Maternal contributions from the uterine endometrial luminal (LE) and glandular (GE) epithelia are unequivocally required to support ruminant conceptus growth and development, elongation and implantation. Therefore, studies were conducted to examine expression of endometrial genes hypothesized to regulate conceptus development. The first study investigated two genes specifically expressed in the LE and superficial GE of the ovine uterus. Insulin-like growth factor binding protein (IGFBP1) and (IGFBP3) expression was coordinate with ovine conceptus elongation. Treatment with P4 induced and IFNT stimulated IGFBP1, but not IGFBP3; however, IFNT only moderately stimulated IGFBP1, indicating that another conceptus-derived factor stimulates endometrial IGFBP1 expression. IGFBP1 did not affect proliferation of ovine trophectoderm (oTr) cells in vitro, but stimulated their migration and attachment. Results indicated that IGFBP1, but not IGFBP3 is a marker of conceptus elongation in ruminants and stimulates cell migration and attachment. The second study evaluated the effects of pregnancy, P4 and IFNT on expression of hydroxysteroid (11-beta) dehydrogenases (HSD11B1 and HSD11B2), nuclear receptor subfamily 3, group C, member 1 (NR3C1), and prostaglandin-endoperoxide synthase 2 (PTGS2) in the ovine uterus. Expression of HSD11B1 mRNA and PTGS2 protein in endometrial LE and sGE were coordinate with conceptus elongation, while HSD11B2 mRNA was expressed primarily in the conceptus. Further, P4 induced, but IFNT only moderately stimulated HSD11B1. Thus, HSD11B1 expression may be regulated by prostaglandins (PGs) during early pregnancy. The presence of NR3C1 in the ovine uterus implicates cortisol, the main product of HSD11B1, in peri-implantation period events that include elongation of the ovine conceptus. The third study determined in vivo effects of PGs on ovine conceptus elongation and endometrial gene expression. Compared to control ewes, intrauterine infusions of a PTGS2 inhibitor, meloxicam, retarded elongation and decreased expression of elongation-related genes including IGFBP1, IGFBP3, HSD11B1, galectin 15 (LGALS15), solute carrier family 2, member 1 (SLC2A1), gastrin-releasing peptide (GRP), cystatin C (CST3), radical S-adenosyl methionine domain containing 2 (RSAD2), and ISG15 ubiquitin-like modifer (ISG15). Collectively, these studies assessed the effects of pregnancy, P4, IFNT, and PGs on endometrial genes implicated in conceptus growth. These results indicate that IGFBP1 is a marker of conceptus elongation in ruminants and provide novel roles for both cortisol and PGs in endometrial gene expression and conceptus elongation.

This research was conducted to test the hypothesis that select nutrients including glucose, leucine, arginine and glutamine stimulate conceptus development by activating mTOR (mammalian target of rapamycin; HGNC approved gene name: FRAP1, FK506 binding protein 12-rapamycin associated protein 1) signaling pathway. First, temporal changes in quantities of select nutrients (glucose, amino acids, glutathione, calcium, sodium and potassium) in uterine lumenal fluid from cyclic (Days 3 to 16) and pregnant (Days 10 to 16) ewes were determined. Total recoverable glucose, Arg, Gln, Leu, Asp, Glu, Asn, His, beta-Ala, Tyr, Trp, Met, Val, Phe, Ile, Lys, Cys, Pro, glutathione, calcium and sodium was greater in uterine fluid of pregnant compared to cyclic ewes between Days 10 and 16 after onset of estrus. Of note were remarkable increases in glucose, Arg, Leu and Gln in uterine flushings of pregnant ewes between Days 10 and 16 of pregnancy. Second, effects of the estrous cycle, pregnancy, progesterone (P4) and interferon tau (IFNT) on expression of both facilitative (SLC2A1, SLC2A3 and SLC2A4) and sodium-dependent (SLC5A1 and SLC5A11) glucose transporters, cationic amino acid transporters (SLC7A1, SLC7A2 and SLC7A3), neutral amino acid transporters (SLC1A4, SLC1A5, SLC3A1, SLC6A14, SLC6A19, SLC7A5, SLC7A6, SLC7A8, SLC38A3, SLC38A6 and SLC43A2) and acidic amino acid transporters (SLC1A1, SLC1A2 and SLC1A3) in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy as well as in conceptuses from Days 13 to 18 of pregnancy were determined. Among these genes, SLC2A3 and SLC7A6 were detectable only in trophectoderm and endoderm of conceptuses. The abundance of mRNAs for SLC2A1, SLC2A4, SLC5A1, SLC5A11, SLC7A1, SLC7A2, SLC1A4, SLC1A5, SLC43A2 and SLC1A3 changed dynamically in ovine uterine endometria according to day of the estrous cycle and early pregnancy. Expression of mRNAs for SLC2A1, SLC5A11 and SLC7A1 in endometria was induced by P4 and further stimulated by IFNT with shortterm treatment (12 days), while expression of SLC7A1 and SLC1A5 in endometria required long-term treatment (20 days) with P4 and IFNT. Third, effects of the estrous cycle, pregnancy, P4 and IFNT on expression of nitric oxide synthase (NOS1, NOS2 and NOS3), GTP cyclohydrolase (GCH1), ornithine decarboxylase 1(ODC1), insulin-like growth factor II (IGF2), FRAP1 complexes (FRAP1, LST8, MAPKAP1, RAPTOR, RICTOR), regulators (TSC1, TSC2, RHEB) and an effector (EIF4EBP1) of FRAP1 signaling in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy as well as in conceptuses from Days 13 to 18 of pregnancy were determined. All of these genes were expressed in ovine uterine endometrium and conceptuses. Among these genes, expression of NOS1, IGF2, RHEB and EIF4EBP1 changed dynamically due to day of the estrous cycle and early pregnancy. Progesterone stimulated NOS1 and GCH1 expression while IFNT inhibited NOS1 expression in uterine endometria, and P4 and IFNT stimulated expression of RHEB and EIF4EBP1 in uterine endometria. Collectively, these results indicate that: 1) the availability of select nutrients in the ovine uterine lumen increases to support the rapid growth and elongation of the conceptus during the peri-implantation stage of pregnancy; 2) P4 and/or IFNT stimulate(s) glucose and amino acid transporters to facilitate their transport from maternal tissues and/or blood into the uterine lumen during early pregnancy; 3) the FRAP1 cell signaling pathway mediates interactions between the maternal uterus and peri-implantation conceptus and both P4 and IFNT affect this pathway by regulating expression of RHEB and EIF4EBP1. Expression of NOS, ODC1 and IGF2 appear to be linked to FRAP1 signaling in both uteri and peri-implantation conceptuses.