Abstract
Melatonin plays a role as a vasodilator. Vasoactive and angiogenic factors are expressed by placental binucleate cells (BNC) and produce chorionic somatomammotropin (CSH), known to impact fetal and placental growth. We hypothesized that melatonin supplementation and restricted nutrition from mid- to late-gestation would alter CSH concentration and some characteristics of BNC in placenta. At day 50 of gestation, ewes carrying singletons were randomly assigned to a 2 × 2 factorial design and were fed either an adequate (ADQ; 100% NRC; n = 15) or restricted (RES; 60% NRC; n = 15) diet supplemented with 0 (CON, n = 14) or 5 mg of melatonin (MEL; n = 16). Placentomes were collected on day 130 of gestation and preserved in formalin for histological analysis. Cotyledon (COT) were snap frozen for western immunoblotting analyses. Tissue sections were stained using biotinylated Dolichos Biflurus (DBA; a marker of fetal membrane) lectin and fluorescein labeled Texas red-avidin and fluorescein labeled Griffonia Simplifolica (BS) lectin (a marker of BNC). The number, area, and diameter of BNC in COT were determined by image analysis. For immunoblotting, protein was extracted from COT in SDS phosphate buffer, loaded equally, and separated on 12.5% polyacrylamide gels. Protein was transferred to PVDF membranes and incubated with rabbit anti-CSH. Bands were visualized and imaged. Data were analyzed using Proc Mixed procedure of SAS. Melatonin supplementation and restricted nutrition did not affect BNC number, area, or diameter, or CSH protein expression. While we reject our hypothesis that melatonin supplementation and nutrient restriction would alter the CSH concentration and BNC characteristics in COT, we continue to evaluate if the BNC produce angiogenic or vasoactive factors that may influence placental and mammary gland functions in sheep.