75 PLACENTA PROTEIN PROFILE CHARACTERIZATION BY PLACENTOME SIZE AND GESTATIONAL AGE IN CATTLE

Abstract

The placenta plays a key role in normal conceptus development and in mediating effects of the maternal system on the fetus. Changes in the environment or in placental function may affect fetal developmental and lead to what is known as “developmental origins of health and disease.” Thus, the aim of this study was to analyse the protein profile of bovine placentomes of different sizes at distinct gestation ages to determine spatial and temporal differences or similarities in the protein pool of biological significance to the conceptus and the newborn. Samples of placentomes (n = 36) representing small, medium, and large sizes among placentomes at 60, 90, 120, and 160 days of gestation were collected at a local slaughterhouse and freeze-dried for protein analysis. For that, 5 mg of each tissue sample was used for protein extraction with 1% Triton X and sonication for 10 min at 4°C, with protein concentration per sample determined by the Bradford method. A total of 300 μg of protein was subjected to 2D-SDS PAGE electrophoresis (GE Healthcare^®), and gels were stained with Coomassie Blue G-250. Spots were analysed using the PDQuest, with data compared by ANOVA or the Kruskall-Wallis test (SAS). In silico protein identification was performed using ExPASy. On average, 74 proteins were detected in the gels, regardless of placentome sizes and gestational ages. No interactions were detected between placentome sizes and gestational periods. However, differences were observed in the mean number of proteins between small (n = 69), medium (n = 90), and large (n = 64) placentomes (P = 0.002), and between Days 60 (n = 113), 90 (n = 65), 120 (n = 44), and 160 (n = 74) of gestation (P = 0.001). One specific spot (32.45 kDa, 4.6 pI) represented 1.0, 10.8, 12.1, and 2.1% of the intensity of all valid spots for Day 60, 90, 120, and 160 placentomes, respectively, indicating a bell-like expression. For small placentomes, all the spots had similar intensities, whereas for medium placentomes, the 32.45 kDa (4.6 pI) spot represented 3.0% of the intensity of all valid spots. For large placentomes, the 32.45 kDa (4.6 pI) spot and a 46.19 kDa (5.4 pI) spot represented 5.8 and 5.0% of the total intensity for all spots, respectively. Six and 10 spots were differentially identified between placentome sizes and between gestational periods, respectively, from which male-enhanced antigen-1 proteins and angio-associated migratory cell protein were identified by in silico analysis, with the former only present on Day 90 placentomes, and the latter for 120 days of gestation. Further analyzes on the identities of such placental proteins and their profiles is underway, which will be crucial to the comprehension of placentome function and growth during pregnancy, and for the understanding of physiological mechanisms and processes associated with normal conceptus development and life ex utero.