Browsing by Subject "conceptus"
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Item Amino acids, polyamines, and nitric oxide synthesis in the ovine conceptus(Texas A&M University, 2005-08-29) Kwon, Hyuk JungThe objective of this study was to determine concentrations of amino acids and polyamines as well as nitric oxide (NO) and polyamine synthesis in the ovine conceptus (embryo/fetal and associated placental membrane). Ewes were hysterectomized on Days 30, 40, 60, 80, 100, 120, or 140 of gestation to obtain allantoic and amniotic fluids, intercotyledonary placenta, placentomes and uterine endometrium for the analyses. Alanine, citrulline plus glutamine accounted for about 80% of total α-amino acids in allantoic fluid during early gestation. Serine (16.5 mM) contributed about 60% of total α-amino acids in allantoic fluid on Day 140 of gestation. Maximal ornithine decarboxylase (ODC) and arginase activities and highest rates of polyamine and NO synthesis occured in all tissues on Day 40 of gestation. In ovine allantoic and amniotic fluids, polyamines were most abundant during early (Days 40-60) and late (Days 100-140) gestation, respectively. Activity of guanosine 5??-triphosphate-cyclohydrolase I (GTP-CH), and concentrations of NOS cofactors, tetrahydrobiopterin (BH4) and NADPH (nicotinamide adenine dinucleotide), peaked on Day 40 of gestation in placental and endometrial tissues. In these tissues, NO synthesis was positively correlated with total NOS activity, GTP-CH activity, and concentrations of BH4 and NADPH. The physiological significance of these changes was manifested by undernutrition-induced intrauterine growth retardation (IUGR). Maternal undernutrition (50% of National Research Council nutrient requirements) reduced concentrations of total α-amino acids in fetal plasma and fluids, and retarded fetal growth at both mid (Day 78) and late (Day 135) gestation. Concentrations of polyamines in fetal fluids were lower in underfed ewes than in control-fed ewes. Realimentation of underfed ewes between Days 78 and 135 of gestation increased concentrations of total α-amino acids and polyamines in fetal plasma and fluids, when compared with non-realimented ewes. Results of these studies demonstrate metabolic coordination among the several integrated pathways to enable high rates of polyamine and NO synthesis in the placenta and endometrium during early pregnancy. Collectively, our findings may have important implications for both IUGR and fetal origins of adult disease.Item Progesterone regulation of endometrial factors supporting conceptus growth and development in the ovine uterus(Texas A&M University, 2008-10-10) Satterfield, Michael CareyProgesterone is unequivocally required for the establishment and maintenance of pregnancy in all mammals studied. Its known functions are complex and encompass global changes in gene expression. Therefore, studies were conducted to characterize the effects of progesterone on expression of genes for endometrial factors having roles in conceptus growth, implantation and establishment of pregnancy. The first study characterized the effect of an artificially induced early increase in circulating progesterone on conceptus growth and development and regulation of expression of galectin-15 (LGALS15), a recently identified protein secreted by the ovine uterine luminal epithelium (LE). Exogenous progesterone beginning on Day 1.5 post-mating accelerated conceptus development on Days 9 and 12. On Day 12 the conceptus was functionally and morphologically advanced to produce greater quantities of interferon tau (IFNT) than blastocysts from control ewes. Further, the endometrium responded to early progesterone and IFNT with early expression of cathepsin L (CTSL), radical S-adenosyl methionine domain containing 2 (RSAD2), and LGALS15 within the endometrium. The second study identifed structural changes within the luminal epithelium which could alter the flux of factors into and out of the uterine lumen to maintain appropriate fetal/maternal communication. In this study, progesterone reduced quantities of proteins associated with both tight and adherens junctions during the elongation period. IFNT subsequently increased these proteins after conceptus elongation. The third and fourth studies identified progesterone-regulated genes which have been implicated as having importance to implantation in sheep, mouse, and human. WNT signaling was transiently downregulated by progesterone, while members of several growth factor families are upregulated including insulin-like growth factor binding proteins (IGFBPs) 1 and 3, hepatocyte growth factor (HGF) and fibroblast growth factor 7 (FGF7), which may enhance conceptus growth. Collectively, these studies assess the role of progesterone in altering gene uterine expression to establish a favorable environment for conceptus development. The long-term goals of these studies are to establish biomarkers of receptivity to conceptus development and implantation, enhance our understanding of gene and pathway regulation in early pregnancy loss, and identify genes which may be targeted in therapeutic strategies to improve reproductive success in humans and animals.Item Select Nutrients, Secreted Phosphoprotein 1 and Insulin-Like Growth Factor 2: Effects of Trophectoderm of Ovine Conceptuses(2011-08-08) Kim, Jin YoungHistotroph, 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.Item Transcriptional Regulation of Galectin 15 (LGALS15): An Implantation-Related Galectin Uniquely Expressed in the Uteri of Sheep and Goats(2010-10-12) Lewis, Shaye K.Galectins are a family of secreted animal lectins with a high affinity to betagalactosides commonly involved in cellular functions such as apoptosis, adhesion and migration. Galectin 15 (LGALS15), a newest member of the galectin superfamily, has a unique C-terminal RGD sequence and participates in integrin-mediated ovine trophectoderm cell attachment and migration. In the ovine uterus, LGALS15 is expressed only by the endometrial luminal (LE) and superficial glandular (sGE) epithelia, induced by progesterone between Days 10 and 12 of the cycle and pregnancy, and then stimulated by interferon tau (IFNT) from the conceptus after Day 14 of pregnancy. During early pregnancy, the canonical janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is not active in the endometrial LE/sGE. Therefore, IFNT may utilizes a non-canonical signaling pathway to increase transcription of genes, including CST3, CTSL, HIF2A, LGALS15, and WNT7A, specifically in the endometrial LE/sGE. Alternatively, IFNT and progesterone could indirectly affect epithelial gene expression by influencing gene expression in the stroma, which then communicates with the epithelium. Although the LGALS15 gene is present in ovine, caprine and bovine species, it is only expressed in uteri of sheep and goats. Available data shows a tissue- and speciesspecific expression pattern for LGALS15, likely involving multiple layers of transcription regulation in the ruminant endometrium. Further analysis of the LGALS15 5? promoter/enhancer region revealed similar predicted transcription factor binding sites in all three species, including; PU.1, Ets-1, AP1, Sp1, and GRE or PRE sites. Interestingly, the proximal promoter region of the LGALS15 gene in all three species exhibited a conserved Sp1 binding site upstream of an AP1 binding site on both sense and antisense strands, and with similar spacing between binding sites. Sequence analysis revealed key differences in LGALS15 gene structure between ruminant species including the proximity of repetitive DNA sequences to the transcription start site (+1). Bovine LGALS15 has repetitive DNA sequences start at - 145 whereas in ovine or caprine LGALS15 it starts at about -300. The length of the repetitive DNA sequence is similar (~1.2 kb) in the 5' promoter/enhancer region of LGALS15 in all three species. Transient transfection analyses found that repetitive DNA sequences reduced basal promoter activity and responsiveness to treatments. None of the promoter construct showed responsiveness to interferon tau (IFNT). The bovine LGALS15 gene promoter showed no activity under any experimental conditions. The current studies indicate that uterine LGALS15 is expressed in ovine and caprine but not bovine species. Additionally, repetitive DNA sequences found in the promoter region may contribute to modulating the LGALS15 gene expression. Therefore, the ruminant LGALS15 gene, like other galectins, is under tight transcriptional control involving hormones, requisite transcription factors and potentially chromatin remodeling complexes working synergistically for LGALS15 promoter transactivation.