Journal of Comparative Physiology A

https://doi.org/10.1007/s00359-019-01376-8

REVIEW

A new view of sexual differentiation of mammalian brain

Margaret M. McCarthy1 

Received: 29 July 2019 / Revised: 16 October 2019 / Accepted: 23 October 2019

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract
Establishment of enduring sex differences in brain and behavior occurs during pre- or perinatal development, depending on
species. For over 50 years the focus has been on gonadal steroid production by male fetuses and the impact on developing
brain. An increasing awareness of the importance of sex chromosome complement has broadened the focus but identifying
specific roles in development has yet to be achieved. Recent emphasis on transcriptomics has revealed myriad and unexpected
differences in gene expression in specific regions of male and female brains which may produce sex differences, serve a
compensatory role or provide latent sex differences revealed only in response to challenge. More surprising, however, has
been the consistent observation of a central role for inflammatory signaling molecules and immune cells in masculinization
of brain and behavior. The signal transduction pathways and specific immune cells vary by brain region, as does the neu-
roanatomical substrate subject to differentiation, reflecting substantial complexity emerging from what may be a common
origin, the maternal immune system. A working hypothesis integrating these various ideas is proposed.

Keywords  Androgens · Estrogens · Amygdala · Preoptic area · Prostaglandins

Introduction Sex determination begins with chromosome

Sexual differentiation of the brain is a developmental pro-
cess occurring during a restricted critical window which is
operationally defined by the onset of androgen production by
the fetal testis of males. The closing of the critical window
is when the blockade or removal of androgens in males is
without effect or similarly, treating females with testoster-
one is ineffective at inducing masculinization. In the first
scenario the process of masculinization has proceeded to
the point of no return and the same is true for feminization
in the second scenario. Defining the critical period as begin-
ning with the onset of hormone secretion and ending with
loss of sensitivity to hormones frames our view of the entire
process as being driven by hormones. This myopic view
prevents consideration of other plausible biological variables
that may be equally, if not more important, for establishing
and maintaining sex differences in physiology and behavior.
This review will highlight the importance of two such vari-
ables, sex chromosome complement and the immune system.
* Margaret M. McCarthy
mmccarth@umaryland.edu
1
Department of Pharmacology, University of Maryland,
School of Medicine, MD, Baltimore, USA
complement
The biological construct of maleness and femaleness begins
with the establishment of sex chromosome complement in
the fertilized zygote and ends with reproductive competence
of the mature adult (Fig. 1). In mammals, a single gene on
the Y chromosome, Sry (Sex-determining region of the Y),
codes for the testis-determining factor protein (Tdf), which
initiates a cascade of gene expression within days of fer-
tilization that will lead to the creation of a testis from the
bipotential gonadal anlage (Goodfellow and Lovell-Badge
1993). If there is no Y chromosome, i.e. XX, or if the Sry
gene is missing or mutated, the gonadal anlage will proceed
down a developmental pathway that culminates in the forma-
tion of an ovary. As the embryo develops the formation of
the reproductive tract and external genitalia are sculpted to
match the demands created by gonadal phenotype. One of
the most profound consequences of the formation of a testis
versus an ovary is not the production of many gametes ver-
sus only a few or the cyclical versus static production of ster-
oid hormones and creation of a sex specific hormonal milieu.
The most important difference is that one gonad, the testis, is
engineered to release gametes outside the body and therefore
must be transferred to a viable mate, while the other gonad,

13
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Fig. 1  Classic view of sexual differentiation of the brain. Sexual dif-
ferentiation begins with chromosome complement and is driven by
the Sry gene of the Y chromosome directing the formation of a testis
from the bipotential gonad. Testosterone production by the resultant
testis will commence during late gestation and into the early postnatal
period of the rodent and masculinize select regions of the male brain.
A separate process of defeminizaton specific to sex behavior removes
the capacity for female-like receptive behavior (i.e. lordosis) in males.
the ovary, is restricted to releasing gametes inside the body
cavity which then with some assistance hopefully find their
way to the inside of the uterus. In order for those gametes
to merge and become zygotes, in other words be fertilized,
there must be a mechanism by which external gametes also
gain access to the interior of the uterus. Both scenarios
require a series of coordinated behaviors by both sexes that
begin with mate seeking, mate recognition and selection fol-
lowed by copulation.
Sexual differentiation of behavior begins
with the brain
A central question in the early days of behavioral neuroen-
docrinology (not called that at the time), was whether the
behaviors associated with male versus female copulation
were a function of the peripheral apparatus, i.e. a penis ver-
sus a vagina, or controlled internally by the brain. Hard as it
is to believe now, the prevailing and favored theory was that
the body was the determinant of behavior. Thus, if an indi-
vidual found themselves with a penis, regardless of any other
variables, they would attempt to mount and copulate with
other individuals possessing a vagina, and vice versa. More-
over, while hormones were clearly required for adult copula-
tory behavior to be displayed, the actions of the hormones
13
Journal of Comparative Physiology A
The female ovary will develop in the absence of signals from the
Sry-induced gene expression cascade and will remain quiescent until
puberty when cyclical production of estrogens and progestins begins.
There is evidence for a sensitive period for feminization that occurs
slightly later in development. The masculinizing actions of testoster-
one and its aromatized by product, estradiol, are considered organi-
zational and are essential for the activational effects of hormones at
puberty on various reproductive parameters
were considered independent of any organizational effect
during development. This notion was empirically tested by
Phoenix and his trainees and essentially rejected in what is
now considered a land mark paper published in Hormones
and Behavior in 1959 (Phoenix et al. 1959). The experi-
ment was simple, they treated pregnant Guinea pigs with
testosterone and then assessed the copulatory behavior of
the offspring as adults. The critical component was the use
of different doses of testosterone, a high dose that fully mas-
culinized the genitalia of the females in utero so that they
were externally indistinguishable from male littermates,
and a lower dose that did not noticeably impact the geni-
talia. Offspring of both treatment groups were compared
to those from untreated dams. The animals were tested in
two important ways. First was to determine the impact of
prenatal androgen exposure on the display of female sexual
receptivity (lordosis). They found that females exposed to
either high or low dose testosterone did not exhibit lordosis
in response to mounting by a male, thus the behavior change
was independent of the peripheral genitalia. Secondly, they
assessed the propensity of the neonatally exposed animals
to engage in male copulatory behavior, finding that females
exposed to high dose testosterone developmentally where
highly responsive to adult testosterone and vigorously
mounted other females. Interestingly they did not test the
low dose exposed females, presumably because they were
Journal of Comparative Physiology A
so inculcated with the prevailing view that without a penis
no animal would attempt to mate like a male. Nonetheless,
they concluded that the actions of the prenatal hormones
were organizational and enduring. They went on to assert
the notion that the tissues which had been organized devel-
opmentally to modify behavior was probably neural, but they
were “not prepared to suggest whether the site of action is
generalized or localized”. They further speculated that any
effects of androgens on the neural substrate would likely
reflect subtle changes in function as opposed to structure.
The intervening 60 years have proved that wrong and filled
in the gaps by convincingly demonstrating that hormones
have highly localized and varied impact on the developing
brain, playing major roles in determining cell birth, death,
differentiation and connectivity. The impact of steroids
expands beyond neurons, also profoundly influencing astro-
cytes, oligodendrocytes and cells of non-neuronal origin
such as the immune microglia and mast cells. The impact of
steroids is so powerful, complex and widespread one could
argue the field has been held prisoner by the all encompass-
ing effort to understand this class of signaling molecules.
One could also say the field has been myopic in its view,
attributing every and all sex difference in brain or behav-
ior to the action of steroid hormones. Indeed, the power of
hormones was revered as so great that the circulating levels
observed in adult females across the estrus cycle was consid-
ered so remarkable in its power to control brain and behav-
ior that females could not be used as reliable subjects in
experimental neuroscience. We will never know for certain,
but this may have contributed to the fact that within a few
short years the majority of neuroscience research narrowed
its focus to only male subjects (Beery and Zucker 2010).
The study of females was only for purposes of understand-
ing reproduction.
Now we are in a new era. In response to a growing aware-
ness of the negative consequences of not considering sex
as a biological variable during preclinical research, the
NIH created a new mandate that requires all funded studies
include subjects of both sexes and that data be disaggregated
and analyzed for an impact of sex. If an NIH funded investi-
gator insists on using subjects of only one sex in preclinical
research it requires extraordinary justification, i.e. studies
of ovarian cancer can only be done in females for instance.
Attention to the importance of sex as a biological variable
has extended to Canadian and European funding agencies
as well. Some consider this to be over reach and possibly
damaging, particularly in the neurosciences, but others have
embraced the new mandate and are finding unexpected and
surprising differences in the characteristics of various bio-
logical processes. Observations of this sort often lead natu-
rally to questions of “how” does said biological sex differ-
ence come about? There are three generic answers to this
question. Sex differences can occur because; (1) the steroid
hormone milieu is markedly different in reproductively
mature animals, (2) developmental programming by steroids
dictates adult responses or (3) every cell in the body, includ-
ing the brain, is either XX or XY within a given individual
and genes on sex chromosomes could have direct effects. All
three or any combination of two of these is also possible.
Sex differences in the brain involve more
than just hormones: Sex chromosome
complement
Because of the hegemony of hormones, the notion that
genes on the sex chromosomes might contribute to male
and female differences in brain and behavior was largely
ignored until the early 2000’s when Art Arnold made use
of a novel mouse strain in which gonadal sex and genetic
sex could be dissociated (De Vries et al. 2002). Known
as the 4-core-genotype, this mouse strain is engineered
so that the Sry gene is removed from the Y chromosome
and translocated to an autosome. This allows for animals
that are XX-Sry + and XY-Sry-. In other words, it creates
XX animals with testis and XY animals with ovaries. By
comparing endpoints in these mice to those that are XX
with ovaries and XY with testis, the relative contribution of
chromosomal sex versus gonadal sex can be assessed. Many
behaviors have been examined and generally those directly
associated with reproduction, i.e. mating and parenting, are
entirely controlled by the gonads (Arnold et al. 2004). But
many other behaviors and some physiological endpoints are
at least “influenced” by chromosomal sex. One of the most
notable is body weight, which is markedly impacted by chro-
mosomal sex but only if the gonads are removed (Chen et al.
2013). In another particularly remarkable study involving an
animal model of multiple sclerosis (MS), the chromosomal
sex of the immune system and the nervous system were
separated so that individuals were XX in the periphery and
XY in the brain. This revealed that the higher vulnerability
of females to MS was a function of the peripheral immune
system, whereas the more severe disease pathology observed
in males is the result of XY in the nervous system (Du et al.
2014). This reinforces the view that the relationship between
gonadal sex and chromosomal sex can be both contextual
and tissue dependent.
What is missing in our understanding of potential impact
of chromosome complement on sex differences is whether
there is any effect during development. An in vitro exami-
nation of dopamine neuron survival in cells derived from
embryos of the 4-core-genotypes model demonstrates a
clear effect of sex chromosome complement (Carruth et al.
2002), and is consistent with inferential findings in vivo
(Dewing et al. 2003), so there is potential for direct effects
but whether they constitute sexual differentiation. per se,
13

is unclear. The sexual differentiation of the brain is clas-
sically viewed as occurring during a critical period when
testicularly produced androgens in the male fetus gain access
to the CNS and initiate a variety of molecular and cellu-
lar processes that are the manifestation of masculinization.
The critical period begins with the production of steroids
late in gestation in rodents and about mid-gestation in pri-
mates, including humans, and ends within the first few days
of postnatal life or prior to birth, respectively (reviewed in
(McCarthy et al. 2017a). In females there is a parallel sensi-
tive period, which is distinct from a critical period in that it
involves sensitivity to an exogenous substance. In our rodent
models, a newborn female can be injected with testosterone
or its aromatized byproduct estradiol, for up to 6–10 days
postnatally and still be masculinized for some endpoints,
i.e. remain “sensitive”. This has provided an excellent tool
for revealing the mechanisms of brain masculinization as
it is relatively easy to manipulate the hormonal milieu and
other physiological parameters in a newborn pup compared
to a developing fetus, which is further confounded by the
hormonal environment of pregnancy. However, in newborn
males masculinization cannot be blocked or reversed easily
because the process was initiated in utero and like a train
that has left the station, it is difficult to impede its forward
progression. In both cases, natural masculinization in males
or induced masculinization in females, the brain is remark-
ably immature at the time that it is being “programmed”
or “organized”, especially in light of the fact that most of
the physiology and behavior that will be impacted does not
appear until the animals are reproductively mature. The pul-
satile versus cyclic release of luteinizing hormone from the
pituitary is a hallmark of puberty, and sexual behavior is not
expressed until well after that, nor is territorial aggression,
courtship, nest building and maternal aggression. And so
there is tremendous dynamism occurring from the time of
sexual differentiation and sex differences in brain and behav-
ior, but what is constant across this period is sex chromo-
some complement. This makes it difficult to identify effects
that might be restricted to a critical or sensitive period.
Further complicating our ability to attribute developmen-
tal effects to sex chromosome complement is the increas-
ing awareness that autosomes are impacted by whether
they share the nucleus with an XX or XY set. There are
several ways this can occur. First is that when a cell has
an XX compliment, one of the X chromosomes is subject
to inactivation. This inactivation is achieved by epigenetic
repression via DNA methylation and histone modification,
an energetically expensive process that could deplete the
cell of precious enzymes and substrates that may be needed
for other epigenetic regulatory processes, thereby creating
a heterochromatic sink. A second route for sex chromo-
some regulation of autosomes is via microRNAs, which
are particularly abundant on the X chromosome (Pinheiro
13
Journal of Comparative Physiology A
et al. 2011). MicroRNAs provide regulatory control of tran-
scription and a single mRNA might impact multiple genes,
thereby modulating coordinated gene networks. Third, there
are some genes that escape X-inactivation, resulting in a
double dose in females and thereby creating the potential
for a sex difference in gene expression (reviewed in (Arnold
et al. 2016). In addition to being rich in microRNAs, the
X-chromosome is also notably rich in genes relevant to brain
development and neural functioning, as well as immune sys-
tem genes (Bianchi et al. 2012). Lastly, while the Y chro-
mosome is small, it is not completely devoid of genes and
these too could have direct effects on the developing brain.
All of these are in the realm of possibility and none of them
exclude another, but to-date there has been no empirical evi-
dence that sex chromosome complement directly impacts
sexual differentiation of the brain (Fig. 2).
Sex differences in the brain involve more
than just hormones: transcription regulatory
networks (TRNs)
Steroid receptors are nuclear transcription factors and this
logically led to the assumption that androgen receptors
(AR), estrogen receptors (ER) or both would induce a set
of genes that drive the brain masculinization process. This
reasonable and plausible scenario leads to a series of test-
able predictions; (1) there will be sex differences in gene
expression, some genes more highly expressed in males and
some higher in females, (2) treatment of females with tes-
tosterone will recapitulate the pattern seen in males, (3) the
androgen-induced genes will be directly relevant to brain
development, and (4) identifying those genes will solve the
mystery of brain sexual differentiation. An analysis of the
transcriptome of the neonatal preoptic area, arguably the
most sexually dimorphic region of the brain, found equal
numbers of genes expressed at higher levels in males versus
females (Nugent et al. 2015), in direct contradiction to pre-
diction #1. Treating females with testosterone (or estradiol)
does increase the level of some mRNA and protein from
some genes normally higher in males (Quadros et al. 2002),
but just as frequently does not. More importantly, the genes
that are regulated do not seem to have direct relationship
to neural development, thus neither prediction #2 or #3 are
strongly supported. One of the more surprising findings to
emerge in recent years are the profound and pervasive sex
differences in the transcriptome, both developmentally and
in adulthood, in animals and humans (Nugent et al. 2015;
Werling et al. 2016; Labonte et al. 2017). The differences
involve large number of genes, belying the notion that a few
select genes are responsible for the masculinization of the
brain.
Journal of Comparative Physiology A
Fig. 2  Incorporation of sex chromosomes and transcriptomics in sex-
ual differentiation of the brain. In recent years it has become increas-
ingly clear that sex chromosome complement in mammals (XX
vs XY) is an important contributor to sex differences in brain and
behavior. However because of the continuous presence of this modi-
fier since the time of conception, it is difficult to attribute particular
So where do sex differences in the transcriptome come
from and what does it mean? Independent of, or, in tandem
with sex chromosome complement, there is a strong poten-
tial for sex differences in transcription regulatory networks
(TRNs). Transcription factors (TF) interact directly with
DNA or via co-activators and co-repressors in a multi-com-
ponent transcriptional complex. They modulate the activity
at gene promoters and interact with distal regulatory ele-
ments known as enhancers. The binding of individual TF’s
can be weak and imprecise and so cooperativity between
them and associated proteins provides the robust regula-
tion needed to precisely regulate the roughly 20,000 genes
in the mammalian genome. TRNs determine cell fate dur-
ing development and adult maintenance of homeostasis.
How networks are established and maintained is still being
explored experimentally. Network stability is influenced by
both intrinsic (i.e. cell cycle progression) and extrinsic (i.e.
metabolic state) factors (reviewed in (Wilkinson et al. 2017).
Steroid receptors are classical TFs that recognize and inter-
act with palindromic sequences embedded in promoters but
also associate with a variety of co-factors, some specific
to steroid receptors and others more general, such as c-fos
(Barrett 1998; Beato and Klug 2000). Steroids can also influ-
ence TRNs by modulating the activity of DNA methylat-
ing enzymes (Nugent et al. 2015), and histone modifying
enzymes (Matsuda et al. 2011). Evidence that TRNs differ
aspects of sex chromosome complement to the process of sexual dif-
ferentiation. Future studies addressing convergence of sex chromo-
some effects and steroid hormones will be valuable in this regard. A
second emerging concept is sex differences in the transcriptome, and
in particular the potential for differences in networks as opposed to
isolated gene expression
as a function of sex is found in differential gene expression,
alternative splice variants and promoter usage, genome wide
methylation and histone modifications associated with pro-
moter activation in rats, mice and humans (Trabzuni et al.
2013; Ghahramani et al. 2014; Nugent et al. 2015; Shen et al.
2015; Werling et al. 2016; Labonte et al. 2017). Of particular
interest would be to know if TRNs are established devel-
opmentally during the critical period and then maintained
across the life span (Fig. 2).
Sex differences in the brain involve
more than just hormones: Immune
and inflammatory mediators
The cellular and molecular mechanisms mediating the mas-
culinization of specific brain regions has been elucidated in
considerable detail for some regions and a surprising pat-
tern has emerged. It was predicted that factors impacting
cell number, dendritic growth and synapse density would
most likely be growth factors, neurotransmitters and other
modulators of neuronal activity such as ion changes. Instead,
an over preponderance of signaling pathways and cell types
involved in immune and inflammatory responses was discov-
ered to be central to masculinization of multiple endpoints
in multiple brain regions (Fig. 3).
13

Fig. 3  New view of sexual differentiation of the brain involving
immune modulators. Converging evidence from studies exploring the
cellular and molecular mechanisms establishing sex differences in
brain and behavior implicates the innate immune cells of the brain,
microglia, as well as mast cells which are found both in the periphery
and centrally. Males consistently have either more of these immune
cells in specific brain regions or the cells are in a more activated
state and producing inflammatory signaling molecules are engaging
Sex differences in synaptic patterning are
directed by prostaglandins and histamine
The preoptic area was noted above as touting some of the
most robust sex differences in the brain which come in the
form of synaptic patterning and differential cell death. This
brain region is also of interest due to its centrality to the
control of both mating and parenting behavior (Numan 1994;
Hull and Dominguez 2007). The neurons in this region are
replete with estrogen and androgen receptors but the key
players initiating the differentiation of the synaptic pattern
(i.e. more dense dendritic spine synapses in males), are two
types of immune cells, microglia and mast cells (Lenz et al.
2013; Lenz et al. 2018). Microglia are immune cells unique
to the brain and are modified macrophages that take up per-
manent residence there early in embryonic development
(Ginhoux et al. 2013). Mast cells are also immune cells but
originate in the bone marrow and are distributed throughout
the body, particularly at surfaces such as skin, nasal epithe-
lium, mouth eyes etc. They are considered first respond-
ers to any tissue disturbances and are packed with large
secretory granules ready to dump histamine, serotonin and
other signaling molecules (Silver and Curley 2013). They
are also capable of making prostaglandins and cytokines
13
Journal of Comparative Physiology A
in high levels of phagocytosis during the critical period for sexual
differentiation, thereby modulating divers endpoints such as synap-
togenesis and cell survival. Cytokines and associated proteins are also
expressed at higher levels in males and in one brain region result in
the death of neurons that would otherwise survive. Thus there both a
complexity and a consistency in the involvement of the immune sys-
tem in masculinization of brain and behavior in rodents
(Theoharides et al. 2012). In the brain mast cells are usu-
ally found in the meninges but during the critical period for
sexual differentiation there is a population deep in the neu-
ropil of the preoptic area, and there are significantly more
in males than females. At the close of the sensitive period
the numbers drop precipitously in both sexes (Lenz et al.
2018). During the time that mast cells are residing within
the neuropil, they are also constitutively degranulating in
males and releasing histamine. Nearby microglia respond to
the released histamine with increased prostaglandin produc-
tion, in particular PGE2, which is usually associated with
fever (Lazarus et al. 2007), but also has signaling capac-
ity through G-protein coupled receptors linked to adenyl
cyclase, cAMP production and activation of protein kinase A
(PKA) (Sugimoto and Narumiya 2007). In the preoptic area
this activated PKA phosphorylates a subunit of the AMPA
glutamate receptor and this in turn promotes the traffick-
ing of the receptor to the membrane and, via mechanisms
not entirely understood, promotes the formation and stabi-
lization of excitatory dendritic spine synapses (Lenz et al.
2011). The increase in dendritic spines endures into adult-
hood and positively correlates with the expression of male
sexual behavior (Wright et al. 2008). The preoptic area is
essential for male sexual behavior and if it is damaged or
Journal of Comparative Physiology A
lesioned males lose interest in copulating. The increase in
excitatory input created by generating a higher density of
glutamatergic synapses promotes mating by enhancing the
neural responsiveness to salient cues from receptive females.
The critical point is that two immune cells, microglia and
mast cells, are essential to the establishment of this node in
the neural circuit controlling male copulation. In support of
this conclusion, depleting microglia selectively during the
critical period completely abolishes the capacity for mating
in males when they mature, but has no impact on females
(VanRyzin et al. 2016).
Sex differences in cell death are directed
by immune signaling
In addition to a sex difference in synaptic patterning in the
preoptic area are volumetric differences in which a subnu-
cleus is larger in one sex. The most famous such nucleus is
actually named for that fact, the sexually dimorphic nucleus
of the preoptic area, or more simply the SDN (Gorski et al.
1980). The topic of intense study, the SDN is arguably the
most robust volumetric sex difference in the brain and its dis-
covery in the 1970’s heralded a new era of connecting brain
morphology to physiology and behavior. It also provided the
first opportunity to identify the specific cellular mechanisms
by which steroids mediate sexual differentiation. Ironically,
attempts at both have largely failed. The precise function of
the SDN remains unclear although it is strongly implicated
in partner preference (Roselli and Balthazart 2011). As to
mechanism, we know that the SDN starts with essentially
the same number of neurons in males and females but they
die in females due to lack local of estradiol, aromatized from
peripheral androgens in males (Jacobson and Gorski 1981;
Davis et al. 1996a). But HOW estradiol is providing survival
support to the SDN neurons in males has been a mystery,
and many obvious answers such as regulation of cell death
and survival pathways have been largely negative (Forger
2009). The possibility that immune cells such as microglia
and mast cells may be key mediators of SDN volume is an
active area of investigation.
A second nucleus within the preoptic area also differs
in volume but in the opposite direction, being larger in
females than males (Davis et al. 1996b). The anteroventral
periventricular nucleus, or more simply the AVPV, is a key
node for the control of LH release from the pituitary by
controlling the activity of GnRH neurons (Gu and Simerly
1997; Clarkson and Herbison 2006; Polston and Simerly
2006). The pattern of LH release is sexually dimorphic,
being continuously pulsatile in males but cyclical in
females and characterized by a surge that is essential for
inducing ovulation. It requires markedly distinct control
of GnRH neuronal activity to achieve these diverse ends
and this control is established during the critical period
for sexual differentiation. If a neonatal female is treated
with a large dose of testosterone or its aromatized end
product, estradiol, she will be sterile as an adult (Gorski
and Barraclough 1963). The steroid-induced sterility is
not because of any fault with the ovary or the pituitary
but rather the loss of the synchronized firing of the GnRH
neurons required for a surge of LH from the pituitary to
induce ovulation. The follicules of the ovary develop per-
fectly well, they simply cannot release the ova, nor do they
form the corpus luteum required to maintain pregnancy.
The firing rate of GnRH neurons is not controlled by direct
action of estrogens because they do not express estrogen
receptors (Shivers et al. 1983). Instead it is the afferent
input to the GnRH neurons which is critical, and an essen-
tial node for the synchronization required for the LH surge
is the AVPV. The AVPV is a region of the preoptic area
and contains a variety of different cell types. Dopamine
neurons residing here are more numerous in females than
males (Simerly et al. 1997), but they are only a small per-
centage of the population (which is not to suggest they
are unimportant). Inhibitory GABAergic neurons in this
region all express estrogen receptors, and some have a dual
identity as glutamatergic as well, but regardless there are
more of them in females and they project monosynapti-
cally onto GnRH neurons (Ottem et al. 2004). The AVPV
is just one part of a complex intercalating network of col-
lections of cells that control GnRH activity but in a very
simplistic sense the more neurons in the AVPV the greater
the potential to induce the surge of LH necessary for ovu-
lation. The number of neurons in the AVPV is established
during the critical period for sexual differentiation, just as
with the SDN, but in this case estradiol appears to induce
cell death, thereby reducing the number of surviving cells
in males (Forger 2006).
That estradiol is acting as a cell killer instead of a
cell survival factor is surprising, but what is even more
intriguing is that the process involves signaling pathways
normally associated with inflammation (Krishnan et al.
2009). Tumor necrosis factor-alpha, or Tnfα, is a cytokine
involved in the acute phase of an inflammatory response,
and it acts through two related receptors, Tnfr1 and Tnfr2.
Tnfr1 is directly associated with cell death, but it is Tnfr2
that is critical to cell survival in the AVPV of females.
The impact of Tnfα is indirect via phosphorylation of
NFκB, which increases the cell survival factor Bcl2. In
males, estrogens induce yet another protein, Traip, which
inhibits NFκB and allows for increased expression of the
Bax mediated cell death pathway and thereby reducing the
overall cell population of the AVPV selectively in males
(reviewed in (Petersen et al. 2012).
13

Sex differences in cell death are directed
by immune cells
The amygdala is a complex collection of subnuclei
involved in a variety of social and emotional behaviors
ranging from juvenile social play, to mating to fear mem-
ories. Play behavior in rats is expressed predominantly
during the period between weaning and puberty, and is
often seen more frequently in males but depends upon con-
text (Bredewold et al. 2014; Argue and McCarthy 2015).
The medial amygdala has long been identified as a brain
region critical to this sex difference (Meaney and McE-
wen 1986), but how that is the case was unknown until
microglia were considered as potential sculptures of the
neural circuit of play. In developing males, the microglia
of the amygdala are highly phagocytic and they selectively
target newly born astrocytes (VanRyzin et al. 2019). The
ability of microglia to engulf and kill new born cells in
a directed manner is a recently discovered phenomenon
referred to as “phagoptosis”, and about which we still have
much to learn (Brown and Neher 2014). In the develop-
ing amygdala, newly born astrocytes express proteins on
their surface that are interpreted by microglia as “eat me”
signals, as opposed to “don’t eat me” signals found on
other cells. In males, the number of astrocytes express-
ing eat me signals is higher, and they are therefore more
frequently consumed. The end result is a reduced popula-
tion of astrocytes in the male medial amygdala compared
to females, and this then correlates with greater neuronal
activation during play bouts, which occur some 3–4 weeks
later (VanRyzin et al. 2019). Thus, once again, the immune
system is the key determinant of neural circuit construc-
tion that will impact sex differences in physiology and
behavior as the animal matures.
A new view of sexual differentiation
of the brain
Based on the above observations, we propose the novel
working hypothesis that neuroinflammatory mediators and
immune cells are primary drivers of the masculinization
of specific brain regions and behaviors, while steroids
are either enablers that act to augment and enhance the
inflammatory signaling, or perhaps are actually acting in
an anti-inflammatory capacity in order to prevent runa-
way inflammation. But this raises the essential question of
why do males have more of these signaling molecules and
immune cells than females? There are multiple potential
sources, beginning with a direct effect of testosterone or
estradiol on the transcription of inflammation associated
13
Journal of Comparative Physiology A
genes. This does seem to be the case for the genes cod-
ing for the COX-1 and COX-2 enzymes (Amateau and
McCarthy 2004), but there are no other clear demonstra-
tions of direct steroid-hormone induced immune related
gene expression. On a more global level immune-related
genes appear to be epigenetically repressed in females
as opposed to males, suggesting higher overall immune
related transcription in regions of male brain (McCarthy
et al. 2017b). This is puzzling given that androgens are
generally anti-inflammatory, not pro-inflammatory. A
second source is the sex chromosome complement, with
the X chromosome being enriched in immune associated
genes (Bianchi et al. 2012). As discussed above, there are
many genes that escape X-inactivation, resulting in a dou-
ble dose in females, which would be counter to the notion
of higher immune activation in males. Lastly there is the
potential that the source is external to the individual and
instead derives from the maternal immune system. During
pregnancy the maternal immune system must be repressed
in order for successful growth of the fetus. However, there
is increasing evidence that repression against male fetuses
is less strong than that against female fetuses (reviewed
in (McCarthy 2019). Flipping that around, the maternal
immune system is more apt to recognize a male fetus as
foreign and mount a reaction against it. This could result in
increased levels of diffusible inflammatory signals, such as
prostaglandins or cytokines, in the male fetuses, or could
evoke a reactive immune response from the male fetus
itself. Lastly, there is the potential for maternal immune
cells gaining access to the male fetus and even invading
the brain. Demonstrating any or all of these will be tech-
nically challenging for many reasons. One of the biggest
is separating what is maternal in origin from what is fetal
in origin. Equally daunting is the accurate identification
and quantification of immune cells in the nervous system
as these cells are both small and rare. Overcoming these
hurdles will provide essential information for determining
the veracity of this new view of sexual differentiation of
the brain.
Funding This work was supported by RO1MH52716 and
R01DA039062 to MMM.
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