149 EXPRESSION OF ANGIOGENIC FACTORS AND THEIR MAJOR RECEPTORS IN THE SHEEP PLACENTA THROUGHOUT THE EARLY PREGNANCY

Embryonic losses are high in mammals, with more than 30% of fertilized eggs not resulting in an offspring. The development of the placenta is critical for normal fetal growth and development as it provides for exchange of respiratory gases, nutrients, and wastes between the fetal and maternal systems. Placental vascular development determines the rate of placental blood flow, which is a primary determinant of placental function. Recent studies suggest that vascular endothelial growth factor (VEGF), its receptors (VEGFR), along with angiopoietins (Ang-1 and Ang-2) and their common receptor Tie-2, are major placental angiogenic factors, along with fibroblast growth factor-2 (FGF-2) and its receptor (FGFR). To evaluate the patterns of placental expression of these factors during early placental development, gravid uteri were obtained from ewes (n = 6 per day) on Days 12, 18, 24, 30, and 40 of gestation (day of mating = Day 0). At slaughter the uterine and embryonic tissues were weighed and representative samples of utero-placenta (CAR - caruncle, maternal placenta; ICAR - intracarunclar, endometrium; FM, fetal membranes) were snap frozen on dry ice and analyzed for relative mRNA levels by real-time RT-PCR (ABI Prism 7000, Sequence Detection System, Applied Biosystems, Monza, Italy) of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-1 (VEGFR-1), vascular endothelial growth factor receptor-2 (VEGFR-2), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), receptor for both angiopoietins (Tie-2), fibroblast growth factor-2 (FGF-2), and fibroblast growth factor receptor (FGFR). The data were analyzed by nonlinear procedures using proc reg of SAS (SAS Institute, Inc., Cary, NC, USA). In CAR, the data showed the exponential increase from Days 12 to 40 in mRNA expression for VEGFR-1 (P < 0.0004; 0.04398e0.08794�day), VEGFR-2 (P < 0.01; 0.119208e0.06537�day), Ang-1 (P < 0.005; 0.00488e0.10881�day), Ang-2 (P < 0.0001; 0.01591e0.07864�day), Tie-2 (P < 0.03; 0.00488e0.06852�day), and FGFR (P < 0.08; 0.24577e0.04721�day). In the CAR, we also observed an exponential decrease in mRNA concentration for VEGF (P < 0.05; 28.193e-1.0719�day). In ICAR, we observed an exponential increase in mRNA concentration for VEGF (P < 0.05; 1.11685e0.06865�day), VEGFR-1 (P < 0.07; 0.09853e0.0383�day), Ang-1 (P < 0.09; 0.009318e0.05711�day), and Ang-2 (P < 0.004; 0.012647e0.09973�day). For FM, no changes in mRNA levels were observed from Days 12 to 40, but levels of all mRNAs were similar to those in CAR and ICAR. Based on the patterns of mRNA expression, these data indicate that these angiogenic factors may play an important role in early placental angiogenesis in sheep. This work was supported by NIH grant HL64141 to LPR and DAR.