Gestational dexamethasone exposure impacts hippocampal excitatory synaptic transmission and learning and memory function with transgenerational effects.

The formation of learning and memory is regulated by synaptic plasticity in hippocampal neurons. Here we explored how gestational exposure to dexamethasone, a synthetic glucocorticoid commonly used in clinical practice, has lasting effects on offspring's learning and memory. Adult offspring rats of prenatal dexamethasone exposure (PDE) displayed significant impairments in novelty recognition and spatial learning memory, with some phenotypes maintained transgenerationally. PDE impaired synaptic transmission of hippocampal excitatory neurons in offspring of F1 to F3 generations, and abnormalities of neurotransmitters and receptors would impair synaptic plasticity and lead to impaired learning and memory, but these changes failed to carry over to offspring of F5 and F7 generations. Mechanistically, altered hippocampal miR-133a-3p-SIRT1-CDK5-NR2B signaling axis in PDE multigeneration caused inhibition of excitatory synaptic transmission, which might be related to oocyte-specific high expression and transmission of miR-133a-3p. Together, PDE affects hippocampal excitatory synaptic transmission, with lasting consequences across generations, and CDK5 in offspring's peripheral blood might be used as an early-warning marker for fetal-originated learning and memory impairment.

MiR-17-5p/FOXL2/CDKN1B signal programming in oocytes mediates transgenerational inheritance of diminished ovarian reserve in female offspring rats induced by prenatal dexamethasone exposure.

Prenatal dexamethasone exposure (PDE) induces long-term reproductive toxicity in female offspring. We sought to explore the transgenerational inheritance effects of PDE on diminished ovarian reserve (DOR) in female offspring. Dexamethasone was subcutaneously administered into pregnant Wistar rats from gestational day 9 (GD9) to GD20 to obtain fetal and adult offspring of the F1 generation. F1 adult females were mated with normal males to produce the F2 generation, and the F3 generation. The findings showed decrease of serum levels of anti-Müllerian hormone (AMH) that in the PDE group, decrease in number of primordial follicles, and upregulation of miR-17-5p expression before birth in F1 offspring rats. Expression of cyclin-dependent kinase inhibitor 1B (CDKN1B) and Forkhead Box L2 (FOXL2) were downregulated, and binding of FOXL2 and the CDKN1B promoter region was decreased in PDE groups of the F1, F2, and F3 generations. In vitro intervention experiments showed that glucocorticoid receptor (GR) was involved in activity of dexamethasone. These findings indicate that PDE can activate GR in fetal rat ovary and induce DOR of offspring, and its heritability is mediated by the cascade effect of miR-17-5p/FOXL2/CDKN1B. Increase in miR-17-5p expression in oocytes is the potential molecular basis for transgenerational inheritance of PDE effects.

Prenatal dexamethasone exposure induces anxiety- and depressive-like behavior of male offspring rats through intrauterine programming of the activation of NRG1-ErbB4 signaling in hippocampal PV interneurons.

Dexamethasone is a commonly used synthetic glucocorticoid in the clinic. As a compound that can cross the placental barrier to promote fetal lung maturation, dexamethasone is extensively used in pregnant women at risk of premature delivery. However, the use of glucocorticoids during pregnancy increases the risk of neurodevelopmental disorders. In the present study, we observed anxiety- and depressive-like behavior changes and hyperexcitability of hippocampal neurons in adult rat offspring with previous prenatal dexamethasone exposure (PDE); the observed changes were related to in utero damage of parvalbumin interneurons. A programmed change in neuregulin 1 (NRG1)-Erb-b2 receptor tyrosine kinase 4 (ErbB4) signaling was the key to the damage of parvalbumin interneurons in the hippocampus of PDE offspring. Anxiety- and depressive-like behavior, NRG1-ErbB4 signaling activation, and damage of parvalbumin interneurons in PDE offspring were aggravated after chronic stress. The intervention of NRG1-ErbB4 signaling contributed to the improvement in dexamethasone-mediated injury to parvalbumin interneurons. These results suggested that PDE might cause anxiety- and depressive-like behavior changes in male rat offspring through the programmed activation of NRG1-ErbB4 signaling, resulting in damage to parvalbumin interneurons and hyperactivity of the hippocampus. Intrauterine programming of neuregulin 1 (NRG1)-Erb-b2 receptor tyrosine kinase 4 (ERBB4) overactivation by dexamethasone mediates anxiety- and depressive-like behavior in male rat offspring.

Programming changes of hippocampal miR-134-5p/SOX2 signal mediate the susceptibility to depression in prenatal dexamethasone-exposed female offspring.

Depression is a neuropsychiatric disorder and has intrauterine developmental origins. This study aimed to confirm the depression susceptibility in offspring rats induced by prenatal dexamethasone exposure (PDE) and to further explore the intrauterine programming mechanism. Wistar rats were injected with dexamethasone (0.2 mg/kg·d) subcutaneously during the gestational days 9-20 and part of the offspring was given chronic stress at postnatal weeks 10-12. Behavioral results showed that the adult PDE female offspring was susceptible to depression, accompanied by increased hippocampal miR-134-5p expression and decreased sex-determining region Y-box 2 (SOX2) expression, as well as disorders of neural progenitor cells proliferation and hippocampal neurogenesis. The PDE female fetal rats presented consistent changes with the adult offspring, accompanied by the upregulation of glucocorticoid receptor (GR) expression and decreased sirtuin 1 (SIRT1) expression. We further found that the H3K9ac level of the miR-134-5p promoter was significantly increased in the PDE fetal hippocampus, as well as in adult offspring before and after chronic stress. In vitro, the changes of GR/SIRT1/miR-134-5p/SOX2 signal by dexamethasone were consistent with in vivo experiments, which could be reversed by GR receptor antagonist, SIRT1 agonist, and miR-134-5p inhibitor. This study confirmed that PDE led to an increased expression level as well as H3K9ac level of miR-134-5p by activating the GR/SIRT1 pathway in the fetal hippocampus and then inhibited the SOX2 expression. The programming effect mediated by the abnormal epigenetic modification could last from intrauterine to adulthood, which constitutes the intrauterine programming mechanism leading to hippocampal neurogenesis disorders and depression susceptibility in female offspring. Intrauterine programming mechanism for the increased depressive susceptibility in adult female offspring by prenatal dexamethasone exposure (PDE). GR, glucocorticoid receptor; SIRT1, sirtuin 1; SOX2, sex-determining region Y-box 2; NPCs, neuroprogenitor cells; H3K9ac, histone 3 lysine 9 acetylation; GRE, glucocorticoid response element.

miRNA320a-3p/RUNX2 signal programming mediates the transgenerational inheritance of inhibited ovarian estrogen synthesis in female offspring rats induced by prenatal dexamethasone exposure.

Our previous studies found that prenatal dexamethasone exposure could cause abnormal follicular development in fetal rats. This study intends to observe the transgenerational inheritance effects of ovarian estrogen inhibition in offspring exposed to dexamethasone (0.2 mg/kg • d) from gestational day 9 (GD9) to GD20 in Wistar rats, and explore the intrauterine programming mechanisms. Prenatal dexamethasone exposure reduced the expression of ovarian cytochrome P450 aromatase (P450arom), the level of serum estradiol (E2) and the number of primordial follicles, while increased the number of atresia follicles before and after birth in F1 offspring rats. At the same time, the expression of miRNA320a-3p in F1 ovaries was down-regulated, and RUNX2 expression increased significantly. These changes were continued to F2 and F3 generations, accompanied by consistently down-regulated miRNA320a-3p expression in oocyte of F1 and F2 adult offspring. In vitro, fetal rat ovaries and KGN human ovarian granulosa cells were treated with dexamethasone. It showed that dexamethasone decreased miRNA320a-3p and P450arom expression, as well as E2 synthesis, and increased RUNX2 expression. All these effects could be reversed by the GR antagonist RU486. The overexpression of miRNA320a-3p in vitro could also reverse the effects of dexamethasone on RUNX2, P450arom, and E2 levels. The dual-luciferase reporter gene experiment further confirmed the direct targeted regulation of miRNA320a-3p on RUNX2. These results indicate that prenatal dexamethasone exposure induces ovarian E2 synthesis inhibition mediated by the GR/miRNA320a-3p/RUNX2/P450arom cascade signal in fetal rat ovary, which has transgenerational inheritance effects and may related to the inhibited miRNA320a-3p expression in oocyte.

The combination of hprt and gapdh is the best compound reference genes in the fetal rat hippocampus.

Hippocampus, as an important organ of central memory storage and spatial orientation, has been studied increasingly in recent years. The expression of reference genes in the hippocampus of adult rats, which are commonly used in the quantitative real-time polymerase chain reaction (qRT-PCR), is unstable in the fetal hippocampus and may not be suitable for the fetal period. Therefore, this study intends to screen and determine the optimal compound reference genes in the fetal rat hippocampus. Based on the literature, we selected five housekeeping genes (HKGs), including glyceraldehyde 3-phosphate dehydrogenase (gapdh), actin beta (ß-actin), hypoxanthine phosphoribosyltransferase (hprt), 18s ribosomal RNA (18s rRNA), and cyclophilin B (cypB). We analyzed the expression of them under physiological conditions in the fetal rat hippocampus using BestKeeper, GeNorm, and NormFinder, to select the most stable compound reference genes. Furthermore, to verify the stability of the compound reference genes, we analyzed the expression of reference genes in the fetal rat hippocampus under the pathological model of prenatal dexamethasone exposure (PDE). Finally, we evaluated the accuracy of compound reference genes through detecting the expression of fetal rat hippocampal brain-derived neurotrophic factor (BDNF) under PDE model. This study determined that the combination of gapdh and hprt was the most stable and suitable compound reference genes in the fetal rat hippocampus. There was no significant difference between male and female fetal rats. We provided the support of accurate and reliable reference genes for the further study of diseases related to the fetal hippocampus.

Intrauterine RAS programming alteration-mediated susceptibility and heritability of temporal lobe epilepsy in male offspring rats induced by prenatal dexamethasone exposure.

Partial temporal lobe epilepsy (TLE) has an intrauterine developmental origin. This study was aimed at elucidating the heritable effects and programming mechanism of TLE in offspring rats induced by prenatal dexamethasone exposure (PDE). Pregnant Wistar rats were injected subcutaneously with dexamethasone (0.2 mg/kg day) from gestational day 9 to 20. The F1 and F2 generations of male offspring were administered lithium pilocarpine (LiPC) for electroencephalography and video monitoring in epilepsy or behavioral tests. Results showed that the PDE + LiPC group exhibited TLE susceptibility, which continued throughout F2 generation. Expression of hippocampal glucocorticoid receptor (GR), CCAAT enhancer-binding protein α (C/EBPα), intrauterine renin-angiotensin system (RAS) classical pathway related genes, the H3K27ac level in angiotensin-converting enzyme (ACE) promoter, as well as high mobility group box 1 (HMGB1) and toll-like receptor 4 (TLR4) were increased, but glutamate dehydrogenase (GLUD) 1/2 expression were decreased, accompanied by increased glutamate levels in PDE fetal and adult rats, as well as in F1 and F2 offspring of the PDE + LiPC group. These consistent changes were also observed by treating the H19-7 fetal hippocampal cell line with dexamethasone and were reversed by GR inhibitor (RU486) and ACE inhibitor (enalaprilat). Our results confirmed that PDE-induced H3K27ac enrichment in the ACE promoter and enhanced the RAS classic pathway via activating GR-C/EBPα-p300 in utero, which caused changes of the HMGB1 pathway and glutamate excitatory damage. Intrauterine programming mediated by abnormal histone modification of hippocampal ACE could continue to adulthood and even F2 generation, which induced the heritability of TLE in male offspring rats.