Prenatal exposure to dexamethasone alters hippocampal drive on hypothalamic-pituitary-adrenal axis activity in adult male rats

Glucocorticoids are essential for normal hypothalamic-pituitary-adrenal (HPA) axis activity; however, recent studies warn that exposure to excess endogenous or synthetic glucocorticoid during a specific period of prenatal development adversely affects HPA axis stability. We administered dexamethasone (DEX) to pregnant rats during the last week of gestation and investigated subsequent HPA axis regulation in adult male offspring in unrestrained and restraint-stressed conditions. With the use of real-time PCR and RIA, we examined the expression of regulatory genes in the hippocampus, hypothalamus, and pituitary, including corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), glucocorticoid receptors (GR), mineralcorticoid receptors (MR), and 11-beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1), as well as the main HPA axis hormones, adrenal corticotropic hormone (ACTH) and corticosterone (CORT). Our results demonstrate that the DEX-exposed group exhibited an overall change in the pattern of gene expression and hormone levels in the unrestrained animals. These changes included an upregulation of CRH in the hypothalamus, a downregulation of MR with a concomitant upregulation of 11beta-HSD-1 in the hippocampus, and an increase in circulating levels of both ACTH and CORT relative to unrestrained control animals. Interestingly, both DEX-exposed and control rats exhibited an increase in pituitary GR mRNA levels following a 1-h recovery from restraint stress; however, the increased expression in DEX-exposed rats was significantly less and was associated with a slower return to baseline CORT compared with controls. In addition, circulating levels of ACTH and CORT as well as hypothalamic CRH and hippocampal 11beta-HSD-1 expression levels were significantly higher in the DEX-exposed group compared with controls following restraint stress. Taken together, these data demonstrate that late-gestation DEX exposure in rats is associated with persistent changes in both the modulation of HPA axis activity and the HPA axis-mediated response to stress.     

Neonatal exposure to oestrogens alters the protein profiles and gene expression in the genital tract of adult male mice.

After neonatal administration of supraphysiological doses of oestradiol, the concentration of tissue proteins, in adult mice, was significantly reduced by 39, 45 and 56% in epididymis, vas deferens and seminal vesicle respectively. The protein profiles showed persistent alterations. In epididymis, 4 protein bands were differentially increased (14.4, 43 and 67 kDa) or reduced (24 kDa) in oestrogenized males. In vas deferens, 4 proteins were increased (14.4, 49,67 and 76 kDa) and one (34 kDa) virtually absent. In seminal vesicle, about 20 proteins of varying molecular weights (12-140 kDa) were differentially increased or decreased. Testosterone substitution, at adulthood, was unable to reverse these effects. Treatments with oestradiol during adult life induced persistent alterations in the protein profiles of the 3 organs but, in contrast to neonatal treatment, these alterations could be reversed by androgen therapy. A cDNA library has been constructed with RNA prepared from adult seminal vesicle and screened by differential hybridization. Neonatal oestrogenization strongly reduced the abundance of some mRNA species. Eleven recombinants containing putative oestrogen-sensitive sequences were isolated. Two of them, having an insert of about 500 base pairs, were used for dot-blot hybridization. Results showed that the two clones contained sequences which were differently regulated by androgens.     

Prenatal alcohol exposure alters phosphorylation and glycosylation of proteins in rat offspring liver

To gain more insights into the translational and PTM that occur in rat offspring exposed to alcohol in utero, 2‐D PAGE with total, phospho‐ and glycoprotein staining and MALDI‐MS/MS and database searching were conducted. The results, based on fold‐change expression, revealed a down‐regulation of total protein expression by prenatal alcohol exposure in 7‐day‐old and 3‐month‐old rats. There was an up‐regulation of protein phosphorylation but a down‐regulation of glycosylation by prenatal alcohol exposure in both age groups. Of 31 protein spots examined per group, differentially expressed proteins were identified as ferritin light chain, aldo‐keto reductase, tumor rejection antigen gp96, fructose‐1,6‐bisphosphatase, glycerol‐3‐phosphate dehydrogenase, malate dehydrogenase, and γ‐actin. Increased phosphorylation was observed in proteins such as calmodulin, gluthatione S‐transferase, glucose regulated protein 58, α‐enolase, eukaryotic translation elongation factor 1 β‐2, riboprotein large P2, agmatinase, ornithine carbamoyltransferase, quinolinate phosphoribosyltransferase, formimidoyltransferase cyclodeaminase, and actin. In addition, glycosylation of adenosine kinase, adenosylhomocysteine hydrolase, and 3‐hydroxyanthranilate dioxygenase was reduced. Pathways affected by these protein alterations include cell signaling, cellular stress, protein synthesis, cytoskeleton, as well as glucose, aminoacid, adenosine and energy metabolism. The activity of the gluconeogenic enzyme fructose‐1,6‐bisphosphatase was elevated by prenatal alcohol. The observations may have important physiological implications.     

GT-repeat polymorphism in the heme oxygenase-1 gene promoter and the risk of carotid atherosclerosis related to arsenic exposure

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival and apoptosis. The SR supplies Ca²⁺ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP₃Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it has been demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload during hypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. We postulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca²⁺ signaling, causing apoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. We examined the distribution of IP₃Rs in cardiomyocytes via immunofluorescence and Western blotting and found that type 3 IP₃Rs were located in the SR. [Ca²⁺]i, [Ca²⁺]_m and [Ca²⁺]_SR were determined using Fluo-4, x-rhod-1 and Fluo 5N, respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laser confocal microscopy. We found that activation of CaR reduced [Ca²⁺]_SR, increased [Ca²⁺]_i and [Ca²⁺]_m and decreased the mitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage of BAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c from mitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activation causes Ca²⁺ release from the SR into the mitochondria through IP₃Rs and induces cardiomyocyte apoptosis during hypoxia/reoxygenation.     

Prenatal exposure to alcohol alters the Golgi apparatus of newborn rat hepatocytes: a cytochemical study

The effect of prenatal exposure to ethanol on the Golgi apparatus of newborn rat hepatocytes has been studied cytochemically using several trans-Golgi markers (thiamine pyrophosphatase, uridine diphosphatase, inosine diphosphatase, acid phosphatase, and 5'-nucleotidase) as well as a cis-side marker (osmium impregnation). The amount of cerium phosphate formed in the cytochemical reactions was roughly quantitated by stereologic methods. The Golgi apparatus of about 40% of the hepatocytes appeared disorganized after alcohol treatment, and in the other 60%, the electron density of reaction product deposits for all phosphatases investigated was decreased. 5'-Nucleotidase was completely absent in cisternae of Golgi apparatus of treated cells. In control cells impregnated with osmium tetroxide, reduced osmium compounds were observed in most Golgi cisternae and in nearby vesicles. In contrast, only small vesicles appeared positive in treated hepatocytes. These results suggest that prenatal alcohol exposure alters some Golgi functions. Thus, the decrease in nucleoside diphosphatase and 5'-nucleotidase cytochemical activities after ethanol exposure strongly suggests that this treatment could affect glycosylation in the Golgi apparatus of newborn rat hepatocytes.     

Short-term exposure to nonylphenol induces pancreatic oxidative stress and alters liver glucose metabolism in adult female rats

Nonylphenol is known to have estrogenic properties and has been reported to cause health hazards to animals and humans. The effects of nonylphenol on pancreas are not clearly elucidated. In this study, we sought to evaluate the effects of nonylphenol on the oxidative status of pancreas and consequential effects of nonylphenol on some of the end points of carbohydrate metabolism in the female rats. Rats were administered nonylphenol orally at the doses of 1.5, 15, and 150 mg/kg of body weight per day for 7 days. After 24 h of last dosing, the animals were sacrificed by cervical dislocation. The activities of pancreatic superoxide dismutase and catalase were significantly decreased with a concomitant increase in the levels of H2O2 and lipid peroxidation. Nonylphenol increased plasma insulin levels with a concomitant decrease in the levels of plasma glucose as compared to the control groups of rats. A dose-dependent increase in the activities of liver hexokinase and phosphofructokinase was recorded along with decreased activity of glycogen phosphorylase in liver. Western blot analysis revealed a significant decrease in the levels of GLUT-2. These results show that nonylphenol causes oxidative stress in pancreas and impairs liver glucose homeostasis.     

Prenatal exposure of male mice to diethylstilbestrol alter the expression of the lactotransferrin gene in seminal vesicles

We have previously isolated an estrogen-inducible secretory protein, lactotransferrin (LTF), and a cDNA to its messenger RNA from the uterus of mice. In this report we determined that the level of LTF mRNA is minimal in the seminal vesicles of normal mice. In contrast, expression of LTF mRNA in the seminal vesicles of developmentally estrogenized males was both constitutive and estrogen inducible. The results suggested that this alteration may be an example of atypical gene expression after hormonal manipulation early in development.     

Prenatal exposure to dexamethasone in the mouse induces sex‐specific differences in placental gene expression

Prenatal stress during pregnancy leads to sex‐specific effects on fetal development and disease susceptibility over the life span; however, the origin of sex differences has not been identified. The placenta not only plays a key role in fetal growth and development throughout pregnancy, but also affects the fetal programming underlying subsequent adult health and accounts. Therefore, sex‐specific adaptation of the placenta may be central to the sex differences in fetal growth and survival. Here, we analyzed the effects of prenatal dexamethasone (Dex) on sex‐specific changes in placental gene expression using RNA‐Seq. Placental tissues from males and females were separated into two developmentally distinct fetal and maternal parts at E11.5 stage. The majority of genes in female placentas were downregulated by prenatal Dex, whereas those were mostly maintained or rather upregulated in male placentas. RNA‐Seq results were validated using independent biological replicates from the same stage and placental tissue samples from E18.5 by realtime PCR assays. Activation of various inflammatory response‐related genes, chemokines and their receptors, particularly in male placentas, strongly implies that prenatal Dex exposure causes sex‐specific physiological responses that can lead to inflammatory diseases involving vascular pathology.     

Identification of factors contributing to variability in a blood-based gene expression test

Background

Corus CAD is a clinically validated test based on age, sex, and expression levels of 23 genes in whole blood that provides a score (1–40 points) proportional to the likelihood of obstructive coronary disease. Clinical laboratory process variability was examined using whole blood controls across a 24 month period: Intra-batch variability was assessed using sample replicates; inter-batch variability examined as a function of laboratory personnel, equipment, and reagent lots.

Methods/Results

To assess intra-batch variability, five batches of 132 whole blood controls were processed; inter-batch variability was estimated using 895 whole blood control samples. ANOVA was used to examine inter-batch variability at 4 process steps: RNA extraction, cDNA synthesis, cDNA addition to assay plates, and qRT-PCR. Operator, machine, and reagent lots were assessed as variables for all stages if possible, for a total of 11 variables. Intra- and inter-batch variations were estimated to be 0.092 and 0.059 Cp units respectively (SD); total laboratory variation was estimated to be 0.11 Cp units (SD). In a regression model including all 11 laboratory variables, assay plate lot and cDNA kit lot contributed the most to variability (p = 0.045; 0.009 respectively). Overall, reagent lots for RNA extraction, cDNA synthesis, and qRT-PCR contributed the most to inter-batch variance (52.3%), followed by operators and machines (18.9% and 9.2% respectively), leaving 19.6% of the variance unexplained.

Conclusion

Intra-batch variability inherent to the PCR process contributed the most to the overall variability in the study while reagent lot showed the largest contribution to inter-batch variability.     

Prenatal exposure to ethanol causes partial diabetes insipidus in adult rats

Chronic consumption of ethanol in adult rats and humans leads to reduced AVP-producing neurons, and prenatal ethanol (PE) exposure has been reported to cause changes in the morphology of AVP-producing cells in the suprachiasmatic nucleus of young rats. The present studies further characterize the effects of PE exposure on AVP in the young adult rat, its hypothalamic synthesis, pituitary storage, and osmotically stimulated release. Pregnant rats were fed a liquid diet with 35% of the calories from ethanol or a control liquid diet for days 7-22 of pregnancy. Water consumption and urine excretion rate were measured in the offspring at 60-68 days of age. Subsequently, the offspring were infused with 5% NaCl at 0.05 ml.kg(-1).min(-1) with plasma samples taken before and at three 40-min intervals during infusion for measurement of AVP and osmolality. Urine output and water intake were approximately 20% greater in PE-exposed rats than in rats with no PE exposure, and female rats had a greater water intake than males. The relationship between plasma osmolality and AVP in PE-exposed rats was parallel to, but shifted to the right of, the control rats, indicating an increase in osmotic threshold for AVP release. Pituitary AVP was reduced by 13% and hypothalamic AVP mRNA content was reduced by 35% in PE-exposed rats. Our data suggest that PE exposure can cause a permanent condition of a mild partial central diabetes insipidus.     

Prenatal ethanol exposure alters the expression of period genes governing the circadian function of beta-endorphin neurons in the hypothalamus

Sleep-wake disturbances and stress hyper-responsiveness have been observed in human neonates, children and adolescents who were exposed to alcohol during the prenatal period. Using the laboratory rat as an animal model, we investigated whether fetal ethanol exposure during gestational days 10-21 affects the circadian function of the stress-axis regulatory beta-endorphin neurons in the hypothalamus. Fetal ethanol-exposed rats showed abnormality in the circadian expression of proopiomelanocortin (POMC) mRNA encoding the peptide beta-endorphin in the arcuate nucleus of the hypothalamus during the adult period. These rats also showed altered circadian expression of the clock governing Period genes rPer1, rPer2 and rPer3, in the arcuate nucleus, and rPer1 and rPer 2 mRNA levels in the suprachiasmatic nucleus. Laser captured microdissection analysis identified constitutive expression of rPer1, rPer2 and rPer3 genes in beta-endorphin-containing neurons. These data suggest for the first time that fetal exposure to ethanol significantly alters the clock mechanisms governing the circadian function of beta-endorphin neurons.     

Prenatal glucocorticoid exposure in the sheep alters renal development in utero: implications for adult renal function and blood pressure control

Treatment of the pregnant ewe with glucocorticoids early in pregnancy results in offspring with hypertension. This study examined whether glucocorticoids can reduce nephron formation or alter gene expression for sodium channels in the late gestation fetus. Sodium channel expression was also examined in 2-mo-old lambs, while arterial pressure and renal function was examined in adult female offspring before and during 6 wk of increased dietary salt intake. Pregnant ewes were treated with saline (SAL), dexamethasone (DEX; 0.48 mg/h) or cortisol (CORT; 5 mg/h) over days 26-28 of gestation (term = 150 days). At 140 days of gestation, glomerular number in CORT and DEX animals was 40 and 25% less, respectively, compared with SAL controls. Real-time PCR showed greater gene expression for the epithelial sodium channel (α-, β-, γ-subunits) and Na(+)-K(+)-ATPase (α-, β-, γ-subunits) in both the DEX and CORT group fetal kidneys compared with the SAL group with some of these changes persisting in 2-mo-old female offspring. In adulthood, sheep treated with dexamethasone or cortisol in utero had elevated arterial pressure and an apparent increase in single nephron glomerular filtration rate, but global renal hemodynamics and excretory function were normal and arterial pressure was not salt sensitive. Our findings show that the nephron-deficit in sheep exposed to glucocorticoids in utero is acquired before birth, so it is a potential cause, rather than a consequence, of their elevated arterial pressure in adulthood. Upregulation of sodium channels in these animals could provide a mechanistic link to sustained increases in arterial pressure in cortisol- and dexamethasone-exposed sheep, since it would be expected to promote salt and water retention during the postnatal period.     

Prenatal nicotine alters vigilance states and AchR gene expression in the neonatal rat: implications for SIDS

Maternal smoking is a major risk factor for sudden infant death syndrome (SIDS). The mechanisms by which cigarette smoke predisposes infants to SIDS are not known. We examined the effects of prenatal nicotine exposure on sleep/wake ontogenesis and central cholinergic receptor gene expression in the neonatal rat. Prenatal nicotine exposure transiently increased sleep continuity and accelerated sleep/wake ontogeny in the neonatal rat. Prenatal nicotine also upregulated nicotinic and muscarinic cholinergic receptor mRNAs in brain regions involved in regulating vigilance states. These findings suggest that the nicotine contained in cigarette smoke may predispose human infants to SIDS by interfering with the normal maturation of sleep and wake.     

Prenatal exposure to low levels of carbon monoxide alters sciatic nerve myelination in rat offspring

Prenatal exposure to low concentrations of carbon monoxide (CO, 75 and 150 ppm from day 0 to day 20 of gestation), resulting in maternal blood HbCO concentrations equivalent to those maintained by human cigarette smokers, leads to subtle myelin alterations in the sciatic nerve of male rat offspring. The rapid growth spurt in pup body weight was related to the period of maximal increase in myelin sheath thickness in both control and CO-exposed animals. A significant reduction in myelin sheath thickness of sciatic nerve fibers, paralleled by changes in the frequency distribution, occurred in both 40- and 90-day-old rats exposed in utero to CO (75 and 150 ppm). Myelin deficit observed in 75 and 150 ppm CO-exposed animals showed up only after the major spurt in myelination but not early during development. The subtle myelin alterations observed in CO-exposed offspring were not accompanied by changes in developmental pattern of axon diameters and did not result in a gross impairment of motor activity. These results suggest that the myelination process is selectively targeted by a prenatal exposure model simulating the CO exposure observed in human cigarette smokers.