Functional Roles of Histotroph (Arginine, Secreted Phosphoprotein 1, and Fructose) in Trophectoderm Development in Mammalian Conceptuses during the Peri-Implantation Period of Pregnancy

The greatest limitation to reproductive performance in most mammals, including humans, is embryonic mortality, which, in general, claims 20%-40% of the embryos during the peri-implantation period of pregnancy. During that stage of pregnancy, the dialogue between the mammalian conceptus (embryo/fetus and associated membranes) and maternal uterus involves signaling for pregnancy recognition and maintenance of pregnancy as the stage is set for implantation and placentation that precedes fetal development. Uterine epithelial cells secrete and/or transport a wide range of molecules, including nutrients, collectively referred to as histotroph, that are transported into the fetal-placental vascular system to support survival, growth and development of conceptuses during pregnancy in mammals. Therefore, studies were conducted to investigate functional roles of selected components in uterine histotroph and their relationships with mechanistic target of rapamycin (MTOR) cell signaling cascades in survival, growth and development of conceptuses during the peri-implantation period of pregnancy using sheep as the animal model for the research. The first three studies investigated the functional roles of polyamines, arginine and nitric oxide (NO), respectively, in ewes by in vivo translational knockdown of mRNA of ODC1 (the key enzyme for classical de novo biosynthesis of polyamines), SLC7A1 (the major arginine transporter), and NOS3 (the major isoform of enzyme for NO synthesis) in ovine conceptus trophectoderm (Tr) using morpholino antisense oligonucleotides (MAO). In the MAO-ODC1 ewes, only 50% of the conceptuses were morphologically and functionally abnormal. Further analyses revealed an alternative pathway for polyamine biosynthesis via arginine decarboxylase (ADC) and agmatinase (AGMAT) that rescued the other 50% of the MAO-ODC1 conceptuses from polyamine deficiency. In the MAO-SLC7A1 ewes, depriving the conceptus of arginine resulted in retarded development of conceptuses and a significant decrease in production of interferon tau (IFNT), the pregnancy recognition signal in ruminants. In MAO-NOS3 ewes, reduced NO production was sufficient to retard development of the elongating ovine conceptuses. However, those conceptuses produce normal amounts of IFNT. The fourth study revealed that arginine stimulates proliferation and IFNT production by ovine Tr (oTr1) cells via NO and polyamine-TSC2-MTOR signaling pathways, particularly the pathway involving polyamine biosynthesis. The fifth and sixth studies demonstrated that arginine and secreted phosphoprotein 1 (SPP1) act cooperatively to increase proliferation, migration and adhesion of oTr1 cells via activation of the PDK1-Akt-TSC2-MTORC1 signaling cascade and/or focal adhesion-MTORC2 mediated cytoskeleton reorganization. The seventh study demonstrated the biological functions of fructose that affect proliferation of oTr1 cells via O-GlcNAcylation-mediated phosphorylation for activation of the Akt-TSC2-MTOR signaling cascade. Collectively, results from the present studies indicated the functional roles of each of the selected components of histotroph, including polyamines, arginine, NO SPP1, and fructose that independently and cooperatively activate nutrient sensing cell signaling pathways, including MTOR, the master regulator of cell growth, for survival, growth and development of conceptuses, as well as for optimization of culture media for in vitro studies of conceptus development