Endothelial (typeIII) nitric oxide synthase (eNOS) contributes to the maintenance of low vascular resistance in the fetal-placental circulation, and has been localized to the placental vasculature and trophoblastic tissue. Little is known about eNOS expression in early placental development with regard to normal and growth restricted pregnancies. Exposure of the ovine pregnancy to heat stress (HS) beginning early in gestation results in a marked reduction of fetal and placental growth near term. Our objective was to evaluate cotyledon eNOS protein expression in this ovine model of placental insufficiency. Beginning at 35 days gestation (term=147 days), 4 ewes were exposed to heat stress (HS) in an environmental chamber, and 4 control ewes were housed in ambient conditions. Sheep were euthanized at 85-93 days gestation. Fetal and placentome weights were recorded. The placentomes were separated into caruncle and cotyledon components. Western blot analysis was performed with a monoclonal antibody against eNOS on crude homogenates of a whole cotyledon from each animal (25μg of total protein). Immunohistochemistry (IHC) for eNOS was performed on paraffin sections. Histology on H&E stained sections was used to assess vessel density (point counts) and vessel diameter (2 × vessel wall thickness/vessel diameter). HS fetuses weighed less than the control fetuses (457.3±49.2gm vs. 631.7±21.8gm; p<0.05). There were no differences between HS and control groups for gestational age (89±1.6 days vs. 90±2.0 days; p=0.8) nor for placentome weights (554.9±122gm vs. 288.8±61.1gm; p=0.09). HS cotyledons demonstrated a 2-fold decrease in eNOS protein content compared to controls (p<0.03). IHC localized eNOS to the vascular endothelium. Histologic evaluation of the HS cotyledons revealed a pattern of decreased fetal to maternal stromal tissue which is suggestive of accelerated placental maturation. Vessel diameter and number of fetal surface vessels were similar between groups (p=0.8). We conclude that HS exposure early in the ovine pregnancy results in a reduction of fetal growth and eNOS protein expression in the cotyledon. We speculate that developmental abnormalities in the fetoplacental vasulature results in reduced eNOS production.
