Metabolic Brain Disease

https://doi.org/10.1007/s11011-021-00732-5

ORIGINAL ARTICLE

The role of sex-differentiated variations in stress hormones,

antioxidants, and neuroimmune responses in relation to social
interaction impairment in a rodent model of autism
Sameera Abuaish 1 & Norah M. Al-Otaibi 2 & Kawther Aabed 2 & Turki S. Abujamel 3,4 & Saleha Ahmad Alzahrani 2 &
Sohailah Masoud Alotaibi 2 & Ramesa Shafi Bhat 5 & Shaista Arzoo 6 & Afaf El-Ansary 7

Received: 2 January 2021 / Accepted: 5 April 2021

# The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021

Abstract
Males are more likely to develop autism as a neurodevelopmental disorder than females, but the mechanisms underlying male
susceptibility are not fully understood. In this paper, we used a well-characterized propionic acid (PPA) rodent model of autism to
study sex differences in stress hormones, antioxidants’ status, and the neuroimmune response that may contribute to the prepon-
derance of autism in males. Sprague Dawley rats of both sexes were divided into a saline-treated group as controls and PPA-
treated groups, receiving 250 mg/kg of PPA per day for three days. Animals’ social behavior was examined using the three-
chamber social test. Hormones (ACTH, corticosterone, melatonin, and oxytocin), oxidative stress biomarkers (glutathione,
glutathione-S-transferase, and ascorbic acid), and cytokines (IL-6, IL-1α, IL-10, and IFNγ) were measured in the brain tissue
of all the animals. The results showed a sex dimorphic social response to PPA treatment, where males were more susceptible to
the PPA treatment and exhibited a significant reduction in social behavior with no effects observed in females. Also, sex
differences were observed in the levels of hormones, antioxidants, and cytokines. Female rats showed significantly higher
corticosterone and lower oxytocin, antioxidants, and cytokine levels than males. The PPA treatment later modulated these
baseline differences. Our study indicates that the behavioral manifestation of autism in PPA-treated males and not females could
be linked to neural biochemical differences between the sexes at baseline, which might play a protective role in females. Our
results can contribute to early intervention strategies and treatments used to control autism, an increasingly prevalent disorder.

Keywords Autism . Sex differences . Oxidative stress . Cytokines . Social interaction . Stress hormones

* Afaf El-Ansary Introduction

afafkelansary@gmail.com

1
Autism spectrum disorder (ASD) is a neurodevelopmental
Department of Basic Sciences, College of Medicine, Princess Nourah
bint Abdurahman University, Riyadh, Saudi Arabia
disorder with a high prevalence of 1 in every 54 children
2
(Maenner et al. 2016). It is usually characterized by persistent,
Department of Biology, College of Science, Princess Nourah bint
Abdulrahman University, Riyadh, Saudi Arabia
lifelong symptoms that present clinically as social interaction
3
deficits and repetitive behaviors. Regardless of the remarkable
Vaccines and Immunotherapy Unit, King Fahd Medical Research
Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia
heterogeneity in the clinical presentation and severity of ASD,
4
one of the most exceptional replicated findings is that males
Department of Medical Laboratory Technology, Faculty of Applied
Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi
are more susceptible to developing autistic characteristics than
Arabia females. The male: female ratio for ASD has been recorded to
5
Department of Biochemistry, College of Science, King Saud
be 4.5:1 (Christensen et al. 2016).
University, Riyadh, Saudi Arabia Propionic acid (PPA) is a gut metabolic end product, and
6
Department of Food Science and Nutrition, King Saud University,
its production is linked to clostridiales and other bacteria
Riyadh, Saudi Arabia known collectively as propionibacteria. In individuals with
7
Central Laboratory, Female Center for Medical Studies and Scientific
ASD, exposure to excessive antibiotics results in reformed
Section, King Saud University, P O Box 22452, Riyadh 11495, microbial composition, which induces dysbiosis and systemic
Saudi Arabia

inflammation and leads to the pathophysiology of gastrointes-
tinal illnesses and autistic features (Navarro et al. 2016). ASD-
like rat models have been generated using various routes of
PPA administration, such as subcutaneous (500 mg/kg),
intragastric gavage (250 mg/kg), intraperitoneal (250 mg/kg),
and intracerebroventricular (4 uL of 0.26 M PPA, pH 7.5).
Following PPA administration, model rats exhibited neuroin-
flammation, oxidative stress, mitochondrial dysfunction as
well as abnormal neurobehaviors, such as repetitive and im-
paired social interactions (Shultz et al. 2008; El-Ansary et al.
2011&El-Ansary et al. 2012; Nankova et al. 2014; MacFabe
et al. 2011; Choi et al. 2018). Interestingly, PPA as a microbial
metabolite can lead to remarkable physiological and behav-
ioral alterations, including hypothalamic-pituitary-adrenal
(HPA) axis activation and altered immune function activity
and neurotransmission system (Rea et al. 2016; Pacharra
et al. 2016).
The stress response involves a series of neural actions that
stimulate the hypothalamic-pituitary-adrenal (HPA) axis.
Activation of the HPA axis excites neuroendocrine signaling,
starting with the release of corticotropin-releasing factor
(CRF) from the paraventricular nucleus (PVN) of the hypo-
thalamus. CRF stimulates the release of adrenocorticotropin
(ACTH) from the anterior pituitary, which in turn induces the
manufacture and secretion of glucocorticoid hormones from
the adrenal glands. Interestingly, this neuroendocrine response
to stress is usually accompanied by the release of oxytocin
from the posterior pituitary terminals (Lang et al. 1983;
Bülbül et al. 2011; Neumann et al. 2000). The mechanisms
underlying oxytocin’s regulatory influence on HPA reactivity
are still being investigated; however, endogenous and exoge-
nous elevations of oxytocin are associated with reductions in
CRF, ACTH, and cortisol (Bülbül et al. 2011).
It is well known that the levels of corticosterone circulating
in rats are indirectly related to the differences in response that
the sexes have to different stressors as baseline levels of cor-
ticosterone are much higher in females than in males (Kitay
1961; Critchlow et al. 1963). Corticosterone begins to in-
crease in the early hours of the day, peaks after rats wake
up, and declines during the remainder of the day (Debono
et al. 2009).
Circulating female sex hormones appear to contribute to
sex differences in response to stress. During the proestrus
phase of the estrous cycle, when ovarian hormones are higher,
corticosterone levels are concomitantly elevated (Carey et al.
1995; Atkinson and Waddell 1997).
Circulating gonadal hormones including androgens, estro-
gens (E2), and progesterone (P) have the potential to affect
HPA axis activity and modulate oxytocin neuron function,
which may contribute to sex differences in response to stress.
In general, testosterone reduces stress-induced corticosterone
release, partially through the inhibition of CRF expression.
The effects of female sex hormones on HPA axis activity are
Metab Brain Dis
more complex. In rodents, estradiol and progesterone inhibit
the negative feedback of corticosterone, which may impair the
downregulation of the stress response (Love 2018). In general,
oxytocin inhibits HPA axis reactivity in both females and
males; however, oxytocinergic effects on stress system reac-
tivity are greatly affected by the sex steroid hormones
(Dumais and Veenema 2016; Neumann 2008).
Sexual dimorphisms regarding oxidative stress and GSH–
antioxidant responses have been described in numerous ani-
mal studies and humans. The factors involved in these sex
differences are just beginning to become apparent but seem
to include genetic factors together with the levels of oxidative
stress and sex hormones. These sex differences in oxidative
stress/antioxidant status biology seem to be related to a wide
range of pathologies and diseases (Wang et al. 2020).
The immune and neuroendocrine systems are inter-related
via a complex network in which hormones, antigens, recep-
tors, cytokines, antibodies, and neuropeptides control the im-
mune response in connection with neuroendocrine alterations
while preserving homeostasis (Besedovsky and del Rey 1996,
2000). Cytokines are highly inducible proteins that mediate
intercellular immune, endocrine, and nervous system
crosstalk.
These considerations were our motivation for measuring
the sex differences in selected behavioral and biochemical
variables related to stress, immune, and oxidative responses
in a control group and a rodent model of ASD. The results
should help with understanding both the vulnerability of
males to developing autistic characteristics and the corre-
sponding protective mechanisms in females.
Materials and methods
Animals
Twenty-eight-day-old Sprague Dawley rats (14 males and 14
females) weighing approximately 80–120 g were obtained
from in-house breeding from different litters and used to test
the sex differences in response to propionic acid (PPA) neu-
rotoxicity under normal physiological conditions. The animals
were housed in cages (3–4 animals per cage) under standard
laboratory conditions. At the start of the study, all the animals
were accustomed to controlled temperature (2°C2 ± 2 °C) and
light (12 h light/12 h darkness) conditions. They had access to
water and food ad libitum. The entire experiment was carried
out in the Center for Laboratory Animals and Experimental
Surgery of the Pharmacy College of King Saud University,
Riyadh, Kingdom of Saudi Arabia. The experimental protocol
was approved by the animal research ethics committee of
King Saud University (IRB No. KSU-SE-19-61) and the re-
search ethics committee of Prince Norah University, Riyadh
(IRB No. 19–0103).
Metab Brain Dis
Experimental design
Two groups of rats, each consisting of 14 rats (7 males and 7
females), were used. Group 1, the control group, was given
1 ml of normal saline (NS) for 25 days. Group 2, the PPA-
treated group, was given an oral dose (250 mg/kg) of PPA for
3 days as a treated neurotoxic dose; after 3 days, the animals in
this group were given 1 ml of NS for 22 days (Fig. 1).
Social interaction behavioral measurement
The male and female rats were tested for social behavior using
a three-chamber social test (Schwartzer et al. 2013; Lo et al.
2015; Fujita et al. 2020). The test was conducted within a clear
rectangular Plexiglass box (of dimensions 80 cm × 40 cm ×
40 cm) divided into three chambers with walls that had
15 cm × 15-cm doors with removable sliders to allow the an-
imals to pass through. Between tests, the three-chamber box
was cleaned with 70% ethanol and dried with paper towels,
and then let to air dry to avoid the rats being exposed to
ethanol before the test. The animals were transferred to the
test room one hour before the trial for acclimatization. The test
animal was picked up gently from the cage and placed in the
center chamber with the two doorways of the box closed and
was allowed to explore for 5 min while recording behavior
with video camera. After 5 min, both doorways were opened
immediately, and a novel same-sex probe rat was placed in
one of two restraining cups located on each side of the box.
After that, the subject rat was allowed to explore all three
chambers freely for 10 min. The probe rat position was alter-
nated between animals to avoid side preference. BORIS
7.9.16 software was used for coding behavior. Time spent
and frequency entering each chamber were scored. Social in-
teraction was defined by the focal animal orienting towards
and investigating the cup holding the probe animals through
sniffing or rearing against it. The times and duration the focal
animal engaged in this behavior was scored.
Fig. 1 Experimental timeline
Biochemical analysis
Brain tissue collection and homogenization
At the end of the experiment, the rats were euthanized by
inhalation of an overdose of anesthetic (Sevoflurane). The
brain was rapidly removed from the skull and flash frozen
on dry ice, and then stored at −80 °C. Before the biochemical
assay, the brain was dissected into small pieces and complete-
ly homogenized in 10 w/v volumes of double-distilled water.
Measurement of the selected biochemical markers, including
glutathione (GSH), glutathione-S-transferase (GST), and
ascorbic acid, in the brain homogenates of the study groups,
was performed using a spectrophotometric and multiplex as-
say system.
Reduced glutathione (GSH)
The assay of GSH was carried out in serum according to the
method of Beutler (1963). This method is based on the devel-
opment of a relatively stable yellow color when 5,5-dithiobis-
2-nitrobenzoic acid (DTNB) is added to sulphydryl
compounds.
Glutathione-S-Transferase
This reaction was measured by observing the conjugation of
1-chloro, 2,4-dinitrobenzene (CDNB) with reduced glutathi-
one (GSH). This was done by observing the increase in O.D at
340 nm.
Ascorbic acid
Ascorbic acid was assayed according to the method of Jagota
and Dani (1982). This method is based on the interaction
between ascorbic acid and Folin-phenol at 760 nm.

Assay of stress hormones and oxytocin
The detection and quantification of ACTH, corticosterone,
and oxytocin were performed using a magnetic bead-based
multiplex assay system. The multiplex system is capable of
measuring a variety of variables using the same sample.
(Luminex® magnetic beads) were purchased from the Al-
Aalmiyyah Trading Company. The assay was conducted ac-
cording to the manufacturer’s procedure.
Cytokine analysis
The detection and quantification of the cytokines (IL-6 IL-1α,
IL-10, and IFNγ) were also performed using the magnetic
bead-based multiplex assay system. A rat cytokine/
chemokine 27-plex kit (Luminex® magnetic beads) were pur-
chased from the Al-Aalmiyyah Trading Company. The assay
was again conducted according to the manufacturer’s
procedure.
Statistical analysis
SPSS version 16.0 was used for data analysis; the results were
expressed as mean ± SD. The data were checked for normality
using the Shapiro-Wilk test. Not more than one outlier was
excluded to achieve normality for some of the measures.
Extreme outliers were determined using box plots and re-
moved when they were more than 3X the data’s interquartile
range (Ghasemi and Zahediasl 2012). Animal behavior anal-
ysis and brain biochemical assays were done by two-way
ANOVA (Sex x Treatment) followed by a Tukey’s post hoc
analysis for multiple comparisons with differences considered
significant at P ≤ 0.05. The effect size was calculated using
partial eta squared (pη2), with η2ρ ≥ 0.06 and ≥ 0.14 indicating
a medium and large effect size, respectively (Lakens 2013).
Results
Animal social behavior
During the 10 min three-chamber social test, PPA treatment
affected the overall social behavior of animals (social interac-
tion frequency: F (1, 22) = 9.827, P = 0.005, η2ρ = 0.31; per-
cent of time in social chamber: F (1, 22) = 4.742, P = 0.04,
η2ρ = 0.11). More interestingly, our data indicate that PPA
treatment affected males and females differently (social inter-
action frequency: sex x treatment interaction (F (1, 22) =
5.893, P = 0.023, η2ρ = 0.21); social interaction duration: sex
x treatment interaction (F (1, 22) = 3.98, P = 0.05, η2ρ = 0.15);
percent time in social chamber: sex x treatment interaction (F
(1, 22) = 4.742 P = 0.04, η2ρ = 0.11). Specifically, PPA treated
male animals were less social compared to all other groups
Metab Brain Dis
measured by frequency of social interaction (Tukey’s post hoc
P ≤ 0.01; Fig. 2a) and percent of total spent in the social cham-
ber (Tukey’s post hoc P ≤ 0.05; Fig. 2c). PPA treated males
also spent less time interacting with the probe animal com-
pared to females in both groups (Tukey’s post hoc P ≤ 0.01,
Fig. 2b). Although PPA treated males spent less time in social
interaction relative to saline treated animals, the difference did
not reach significance (Tukey’s post hoc P = 0.1). In addition,
PPA treatment affected the time spent immobile (F (1, 22) =
5.486, P = 0.02, η2ρ = 0.08). This effect was mainly present in
male animals (sex x treatment interaction (F (1, 22) = 4.234,
P = 0.05, η2ρ = 0.06), with PPA males spending more time
being immobile (still) during the test compared to all other
groups (Tukey’s post hoc P ≤ 0.05; Fig. 2d). Social behavior
of female animals on the other hand was not affected by the
PPA treatment when compared to the saline group (Tukey’s
post hoc P > 0.05).
Hormone levels
We also investigated the effects of PPA treatment on a number
of hormones in the brain including stress hormones, oxytocin,
and melatonin. There were no significant differences observed
between the sexes (F (1, 10) = 3.132, P = 0.10) or treatments
(F (1, 10) = 2.579, P = 0.13) in the levels of ACTH (Fig. 3a).
Corticosterone levels (Fig. 3b), on the other hand, were lower
in males overall (F (1, 11) = 7.665, P = 0.02, η2ρ = 0.41), with
control males showing a trend of lower levels than control
females (Tukey’s post hoc: P = 0.06). In addition, PPA treat-
ment decreased corticosterone levels overall (F (1, 11) =
7.809, P = 0.02, η2ρ = 0.41), with PPA treated females show-
ing a trend of lower levels than control females (Tukey’s post
hoc: P = 0.06). There were no significant differences observed
between the sexes (F (1, 10) = 2.043, P = 0.20) or treatments
(F (1, 10) = 0.4812, P = 0.5) in the levels of melatonin (Fig.
3c). oxytocin levels (Fig. 3d) were affected by PPA treatment
in males and females (sex x treatment interaction: F (1, 10) =
6.845, P = 0.02, η2ρ = 0.40), where control males had signifi-
cantly higher oxytocin levels than control females (Tukey’s
post hoc: P = 0.05), which is a difference that is not observed
after PPA treatment between the sexes.
Oxidative stress biomarkers levels
We evaluated the levels of a number of biomarkers related to
oxidative stress in the brain of male and female animals and
their response to PPA treatment. We did not observe any dif-
ferences in the levels of glutathione (Fig. 4a) between the
sexes (F (1, 19) = 0.0379, P > 0.05) or in response to PPA (F
(1, 19) = 0.5867, P > 0.05). However, glutathione S-
transferase (GST) levels (Fig. 4b) were affected by PPA treat-
ment in males and females (sex x treatment interaction: F (1,
20) = 9.395, P = 0.006, η2ρ = 0.34). PPA treatment in males
Metab Brain Dis

Fig. 2 Effect of Propionic acid

(PPA) treatment on social behav-
ior during the 10-min three-
chamber social test in male and
female rats. (a) Frequency of so-
cial interaction. (b) Duration of
social interaction. (c) The ratio of
time spent in the social chamber
relative to total time. (d) Time
spent immobile during the test.
PPA n = 6–7/sex, Saline n = 6–7/
sex. Data presented are means ±
standard error. Treatment effect:
**P ≤ 0.01, *P ≤ 0.05; Sex effect:
**P ≤ 0.01, *P ≤ 0.05. Multiple
comparisons by Tukey’s post hoc
test ***P ≤ 0.001, **P ≤ 0.01,
*P ≤ 0.05, #P = 0.1

specifically decreased GST levels compared to control males,
and PPA-treated females (Tukey’s post hoc: P ≤ 0.05). In ad-
dition, PPA treatment increased the levels of ascorbic acid (F
(1, 22) = 10.42, P = 0.004, η2ρ = 0.32; Fig. 4c). This effect was
observed specifically in females (sex x treatment interaction: F
(1, 22) = 21.18, P = 0.0001, η2ρ = 0.49), with PPA treated fe-
males having significantly higher levels than control females
(Tukey’s post hoc: P ≤ 0.0001), which also had lower levels
relative to control males (Tukey’s post hoc: P ≤ 0.01).
Cytokines levels
We investigated the effect of PPA treatment on levels of pro-
inflammatory cytokines in the brain of male and female ani-
mals. We found a main effect of sex (F (1, 11) = 5.773, P =
0.03, η2ρ = 0.34) in IL-1α levels (Fig. 5a), where control
males had significantly higher levels than control females
(Tukey’s Post hoc: P ≤ 0.05). This sex differences were not
present after PPA treatment, however (sex x treatment inter-
action: F (1, 11) = 5.760, P = 0.03, η2ρ = 0.34, Tukey’s Post
hoc: P > 0.05). We also observed significantly higher levels
IL-6 (Fig. 5b), another pro-inflammatory cytokine, in males
compared to females independent of PPA treatment (Sex
effect: F (1, 11) = 13.09, P = 0.004, η2ρ = 0.54). IFNγ levels
(Fig. 5c) were affected in PPA treated males and females (sex
x treatment interaction: F (1, 11) = 13.46, P = 0.004, η2ρ =
0.55). PPA males had higher IFNγ levels than PPA treated
females (Tukey’s Post hoc: P = 0.056). Further, females had
lower IFNγ levels in response to PPA treatment compared to
saline control treatment (Tukey’s Post hoc: P = 0.056).
We also examined PPA treatment’s effect on levels of the
anti-inflammatory cytokine IL-10 (Fig. 5d) in the brain of
male and female animals. We found that males overall had
significantly higher levels of IL-10 than females independent
of treatment (sex effect: F (1, 11) = 6.716, P = 0.02, η2ρ =
0.38).
Discussion
Sex-specific differences exist in relation to several disorders.
In particular, by a ratio of 4:1, autism is much more common
in males than females. However, there is a lack of understand-
ing of the sex bias in ASD. In an attempt to gain a better
understanding of this sex bias, in this study, a comparison
between autistic characteristics such as social behavior,
 Metab Brain Dis

Fig. 3 Effect of Propionic acid

(PPA) treatment on brain hor-
mones in male and female rats. (a)
ACTH. (b) Corticosterone. (c)
Melatonin. (d) Oxytocin. PPA
n = 3–4/sex, Saline n = 3–4/sex.
Data presented are means ± stan-
dard error. Treatment effect: *p ≤
0.05. Sex effect: *p ≤ 0.05.
Multiple comparisons by Tukey’s
post hoc test *P ≤ 0.05, #P ≤ 0.06

Fig. 4 Effect of Propionic acid

(PPA) treatment on brain oxida-
tive stress biomarkers in male and
female rats. (a) Glutathione. (b)
Glutathione S-transferase (GST).
(c) Ascorbic acid. PPA n = 4–6/
sex, Saline n = 6–7/sex. Data pre-
sented are means ± standard error.
Treatment effect: **p ≤ 0.01.
Multiple comparisons by Tukey’s
post hoc test ****P ≤ 0.0001,
**P ≤ 0.01, *P ≤ 0.05
Metab Brain Dis

Fig. 5 Effect of Propionic acid

(PPA) treatment on brain cyto-
kines in male and female rats. (a)
IL-1α. (b) IL-6. (c) IFNγ. (d) IL-
10. PPA n = 4/sex, Saline n = 3–4/
sex. Data presented are means ±
standard error. Sex effect: **p ≤
0.01, *p ≤ 0.05. Multiple com-
parisons by Tukey’s post hoc test
*P ≤ 0.05, #P ≤ 0.08

response to stress, oxidative stress, and immune response was
made between male and female rats.
Sex differences in social behavior impairment in
response to PPA as stressor
We first investigated the impact of PPA treatment in inducing
social behavior impairments that is a feature in animal models
of autism, such as reduced social investigation and interaction
of a novel animal in the environment (Shams et al. 2019; Choi
et al. 2018; Shultz et al. 2008). The three-chamber social test
measures the innate preference of rodents to interact with a
social stimulus over an inanimate object and it has been used
extensively to evaluate social impairment in rodent models of
autism (Rein et al. 2020). We report that only males exposed
to PPA treatment exhibited impaired social behavior indicated
by their significantly lower frequency of social interaction
with the novel animal and significantly reduced time spent
in the chamber containing the novel animal (Fig. 2). This
can find support in the recent work of Paudel et al. (2020)
which recorded significantly decreased social interaction on
the 22th to 28th day post oral administration of 250 mg/kg of
PPA for 3 days as compared to control group. This could help
to suggest that orally administered PPA, used in the current
study, induces persistent biochemical and behavioral autistic
features through the gut-brain axis much higher than the cen-
tral intracerebroventricular (ICV) treatment method (MacFabe
et al. 2011; El-Ansary and Shaker 2013; Choi et al. 2018).
This impairment was not observed in the female animals ex-
posed to the same PPA treatment, which indicate that males
are more sensitive to the PPA neurotoxicity than females.
Impairment of social behavior in males has been reported in
other PPA treatment models of autism (intracerebroventricular
or intraperitoneal injections) (Shams et al. 2019; Choi et al.
2018; Shultz et al. 2008), however there are limited studies
investigating the sex-dimorphic behavioral response to the
PPA treatment in these animals. Additionally, we found that
PPA treated males also were more still or immobile during the
test than all other groups. This could be indicative of increased
stress and anxiety-like behavior, which has been reported in
other PPA models of autism (Shams et al. 2019). The unal-
tered social behavior of female rats suggests that females have
a better adaptive capability to environmental insults than
males.
Sex differences in stress response
Figure 3 presents the levels of ACTH, corticosterone,
melatonin and oxytocin in control and PPA- treated male
and female rats. The significantly higher levels of corti-
costerone in healthy female rats compared to males can be
explained by the difference in response of the two sexes
to CRF’s regulatory effects on the HPA axis. Specifically,
it is well documented that giving an intravenous injection
of CRF to primates’ increases corticosterone levels more
in females than in males (Johnson et al. 1996; Sanchez

et al. 2010). This sex difference is related to the male sex
hormone, dihydrotestosterone, which inhibits the dis-
charge of CRF-stimulated corticosterone in male monkeys
(Gallucci et al. 1993; Toufexis and Wilson 2012). The
recorded non-significant difference in ACTH level be-
tween female and male rats (Fig. 3) is in good agreement
with the early work of Gallucci et al. (1993), which
showed that CRF injections did not induce higher
ACTH levels in females than males and in which it was
proposed that there were sex differences in the sensitivity
of the adrenal response to ACTH but not of the anterior
pituitary to CRF (Sanchez et al. 2010; Toufexis and
Wilson 2012). There appears to be no significant differ-
ence in the simple neuroendocrine signaling of stress re-
sponses between males and females (Chrousos 2000).
Both sexes experience a cascade of hormonal signaling
in response to threats that appears to begin with the rapid
release of oxytocin, melatonin, CRF, and probably other
hormones produced in the paraventricular nucleus of the
hypothalamus. Direct neural activation of the adrenal me-
dulla prompts the release of norepinephrine and epineph-
rine concomitant with sympathetic responses (Chrousos
2000). In the present study, the highly significant differ-
ences between the responses of male and female rats to
PPA stress can be also related to the remarkable higher
levels of melatonin in PPA-treated females compared to
PPA-treated males (Fig. 3). This can be easily explained
by considering three important facts. First, the neuropro-
tective effects of melatonin in female rats (Gomaa et al.
2017). Second, there is a functional relationship and feed-
back regulation between the pineal gland as secretory site
of melatonin and the testes. (Yilmaz et al. 2000; Hastings
et al. 2018; McHenry et al. 2014); in contrast, estrogen
has a pronounced modulating effect on oxytocin
(McCarthy 1995). Estrogen is also known to enhance
the anxiolytic properties of oxytocin (Windle et al.
1997). Finally, the observed increase in oxytocin in
PPA-treated females together with the near significant de-
crease in corticosterone (P < 0.077) is supported by the
results of the study of Uvnäs-Moberg (1997), in which
the exogenous administration of oxytocin in rats resulted
in decreased pain sensitivity and decreased corticosteroid
levels in females, suggestive of a weak stress response
(Uvnäs-Moberg 1997). The more or less normal social
behavior in PPA-treated females can be attributed to the
remarkable elevation of oxytocin in response to stress.
Oxytocin is directly related to mammalian social behav-
ior, which comprises attachment, communication, and so-
cial cognition (Jiang and Platt 2018). The connections
between HPA and behavioral responses to stress in male
and female animals are different, which demonstrates that
there are sex differences in the signaling that regulates the
stress response. Although the exact mechanisms remain
Metab Brain Dis
unidentified, female sex hormones play a critical role in
generating these sex differences regarding the effects of
stress on CNS processes.
Sex differences in oxidative stress and antioxidant
response
Sex differences in GSH metabolism during early development
can profoundly impact a large number of physiological pro-
cesses. Decreases in brain GSH level have been found in as-
sociation with numerous neurodevelopmental disorders, in-
cluding autism (James et al. 2004; Gawryluk et al. 2011;
Rose et al. 2012). Fig. 4 shows the significantly lower levels
of GSH, GST, and vitamin C that were found in healthy fe-
male compared to healthy male rats. Although these results
differ from those of many studies, they are supported by
others. The livers of fetal male sheep were found to have a
higher GSH content than their female twins; the same was also
found in skeletal muscle, indicating increased oxidative stress
during female fetal development. Moreover, microarray anal-
ysis revealed that GSTM mRNA levels in the kidneys of male
rats were twice as high as in females (Kwekel et al. 2013).
GST plays a key role in cellular detoxification after exposure
to oxidative stress, xenobiotics, and harmful compounds.
GST, sexual dimorphisms have been observed in multiple
organs, species, and disease models (Snyder and Maddison
1997). In spite of the lower GSH, GST, and vitamin C found
in healthy female rats compared to males, it was very interest-
ing to observe the markedly different responses to the PPA
challenge compared to males (Fig. 4). Although PPA-treated
male rats exhibited behavioral changes concomitant with a
significant reduction of GST levels and non-significant deple-
tion of GSH and vitamin C, females exhibited a completely
different response to PPA toxicity, which presented as a sig-
nificant elevation of the three measured variables (GSH, GST,
and vitamin C). This response in females can easily be related
to the antioxidant effects of estrogen, which, in addition to its
direct free-radical scavenging activity, has a marked effect on
the up-regulation of antioxidant enzymes such as GST, GSH-
synthesizing enzymes, and vitamin C-synthesizing enzymes
(Ruiz-Larrea et al. 1997; Borrás et al. 2005; Hoang et al.
2009).
The significant increase in antioxidant status in response to
PPA administration in females but not in males can easily be
related to earlier work by Al-Suwailem et al. (2018), which
demonstrated the role of female sex hormones (estrogen and
progesterone) in avoiding glutamate excitotoxicity in PPA-
treated female rats. As a metabolite of progesterone,
allopregnanolone has an anti-epileptic effect through binding
to γ-aminobutyric acid A (GABAA) receptors and the elici-
tation of an inhibitory current (Greenfield Jr 2013).
Furthermore, estrogens have been reported to decrease the
mortality rate due to epileptic seizures (Hoffman et al. 2003;
Metab Brain Dis
Velíšková and DeSantis 2013). Endogenous 17β-estradiol
may play a role in protecting neurons from glutamate
excitotoxicity as an etiological mechanism in autism.
Sex differences in neuroimmune response
There is strong evidence for both qualitative and quantitative
sex differences in neuroimmune response to a systemic immune
challenge. These differences present as different roles for
neuroimmune cells and cytokines in females and males.
Cytokines are known to play different roles in the brain of
males and females. For example, whereas hippocampal IL-2
impairs neurogenesis only in males (Beck Jr et al. 2005), IL-
13 mediates symptoms of multiple sclerosis in female rodents
but not in males (Sinha et al. 2008). Males and females, there-
fore, exhibit different functional correlates of neuroimmune
signaling, perhaps due to sex-specific patterns of cytokine acti-
vation in the brain. In general, the female immune system when
compared to the male immune system has several protective
factors that induce a stronger neuroimmune response (Klein
and Flanagan 2016; Fransen et al. 2017). In the present study,
the notable differences in the levels of Il-1α, Il-6, Il-10, and
IFNγ between male and female control rats imply that there
are sex differences in the neuroimmune response (Fig. 5).
In the current study, females and males showed different
immune responses to PPA neurotoxicity (Fig. 5). IL1-α, IL-6,
and IL-10 showed non-significant but different responses to
the PPA neurotoxic effects, whereas there was a remarkable
elevation of IFN-γ in males and a response to fairly significant
reduction in females. These results can be explained by con-
sidering that male and female rats exhibit different functional
correlates of neuroimmune signaling with different patterns of
cytokine activation in the brain (Pyter et al. 2013; Hudson
et al. 2014). In our study, the altered levels of IL-6 and IL-
10 in the brain homogenates of PPA-treated males were mark-
edly different than those in PPA-treated females. Testosterone
and its receptors can initiate the production of IL-6 through
binding to the IL-6 promoter and stimulating its transcription
(Dong et al. 2017). Sex differences in immune responses re-
sult in different susceptibilities to neurodevelopmental disor-
ders. In the present study, following the PPA injection, the
female rats had much higher levels of IL-10, an anti-
inflammatory cytokine (Fig. 5). Increased IL-10 may be one
of the protective mechanisms present in females. This sugges-
tion is supported by consideration of the work of Villa et al.
(2015), in which the anti-inflammatory effect of estrogen
through activation of the IL-10 premotor was recorded.
Because of the known inhibitory actions of IL-10 on Th1 cells
and monocyte/macrophages, these high IL-10 levels favor the
non-pathogenic Th2 pathway over the Th1-mediated autoim-
mune pathway. This might explain the resistance of female
rats to the development of behavioral and biochemical autistic
features after exposure to PPA. Interestingly, it has also been
reported that the female gut shows a greater abundance of anti-
inflammatory bacteria such as Bifidobacterium and
Lactobacillus (Heberling et al. 2013).
Interestingly, the negative correlations between Il-6 as pro-
inflammatory marker and both melatonin and oxytocin as
estrogen-induced neuroprotective hormones in PPA-treated
female rats can explain the lesser vulnerability of females to
develop autistic features as they have much higher functioning
immune system with higher IL-10 and lower IL-6.
Conclusion
This study clarifies, to some extent, the biochemical aspects of
the increased vulnerability of male rats to developing persis-
tent behavioral and biochemical autistic features in response
to the administration of a neurotoxic dose of PPA. Marked
differences in the levels of stress hormones, antioxidant status,
and neuroimmune response were observed between male and
female healthy control, and rodent models of autism.
Understanding the sex differences in autism and the protective
role of female sex hormones could help with the development
of possible protective or early intervention strategies that
could be used to control this increasingly prevalent disorder.
Acknowledgments This research project was supported by the Deanship
of Scientific Research, Princess Nourah Bint Abdulrahman University,
Grant number RGP-1441-0027. The authors thank the Deanship of
Scientific Research and RSSU at King Saud University for the technical
support.
Availability of data and materials The datasets generated analyses dur-
ing the current study are available from the corresponding author on
reasonable request.
Author’s contributions Conceptualization: AE.
Formal analysis: SA.
Funding acquisition: KA.
Investigation: SA, NMA, SAA.
Methodology: RSB, SMA, SA.
Supervision: AE, KA.
Writing – original draft: AE.
Writing – review & editing: TSA, SA.
Funding This research project was supported by the Deanship of
Scientific Research, Princess Nourah Bint Abdulrahman University.
Declarations
Ethical approval Ethical approval from the Institutional Review Board
were obtained.
Consent of participants NA
Consent for publication All authors read the manuscript and agree to
publish.

Competing interests The authors declare no potential conflicts of inter-
est with respect to the authorship and/or publication of this article.
References
Al-Suwailem E, Abdi S, El-Ansary A (2018) Sex differences in the glu-
tamate signaling pathway in juvenile rats. J Neurosci Res 96(3):
459–466
Atkinson HC, Waddell BJ (1997) Circadian variation in basal plasma
corticosterone and adrenocorticotropin in the rat: sexual dimorphism
and changes across the estrous cycle. Endocrinology 138(9):3842–
3848
Beck RD Jr, Wasserfall C, Ha GK, Cushman JD, Huang Z, Atkinson
MA, Petitto JM (2005) Changes in hippocampal IL-15, related cy-
tokines, and neurogenesis in IL-2 deficient mice. Brain Res 1041(2):
223–230
Besedovsky HO, del Rey AZ (2000) The cytokine-HPA axis feedback
circuit. Z Rheumatol 59(2):II26–II30
Besedovsky HO, del Rey A (1996) Immune-neuro-endocrine interac-
tions: facts and hypotheses. Endocr Rev 17(1):64–102
Beutler E (1963) Improved method for the determination of blood gluta-
thione. J Lab Clin Med 61:882–888
Borrás C, Gambini J, Gómez-Cabrera MC, Sastre J, Pallardó FV, Mann
GE, Viña J (2005) 17β-oestradiol up-regulates longevity-related,
antioxidant enzyme expression via the ERK1 and ERK2 [MAPK]/
NFκB cascade. Aging Cell 4(3):113–118
Bülbül M, Babygirija R, Cerjak D, Yoshimoto S, Ludwig K, Takahashi T
(2011) Hypothalamic oxytocin attenuates CRF expression via
GABAA receptors in rats. Brain Res 1387:39–45
Carey MP, Deterd CH, De Koning J, Helmerhorst F, De Kloet ER (1995)
The influence of ovarian steroids on hypothalamic-pituitary-adrenal
regulation in the female rat. J Endocrinol 144(2):311–321
Choi J, Lee S, Won J, Jin Y, Hong Y, Hur T-Y, Kim J.H., Lee S.R., Hong
Y. (2018) Pathophysiological and neurobehavioral characteristics of
a propionic acidmediated autism-like rat model. PLoS One 13(2):
e0192925. https://doi.org/10.1371/journal. pone.0192925
Christensen DL, Baio J, Van Naarden Braun K, Bilder D, Charles J,
Constantino JN, Daniels J, Durkin MS, Fitzgerald RT, Kurzius-
Spencer M, Lee L-C, Pettygrove S, Robinson C, Schulz E, Wells
C, Wingate MS, Zahorodny W, Yeargin-Allsopp M, Centers for
Disease Control and Prevention (CDC) (2016) Prevalence and char-
acteristics of autism spectrum disorder among children aged 8
years—autism and developmental disabilities monitoring network,
11 sites, United States, 2012. MMWR Surveill Summ 65(3):1–23.
https://doi.org/10.15585/mmwr.ss6503a1
Chrousos GP (2000) The role of stress and the hypothalamic–pituitary–
adrenal axis in the pathogenesis of the metabolic syndrome: neuro-
endocrine and target tissue-related causes. Int J Obes 24(2):S50–S55
Critchlow V, Liebelt RA, Bar-Sela M, Mountcastle W, Lipscomb HS
(1963) Sex difference in resting pituitary-adrenal function in the
rat. American Journal of Physiology-Legacy Content 205(5):807–
815
Debono M, Ghobadi C, Rostami-Hodjegan A, Huatan H, Campbell MJ,
Newell-Price J, Darzy K, Merke DP, Arlt W, Ross RJ (2009)
Modified-release hydrocortisone to provide circadian cortisol pro-
files. The Journal of Clinical Endocrinology & Metabolism 94(5):
1548–1554
Dong H, Xu J, Li W, Gan J, Lin W, Ke J, Jiang J, du L, Chen Y, Zhong X,
Zhang D, Yeung SCJ, Li X, Zhang H (2017) Reciprocal androgen
receptor/interleukin-6 crosstalk drives oesophageal carcinoma pro-
gression and contributes to patient prognosis. J Pathol 241(4):448–
462. https://doi.org/10.1002/path.4839
Metab Brain Dis
Dumais KM, Veenema AH (2016) Vasopressin and oxytocin receptor
systems in the brain: sex differences and sex-specific regulation of
social behavior. Front Neuroendocrinol 40:1–23
El-Ansary AK, Al-Daihan SK, El-Gezzery AR. (2011) On the protective
effect of omega-3 against propionic acid-induced neurotoxicity in
rat pups. Lipids Health Dis. 10: 142. https://doi.org/10.1186/1476-
511X-10-142 PMID: 21854591
El-Ansary AK, Ben Bacha AB, Kotb M. (2012) Etiology of autistic
features: the persisting neurotoxic effects of propionic acid. J
Neuroinflammation. 9: 74. https://doi.org/10.1186/1742-2094-9-74
PMID: 22531301
El-Ansary AK, Shaker GH (2013) Rizk MZ role of gut–brain axis in the
aetiology of neurodevelopmental disorders with reference to autism.
J Clin Toxicol S6:005
Fransen F, van Beek AA, Borghuis T, Meijer B, Hugenholtz F, van der
Gaast-de Jongh C, de Vos P (2017). The impact of gut microbiota on
gender-specific differences in immunity. Front immunol 8(JUN),
754. https://doi.org/10.3389/fimmu.2017.00754
Fujita Y, Fujita A, Ishima T, Hirai A, Suzuki S, Suganuma H, Hashimoto
K (2020). Dietary intake of glucoraphanin during pregnancy and
lactation prevents the behavioral abnormalities in the offspring after
maternal immune activation. Neuropsychopharmacol Rep
npr2.12112. https://doi.org/10.1002/npr2.12112
Gallucci WT, Baum A, Laue L, Rabin DS, Chrousos GP, Gold PW,
Kling MA (1993) Sex differences in sensitivity of the
hypothalamic-pituitary-adrenal axis. Health Psychol 12(5):420–425
Gawryluk JW, Wang J-F, Andreazza AC, Shao L, Young LT (2011)
Decreased levels of glutathione, the major brain antioxidant, in
post-mortem prefrontal cortex from patients with psychiatric disor-
ders. Int J Neuropsychopharmacol 14(1):123–130
Ghasemi A, Zahediasl S (2012) Normality tests for statistical analysis: a
guide for non-statisticians. International Journal of Endocrinology
and Metabolism 10(2):486–489. https://doi.org/10.5812/ijem.3505
Gomaa AM, Galal HM, Abou-Elgait AT (2017) Neuroprotective effects
of melatonin administration against chronic immobilization stress in
rats. International journal of physiology, pathophysiology and phar-
macology 9(2):16–27
Greenfield LJ Jr (2013) Molecular mechanisms of antiseizure drug activ-
ity at GABAA receptors. Seizure 22(8):589–600
Hastings WJ, Chang A-M, Ebstein RP, Shalev I (2018) Neuroendocrine
stress response is moderated by sex and sex hormone receptor poly-
morphisms. Horm Behav 106:74–80
Heberling C, Dhurjati P, Sasser M (2013) Hypothesis for a systems con-
nectivity model of autism spectrum disorder pathogenesis: links to
gut bacteria, oxidative stress, and intestinal permeability. Med
Hypotheses 80(3):264–270. https://doi.org/10.1016/j.mehy.2012.
11.044
Hoang YD, Nakamura BN, Luderer U (2009) Follicle-stimulating hor-
mone and estradiol interact to stimulate glutathione synthesis in rat
ovarian follicles and granulosa cells. Biol Reprod 81(4):636–646
Hoffman GE, Moore N, Fiskum G, Murphy AZ (2003) Ovarian steroid
modulation of seizure severity and hippocampal cell death after
kainic acid treatment. Exp Neurol 182(1):124–134
Hudson SP, Jacobson-Pick S, Anisman H (2014) Sex differences in be-
havior and pro-inflammatory cytokine mRNA expression following
stressor exposure and re-exposure. Neuroscience 277:239–249
Jagota SK, Dani HM (1982). A new colorimetric technique for the esti-
mation of vitamin C using Folin phenol reagent. Anal Biochem,
127(1), 178–182. https://doi.org/10.1016/0003-2697(82)90162-2
James SJ, Cutler P, Melnyk S, Jernigan S, Janak L, Gaylor DW,
Neubrander JA (2004) Metabolic biomarkers of increased oxidative
stress and impaired methylation capacity in children with autism.
Am J Clin Nutr 80(6):1611–1617
Jiang Y, Platt ML (2018) Oxytocin and vasopressin increase male-
directed threats and vocalizations in female macaques. Sci Rep
8(1):1–14
Metab Brain Dis
Johnson EO, Kamilaris TC, Carter CS, Calogero AE, Gold PW,
Chrousos GP (1996) The biobehavioral consequences of psycho-
genic stress in a small, social primate (Callithrix jacchus jacchus).
Biol Psychiatry 40(5):317–337
Kitay JI (1961) Sex differences in adrenal cortical secretion in the rat.
Endocrinology 68(5):818–824
Klein SL, Flanagan KL (2016, October 1) Sex differences in immune
responses. Nat Rev Immunol 16:626–638. https://doi.org/10.1038/
nri.2016.90
Kwekel JC, Desai VG, Moland CL, Vijay V, Fuscoe JC (2013) Sex
differences in kidney gene expression during the life cycle of F344
rats. Biol Sex Differ 4(1):1–13
Lakens D (2013) Calculating and reporting effect sizes to facilitate cu-
mulative science: a practical primer for t-tests and ANOVAs. Front
Psychol 4:863
Lang RE, Heil JWE, Ganten D, Hermann K, Unger T, Rascher W (1983)
Oxytocin unlike vasopressin is a stress hormone in the rat.
Neuroendocrinology 37(4):314–316
Lo S-C, Scearce-Levie K, Sheng M (2015) Characterization of social
behaviors in caspase-3 deficient mice OPEN. Nat Publ Group 6.
https://doi.org/10.1038/srep18335
Love TM (2018) The impact of oxytocin on stress: the role of sex. Curr
Opin Behav Sci 23:136–142
MacFabe DF, Cain NF, Boon F, Ossenkopp KP, Cain DP (2011). Effects
of the enteric bacterial metabolic product propionic acid on object-
directed behavior, social behavior, cognition, and neuroinflamma-
tion in adolescent rats: relevance to autism spectrum disorder. Behav
Brain Res 217(1), 47-54MacFabe, D. F., Cain, N. E., Boon, F.,
Ossenk. https://doi.org/10.1016/j.bbr.2010.10.005
Maenner MJ, Shaw KA, Baio J, et al. (2016) Prevalence of Autism
Spectrum Disorder Among Children Aged 8 Years — Autism and
Developmental Disabilities Monitoring Network, 11 Sites, United
States. MMWR Surveill Summ 2020, 69 (No. SS-4): 1–12. https://
www.cdc.gov/mmwr/volumes/69/ss/ss6904a1.htm
McCarthy MM (1995) Estrogen modulation of oxytocin and its relation
to behavior. Adv Exp Med Biol 395:235–245
McHenry J, Carrier N, Hull E, Kabbaj M (2014) Sex differences in anx-
iety and depression: role of testosterone. Front Neuroendocrinol
35(1):42–57
Nankova BB, Agarwal R, Macfabe DF, La Gamma EF. (2014) Enteric
bacterial metabolites propionic and butyric acid modulate gene ex-
pression, including CREB-dependent catecholaminergic neurotrans-
mission, in PC12 cells—possible relevance to autism spectrum dis-
orders. PLoS One. 9: e103740. https://doi.org/10.1371/journal.
pone.0103740 PMID: 25170769
Navarro F, Liu Y, Rhoads JM. (2016) Can probiotics benefit children
with autism spectrum disorders? World J Gastroenterol. 22:10093–
10102. https://doi.org/10.3748/wjg.v22.i46.10093 PMID:
28028357
Neumann ID (2008) Brain oxytocin: a key regulator of emotional and
social behaviours in both females and males. J Neuroendocrinol
20(6):858–865
Neumann ID, Krömer SA, Toschi N, Ebner K (2000) Brain oxytocin
inhibits the (re) activity of the hypothalamo–pituitary–adrenal axis
in male rats: involvement of hypothalamic and limbic brain regions.
Regul Pept 96(1–2):31–38
Paudel R, Raj K, Gupta YK, Singh S (2020) Oxiracetam and Zinc
Ameliorates Autism-Like Symptoms in Propionic Acid Model of
Rats. Neurotoxicity Research 37. https://doi.org/10.1007/s12640-
020-00169-1
Pyter LM, Kelly SD, Harrell CS, Neigh GN (2013) Sex differences in the
effects of adolescent stress on adult brain inflammatory markers in
rats. Brain Behav Immun 30:88–94
Pacharra M, Schäper M, Kleinbeck S, Blaszkewicz M, Golka K, Thriel C
(2016) Neurobehavioral effects of exposure to propionic acid
revisited—does psychosocial stress interfere with distractive effects
in volunteers? NeuroToxicology. 55:102–111. https://doi.org/10.
1016/j.neuro.2016.05.019
Rea K, Dinan TG, Cryan JF (2016) The microbiome: a key regulator of
stress and neuroinflammation. Neurobiology of stress 4:23–33.
https://doi.org/10.1016/j.ynstr.2016.03.001
Rein B, Ma K, Yan ZA (2020) Standardized social preference protocol
for measuring social deficits in mouse models of autism. Nat Protoc
15(10):3464–3477. https://doi.org/10.1038/s41596-020-0382-9
Rose S, Melnyk S, Pavliv O, Bai S, Nick TG, Frye RE, James SJ (2012)
Evidence of oxidative damage and inflammation associated with
low glutathione redox status in the autism brain. Transl Psychiatry
2(7):e134. https://doi.org/10.1038/tp.2012.61
Ruiz-Larrea MB, Leal AM, Martin C, Martinez R, Lacort M (1997)
Antioxidant action of estrogens in rat hepatocytes. Rev Esp Fisiol
53(2):225–229
Sanchez MM, Mccormack K, Grand AP, Fulks R, Graff A, Maestripieri
D (2010) Effects of sex and early maternal abuse on adrenocortico-
tropin hormone and cortisol responses to the corticotropin-releasing
hormone challenge during the first 3 years of life in group-living
rhesus monkeys. Dev Psychopathol 22(1):45–53
Schwartzer JJ, Careaga M, Onore CE, Rushakoff JA, Berman RF,
Ashwood P (2013) Maternal immune activation and strain specific
interactions in the development of autism-like behaviors in mice.
Transl Psychiatry 3(3):e240. https://doi.org/10.1038/tp.2013.16
Shams S, Foley KA, Kavaliers M, MacFabe DF, Ossenkopp K-P (2019)
Systemic treatment with the enteric bacterial metabolic product
propionic acid results in reduction of social behavior in juvenile rats:
contribution to a rodent model of autism spectrum disorder. Dev
Psychobiol 61(5):688–699. https://doi.org/10.1002/dev.21825
Shultz SR, MacFabe DF, Ossenkopp KP, Scratch S, Whelan J, Taylor R,
Cain DP (2008) Intracerebroventricular injection of propionic acid,
an enteric bacterial metabolic end-product, impairs social behavior
in the rat: implications for an animal model of autism.
Neuropharmacology 54(6):901–911. https://doi.org/10.1016/j.
neuropharm.2008.01.013
Sinha S, Kaler LJ, Proctor TM, Teuscher C, Vandenbark AA, Offner H
(2008) IL-13-mediated gender difference in susceptibility to auto-
immune encephalomyelitis. J Immunol 180(4):2679–2685. https://
doi.org/10.4049/jimmunol.180.4.2679
Snyder MJ, Maddison DR (1997) Molecular phylogeny of glutathione-S-
transferases. DNA Cell Biol 16(11):1373–1384
Toufexis DJ, Wilson ME (2012) Dihydrotestosterone differentially mod-
ulates the cortisol response of the hypothalamic–pituitary–adrenal
axis in male and female rhesus macaques, and restores circadian
secretion of cortisol in females. Brain Res 1429:43–51
Uvnäs-Moberg K (1997) Oxytocin linked antistress effects–the relaxation
and growth response. Acta Physiol Scand Suppl 640:38–42
Velíšková J, DeSantis KA (2013) Sex and hormonal influences on sei-
zures and epilepsy. Horm Behav 63(2):267–277
Villa A, Rizzi N, Vegeto E, Ciana P, Maggi A (2015) Estrogen acceler-
ates the resolution of inflammation in macrophagic cells. Sci Rep 5:
15224
Wang L, Ahn YJ, Asmis R (2020) Sexual dimorphism in glutathione
metabolism and glutathione-dependent responses. Redox Biol 31:
101410
Windle RJ, Shanks N, Lightman SL, Ingram CD (1997) Central oxytocin
administration reduces stress-induced corticosterone release and
anxiety behavior in rats. Endocrinology 138(7):2829–2834
Yilmaz B, Kutlu S, Mogulkoç R, Canpolat S, Sandal S, Tarakçi B,
Kelestimur H (2000) Melatonin inhibits testosterone secretion by
acting at hypothalamo-pituitary-gonadal axis in the rat. Neuro
Endocrinol Lett; 21(4):301–306. PMID: 11455362
Publisher’s note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.