Evidence for metabolism of creatine by the conceptus, placenta, and uterus for production of ATP during conceptus development in pigs.

In pigs, the majority of embryonic mortality occurs when free-floating conceptuses (embryos/fetuses and associated placental membranes) elongate and the uterine-placental interface undergoes folding and develops areolae. Both periods involve proliferation, migration, and changes in morphology of cells that require ATP. We hypothesize that insufficient ATP in conceptus and uterine tissues contributes to conceptus loss in pigs. Creatine is stored in cells as phosphocreatine (PCr) for ATP regeneration through the creatine (Cr)-creatine kinase (CK)-PCr pathway. However, the expression of components of this pathway in pigs has not been examined throughout gestation. Results of qPCR analyses indicated increases in AGAT, GAMT, CKM, CKB, and SLC6A8 mRNAs in elongating porcine conceptuses and immunofluorescence microscopy localized GAMT, CKM, and CKB proteins to the trophectoderm of elongating conceptuses, to the columnar chorionic epithelial cells at the bottom of chorioallantoic troughs, and to endometrial luminal epithelium (LE) at the tops of the endometrial ridges of uterine-placental folds on Days 40, 60, and 90 of gestation. GAMT protein is expressed in endometrial LE at the uterine-placental interface, but immunostaining is more intense in LE at the bottoms of the endometrial ridges. Results of this study indicate that key elements of the pathway for creatine metabolism are expressed in cells of the conceptus, placenta, and uterus for potential production of ATP during two timepoints in pregnancy with a high demand for energy; elongation of the conceptus for implantation and development of uterine-placental folding during placentation.

Temporal and spatial expression of aquaporins 1, 5, 8, and 9: Potential transport of water across the endometrium and chorioallantois of pigs.

INTRODUCTION: The uterus and placenta transport water during pregnancy recognition signaling, conceptus implantation, and placental development/placentation. This is likely influenced by aquaporins (AQPs) in the reproductive tract. This study determined mRNA and cell-type specific expression of AQP 1, 5, 8, and 9 proteins in the porcine uterus and placenta. METHODS: Porcine uteri and Chorioallantois were subjected to real-time PCR and immunofluorescence microscopy. RESULTS: AQP1 mRNA was maximal by Day 25 in endometrium and remained stable thereafter. AQP1 mRNA did not change in chorioallantois. AQP1 protein localized to erythrocytes and endothelium of the endometrium and allantois, and to smooth muscle of the myometrium. AQP5 protein localized to apical and lateral surfaces of the chorionic epithelia of areolae, but mRNA did not change in chorioallantois. AQP8 mRNA was high in the endometrium from Days 15 through 60 of gestation, and protein localized to multiple cell types within the endometrium and chorioallantois. AQP9 mRNA was highest in the endometrium on Days 10, 12 and 25, but did not change in the chorioallantois. AQP9 protein localized to the apical surface of endometrial luminal epithelial cells during early pregnancy, with a shift towards the basal surface later. AQP9 protein was observed in the allantoic epithelium. DISCUSSION: Results reveal pigs can potentially use AQP1, AQP5, AQP8, and AQP9 to transport water from the endometrial bloodstream to the allantoic bloodstream or allantoic fluid. The reverse is also possible and may explain the mechanism for changing volumes of allantoic fluid and hydration of allantoic connective tissues during pregnancy.

Dietary supplementation with L-arginine between days 14 and 25 of gestation enhances NO and polyamine syntheses and the expression of angiogenic proteins in porcine placentae.

Dietary supplementation with 0.4 or 0.8% L-arginine (Arg) to gilts between days 14 and 25 of gestation enhances embryonic survival and vascular development in placentae; however, the underlying mechanisms are largely unknown. This study tested the hypothesis that Arg supplementation stimulated placental expression of mRNAs and proteins that enhance angiogenesis, including endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), placental growth factor (PGF), GTP cyclohydrolase-I (GTP-CH1), ornithine decarboxylase (ODC1), and vascular endothelial growth factor receptors 1 and 2 (VEGFR1 and VEGFR2). Beginning on the day of breeding, gilts were fed daily 2 kg of a corn-soybean meal-based diet supplemented with 0.0 (control), 0.4, or 0.8% Arg. On day 25 of gestation, gilts were hysterectomized to obtain uteri and conceptuses for histochemical and biochemical analyses. eNOS and VEGFR1 proteins were localized to endothelial cells of maternal uterine blood vessels and to the uterine luminal epithelium, respectively. Compared with the control, dietary supplementation with 0.4 or 0.8% Arg increased (P < 0.05) the amounts of nitrite plus nitrate (NOx; oxidation products of NO) and polyamines in allantoic and amniotic fluids, concentrations of NOx, tetrahydrobiopterin (BH4, an essential cofactor for all NOS isoforms) and polyamines in placentae, as well as placental protein abundances of GTP-CH1 (the key enzyme for BH4 production) and ODC1 (the key enzyme for polyamine synthesis). Placental  mRNA levels for GTP-CH1, eNOS, PGF, VEGF, and VEGFR2 increased in response to both 0.4% and 0.8% Arg supplementation. Collectively, these results indicate that dietary Arg supplementation to gilts between days 14 and 25 of pregnancy promotes placental angiogenesis by increasing the expression of mRNAs and proteins for angiogenic factors as well as NO and polyamine syntheses.

A Role for Fructose Metabolism in Development of Sheep and Pig Conceptuses.

The period of conceptus (embryo and extraembryonic membrane) development between fertilization and implantation in mammalian species is critical as it sets the stage for placental and fetal development. The trophectoderm and endoderm of pre-implantation ovine and porcine conceptuses undergo elongation, which requires rapid proliferation, migration, and morphological modification of the trophectoderm cells. These complex events occur in a hypoxic intrauterine environment and are supported through the transport of secretions from maternal endometrial glands to the conceptus required for the biochemical processes of cell proliferation, migration, and differentiation. The conceptus utilizes glucose provided by the mother to initiate metabolic pathways that provide energy and substrates for other metabolic pathways. Fructose, however, is in much greater abundance than glucose in amniotic and allantoic fluids, and fetal blood during pregnancy. Despite this, the role(s) of fructose is largely unknown even though a switch to fructosedriven metabolism in subterranean rodents and some cancers are key to their adaptation to hypoxic environments.

Cell-Specific Expression of Enzymes for Serine Biosynthesis and Glutaminolysis in Farm Animals.

During the peri-implantation period, conceptuses [embryo and placental membranes, particularly the trophectoderm (Tr)] of farm animals (e.g., sheep and pigs) rapidly elongate from spherical to tubular to filamentous forms. In concert with Tr outgrowth during conceptus elongation, the Tr of sheep and pig conceptuses attaches to the endometrial luminal epithelium (LE) to initiate placentation. In sheep, binucleate cells (BNCs) begin to differentiate from the mononuclear trophectoderm cells and migrate to the endometrial LE to form syncytial plaques. These events require Tr cells to expend significant amounts of energy to undergo timely and extensive proliferation, migration and fusion. It is likely essential that conceptuses optimally utilize multiple biosynthetic pathways to convert molecules such as glucose, fructose, and glutamine (components of histotroph transport by sheep and pig endometria into the uterine lumen), into ATP, amino acids, ribose, hexosamines and nucleotides required to support early conceptus development and survival. Elongating and proliferating conceptus Tr cells potentially act, in a manner similar to cancer cells, to direct carbon generated from glucose and fructose away from the TCA cycle for utilization in branching pathways of glycolysis, including the pentose phosphate pathway, one-carbon metabolism, and hexosamine biosynthesis. The result is a limited availability of pyruvate for maintaining the TCA cycle within mitochondria, and Tr cells replenish TCA cycle metabolites via a process known as anaplerosis, primarily through glutaminolysis to convert glutamine into TCA cycle intermediates. Here we describe the cell-specific expression of enzymes required for serine biosynthesis, one-carbon metabolism and glutaminolysis at the uterine-placental interface of sheep and pigs, and propose that these biosynthetic pathways are essential to support early placental development including Tr elongation, cell migration, cell fusion and implantation by ovine and porcine conceptuses.

Pig conceptuses secrete interferon gamma to recruit T cells to the endometrium during the peri-implantation period†.

The emerging paradigm in the immunology of pregnancy is that implantation of conceptuses does not progress in an immunologically suppressed environment. Rather, the endometrium undergoes a controlled inflammatory response during implantation as trophectoderm of elongating and implanting pig conceptuses secrete the pro-inflammatory cytokine interferon gamma (IFNG). Results of this study with pigs revealed: (1) accumulation of immune cells and apoptosis of stromal cells within the endometrium at sites of implantation during the period of IFNG secretion by conceptuses; (2) accumulation of proliferating cell nuclear antigen (PCNA)-positive T cells within the endometrium at sites of implantation; (3) significant increases in expression of T cell co-signaling receptors including programmed cell death 1 (PDCD1), CD28, cytotoxic T-lymphocyte associated protein 4 (CTLA-4), and inducible T cell co-stimulator (ICOS), as well as chemokines CXCL9, 10, and 11 within the endometrium at sites of implantation; (4) significant increases in T cell co-signaling receptors, PDCD1 and ICOS, and chemokine CXCL9 in the endometrium of cyclic gilts infused with IFNG; and (5) identification of CD4+ (22.59%) as the major T cell subpopulation, with minor subpopulations of CD8+ (1.38%), CD4+CD25+ (1.08%), and CD4+CD8+ (0.61%) T cells within the endometrium at sites of implantation. Our results provide new insights into the immunology of implantation to suggest that trophectoderm cells of pigs secrete IFNG to recruit various subpopulations of T cells to the endometrium to contribute to a controlled inflammatory environment that supports the active breakdown and restructuring of the endometrium in response to implantation of the conceptus.

Steroids Regulate SLC2A1 and SLC2A3 to Deliver Glucose Into Trophectoderm for Metabolism via Glycolysis.

The conceptuses (embryo/fetus and placental membranes) of pigs require energy to support elongation and implantation, and amounts of glucose and fructose increase in the uterine lumen during the peri-implantation period. Conceptuses from day 16 of pregnancy were incubated with either 14C-glucose or 14C-fructose and amounts of radiolabeled CO2 released from the conceptuses measured to determine rates of oxidation of glucose and fructose. Glucose and fructose both transport into conceptuses, and glucose is preferentially metabolized in the presence of fructose, whereas fructose is actively metabolized in the absence of glucose and to a lesser extent in the presence of glucose. Endometrial and placental expression of glucose transporters SLC2A1, SLC2A2, SCL2A3, and SLC2A4 were determined. SLC2A1 messenger RNA (mRNA) and protein, and SLC2A4 mRNA were abundant in the uterine luminal epithelium of pregnant compared to cycling gilts, and increased in response to progesterone and conceptus-secreted estrogen. SLC2A2 mRNA was expressed weakly by conceptus trophectoderm on day 15 of pregnancy, whereas SLC2A3 mRNA was abundant in trophectoderm/chorion throughout pregnancy. Therefore, glucose can be transported into the uterine lumen by SLC2A1, and then into conceptuses by SLC2A3. On day 60 of gestation, the cell-specific expression of these transporters was more complex, suggesting that glucose and fructose transporters are precisely regulated in a spatial-temporal pattern along the uterine-placental interface of pigs to maximize hexose sugar transport to the pig conceptus/placenta.

Cellular events during ovine implantation and impact for gestation.

The establishment of pregnancy in sheep includes elongation of the blastocyst into a filamentous conceptus, pregnancy recognition, production of histotroph, attachment of the conceptus to the endometrium for implantation, and development of synepitheliochorial placentation. These processes are complex, and this review describes some of the molecular events that underlie and support successful pregnancy. The free-floating sheep blastocyst elongates into a filamentous conceptus and metabolizes, or is responsive to, molecules supplied by the endometrium as histotroph. Amongst these molecules are SPP1, glucose and fructose, and arginine that stimulate the MTOR nutrient sensing system. The placental trophectoderm of elongating conceptuses initiate pregnancy recognition and implantation. The mononucleate cells of the trophectoderm secrete IFNT, which acts on the endometrial LE to block increases in estrogen receptor α to preclude oxytocin receptor expression, thereby preventing oxytocin from inducing luteolytic pulses of PGF2α. In addition, IFNT increases expression of IFN stimulated genes in the endometrial stroma, including ISG15, a functional ubiquitin homologue. Implantation is the initial step in placentation, and includes sequential pre-contact, apposition, and adhesion phases. Implantation in sheep includes downregulation of Muc1 and interaction of GLYCAM1, galectin 15 (LGALS15) and SPP1 with lectins and integrins (αvß3). Sheep have synepitheliochorial placentation in which mononucleate trophectoderm cells fuse to form binucleate cells (BNCs). BNCs migrate and fuse with endometrial LE cells to form trinucleate syncytial cells, and these syncytia enlarge through continued BNC fusion to form syncytial plaques that form the interface between endometrial and placental tissues within the placentome. The placentae of sheep organize into placentomal and interplacentomal regions. In placentomes there is extensive interdigitation of endometrial and placental tissues to provide hemotrophic nutrition to the fetus. In interplacentomal regions there is epitheliochorial attachment of endometrial LE to trophectoderm, mediated through focal adhesion assembly, and areolae that take up histotroph secreted by endometrial GE.