4.2 The low level of maternally derived glucocorticoid exposure
in the PDE fetal rats and its maternal-fetal interaction mechanism
In humans, the weight and size of the maternal adrenal gland don’t
change significantly during pregnancy. However, the level of free
glucocorticoid in maternal blood is still increased by 2-4 times in the
third trimester compared with
pre-pregnancy(Nolten et al. , 1981;
Rainey et al. , 2004). This
indicates that the glucocorticoid synthesis function of the maternal
adrenal is enhanced during pregnancy. The development of the fetal
adrenal gland is a unique process. Although the fetal adrenal gland can
synthesize a small amount of glucocorticoid through progesterone
(Macnaughton et al. , 1977), thede novo glucocorticoid synthesis pathway in the fetal adrenal
gland was established in the third trimester
(Mesiano et al. , 1997). Therefore,
the amount of glucocorticoid secreted by the fetal adrenal gland is
relatively less than that of the mother, and the fetal blood
glucocorticoid in the intrauterine
period is mainly derived from maternal blood.
In vivo , we found dexamethasone directly inhibited the maternal
adrenal steroidogenesis rather than inhibiting the HPA axis. Further,
the serum corticosterone levels were also decreased significantly in the
PDE fetal rats. It is known that the maternal glucocorticoid levels
during pregnancy are 5-10 times higher than that of the fetus, and the
concentration gradient is maintained by the placental glucocorticoid
barrier, including P-glycoprotein and 11β-HSDs system
(Zhu et al. , 2019). In this study,
we observed PDE opened the placental glucocorticoid barrier. Moreover,
we also confirmed that there is a positive correlation between maternal
and fetal serum corticosterone levels. In summary, we demonstrated the
low exposure to maternal glucocorticoid in the PDE fetal rats was mainly
due to the decrease of maternal serum glucocorticoid level and the
opening of the placental glucocorticoid barrier, indicating the presence
of a maternal-fetal interaction mechanism.
4.3 The positive programming of GC-IGF1 axis mediated thegender difference in
adrenal function of the PDE offspring after birth
There is a common phenomenon of maternal glucocorticoid overexposure
induced by the prenatal adverse environment, such as xenobiotics
exposure (Morales-Rubio et al. ,
2019), hypoxia (Cuffe et al. ,
2014), and stress (Buss et al. ,
2012). Our previous research found that the negative regulation change
of the GC-IGF1 axis was involved in the adrenal developmental
programming and homeostasis alterations in offspring with high
maternally derived glucocorticoid exposure
(Huang et al. , 2015;
He et al. , 2019). However, in this
study, the serum corticosterone levels decreased in the PDE male
offspring, accompanied by the persistent decrease in the H3K27ac level
of the IGF1 promoter region, IGF1 signaling pathway and adrenal
steroidogenesis. It should be noted that chronic stress significantly
increased the serum corticosterone level in adult offspring of the
control group, while the above indicators decreased. The serum
corticosterone levels were markedly increased in the PDE adult offspring
with chronic stress; meanwhile, the above-accompanied indicators were
significantly enhanced. In vitro,the lower physiological
concentrations of cortisol decreased the IGF1 H3K27ac and expression
levels as well as intracellular cortisol production, and the
over-expression of GRα could reverse these inhibitory effects of
cortisol. In conclusion, PDE-induced maternal glucocorticoid
low-exposure caused adrenal insufficiency of the male offspring ratsvia “positive programming” of the GC-IGF1 axis.
Exposure to the same environment during pregnancy has different effects
on the growth and development in individuals and the occurrence time,
course, and outcome of chronic diseases in the offspring of different
genders (van Abeelen et al. , 2011;
Waddell et al. , 2012;
Schaafsma et al. , 2017). Clinic
and animal studies reported that the gender difference in catch-up
growth patterns is related to the differences in sex hormones in
individuals (Perry et al. , 2008).
Accumulating evidence has shown that IGF1 plays a vital role in the
catch-up growth of mammals, and estrogen can promote IGF1 expression
through estrogen receptors (Hewittet al. , 2019). This study also sought to determine whether the
adrenal development in PDE female offspring is different from that of
the males and whether it is also regulated by positive programming of
the GC-IGF1 axis. In this study, we found the serum corticosterone level
of the PDE male offspring decreased after birth, accompanied by
continuous inhibition of adrenal IGF1 signaling pathway and
steroidogenic function. However, the serum corticosterone level of the
PDE females increased after birth (Fig S2), accompanied by continuous
enhancement of the above indicators. This suggested that positive
programming of the GC-IGF1 axis is also involved in the PDE female
adrenal development, and the enhanced female adrenal steroidogenic
function after birth might be related to the high level of estrogen to
promote IGF1 expression via estrogen receptor.
4.4 GRα/miR-370-3p/Sirt3 signal mediated the lower
physiologicalconcentrations of
glucocorticoid-induced positive programming of the adrenal GC-IGF1 axis
In this study, the levels of H3K27ac of the adrenal IGF1 promoter region
decreased continuously in the PDE offspring rats. Further, we found that
the expression of adrenal Sirt3 significantly increased in the PDE fetal
rats (Fig S3). Mitochondria are the main organelles for adrenal
steroidogenesis, and Sirt3 is the main histone deacetylase in
mitochondria (Onyango et al. ,
2002). We observed that the adrenal Sirt3 expression markedly increased
in the PDE offspring rats, while its expression relatively decreased
with chronic stress in adulthood. Studies have reported that Sirt3 is
targeted by miRNAs (Zhang et al. ,
2018). We found that the adrenal miR-370-3p expression reduced in the
PDE fetal rats by whole-genome sequence analysis. Meanwhile, miRWalk
bioinformatics prediction revealed that the 3’UTR of Sirt3 mRNA had a
miR-370-3p target site. In vivo , the adrenal miR-370-3p
expression significantly reduced in the PDE offspring, while its
expression relatively increased with chronic stress in adulthood.In vitro, the low physiological concentrations of cortisol
concentration-dependently inhibited the miR-370-3p expression and
promoted Sirt3 expression. Further, Sirt3 knockdown or miR-370-3p mimics
significantly reversed the inhibitory effect of low physiological
concentration of cortisol on the H3K27ac and expression levels of IGF1,
and miR-370-3p mimics also reversed its promotive effect on Sirt3
expression. Additionally, the luciferase reporter analysis showed that
miR-370-3p could directly target Sirt3. It indicated the low
physiological concentrations of glucocorticoid downregulated miR-370-3p
to target up-regulation of Sirt3, thereby reducing the H3K27ac and
expression levels of IGF1 in NCI-H295R cells.
Glucocorticoids mainly exert biological effects through GR, and GR can
regulate the expression of non-coding RNA, including miRNA
(Tessel et al. , 2010). In this
study, we found that the increased expression of fetal adrenal GRαin utero was related to the fetal blood dexamethasone exposure,
while the adrenal GRα expression alterations after birth was consistent
with the serum corticosterone concentration. Moreover, in vitro,the low physiological concentrations of cortisol inhibited the
expression of GRα and miR-370-3p, while over-expression of GRα reversed
the inhibitory effect of cortisol on miR-370-3p expression. Above all,
it indicated the GRα/miR-370-3p/Sirt3 signal mediated the low
physiological concentrations of endogenous glucocorticoids on the
positive programming of the adrenal GC-IGF1 axis (Fig 8).
In addition, we were interested in whether the suppression of
dexamethasone on adrenal IGF1 expression could last until after birth.
We found that the H3K9ac and H3K27ac levels of the adrenal IGF1 promoter
region decreased in the PDE fetal rats, but only the H3K27ac level
decreased after birth. In vitro , dexamethasone significantly
reduced the H3K9ac level of the IGF1 promoter region. It suggested
dexamethasone could inhibit adrenal IGF1 expression by reducing the
H3K9ac level of the IGF1 promoter region in utero, but this
effect could not continue after birth (without dexamethasone exposure).
5 Conclusions
This study confirmed that PDE
inhibited maternal adrenal function and opened placental glucocorticoid
barrier, leading to a low exposure of maternally derived glucocorticoid
in the fetal rats. The low level of intrauterine endogenous
glucocorticoids could cause adrenal insufficiency in adult male
offspring rats, which is mediated by the GRα/miR-370-3p/Sirt3 signal
through epigenetic positive programming of the GC-IGF1 axis.
However, the inhibitory effect of
dexamethasone on adrenal function can’t last after birth. It remains to
think the causes of gender differences in adrenal function of PDE
offspring after birth. This study for the first time systematically
confirmed that synthetic glucocorticoids (e.g., dexamethasone) could
alter the adrenal development programming and homeostasis in offspring
by inhibiting the maternal adrenal function, and it provides an
important theoretical and experimental evidence for the comprehensive
explanation of maternal adrenal influence on the offspring development
and the disease susceptibility.