Daily ethanol exposure during late ovine pregnancy: physiological effects in the mother and fetus in the apparent absence of overt fetal cerebral dysmorphology

High levels of ethanol (EtOH) consumption during pregnancy adversely affect fetal development; however, the effects of lower levels of exposure are less clear. Our objectives were to assess the effects of daily EtOH exposure (3.8 USA standard drinks) on fetal-maternal physiological variables and the fetal brain, particularly white matter. Pregnant ewes received daily intravenous infusions of EtOH (0.75 g/kg maternal body wt over 1 h, 8 fetuses) or saline (8 fetuses) from 95 to 133 days of gestational age (DGA; term ～145 DGA). Maternal and fetal arterial blood was sampled at 131-133 DGA. At necropsy (134 DGA) fetal brains were collected for analysis. Maternal and fetal plasma EtOH concentrations reached similar maximal concentration (～0.11 g/dl) and declined at the same rate. EtOH infusions produced mild reductions in fetal arterial oxygenation but there were no changes in maternal oxygenation, maternal and fetal Pa(CO(2)), or in fetal mean arterial pressure or heart rate. Following EtOH infusions, plasma lactate levels were elevated in ewes and fetuses, but arterial pH fell only in ewes. Fetal body and brain weights were similar between groups. In three of eight EtOH-exposed fetuses there were small subarachnoid hemorrhages in the cerebrum and cerebellum associated with focal cortical neuronal death and gliosis. Overall, there was no evidence of cystic lesions, inflammation, increased apoptosis, or white matter injury. We conclude that daily EtOH exposure during the third trimester-equivalent of ovine pregnancy has modest physiological effects on the fetus and no gross effects on fetal white matter development.     

Alcohol exposure during late gestation adversely affects myocardial development with implications for postnatal cardiac function

Prenatal exposure to high levels of ethanol is associated with cardiac malformations, but the effects of lower levels of exposure on the heart are unclear. Our aim was to investigate the effects of daily exposure to ethanol during late gestation, when cardiomyocytes are undergoing maturation, on the developing myocardium. Pregnant ewes were infused with either ethanol (0.75 g/kg) or saline for 1 h each day from gestational days 95 to 133 (term ～145 days); tissues were collected at 134 days. In sheep, cardiomyocytes mature during late gestation as in humans. Within the left ventricle (LV), cardiomyocyte number was determined using unbiased stereology and cardiomyocyte size and nuclearity determined using confocal microscopy. Collagen deposition was quantified using image analysis. Genes relating to cardiomyocyte proliferation and apoptosis were examined using quantitative real-time PCR. Fetal plasma ethanol concentration reached 0.11 g/dL after EtOH infusions. Ethanol exposure induced significant increases in relative heart weight, relative LV wall volume, and cardiomyocyte cross-sectional area. Ethanol exposure advanced LV maturation in that the proportion of binucleated cardiomyocytes increased by 12%, and the number of mononucleated cardiomyocytes was decreased by a similar amount. Apoptotic gene expression increased in the ethanol-exposed hearts, although there were no significant differences between groups in total cardiomyocyte number or interstitial collagen. Daily exposure to a moderate dose of ethanol in late gestation accelerates the maturation of cardiomyocytes and increases cardiomyocyte and LV tissue volume in the fetal heart. These effects on cardiomyocyte growth may program for long-term cardiac vulnerability.     

The oncogene Trop2 regulates fetal lung cell proliferation

The factors regulating growth of the developing lung are poorly understood, although the degree of fetal lung expansion is critical. The oncogene Trop2 (trophoblast antigen 2) is upregulated during accelerated fetal lung growth, and we hypothesized that it may regulate normal fetal lung growth. We investigated Trop2 expression in the fetal and neonatal sheep lung during accelerated and delayed lung growth induced by alterations in fetal lung expansion, as well as in response to glucocorticoids. Trop2 expression was measured using real-time PCR and localized spatially using in situ hybridization and immunofluorescence. During normal lung development, Trop2 expression was higher at 90 days gestational age (GA; 4.0 ± 0.8) than at 128 days GA (1.0 ± 0.1), decreased to 0.5 ± 0.1 at 142 days GA (full term ～147 days GA), and was positively correlated to lung cell proliferation rates (r = 0.953, P < 0.005). Trop2 expression was regulated by fetal lung expansion, but not by glucocorticoids. It was increased nearly threefold by 36 h of increased fetal lung expansion (P < 0.05) and was reduced to ～55% of control levels by reduced fetal lung expansion (P < 0.05). Trop2 expression was associated with lung cell proliferation during normal and altered lung growth, and the TROP2 protein colocalized with Ki-67-positive cells in the fetal lung. TROP2 was predominantly localized to fibroblasts and type II alveolar epithelial cells. Trop2 small interfering RNA decreased Trop2 expression by ～75% in cultured fetal rat lung fibroblasts and decreased their proliferation by ～50%. Cell viability was not affected. This study demonstrates that TROP2 regulates lung cell proliferation during development.     

Effects of prenatal ethanol exposure on the lungs of postnatal lambs

Prenatal ethanol exposure increases collagen deposition and alters surfactant protein (SP) expression and immune status in lungs of near-term fetal sheep. Our objectives were to determine 1) whether these prenatal effects of repeated gestational ethanol exposure persist after birth and 2) whether surfactant phospholipid composition is altered following prenatal ethanol exposure. Pregnant ewes were chronically catheterized at 90 days of gestational age (DGA) and given a 1-h daily infusion of ethanol (0.75 g/kg, n = 9) or saline (n = 7) from 95 to 135 DGA; ethanol administration ceased after 135 DGA. Lambs were born naturally at full term (146 ± 0.5 DGA). Lung tissue was examined at 9 wk postnatal age for alterations in structure, SP expression, and inflammation; bronchoalveolar lavage fluid was examined for alterations in surfactant phospholipid composition. At 134 DGA, surfactant phospholipid concentration in amniotic fluid was significantly reduced (P < 0.05) by ethanol exposure, and the composition was altered. In postnatal lambs, there were no significant differences between treatment groups in birth weight, postnatal growth, blood gas parameters, and lung weight, volume, tissue fraction, mean linear intercept, collagen content, proinflammatory cytokine gene expression, and bronchoalveolar lavage fluid surfactant phospholipid composition. Although SP-A, SP-B, and SP-C mRNA levels were not significantly different between treatment groups, SP-D mRNA levels were significantly greater (P < 0.05) in ethanol-treated animals; as SP-D has immunomodulatory roles, innate immunity may be altered. The adverse effects of daily ethanol exposure during late gestation on the fetal lung do not persist to 2 mo after birth, indicating that the developing lung is capable of repair.     

VDUP1: a potential mediator of expansion-induced lung growth and epithelial cell differentiation in the ovine fetus

The degree of fetal lung expansion is a critical determinant of fetal lung growth and alveolar epithelial cell (AEC) differentiation, although the mechanisms involved are unknown. As VDUP1 (vitamin D3-upregulated protein 1) can modulate cell proliferation, can induce cell differentiation, and is highly expressed in the lung, we have investigated the effects of fetal lung expansion on VDUP1 expression and its relationship to expansion-induced fetal lung growth and AEC differentiation in fetal sheep. Alterations in fetal lung expansion caused profound changes in VDUP1 mRNA levels in lung tissue. Increased fetal lung expansion significantly reduced VDUP1 mRNA levels from 100+/-8% in control fetuses to 37+/-4, 46+/-4, and 45+/-9% of control values at 2, 4, and 10 days of increased fetal lung expansion, respectively. Reduced fetal lung expansion increased VDUP1 mRNA levels from 100+/-16% in control fetuses to 162+/-16% of control values after 7 days. VDUP1 was localized to airway epithelium in small bronchioles, AECs, and some mesenchymal cells. Its expression was inversely correlated with cell proliferation during normal lung development (R2=0.972, P<0.002) as well as in response to alterations in fetal lung expansion (R2=0.956, P<0.001) and was positively correlated with SP-B expression during normal lung development (R2=0.803, P<0.0001) and following altered lung expansion (R2=0.817, P<0.001). We suggest that VDUP1 may be an important mediator of expansion-induced lung cell proliferation and AEC differentiation in the developing lung.