Effects Of Pregnancy Intermittent Hypoxia Or Restraint And Combination Of Both On The HPA Axis And Anxiety-like Behavior In Rat Offspring

The early life adverse environments pose a profound effect on physiological function of fetus and offspring. There are epidemiological investigations and clinic researchs suggest maternal stress has an effect on the endocrine function and behavioral response of offspring, induces disorders or susceptibility to diseases. HPA axis is a nerve-endocrine network which consists of hypothalamic, pituitary and adrenal, involved in the regulation of a varity of physiological function and stress response and plays an important role in neuroendocrine, immune, autonomic nerve and behavioral response. Pregnancy stresses influences the physiological function of HPA axis in offspring, dysfunction of HPA axis is associated with the abnormal physiological function of offspring.Pregnant maternal hypoxia stress is a common clinical pathological and physiological phenomenon. For example, apnea, asthma, infarct, cardiovascular disease, anemia, hypertension, umbilical occlusion, little placental, uterus blood flow decrease, smoking, drinking, and live in the high altitude that oxygen supply is deficient for pregnant animal and oxygen concentration decrease in organ and tissue, which will have an impact on the fetus and offspring, and eventually induced pathological symptom and abnormal behavioral response in offspring.In this study, we established a model that the pregnant rats were given simulated intermittent normobaric hypoxia (5 km, 10.8% O₂) (Hypo), restraint (Res) and combination of both (Hypo+Res) throughout the pregnant period, 4 hours per day. We examined the change of the body weight gain in postnatal early rats, basal level of HPA axis and the level of response to stress in early (P35) and late puberty (P65) male rats; and anxiety-like behavior of adult (P120) male rats. The protein of CRH, CRHR1 and CRHR2 in the hypothalamic PVN were detected by immunohistochemistry (IHC). The colocalization of CRH family in PVN and locus ceruleus (LC) of brain stem was studied by confocal immunofluroscence. CRHR1 mRNA and CRHR2 mRNA in pituitary were measured by in situ hybridization (ISH) and reverse-transcript polymerase chain reaction (RT-PCR). The elevated-plus maze (EPM) was applied to test the anxiety-like behavior and the high performance liquid chromatography (HPLC) was applied to measure noradrenaline (NE) and dopamine (DA) in LC of brain stem, and levels of plasma ACTH and CORT were measured by radioimmunoassay (RIA) and fluorimetric method respectively. Results:1. Effects of pregnancy intermittent Hypo, Res and Hypo+Res on the birth weight and body weight gain of rats offspring.The birth weight was significantly decreased in rat offspring after pregnancy intermittent Hypo, Res and Hypo+Res. There was no change on the maternal body weight gain, gestation length, litter size or proportion of male and females. The body weight gain of male rat offspring was significantly decreased in postnatal 1-2 w and female was inl-3w after pregnancy intermittent Hypo, Res and Hypo+Res. After that it returned to the control. The body weight of the male was higher than that of the female.2. Effects of pregnancy intermittent Hypo, Res and Hypo+Res on basal levels of HPA axis in early and late puberty rats.2.1 Early puberty (P35)There was no change of the basal levels of CRH peptides in PVN of early puberty male rats after pregnancy intermittent Hypo, Res and Hypo+Res. Compared with control or Hypo+Res group, basal levels of CRHR1 and CRHR2 peptides were significantly decreased in Hypo and Res groups. The basal levels of CRHR1 mRNA in pituitary were significantly decreased among all stress groups, CRHR2 mRNA was significantly decreased in Hypo and Hypo+Res groups, but was no change in Res group. Compared with control, CORT basal level in Hypo+Res group was significantly decreased, but was no change in Hypo or Res group.2.2 Late puberty (P65)There was no change of the basal levels of CRH or CRHR1 peptides in PVN of late puberty male rats after pregnancy intermittent Hypo, Res and Hypo+Res. Compared with control or Hypo group, the basal levels of CRHR1 or CRHR2 peptides were markedly decreased in Hypo+Res. Compared with control, Res or Hypo+Res group, the basal levels of CRHR1 mRNA in pituitary were significantly decreased in Hypo, CRHR2 mRNA was significantly decreased in Hypo or Res, but a significant increase in Hypo+Res (vs. control). ACTH basal levels were significantly increased in Hypo+Res (vs. control or Hypo group), CORT basal levels were significantly increased among Hypo, Res or Hypo+Res group compared with controls.3. Effects of pregnancy intermittent Hypo, Res and Hypo+Res on the HPA axis activity after stress in early and late puberty rats.3.1 Early puberty There was no change of the levels of CRH peptides in PVN of early puberty male rats offspring after exposed to the corresponding stress (vs. control). The levels of CRH peptides in PVN of Res and Hypo+Res groups were both markedly decreased (vs. corresponding group in None-PS). CRHR1 peptides levels in Res group were significantly increased (vs. control or corresponding group in None-PS). CRHR1 peptides levels in Hypo+Res group were significant decreased (vs. control). CRHR2 peptides levels in Hypo group were significantly increased (vs. control), but there was change for Res or Hypo+Res group.CRHR1 mRNA in pituitary was significantly decreased in Hypo, Res and Hypo+Res group (vs. control). CRHR1 mRNA was significantly decreased in Hypo group, but increased in Hypo+Res group (vs. corresponding group in None-PS). CRHR2 mRNA were significantly increased in Hypo group (vs. control) and Res group (vs. corresponding group in None-PS). Plasma CORT levels were significantly increased after stress exposure (vs. controls).3.2 Late pubertyPregnancy intermittent Hypo, Res or Hypo+Res exposure significantly decreased the levels of CRH peptides in PVN of late puberty male rats. CRH peptides levels in Hypo+Res group were markedly decreased (vs.corresponding group in None-PS). Although there was no change in Hypo, Res or Hypo+Res group (vs.control), CRHR1 peptides levels were significantly increased (vs. corresponding group in None-PS). CRHR2 peptides levels in Hypo+Res group were significantly increased (vs. control), but there was no change in Hypo or Res group.CRHR1 mRNA in pituitary was significantly decreased in Hypo+Res group and a trend for Res group (vs. control). CRHR2 mRNA was significantly increased in Hypo group (vs. control or corresponding group in None-PS) and Hypo+Res group (vs. corresponding group in None-PS). Plasma ACTH levels were greatly increased in Hypo and Res group (vs. control) and Hypo+Res group (vs. control or corresponding group in None-PS). Plasma CORT levels were significantly increased (vs. control).4. Comparison of HPA axis reactivity of early and late puberty rat offspring in pregnancy stress and none-pregnacy stress (none PS)4.1 Early puberty (P35)CRH peptides in PVN of Res and Hypo+Res groups were significantly decreased (vs. the corresponding group None-PS). CRHR1 peptides in Res group were significantly increased, and decreased in Hypo group (vs. the corresponding group in None-PS). CRHR1 mRNA in pituitary were significantly decreased in Hypo, and increased in Hypo+Res group (vs. the corresponding group in None-PS). CRHR2 mRNA in Hypo group were significantly increased (vs. control), and increased in Res group (vs. the corresponding group in None-PS).4.2 Late puberty (P65)CRH peptides in PVN of late puberty male rats were significantly decreased after pregnancy intermittent Hypo+Res stress (vs. the corresponding group in None-PS). CRHR1 peptides were significantly decrease in all groups (vs. None-PS); and CRHR2 peptides in Hypo+Res group were enhanced (vs. the corresponding group in None-PS), but was no change in Hypo or Res group. CRHR1 mRNA in pituitary were significantly decreased in Hypo group (vs. None-PS), and CRHR2 mRNA in Hypo+Res group were significant increased (vs. the corresponding group in None-PS). Plasma ACTH levels in Hypo+Res group were markedly increased (vs. the corresponding group in None-PS).5. Effects of pregnancy intermittent Hypo, Res and Hypo+Res on the anxiety-like behavior in adult male rats (P120).Pregnancy intermittent Hypo, Res or Hypo+Res exposure affects the HPA axis and behavior in adult male rats. CRH and CRHR1 protein in PVN of the hypothalamus significantly increased, but CRHR2 significantly decreased. By confocal immunofluroscence, CRH and CRHR1, UCN I and CRHR2, CRH and CRHR2, UCN III and CRHR2 in PVN of the hypothalamus, and CRH and CRHR1 in LC were colocalized in Hypo+Res group. Pituitary CRHR1 and CRHR2 mRNA were markedly enhanced in PS (pregnancy stress) offspring. Plasma ACTH, CORT levels and adrenal weight were significantly increased (vs.control). Also, the norepinephrine and dopamine levels significantly increased in the locus coeruleus, the highest level was in Hypo+Res offspring. Pregnancy intermittent Hypo, Res or Hypo+Res significantly increased anxiety-like behavior of adult male rats, the percentage of open arm entries in the elevated-plus maze test markedly decreased in rats offspring from all stressed groups.Conclusion:1. Pregnancy intermittent Hypo, Res or combination of both decreases the birth weight and body weight gain in postnatal early life.2. Pregnancy intermittent Hypo, Res or combination of both decrease HPA axis basal activity in early puberty, but increase in late puberty rat offspring. 3. Pregnancy intermittent Hypo, Res or combination of both alter the CRFR1 in the PVN as well as the drive on the cascade of responses of the HPA axis, resulting in an increase of anxiety-like behavior in adult male rat offspring. The colocalization of CRH and CRHR1, UCN I and CRHR2, CRH and CRHR2, UCN III and CRHR2 in PVN of the hypothalamus, and CRH and CRHR1 in LC in Hypo+Res group are involved in the proceeding, and it maybe drive from HPA axis and NE-sympathetic nerve system. The results indicate that CRH regulates its synthesis and release through integration of CRHR1 and CRHR2 in offspring after pregnancy intermittent Hypo, Res or combination of both.