Effects of mTORC2 on Ovine Conceptus Elongation and Implantation.

Specific components of uterine histotroph, including glucose, leucine, arginine, glutamine and secreted phophoprotein 1 (SPP1) activate the mammalian target of rapamycin (MTOR) nutrient sensing pathway. MTORC1 is associated with cell proliferation and mRNA translation, whereas MTORC2 is associated with cell migration, cytoskeletal organization, and cell survival. We demonstrated that arginine (Arg) activates MTORC1 signaling to stimulate proliferation and migration of ovine trophectoderm (oTr1) cells. Therefore, this study focused on interactions between Arg and SPP1 that stimulate MTORC2 to affect cytoskeletal organization in ovine conceptuses. In Experiment 1, ewes were hysterectomized on Days 13, 14, 15, and 16 of pregnancy and uterine tissue frozen in OCT for immunofluorescence (IF) staining and in 4% paraformaldeyde for immunohistochemistry (IHC). Rictor, a protein unique to the mTORC2 complex, was detected in endoderm and to a lesser extent trophectoderm of conceptuses using IHC and western blotting and rictor mRNA was detected by PCR. Using IF, cytokeratin and vimentin were detected in trophectoderm and uterine luminal, and stromal (ST) cells respectively. Alpha smooth muscle actin and desmin, markers of smooth muscle, were expressed in uterine ST cells by Day 15 of pregnancy indicating their transition to a myofibroblast phenotype. The myofibroblast transition was more evident in cauncular than intercaruncular ST cells. SPP1, αv integrin, α-parvin and integrin linked kinase (ILK), all components of focal adhesions at the interface between the chorion and uterine LE by mid-pregnancy, were examined by IF. SPP1 was abundant on both trophectoderm and uterine LE, whereas αv, α-parvin and ILK were detected in trophectoderm and uterine LE in low abundance. However, when oTr1 cells were cultured and immunostained for αv and α-parvin a punctuate signal was detected at the base of the cells characteristic of focal adhesion assembly. An intense fluorescence signal for phalloidin, a stain for F-actin, was observed in uterine LE and myometrium, but the signal was low in trophectoderm; however, oTr1 cells in culture had highly organized actin filaments. In Experiment 2, Alzet osmotic pumps were loaded with 2 ml of either 100 nM Rapamycin (MTOC1 inhibitor), 50 µM Torin1 (mTORC2 inhibitor), 50 µM Blebbistatin (myosin II inhibitor), or vehicle (5% DMSO) and delivered continuously into the uterine lumen from Days 7 to 15 of pregnancy when ewes were hysterectomized. Uteri were flushed to recover conceptuses and assess effects of treatment on conceptus morphology and secretion of interferon tau (IFNT). Vehicle infused ewes had 4 elongated and 1 developmentally retarded conceptus. Blebbistatin infused ewes had 3 elongated, but fragmented, and 2 developmentally retarded conceptuses. Rapamycin infused ewes had 4 elongated conceptuses and no retarded conceptuses. Torin1 infused ewes had 5 elongated, but very fragile conceptuses and 1 developmentally retarded conceptus. IFNT in uterine flushings was only less (P<0.05) in Blebbistatin-treated ewes compared to control ewes. These results indicated that components of MTORC2 are expressed in uterine and conceptus tissues, but inhibitors of MTORC1 and MTORC2 did not suppress elongation of conceptuses during the peri-implantation period of pregnancy in ewes. This project was supported by AFRI Grant 2010-03220 from the USDA National Institute of Food and Agriculture.

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