and a Y chromosome from the sperm, thereby differentiating him

genetically and phenotypically from a developing female (Painter 1923). As cells

multiply and the zygote matures into an embryo, then fetus, and eventually a

newborn baby, the presence of either XX or XY chromosomes is maintained within

each and every cell, and therefore each cell sustains an autonomous chromosomal

sex throughout the lifespan that is established at the moment of conception.

Sexual differentiation is the process by which the developing embryo becomes

male or female after sex determination; and this important process occurs quite early

in embryonic development. Sexual differentiation is the process by which primary

sex characteristics develop, including the development of the gonads and sex organs,

but it is also the process by which many sex differences in the brain are established

(Fig. 1). While it is a distinct process, sexual differentiation is dependent upon the

process of sex determination. In particular, the sex-determining region of the Y

chromosome, known as the Sry gene, is critical for the process of sexual differ-

entiation of males. The Sry gene encodes for the protein known as testis-determining

factor (TDF). TDF initiates the differentiation of steroidogenic precursor cells and

primordial germ cells within the developing gonads into Sertoli cells, resulting in the

Sexual Differentiation and Sex Differences in Neural Development

formation of testes (Fig. 1). Developing females (XX) do not have Y chromosomes

and therefore, do not have the SRY gene. Consequently, devoid of TDF expression,

the precursor cells of the developing gonadal organs differentiate into granulosa

cells, resulting in the formation of ovaries (Sekido 2014; Fig. 1).

Prior to sexual differentiation and Sry gene expression, the developing gonads are

“bipotential,”meaning they have the ability to develop into either testes or ovaries.

Importantly, expression of the Sry gene in developing males must occur within a

sensitive window during embryogenesis in order for the bipotential gonads to

differentiate into testes as opposed to ovaries (Taketo et al. 2005). The long-held

belief was that without the presence of the Sry gene, the precursor cells of

the developing gonads passively become ovaries (Haseltine and Ohno 1981;

Fig. 1 Sex determination and sexual differentiation of the brain. Sex determination occurs at the

moment of conception, when the embryo receives either two X chromosomes (female embryo) or

an X and a Y chromosome (male embryo) from the mother and the father. If the developing embryo

has a Y chromosome, this chromosome contains the sex-determining region of the Y chromosome

(SRY gene) that is expressed early in development within speciﬁc tissues, in particular the

undifferentiated gonads. The SRY gene encodes for a protein known as the testes-determining

factor, which differentiates the gonads into a testis early in development. In the absence of the Y

chromosome, in females, the gonads differentiate into ovaries. Later, the testes begin to secrete

testosterone and dihydrotestosterone (DHT), which differentiate the body and the brain into a male

phenotype. In particular, the external genitalia are masculinized; the Wolfﬁan duct develops into the

ducts, epididymis, vas deferens, and seminal vesicles; and, importantly for this chapter, the brain is

masculinized. In the absence of any testosterone secretion, the external genitalia develops as female,

the Müllerian duct develops as the uterus and fallopian tubes, and the brain is feminized

A. Turano et al.

Jost 1947). Contrary to this long-held belief, however, sex reversal studies investi-

gating the result of XX, loss-of-function and XY, gain-of-function mutations on

speciﬁc sex-linked genes including Wnt4,β-catenin,R-spondin1, and Foxl2 have

found that differentiation of the bipotential gonads into ovaries also requires the

active repression of testis formation (Chassot et al. 2008; Maatouk and Capel 2008;

Pailhoux et al. 2001; Sekido 2014; Vainio et al. 1999). These ﬁndings in particular

highlight the complex process of gonadal differentiation in males and females.

Importantly, the brain is also differentiated as either male or female via active and

distinct processes; however, it has proven to be more difﬁcult to discern the

mechanisms by which the developing brain actively differentiates as female,

which we will discuss in further detail in Sect. 6.

2.2 Sex Hormones Induce Sexual Differentiation of the Body

Once sexually differentiated, the male testes begin to secrete important molecules for

the continued sexual differentiation of the body and the brain; thus, the process of

gonadal differentiation is not concurrent with the process of brain sexual differ-

entiation, but rather one process sequentially follows the other (Fig. 1). Because the

process of sexual differentiation of the body and brain occurs at various points after

sex determination and the formation of the gonads, disruption in one or more of these

processes can result in disorders of sexual development wherein the “sex”of the

differentiated body or brain does not “match the sex”of the already differentiated

gonads. This may result in ambiguous genitalia or a dysmorphic gender identity,

both of which comprise an important, but distinct area of research from what we will

cover here.

In males, the testes produce an early surge of androgens, including testosterone

and dihydrotestosterone (DHT), that leads to the development of sex-related organs,

such as the epididymis, vas deferens, and male genitalia (Fig. 1). The testes also

secrete anti-Müllerian hormone, which is responsible for actively repressing

the Müllerian duct. The Müllerian duct is a primordial structure necessary for the

development of the female sex organs; thus its suppression is necessary for the

complete and correct development of male as opposed to female sex organs (Josso

et al. 1993). As the Müllerian duct regresses in developing males, the Wolfﬁan duct

grows into the male accessory organs (Fig. 1). In contrast to developing males,

developing females possess ovaries. Like the testes, the ovaries also secrete sex

hormones; however, sex hormone secretion from the ovaries occurs at a much later

time –at the onset of puberty (Sekido 2014). Thus, in the complete absence of

perinatal androgen exposure, the Wolfﬁan duct regresses, and the Müllerian duct

develops, opposite to the ongoing processes that occur in developing males. Speciﬁ-

cally, in the absence of early-life androgens and anti-Müllerian hormone, female sex

organs, such as the oviduct, uterus, and female genitalia, begin to develop (Jost

1947; Fig. 1). As the appearance of these outwardly distinct male or female sex

organs begins to develop, we are able to determine the sex of a developing fetus in

Sexual Differentiation and Sex Differences in Neural Development

the womb during an ultrasound –often a monumental event when anticipating the

arrival of a soon-to-be bumbling baby.

2.3 Sex Hormones Induce Sexual Differentiation of the Brain

Sexual differentiation does not end at the level of the gonads and sex organs. This

process of becoming male or female also involves the sexual differentiation of the

brain (Fig. 1). Like the gonads, the brain is initially a bipotential organ, meaning that

it has the potential to be either masculinized or feminized. The presence (or absence)

of androgens during perinatal development “organizes”certain structures and func-

tions of the brain in a sexually dimorphic manner. The consequences of this

organization become most apparent following “activation”of these sexually dimor-

phic brain regions during puberty, which is induced by the production of the sex

hormones testosterone (males) and estrogen and progesterone (females). As a result,

this theory of sexual differentiation of the brain became known as the organizational-

activational hypothesis (Phoenix et al. 1959; Fig. 2) that sex differences in the brain

are organized during early brain development and then activated by circulating

Fig. 2 The organizational-activational hypothesis of sex differences in the rodent brain. Testos-

terone levels peak around birth in the developing male rodent and naturally decrease within a few

days after birth. During that time, the testosterone permanently masculinizes the brain, organizing

the size and structure of certain brain regions necessary to perform male sex behavior. In the

absence of testosterone, the brain is feminized, permanently organizing the size and structures of the

brain regions necessary to perform female sex behavior. At puberty, the testes and the ovaries begin

to secrete sex steroid hormones. Males gradually increase production of testosterone, the levels of

which are maintained at a relatively consistent point. Females show varying levels of estradiol and

progesterone throughout the estrous cycle necessary for ovulation. The production of sex steroid

hormones at puberty activates brain regions that were organized during development, thereby

producing sex-speciﬁc sexual behaviors necessary for reproduction

A. Turano et al.

hormones after puberty. Demonstrating this theory, Phoenix et al. (1959)ﬁrst

ovariectomized female neonatal guinea pigs and then treated them with testosterone

during the “organizational”period of brain development. Following what would

have been the onset of puberty, female guinea pigs were treated with a second

injection of testosterone, and they subsequently exhibited male-typical, as opposed

to female-typical, sex behavior (Phoenix et al. 1959). Testosterone given to the

ovariectomized neonatal female guinea pig was sufﬁcient to masculinize the brain of

the genetically female (XX) guinea pig, causing her to display male-typical sex

behaviors following a second testosterone injection later in life. Notably, this process

of testosterone-induced sexual differentiation of the brain is conserved across many

species including other rodents, birds, and primates, including humans.

The mechanisms by which certain brain regions are organized in a sexually

dimorphic manner, resulting in sex-speciﬁc behavior, involve speciﬁc processes

that must occur at speciﬁc stages of neural development. In accordance with the

organizational-activational hypothesis and the experiments by Phoenix and col-

leagues in 1959, studies by Gorski and colleagues in 1978 indicated that ovariecto-

mized female rats also exhibited male-typical sex behavior following a pubertal

testosterone injection, if they had been exposed to testosterone early in life. Inter-

estingly, they proposed that this behavior was due to increases in the number of cells

within a brain region known as the sexually dimorphic nucleus of the preoptic area

(SDN-POA; Gorski et al. 1978). The SDN-POA is larger in males than in females,

indicative of its role in male-typical sex behavior. Concurrently, castrated males (i.e.,

devoid of perinatal testosterone exposure) exhibited no sex behavior following an

injection with testosterone in puberty and showed decreases in the number of cells

within the SDN-POA (Murakami and Arai 1989). Therefore, androgens are neces-

sary to prevent ongoing programmed cell death, or apoptosis, in the SDN-POA

during early postnatal brain development, and in doing so, androgens organize the

size and structure of the SDN-POA in a sexually dimorphic manner (Dodson and

Gorski 1993). The presence or absence of early-life androgens impacts the size of the

SDN-POA, as well as a number of other sexually dimorphic brain regions (Fig. 3);

and in doing so, it permanently affects the behavioral output of the animal in a

sex-speciﬁc manner later in life.

Notably, testosterone secreted by the testes is converted to estradiol in the

developing male rodent brain, by the p450 enzyme, aromatase. While testosterone’s

action on the androgen receptor contributes to masculinization of the brain, estra-

diol’saction on estrogen receptors (in particular, estrogen receptor α) is also

important for the masculinization and defeminization of the developing male rodent

brain (Arnold and Gorski 1984). During this critical period of sexual differentiation

of the brain, female rodents may be exposed to exogenous estrogens from the

mother, but, female rodents are protected from any possible estradiol-induced

changes in the developing brain by the high afﬁnity alpha-fetoprotein (AFP) binding

protein, which binds any possible circulating estrogens during the early develop-

mental period, thus protecting females against potential masculinization and

defeminization of the brain by maternal estrogens (Bakker et al. 2006).

Sexual Differentiation and Sex Differences in Neural Development

Fig. 3 The basic processes of neural development that occur in a sex-speciﬁc manner. (a) Proliferation of cells or neurogenesis (the generation of new neurons)

occurs in a sexually dimorphic manner in brain regions including the anteroventral periventricular nucleus (AVPV), the hippocampus, and the amygdala. (b)

Differentiation of neural progenitor cells into either neurons or glia can occur in a sex-dependent manner in brain regions such as the amygdala and the

anteroventral periventricular nucleus (AVPV). (c) Testosterone or estradiol converted from testosterone promotes the outgrowth of axons seeking new synaptic

connections in the amygdala and the AVPV. (d) In the preoptic area (POA) and the ventromedial nucleus of the hypothalamus (VMN), estradiol converted from

A. Turano et al.

testosterone promotes dendritic spine synapses during postnatal brain development. (e) In the spinal nucleus of the bulbocavernosus (SNB) muscle, testosterone

promotes axon survival of neurons projecting to the muscle. In the absence of testosterone, in females, the synapses are naturally eliminated during postnatal

development. (f) Naturally occurring cell death, apoptosis occurs in a sexually dimorphic manner to produce sex differences in the size of certain brain nuclei. In

brain regions where the size is larger in males than in females (M >F), testosterone promotes the survival of the neurons and prevents apoptosis from occurring.

In brain regions where the size is larger in females than in males (F >M), testosterone promotes naturally occurring cell death during neural development

Sexual Differentiation and Sex Differences in Neural Development

The seminal study by Phoenix and colleagues in 1959, and subsequent studies by

Gorski and colleagues, provided the ﬁrst evidence of gonadal hormone-induced

sexual differentiation of the brain and behavior. This work led to an intense

investigation into the cellular mechanisms by which hormones can produce

sex-speciﬁc phenotypes of the brain and behavior during early brain development.

One of the best-known examples of sex differences in brain structure producing sex

differences in behavior is observed in birds that produce song. Male birds that

produce song as a way to attract female mates have signiﬁcantly larger nuclei

compared to females within two main neural pathways important for the learning

and the production of song (Jordan et al. 1988; Nottebohm and Arnold 1976;

Nottebohm et al. 1982). For example, the “high vocal center”(HVC) and the robust

nucleus of the archistriatum (RA) are two sexually dimorphic nuclei within the

efferent motor pathway responsible for the production of male bird song. Both

regions are three to six times larger in males compared to females (Nottebohm and

Arnold 1976; Nottebohm 1991). The neurons of these nuclei were later found to be

more numerous, larger in size, and more scattered in males than in females (Arnold

and Gorski 1984; Mathews et al. 1988). If females are treated with estradiol early

post-hatching and then treated with either testosterone or dihydrotestosterone (DHT)

as adults, the HVC and the RA become similar in size to that of the males, and the

treated females are then able to produce song (Gurney and Konishi 1980).

Characterization of the sexually dimorphic nuclei controlling song production in

birds provided some of the ﬁrst direct evidence linking hormone-induced changes in

brain structure to sexually dimorphic behavior but also provided converging support

for the organizational-activational hypothesis of sexual differentiation of the brain.

That said, evidence from these studies also suggested that the organizational and

activational effects of hormones cannot completely account for the sexual differen-

tiation of these brain nuclei. For example, male birds that are castrated as hatchlings,

prior to the secretion of testosterone, sing normally as adults, suggesting the possi-

bility that activation of genes on the avian sex chromosomes may also induce

masculinization of the brain in song birds (Arnold 2003; Wade et al. 2001).

2.4 Genetic Sex Differences Impact the Developing Brain

While the evidence above elucidates androgens as an essential mediator of sexual

differentiation of the brain, it is also important to keep in mind that each cell’s

intrinsic sex may also have the ability to contribute to the organizational-activational

hypothesis, as the presence of either XX or XY chromosomes is maintained within

every cell. In other words, organization of sexually dimorphic brain regions may not

rely on gonadal sex hormones alone but may also involve sex chromosome com-

plement, such as the direct expression of the Sry gene within male brain cells or sex

differences in gene expression that result from the process of X inactivation within

female brain cells (Arnold and Chen 2009; Carrel and Willard 2005; Dewing et al.

2006). This phenomenon has been investigated using a mouse model described as

A. Turano et al.

the “four core genotypes”(FCG) model, in which gonadal sex and chromosomal sex

are purposely misaligned to examine the impact of each factor either separately or

interdependently (Carruth et al. 2002; De Vries et al. 2002). This phenomenon has

also been investigated in a gynandromorphic ﬁnch, in which sex chromosome

complement was bilaterally split in a bird, but testosterone derived from the one

testis (on the “genetic male”side of the body) could circulate throughout the

bloodstream and inﬂuence the entire organism, including its brain (Agate et al.

2003). Thus, when sex differences were present across both hemispheres of the

brain in the gynandromorphic ﬁnch, despite the similar hormonal exposure, they

could be attributed to sex differences in gene expression that resulted from the avian

sex chromosomes. Notably, this ﬁnch could still produce male song, indicative of the

important role testosterone has in differentiating man regions within male brain and

associated behavior. Taken together, these models have been effective in shedding

light on how sex chromosome complement affects sex differences in gene expres-

sion and phenotypic variation in the brain beyond the scope of sex hormones alone

(Carruth et al. 2002; Xu and Disteche 2006).

It is also important to consider the evolutionary basis underlying the mechanisms

for sexual differentiation of the brain in general. Despite the fact that males and

females each have a unique set of sex chromosomes, the sex chromosomes have an

important, but relatively limited role in the process of sexual differentiation of the

body and brain in many species, including humans (McCarthy and Arnold 2011).

Instead, the sex hormones are the primary mechanism by which sexual differentia-

tion of the body and brain occurs. Sexual differentiation requires the coordinated

differentiation of a wide variety of cells and tissues throughout the body. These cells

and tissues are also mutually dependent upon each other for proper function and

coordinated behavior later in life. As a result, sex hormones may have “taken on”the

important role of sexual differentiation because of their ability to target speciﬁc cells

and tissues throughout the entire body and because of their important role in

sex-speciﬁc physiology and behavior in general. Despite this, there may be many

other processes of neural development that are directly affected by the genes of the

sex chromosomes or by the sexually dimorphic expression of genes independent of

sex hormones that remain to be discovered. Thus, the complexity of sex-speciﬁc

neurodevelopmental and long-term behavioral outcomes resulting from either

chromosomal or hormonal sex, including those that do not apply directly to repro-

ductive or copulatory behavior, continues to be explored.

The organizational-activational hypothesis is supported by sex differences in the

development of a number of brain regions including the SDN-POA (described

above), the spinal nucleus of the bulbocavernosus (SNB), the bed nucleus of the

stria terminalis (BNST), and the anteroventral periventricular nucleus of the hypo-

thalamus (AVPV) (Fig. 3). These brain regions and their associated sex-speciﬁc

behaviors will be described throughout the remaining chapter. Importantly, we will

focus our discussion on the mechanisms by which neurodevelopment is inﬂuenced

by sex hormones to induce these well-known sex differences in the brain, and in the

ﬁnal conclusions of this chapter, we will discuss important future questions and

ideas that arise from our understanding of sex differences in neurodevelopmental

processes.

Sexual Differentiation and Sex Differences in Neural Development

3 Sex Differences in the Size of Brain Nuclei

3.1 The Spinal Nucleus of the Bulbocavernosus

The spinal nucleus of the bulbocavernosus (SNB) was the ﬁrst region identiﬁed in

the rodent central nervous system (CNS) to exhibit an androgen-dependent sex

difference in cell number. Speciﬁcally, males have signiﬁcantly more neurons within

the SNB than females, and treatment of neonatal females with testosterone increases

the number of neurons in the female SNB to that seen in males (Sengelaub and

Forger 2008). Breedlove and Arnold (1980) identiﬁed this collection of motor

neurons, located within the ﬁfth and sixth lumbar segments of the rat spinal cord,

which they named the SNB after the perineal striated muscles these neurons inner-

vate. They observed that these particular motor neurons were more sensitive to

treatment with testosterone than treatment with estradiol, the aromatized product

of testosterone. Interestingly, when they examined genetically male rats with a

testicular feminization mutation that severely reduced the amount of functional

androgen receptors, Breedlove and Arnold (1981) observed a reduction in, or

demasculinization of, the total cell number within the SNB of these mutated animals.

Based on these observations, they concluded that testosterone signaling at the

androgen receptor during development plays an important role in determining the

sex difference in the number of neurons within this nucleus of the CNS.

Nordeen et al. (1985) subsequently identiﬁed an epoch within the perinatal

period –between embryonic day 22 (E22) and postnatal day 10 (P10) –during

which the SNB cell numbers are decreased via naturally occurring apoptotic

processes, and this neurodevelopmental process is more robust in female compared

to male rats, resulting in the sex difference in cell number described above (Fig. 3).

Female rats that were treated with testosterone during the critical period of spinal

cord development lost signiﬁcantly fewer neurons in the SNB, thereby exhibiting a

masculinized SNB cell number (Nordeen et al. 1985). In concordance with these

data, Breedlove and Arnold (1983) demonstrated greater cell death of SNB motor

neuron number when treating male rats with ﬂutamide, an androgen receptor anta-

gonist, which blocks the androgen receptor. Together, these data indicate that

androgens are effective in preventing programmed cell death within the SNB during

perinatal development. Ultimately, this proposed mechanism of sexually dimorphic

cell death is involved in the establishment of the sexually dimorphic size of the SNB,

which is maintained into adulthood and allows for successful male reproductive

behavior, in particular the control of the perineal muscles.

In addition to the impact of neonatal testosterone on naturally occurring apoptosis

in the SNB, Lubischer and Arnold (1995) found that treatment of castrated juvenile

male rats with testosterone delayed naturally occurring synapse elimination in the

spinal cord by increasing the percentage of perineal muscle ﬁbers innervated by

multiple axons, thus increasing the size of the associated motor units. The observed

changes in synapse elimination persisted for at least 12 months after the completion

of testosterone treatment, demonstrating that the effects of testosterone are persistent

A. Turano et al.

and have an important role in the establishment of sex differences in the spinal cord

that are maintained long-term (Lubischer and Arnold 1995).

These data sparked an investigation into the ontogeny of androgen receptor

expression in the muscles innervated by SNB motor neurons; and, in 1997, Jordan

and colleagues deﬁnitively showed that the rat perineal muscles that are innervated

by SNB motor neurons also express androgen receptors. More importantly, the

emergence of androgen receptors on the perineal muscles actually precedes their

emergence on the motor neurons that synapse with the SNB (Jordan et al. 1997). In

fact, in the mouse SNB circuit, androgen receptors were not expressed on SNB

motor neurons until P4, but they were present on the SNB target muscles as early as

E15 (Smith et al. 2012). Ultimately, these discoveries have challenged the notion

that neonatal androgen exposure is necessary for the sex difference in cell number in

the SNB via the direct action of androgens on the motor neurons themselves,

particularly given that androgen receptors were not detected on SNB motor neurons

until P4, a time point outside of the natural surge in testosterone that occurs in

developing males. Instead, androgens’action on the perineal muscles inﬂuences the

way by which these muscles are subsequently innervated by SNB motor neurons at a

later date, and thereby, it is androgens’action on the muscles itself that has a critical

role in establishing the male-biased sex difference in SNB cell number that is

maintained into adulthood. This is an important point to consider when understand-

ing the various mechanisms by which sex differences in the nervous system can be

established. Sex hormones, in this case testosterone, can inﬂuence one set of target

cells (muscle tissue) to, in turn, inﬂuence the innervation, number, and function of

motor neurons in the spinal cord, likely via the androgen-induced structural support

and growth factors that are present at the neuromuscular junction (Fig. 3). These

ﬁndings highlight the idea that sexual differentiation of the nervous system can be

initiated early, at the time of perinatal testosterone exposure, but that the effects can

also be persistent, as testosterone is able to maintain cell survival at a later point in

development, as naturally occurring apoptosis continues postnatally.

3.2 The Anteroventral Periventricular Nucleus

of the Hypothalamus

Unlike many of the other sexually dimorphic brain regions discussed in this chapter,

the anteroventral periventricular nucleus of the hypothalamus (AVPV) is signiﬁ-

cantly larger in females than in males. This particular nucleus of the hypothalamus

plays an important role in gonadotropin release via the robust expression of

kisspeptin, found in a subset of AVPV neurons (Kauffman et al. 2007). Speciﬁcally,

the Kiss1 neurons in the AVPV control a preovulatory surge in luteinizing hormone

(LH) that drives ovulation in females (Hu et al. 2015). Prior to its designation as the

AVPV, this collection of cells was originally identiﬁed by Bleier et al. (1982) as the

medial preoptic nucleus (MPN). In a comparative anatomy study, they discovered

Sexual Differentiation and Sex Differences in Neural Development

that the MPN (or AVPV) of the guinea pig, rat, and hamster contained more cells in

adult females compared to adult males (Bleier et al. 1982). The work of Bleier and

colleagues established that the sexually dimorphic nature of this speciﬁc brain region

is, indeed, conserved across a number of species, corresponding with the later

discovery of the AVPV’s important role in female sex behaviors and the control of

ovulation.

In previous sections, we discussed how neonatal androgen receptor signaling

mediated the establishment of a sex difference in cell number within the SNB and the

SDN-POA via its impact on cell survival –by preventing naturally occurring

apoptosis. Similar to these two brain regions, sex-speciﬁc cell death is also the

cause of differences in cell number within the AVPV. In developing rats, the

AVPV undergoes neurogenesis between E13 and E18, and treatment of females

with testosterone or estradiol does not affect the number of newly born neurons in

the AVPV (Sumida et al. 1993). In contrast, treatment with either testosterone or

estradiol increases subsequent apoptosis in the AVPV, resulting in a sex difference

in the size of the AVPV by the time it was examined at E21 (Sumida et al. 1993;

Fig. 3). Notably, this pro-apoptotic effect of testosterone in the AVPV is contrary to

the role of neonatal testosterone signaling in the SNB, which protects against

apoptosis (Arai et al. 1996; Sumida et al. 1993). Furthermore, the data indicate

that the aromatization of testosterone into estradiol and thus the actions of estradiol,

in particular, contribute to increased cell death in the developing male AVPV. These

data are corroborated by the high level of estrogen receptor alpha (ERα) expression

on the cells of the AVPV (Shughrue et al. 1997). Bax, a protein that promotes

apoptosis, is found at higher concentrations in the developing male AVPV; while

Bcl-2, a protein that promotes cell survival, is found at lower levels in the developing

male AVPV (Tsukahara 2009). Thus, estradiol aromatized from testosterone results

in the sexually dimorphic expression of these pro- and anti-apoptotic genes (see

Forger 2006 for a comprehensive review), pushing the newly born neurons in

the male AVPV toward a fate of cell death during this embryonic period of

neurodevelopment (Fig. 3).

It is also important to note that the AVPV controls the LH surge in adult females

as a result of its ability to integrate hormonal signals with circadian signals, via the

function of kisspeptin neurons localized in this brain region (Simerly 2002). The LH

surge is the result of a neuroendocrine positive feedback loop between kisspeptin

and estradiol, which is only fully functional after puberty (Clarkson et al. 2009). This

positive feedback loop of hormones acting on the AVPV results in the addition of

even more cells to the female AVPV. Thus, similar to the mechanisms and timeline

of sexual differentiation of the SNB, the sex difference in cell number in the AVPV

is initially established during the prenatal period via apoptosis; however, there

remain additional changes to cell number during puberty that are necessary for the

establishment and maintenance of sex differences in the adult AVPV necessary to

control female ovulation. Interestingly, Mohr et al. (2016) found that this increase in

cell number in the AVPV at puberty not only includes the addition of new neurons

but also includes the addition of glial cells, indicating that both neurons and glia

appear to be contributing to the establishment and maintenance of sex differences in

A. Turano et al.

the size and function of the AVPV (Mohr et al. 2016; Fig. 3). This important idea

that various cell types in the developing brain contribute to sex-dependent develop-

ment and function of the brain is seen in other examples that will be discussed in

Sect. 4.

3.3 The Bed Nucleus of the Stria Terminalis

The bed nucleus of the stria terminalis (BNST) is located at one end of the stria

terminalis, which is the bundle of axon ﬁbers that connects the BNST to the

amygdala. Because of its structure and its role in social and emotional behaviors,

the BNST has been described as the “extended amygdala”(Lebow and Chen 2016).

Though the BNST forms a continuum with amygdaloid structures, it is now clear

that there are at least two distinct regions, including the medial division containing

the medial amygdaloid nucleus and the medial BNST, as well as a central division

containing projections between the central amygdaloid nucleus and the lateral BNST

(reviewed in Crestani et al. 2013). The BNST contains approximately 20 distinct

nuclei that are characterized based on the various cell types and their distinct pro-

jections to other brain regions. Characterization of the various cell types and the

afferent and efferent projections of the BNST (e.g., limbic structures, hypothalamic

structures, and brain stem nuclei) suggests that it serves as a critical relay for a

variety of neuronal circuits and other systems (e.g., neuroendocrine) to coordinate

activity related to the physiology and behavior of the organism. The BNST neurons

contain a variety of neurotransmitter and neuropeptides and their respective recep-

tors including vasopressin, glutamate, GABA, noradrenaline, acetylcholine, nitric

oxide, cannabinoids, and corticotropin-releasing factor (Crestani et al. 2013).

There is a signiﬁcant sex difference in the size of the principle nucleus of the

BNST (pBNST) in addition to sexually dimorphic innervation of vasopressin neu-

rons into the BNST. Vasopressin has been studied extensively in the BNST for its

role in various physiological and behavioral functions (see Sect. 4.3). For this

reason, much of our understanding of the functional role of the BNST in males

and females throughout brain development has its foundation in the vasopressin

system. Males have signiﬁcantly more vasopressin immunoreactivity, mRNA

expression, biosynthetic capacity, and a greater ﬁber density within the BNST

compared to females (Bales et al. 2007; Miller et al. 1989a,b). One of the main

vasopressin receptors, V1a, is more abundant in the BNST of males compared to

females in the medial posterior BNST, but not in the lateral dorsal or lateral posterior

BNST, suggesting that there is considerable heterogeneity within the subnuclei of

this region for V1a receptor distribution (Dumais and Veenema 2016). Vasopressin

neurons in the medial BNST were identiﬁed as the source of the sexually dimorphic

vasopressin ﬁber innervation of the brain in the mid- to late 1980s with the discovery

that males have nearly twice as many vasopressin-immunoreactive ﬁbers than

females (Fig. 3). Additionally, Miller et al. (1989a,b) determined that males have

signiﬁcantly more vasopressin-expressing cells and more vasopressin grains per cell

Sexual Differentiation and Sex Differences in Neural Development

than females. These data indicate that the sex differences in vasopressin BNST ﬁber

densities are due to males having more vasopressin-expressing neurons as well as

increased biosynthetic capacity on a per cell basis compared to females.

Progesterone is also important for establishing the sexually dimorphic expression

of vasopressin cells in the BNST. Testosterone, converted to estradiol, can induce

the synthesis of progesterone in the brain during postnatal development (Shughrue

et al. 1992), but more so, it is thought that progesterone from the pregnant mother

may induce sex differences in the developing fetal brain if sex differences are present

in the expression of progesterone receptors in the brain (Wagner et al. 1998). In fact,

there is a striking sex difference in progestin receptor (PR) expression on vasopressin

cells in the BNST (Quadros et al. 2002), and subcutaneous injection of progesterone

reduces the number of vasopressin mRNA-expressing cells in the BNST. To deter-

mine the role of PRs in the development of sex differences in the vasopressin system,

Rood et al. (2008) compared PR

lacZ

reporter mice to wild-type (WT) mice. PR

lacZ

reporter mice have a functional disruption in the PR gene that renders them unable to

respond to circulating progesterone. Surprisingly, PR

lacZ

reporter mice showed a

similar sex difference in the number of vasopressin-expressing cells in the BNST as

WT mice, suggesting that PRs do not play a major role in sexual differentiation of

vasopressin cells in the BNST; however, additional data from this study indicated

that PRs have a role in establishing the density of projections to brain regions that are

innervated by BNST originating vasopressin neurons (Rood et al. 2008). These

ﬁndings highlight the more “unconventional”mechanisms by which sex differences

in the developing brain can be established, in this case via the function of the

progesterone receptor. While it is clear that the BNST is sexually dimorphic, with

the vasopressin system playing an integral role in this sex difference, it remains to be

determined how disruptions in the development of the BNST can affect brain

development and sex-speciﬁc behavior later in life.

4 Sexual Differentiation of the Brain Requires

Communication Across and Involvement of Multiple Cell

Types in the Brain

As described in the previous sections, the sex of an organism is determined at the

moment of conception, and from that point of determination, every cell in the

organism has a sex. The sex chromosomes and, more importantly, sex hormones

differentiate the developing brain, body, and behavior of the organism early in

development. These early-life organizational and activational effects of sex hor-

mones “program”the function of various cells throughout the nervous system. As a

result, it is important that we also understand the cellular mechanisms by which

basic sex differences in the brain are established, as this may provide additional

insight into the cellular mechanisms resulting in disease processes that often occur in

a sex-speciﬁc manner throughout neurodevelopment.

A. Turano et al.

The brain is arguably one of the most active endocrine organs as it produces and

responds to a diverse array of chemical messengers. Chemical communication, such

as the kind that occurs in the endocrine system, via hormones and their cognate

receptors, is integral to all levels of biological organization, from the mediation of

intracellular processes to communication between organs and even communication

between individuals. Traditionally, chemical messengers have been classiﬁed into

separate systems, mainly the nervous system (i.e., neurotransmitters), endocrine

system (i.e., hormones), and the immune system (i.e., cytokines). However, it has

become increasingly clear that there is substantial overlap and intercellular commu-

nication between these systems. In fact, many neurons express receptors for hor-

mones and cytokines; at the same time, many immune cells express receptors for

neurotransmitters and hormones. With this knowledge in mind, it is easy to imagine

how the sex hormones responsible for sexual differentiation are also capable of

affecting neural development via their actions on multiple cell types and various

chemical messenger systems to differentiate the brain and determine neural function.

4.1 An Immune Molecule and a Neurotransmitter Coordinate

Masculinization of the Preoptic Area

The medial preoptic area (POA) in the rodent brain is a sexually dimorphic cluster of

cells, located in the preoptic nucleus (Gorski 1978). Discussed earlier, the sexually

dimorphic nucleus (SDN), a small cluster of cells of the POA, is ﬁve to seven times

larger in males compared to females and is necessary for the appropriate expression

of appetitive and consummatory male sex behavior (De Jonge et al. 1989;

Houtsmuller et al. 1994; Jarzab et al. 1990; Jeong et al. 2008; Todd et al. 2005).

In addition to this macroscopic, structural difference in the size of the SDN-POA, the

neurons throughout the POA of males also have two to three times more dendritic

spines than females. Within the ﬁrst 2 weeks of postnatal life, male rats have a higher

density of spine synapses along the dendrite on neurons in the POA compared to

female rats. Moreover, astrocytes in the male POA have longer processes with a

greater amount of branching than females (Amateau and McCarthy 2002a,b).

Masculinization of the POA is dependent on testosterone, which is converted to

estradiol in the rodent brain during the critical period of sexual differentiation. While

it is clear that naturally occurring apoptosis plays a critical role in establishing the

SDN-POA, another question remained –how does estradiol induce these structural

and synaptic changes to the neurons of the male POA? The answer, which was

surprising at the time, is that these changes are induced through increases in the

synthesis of prostaglandin E2 (PGE2), a pro-inﬂammatory signaling molecule.

PGE2 is derived from arachidonic acid following cyclinization by the cyclooxy-

genase enzymes COX-1 and COX-2, and PGE2 is best known for its role in fever

production and inﬂammation. Amateau and McCarthy (2004) determined that acti-

vation of estrogen receptors in the POA upregulates the production of COX-1 and

Sexual Differentiation and Sex Differences in Neural Development

COX-2 leading to a nearly sevenfold increase in PGE2. Notably, PGE2 has been

shown to be synthesized by other cells, such as microglia and astrocytes, within the

POA via activation of glial adenosine receptors by ATP (Fiebich et al. 2014);

however, Amateau and McCarthy (2004) showed that the increase in PGE2 synthe-

sis in the POA was speciﬁc to neurons. Neuronal PGE2 then induces the growth of

neuronal dendritic spines via the release of glutamate and activation of AMPA

receptors (Amateau and McCarthy 2004). Astrocytes are also responsive to estra-

diol; they release glutamate following stimulation with PGE2, and their morphology

is sexually dimorphic in the POA, as described above (Amateau and McCarthy

2002a; Bezzi et al. 1998). Thus, astrocytes play a critical role in the establishment of

sexually dimorphic synaptic connectivity within the POA (Fig. 3). Treating females

with estradiol or PGE2 produces male-like patterns of dendritic spines, and blocking

COX-1 or COX-2 in males leads to female-like patterns of dendritic spines in the

POA that are maintained into adulthood (Amateau and McCarthy 2002a,2004;

Wright et al. 2008). Furthermore, PGE2 receptors 2 and 4 (EP2 and EP4) have

been shown to be the primary contributors to the masculinizing effects of PGE2 in

the POA (Wright et al. 2008). Speciﬁcally, activation of EP2 or EP4 leads to

increases in protein kinase A (PKA), and if PKA signaling is disrupted during

neonatal development, the masculinization of the POA is blocked (Wright and

McCarthy, unpublished observation). Taken together, these data highlight a unique

multicellular signaling process through which sex steroid hormones can sexually

differentiate the developing brain.

4.2 Sexual Differentiation Across the Synapse

in the Ventromedial Nucleus of the Hypothalamus

A central region of the hypothalamus, the ventromedial nucleus (VMN), is a brain

region necessary for female sex behavior (Pfaff and Sakuma 1979). The VMN is

characterized by its oval shape and its sparse, thin dendrites (Millhouse 1973).

Similar to the POA, the male VMN neurons contain more than three times as

many dendritic spines and shaft synapses as those measured on female VMN

neurons (Matsumoto and Arai 1983). This sex difference in synaptic connectivity

is detected as early as postnatal day 2 and is maintained in the rodent brain until

postnatal day 100 (Matsumoto and Arai 1986; Pozzo Miller and Aoki 1991),

consistent with the long-term organizational effects of estradiol in the developing

brain. This masculinized pattern of dendritic spine morphology in the VMN can be

induced in females by treatment with either testosterone or its metabolite, estradiol,

within the ﬁrst few days of life. In adulthood, the female estrous cycle or exogenous

estradiol treatment causes changes in the dendritic spine density and patterning of the

VMN, which correlate with changes in female sex receptivity (Frankfurt et al. 1990),

indicating that changes in the synaptic connectivity of the VMN are a critical mech-

anism of inducing or constraining female sex behavior.

A. Turano et al.

Glutamate is the primary neurotransmitter in the VMN (Ziegler et al. 2002), and

estradiol mediates the changes in neuronal morphology in the developing VMN by

directly enhancing glutamate release from these developing neurons (Schwarz et al.

2008). During the critical period of sexual differentiation, estradiol exposure induces

the activation of PI3 kinase within only 3 h, which leads to a cascade of events that

can induce the defeminization of the synaptic connections in the VMN and the

defeminization of sexual behavior in adulthood. Speciﬁcally, activation of PI3

kinase in the developing VMN increases the release of glutamate from presynaptic

terminals within this region. This, in turn, activates glutamatergic receptors, activat-

ing MAP kinase and masculinizing the dendritic spine number measured in the

VMN (Schwarz et al. 2008; Fig. 3). Blocking any one of these steps, including PI3

kinase activation, MAP kinase activation, or AMPA/NMDA glutamate receptor

activation, completely prevents the increase in dendritic spine proteins seen follow-

ing estradiol treatment of the female rat pup and ultimately prevents the defemini-

zation of the brain and behavior later in life. Speciﬁcally, blocking NMDA receptors

during the critical period of sexual differentiation blocks estradiol-induced defemi-

nization of behavior, which results in the production of an adult rat that is capable of

expressing both male and female sex behavior (Schwarz and McCarthy 2008). These

ﬁndings describing sexual differentiation of the VMN indicate that the effect of the

sex hormone estradiol is not limited to the cells that express the estrogen receptor but

rather that estradiol-induced masculinization in the VMN initiates the coordination

of synaptic connections and entire circuits that control male sex behavior. As stated

above, the fact that estradiol activates PI3 kinase in the developing VMN within 3 h

after estradiol exposure indicates that the effects of estradiol on modulating neuro-

transmitter function to initiate the process of sexual differentiation within the VMN

occur in a relatively rapid time course. The relatively rapid time course of estradiol’s

effects suggests that the developing circuitry is extremely sensitive to the organiza-

tional effects of estradiol exposure during the critical period of sexual differentiation,

and this is likely the case for other brain regions that undergo sexual differentiation

during this brief and critical developmental window.

4.3 Development of Neuropeptides Involved in Sex-Speciﬁc

Behaviors

Vasopressin and oxytocin are neuropeptides involved in the regulation of many

sexually dimorphic social behaviors including social recognition, pair bonding, and

social cognition in various species (Ross and Young 2009; Veenema and Neumann

2008). These sex differences in behavior are the result of sexually dimorphic

innervation of these two neuropeptides within speciﬁc brain regions, which are

established during brain development. The vasopressin system was ﬁrst shown to

be sexually dimorphic in 1981 by de Vries and colleagues, who discovered that

males have more vasopressin-immunoreactive ﬁbers in the lateral septum (LS) and

Sexual Differentiation and Sex Differences in Neural Development

lateral habenula than females and approximately 90% of vasopressin cells in the

BNST (see Sect. 3.3) express androgen receptors, the absolute number of which is

signiﬁcantly greater in males compared to females (Bales et al. 2007; de Vries et al.

1981). Previous work has shown that gonadectomy of neonatal male rats signiﬁ-

cantly reduces vasopressin ﬁber density later in life, indicating that androgens are

necessary for the organization of the sexually dimorphic vasopressin system in the

BNST (De Vries et al. 1983). Interestingly, administration of testosterone to gonad-

ectomized males or intact females during the ﬁrst, second, or even third postnatal

week can induce masculinization of the vasopressin system. These ﬁndings suggest

that not only are androgens necessary for the organization of this particular neuro-

peptide system in males but that vasopressin neurons in the female brain are also

sensitive to the effects of androgens. Gonadectomy of adult males and ovariectomy

of adult females cause vasopressin immunostaining in the BNST to disappear almost

completely within several weeks post-surgery. This ﬁnding suggests that vasopres-

sin expression in the BNST is responsive to circulating steroid hormones even into

adulthood and indicates that males and females may respond differently to testo-

sterone (or its metabolites) in order to maintain the sexually dimorphic features of the

vasopressin system in adulthood. In fact, later studies investigating this possibility

found that when adult male and female rats were gonadectomized or ovariecto-

mized, respectively, and then subsequently administered equal doses of testosterone

(i.e., via a subcutaneous implant), the sexual dimorphism in the number of vaso-

pressin cells was maintained in the BNST, supporting the hypothesis that males and

females show different sensitivities to either testosterone or the metabolites of

testosterone (De Vries et al. 1994). It is likely that this sex difference in sensitivity

to androgens in the BNST is established early in life by perinatal testosterone

exposure. Indeed, the testosterone metabolites estradiol and 5α-dihydrotestosterone

(DHT) can affect vasopressin cell number in a sex-dependent manner, such that

treatment with estradiol increases the number of vasopressin cells in the BNST in

males and females compared to controls and DHT-treated animals; however, this

effect was greater in males than females (De Vries et al. 1994). Additionally, males

treated with estradiol and DHT combined had signiﬁcantly more vasopressin cells in

the BNST than when treated with estradiol alone, while this same effect was not true

for females. These data reiterate the fact that, in addition to the circulating hormones

of the neuroendocrine system, every cell has a sex, which means that male and

female cells have the potential to respond differently to the same stimulus, thus

impacting the brain and behavior in a sexually dimorphic manner.

The ﬁnding that the vasopressin system is more prominent in males compared to

females suggests that males, more than females, may rely on this neuropeptide for

the regulation of social behavior. In support of this notion, injections of vasopressin

enhanced social recognition in both males and females, but injection of a vasopressin

receptor antagonist prevented social recognition in males only (Bluthe et al. 1994;

Dantzer and Bluthe 1993). Additionally, vasopressin mRNA expression in the

paraventricular nucleus of the hypothalamus (PVN) was determined to be positively

correlated with the amount of social play behavior in juvenile males, but not females

(Paul et al. 2014). Indeed, vasopressin has been shown to regulate some behaviors

A. Turano et al.

differently in males and females. For example, vasopressin administered to males

stimulates aggressive behavior, while this same administration of vasopressin

inhibited aggressive behavior in females (Terranova et al. 2016). Furthermore,

dominance in males (i.e., more aggressive males) is strongly associated with acti-

vation of vasopressin cell bodies in the supraoptic nucleus of the hypothalamus

compared to subordinate or control males; however, in females, activation of

vasopressin cell bodies was observed in dominant and subordinate females to the

same degree, which were both higher than that observed in control females

(Terranova et al. 2016). These ﬁndings indicate that the vasopressin system exhibits

a fundamental sex difference in the regulation of aggressive behavior, whereby the

male vasopressin system shows a strong correlation to dominance status, but the

female vasopressin system is associated with social interaction more generally.

While the exact cellular mechanisms underlying the sexually dimorphic innervation

of the vasopressin system established by early androgen exposure are not known, it

is clear that this neuropeptide is integral to the expression of a myriad of sexually

dimorphic behaviors expressed later in life.

In contrast to vasopressin, oxytocin-synthesizing cells are greater in number in

females compared to males across a number of brain regions including the PVN,

lateral hypothalamus, lateral septum (LS), POA, and the BNST in several species.

Furthermore, the number of oxytocin-immunoreactive ﬁbers in the LS and BNST is

signiﬁcantly greater in female compared to male mice (Haussler et al. 1990) and in

the lateral hypothalamus of mandarin voles (Qiao et al. 2014); however, others have

found no sex differences in oxytocin mRNA expression (Dumais et al. 2013) nor in

the number of oxytocin neurons in humans (Wierda et al. 1991). It has been shown,

however, that administration of oxytocin or the oxytocin receptor antagonist can

increase oxytocin immunoreactivity in the PVN of female prairie voles, but not male

prairie voles (Yamamoto et al. 2004). Furthermore, when given neonatally, oxytocin

increased ERαimmunoreactivity in the ventromedial hypothalamus, and oxytocin

receptor antagonist decreased ERαimmunoreactivity in the POA of females, but not

males (Yamamoto et al. 2006). Unfortunately, there has been surprisingly little

investigation into the development of the oxytocin system in males and females,

so there is still much that remains to be elucidated regarding the neuroendocrine

regulation of oxytocin in the brain. It is also important to note that while less is

known about the oxytocin system in the brain, there is an abundance of evidence

demonstrating the sex-speciﬁc effects of oxytocin on behavior (for a review see

Caldwell 2017). For example, neonatal oxytocin exposure facilitates mate-guarding

behavior (a component of pair bonding that increases aggression and reduces social

behavior toward other voles) in female, but not male prairie voles (Bales and Carter

2003), and ICV administration of oxytocin was shown to facilitate partner preference

formation in females, but not males (Insel and Hulihan 1995; Williams et al. 1994;

Winslow et al. 1993).

Despite the evidence indicating that oxytocin may play a more prominent role in

females, Yao et al. (2017) found that oxytocin can impact certain social behaviors in

adult male, but not female, mice. Adult male mice that lack oxytocin signaling did

not prefer a female over a male social partner in a 3-chamber-social-investigation

Sexual Differentiation and Sex Differences in Neural Development

paradigm, as is typically observed in wild-type male mice. In contrast, no changes in

partner preference were observed in adult female mice with altered oxytocin signal-

ing. Also in contrast with the data described above, vasopressin did not impact

partner preference in either adult male or female mice within this speciﬁc paradigm.

Further examination indicated that social recognition in adult male mice appears to

be exclusively dependent upon oxytocin signaling in the medial amygdala, as

endogenous vasopressin was unsuccessful in deterring the negative effects of altered

oxytocin signaling on male partner preference. Notably, the authors indicated that

this male-speciﬁc effect of oxytocin on social partner preference is likely the result of

oxytocin rapidly modulating the sensory responses of aromatase neurons to social

cues; therefore, the basis for these contrasting results likely stems from the fact that

while oxytocin-synthesizing cells are greater in number in females compared to

males in the PVN, lateral hypothalamus, LS, POA, and the BNST, there are a greater

number of aromatase-expressing neurons in the medial amygdala of male mice,

compared to female mice (Wu et al. 2009; Yao et al. 2017). Importantly, these results

highlight the idea that sex differences in the development of neural circuits may

occur via neuropeptides (hormones or neurotransmitter systems) that are not directly

inﬂuenced by the traditional testosterone-mediated mechanisms of sexual

differentiation.

4.4 Microglia Induce Sex-Speciﬁc Neuronal Development

Beyond the “traditional”neural cells and sexually dimorphic circuits that have been

investigated for many years, the past decade or so has also uncovered sex differences

in the immune cells of the brain. For example, microglia, the resident immune cells

of the brain, are critical for many normal neurodevelopmental processes including

supporting neurogenesis, pruning spurious synaptic connections, and phagocytosing

apoptotic cells (Boulanger 2009; Deverman and Patterson 2009). During early

development, males have more microglia than females in certain brain regions,

including the hippocampus, the parietal cortex, and the POA (Lenz and McCarthy

2014; Schwarz et al. 2012). Interestingly, this sexually dimorphic proﬁle of

microglia shifts dramatically throughout development, with females having more

microglia with thick processes or stout phenotype than males as adults (Schwarz

et al. 2012). Given the striking sex difference in microglia number across multiple

brain regions, and given their importance in neural development, investigators have

sought to determine the developmental proﬁle of microglia (Hanamsagar et al. 2017)

and whether microglia have an active role in the sexual differentiation of the

developing brain. A recent study found that during hormone-induced sexual differ-

entiation of the brain, microglia in the female hippocampus have signiﬁcantly more

phagocytic cups than those in the male hippocampus (Nelson et al. 2017). When

females are treated with estradiol, the number of phagocytic cups is reduced to male

numbers (Nelson et al. 2017). In the POA, males have twice as many amoeboid

microglia as females, and estradiol treatment of female rat pups masculinizes

A. Turano et al.

microglia number and morphology in the POA (Lenz et al. 2013). Furthermore,

inhibiting microglia prevents the normal estradiol-induced masculinization of den-

dritic spines in the POA and adult copulatory behavior in males, indicating that

microglia are also an important cellular mediator of the process of brain sexual

differentiation (Lenz et al. 2013).

As the immune cells of the brain, microglia also respond to immune activation or

injury with rapid changes in morphology and the release of cytokines, processes

critical to maintaining homeostasis in the brain. Emerging literature in humans

suggests that early-life immune dysregulation leads to cognitive and behavioral

disorders that persist throughout the lifespan (Bilbo and Schwarz 2009; Frick et al.

2013; Maezawa et al. 2011; Sheridan et al. 2014) and many of these disorders show a

distinct male bias (e.g., autism spectrum disorders [ASD], ADHD, learning disabil-

ities, etc.; Schwarz and Bilbo 2012). Animal models investigating the effects of

early-life immune activation have found several sex-speciﬁcdeﬁcits in behavior. For

example, prenatal exposure to lipopolysaccharide (LPS; which activates microglia

via TLR-4 receptors) produces deﬁcits in social play behavior in juvenile males, but

not females (Taylor et al. 2012). Prenatal exposure to stress, which activates the

neuroimmune system, induces behavioral impairments including hyperactivity,

behavioral despair, and anhedonia in males, but not females (Bronson and Bale

2014; Mueller and Bale 2008). Collectively, the ﬁndings presented here highlight the

intricate communication that must occur between many cell types in the endocrine

system, nervous system, and immune system to allow for the development of a

sexually dimorphic brain and sex-speciﬁc expression of behavior.

4.5 Sex Differences in Cell Genesis and Fate During

the Development of Cognitive Circuits

Many of the brain regions described thus far are essential for controlling a variety of

sexually dimorphic sexual and social behaviors. There are many other brain regions

whose functions are very important, and yet no overt sex differences in these

associated behaviors have been found. For example, the hippocampus and the

amygdala are both important for the formation of memory and cognition, yet there

are no overt sex differences in the basic function of these circuits. That is, both males

and females are able to perform complex cognitive tasks and remember things to a

similar degree. That said, there are sex differences in the development of the

hippocampus and amygdala that have been identiﬁed and that are worth noting in

this chapter, mainly because of their potential role in the etiology or the prevalence

of neurodevelopmental disorders that are associated with deﬁcits or alterations in

learning, cognition, or emotion and that also display strong sex biases.

During postnatal brain development, the male hippocampus is only slightly larger

than the female hippocampus, even after controlling for sex differences in body or

head size (McCarthy and Konkle 2005), and this is thought to be the result of more

Sexual Differentiation and Sex Differences in Neural Development

neurons and glia in the male compared to the female hippocampus (Conejo et al.

2003; Hilton et al. 2003; Nunez et al. 2003). Consistent with this idea, there is a

signiﬁcant sex difference in the number of newly born cells in the developing rat

hippocampus, with the neonatal male rostral CA1 and DG having more proliferating

cells than the numbers observed in females. Both androgen and estrogen treatment

increased the number of proliferating cells in females to the numbers seen in

males underscoring, once again, the sexually dimorphic role of testosterone in

neurodevelopment (Zhang et al. 2008). These data highlight the fact that, even

though the size of the hippocampus is only slightly larger in males than females,

there is a striking and signiﬁcant sex difference in the proliferation of newly born

cells during a time when cell genesis, in particular neurogenesis, in the developing

brain and hippocampus is most robust. Notably, this sex difference in cell prolifer-

ation is an effect that is driven by prenatal androgen exposure (Fig. 3), and more

importantly, it is an effect that may have long-lasting consequences for the function

of this structure throughout development.

An adjacent brain region in the temporal lobe is the amygdala, which has an

important role in the processing of emotional stimuli and learning. The amygdala

also inﬂuences a number of sexually dimorphic behaviors including juvenile rough

and tumble play (which is greater in males and inﬂuenced by prenatal testosterone

exposure), and it is also involved in mating, parenting, aggression, and territoriality,

all of which are sexually dimorphic. The overall size of the medial amygdala is larger

in males than in females (similar to the hippocampus; Cooke 2006); however, in

contrast to the hippocampus, the female amygdala has signiﬁcantly more cell

proliferation than males during early postnatal brain development. Interestingly,

these cells differentiate into astrocytes as opposed to neurons (Krebs-Kraft et al.

2010; Fig. 3). This sex difference is the result of differential endogenous cannabi-

noid signaling in males and females that inﬂuences the likelihood of ongoing cell

proliferation and ultimately the fate of these cells as astrocytes.

To date, the question remains, what is the role of these sex differences in cell

number in both the hippocampus and the amygdala? It is likely that they have an

important role in generating sex differences in social and perhaps also cognitive

behaviors; however, that remains to be fully determined. Importantly, there are times

when a sex difference occurs in a brain region in order to eliminate sex differences in

behavior and in order to compensate for a sex difference in another coordinating

brain region (De Vries 2004). This may be particularly important for neural func-

tions or behaviors that are essential, like cognition (De Vries 2004), and may be one

explanation for the sex differences seen here, particularly in the hippocampus.

Perhaps more importantly, researchers should consider how these sex differences

in neural development may confer risk for deﬁcits in social or cognitive function,

particularly following a trauma or a challenge that perturbs cell genesis during early

brain development, an effect that may persist throughout the lifespan. In this case,

one sex may be more vulnerable to the trauma or challenge, resulting in a sex bias in

cognitive, social, or emotional disorders.

A. Turano et al.

5 A Whole Body Perspective on Sex Differences in Neural

Development

In accordance with the idea that every cell has a sex, there is accumulating evidence

that all organs are sexually differentiated and that even peripheral organs may affect

brain development (for a recent review, see de Vries and Forger 2015). The classic

example of peripheral organs affecting the brain is the basic process of sexual

differentiation of the brain via hormones released from the gonads, which act on

multiple cell types and receptors in the brain during development, as we described

above. The idea that sex differences in the body and brain are an important factor

affecting the health of an organism has been somewhat slow to be accepted, but

ignoring this could have detrimental effects for understanding disparate health out-

comes in men and women. In fact, there are well-known sex differences in vulner-

ability to disease, and in some cases sex is, perhaps, the most predictive factor

contributing to the prevalence of a disorder. Moreover, many of these disorders have

their origins in early development; thus an understanding of how other organs may

interact with ongoing processes that affect sexual differentiation of the brain is key to

elucidating mechanisms governing the neuroendocrine regulation of neural devel-

opment and behavior. Here we will present just a few examples of the sex differences

in peripheral organs and how they may affect brain function (Fig. 4).

Fig. 4 Examples of how peripheral organs can inﬂuence sex-speciﬁc development of the brain. Sex

differences in various peripheral tissues and organs, including the gut microbiome, the placenta, and

the liver, can produce sex-speciﬁcinﬂuences on the brain that consequently inﬂuence neural

development in a sex-speciﬁc manner

Sexual Differentiation and Sex Differences in Neural Development

5.1 The Gut Microbiome

Communication between the gut microbiome and the brain is now recognized as an

integral factor that impacts brain development and behavior. Following bacterial

inoculation of the infant with the maternal vaginal microbiome at birth, communi-

cation between these ﬂourishing gut microbes and the brain can function to integrate

various signals from the body (e.g., autonomic, neuroendocrine, etc.) that in turn

impact the brain. Importantly, the microbiome is highly dynamic, and alterations in

its composition occur throughout development, particularly during critical times of

sexual differentiation of the brain (Borre et al. 2014). Indeed, sex differences in the

gut microbiome emerge during puberty and are actively maintained into adulthood

(Yurkovetskiy et al. 2013). Speciﬁcally, the female microbiome stays relatively

similar to that of the prepubertal male and female microbiome, whereas the male

microbiome begins to diverge during puberty and exhibits a distinctive phenotype by

adulthood (Yurkovetskiy et al. 2013). Interestingly, if the surge in testosterone

during puberty is prevented via gonadectomy in males, this masculinization of the

gut microbiota is eliminated in adulthood. Furthermore, transfer of the microbiome

of an adult male to a pubertal female completely masculinizes the microbiota

composition, metabolomic proﬁle, and produces an increase in testosterone levels

that persist into adulthood suggesting that the differentiation of the microbiome

during development subsequently impacts neural and physiological function

(Markle et al. 2013). If, however, the antiandrogen ﬂutamide is administered with

the microbiome transfer, all of these changes are prevented in females demonstrating

the important, mechanistic role of testosterone in producing this microbiome-

induced masculinization (Fig. 4). In a well-established mouse model of autism

spectrum disorder (i.e., BTBR inbred mice), it was found that, while males and

females have signiﬁcantly altered microbiome composition compared to control

mice, these alterations are sex-speciﬁc (Coretti et al. 2017). These mice also display

sex differences in their inﬂammatory proﬁle in colon tissue, with males showing

signiﬁcantly higher levels of IL-6 and CD11c compared to females and control mice.

In another model of ASD, male mice, but not female mice, that are treated with

valproic acid on gestational day 11 show reduced social interactions and increased

gut inﬂammation (de Theije et al. 2014a). Additionally, valproic acid signiﬁcantly

alters the composition of the gut microbiota, and the levels of social interaction

correlate with metabolites of the microbiota (de Theije et al. 2014b). These data

suggest that the gut microbiome is capable of impacting the developing brain and are

quite intriguing given the increased prevalence of ASD in males and autism’s

associated risk with early-life inﬂammation.

5.2 The Liver

Approximately 72% of genes that have been examined in the liver show sex

differences in their expression. Given the important role of the liver and many of

A. Turano et al.

these genes in drug metabolism, this striking sex difference can in turn inﬂuence the

amount of drug that is delivered to the brain in a sex-speciﬁc manner (Yang et al.

2006). In fact, the best-known example of sex differences in liver function is drug

metabolism, which leads to differences in pharmacodynamics and pharmacokinetics

between males and females (Waxman and Holloway 2009). Sex differences in liver

metabolism can have profound effects on brain function in adults. For example, the

prescription drug zolpidem, which acts as a sleep aid by binding to GABA

receptors in the brain, was found to have a signiﬁcantly lower clearance rate in

women than men (Greenblatt et al. 2014), making it necessary to decrease the dosage

by 50% for women. Despite the fact that 6–7% of drugs that have examined sex as a

variable have shown a large difference in pharmacokinetics between men and

women, there is still very little known about how sex differences in drug metabolism

by the liver inﬂuence how drugs affect male and female brains. The liver secretes

steroid-binding proteins and enzymes that metabolize circulating gonadal hormones,

which inﬂuence the available levels of these hormones in the periphery of males and

females. Exactly how these liver enzymes alter the amount of gonadal hormones that

reach the brain to affect brain development in males and females is not yet known.

The maternal liver also synthesizes proteins that are critical for the development of

the fetus (Roy and Chatterjee 1983); however, it is not clear exactly how these

proteins affect long-term brain development of the fetus.

Furthermore, sexually dimorphic gene expression in the liver is regulated by

growth hormone-dependent transcription factors (Legraverend et al. 1992), which is

the result of a signiﬁcant sex difference in the expression of growth hormone itself.

Therefore, not only can sexually dimorphic genes of the liver differentially impact

brain function, but the brain (via sex differences in growth hormone expression) may

also impact the sexually dimorphic expression of genes in the liver, thereby creating

an interesting “brain affects liver, liver affects brain”mechanism. Still, sex differ-

ences in liver gene expression are also, in part, regulated by growth hormone-

independent transcription factors, suggesting that the sex differences in liver gene

expression extend beyond sex differences in growth hormone expression alone

(Yang et al. 2006). Clearly, more work is necessary to provide a complete under-

standing of the intricate processes by which peripheral tissues can impact sex

differences in the brain.

5.3 The Placenta

Beyond the developing fetus, the placenta is comprised of both maternal and fetal

cells. As a result, the placenta also has a sex (i.e., that of the developing fetus) that

has been shown to inﬂuence the relative risk or resilience of the fetus to maternal

insults in a sex-speciﬁc manner (Clifton 2010; Gabory et al. 2013). For example,

when pregnant rats are exposed to a stressor during early gestation, male but not

female placentas show increases in IL-6 and IL-1βgene expression. Importantly,

following this stress, male but not female offspring display anhedonia, adverse stress

Sexual Differentiation and Sex Differences in Neural Development

responsivity, and altered responses to selective serotonin reuptake inhibitors as

adults (Mueller and Bale 2008), and blocking the action of these cytokines in the

male placenta blunts the stress phenotype in adulthood, suggesting that the changes

in immune gene expression in the placenta are the mechanistic source of these

changes in the developing brain. The O-glycotransferase enzyme, O-GlcNAc trans-

ferase (OGT), is important for regulating gene expression through chromatin

remodeling in the placenta and is naturally lower in the male placenta compared to

the female placenta. Howerton and Bale (2014) showed that key features of the male

offspring that were subjected to early prenatal stress were recapitulated in males that

had genetically reduced levels of OGT in the placenta including HPA axis

dysregulation, reduced postpubertal growth, and hypothalamic mitochondrial dys-

function. Furthermore, females in this model, instead, had a blunted HPA axis

phenotype indicating sex-speciﬁc programming of placental OGT and supporting

the possible involvement of placental X- and Y-linked genes in determining stress

axis programming in adulthood (Howerton and Bale 2014; Fig. 4).

These are just a few known examples by which peripheral tissues can inﬂuence

the development and function of the brain in a sex-speciﬁc manner. Given that there

are many sex differences in the function of tissues and organs throughout the body,

an important area of research is to better understand how these tissues and organs

may also impact sex differences in the development of the brain, during normal brain

development, and also following early-life challenges (e.g., stress, infection, expo-

sure to drugs of abuse) or during disease states that affect the function of these

organs.

6 Epigenetics and the Sexual Differentiation of the Brain

The mechanisms described thus far that contribute to sexual differentiation of the

developing brain involve both the direct actions of sex hormones, as well as the

direct actions of the different genes located on the sex chromosomes that are inherent

in each cell (Arnold 2009; Gorski et al. 1978). Importantly, gene expression plays a

critical role in both of these interdependent mechanisms of brain organization

throughout early development. Therefore, it is perhaps not surprising that changes

in gene expression, induced via epigenetic modiﬁcations, also contribute to sexual

differentiation of the developing brain.

Epigenetics refers to the mechanisms that change gene expression via the proteins

or enzymes that moderate gene expression –mechanisms including modiﬁcations to

DNA methylation, histones, or DNA-binding proteins. These modiﬁcations take

place without causing signiﬁcant changes to the core DNA sequence (Menger et al.

2010; Nugent and McCarthy 2011), but rather these changes determine whether and

under what circumstances genes are transcribed or silenced. Epigenetic changes can

be induced as a result of the interactions between an organism’s genome and changes

in the organism’s internal or external environment. Notably, gonadal hormones,

which are key regulators of our internal environment, can signiﬁcantly impact

A. Turano et al.

epigenetic modiﬁcations to the DNA (Auger and Auger 2011; Nugent et al. 2015).

Therefore, given the sexually dimorphic nature of gonadal hormone secretion,

epigenetic processes are likely contributors in the process of sexual differentiation,

thereby determining in which sex and under what circumstances certain genes are

expressed in the body and the brain. While we have a strong understanding of how

sex differences in the brain are established, our understanding of how these sex

differences in the brain are maintained throughout the lifespan requires further

examination (McCarthy et al. 2009). Fortunately, epigenetic modiﬁcations of the

DNA, which are capable of inducing lifelong and even transgenerational changes in

gene expression (Roth et al. 2010), can and have been examined to answer this

exciting question of how sex differences may be established and maintained in the

developing brain.

We previously discussed the sexually dimorphic nature of the bed nucleus of the

stria terminalis (BNST), wherein the number of cells and the overall volume of the

primary nucleus of this brain region are signiﬁcantly greater in the male compared to

the female brain. This sex difference in the size of the BNST is a result of the

neuroprotective effects of testosterone in the developing male brain, which prevents

naturally occurring cell death in this brain region (Guillamon et al. 1988); however,

the mechanism by which testosterone protects neurons from this naturally occurring

apoptosis was previously not well understood. To address this gap in the research,

Murray et al. (2009) examined the role of epigenetic changes in the brain, speciﬁ-

cally by manipulating histone acetylation. Histone acetylation, via histone

acetyltransferases (HATs), is the process by which an acetyl group is added to the

lysine residues at the N-terminus of a histone protein, essentially “loosening”the

DNA from a tightly wrapped spool. Loosening the DNA from the histone increases

the opportunity for gene transcription. Conversely, histone deacetylases (HDACs)

decrease the opportunity for gene transcription by removing that same acetyl group

from the histone protein, keeping the DNA tightly wrapped around the histone.

HDAC activity can be blocked by the administration of HDAC inhibitors, like

valproic acid (VPA), thus resulting in increased histone acetylation and increased

gene transcription. When Murray et al. (2009) treated newborn male mice with VPA,

not only was histone acetylation increased throughout the brain, but the

neuroprotective effect of testosterone on cell number in the BNST was prevented.

In other words, the VPA-treated males had reduced BNST volumes. This effect was

also observed in newborn female mice that had been treated with testosterone and

thus, prior to VPA treatment, had BNST volumes equivalent to developing males

(Murray et al. 2009). Therefore, the masculinization of the BNST, via neonatal

testosterone exposure, also involves histone deacetylation, or decreased gene expres-

sion, within this brain region that persists even after the initial testosterone exposure.

These ﬁndings highlight the idea that androgens can disrupt an ongoing

neurodevelopmental process, in this case apoptosis, by disrupting the expression

of genes and thereby inducing a sex difference in the brain. Importantly, when these

genes are turned back on by HDAC inhibitors, like VPA, the sex difference can be

reversed (Fig. 5). Still, the question remains of whether or not this sex difference can

also be modulated outside the critical period of sexual differentiation. One might

Sexual Differentiation and Sex Differences in Neural Development

Fig. 5 Epigenetic mechanisms by which sex differences in the brain are established during neural development. (1) In males, testosterone converted to estradiol

can activate histone deacetylase (HDAC) resulting in the deacetylation of histones in the bed nucleus of the stria terminalis (BNST), which represses gene

expression necessary for programmed cell death in this brain region. (2) In females, in the absence of testosterone exposure, acetylation of histones increases

gene expression necessary for the programmed cell death in the female BNST. (3a) In males, testosterone converted to estradiol inhibits DNA methyltransferase

(Dnmt) in the preoptic area (POA) which results in the removal of methyl groups from the cytosines (C) of the DNA. (3b) In the absence of methylation in the

POA, genes necessary for masculinization of the brain and sexual behavior are expressed. (4a) In females, Dnmt is constitutively active in the POA, in the

absence of testosterone. (4b) Methylation of the DNA is maintained by Dnmt activity, which results in the repression of genes necessary for masculinization of

the POA and behavior

A. Turano et al.

assume that apoptosis in the BNST that occurs during the period of neuro-

development occurs exclusively during this critical period. However, it is also

possible that testosterone permanently alters the likelihood that the genes necessary

for programed cell death in the BNST are expressed, making it possible for gene

transcription to be turned on outside of the critical period of sexual differentiation.

If so, could cell death again be induced, thereby reversing the sex difference in the

size of this nucleus?

This fascinating question may be answered in part by a more recent study that

examined the epigenetic mechanisms by which sex differences in the structure and

function of the POA are established and maintained. As mentioned previously, the

POA exhibits a striking sex difference in the structure and function of its neurons

that is established as a result of perinatal testosterone exposure and is necessary for

the maintenance and expression of sex differences in sexual behavior later in life

(Amateau and McCarthy 2004). A study by Nugent et al. (2015) found that andro-

gens, converted to estradiol in the rodent brain, induce this sex difference in the POA

via the inhibition of DNA methyltransferase (Dnmt) activity. Dnmt is an enzyme that

methylates DNA, which is thought to silence gene transcription. Thus, androgen

exposure during the critical period of sexual differentiation induces the expression of

certain genes that are necessary to masculinize the POA by inhibiting the process of

DNA methylation. This effect may be maintained as the developing POA matures,

particularly given that others have shown that testosterone (or estradiol) can induce a

global decrease in DNA methylation that is maintained as cells multiply and mature

(Bramble et al. 2016). Furthermore, Nugent et al. (2015) found that when Dnmt3

activity was conditionally knocked out later in life (after the critical period of sexual

differentiation), in the absence of Dnmt3 and decreases in DNA methylation in the

POA, genes necessary for masculinization of the POA could be turned on, resulting

in masculinization of the brain and displays of male sex behavior. These data

highlight a few important points: (1) in the female brain, methylation represses the

genes necessary to induce masculinization of the POA; (2) during the critical period

of sexual differentiation, testosterone (via estradiol) induces the transcription of

genes that are actively epigenetically repressed by Dnmts in the female brain; and

(3) masculinization can be induced later in life if sufﬁcient gene transcription is

re-induced in the POA, via the knockout of Dnmt3, which was actively maintaining

DNA methylation in the female brain. Furthermore, these data suggest that during

the critical period of sexual differentiation, Dnmt activity is particularly sensitive to

androgen exposure, but outside of this critical period, Dnmt activity is not as

responsive to androgen exposure and thus, estrogen exposure as well. Therefore,

one might conclude that the ability of Dnmt to be inhibited by testosterone (via

estrogen) may be one mechanism by which the critical period of sexual differenti-

ation “closes,”and the fate of the male and female brain is ﬁnalized.

While the relationship between neonatal testosterone and histone acetylation or

DNA methylation within the BNST and POA, respectively, has proven to be fairly

straightforward, the same cannot be said for other epigenetic changes that may

contribute to the sexual differentiation of the brain. For example, contradictory

evidence has emerged for how gonadal hormone exposure and maternal behavior

Sexual Differentiation and Sex Differences in Neural Development

may impact DNA methylation of estrogen receptor alpha (ERα) in the rodent POA.

ERαexpression in the rodent POA is critical to the organization of typical sex

behavior in both male and female rats. In combination with estradiol, which is

converted from testosterone by aromatase, ERαinitiates defeminization and mascu-

linization of the POA (Kudwa et al. 2006; McCarthy et al. 2008). Interestingly,

maternal behavior can impact the methylation of the ERαgene in the neonatal pup

(Cameron et al. 2008), thereby impacting masculinization and defeminization of the

brain and leading to male sexual behavior or female maternal behavior later in life.

The impact of maternal behavior on ERαmethylation differed depending on the

whether the behavior was simulated as opposed to naturally occurring (Kurian et al.

2010; Lenz et al. 2013); and moreover, sex differences in the methylation of the ERα

gene were largely dependent upon the developmental time point at which they were

measured (Schwarz et al. 2010). Overall, these divergent results complicate our

understanding of the ways in which DNA methylation and possibly other epigenetic

modiﬁcations may be involved in the maintenance of sex differences in the brain.

There is still so much to be explored in the realm of epigenetic changes and their

potential impact on the establishment and maintenance of sex differences in the

brain. Importantly, this new ﬁeld of research has highlighted that sexual differenti-

ation of the brain requires large, complex networks of gene transcription. It has long

been thought that hormone-induced sexual differentiation of the brain involved the

transcription of genes that exclusively had a hormone response element (HRE).

Rather, we now know that hormones such as androgens (and estrogens metabolized

from testosterone) can modulate epigenetic mechanisms such as histone acetylation

and DNA methylation, which in turn produce a concert of changes in gene expres-

sion –all of which are likely necessary for the masculinization of the brain. These

ﬁndings also have the potential to increase our understanding of the mechanisms by

which the brain is feminized. While many studies have used testosterone treatment

of female rodents to induce the process of brain masculinization and examine the

expression of genes induced by testosterone during the critical period of sexual

differentiation (Armoskus et al. 2014a,b; Nakachi et al. 2015), it has been difﬁcult to

identify the genes that are actively involved in the process of brain feminization,

given that there is no known factor that initiates the process at one speciﬁc time. Now

that research indicates that feminization of the brain requires active repression of

masculinization via DNA methylation (Nugent et al. 2015), it is possible that we

may ﬁnally be able to identify the genes that are explicitly regulated in the process of

brain feminization during this time by exploring the genes that are targeted for

methylation by androgens (Shen et al. 2015). From highly recognized mechanisms

of gene silencing due to the sexually dimorphic process of X inactivation (Deng et al.

2014) to the understudied impact of sex-speciﬁc microRNAs (miRNAs) on post-

transcriptional silencing of genes or even steroid hormone-related genes (Cochrane

et al. 2011), the exciting ﬁeld of epigenetic research still has a long way to go, but it

may be the key to unlocking a more complete understanding of the processes that

differentiate the male and female brain.

A. Turano et al.

7 Conclusions

Sexual differentiation of the brain is an important neurodevelopmental process that

results in the sex-speciﬁc development of neural structures and circuits that control

sexual behavior and associated physiology. Studying the neural mechanisms of

sexual differentiation not only informs our understanding of how these sex differ-

ences in the brain are established but also increases our understanding of how gender

identity and sexual orientation may be inﬂuenced. In addition, our understanding of

sexual differentiation has informed our general understanding of how behavioral

circuits develop during critical periods of life. It has also informed our understanding

of how sex steroid hormones permanently inﬂuence the function of the brain and

how various cell types communicate with each other during early brain development

to shape the sexually dimorphic brain. There are still many aspects of sexual

differentiation that have yet to be determined. In particular, these primary questions

remain: what are the mechanisms underlying brain feminization? What are the

genes, proteins, or cellular processes that actively shape the development of the

female brain? As mentioned earlier, if we could potentially identify genes that are

suppressed (e.g., methylated) or downregulated by testosterone exposure, this might

help to identify the genes that induce brain feminization.

Studies of sexual differentiation of the developing brain have revealed the

mechanisms by which hypothalamic and forebrain structures are differentiated in

order to control sexual and social behaviors. An important future area of research

will be to understand whether there are also sex differences in the development of

other circuits that are important for cognition and emotion. Alternatively, if there are

no sex differences in the development of these circuits, perhaps we can understand

how events that disrupt or perturb development can disrupt the development of

cognitive and emotional circuits in a sex-dependent manner, or disrupt the neuro-

transmitters systems that control emotion. In this way, perhaps we can understand

the sex bias in neurodevelopmental disorders such as autism, schizophrenia, and

generalized or pervasive developmental disorders that affect the development and

cognitive functioning of young boys more than girls.

Along those lines, we have yet to understand the full ontogeny of sex differences

in the brain from prenatal to postnatal stages of life and throughout pubertal and

adolescent development. Given that sexual differentiation of the brain permanently

organizes neural development in one direction (male) or the other (female), sexual

differentiation of the brain can thus determine the fate and function of cells through-

out each of these stages of life. To that end, we have yet to understand how the cells

maintain a memory of their sex, particularly as it was inﬂuenced by sex hormones

during early brain development. This “memory”of the cell’s“hormonal sex”

continuously inﬂuences the function of the cells and the expression of genes

throughout the lifespan.

In conclusion, as we understand more about the processes underlying sexual

differentiation, we can inform our general understanding of neural development and

the formation of functional neural circuits. A secondary and important goal of

Sexual Differentiation and Sex Differences in Neural Development

understanding sex differences in neural development is to understand the possible

mechanisms by which mental health and neurodevelopmental disorders arise,

particularly those that have origins in early development and have a well-known

sex bias.

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Sex Differences: Neurodevelopment

Chapter

Jan 2023

Insights into Sex and Gender Differences in Brain and Psychopathologies Using Big Data

Article

Full-text available

Aug 2023

The societal implication of sex and gender (SG) differences in brain are profound, as they influence brain development, behavior, and importantly, the presentation, prevalence, and therapeutic response to diseases. Technological advances have enabled speed up identification and characterization of SG differences during development and in psychopathologies. The main aim of this review is to elaborate on new technological advancements, such as genomics, imaging, and emerging biobanks, coupled with bioinformatics analyses of data generated from these technologies have facilitated the identification and characterization of SG differences in the human brain through development and psychopathologies. First, a brief explanation of SG concepts is provided, along with a developmental and evolutionary context. We then describe physiological SG differences in brain activity and function, and in psychopathologies identified through imaging techniques. We further provide an overview of insights into SG differences using genomics, specifically taking advantage of large cohorts and biobanks. We finally emphasize how bioinformatics analyses of big data generated by emerging technologies provides new opportunities to reduce SG disparities in health outcomes, including major challenges.

Association of perinatal factors with suspected developmental delay in urban children aged 1-36 months - a large-scale cross-sectional study in China

Article

Full-text available

Jan 2023
BMC Pediatr

Background: Studies on perinatal risk factors and the developmental delay of children have been inconclusive and few studies have assessed the association between infants and toddlers' body mass index (BMI) and developmental outcomes. Methods: We conducted a cross-sectional study of children aged 1-36 months who had a routine physical examination in the child health departments of hospitals from March 2018 to November 2021 in 16 provinces, 4 autonomous regions and 2 municipalities directly under the central government by using the Infant Toddler Growth Development Screening Test (ITGDST). Normal children were defined as those with scores ≥ mean - 2 standard deviations (SD), while children with developmental delay were those with scores < mean-2SD in terms of overall development, gross motor, fine motor and language development. Binary logistic regression was used to analyze the risk factors of gross motor, fine motor, language and overall neurodevelopment. Results: After removing some provinces with a small sample size and children with incomplete data, 178,235 children with 12 complete variables were included in the final analysis. The rate of overall developmental delay was 4.5%, while 12.5% of children had at least one developmental delay aspect. Boys, parity, advanced maternal age, multiple birth, cesarean section, neonatal injury, family heredity history, microcephaly, abnormal BMI at birth and at physical examination after controlling the confounding of other factors had a significant effect on development delay (overall neurodevelopment, gross motor, fine motor or language development). Per capita gross domestic product was a protective factor for the children's neuropsychological development. Conclusions: This study reveals significant associations of perinatal factors and BMI with developmental delay in the Chinese children aged 1-36 months, which may be crucial for early intervention.

Modeling sex differences in humans using isogenic induced pluripotent stem cells

Article

Full-text available

Nov 2022

Biological sex is a fundamental trait influencing development, reproduction, pathogenesis, and medical treatment outcomes. Modeling sex differences is challenging because of the masking effect of genetic variability and the hurdle of differentiating chromosomal versus hormonal effects. In this work we developed a cellular model to study sex differences in humans. Somatic cells from a mosaic Klinefelter syndrome patient were reprogrammed to generate isogenic induced pluripotent stem cell (iPSC) lines with different sex chromosome complements: 47,XXY/46,XX/46,XY/45,X0. Transcriptional analysis of the hiPSCs revealed novel and known genes and pathways that are sexually dimorphic in the pluripotent state and during early neural development. Female hiPSCs more closely resembled the naive pluripotent state than their male counterparts. Moreover, the system enabled differentiation between the contributions of X versus Y chromosome to these differences. Taken together, isogenic hiPSCs present a novel platform for studying sex differences in humans and bear potential to promote gender-specific medicine in the future.

Mate Choice, Sex Roles and Sexual Cognition: Neuronal Prerequisites Supporting Cognitive Mate Choice

Article

Full-text available

Nov 2021

Theodora Fuss

Across taxa, mate choice is a highly selective process involving both intra- and intersexual selection processes aiming to pass on one’s genes, making mate choice a pivotal tool of sexual selection. Individuals adapt mate choice behavior dynamically in response to environmental and social changes. These changes are perceived sensorily and integrated on a neuronal level, which ultimately leads to an adequate behavioral response. Along with perception and prior to an appropriate behavioral response, the choosing sex has (1) to recognize and discriminate between the prospective mates and (2) to be able to assess and compare their performance in order to make an informed decision. To do so, cognitive processes allow for the simultaneous processing of multiple information from the (in-) animate environment as well as from a variety of both sexual and social (but non-sexual) conspecific cues. Although many behavioral aspects of cognition on one side and of mate choice displays on the other are well understood, the interplay of neuronal mechanisms governing both determinants, i.e., governing cognitive mate choice have been described only vaguely. This review aimed to throw a spotlight on neuronal prerequisites, networks and processes supporting the interaction between mate choice, sex roles and sexual cognition, hence, supporting cognitive mate choice. How does neuronal activity differ between males and females regarding social cognition? Does sex or the respective sex role within the prevailing mating system mirror at a neuronal level? How does cognitive competence affect mate choice? Conversely, how does mate choice affect the cognitive abilities of both sexes? Benefitting from studies using different neuroanatomical techniques such as neuronal activity markers, differential coexpression or candidate gene analyses, modulatory effects of neurotransmitters and hormones, or imaging techniques such as fMRI, there is ample evidence pointing to a reflection of sex and the respective sex role at the neuronal level, at least in individual brain regions. Moreover, this review aims to summarize evidence for cognitive abilities influencing mate choice and vice versa. At the same time, new questions arise centering the complex relationship between neurobiology, cognition and mate choice, which we will perhaps be able to answer with new experimental techniques.

Prenatal vitamin B12 status and cognitive functioning in children at 4 years of age: The ECLIPSES Study

Article

Nov 2023
MATERN CHILD NUTR

Maternal vitamin B12 deficiency has been associated with disturbed cognitive functioning in offspring at different ages during childhood. However, this association has not been explored in pre‐school‐age children. The objective of this study was to examine the association between maternal vitamin B12 levels at the beginning and end of pregnancy and cognitive functioning in their children at 4 years of age. This longitudinal prospective study included a subsample of pregnant women and their children aged 4 years ( n = 249) who participated in the ECLIPSES Study conducted in the province of Tarragona, Spain, from 2013 to 2017. Maternal vitamin B12 concentrations were determined in the first and third trimesters, and sociodemographic, nutritional and psychological data were collected. The children's cognitive functioning was assessed using the Wechsler Preschool and Primary Scale of Intelligence (WPPSI‐IV) and subtests of the Neuropsychological Assessment of Development (NEPSY‐II). The multivariable models showed a significant relationship between vitamin B12 and the working memory index in the first trimester of the pregnancy but not in the third trimester. Children of mothers in the second vitamin B12 level tertile (314–413 pg/mL) ( β = 6.468, 95% confidence interval [CI]: = 2.054, 10.882, p = 0.004) and third vitamin B12 level tertile (≥414 pg/mL) ( β = 4.703, 95% CI: = 0.292, 9.114, p = 0.037) scored higher in the working memory index of the WPPSI‐IV than the children of mothers with vitamin B12 levels in the first tertile (<314 pg/mL). Maintaining an adequate level of maternal vitamin B12 during early pregnancy contributes to improved performance in childhood working memory at 4 years of age.

Male-specific vasotocin expression in the medaka tuberal hypothalamus: Androgen dependence and probable role in aggression

Article

Nov 2023
MOL CELL ENDOCRINOL

Sex/Gender Differences in Cognitive Abilities

Article

Jul 2023
NEUROSCI BIOBEHAV R

Sex/gender differences in cognitive sciences are riddled by conflicting perspectives. At the center of debates are clinical, social, and political perspectives. Front and center, evolutionary and biological perspectives have often focused on 'nature' arguments, while feminist and constructivist views have often focused on 'nurture arguments regarding cognitive sex differences. In the current narrative review, we provide a comprehensive overview regarding the origins and historical advancement of these debates while providing a summary of the results in the field of sexually polymorphic cognition. In so doing, we attempt to highlight the importance of using transdisciplinary perspectives which help bridge disciplines together to provide a refined understanding the specific factors that drive sex differences a gender diversity in cognitive abilities. To summarize, biological sex (e.g., birth-assigned sex, sex hormones), socio-cultural gender (gender identity, gender roles), and sexual orientation each uniquely shape the cognitive abilities reviewed. To date, however, few studies integrate these sex and gender factors together to better understand individual differences in cognitive functioning. This has potential benefits if a broader understanding of sex and gender factors are systematically measured when researching and treating numerous conditions where cognition is altered.

Chromosomal and gonadal factors regulate microglial sex effects in the aging brain

Article

Full-text available

Feb 2023
BRAIN RES BULL

Biological sex contributes to phenotypic sex effects through genetic (sex chromosomal) and hormonal (gonadal) mechanisms. There are profound sex differences in the prevalence and progression of age-related brain diseases, including neurodegenerative diseases. Inflammation of neural tissue is one of the most consistent age-related phenotypes seen with healthy aging and disease. The pro-inflammatory environment of the aging brain has primarily been attributed to microglial reactivity and adoption of heterogeneous reactive states dependent upon intrinsic (i.e., sex) and extrinsic (i.e., age, disease state) factors. Here, we review sex effects in microglia across the lifespan, explore potential genetic and hormonal molecular mechanisms of microglial sex effects, and discuss currently available models and methods to study sex effects in the aging brain. Despite recent attention to this area, significant further research is needed to mechanistically understand the regulation of microglial sex effects across the lifespan, which may open new avenues for sex informed prevention and treatment strategies.

Lactational exposure to venlafaxine provokes late repercussions on reproductive parameters in male rat offspring

Article

Sep 2022

Exposure to selective serotonin reuptake inhibitors can affect hormone‐dependent processes, such as the brain sexual differentiation. Since the use of these antidepressants cause concern during lactation, we evaluated the possible effects of venlafaxine on lactational exposure and its late repercussions on reproductive parameters in male rats. Lactating rats were exposed to venlafaxine (3.85, 7.7, or 15.4 mg/kg/body weight; gavage), from lactational day 1 to 20. Venlafaxine and O‐desmethylvenlafaxine residues were found in all milk samples of dams treated, demonstrating the lactational transfer of this antidepressant to the offspring. Although the maternal behavior was normal, the dams presented an increase in urea and uric acid levels in the groups treated with 7.7 and 15.4, respectively, as well as a spleen weight increased in the 3.85, and 15.4 groups. The male offspring showed a decrease in play behavior parameters in the intermediate dose group. Sperm analysis indicated a reduction in sperm motility in all treated groups. The androgen receptor expression in the hypothalamus was decreased in the highest dose group, although the sexual behavior had not been affected. In conclusion, venlafaxine was transferred through breast milk and promoted changes in play behavior, sperm quality, and hypothalamic AR content, which may indicate an incomplete masculinization of the brain of male offspring. Since the use of SNRIs during lactation cause concern, we evaluated the possible effects of the antidepressant venlafaxine on reproductive parameters in male rats. Lactating rats were exposed to venlafaxine (3.85, 7.7, or 15.4 mg/kg/body weight; gavage), from lactational day 1 to 20. Venlafaxine treatment caused mild signs of maternal toxicity and altered important parameters evaluated in the male offspring, such as sperm parameters and AR expression in the hypothalamus. Thus, venlafaxine exposure was detrimental in these conditions.

Oxytocin signaling in the medial amygdala is required for sex discrimination of social cues

Article

Full-text available

Dec 2017
eLife

The neural control of social behaviors in rodents requires the encoding of pheromonal cues by the vomeronasal system. Here we show that the typical preference of male mice for females is eliminated in mutants lacking oxytocin, a neuropeptide modulating social behaviors in many species. Ablation of the oxytocin receptor in aromatase-expressing neurons of the medial amygdala (MeA) fully recapitulates the elimination of female preference in males. Further, single- unit recording in the MeA uncovered significant changes in the sensory representation of conspecific cues in the absence of oxytocin signaling. Finally, acute manipulation of oxytocin signaling in adults is sufficient to alter social interaction preferences in males as well as responses of MeA neurons to chemosensory cues. These results uncover the critical role of oxytocin signaling in a molecularly defined neuronal population in order to modulate the behavioral and physiological responses of male mice to females on a moment-to-moment basis.

Article

Full-text available

Mar 2017

Alterations of microbiota-gut-brain axis have been invoked in the pathogenesis of autism spectrum disorders (ASD). Mouse models could represent an excellent tool to understand how gut dysbiosis and related alterations may contribute to autistic phenotype. In this study we paralleled gut microbiota (GM) profiles, behavioral characteristics, intestinal integrity and immunological features of colon tissues in BTBR T + tf/J (BTBR) inbred mice, a well established animal model of ASD. Sex differences, up to date poorly investigated in animal models, were specifically addressed. Results showed that BTBR mice of both sexes presented a marked intestinal dysbiosis, alterations of behavior, gut permeability and immunological state with respect to prosocial C57BL/6j (C57) strain. Noticeably, sex-related differences were clearly detected. We identified Bacteroides, Parabacteroides, Sutterella, Dehalobacterium and Oscillospira genera as key drivers of sex-specific gut microbiota profiles associated with selected pathological traits. Taken together, our findings indicate that alteration of GM in BTBR mice shows relevant sex-associated differences and supports the use of BTBR mouse model to dissect autism associated microbiota-gut-brain axis alteration.

Sex-Specific Effects of Testosterone on the Sexually Dimorphic Transcriptome and Epigenome of Embryonic Neural Stem/Progenitor Cells OPEN

Article

Full-text available

Nov 2016

The mechanisms by which sex differences in the mammalian brain arise are poorly understood, but are influenced by a combination of underlying genetic differences and gonadal hormone exposure. Using a mouse embryonic neural stem cell (eNSC) model to understand early events contributing to sexually dimorphic brain development, we identified novel interactions between chromosomal sex and hormonal exposure that are instrumental to early brain sex differences. RNA-sequencing identified 103 transcripts that were differentially expressed between XX and XY eNSCs at baseline (FDR = 0.10). Treatment with testosterone-propionate (TP) reveals sex-specific gene expression changes, causing 2854 and 792 transcripts to become differentially expressed on XX and XY genetic backgrounds respectively. Within the TP responsive transcripts, there was enrichment for genes which function as epigenetic regulators that affect both histone modifications and DNA methylation patterning. We observed that TP caused a global decrease in 5-methylcytosine abundance in both sexes, a transmissible effect that was maintained in cellular progeny. Additionally, we determined that TP was associated with residue-specific alterations in acetylation of histone tails. These findings highlight an unknown component of androgen action on cells within the developmental CNS, and contribute to a novel mechanism of action by which early hormonal organization is initiated and maintained.

Serotonin and arginine-vasopressin mediate sex differences in the regulation of dominance and aggression by the social brain

Article

Full-text available

Nov 2016
P NATL ACAD SCI USA

Significance There are profound sex differences in the expression of social behavior and in the incidence of many psychiatric disorders, and yet little is known about how the brain mechanisms underlying these phenomena differ in females and males. Here, we report that serotonin (5-HT) and arginine–vasopressin (AVP) act in opposite ways within the hypothalamus to regulate dominance and aggression in females and males. Dominance and aggression are promoted by 5-HT in females and by AVP in males. Because dominance and aggressiveness have been linked to the resistance to stress-related psychiatric disorders, these disorders may be more effectively treated with 5-HT–targeted drugs in females and AVP-targeted drugs in males.

Generation of a microglial developmental index in mice and in humans reveals a sex difference in maturation and immune reactivity

Article

Jun 2017
GLIA

Evidence suggests many neurological disorders emerge when normal neurodevelopmental trajectories are disrupted, i.e., when circuits or cells do not reach their fully mature state. Microglia play a critical role in normal neurodevelopment and are hypothesized to contribute to brain disease. We used whole transcriptome profiling with Next Generation sequencing of purified developing microglia to identify a microglial developmental gene expression program involving thousands of genes whose expression levels change monotonically (up or down) across development. Importantly, the gene expression program was delayed in males relative to females and exposure of adult male mice to LPS, a potent immune activator, accelerated microglial development in males. Next, a microglial developmental index (MDI) generated from gene expression patterns obtained from purified mouse microglia, was applied to human brain transcriptome datasets to test the hypothesis that variability in microglial development is associated with human diseases such as Alzheimer's and autism where microglia have been suggested to play a role. MDI was significantly increased in both Alzheimer's Disease and in autism, suggesting that accelerated microglial development may contribute to neuropathology. In conclusion, we identified a microglia-specific gene expression program in mice that was used to create a microglia developmental index, which was applied to human datasets containing heterogeneous cell types to reveal differences between healthy and diseased brain samples, and between males and females. This powerful tool has wide ranging applicability to examine microglial development within the context of disease and in response to other variables such as stress and pharmacological treatments.

Oxytocin and Vasopressin: Powerful Regulators of Social Behavior

Article

May 2017

Heather K Caldwell

For many, the terms oxytocin and vasopressin immediately evoke images of animals interacting with one another, as both of these neuropeptides have been implicated as being part of the neurochemical "glue" that socially binds animals. However, social environments and social interactions are complex and include behaviors that bring animals together as well as behaviors that keep animals apart. It is at the intersection of social context, social experience, and an individual's sex that oxytocin and vasopressin act to modulate social behavior and social cognition. In this review, this complexity will be explored across mammalian species, with a focus on social memory, cooperative behaviors, and competitive behaviors. Implications for humans as well as future directions will also be considered.

Sex differences in microglial phagocytosis in the neonatal hippocampus

Article

Mar 2017

Microglia regulate brain development through many processes, such as promoting neurogenesis, supporting cell survival, and phagocytizing progenitor, newly-born, and dying cells. Many of these same developmental processes show robust sex differences, yet very few studies have assessed sex differences in microglia function during development. Hormonally-induced sexual differentiation of the brain occurs during the perinatal period, thus we examined sex differences in microglial morphology, phagocytosis, and proliferation in the hippocampus during the early postnatal period. We found that the neonatal female hippocampus had significantly more microglia with phagocytic cups than the male hippocampus. We subsequently found that female microglia phagocytized more neural progenitor cells and healthy cells compared to males, but there were no sex differences in the number of newly-born or dying cells targeted by microglial phagocytosis. We found that the number of phagocytic microglia in females was reduced to male-typical levels by treatment with estradiol, the hormone responsible for masculinizing the rodent brain. Females also had higher expression of several phagocytic pathway genes in the hippocampus compared to males. In contrast to robust sex differences in phagocytic microglia, we found no sex differences in the number of microglia with amoeboid, transitioning, or ramified morphologies or differences in three-dimensional reconstructions of microglial morphology. While we did not find a baseline sex difference in microglial proliferation during or following the prenatal gonadal hormone surge in males, we found that estradiol treatment increased microglia proliferation in females. Overall, these data show that there are important sex differences in microglia function in the hippocampus during the early neonatal period.

Transcriptional regulation of rat P-450 2C gene subfamily members by the sexually dimorphic pattern of growth hormone secretion.

Article

Feb 1992

The onset of the sexually dimorphic pattern of GH secretion and increased hepatic GH-binding capacity in rats at puberty is temporally correlated with the developmental induction of three hepatic cytochrome P-450s with steroid hydroxylase activity, P-450 IIC11, P-450 IIC12, and P-450 IIC13, and one cytochrome P-450 with vitamin A hydroxylase activity, P-450 IIC7. In this study we demonstrate that expression of the 2C11, 2C12, and 2C13 genes is modulated by GH at the level of transcriptional initiation both in vivo and in primary cultures of adult hepatocytes. In an effort to define the minimum sequence responsible for the inductive effects of GH, we have analyzed the ability of a 0.7-kilobase fragment isolated from the 5'-flank of the 2C12 gene, including the natural promoter, to drive transcription of a 320-basepair G-less cassette in vitro. We were unable to detect any substantial difference in RNA polymerase-II-dependent transcriptional efficiency toward the 2C12 promoter between liver nuclear extracts...

Sexual Differentiation of the Brain

Article

Oct 1978

Roger A. Gorski

Neurons and Glial Cells Are Added to the Female Rat Anteroventral Periventricular Nucleus During Puberty

Article

May 2016
ENDOCRINOLOGY

The anteroventral periventricular nucleus (AVPV) orchestrates the neuroendocrine positive feedback response that triggers ovulation in female rodents. The AVPV is larger and more cell-dense in females than in males, and during puberty, only females develop the capacity to show a positive feedback response. We previously reported a potential new mechanism to explain this female-specific gain of function during puberty, namely a female-biased sex difference in the pubertal addition of new cells to the rat AVPV. Here we first asked whether this sex difference is due to greater cell proliferation and/or survival in females. Female and male rats received the cell birthdate marker bromodeoxyuridine (BrdU; 200 mg/kg, ip) on postnatal day (P)30; brains were collected at short and long intervals after BrdU administration to assess cell proliferation and survival, respectively. Overall, females had more BrdU-ir cells in the AVPV than did males, with no sex differences in the rate of cell attrition over time. Thus, the sex difference in pubertal addition of AVPV cells appears to be due to greater cell proliferation in females. Next, to determine the phenotype of pubertally born AVPV cells, daily BrdU injections were given to female rats on P28-56 and tissue was collected on P77 to assess colocalization of BrdU and markers for mature neurons or glia. Of the pubertally born AVPV cells, ∼15% differentiated into neurons, ∼19% into astrocytes, and ∼23% into microglia. Thus, both neuro- and gliogenesis occur in the pubertal female rat AVPV and potentially contribute to maturation of female reproductive function.

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