Showing papers by "Fuller W. Bazer published in 2009"

Arginine metabolism and nutrition in growth, health and disease

Abstract: l-Arginine (Arg) is synthesised from glutamine, glutamate, and proline via the intestinal-renal axis in humans and most other mammals (including pigs, sheep and rats). Arg degradation occurs via multiple pathways that are initiated by arginase, nitric-oxide synthase, Arg:glycine amidinotransferase, and Arg decarboxylase. These pathways produce nitric oxide, polyamines, proline, glutamate, creatine, and agmatine with each having enormous biological importance. Arg is also required for the detoxification of ammonia, which is an extremely toxic substance for the central nervous system. There is compelling evidence that Arg regulates interorgan metabolism of energy substrates and the function of multiple organs. The results of both experimental and clinical studies indicate that Arg is a nutritionally essential amino acid (AA) for spermatogenesis, embryonic survival, fetal and neonatal growth, as well as maintenance of vascular tone and hemodynamics. Moreover, a growing body of evidence clearly indicates that dietary supplementation or intravenous administration of Arg is beneficial in improving reproductive, cardiovascular, pulmonary, renal, gastrointestinal, liver and immune functions, as well as facilitating wound healing, enhancing insulin sensitivity, and maintaining tissue integrity. Additionally, Arg or l-citrulline may provide novel and effective therapies for obesity, diabetes, and the metabolic syndrome. The effect of Arg in treating many developmental and health problems is unique among AAs, and offers great promise for improved health and wellbeing of humans and animals.

Comparative aspects of implantation

Abstract: Uterine receptivity to implantation of blastocysts in mammals includes hatching from zona pellucida, precontact with uterine luminal (LE) and superficial glandular (sGE) epithelia and orientation of blastocyst, apposition between trophectoderm and uterine LE and sGE, adhesion of trophectoderm to uterine LE/sGE, and, in some species, limited or extensive invasion into the endometrial stroma and induction of decidualization of stromal cells. These peri-implantation events are prerequisites for pregnancy recognition signaling, implantation, and placentation required for fetal-placental growth and development through the remainder of pregnancy. Although there is a range of strategies for implantation in mammals, a common feature is the requirement for progesterone (P(4)) to downregulate expression of its receptors in uterine epithelia and P(4) prior to implantation events. P(4) then mediates its effects via growth factors expressed by stromal cells in most species; however, uterine luminal epithelium may express a growth factor in response to P(4) and/or estrogens in species with a true epitheliochorial placenta. There is also compelling evidence that uterine receptivity to implantation involves temporal and cell-specific expression of interferon (IFN)-stimulated genes that may be induced directly by an IFN or induced by P(4) and stimulated by an IFN. These genes have many roles including nutrient transport, cellular remodeling, angiogenesis and relaxation of vascular tissues, cell proliferation and migration, establishment of an antiviral state, and protection of conceptus tissues from challenges by the maternal immune cells.

Progesterone-Regulated Changes in Endometrial Gene Expression Contribute to Advanced Conceptus Development in Cattle

Abstract: The postovulatory rise in circulating progesterone (P4) concentrations is associated with increased pregnancy success in beef and dairy cattle. Our study objective was to determine how elevated P4 alters endometrial gene expression to advance conceptus development. Synchronized heifers were inseminated (Day 0) and randomly assigned to pregnant high P4 or to pregnant normal P4. All high P4 groups received a P4-release intravaginal device on Day 3 after insemination that increased P4 concentrations up to Day 7 (P < 0.05). Tissue was collected on Day 5, 7, 13, or 16 of pregnancy, and endometrial gene expression was analyzed using the bovine Affymetrix (Santa Clara, CA) microarrays. Microarray analyses demonstrated that the largest number of P4-regulated genes coincided with the day when the P4 profiles were different for the longest period. Genes with the largest fold change increase (such as DGAT2 and MSTN [also known as GDF8]) were associated with triglyceride synthesis and glucose transport, which can be utilized as an energy source for the developing embryo. Temporal changes occurred at different stages of early pregnancy, with the greatest difference occurring between well-separated stages of conceptus development. Validation of a number of genes by quantitative real-time PCR indicated that P4 supplementation advances endometrial gene expression by altering the time (FABP, DGAT2, and MSTN) or duration (CRYGS) of expression pattern for genes that contribute to the composition of histotroph.

Select Nutrients in the Ovine Uterine Lumen. I. Amino Acids, Glucose, and Ions in Uterine Lumenal Flushings of Cyclic and Pregnant Ewes

Abstract: Nutrients in uterine secretions are essential for development and survival of conceptuses (embryo and associated extraembryonic membranes) during pregnancy; however, little is known about changes in the amounts of specific nutrients in the uterine fluids of cyclic and pregnant ruminants. This study determined quantities of glucose, amino acids, glutathione, calcium, sodium, and potassium in uterine lumenal fluid from cyclic (Days 3-16) and pregnant (Days 10-16) ewes. 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 were greater in the uterine fluid of pregnant compared with cyclic ewes between Days 10 and 16. In cyclic ewes, only modest changes in the total amounts of glucose, Asn, Cit, Tyr, Trp, Met, Val, Cys, glutathione, calcium, and potassium were detected between Days 3 and 16. However, in pregnant ewes, amounts of glucose, Arg, Gln, Glu, Gly, Cys, Leu, Pro, glutathione, calcium, and potassium in uterine fluids increased 3- to 23-fold between Days 10 and 14 and remained high to Day 16. Of particular interest were increases in glucose, Arg, Leu, and Gln in uterine flushings of pregnant ewes between Days 10 and 16 of pregnancy. Total amounts of His, ornithine, Lys, Ser, Thr, Ile, Phe, Trp, Met, and Cit in uterine fluids also increased, but to a lesser extent during early pregnancy. These novel results indicate activation of pregnancy-associated mechanisms for transport of nutrients into the uterine lumen, and they provide a framework for future studies of nutrients, including glucose, amino acids, and glutathione, required to activate nutrient-sensing cell signaling pathways for growth, development, and survival of conceptuses, as well as for optimization of culture media for in vitro studies of conceptus development.

Amino acids and gaseous signaling

Abstract: Gases, such as nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H2S), and sulfur dioxide (SO2) are known toxic pollutants in the air. However, they are now recognized as important signaling molecules synthesized in animals and humans from arginine, glycine (heme), and cysteine, respectively. At physiological levels, NO, CO, and SO2 activate guanylyl cyclase to generate cGMP which elicits a variety of responses (including relaxation of vascular smooth muscle cells, hemodynamics, neurotransmission, and cell metabolism) via cGMP-dependent protein kinases. H2S is also a crucial regulator of both neurological function and endothelium-dependent relaxation through cGMP-independent mechanisms involving stimulation of membrane KATP channels and intracellular cAMP signaling. Additionally, NO, CO, and H2S confer cytoprotective and immunomodulatory effects. Moreover, NH3 is a major product of amino acid catabolism and profoundly affects the function of neurons and the vasculature through glutamine-dependent inhibition of NO synthesis. Emerging evidence shows that amino acids are not only precursors for these endogenous gases, but are also regulators of their production in a cell-specific manner. Thus, recent advances on gaseous signaling have greatly expanded our basic knowledge of amino acid biochemistry and nutrition. These exciting discoveries will aid in the design of new nutritional and pharmacological means to prevent and treat major health problems related to developmental biology and nutrient metabolism, including intrauterine growth restriction, preterm birth, aging, neurological disorders, cancer, obesity, diabetes, and cardiovascular disease.

Interferons and uterine receptivity.

Abstract: This article focuses on the potential roles of interferons (IFNs) in establishing uterine receptivity to implantation. A common feature of the peri-implantation period of pregnancy in most mammals is production of type I and/or type II IFNs by trophoblasts that induce and/or stimulate expression of an array of IFN-stimulate genes (ISGs). These effects range from pregnancy recognition signaling in ruminants through IFN tau to effects on cellular functions of the uterus and uterine vasculature. For actions of IFNs, progesterone (P4) is permissive to the expression of many effects and to the expression of ISGs that are induced directly by an IFN or induced by P4 and stimulated by an IFN in a temporal and/or cell-specific manner. Uterine receptivity to implantation is P4 dependent; however, implantation events are preceded by loss of expression of progesterone (PGR) and estrogen (ESR1) receptors by uterine epithelia. Therefore, P4 likely acts via PGR-positive stromal cells to induce expression of fibroblast growth factors-7 and -10 and/or hepatocyte growth factor (progestamedins) that then act via their respective receptors on uterine epithelia and trophectoderm to affect expression of ISGs. The permissive effects of P4 on the expression of ISGs and the effects of P4 to induce and IFNs to stimulate gene expression raise the question of whether uterine receptivity to implantation requires P4 and IFN to activate unique, but complementary, cell signaling pathways. Uterine receptivity to implantation, depending on species, involves changes in the expression of genes for the attachment of trophectoderm to the uterine lumenal epithelium (LE) and superficial glandular epithelium (sGE), modification of the phenotype of uterine stromal cells, the silencing of PGR and ESR1 genes, the suppression of genes for immune recognition, alterations in membrane permeability to enhance conceptus-maternal exchange of factors, increased vascularity of the endometrium, activation of genes for transport of nutrients into the uterine lumen, and enhanced signaling for pregnancy recognition. Differential expression of genes by uterine LE/sGE, mid- to deep-glandular epithelia (GE), and stromal cells in response to P4 and IFNs is likely to influence uterine receptivity to implantation in most mammals. Understanding the roles of IFNs in uterine receptivity for implantation is necessary to develop approaches to enhance reproductive health and fertility in humans and domestic animals.

Select nutrients in the ovine uterine lumen. ii. glucose transporters in the uterus and peri-implantation conceptuses

Abstract: Total glucose in ovine uterine lumenal fluid increases 6-fold between Days 10 and 15 of gestation, but not the estrous cycle; however, mechanisms for glucose transport into the uterine lumen and uptake by conceptuses (embryo/fetus and associated membranes) are not established. This study determined the 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 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 10 to 20 of pregnancy. The SLC2A1 and SLC5A1 mRNAs and proteins were most abundant in uterine luminal epithelia and superficial glandular epithelia (LE/sGE), whereas SLC2A4 was present in stromal cells and glandular epithelia (GE). SLC5A11 mRNA was most abundant in endometrial GE, whereas SLC2A3 mRNA was not detectable in endometria. SLC2A1, SLC2A3, SLC2A4, SLC5A1, and SLC5A11 were expressed in the trophectoderm and endoderm of conceptuses. Steady-state levels of SLC2A1, SLC5A1, and SLC5A11 mRNAs, but not SLC2A4 mRNA, were greater in endometria from pregnant than from cyclic ewes. Progesterone increased SLC2A1, SLC5A11, and SLC2A4 mRNAs in the LE/sGE and SLC5A1 in the GE of ovariectomized ewes. Expression of SLC5A1 was inhibited by ZK136,317 (progesterone receptor antagonist), and the combination of ZK136,317 and IFNT further decreased expression in GE. In constrast, P4 induced and IFNT stimulated expression of SLC2A1 and SLC5A11, and these effects were blocked by ZK136,317. Results of this study indicate differential expression of facilitative and sodium-dependent glucose transporters in ovine uteri and conceptuses for transport and uptake of glucose, and that P4 or P4 and IFNT regulate their expression during the peri-implantation period of pregnancy.

Select Nutrients in the Ovine Uterine Lumen. III. Cationic Amino Acid Transporters in the Ovine Uterus and Peri-Implantation Conceptuses

Abstract: Arginine is an essential amino acid for conceptus (embryo/fetus and trophoblast/placenta) growth and development; however, the mechanisms for arginine transport into the uterine lumen and uptake by conceptuses are largely unknown. In this study, expression of System y(+) (SLC7A1, SLC7A2, and SLC7A3) cationic amino acid transporters in uteri of cyclic and pregnant ewes and conceptuses was studied, and effects of pregnancy, progesterone (P4), and interferon tau (IFNT) on their expression were investigated. SLC7A1 mRNA was most abundant in endometrial luminal (LE) and superficial glandular (sGE) epithelia on Day 16 of the estrous cycle and on Days 16-20 of pregnancy, whereas SLC7A2 mRNA was most abundant in LE and mid to deep glandular (GE) epithelia on Days 14-20 of gestation. Expression of SLC7A1 and SLC7A2 was enhanced in pregnant ewes in a cell-specific manner, but abundance of SLC7A3 was not affected by day of the estrous cycle or by pregnancy status. SLC7A1, SLC7A2, and SLC7A3 mRNAs were expressed in trophectoderm and endoderm of conceptuses. In ovariectomized ewes, short-term treatment of ewes with P4 and IFNT did not affect endometrial SLC7A1 mRNA, while long-term treatment with P4 stimulated SLC7A1 in LE and GE, and IFNT tended to increase SLC7A1 abundance in LE. SLC7A2 mRNA abundance increased 4.1-fold in response to short-term P4 treatment and an additional 1.7-fold by IFNT primarily in endometrial LE/sGE, and these effects were ablated by a P4 receptor antagonist. These results indicate that coordinate changes in SLC7A1, SLC7A2, and SLC7A3 expression in uterine endometria and conceptuses are likely important in transport of arginine that is critical to conceptus growth, development, and survival.

Select Nutrients in the Ovine Uterine Lumen. IV. Expression of Neutral and Acidic Amino Acid Transporters in Ovine Uteri and Peri-Implantation Conceptuses

Abstract: The availability of specific neutral and acidic amino acids in the uterine lumen of ewes increased significantly during the peri-implantation period, but mechanisms for their transport into the uterine lumen and uptake by conceptuses are not established in any species. In this study, effects of pregnancy, progesterone (P4), and interferon tau (IFNT) on expression of neutral and acidic amino acid transporters in uteri of cyclic and pregnant ewes and conceptuses were studied. SLC1A2, SLC1A3, SLC3A1, SLC6A14, SLC6A19, SLC7A6, SLC38A3, and SLC38A6 mRNAs were only weakly expressed in the ovine endometrium. However, SLC1A4, SLC1A5, SLC7A8, and SLC43A2 mRNAs were detectable in uterine luminal epithelia (LE), superficial glandular epithelia (sGE), and/or glandular epithelia (GE). SLC1A1 and SLC7A5 mRNAs were most abundant in LE/sGE and GE. SLC1A3 and SLC38A4 mRNAs were most abundant in uterine stroma. SLC38A6 mRNA was detected only in cells with a stromal distribution suggesting immune lineage. SLC1A5 mRNA was expressed primarily in LE/sGE and stromal cells, and it was more abundant in uteri of pregnant ewes (day x status interaction; P < 0.05). Furthermore, P4 induced and IFNT further stimulated SLC1A5 expression in LE/sGE. Endometrial SLC1A1, SLC7A5, and SLC43A2 mRNAs demonstrated both temporal and cellSLC-specific changes. Several mRNAs were detectable in trophectoderm (SLC6A19, SLC7A5, SLC7A6, and SLC43A2), while others were more abundant in endoderm (SLC1A4, SLC1A5, SLC6A19, SLC7A5, SLC7A6, SLC7A8, and SLC43A2) of conceptuses. These results document coordinate changes in expression of transporters that are likely responsible for increases in amounts of neutral and acidic amino acids in the uterine lumen to support conceptus growth, development, and survival.

Heterotopic uterus transplantation in a swine model.

Abstract: Background. The aim of our study was to examine the feasibility of allogeneic uterine transplantation in a large animal model. Methods. We performed heterotopic uterine transplants in genetically defined mini-pigs. Immunosuppression was tacrolimus administered intravenously for the first 12 days posttransplantation followed by oral cyclosporine maintenance immunosuppression. The graft was transplanted heterotopically in the lower abdominal cavity of the recipient. The vaginal vault was exteriorized as a stoma in the lower right abdominal wall. The uterine grafts were followed with endoscopies and biopsies. Results. Ten transplants were performed. Follow-up was until July 2008. At the end of the follow-up period, 5 animals were alive and healthy, 0.5 to 12 months posttransplantation. There were 5 deaths due to pneumonia (n=1), intussusception of the graft (n=1), cardiorespiratory arrest during anesthesia (n=1), and complications of the stoma (n=2). Acute rejections of the graft presented during the 2nd and 3rd month posttransplantation were treated successfully with increase of the maintenance immunosuppression and steroids. Other complications included prolapse and infections of the graft stoma. Pathological changes seen in the endometrial biopsies included acute rejection and acute endometritis. Conclusion. These findings demonstrate that successful uterus transplantation in a large animal model (miniature swine) is feasible using this heterotopic model, and it can be useful for the study of these transplants.

Discovery of candidate genes and pathways in the endometrium regulating ovine blastocyst growth and conceptus elongation

Abstract: Establishment of pregnancy in ruminants requires blastocyst growth to form an elongated conceptus that produces interferon tau, the pregnancy recognition signal, and initiates implantation. Blastocyst growth and development requires secretions from the uterine endometrium. An early increase in circulating concentrations of progesterone (P4) stimulates blastocyst growth and elongation in ruminants. This study utilized sheep as a model to identify candidate genes and regulatory networks in the endometrium that govern preimplantation blastocyst growth and development. Ewes were treated daily with either P4 or corn oil vehicle from day 1.5 after mating to either day 9 or day 12 of pregnancy when endometrium was obtained by hysterectomy. Microarray analyses revealed many differentially expressed genes in the endometria affected by day of pregnancy and early P4 treatment. In situ hybridization analyses revealed that many differentially expressed genes were expressed in a cell-specific manner within the endometrium. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) was used to identify functional groups of genes and biological processes in the endometrium that are associated with growth and development of preimplantation blastocysts. Notably, biological processes affected by day of pregnancy and/or early P4 treatment included lipid biosynthesis and metabolism, angiogenesis, transport, extracellular space, defense and inflammatory response, proteolysis, amino acid transport and metabolism, and hormone metabolism. This transcriptomic data provides novel insights into the biology of endometrial function and preimplantation blastocyst growth and development in sheep.

Conceptus-uterus interactions in pigs: endometrial gene expression in response to estrogens and interferons from conceptuses.

Abstract: This review highlights information on conceptus-uterus interactions in the pig with respect to uterine gene expression in response to estrogens and interferons (IFNs) secreted from elongating conceptuses. Pig conceptuses release estrogens for pregnancy recognition, but also secrete IFNs that do not appear to be antiluteolytic. Estrogens and IFNs induce expression of largely non-overlapping sets of genes, and evidence suggests that pig conceptuses orchestrate essential events of early pregnancy including pregnancy recognition signaling, implantation and secretion of histotroph by precisely controlling temporal and spatial (cell-specific) changes in uterine gene expression through initial secretion of estrogens, followed by cytokines including IFNG and IFND. By Day 12 of pregnancy, estrogens increase the expression of multiple genes in the uterine luminal epithelium including SPP1, STC1, IRF2 and STAT1 that likely have roles for implantation. By Day 15 of pregnancy, IFNs upregulate a large array of IFN responsive genes in the underlying stroma and glandular epithelium including ISG15, IRF1, STAT1, SLAs and B2M that likely have roles in uterine remodeling to support placentation.

Intravenous Administration of l-Citrulline to Pregnant Ewes Is More Effective Than l-Arginine for Increasing Arginine Availability in the Fetus

Abstract: l-Arginine administration may be useful for the treatment of intrauterine growth restriction, but concerns remain about effective precursors for administration into pregnant dams. Therefore, we used an ovine model to test the hypothesis that infusion of l-citrulline into the maternal circulation increases l-arginine availability to the fetus. On d 135 ± 1 of gestation, ewes received an i.v. bolus dose of l-citrulline (155 μmol/kg body weight) or the same dose of l-arginine-HCl. Maternal and fetal arterial blood samples were obtained simultaneously at −120, −60, 0, 5, 15, 30, 60, 120, 180, and 240 min relative to the time of amino acid administration. Concentrations of arginine in maternal plasma increased to peak values within 5 min after its injection in ewes and declined rapidly thereafter, whereas concentrations of arginine in fetal plasma increased between 15 and 30 min and returned to baseline values by 60 min. In contrast, administration of citrulline increased concentrations of citrulline and arginine in maternal and fetal plasma between 5 and 60 min and values remained elevated thereafter. The differential pharmacokinetics for arginine compared with citrulline infusion was consistent with the observation that the half-life of citrulline was twice that of arginine in ewes. We conclude that i.v. administration of citrulline is more effective than arginine in sustaining high concentrations of arginine in the maternal and fetal circulations of pregnant ewes. These novel findings provide support for studies of the clinical use of arginine and citrulline as therapeutic means to prevent or ameliorate fetal growth retardation in mammals.

Farm Animal Research in Crisis

Abstract: The annual economic value of livestock and poultry sales in the United States currently exceeds $132 billion ( 1 ), yet only about 0.04% ($32.15 million) ( 2 ) of the $88 billion Department of Agriculture (USDA) budget in fiscal year 2007 ( 3 ) was allocated to its competitive grants program for research that directly involves agriculturally important domestic animals. By contrast, the Department of Health and Human Services (DHHS) apportioned 4.1% ($29.5 billion) of its $716 billion budget in fiscal year 2008 to the National Institutes of Health (NIH) of which ∼80% supported extramural research ( 4 ). Whether this direct comparison between USDA and DHHS is appropriate may be debatable; still, it clearly illustrates the huge disparity in total budget available for research grants focused on animal agriculture, about 1/918th that for human health. The private sector does invest in agricultural research and development, but, understandably, such funds are highly focused on commercial interests and not on basic research of the kind we discuss.

Insulin-like growth factor binding protein-1 in the ruminant uterus: potential endometrial marker and regulator of conceptus elongation.

Abstract: Establishment of pregnancy in ruminants requires conceptus elongation and production of interferon-tau (IFNT), the pregnancy recognition signal that maintains ovarian progesterone (P4) production. These studies determined temporal and spatial alterations in IGF binding protein (IGFBP)-1 and IGFBP3 in the ovine and bovine uterus; effects of P4 and IFNT on their expression in the ovine uterus; and effects of IGFBP1 on ovine trophectoderm cell proliferation, migration, and attachment. IGFBP1 and IGFBP3 were studied because they are the only IGFBPs specifically expressed by the endometrial luminal epithelia in sheep. In sheep, IGFBP1 and IGFBP3 expression was coordinate with the period of conceptus elongation, whereas only IGFBP1 expression was coordinate with conceptus elongation in cattle. IGFBP1 mRNA in the ovine endometria was between 5- and 29-fold more abundant between d 12 and 16 of pregnancy compared with the estrous cycle and greater on d 16 of pregnancy than nonpregnancy in the bovine uterus. In sheep, P4 induced and IFNT stimulated expression of IGFBP1 but not IGFBP3; however, the effect of IFNT did not mimic the abundant increase observed in pregnant ewes. Therefore, IGFBP1 expression in the endometrium is regulated by another factor from the conceptus. IGFBP1 did not affect the proliferation of ovine trophectoderm cells in vitro but did stimulate their migration and mediate their attachment. These studies reveal that IGFBP1 is a common endometrial marker of conceptus elongation in sheep and cattle and most likely regulates conceptus elongation by stimulating migration and attachment of the trophectoderm.

Stanniocalcin 1 is a luminal epithelial marker for implantation in pigs regulated by progesterone and estradiol.

Abstract: Stanniocalcin 1 (STC1) is a glycoprotein that decreases calcium and increases phosphate in cells/tissues. This investigation examined endocrine regulation of STC1 in endometria of pigs during the estrous cycle and pregnancy. STC1 mRNA was present exclusively in luminal epithelium (LE) between d 12 and 15 of the estrous cycle, increased between d 12 and d 20, and was not detectable by d 30 of pregnancy. STC1 protein was also detected in uterine flushings. To determine effects of estrogen and progesterone, pigs were ovariectomized and treated with these hormones alone or together. Progesterone, but not estrogen, induced STC1 in LE. Cotreatment with progesterone and estrogen further stimulated STC1 over progesterone alone. To determine effects of pseudopregnancy, nonpregnant gilts were given daily injections of estradiol benzoate from d 11 to d 14. STC1 was not expressed in LE on d 90 of pseudopregnancy, suggesting that the estradiol given to induce pseudopregnancy and/or long-term exposure to progesterone are required for down-regulation of STC1. To determine effects of long-term progesterone, without effects of estradiol, pigs were ovariectomized on d 12, given daily injections of progesterone through d 39, and hysterectomized on d 40 after estrus. STC1 was expressed in LE of progesterone-treated pigs, suggesting that estrogen is involved in down-regulation of STC1. We conclude that STC1 is induced in LE by progesterone and further stimulated by estrogen, and its down-regulation in LE by d 25 likely requires exposure of the progestinized uterus to estrogen. The temporal and cell type-specific expression of STC1 makes this gene a unique marker for implantation in pigs.

Select Nutrients in the Ovine Uterine Lumen. V. Nitric Oxide Synthase, GTP Cyclohydrolase, and Ornithine Decarboxylase in Ovine Uteri and Peri-Implantation Conceptuses

Abstract: Nitric oxide (NO) and polyamines are critical for implantation and development of conceptuses (embryo and extraembryonic membranes), but mechanisms regulating their biosynthesis in uteri and conceptuses are largely unknown. This study determined the effects of the estrous cycle, pregnancy, progesterone, and interferon tau (IFNT) on expression of NO synthases (NOS1, NOS2, and NOS3), guanosine triphosphate (GTP) cyclohydrolase (GCH1, the key enzyme in de novo synthesis of tetrahydrobiopterin, a cofactor for NO production), and ornithine decarboxylase (ODC1) in uterine endometria in cyclic ewes (Days 10-16) and pregnant ewes (Days 10-20). The mRNAs and proteins for NOS1 and ODC1 were most abundant in uterine luminal (LE) and superficial glandular (sGE) epithelia, and abundance was affected by day of estrous cycle and early pregnancy. NOS2, GCH1, and NOS3 mRNAs were detected in very low abundance in uterine epithelia and stromal cells in both cyclic and pregnant ewes. NOS1 mRNA also was expressed very weakly in conceptuses, whereas NOS3 mRNA was abundant in the trophectoderm and endoderm of conceptuses, as were total NOS1 and NOS3 proteins, inhibitory p-NOS1 protein, and stimulatory p-NOS3 protein. GCH1 mRNA was abundant in the trophectoderm and endoderm of conceptuses between Days 13 and 15 of pregnancy and then decreased thereafter, whereas ODC1 mRNA abundance increased in conceptuses between Days 13 and 18 of pregnancy. GCH1 protein was localized primarily in the nuclei of trophectoderm and endoderm, and its abundance decreased after Day 14 of pregnancy, whereas ODC1 protein was more abundant in the trophectoderm than in the endoderm between Days 13 and 18 of pregnancy. Progesterone stimulated NOS1 and GCH1 expression in LE/sGE and glandular epithelia, whereas IFNT inhibited NOS1 expression in these cell types. Thus, biosynthesis of NO and polyamines in ovine uterine endometria and conceptuses is potentially regulated at transcriptional, translational, and posttranslational levels to favor conceptus development and implantation.

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

Abstract: FRAP1 (FK506-binding protein 12-rapamycin complex-associated protein 1), a component of the nutrient-sensing cell signaling pathway, is critical for cell growth and metabolism. The present study determined expression of FRAP1 and associated members of the mTORC1 and mTORC2 cell signaling pathways in uteri of cyclic and pregnant ewes and conceptuses, as well as effects of pregnancy, progesterone (P4), and interferon tau (IFNT) on their expression. The mRNAs for FRAP1, LST8, MAPKAP1, RAPTOR, RICTOR, TSC1, TSC2, RHEB, and EIF4EBP1 were localized to luminal, superficial glandular, and glandular epithelia and stromal cells of uteri from cyclic and pregnant ewes, as well as trophectoderm and endoderm of conceptuses between Days 13 and 18 of pregnancy. The abundance of FRAP1, RAPTOR, RICTOR, TSC1, and TSC2 mRNAs in endometria was unaffected by pregnancy status or by day of the estrous cycle or pregnancy; however, levels of LST8, MAPKAP1, RHEB, and EIF4EBP1 mRNA increased in endometria during early pregnancy. In ovariectomized ewes, P4 and IFNT stimulated expression of RHEB and EIF4EBP1 in uterine endometria. Total endometrial FRAP1 protein and phosphorylated FRAP1 protein levels were affected by pregnancy status and by day after onset of estrus, and phosphorylated FRAP1 protein was detected in nuclei of uterine epithelia and conceptuses. In endometria of pregnant ewes, increases in abundance of mRNAs for RICTOR, RHEB, and EIF4EBP1, as well as RHEB protein, correlated with rapid conceptus growth and development during the peri-implantation period. These results suggest that the FRAP1 cell signaling pathway mediates interactions between the maternal uterus and peri-implantation conceptuses and that P4 and IFNT affect this pathway by regulating expression of RHEB and EIF4EBP1.

Progesterone and interferon tau regulate leukemia inhibitory factor receptor and IL6ST in the ovine uterus during early pregnancy.

Abstract: The actions of leukemia inhibitory factor (LIF) via LIF receptor (LIFR) and its co-receptor, IL6 signal transducer (IL6ST), are implicated in uterine receptivity to conceptus implantation in a number of species including sheep. The present study determined the effects of the estrous cycle, pregnancy, progesterone (P4), and interferon tau (IFNT) on the expression of LIFR and IL6ST in the ovine uterus. LIFR mRNA and protein were localized to the endometrial luminal (LE) and superficial glandular epithelia (sGE), whereas IL6ST mRNA and protein were localized primarily in the middle to deep GE. Both LIFR and IL6ST mRNAs and protein were more abundant in pregnant than cyclic ewes and increased from days 10 to 20 of pregnancy. Treatment of ovariectomized ewes with P4 and/or infusion of ovine IFNT increased LIFR and IL6ST in endometrial LE/sGE and GE respectively. Co-expression of LIFR and IL6ST as well as phosphorylated STAT3 was observed only in the upper GE of the endometrium as well as in the conceptus trophectoderm on days 18 and 20. In mononuclear trophectoderm and GE cells, LIF elicited an increase in phosphorylated STAT3 and MAPK3/1 MAPK proteins. Collectively, these results suggest that LIFR and IL6ST are both stimulated by IFNT and regulated by P4 in a complex stage- and cell-specific manner, and support the hypothesis that LIF exerts effects on the endometrial GE as well as conceptus trophectoderm during early pregnancy in sheep. Thus, LIF and STAT3 may have biological roles in endometrial function and trophectoderm growth and differentiation.

Transplacental Transfer of Iron in the Water Buffalo (Bubalus bubalis): Uteroferrin and Erythrophagocytosis

Abstract: The objectives of this investigation were to understand transplacental transport of iron by secreted uteroferrin (UF) and haemophagous areas of water buffalo placenta and clarify the role(s) of blood extravasation at the placental-maternal interface. Placentomes and interplacentomal region of 51 placentae at various stages of gestation were fixed, processed for light and transmission electron microscopy, histochemistry and immunohistochemistry. Haemophagous areas were present in placentomes collected between 4 and 10 months of pregnancy. Perl's reaction for ferric iron was negative in placentomes, but positive in endometrial glands. Positive staining for UF indicated areas in which it was being taken up by phagocytosis and/or fluid phase pinocytosis in areolae of the interplacentomal mesenchyme, with little staining in endometrial stroma. Imunohistochemistry detected UF in trophectoderm of haemophagous regions of placentomes and in other parts of the foetal villous tree, but the strongest immunostaining was in the epithelial cells and lumen of uterine glands. Ultrastructural analyses indicated that erythrophagocytosis was occurring and that erythrocytes were present inside cells of the chorion that also contained endocytic vesicles and caveolae. Results of this study indicate that both the haemophagous areas of placentomes and the areolae at the interface between chorion and endometrial glands are important sites for iron transfer from mother to foetal-placental tissues in buffalo throughout pregnancy.

Progesterone and interferon tau-regulated genes in the ovine uterine endometrium: identification of periostin as a potential mediator of conceptus elongation

Abstract: During early pregnancy in ruminants, progesterone (P(4)) and interferon tau (IFNT) act on the endometrium to regulate genes hypothesized to be important for conceptus development and implantation. The present study was conducted to verify several candidate genes (actin alpha-2, smooth muscle, aorta (ACTA2), collagen, type III, alpha-1 (COL3A1), periostin (POSTN), secreted protein acidic cysteine-rich (SPARC), S100 calcium-binding protein A2 (S100A2), STAT5A and transgelin (TAGLN)) regulated by pregnancy, P(4), and/or IFNT in the endometrium determined using a custom ovine cDNA array. S100A2 mRNA was detected primarily in endometrial epithelia and conceptuses. S100A2 mRNA increased in endometrial epithelia from days 10 to 16 in cyclic ewes and from days 10 to 14 in pregnant ewes and declined thereafter. The abundance of S100A2 mRNA was less in endometrial luminal epithelium of IFNT-infused ewes receiving P(4). Expression of COL3A1, SPARC, ACTA2, and TAGLN was independent of pregnancy, P(4), or IFNT. POSTN mRNA was detected primarily in compact stroma of intercaruncular and caruncular endometria, but not in the conceptus. Endometrial POSTN mRNA increased between days 12 and 14 in pregnant but not cyclic ewes, and POSTN mRNA was more abundant in uterine stroma of ewes treated with P(4). POSTN protein was detected in uterine flushings of pregnant ewes and found to mediate attachment and stimulate migration of ovine trophectoderm cells in vitro. These results support the ideas that POSTN and S100A2 are regulated by P(4) and IFNT respectively, and that POSTN is involved in conceptus elongation during early pregnancy.

Immunohistochemical and histochemical identification of proteins and carbohydrates in the equine endometrium: reaction patterns in the cycling mare.

Abstract: Little is known about the stage of estrus cycle-dependent variations of uterine secretions of the more and most published results were based on evaluation of uterine flushings. Hence, the aim of this study was to define patterns of expression of different endometrial proteins throughout the estrus cycle using newly established immunohistochemical methods and endometrial biopsies. Therefore, endometrial biopsies were collected from three mares on defined days of the estrus cycle with known concentrations of estradiol and progesterone in serum. The proteins uteroglobin, uteroferrin, calbindin(D9k) and uterocalin were examined using polyclonal antibodies. Furthermore glycogen was identified using the PAS-reaction prior to and after a-amylase digestion. During the estrus cycle expression of the examined proteins, as well as glycogen, revealed a typical, progesterone-dependent pattern. In the early- and mid-interestrus there is a simultaneous increase in expression of uterocalin and glycogen staining intensity associated with increasing concentrations of progesterone in peripheral blood. The maximal expression of calbindin(D9k) was also in association with high levels of circulating progesterone in mid-interestrus. In contrast, secretion of both uteroglobin and uteroferrin was simultaneously associated with the decrease in circulating concentrations of progesterone in mid-to late-interestrus. However, it has to be emphasized that distinct among mare differences were obvious. This new method enables pathologists to interpret changes in the uterine secretion pattern associated with fertility reducing diseases by using a single endometrial biopsy

Select nutrients and glucose transporters in pig uteri and conceptuses.

Abstract: Glucose present in the intrauterine environment can be metabolized, activate cell signaling pathways or be converted to a "storage" form. Total recoverable glucose in uterine fluid of pregnant, but not cyclic pigs increases from Day 12 after onset of estrus in concert with conceptus elongation (Bazer et al. 1991). Transport of glucose into the ovine uterus and its uptake by conceptuses involves sodium-dependent and facilitative glucose transporters (Gao et al. 2009). Glucose can activate FRAP1/mTOR "nutrient sensing" pathway in which protein kinases activate p70S6 through phosphorylation to increase translation of 5'TOP mRNAs (terminal oligopyrimidine tract) (Wen et a/. 2005). Activated FRAP1 also regulates differentiation of trophectoderm (Tr) via Ras transformation by phosphorylating eukaryotic initiation factor 4E binding protein 1 (eIF4EBP1), a translational repressor of CAP-dependent translation (De Benedetti & Rhoads 1990). Select nutrients that stimulate FRAP1 activity in Tr include glucose, arginine (Arg), leucine (Leu) and glutamine (Gln) which may increase expression of IGF2, ODC and NOS mRNAs (Nielsen et al. 1995; Kimball et al. 1999; Martin & Sutherland 2001) which are required for conceptus development, differentiation and implantation through effects on production of NO (Kaliman et al. 1999) and polyamines (Van Winkle & Campione 1983). FRAP1 null mice die shortly after implantation due to impaired cell proliferation and hypertrophy in both the embryonic disc and Tr (Murakami et al. 2004). There are 14 isoforms of facilitative glucose transporters and 6 sodium-dependent glucose transporters. Of these, SLC2A I, SLCSA1 and SLCSA1 I mRNAs are most abundant in endometria and SLC2A3 is uniquely expressed by ovine conceptus Tr and endoderm (Gao et al. 2009). The objective of this study with sexually mature giks was to identify effects of pregnancy, long-term treatment of ovariectomized gilts with progesterone (P4) and estradiol-induced pseudopregnancy (PP) on changes in amounts of select nutrients (glucose, Arg, Leu and Gln) in uterine fluid and expression of glucose transporters in endometria and conceptuses. Experiment 1 determined effects of day of the estrous cycle and pregnancy on total recoverable glucose, Arg, Leu and Gln in uterine flushings from gilts on Days 5, 9, 12 and 15 of the estrous cycle (Cy) and Days 9, 10, 12, 13, 14 and 15 of pregnancy (Px). Total recoverable glucose, Arg, Leu and Gln increased (P< 0.05) with day in Cy and Px gilts, but only Arg increased more in Px than Cy ewes (day x pregnancy status; P<0.05) between Days 12 and 15. Experiment 2 determined recoverable amounts of selected nutrients in uterine flushings of gilts ovariectomized on Day 12 and treated daily with either corn oil (OVX-CO; n —4) or 200 mg progesterone (OVX-P4;n —5) to Day 39 and hysterectomized on Day 40. Values (mean +SEM; nmol) were greater for OVX-P4 than OVX-CO gilts for glucose (4,955+2,534 vs 726+ 133), Arg (207,112 + 160,979 vs 7,409+ 2,877) and Leu (248,255 + 178,599 vs 13,983+ 5,225), but differences were not significant due to high variability and small sample size. Experiment 3 determined amounts of selected nutrients in uterine flushings of gilts on Day 90 of pseudopregnancy (PP) induced by treatment with 5 mg/day estradiol benzoate on Days

Interferon Tau in the Ovine Uterus

Abstract: Gwonhwa Song*, Jae Yong Han*, Thomas E. Spencer** and Fuller W. Bazer*, **WCU Biomodulation Major, Department of Agricultural Biotechnology, and Research Institute for Agriculture and Life Sciences, Seoul National University, Korea*, Department of Animal Science, Center for Animal Biotechnology and Genomics, Texas A&M University, 442 Kleberg Center, 2471 TAMU, College Station, Texas 77843-2471, USA**ABSTRACTThe peri-implantation period in mammals is critical with respect to survival of the conceptus(embryo/fetus and associated extraembryonic membranes) and establishment of pregnancy. During this period of pregnancy, reciprocal communication between ovary, conceptus, and endometrium is required for successful implantation and placentation. At this time, interferon tau(IFNT) is synthesized and secreted by the mononuclear trophectodermal cells of the conceptus between days 10 and 21~25. The actions of IFNT to signal pregnancy recognition and induce or increase expression of IFNT-stimulated genes (ISGs), such as ISG15 and OAS, are mediated by the Type I IFN signal transduction pathway. This article reviews the history, signaling pathways of IFNT and the uterine expression of several IFNT-stimulated genes during the peri-implantation period. Collectively, these newly identified genes are believed to be critical to unraveling the mechanism(s) of reciprocal fetal-maternal interactions required for successful implantation and pregnancy. (Key words :Interferon tau, Progesterone, Sheep, Uterus, Implantation)