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Page 16 Yanguas-Casás. Neuroimmunol Neuroinflammation 2020;7:13-22 I http://dx.doi.org/10.20517/2347-8659.2019.31

Microglia and sexual differentiation of the brain
Sexual differentiation of the brain is orchestrated by sex chromosomes, gonadal hormones, and early
postnatal environment. X chromosome contains the largest number of immune-related genes in the human
genome, including Toll-like receptor pathways (BTK, IRAK1, and IKKγ) and microRNAs involved in
immune regulation [76-78] .

X chromosome inactivation to match gene expression levels between males and females is not random,
as previously thought. Indeed, it is the paternal X chromosome that is consistently inactivated in neonatal
[79]
brains . Fifteen percent of the genes in the X chromosome, particularly immune-related genes such as
toll-like receptor 7 (Tlr7), escape inactivation in females [80,81] . TLR7 is implicated in miRNA-mediated
increased TNFα release, and different expression of Tlr7 in females may contribute to intrinsic differences
[82]
in immune response . Therefore, male and female microglia are differentially influenced by these factors
[83]
since early developmental stages .
Sex hormones are likely key players in microglia sexual differentiation, independently of their genetic
background. Microglia physiologically express steroid hormone receptors, and are therefore sensitive to
[84]
the effects of both estrogens and testosterone . Indeed, hormones are necessary to establish initial sex
[30]
differences in microglia. Studies by Villa et al. showed that masculinization of female brain at E2 in mice
resulted in transcriptionally male microglia in adulthood in those females. Indeed, once differentiated,
microglia retain their sex-specific transcriptional profiles even after transplantation into the brain of the
[30]
opposite sex in adulthood .

Interestingly, young adult female microglia maintain their sex differences in the absence of hormones,
as their transcriptome is not drastically affected after ovariectomy . However, hormone depletion in
[30]
aged female mice (over 13 months old) induces profound transcriptome changes in these cells, with
increased inflammatory phenotypes [85,86] . Further studies are required to determine if changes in circulating
hormones during aging are responsible for these differences. It would be especially relevant to determine
the relevance of this in the incidence of neurodegenerative disorders in women, as these often appear in the
postmenopausal period.

In addition, early pre- and postnatal environment is key in the sexual differentiation of the brain.
Development at this point involves rapid myelination of neuronal fibers and synaptogenesis, arborization,
and pruning. This time of extensive growth is also a critical period where environmental factors, such
as nutritional factors (folate and palmitic acid), early postnatal stress, or smoking, can influence optimal
CNS development [87,88] . Indeed, some functional sex differences in early postnatal microglia are lost upon
exposure to palmitic acid .
[89]
Basal sex differences in microglia functional responses
There are well described sex differences in microglia in the male and female brain. These differences
range from cell density and morphology to different transcription profiles and functions. Transcriptomic
data have shown that microglia transcriptome during brain development is characterized by temporal
maturation steps that follow different trajectories in males and females: male microglia are developmentally
delayed compared with female microglia, starting from E18 [90,91] . Besides, the maturation process has
features that resemble the pro-inflammatory activation programs typical of adult cells. This is of special
relevance, as it suggests a higher sensitivity to inflammatory events in male microglia, which could lead to a
faster aging of these cells and affect the risk of disorders [Figure 1].
[91]

Microglia density varies significantly across different subregions in the brain in a spatiotemporal fashion. In
early developmental stages, microglia density in specific brain areas such as the hippocampus is higher in