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.