Functional Roles of Arginine During the Peri-Implantation Period of Pregnancy. III. Arginine Stimulates Proliferation and Interferon Tau Production by Ovine Trophectoderm Cells via Nitric Oxide and Polyamine-TSC2-MTOR Signaling Pathways

In mammal species, arginine is a multifunctional amino acid required for survival, growth, and development of conceptuses (embryo/fetus and associated extraembryonic membranes) during the peri-implantation period of pregnancy. However, functional roles of arginine with respect to it being a substrate for production of nitric oxide (NO) and polyamines on trophectoderm cell proliferation and function remain largely unknown. To systematically assess roles of arginine in conceptus development and its effect on interferon tau (IFNT) production for pregnancy recognition signaling in ruminants, an established ovine trophectoderm (oTr1) cell line isolated from Day-15 ovine conceptuses were used to determine their response to arginine, putrescine, and NO donors, as well as their associated inhibitors. Arginine at physiological concentration (0.2 mM) stimulated maximum oTr cell proliferation (increased 2.0-fold at 48 h and 2.6-fold at 96 h; P < 0.05), stimulated IFNT production (IFNT/cell increased 3.1-fold; P < 0.05), and increased total protein per cell by more than 1.5-fold (P < 0.05). It also increased phosphorylated tuberous sclerosis protein (p-TSC2) and phosphorylated mechanistic target of rapamycin (MTOR) abundance by more than 2.7- and 4.3-fold (P < 0.0001) after long-term incubation, respectively. When N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME; NO synthase inhibitor), DL-alpha-difluoromethylornithine hydrochloride hydrate (DFMO; ornithine decarboxylase inhibitor), and the combination (L-NAME + DFMO) were added, the effects of arginine on cell proliferation was reduced by 10.7%, 16.1%, and 22.3% (P < 0.05) at 48 h, and 15.3%, 27.2%, and 39.1% (P < 0.05) at 96 h of incubation, respectively, but values remained 1.5-fold higher (P < 0.05) than for the arginine-free control, which suggests that arginine, per se, serves as a growth factor. Both putrescine and NO stimulate cell proliferation via activation of the TSC2-MTOR signaling cascade, whereas only putrescine increased IFNT production. Collectively, our results indicate that arginine is essential for oTr1 cell proliferation and IFNT production via the NO/polyamine-TSC2-MTOR signaling pathways, particularly the pathway involving polyamine biosynthesis.