Select Nutrients, Secreted Phosphoprotein 1 and Insulin-Like Growth Factor 2: Effects of Trophectoderm of Ovine Conceptuses

Histotroph, secretions from luminal (LE), superficial glandular (sGE) and glandular (GE) epithelia and molecules selectively transported into the uterine lumen, are essential for peri-implantation ovine conceptus development and maternal recognition of pregnancy. Among them, several components of histotroph including nutrients, cell matrix proteins and growth factors may activate mTOR (mammalian target of rapamycin; also known as FRAP1) to stimulate hypertrophy, hyperplasia, and/or migration of conceptus trophectoderm cells, as well as expression of IFNT for pregnancy recognition and critical proteins for conceptus development. Therefore, studies were conducted to examine effects of select nutrients (arginine, leucine, glutamine and glucose), IGF2 and SPP1 on mTOR signal transduction pathways and determine their biological effects on proliferation, migration and/or attachment of ovine trophectoderm (oTr) cells and conceptuses (embryo and it extra-embryonic membranes). The first study defined the expression of IGF2, RPS6K, phosphorylated AKT, RPS6K, P38 and ERK1/2 MAPK by the uterus and conceptus during the periimplantation period. In addition, effects of IGF2 on the PI3K signaling pathway were evaluated using oTr cells isolated from Day 15 conceptuses. IGF2 was most abundant in compact stroma of endometrial caruncles and also present in all cells of the conceptus, but particularly abundant in the endoderm and yolk sac. Phosphorylated AKT1, RPS6K, P38 and ERK1/2 proteins were abundant in nuclei of endometrial LE and conceptus trophectoderm. IGF2 activated multiple cell signaling pathways including PDK/AKT/mTOR/RPS6K and MAPKs that are critical to survival, growth and migration of the ovine trophoblast cells. The second study demonstrated the multifunctional effects of secreted phosphoprotein 1 (SPP1) on oTr cells including cell signaling transduction, migration, and adhesion. Novel results of this study indicated that SPP1 binds ?v?3 and ?5?1 integrins to activate PI3K/mTOR/RPS6K, MAPK as well as crosstalk between mTOR and MAPK pathways that are essential for expansion and elongation of conceptuses and attachment of trophectoderm to uterine LE during implantation. The third study identified effects of arginine (Arg), leucine (Leu), glutamine (Gln) and glucose on oTr cells. Arg, Leu and glucose, but not Gln, activated PI3KAKT1 and mTOR-RPS6K-RPS6 signaling pathways. Arg, Leu and glucose increased abundance of p-RPS6K in nuclei and p-RPS6 in cytoplasm of oTr cells. In addition, results of this study demonstrated that Arg and Leu are remarkably stimulatory to cell proliferation and migration. The fourth study determined effects of Arg on signal transduction pathways and oTr cell proliferation, as well as inhibition of oTr cell proliferation by L-NAME (an inhibitor of NOS) or Nor-NOHA (an inhibitor of arginase) on oTr cells. Arg increased p-mTOR, RPS6K and 4EBP1 protein and also increased protein synthesis and reduced protein degradation in oTr cells. Both NO and polyamines enhanced cell proliferation in a dose-dependent manner. The effects of Arg were partially inhibited by both L-NAME and Nor-NOHA. These results indicate that Arg enhances production of polyamines and NO and activates the mTOR-FRAP1-RPS6K-RPS6 signaling pathway to stimulate proliferation of oTr. The fifth study identified differential effects of Arg, Leu, Gln and glucose on gene expression and protein translation in explants cultures of ovine conceptuses. Expression of mRNAs was not affected by treatments with the select nutrients; however, Arg, Leu, Gln and glucose increased abundance of total and phosphorylated forms of mTOR, RPS6K, 4E-BP1 and RPS6. Arg, Leu, Gln and glucose also increased the amounts of NOS and ODC1, but only Arg stimulated a significant increase in abundance of IFNT. Collectively, these studies indicated that IGF2, SPP1 and select nutrients activate mTOR cell-signaling pathways that converge on AKT1 and that are likely critical to mechanism(s) responsible for survival, elongation an development of conceptuses. A more complete understanding of this mechanism will be important to development of strategies to reduce early embryonic losses in ruminants and and in other species including humans.