| Epidemiological studies have indicated that exposure to stress during pregnancy may result in an increased vulnerability of numerous mood disorders in the offspring, including anxiety, depression, schizophrenia and autism, and behavior alteration. There is compelling evidence that many neurological disorders which become apparent after birth have their origins during fetal life. The mechanism through which fetal antecedents contribute to disease development is not known but likely involves a complex interaction between environment and genetics. One potential mechanism whereby stress can influence fetal development and programming is through epigenetics, including DNA methylation. Recent work has shown that early life stress in rodents and humans can change methylation patterns at specific loci of the genomic DNA, which in turn permanently alter gene expression in the brain and induce increases in anxiety or depression behavior in the adult.Maternal hypoxia in pregnancy has been reported to be one of the most important putative noxious signals occurring during development, which has long lasting consequences for the fetus, infant and adult. The fetal may experience hypoxia stress under a variety of conditions, such as pregnancy at high altitude, pregnancy with anemia, cord compression, hypertension disorder, obstructive sleep apnea and preeclampsia. Moreover, pregnant women with these diseases often suffer from intrauterine intermittent hypoxia. We previously reported that intermittent hypoxia during pregnancy induced a high activity of hypothalamo-pituitary adrenal axis (HPA) and anxiety-like behavior in the adult offspring that linked to the activation of corticotroping-releasing hormone type-1receptor (CRHR1) expression in the brain hypothalamus paraventricular nucleus (PVN). Recently it has been reported that Stress experience early in pregnancy may contribute to sex-specific programming of offspring emotionality through impacts on placental function and fetal neurodevelopment, and recently study showed that prenatal stress had a differential sex-dependent effect on the expression of CRH and its receptors in the PVN and amygdala of rats. However, there are still questions of how the effect of maternal stress influences the offspring behavior and why was performed with a sex-specific manner. So we hypothesize that the sex-dependent differences on DNA methylation of Crhrl gene in early life and adulthood subjected to gestational intermittent hypoxia (GIH) and the epigenetic modification of Crhrl gene promoter correlates to the activation of CRHR1mRNA in PVN of offspring to induce anxiety-like behavior.Here we reported that maternal gestational intermittent hypoxia (GIH)(hypobaric high-altitude hypoxia of5km, equivalent to-10.8%O2at sea level,4h/d) for21days, produced an anxiety-like behavior in P90male offspring not females that may correlate with a significant increase of CRHR1mRNA expression in the PVN of P90males and besides GIH induced an increased CRHR1mRNA in the hypothalamus of E19males. At E19female embryos, there was a decreased mRNA expression of CRHR1and an increase of CRHR2mRNA in corresponding with the decline CRFR1protein expression and the enhanced CRFR2protein levels, and the p-ERKl/2protein expression was simultaneously elevated. It was indicated that CRFR2might be involved in the down-regulation of CRFR1in the hypothalamus area, and following with the activation of Ras-MAPK signaling pathway.Effects of GIH on mRNA expression of CRHR1and CRHR2were measured by qPCR in the PVN, pituitary and CeA after birth. In the PVN, GIH reduced the CRHR1mRNA expression of female offspring at P7, but no change at other days. However, there was no change of CRHR1expression in male offspring at P1, P7, P14and P21. GIH enhanced the CRHR2mRNA expression of females at P14, but no change at other days. And no change of CRHR2mRNA expression was found in male offspring at P1, P7, P14and P21. At P7, the relative change of CRHR1and CRHR2was decreased in both male and female offspring. In the pituitary, GIH reduced the CRHR1mRNA expression of male offspring at P1and P14, but no change at other days. And there was no change of CRHR1expression in female offspring at P1, P7, P14and P21. GIH enhanced the CRHR2mRNA expression of males at P7, but no change at other days. And no change of CRHR2mRNA expression was found in female offspring at P1, P7, P14and P21. The relative change of CRHR1and CRHR2was decreased in male offspring, but increased in females at P1but decreased in both male and female offspring at P7. In the CeA, GIH reduced the CRHR1mRNA expression of male offspring at P1and increase the CRHR1expression at P14, but no change at other days. And there was a decrease of CRHR1expression in female offspring at P7, but no change at other days. GIH enhanced the CRHR2mRNA expression of females at P1, P7and P14, but no change at P21. And no change of CRHR2mRNA expression was found in male offspring at P1, P7, P14and P21. At P1, the relative change of CRHR1and CRHR2was decreased in female offspring, but no change in males. The relative change of CRHR1and CRHR2was decreased at P7and P14in both male and female offspring. At P21, the relative change of CRHR1and CRHR2was decreased only in male offspring. In addition, we used the software of MethPrimer-Design Primers for Methylation PCRs to analyze the CpG island status within the promoter region of rat Crhrl gene, and we found that there were two CpG islands (island-1and island-2) in the Crhrl gene promoter. Island1was located between the-607and-502bp relative to+1, island2was located between the-6and+429bp relative to+1. The results using duo-luciferase reporter gene showed that the first CpG island functioned as the major regulation domain of Crhrl transcription activity. Then, we analyzed9specific CpG dinucleotides within the first CpG island of Crhrl promoter in vivo, a significant decrease in methylation at-535cytosine was evident in the hypothalamus of male embryos at embryonic day19(E19), which both increased CRFR1mRNA and protein expression, while there was an increased in methylation at-547cytosine in females in corresponding with the down-regulation of CRHR1mRNA and protein expression. During the postnatal development, only-535cytosine methylation level was significantly decreased in the paraventricular nucleus of hypothalamus (PVN) of GIH male offspring at P1, P14and P90. And the methylation level of four sites (-587,-561,-547and-544cytosines) had been all decreased in P90GIH male offspring, while there was no change in GIH female offspring. In the pituitary, there was a declined methylation level at three cytosine sites (-547,-544and-535cytosines) in GIH male offspring at P1, while the female offspring showed one site (-544cytosine) demethylation at P1, and there was an increase in methylation at-561cytosine at P14and P90. In the CeA, the enhancement of methylation in-552,-547,-544and-535cytosines were found both in male and female offspring at P1, and the enhancement of methylation in-579,-552and-547cytosines were found only in male offspring at P90. Furthermore, the decreased methylation at CpG sites within the island-1may modulated by a decreased expression of DNMT3b in the PVN of P90males and hypothalamus of E19embryo. In vitro the basal activity of Crhrl promoter was notably attenuated by a mutation at the-547CpG site in both cell lines, suggesting that the transcription of Crhrl could be affected by the sequence of this domain through binding specific transcription factors. Methylation of six CpG site mutation, such as-604,-587,-579,-547,-544, and-535site, could not reduce the Crhrl promoter activity respectively as the non-mutation methylation effect, but methylation of-561,-552and-537CpG site mutation still substantially downregulated its activity in both cell lines, indicating that methylation of-604,-587,-579,-547,-544, and-535site may be involvement in the regulation of the CRHR1expression. All data indicated that multiple events was involved in the eptigentic regulation of Crhrl gene, despite they were not embryonic origin.In summary, our present study show that GIH induces anxiety-like behavior of the adult offspring, and this behavioral change is sex-difference, which is closely associated with sex-dependent expression of CRHR1mRNA in the PVN but not in the CeA of adult male and female offspring. Our study strongly provide the evidence that GIH produces alterations of Crhrl gene methylation principally modulated by the DNMT3b, implying a mechanism of sex-biased manner for the effects on Crhrl gene expression related with the anxiety-like behavior in the GIH adult offspring. Our findings suggest that the vulnerability for hypoxia-induced anxiety in offspring could occur through the sex-differential epigenetic modification of Crhrl genomic regions that are implicated in the regulation of stress pathway programming specific to the maternal stress response. |
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