Showing papers by "Gregory A. Johnson published in 2022"

Insights into the Regulation of Implantation and Placentation in Humans, Rodents, Sheep, and Pigs.

Abstract: Precise cell-specific spatio-temporal molecular signaling cascades regulate the establishment and maintenance of pregnancy. Importantly, the mechanisms regulating uterine receptivity, conceptus apposition and adhesion to the uterine luminal epithelia/superficial glandular epithelia and, in some species, invasion into the endometrial stroma and decidualization of stromal cells, are critical prerequisite events for placentation which is essential for the appropriate regulation of feto-placental growth for the remainder of pregnancy. Dysregulation of these signaling cascades during this critical stage of pregnancy can lead to pregnancy loss, impaired growth and development of the conceptus, and alterations in the transplacental exchange of gasses and nutrients. While many of these processes are conserved across species, significant variations in the molecular mechanisms governing maternal recognition of pregnancy, conceptus implantation, and placentation exist. This review addresses the complexity of key mechanisms that are critical for the establishment and maintenance of a successful pregnancy in humans, rodents, sheep, and pigs. Improving understanding of the molecular mechanisms governing these processes is critical to enhancing the fertility and reproductive health of humans and livestock species.

Nutritional and Physiological Regulation of Water Transport in the Conceptus.

Abstract: Water transport during pregnancy is essential for maintaining normal growth and development of conceptuses (embryo/fetus and associated membranes). Aquaporins (AQPs) are a family of small integral plasma membrane proteins that primarily transport water across the plasma membrane. At least 11 isoforms of AQPs (AQPs 1–9, 11, and 12) are differentially expressed in the mammalian placenta (amnion, allantois, and chorion), and organs (kidney, lung, brain, heart, and skin) of embryos/fetuses during prenatal development. Available evidence suggests that the presence of AQPs in the conceptus mediates water movement across the placenta to support the placentation, the homeostasis of amniotic and allantoic fluid volumes, as well as embryonic and fetal survival, growth and development. Abundances of AQPs in the conceptus can be modulated by nutritional status and physiological factors affecting the pregnant female. Here, we summarize the effects of maternal dietary factors (such as intakes of protein, arginine, lipids, all-trans retinoic acid, copper, zinc, and mercury) on the expression of AQPs in the conceptus. We also discuss the physiological changes in hormones (e.g., progesterone and estrogen), oxygen supply, nitric oxide, pH, and osmotic pressure associated with the regulation of fluid exchange between mother and fetus. These findings may help to improve the survival, growth, and development of embryo/fetus in livestock species and other mammals (including humans).

Metabolic pathways utilized by the porcine conceptus, uterus, and placenta.

Abstract: Conceptus elongation and early placentation involve growth and remodeling that requires proliferation and migration of cells. This demands conceptuses expend energy before establishment of a placenta connection and when they are dependent upon components of histotroph secreted or transported into the uterine lumen from the uterus. Glucose and fructose, as well as many amino acids (including arginine, aspartate, glutamine, glutamate, glycine, methionine, and serine), increase in the uterine lumen during the peri-implantation period. Glucose and fructose enter cells via their transporters, SLC2A, SLC2A3, and SLC2A8, and amino acids enter the cells via specific transporters that are expressed by the conceptus trophectoderm. However, porcine conceptuses develop rapidly through extensive cellular proliferation and migration as they elongate and attach to the uterine wall resulting in increased metabolic demands. Therefore, coordination of multiple metabolic biosynthetic pathways is an essential aspect of conceptus development. Oxidative metabolism primarily occurs through the tricarboxylic acid (TCA) cycle and the electron transport chain, but proliferating and migrating cells, like the trophectoderm of pigs, enhance aerobic glycolysis. The glycolytic intermediates from glucose can then be shunted into the pentose phosphate pathway and one-carbon metabolism for the de novo synthesis of nucleotides. A result of aerobic glycolysis is limited availability of pyruvate for maintaining the TCA cycle, and trophectoderm cells likely replenish TCA cycle metabolites primarily through glutaminolysis to convert glutamine into TCA cycle intermediates. The synthesis of ATP, nucleotides, amino acids, and fatty acids through these biosynthetic pathways is essential to support elongation, migration, hormone synthesis, implantation, and early placental development of conceptuses.

Osteopontin (OPN)/Secreted Phosphoprotein 1 (SPP1) Binds Integrins to Activate Transport of Ions Across the Porcine Placenta.

Abstract: BACKGROUND Fetal-placental development depends on a continuous and efficient supply of nutrients from maternal blood that are acquired by exchange through the placenta. However, the placenta is a low permeability barrier, and effective transport of substances depends on specific transport mechanisms. Active transport requires that ions or nutrients be moved against an electrical and/or concentration gradient. In pigs, active transport of ions occurs across the chorioallantois placenta to produce an electrochemical gradient that changes throughout gestation. The aim of this study was to utilize Ussing chambers to detect regulation of ion transport across the porcine chorioallantois by a factor(s) within the uterine-placental environment of pigs. METHODS For the measurement of transchorioallantoic voltage potential as an index of ion transport across the placenta, pieces of chorioallantoic tissue from Day 60 of gestation were mounted into the cassettes of Ussing chambers, and treatments were added to the mucasal side of the tissue. Treatments included: (1) media incubated with Day 60 chorioallantois (placenta-conditioned media); (2) osteopontin/secreted phosphoprotein 1 (OPN/SPP1) purified from cow's milk; (3) placenta-conditioned media from which OPN/SPP1 was removed; and (4) recombinant rat OPN with an intact RGD integrin binding sequence or a mutated RAD sequence. Ouabain was added to both sides of the chamber. Immunofluorescence was utilized to localize beta 3 integrin, aquaporin 8 and OPN/SPP1 in porcine placental tissues, and OPN/SPP1 within porcine lung, kidney and small intestine. RESULTS Day 60 chorioallantoic membranes had greater transepithelial voltage in the presence of porcine placenta-conditioned media, indicating that a molecule(s) released from the placenta increased ion transport across the placenta. OPN/SPP1 purified from cow's milk increased ion transport across the placenta. When OPN/SPP1 was removed from placenta-conditioned media, ion transport across the placenta did not increase. Recombinant rat OPN/SPP1 with a mutated RGD sequence that does not bind integrins (RAD) did not increase ion transport across the placenta. Ouabain, an inhibiter of the sodium-potassium ion pump, ablated ion transport across the placenta. CONCLUSIONS The present study documents a novel pericellular matrix role for OPN/SPP1 to bind integrins and increase ion transport across the porcine chorioallantoic placenta.

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

Abstract: 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.

Arginine Promotes the Expression of Aquaporin-3 and Water Transport in Porcine Trophectoderm Cells Through NO- and cAMP-Dependent Mechanisms.

Abstract: BACKGROUND Dietary supplementation with L-arginine (Arg) has been shown to increase the volume of fetal fluids in gestating swine. Aquaporins (AQPs), known as water channel proteins, are essential for embryonic growth and development. It was not known if Arg mediates water transport through AQPs in porcine conceptus trophectoderm (pTr2) cells. METHODS pTr2 cells derived from pregnant gilts on day 12 of gestation were cultured in customized Arg-free Dulbecco's modified Eagle's Ham medium (DMEM) supplemented with either 0.00, 0.25, or 0.50 mM Arg. RESULTS Arg treatment increased water transport and the expression of AQP3, which was abundantly expressed in pTr2 cells at both the mRNA and protein levels. Arg also increased the expression of iNOS and the synthesis of nitric oxide (NO) in pTr2 cells. The presence of Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME; an inhibitor of NO synthase) significantly attenuated the Arg-induced expression of AQP3. Furthermore, 0.50 mM Arg increased the concentrations of cAMP and the abundances of phosphorylated cAMP-dependent protein kinase A (PKA), phosphorylated PKA α/β/γ, and phosphorylated CREB. These effects of Arg were mimicked by Forskolin (a cell-permeable activator of adenylyl cyclase), but inhibited by H-89 (an inhibitor of cAMP-dependent protein kinase). CONCLUSIONS The results of this study demonstrate that Arg regulates AQP3 expression and promotes water transport in pTr2 cells through NO- and cAMP-dependent signaling pathways.

Dietary supplementation with 0.4% L-arginine between days 14 and 30 of gestation enhances NO and polyamine syntheses and water transport in porcine placentae

Abstract: Abstract Background Most embryonic loss in pigs occurs before d 30 of gestation. Dietary supplementation with L -arginine (Arg) during early gestation can enhance the survival and development of conceptuses (embryo/fetus and its extra-embryonic membranes) in gilts. However, the underlying mechanisms remain largely unknown. Methods Between d 14 and 30 of gestation, each gilt was fed daily 2 kg of a corn- and soybean-meal based diet (12% crude protein) supplemented with either 0.4% Arg (as Arg-HCl) or an isonitrogenous amount of L -alanine (Control). There were 10 gilts per treatment group. On d 30 of gestation, gilts were fed either Arg-HCl or L -alanine 30 min before they were hysterectomized, followed by the collection of placentae, embryos, fetal membranes, and fetal fluids. Amniotic and allantoic fluids were analyzed for nitrite and nitrate [NOx; stable oxidation products of nitric oxide (NO)], polyamines, and amino acids. Placentae were analyzed for syntheses of NO and polyamines, water and amino acid transport, concentrations of amino acid-related metabolites, and the expression of angiogenic factors and aquaporins (AQPs). Results Compared to the control group, Arg supplementation increased ( P < 0.05) the number of viable fetuses by 1.9 per litter, the number and diameter of placental blood vessels (+ 25.9% and + 17.0% respectively), embryonic survival (+ 18.5%), total placental weight (+ 36.5%), the total weight of viable fetuses (+ 33.5%), fetal crown-to-rump length (+ 4.7%), and total allantoic and amniotic fluid volumes (+ 44.6% and + 75.5% respectively). Compared to control gilts, Arg supplementation increased ( P < 0.05) placental activities of GTP cyclohydrolase-1 (+ 33.1%) and ornithine decarboxylase (+ 29.3%); placental syntheses of NO (+ 26.2%) and polyamines (+ 28.9%); placental concentrations of NOx (+ 22.5%), tetrahydrobiopterin (+ 21.1%), polyamines (+ 20.4%), cAMP (+ 27.7%), and cGMP (+ 24.7%); total amounts of NOx (+ 61.7% to + 96.8%), polyamines (+ 60.7% to + 88.7%), amino acids (+ 39% to + 118%), glucose (+ 60.5% to + 62.6%), and fructose (+ 41.4% to + 57.0%) in fetal fluids; and the placental transport of water (+ 33.9%), Arg (+ 78.4%), glutamine (+ 89.9%), and glycine (+ 89.6%). Furthermore, Arg supplementation increased ( P < 0.05) placental mRNA levels for angiogenic factors [ VEGFA120 (+ 117%), VEGFR1 (+ 445%), VEGFR2 (+ 373%), PGF (+ 197%), and GCH1 (+ 126%)] and AQPs [ AQP1 (+ 280%), AQP3 (+ 137%), AQP5 (+ 172%), AQP8 (+ 165%), and AQP9 (+ 127%)]. Conclusion Supplementing 0.4% Arg to a conventional diet for gilts between d 14 and d 30 of gestation enhanced placental NO and polyamine syntheses, angiogenesis, and water and amino acid transport to improve conceptus development and survival.

Creatine metabolism at the uterine–conceptus interface during early gestation in sheep

Abstract: Abstract Ruminant conceptuses that elongate and attach to the uterine luminal epithelium (LE) to establish pregnancy require a large amount of adenosine triphosphate (ATP). The creatine (Cr)–creatine kinase (CK)–phosphocreatine (PCr) system re-generates ATP in dividing and migrating cells such as the conceptus trophectoderm cells. However, little is known about metabolism of Cr within uterine and conceptus tissues in livestock species during early gestation. In this study, Suffolk ewes were ovariohysterectomized on Days 9, 12, 15, 16, 17, 18, 20, or 21 of pregnancy (n = 2–5 animals/per day) to investigate metabolites, mRNAs, and proteins of the Cr–CK–PCr system at uterine–conceptus interface. Amounts of Cr and guanidinoacetate (GA) in uterine flushings increased between Days 12 and 17 of pregnancy. Endometrial expression of mRNAs for GA formation (AGAT), Cr synthesis (GAMT), and Cr/PCr utilization (CKB) was greater on Days 17 and 21 than on Days 9 and 12 of pregnancy. Immunoreactive AGAT was detected in uteri only on Day 21 but not in uteri or conceptuses at earlier days of pregnancy. GAMT, SLC6A8, and CKs were expressed in uterine luminal and glandular epithelia. Immunoreactive CKs (CKB, CKM, and CKMT1) appeared greater on Day 9 than Day 17 of pregnancy. Immunoreactive GAMT and CKs appeared greater in trophectoderm of conceptuses on Day 20 than on Day 15 of pregnancy, whereas the opposite was observed for that of SLC6A8. This study provides insights into cell-, tissue-, and time-specific metabolism of Cr at the uterine–conceptus interface suggesting a role for the Cr–CK–PCr system in ovine conceptus development and implantation. Summary Sentence Creatine is abundant in the histotroph, and the proteins required for synthesis and transport of creatine are expressed in uteri and conceptuses during early gestation in sheep indicating a role for creatine metabolism in conceptus development.

Ovine conceptus tissue metabolizes fructose for metabolic support during the peri-implantation period of pregnancy

Abstract: Abstract Roles of fructose in elongating ovine conceptuses are poorly understood, despite it being the major hexose sugar in fetal fluids and plasma throughout gestation. Therefore, we determined if elongating ovine conceptuses utilize fructose via metabolic pathways for survival and development. Immunohistochemical analyses revealed that trophectoderm and extra-embryonic endoderm express ketohexokinase and aldolase B during the peri-implantation period of pregnancy for conversion of fructose into fructose-1-phosphate for entry into glycolysis and related metabolic pathways. Conceptus homogenates were cultured with 14C-labeled glucose and/or fructose under oxygenated and hypoxic conditions to assess contributions of glucose and fructose to the pentose cycle (PC), tricarboxylic acid cycle, glycoproteins, and lipid synthesis. Results indicated that both glucose and fructose contributed carbons to each of these pathways, except for lipid synthesis, and metabolized to pyruvate and lactate, with lactate being the primary product of glycolysis under oxygenated and hypoxic conditions. We also found that (1) conceptuses preferentially oxidized glucose over fructose (P < 0.05); (2) incorporation of fructose and glucose at 4 mM each into the PC by Day 16 conceptus homogenates was similar in the presence or absence of glucose, but incorporation of glucose into the PC was enhanced by the presence of fructose (P < 0.05); (3) incorporation of fructose into the PC in the absence of glucose was greater under oxygenated conditions (P < 0.01); and (4) incorporation of glucose into the PC under oxygenated conditions was greater in the presence of fructose (P = 0.05). These results indicate that fructose is an important metabolic substrate for ovine conceptuses. Summary Sentence Fructose and glucose are substrates for the pentose cycle and tricarboxylic acid cycle in ovine conceptuses.

278 Awardee Talk: Progesterone Regulates the Abundance of Mrnas and Proteins Involved in Polyamine Metabolism in the Sheep Endometrium

Abstract: Agmatine and polyamines (putrescine, spermidine, and spermine), metabolites of L-arginine, are important for development of mammalian conceptuses. The objective of this study was to determine if exogenous progesterone administered during the pre-implantation period, which accelerates conceptus development, alters the expression of mRNAs and proteins involved in the metabolism of agmatine and polyamines in the sheep uterus during pregnancy. Mature Suffolk ewes were bred to fertile rams and assigned randomly to be treated with daily i.m. injections of either 1ml corn oil (CO;n=28) or 25mg progesterone in corn oil (P4;n=20) from Day 1.5 through Day 8 after breeding (Day 0). Ewes were hysterectomized on Day 9, 12, or 125 of gestation and endometrial samples were collected. The expression of candidate mRNAs was quantified using qPCR and proteins localized using immunohistochemistry. On Day 9 of pregnancy, the expression of mRNAs for AMD1 (P < 0.01), ASL (P < 0.01), and SLC12A8 (P < 0.05) was greater in endometria of P4-treated compared to CO-treated ewes. The expression for MAT2B mRNA was greater (P < 0.001) for P4-treated compared to CO-treated ewes on both Days 9 and 12. On Day 125 of gestation, endometria from P4-treated ewes tended to have greater expression of SAT1 (P < 0.10) mRNA compared to CO-treated ewes. In contrast, the expression of AMD1(P < 0.01), MAT2B (P < 0.05), OAT (P < 0.05), SMOX (P < 0.05), and SLC12A8 (P < 0.05) mRNAs was lower in endometria from P4-treated compared to CO-treated ewes on Day 125. Exogenous P4 appeared to increase the relative abundance of SAT1 and SMOX proteins in the uterine luminal and glandular epithelia on Day 125 of pregnancy. These results suggest that early administration of progesterone may alter the biosynthesis of agmatine and polyamines in the sheep uterus during the pre-implantation period of pregnancy with potential ‘programming’ effects manifested in late pregnancy.