Progress 09/01/06 to 08/31/10
Outputs
OUTPUTS: L-arginine (Arg) in the uterine lumen of ewes increases significantly between Days 10 and 15 of pregnancy. It is an essential substrate for synthesis of nitric oxide (NO), by nitric oxide synthase (NOS), and of polyamines, by ornithine decarboxylase (ODC1), via arginase required for growth and development of conceptuses. Study 1 used an established ovine trophectoderm cell line (oTr cells) to examine effects of Arg metabolism of both NO and polyamines to affect cell proliferation. These effects were blocked in the presence of inhibitors for NOS and arginase. Results indicated that Arg enhanced cell proliferation via NOS and NO, whereas effects on oTr cell proliferation mediated via arginase, ODC1 and polyamines were greater. Study 2 examined effects of Arg, Leucine (Leu), Glutamine (Gln) and glucose on gene expression in explant cultures of Day 16 ovine conceptuses. The mRNAs and proteins for MTOR (mammalian target of rapamycin), RPS6 (ribosomal protein S6), RPS6K (RPS6 kinase), and eukaryotic translation initiation factor 4E-binding protein 1 (EIF4EBP1), as well as NOS2, NOS3, ODC1, GTP cyclohydrolase I (GCH1) and interferon tau (IFNT) were analyzed using RT-PCR and immunoblotting. MTOR, RPS6K, EIF4EBP1, RPS6, ODC1, NOS2, NOS3 and IFNT mRNAs were not affected by any select nutrient, but GCH1 mRNA increased (P<0.05) in response to Arg. In contrast, all select nutrients increased (P<0.05) translation of mRNAs for MTOR, RPS6, RPS6K, and EIF4EBP1 proteins. Arg and glucose increased (P<0.01) total and phosphorylated forms of ODC1, NOS2 and GCH1, but Leu and Gln failed to increase ODC1 and NOS proteins, but only Arg increased (P<0.01) IFNT protein. These results indicate that Arg, Leu, Gln and glucose differentially increase the abundance of NOS2, NOS3, ODC1, GCH1 and IFNT proteins in ovine conceptuses. The increase in GCH1 mRNA and protein is significant as GCH1 is a rate-limiting enzyme for synthesis of tetrahydrobiopterin, an essential cofactor for all isoforms of NOS. The results of these studies provide insight into mechanisms whereby select nutrients act to increase transcription and/or translation of mRNAs for molecules that affect conceptus growth and development, and pregnancy recognition signaling during the peri-implantation period of pregnancy. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The information reported here is directed toward the academic community to advance understanding of the role of select nutrients transported into the uterine lumen to stimulate growth, development and survival of the conceptus (embryo and its associated membranes). The results are directly applicable to the sheep industry, but also likely relevant to other livestock industries such as cattle and swine. There is emerging evidence that dietary supplementation of diets with select nutrients, particularly arginine, enhance growth and development of the fetal-placental tissues and improves litter size and pregnancy rates in swine, sheep and rats. The development of rumen-protected select nutrients such as arginine is needed so that the effects of this dietary supplements with this amino acid can be determined with respect to reproductive efficiency in dairy cattle and beef cattle, as well as sheep and goats. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results of this long-term study indicate that arginine, leucine, glutamine and glucose are selectively transported into the uterine lumen of ewes during early pregnancy to support growth and development of the conceptus (embryo and its associated membranes). This due to effects of progesterone, the hormone of pregnancy, and interferon tau, the pregnancy recognition signal, acting in concert to stimulate expression of transporters that move these nutrients into the uterine lumen. The nutrients act through a nutrient sensing pathway called mammalian target of rapamycin to stimulate proliferation, migration and translation of specific mRNAs for generating nitric oxide and polyamines that not only stimulate conceptus growth and development, but also stimulate growth of blood vessels and enhance secretion of interferon tau. Given this knowledge, translational studies are being conducted to determine effects of dietary supplements that increase availability of select nutrients, particularly arginine, for transport into the uterine lumen to stimulate development and survival of conceptuses, as well as growth of blood vessels in the uterus and placenta to increase growth and development of the fetus. This will increase reproductive efficiencies and profitability of sheep enterprises and likely that of other livestock enterprises in animal agriculture.
Publications
- Gao H, Wu G, Spencer TE, Johnson GA, Bazer FW. Select nutrients in the ovine uterine lumen: III Cationic amino acid transporters in the ovine uterus and peri-implantation conceptuses. Biol Reprod 2009; 80:602-609
- Gao H, Wu G, Spencer TE, Johnson GA, Bazer FW. Select Nutrients in the Ovine Uterine Lumen: IV Expression of Neutral and Acidic Amino Acid Transporters in Ovine Uteri and Periimplantation Conceptuses. Biol Reprod 2009;80: 1196-1208
- Gao H, Wu G, Spencer TE, Johnson GA, Bazer FW. Select Nutrients in the Ovine Uterine Lumen: V Nitric Oxide Synthase, GTP Cyclohydrolase and Ornithine Decarboxylase in Ovine Uteri and Periimplantation Conceptuses. Biol Reprod 2009; 81:67-76
- Gao H, Wu G, Spencer TE, Johnson GA, Bazer FW. Select Nutrients in the Ovine Uterine Lumen: VI. Expression of FK506-Binding Protein 12-Rapamycin Complex-Associated Protein 1 (FRAP1) and regulators and effectors of mTORC1 and mTORC2 complexes in ovine uteri and conceptuses. Biol Reprod 2009; 81:87-100
- Bazer FW, Spencer TE, Johnson GA, Burghardt RC, Wu G. Comparative aspects of implantation. Reproduction 2009;138:195-209
- Bazer FW, Wu G, Spencer TE, Johnson GA, Burghardt RC, Bayless K. Novel Pathways for Implantation and Establishment and Maintenance of Pregnancy in Mammals. Mol Hum Reprod 2010;16:135-152
- Satterfield MC, Gao H, Li X, Wu G, Johnson GA, Spencer TE, Bazer FW. Select nutrients and their associated transporters are increased in the ovine uterus following early progesterone administration. Biol Reprod. 2010;82:224 231
- Kim J, Erikson DW, Burghardt RC, Spencer TE, Wu G, Bayless KJ, Johnson GA, Bazer FW. Secreted phosphoprotein 1 binds integrins to initiate multiple cell signaling pathways, including FRAP1/mTOR, to support attachment and force-generated migration of trophectoderm cells. Matrix Biology 2010;29:369-382
- Kim J, Burghardt RC, Wu G, Johnson GA, Spencer TE, Bazer FW. Select Nutrients in the ovine uterine lumen: VII Effects of arginine, leucine, glutamine and glucose on trophectodem cell signaling, proliferation and migration. Biol Reprod 2010;(In Press)
- Kim J, Burghardt RC, Wu G, Johnson GA, Spencer TE, Bazer FW. Select Nutrients in the ovine uterine lumen: VIII Arginine stimulates proliferation of ovine trophectoderm cells through mTOR RPS6K RPS6 signaling cascade and synthesis of nitric oxide and polyamines. Biol Reprod 2010; (In Press)
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Arginine (Arg), an essential nutrient for fetal-placental and neonatal growth and development via its role in nitric oxide signaling and polyamine synthesis, increases in the uterine lumen of pregnant, but not cyclic ewes between Days 10 and 16 post-estrus/mating. Thus, mechanisms for transport of Arg into the uterine lumen and uptake by conceptuses were studied and results revealed expression of system y+ members SLC7A1, SLC7A2 and SLC7A3 in uterine luminal (LE) and superficial glandular (sGE) epithelia and stromal Cells. Expression of SLC7A1 and SLC7A2 mRNAs was greater in pregnant than cyclic ewes (day x status, P<0.01) and both increased (P<0.01) between Days 10 and 20 of pregnancy. SLC7A1, SLC7A2, and SLC7A3 mRNAs were also detected in trophectoderm and endoderm of conceptuses from Days 13 to 20 of pregnancy. It was also determined that SLC7A2 expression in endometrial LE/sGE was induced by progesterone (P4) and stimulated by interferon tau (IFNT) and that increases in endometrial SLC7A1 and SLC7A2 expression were coordinate with increases in concentrations of Arg in the uterine lumen. The next study tested the hypothesis that arginine (Arg), leucine (Leu), glutamine (Gln) and glucose have differential effects on hypertrophy, hyperplasia and differentiated functions critical to ovine conceptus development by determining if each of these nutrients could activate the phosphatidylinositol 3-kinase (PI3K)/proto-oncogenic protein kinase 1 (AKT1) and/or FK506 binding protein 12-rapamycin associated protein 1 (FRAP1; also known as mammalian target of rapamycin or mTOR)/ribosomal protein S6 kinase (RPS6K) signaling pathway. In Study One, it was determined that Arg, Leu and glucose significantly (P<0.05) increased: 1) phosphorylated AKT1 (p-AKT1) within 15 min; 2) phosphorylated-glycogen synthase kinase 3 beta (p-GSK3B) within 15 min; and 3) p-FRAP1 and p-RPS6K increase between 0 and 15 min and remained elevated to 60 min. In Study Two, Day 16 conceptuses were dissected in to pieces and about 100 mg of each conceptus was cultured with Arg (0.2 mM), Leu (0.2 mM), Gln (0.4 mM) or glucose (2 mM) in 2 ml of customized medium. Western blot analyses of whole conceptus extracts with antibody to p-RPS6K revealed that Arg, Leu, Gln and glucose increased (P<0.05) p-RPS6K. These results indicate that Arg, Leu, Gln and glucose coordinately activate PI3K-AKT1 and FRAP1-RPS6K signaling pathways in ovine trophectoderm cells and conceptuses, and support the hypothesis that components of histotroph activate these cell signaling pathways to stimulate hypertrophy, hyperplasia, and/or migration of conceptus trophectoderm. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The information reported is directed to the academic community to stimulate research on effects of specific nutrients that affect development and survival of conceptuses in sheep and perhaps other species. It is also expected that this information is of value to livestock producers and to industry for formulation of rations to enhance reproductive efficiencies in livestock. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Results obtained in 2008 indicate that arginine, glutamine, leucine and glucose, which only increase in the uterine lumen of pregnant ewes, stimulate the nutrient cell sensing pathway that can stimulate proliferation, migration and differentiated functions of trophectoderm cells. Further, transporters for arginine expressed by uterine epithelia and conceptus trophectoderm were identified and on, SLC7A2, was shown to be induced by progesterone and stimulated by interferon tau. The results suggest that supplementation of diets of ruminants with select nutrients may enhance embryonic development and survival.
Publications
- Bazer, F.W., Burghardt, R.C., Johnson, G.A., Spencer, T.E. and Wu, G. 2008. Interferons and progesterone for establishment and maintenance of pregnancy: Interactions among novel cell signaling pathways. Reprod. Biol. 8:179-211.
- Kim, J., Song, G., Gao, H., Farmer, J.L., Satterfield, M.C., Burghardt, R.C., Wu, G., Johnson, G.A., Spencer, T.E. and Bazer, F.W. 2008. Insulin-like growth factor 2 (IGF2) activates PI3K-AKT1 and MAPK cell signaling pathways and stimulates proliferation and migration of ovine trophectoderm cells. Endocrinology 149:3085-3094.
- Gao, H., Wu, G., Spencer, T.E., Johnson, G.A., Li, X. and Bazer, F.W. 2009. Select nutrients in the ovine uterine lumen: I. Amino acids, glucose and ions in uterine lumenal fluid of cyclic and pregnant ewes. Biol. Reprod. 80:86-93.
- Gao, H., Wu, G., Spencer, T.E., Johnson, G.A. and Bazer, F.W. 2009. Select Nutrients In The Ovine Uterine Lumen: II. Glucose Transporters in the Uterus and Peri-Implantation Conceptuses. Biol. Reprod. 80:94-104.
- Gao, H., Wu, G., Spencer, T.E., Johnson, G.A. and Bazer, F.W. 2009. Select Nutrients In The Ovine Uterine Lumen: II. Glucose Transporters in the Uterus and Peri-Implantation Conceptuses. Biol. Reprod. 80:94-104.
- Gao, H., Wu, G., Spencer, T.E., Johnson, G.A. and Bazer, F.W. 2009. Select nutrients in the ovine uterine lumen: III Cationic amino acid transporters in the ovine uterus and peri-implantation conceptuses. Biol. Reprod. 80:602-609.
- Gao, H., Wu, G., Spencer, T.E., Johnson, G.A. and Bazer, F.W. 2009. Select Nutrients in the Ovine Uterine Lumen: IV. Expression of Neutral and Acidic Amino Acid Transporters in Ovine Uteri and Periimplantation Conceptuses. Biol. Reprod. 80 1196-1208.
- Gao, H., Wu, G., Spencer, T.E., Johnson, G.A. and Bazer, F.W. 2009. Select Nutrients in the Ovine Uterine Lumen: V. Nitric Oxide Synthase, GTP Cyclohydrolase and Ornithine Decarboxylase in Ovine Uteri and Peri-Implantation Conceptuses. Biol. Reprod. 81:67-76.
- Gao, H., Wu, G., Spencer, T.E., Johnson, G.A. and Bazer, F.W. 2009. Select Nutrients in the Ovine Uterine Lumen: VI. Expression of FK506-Binding Protein 12-Rapamycin Complex-Associated Protein 1 (FRAP1) and regulators and effectors of mTORC1 and mTORC2 complexes in ovine uteri and conceptuses. Biol. Reprod. 81:87-100.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: Conceptus development in ruminants requires components of uterine histotroph which include glucose and selected amino acids, including arginine, which can activate the FRAP1 nutrient sensing cell signaling pathway to stimulate migration, growth, differentiation and gene expression by trophectoderm. Analyses of uterine flushings from cyclic and pregnant ewes on Days 10, 12, 13, 14, 15 and 16 indicated a day by pregnancy interaction (P<0.01) due to increases (P<0.01) in recoverable glucose, glutamine and arginine between Days 13 and 16 and recoverable leucine between Days 12 and 16 of pregnancy. Increases in recoverable glucose in uterine flushings occurred coincidentally with increases in expression of facilitated glucose transporter-1 (SLC2A1) in uterine luminal epithelium which was induced by progesterone (P4) and further stimulated by interferon tau (IFNT), while SLC2A1 and SLC2A3 were expressed by conceptuses2. Expression of CAT2, the cationic amino acid transporter 2 (CAT2) for arginine, in uterine LE and stroma of cyclic ewes was weak, but it was abundant in luminal and superficial glandular epithelia between Days 14 and 20 of pregnancy, and its expression was induced by P4 and stimulated by IFNT. Because early treatment of ewes with P4 (versus corn oil, CO) from Days 1.5 to 9 of pregnancy advances conceptus development, as well as recoverable arginine (5 vs 13 μg; CO vs P4) and glucose (48 vs 76 μg; CO vs P4) in uterine flushings, and expression of SLC2A1 on Day 9 of pregnancy. In vitro experiments with ovine trophectoderm cells revealed that 2 mM arginine stimulates phosphatidylinositol 3-kinase (PI3K) cell signaling through the PI3K p85 alpha regulatory subunit gene (PIK3R1) to increase 3-phosphoinositide-dependent protein kinase 1 (PDPK1), V-akt murine thymoma viral oncogene homolog 1 (AKT1), cAMP response element-binding protein 1 (CREB1), glycogen synthase kinase 3-beta (GSK3B), rapamycin complex-associated protein 1 (FRAP1), eukaryotic translation initiation factor 4e-binding protein 1 (EIF4EBP1) and ribosomal protein S6 kinase (RPS6KB1). Insulin-line growth factor 2 (IFG2) was determined to be abundant in compact stroma of endometrial caruncles of cyclic and pregnant ewes and to transition from the stroma to the luminal epithelium in intercaruncular endomtria between Days 15 and 20 of pregnancy. IGF2 mRNA was also present in all cells of the conceptus,but particularly abundant in endoderm. Effects of IGF2 on the PI3K/FRAP1signaling pathway in mononuclear ovine trophectoderm cells (oTr) revealed that IGF2: 1) increased pPDK1 and pAKT1 by 1.5- and 3-fold, respectively, within 15 min which was maintained for up to 90 min: 2) stimulated a 1.4-fold increase in pGSK3B within 15 min; and 3) increased pFRAP1 and pRPS6K protein by 15 min that was sustained to 90 min. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Collectively, results obtained in 2007 indicate that both IGF2 and arginine can activate cell signaling leading to activation (phosphorylation) of FRAP1 and RPS6K in oTr cells which support the hypothesis that IGF2 is a critical regulator of trophoblast survival, growth and differentiation during early pregnancy. These findings may lead to management practices wherein supplementation of nutrients with arginine may enhance embryonic development and survival to improve reproductive efficiencies in ruminants.
Publications
- No publications reported this period
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Progress 09/01/06 to 08/31/07
Outputs
Mammalian target of rapamycin (mTOR/FRAP1) is a serine/threonine protein kinase that stimulates proliferation, migration and differentiated cell functions and acts as a nutrient sensor responsive to glucose and amino acids. FRAP1 affects growth and proliferation of mouse embryos as FRAP1 null mice experience post-implantation embryonic death. Little is known of uterine factors that stimulate FRAP1 expression. This study tested the hypothesis that nutrients present in ovine uterine secretions stimulate FRAP1 in conceptuses. First, conceptuses from ewes on Days 13, 15, 18 and 20 of gestation (n=3 conceptus/day) were analyzed for total FRAP1 using immunohistochemistry. FRAP1 protein was detected in both trophectoderm and extraembryonic endoderm of the conceptuses and in uterine glandular epithelium. Second, uterine flushings from cyclic (Days 3, 6, 10, 12, 13, 14, 15 and 16) and pregnant (Days 10, 12, 13, 14, 15 and 16) ewes (n=3-5/ewes/day) were analyzed for glucose
(fluorimetric method), amino acids (HPLC), and ions (Ca++, Na+ and K+ by inductively coupled plasma/mass spectrometry). Effects of day (D), pregnancy status (S) and their interaction were determined by least squares analysis of variance. Total recoverable arginine, glutamine, leucine and glucose increased (P<0.01, DxS) by 16-, 42-, 9-, and 12-fold, respectively between Days 10 and 15 of pregnancy, but not between Days 10 and 15 of the estrous cycle. Total recoverable Ca++ was higher in pregnant than cyclic ewes between Days 13 and 16 (P<0.01, DxS), changes in recoverable K+ were affected by Day (P<0.05) and were higher in pregnant ewes (P<0.05, S), but Na+ was not affected by D or S. Results indicate that FRAP1 is expressed by peri-implantation ovine conceptuses and that amino acids, glucose, Ca++ and K+ increase in the uterine environment of peri-implantation ovine conceptuses. These nutrients are now being evaluated for their ability to stimulate FRAP1 and, in turn, conceptus
development.
Impacts
Embryonic mortality is a key impediment to improving reproductive efficiency and profitability in livestock enterprises. This study is to understanding of key hormonal, cellular and molecular mechanisms critical to conceptus (embryo and its membranes) development and survival that are are affected by "select nutrients" defined as glucose, leucine, arginine, glutamine and a uterine secretory protein, secreted phosphoprotein 1 (SPP1). Is is expected that the select nutrients and SPP1 will activate mTOR cell signaling to stimulate trophectoderm to express interferon tau for pregnancy recognition signaling, insulin-like growth factor II for conceptus development, nitric oxide synthase for vasculature development and function, and ornithine decarboxylase for production of polyamines for trophoblast development. The impact will ultimately be improvements in diets to enhance reproductive efficiency in sheep, cattle and swine since the mTOR cell signaling pathway is common to
most if not all livestock species and other mammals.
Publications
- Bazer, F.W., Gao,H., Spencer, T.E., Wu, G., Johnson, G.A. 2006. Mammalian target of rapamycin (FRAP1) in ovine conceptus development. Proc 7th International Symposium on Ruminant Reproduction, Wellington, New Zealand, p 97.
- Gao, H., Spencer,T.E., Wu, G., Johnson, G.A., Bazer, F.W. Mammalian 2006. Target Of Rapamycin In Ovine Conceptus Development. Biol. Reprod. (Special Issue); p123.
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