106 ANGIOGENIC FACTOR mRNA EXPRESSION IN FETAL MEMBRANES IN EARLY PREGNANT SHEEP

Abstract

Maternal and fetal placental development may be compromised by use of assisted reproductive techniques, including cloning, resulting in poor placental angiogenesis and subsequent high embryonic/fetal loss (Palmieri et al. 2007 Placenta 28, 577–584). Before changes in vascular development in placenta from compromised pregnancies can be understood, a detailed knowledge of regulation of angiogenesis in placental tissues from normal pregnancies is necessary. Therefore, this study determined the expression pattern of mRNA for several angiogenic factors and their receptors: vascular endothelial growth factor (VEGF) and receptor (R) 1 and 2; basic fibroblast growth factor (FGF2) and FGFRIIIc; angiopoietin (ANGPT) 1 and 2 and ANGPTR (Tie2); endothelial nitric oxide synthase (eNOS) and NO receptor GUCY1B3 in fetal membranes (FM; fetal placenta) collected on Days 16, 18, 20, 22, 24, 26, 28, and 30 after mating (n = 5 to 8/day). Fetal membranes were snap frozen for evaluation of gene expression using quantitative, real-time RT-PCR. VEGF mRNA was increased (P < 0.05) 2-fold on Days 28 and 30 compared with Days 16, 18, and 20, whereas VEGFR1 mRNA increased (P < 0.05) 25- to 50-fold on Days 28 and 30 compared with Day 16, and VEGFR2 mRNA was greatest (P < 0.05) on Day 22 compared with Days 16, 18, 28, and 30. FGF2 mRNA was 4-fold greater (P < 0.05) on Day 22 compared with Day 16; however, FGF2RIIIc was unchanged from Day 16 through 30. eNOS mRNA was greatest (P < 0.05) on Days 22 and 24 compared with Days 16 and 18, but GUCY1B3 mRNA was greatest (P < 0.05) on Day 18 compared with Days 20, 24, and 28. ANGPT1 mRNA increased (P < 0.05) 40-fold by Days 28 and 30 compared with Days 16 and 18. ANGPT2 mRNA was undetectable on Day 16, and increased (P < 0.05) 5-fold from Days 18 through 30. ANGPTR mRNA was greatest (P < 0.05) on Days 22 and 24 compared with Days 16 and 18. This description of expression of factors potentially regulating early placental angiogenesis during normal pregnancy in sheep will provide the foundation for understanding the dramatic increases in capillary cell proliferation and capillary size we have previously observed (unpublished) and for determining whether placental vascular development is altered in compromised pregnancies.

USDA-NRI grant 2007-01215 to LPR and ATGB; NIH grant HL64141 to LPR and DAR; ND EPSCoR AURA grant to ATGB and MAM; ND Space Grant Fellowship Program award to MAM; and NIH grant P20 RR016741 (INBRE program of the NCRR, NIH).