Harney J P, Mirando M A, Smith L C, Bazer F W
Animal Science Department, University of Florida, Gainesville 32611-0691.
Biol Reprod. 1990 Mar;42(3):523-32. doi: 10.1095/biolreprod42.3.523.
Pig conceptuses and endometrial explants recovered from gilts between Days 10 and 15 of pregnancy were cultured in leucine-deficient or methionine-deficient medium supplemented with 3H-leucine or 35S-methionine, respectively, for 30 h. Conceptus and endometrial tissues from Day 15 of pregnancy were fixed in Bouin's fixative for immunocytochemistry and light microscopy. Conceptus culture medium from Day 15 of pregnancy was pooled, dialyzed, and fractionated by anion exchange and gel filtration chromatography. A family of 3-5 low molecular weight (Mr) acidic (Mr = 19,000-22,000; pI = 5.6-6.5) 3H-leu-labeled proteins were isolated and identified by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), electroblotting, and fluorography. The two major proteins (pCSP-1 and pCSP-2) were excised from a polyvinylidene difluoride transfer membrane, and NH2-terminal amino acids were sequenced. One peptide was sequenced through 33 amino acids and the second, which shared 100% homology, was sequenced through 22 amino acids. Analysis of the larger sequence indicated that it shared 93.9% and 90.9% homology with the first 33 amino acids of human and rabbit plasma retinol-binding protein (RBP), respectively. Analyses of culture medium from pig conceptus incubations by 2D-PAGE and immunoprecipitation with rabbit anti-human RBP serum indicated that immunoreactive RBP was produced between Days 10 and 15 of pregnancy and was present in Day 30 allantoic fluid. Western blotting of enriched fractions of Day 15 conceptus RBP followed by immunostaining indicated that five isoforms of radiolabeled RBP were present. Immunoreactive RBP was detected in trophectoderm and yolk sac of conceptuses and endometrial surface and glandular epithelium at Day 15 of pregnancy. Results from this study demonstrate that pig conceptuses secrete RBP prior to onset of conceptus elongation and throughout the peri-implantation period, which suggests that RBP and associated retinoids influence conceptus development.
从妊娠第10至15天的后备母猪中获取猪胚胎和子宫内膜外植体，分别在补充有3H-亮氨酸或35S-蛋氨酸的亮氨酸缺乏或蛋氨酸缺乏培养基中培养30小时。将妊娠第15天的胚胎和子宫内膜组织固定于Bouin固定液中用于免疫细胞化学和光学显微镜检查。将妊娠第15天的胚胎培养基合并、透析，并通过阴离子交换和凝胶过滤色谱法进行分级分离。通过二维聚丙烯酰胺凝胶电泳（2D-PAGE）、电印迹和荧光自显影法分离并鉴定了一组3至5种低分子量（Mr）酸性（Mr = 19,000 - 22,000；pI = 5.6 - 6.5）的3H-亮氨酸标记蛋白。从聚偏二氟乙烯转移膜上切下两种主要蛋白（pCSP-1和pCSP-2），并对其氨基末端氨基酸进行测序。一种肽段测序了33个氨基酸，另一种与之具有100%同源性的肽段测序了22个氨基酸。对较长序列的分析表明，它与人血浆视黄醇结合蛋白（RBP）和兔血浆视黄醇结合蛋白的前33个氨基酸分别具有93.9%和90.9%的同源性。通过2D-PAGE对猪胚胎培养物培养基进行分析，并使用兔抗人RBP血清进行免疫沉淀，结果表明在妊娠第10至15天产生了免疫反应性RBP，且在妊娠第30天的尿囊液中存在。对妊娠第15天胚胎RBP的富集级分进行蛋白质印迹，随后进行免疫染色，结果表明存在五种放射性标记RBP的异构体。在妊娠第15天，在胚胎的滋养外胚层和卵黄囊中以及子宫内膜表面和腺上皮中检测到免疫反应性RBP。本研究结果表明，猪胚胎在胚胎伸长开始之前以及整个着床前期都会分泌RBP，这表明RBP和相关类视黄醇会影响胚胎发育。
