Center for Animal Biotechnology and Genomics, Department of Animal Science, Texas A&M University, College Station, Texas 77843-2471, USA.
Biol Reprod. 2011 Jan;84(1):62-9. doi: 10.1095/biolreprod.110.085738. Epub 2010 Sep 15.
Histotroph is required for survival and development of ovine conceptuses (embryo and extraembryonic membranes). Results from our laboratory indicate that arginine (Arg), leucine (Leu), glutamine (Gln), and glucose increase in the uterine lumen between Days 10 and 15 of pregnancy, coincident with increases in expression of amino acid and glucose transporters by uterine epithelia as well as trophectoderm and yolk sac of conceptuses and elongation of the conceptus trophectoderm. Therefore, we hypothesized that Arg, Leu, Gln, and glucose have differential effects on hypertrophy, hyperplasia, and differentiated functions of trophectoderm cells that are critical to conceptus development. Primary ovine trophectoderm (oTr) cells isolated from Day 15 conceptuses were serum-starved for 24 h in a customized medium, deprived of select nutrients, and then treated with either Arg, Leu, Gln, or glucose. Western blot analyses of whole oTr cell extracts revealed that Arg, Leu, and glucose, but not Gln, increased phosphorylated AKT1 by 2.8-, 2.5-, and 1.8-fold, respectively, within 15 min, and the increase was maintained to 60 min. Arg, Leu, and glucose also stimulated increases in phosphorylated ribosomal protein S6K (pRPS6K) by 4.2-, 4.7-, and 2.3-fold, respectively, within 15 min, as well as increases in phosphorylated ribosomal protein S6 (pRPS6) between 0 and 30 min posttreatment, that were sustained to 60 min. When oTr cells were treated with Arg, pRPS6K protein increased in nuclei, but this was not observed in nuclei of oTr cells treated with Leu and glucose. Immunocytochemical analyses also revealed abundant amounts of pRPS6 protein in the cytoplasm of oTr cells treated with Arg, Leu, and glucose. Furthermore, Arg and Leu increased proliferation and migration of oTr cells. Collectively, these results indicate that Arg, Leu, and glucose, but not Gln, in histotroph coordinately activate AKT1-mechanistic target of rapamycin and RPS6K-RPS6 cell signaling pathways to stimulate hypertrophy, hyperplasia, and migration of oTr cells.
组织滋养层对于绵羊胚胎（胚胎和胚胎外膜）的存活和发育是必需的。我们实验室的研究结果表明，在妊娠第 10 天至 15 天期间，子宫腔中的精氨酸（Arg）、亮氨酸（Leu）、谷氨酰胺（Gln）和葡萄糖含量增加，同时子宫上皮以及胚胎滋养层和卵黄囊的氨基酸和葡萄糖转运蛋白的表达也增加，胚胎滋养层延长。因此，我们假设 Arg、Leu、Gln 和葡萄糖对滋养层细胞的肥大、增生和分化功能具有不同的影响，而这些功能对胚胎发育至关重要。从第 15 天的胚胎中分离出的原代绵羊滋养层（oTr）细胞在定制培养基中饥饿培养 24 小时，然后用 Arg、Leu、Gln 或葡萄糖处理。整个 oTr 细胞提取物的 Western blot 分析显示，Arg、Leu 和葡萄糖分别在 15 分钟内使磷酸化 AKT1 增加 2.8、2.5 和 1.8 倍，并且这种增加持续到 60 分钟。Arg、Leu 和葡萄糖还分别在 15 分钟内刺激磷酸化核糖体蛋白 S6K（pRPS6K）增加 4.2、4.7 和 2.3 倍，以及在 0 至 30 分钟的处理后磷酸化核糖体蛋白 S6（pRPS6）增加，这种增加持续到 60 分钟。当 oTr 细胞用 Arg 处理时，pRPS6K 蛋白在细胞核中增加，但在用 Leu 和葡萄糖处理的 oTr 细胞中未观察到这种情况。免疫细胞化学分析还显示，用 Arg、Leu 和葡萄糖处理的 oTr 细胞的细胞质中存在大量的 pRPS6 蛋白。此外，Arg 和 Leu 增加了 oTr 细胞的增殖和迁移。综上所述，这些结果表明，组织滋养层中的 Arg、Leu 和葡萄糖，但不是 Gln，可协调激活 AKT1-雷帕霉素靶蛋白和 RPS6K-RPS6 细胞信号通路，从而刺激 oTr 细胞的肥大、增生和迁移。
