Vascularization and Expression of Fibroblast Growth Factor (FGF) 2 and FGF Receptor (FGFR) 2 IIIc Protein in Endometrium During Early Pregnancy in Sheep. (2008)

Uterolacental growth and angiogenesis are critical for normal placental development and function and thus for normal embryonic/fetal growth and development. To determine if uteroplacental development is altered in compromised pregnancies, a detailed knowledge of the expression of growth factors that regulate vascular and nonvascular tissue growth and remodeling in normal pregnancies is required. Therefore, this study evaluated vascular development and localization/quantification of proteins for fibroblast growth factor (FGF) 2 and a major FGF receptor (FGFR) 2 IIIc within endometrium using immunohistochemistry and image analysis. Uterine tissues were collected on days 20, 22, 24, 26, and 30 after mating (n = 5 to 6 ewes/day) and on day 10 after estrus (n=5 ewes; nonpregnant controls). For immunohistochemistry, cross sections of uterine tissues containing endometrium and adjacent fetal membranes were fixed in Carnoys solution or 10% neutral buffered formalin, embedded in paraffin, sectioned (5-micrometers thick), and immunostained to detect factor VIII (a marker of endothelial cells and thus of vascularization), FGF2, and FGFR2 IIIc in caruncular (CAR) and intercaruncular (ICAR) areas. Factor VIII was exclusively detected in the blood vessels in CAR and ICAR. FGF2 and FGFR2 IIIc were immunolocalized in connective tissues and blood vessels of CAR and ICAR and in endometrial glands of ICAR throughout early pregnancy as well as in nonpregnant controls. Compared to nonpregnant controls, Factor VIII protein expression was similar on days 20 to 24 of pregnancy and then increased (P < 0.01) by ~3- to 4-fold on days 26 to 30. FGF2 protein expression on days 20 to 28 of pregnancy was similar to nonpregnant controls and then increased (P < 0.04) by 12-fold on day 30 of pregnancy. Expression of FGFR2 IIIc protein did not change throughout early pregnancy and was similar to that of nonpregnant controls. Across all of the days of early pregnancy examined, expression of FGF2 protein tended (P < 0.07 to P < 0.09) to be correlated with factor VIII expression (r = 0.345) and capillary area density (r = 0.327), and was correlated (P < 0.001 to P < 0.05) with vascular labeling index (r = 0.473), capillary number density (r = -¬0.348), and area per capillary (r = 0.473). Furthermore, expression of factor VIII was correlated (P < 0.001 to P < 0.05) with vascular labeling index (r = 0.569), capillary number density (r = -0.535), and area per capillary (r = 0.480) throughout early pregnancy. Thus, during early pregnancy vascularization of the endometrial tissues increases dramatically after day 24, and endometrial vascular development parallels the changes in expression of protein for FGF2 but not FGFR2 IIIc receptor. It therefore seems likely that FGF2 plays a role in regulating uteroplacental vascular and nonvascular growth during early pregnancy; these data will enable us to determine whether uteroplacental expression of this growth factor is altered in compromised pregnancies. Supported by: NIH grants P20 RR016741 (INBRE program, NCRR) to LPR, and HL64141 to LPR and DAR; USDA-NRI grant 2007-01215 to LPR, PL, GP, and ATG-B; ND EPSCoR AURA grant to ATG-B and MAM; and ND Space Grant Fellowship Program award to MAM.