RESUMO
Fructose, the most abundant hexose sugar in fetal fluids and the blood of sheep and other ungulates and cetaceans, is synthesized from glucose via the polyol pathway in trophectoderm and chorion. However, the cell-specific and temporal expression of enzymes for the synthesis and metabolism of fructose in sheep conceptuses (embryo and placental membranes) and placentomes has not been characterized. This study characterized key enzymes involved in fructose synthesis and metabolism by ovine conceptuses throughout pregnancy. Day 17 conceptuses expressed mRNAs for the polyol pathway (SORD and AKR1B1) and glucose and fructose metabolism (HK1, HK2, G6PD, OGT, and FBP), but not those required for gluconeogenesis (G6Pase or PCK). Ovine placentomes also expressed mRNAs for SORD, AKR1B1, HK1, and OGT. Fructose can be metabolized via the ketohexokinase (KHK) pathway, and isoforms, KHK-A and KHK-C, were expressed in ovine conceptuses from Day 16 of pregnancy and placentomes during pregnancy in a cell-specific manner. The KHK-A protein was more abundant in the trophectoderm and cotyledons of placentomes, while KHK-C protein was more abundant in the endoderm of Day 16 conceptuses and the chorionic epithelium in placentomes. Expression of KHK mRNAs in placentomes was greatest at Day 30 of pregnancy (P < 0.05), but not different among days later in gestation. These results provide novel insights into the synthesis and metabolism of fructose via the uninhibited KHK pathway in ovine conceptuses to generate ATP via the tricarboxylic cycle, as well as substrates for the pentose cycle, hexosamine biosynthesis pathway, and one-carbon metabolism required for conceptus development throughout pregnancy.
Assuntos
Frutose , Glucose , Placenta , Animais , Feminino , Frutose/metabolismo , Gravidez , Ovinos/metabolismo , Glucose/metabolismo , Placenta/metabolismo , Redes e Vias Metabólicas/genética , Embrião de Mamíferos/metabolismoRESUMO
Lactate, an abundant molecule in fetal fluids and blood of mammalian species, is often overlooked as a metabolic waste product generated during pregnancy. Most of the glucose and fructose consumed by ovine conceptuses is converted to lactate, but proteins involved in lactate metabolism and transport have not been investigated. This study characterized total lactate produced by ovine conceptuses throughout gestation, as well as expression of mRNAs and proteins involved in lactate metabolism. Lactate increased in abundance in the uterine lumen during the preimplantation period and was more abundant than pyruvate. The abundance of lactate in allantoic and amniotic fluids increased with advancing days of gestation and most abundant on Day 125 of pregnancy (P < 0.05). Lactate dehydrogenase subunits A (converts pyruvate to lactate) and B (converts lactate to pyruvate) were expressed by conceptuses throughout gestation. Lactate is transported via monocarboxylic acid transporters SLC16A1 and SLC16A3, both of which were expressed by the conceptus throughout gestation. Additionally, the interplacentomal chorioallantois from Day 126 expressed SLC16A1 and SLC16A3 and transported lactate across the tissue. Hydrocarboxylic acid receptor 1 (HCAR1), a receptor for lactate, was localized to the uterine luminal and superficial glandular epithelia of pregnant ewes throughout gestation and conceptus trophectoderm during the peri-implantation period of gestation. These results provide novel insights into the spatiotemporal profiles of enzymes, transporters, and receptor for lactate by ovine conceptuses throughout pregnancy.
Assuntos
Frutose , Glucose , Ácido Láctico , Animais , Feminino , Gravidez , Ácido Láctico/metabolismo , Ácido Láctico/sangue , Ovinos , Glucose/metabolismo , Frutose/metabolismo , Redes e Vias Metabólicas/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transporte Biológico , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Tissue-nonspecific alkaline phosphatase (TNSALP; encoded by ALPL gene) has a critical role in the regulation of phosphate homeostasis postnatally. However, the utero-placental expression of TNSALP and the role in phosphate transport in pregnancy is poorly understood. Estrous cycles of ewes were synchronized, and ewes were euthanized and hysterectomized on Days 1, 9, or 14 of the estrous cycle or bred to fertile rams and euthanized and hysterectomized on Days 9, 12, 17, 30, 50, 70, 90, 110, or 125 of pregnancy. The expression of ALPL mRNA, immunolocalization of TNSALP protein, and quantification and localization of TNSALP enzymatic activity was performed on ovine endometria and placentomes. Day of the estrous cycle did not alter ALPL mRNA expression or enzymatic activity of TNSALP. TNSALP protein localized to uterine epithelial and stromal cells, blood vessels, myometrium, caruncular, and cotyledonary stroma. TNSALP activity was localized to uterine epithelia, blood vessels, caruncular stroma (from Day 70 of gestation), and the apical surface of chorionic epithelia (from Day 50 of gestation). TNSALP protein and activity localized to the apical surface of uterine epithelia during the estrous cycle and in early pregnancy. Endometrial TNSALP enzymatic activity was downregulated on Days 17 and 30 of gestation (P < 0.05). Expression of ALPL mRNA decreased in late gestation in endometria and placentomes (P < 0.05). TNSALP activity peaked in placentomes on Days 70 and 90 of gestation. Collectively, these results suggest a potential role of TNSALP in the regulation of phosphate transport and homeostasis at the maternal-conceptus interface in ruminants.
Assuntos
Fosfatase Alcalina , Placenta , Gravidez , Ovinos , Animais , Feminino , Masculino , Placenta/metabolismo , Fosfatase Alcalina/metabolismo , Útero/metabolismo , Endométrio/metabolismo , Carneiro Doméstico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fosfatos/metabolismoRESUMO
Progesterone (P4), estradiol (E2), and expression of their receptors (PGR and ESR1, respectively) by cells of the uterus regulate reproductive performance of mammals through effects on secretion and transport of nutrients into the uterine lumen. This study investigated the effect of changes in P4, E2, PGR, and ESR1 on expression of enzymes for the synthesis and secretion of polyamines. Suffolk ewes (n = 13) were synchronized to estrus (Day 0) and then, on either Day 1 (early metestrus), Day 9 (early diestrus), or Day 14 (late diestrus) of the estrous cycle, maternal blood samples were collected, and ewes were euthanized before obtaining uterine samples and uterine flushings. Endometrial expression of MAT2B and SMS mRNAs increased in late diestrus (P < 0.05). Expression of ODC1 and SMOX mRNAs decreased from early metestrus to early diestrus, and expression of ASL mRNA was lower in late diestrus than in early metestrus (P < 0.05). Immunoreactive PAOX, SAT1, and SMS proteins were localized to uterine luminal, superficial glandular, and glandular epithelia, stromal cells, myometrium, and blood vessels. Concentrations of spermidine and spermine in maternal plasma decreased from early metestrus to early diestrus and decreased further in late diestrus (P < 0.05). The abundances of spermidine and spermine in uterine flushings were less in late diestrus than early metestrus (P < 0.05). These results indicate that synthesis and secretion of polyamines are affected by P4 and E2, as well as the expression of PGR and ESR1 in the endometria of cyclic ewes.
Assuntos
Estradiol , Progesterona , Feminino , Animais , Ovinos , Estradiol/metabolismo , Poliaminas/metabolismo , Espermidina/metabolismo , Espermina/metabolismo , Rubor/metabolismo , Útero/metabolismo , Receptores de Progesterona/metabolismo , Mamíferos/metabolismoRESUMO
The placenta requires high levels of adenosine triphosphate to maintain a metabolically active state throughout gestation. The creatine-creatine kinase-phosphocreatine system is known to buffer adenosine triphosphate levels; however, the role(s) creatine-creatine kinase-phosphocreatine system plays in uterine and placental metabolism throughout gestation is poorly understood. In this study, Suffolk ewes were ovariohysterectomized on Days 30, 50, 70, 90, 110 and 125 of gestation (n = 3-5 ewes/per day, except n = 2 on Day 50) and uterine and placental tissues subjected to analyses to measure metabolites, mRNAs, and proteins related to the creatine-creatine kinase-phosphocreatine system. Day of gestation affected concentrations and total amounts of guanidinoacetate and creatine in maternal plasma, amniotic fluid and allantoic fluid (P < 0.05). Expression of mRNAs for arginine:glycine amidinotransferase, guanidinoacetate methyltransferase, creatine kinase B, and solute carrier 16A12 in endometria and for arginine:glycine amidinotransferase and creatine kinase B in placentomes changed significantly across days of gestation (P < 0.05). The arginine:glycine amidinotransferase protein was more abundant in uterine luminal epithelium on Days 90 and 125 compared to Days 30 and 50 (P < 0.01). The chorionic epithelium of placentomes expressed guanidinoacetate methyltransferase and solute carrier 6A13 throughout gestation. Creatine transporter (solute carrier 6A8) was expressed by the uterine luminal epithelium and trophectoderm of placentomes throughout gestation. Creatine kinase (creatine kinase B and CKMT1) proteins were localized primarily to the uterine luminal epithelium and to the placental chorionic epithelium of placentomes throughout gestation. Collectively, these results demonstrate cell-specific and temporal regulation of components of the creatine-creatine kinase-phosphocreatine system that likely influence energy homeostasis for fetal-placental development.
Assuntos
Creatina , Placenta , Gravidez , Feminino , Animais , Ovinos , Placenta/metabolismo , Creatina/metabolismo , Guanidinoacetato N-Metiltransferase/metabolismo , Fosfocreatina/metabolismo , Creatina Quinase/metabolismo , Trifosfato de Adenosina/metabolismo , ArgininaRESUMO
Highly proliferative cells rely on one carbon (1C) metabolism for production of formate required for synthesis of purines and thymidine for nucleic acid synthesis. This study was to determine if extracellular serine and/or glucose and fructose contribute the production of formate in ovine conceptuses. Suffolk ewes (n = 8) were synchronized to estrus, bred to fertile rams, and conceptuses were collected on Day 17 of gestation. Conceptuses were either snap frozen in liquid nitrogen (n = 3) or placed in culture in medium (n = 5) containing either: 1) 4 mM D-glucose + 2 mM [U-13C]serine; 2) 6 mM glycine + 4 mM D-glucose + 2 mM [U-13C]serine; 3) 4 mM D-fructose + 2 mM [U-13C]serine; 4) 6 mM glycine + 4 mM D-fructose + 2 mM [U-13C]serine; 5) 4 mM D-glucose + 4 mM D-fructose + 2 mM [U-13C]serine; or 6) 6 mM glycine + 4 mM D-glucose + 4 mM D-fructose + 2 mM [U-13C]serine. After 2 h incubation, conceptuses in their respective culture medium were homogenized and the supernatant analyzed for 12C- and 13C-formate by gas chromatography and amino acids by high performance liquid chromatography. Ovine conceptuses produced both 13C- and 12C-formate, indicating that the [U-13C]serine, glucose, and fructose were utilized to generate formate, respectively. Greater amounts of 12C-formate than 13C-formate were produced, indicating that the ovine conceptus utilized more glucose and fructose than serine to produce formate. This study is the first to demonstrate that both 1C metabolism and serinogenesis are active metabolic pathways in ovine conceptuses during the peri-implantation period of pregnancy, and that hexose sugars are the preferred substrate for generating formate required for nucleotide synthesis for proliferating trophectoderm cells.
Assuntos
Interferon Tipo I , Serina , Gravidez , Ovinos , Animais , Feminino , Masculino , Glucose , Frutose , Carneiro Doméstico/metabolismo , Glicina , FormiatosRESUMO
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.
Assuntos
Creatina , Proteínas da Gravidez , Gravidez , Ovinos , Animais , Feminino , Creatina/metabolismo , Proteínas da Gravidez/metabolismo , Útero/metabolismo , Implantação do Embrião , Endométrio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trifosfato de Adenosina/metabolismoRESUMO
The one-carbon metabolism (OCM) pathway provides purines and thymidine for synthesis of nucleic acids required for cell division, and S-adenosyl methionine for polyamine and creatine syntheses and the epigenetic regulation of gene expression. This study aimed to determine if serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in the OCM pathway, is critical for ovine trophectoderm (oTr) cell function and conceptus development by inhibiting translation of SHMT2 mRNA using a morpholino antisense oligonucleotide (MAO). In vitro treatment of oTr cells with MAO-SHMT2 decreased expression of SHMT2 protein, which was accompanied by reduced proliferation (P = 0.053) and migration (P < 0.05) of those cells. Intrauterine injection of MAO-SHMT2 in ewes on Day 11 post-breeding tended to decrease the overall pregnancy rate (on Days 16 and 18) compared with MAO-control (3/10 vs. 7/10, P = 0.07). The three viable conceptuses (n = 2 on Day 16 and n = 1 on Day 18) recovered from MAO-SHMT2 ewes had only partial inhibition of SHMT2 mRNA translation. Conceptuses from the three pregnant MAO-SHMT2 ewes had similar levels of expression of mRNAs and proteins involved in OCM as compared with conceptuses from MAO-control ewes. These results indicate that knockdown of SHMT2 protein reduces proliferation and migration of oTr cells (in vitro) to decrease elongation of blastocysts from spherical to elongated forms. These in vitro effects suggest that increased embryonic deaths in ewes treated with MAO-SHMT2 are the result of decreased SHMT2-mediated trophectoderm cell proliferation and migration supporting a role for the OCM pathway in survival and development of ovine conceptuses.
Assuntos
Implantação do Embrião , Epigênese Genética , Gravidez , Ovinos , Animais , Feminino , Implantação do Embrião/fisiologia , Biossíntese de Proteínas , Embrião de Mamíferos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Oligonucleotídeos Antissenso/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Monoaminoxidase/genética , Monoaminoxidase/metabolismo , Monoaminoxidase/farmacologiaRESUMO
Progesterone (P4) and interferon tau (IFNT) are important for establishment and maintenance of pregnancy in ruminants. Agmatine and polyamines (putrescine, spermidine, and spermine) have important roles in the survival, growth, and development of mammalian conceptuses. This study tested the hypothesis that P4 and/or IFNT stimulate the expression of genes and proteins involved in the metabolism and transport of polyamines in the ovine endometrium. Rambouillet ewes (n = 24) were surgically fitted with intrauterine catheters on Day 7 of the estrous cycle. They received daily intramuscular injections of 50 mg P4 in corn oil vehicle and/or 75-mg progesterone receptor antagonist (RU486) in corn oil vehicle from Days 8-15, and twice daily intrauterine injections (25 µg/uterine horn/day) of either control serum proteins (CX) or IFNT from Days 11-15, resulting in four treatment groups: (i) P4 + CX; (ii) P4 + IFNT; (iii) RU486 + P4 + CX; or (iv) RU486 + P4 + IFNT. On Day 16, ewes were hysterectomized. The total amounts of arginine, citrulline, ornithine, agmatine, and putrescine in uterine flushings were affected (P < 0.05) by P4 and/or IFNT. P4 increased endometrial expression of SLC22A2 (P < 0.01) and SLC22A3 (P < 0.05) mRNAs. IFNT affected endometrial expression of MAT2B (P < 0.001), SAT1 (P < 0.01), and SMOX (P < 0.05) mRNAs, independent of P4. IFNT increased the abundance of SRM protein in uterine luminal (LE), superficial glandular (sGE), and glandular epithelia (GE), as well as MAT2B protein in uterine LE and sGE. These results indicate that P4 and IFNT act synergistically to regulate the expression of key genes required for cell-specific metabolism and transport of polyamines in the ovine endometrium during the peri-implantation period of pregnancy.
Assuntos
Agmatina , Interferon Tipo I , Agmatina/metabolismo , Agmatina/farmacologia , Animais , Óleo de Milho/metabolismo , Endométrio/metabolismo , Feminino , Interferon Tipo I/metabolismo , Mifepristona , Poliaminas/metabolismo , Gravidez , Proteínas da Gravidez , Progesterona/metabolismo , Proteínas/metabolismo , Putrescina , RNA Mensageiro/metabolismo , Ovinos , Carneiro Doméstico , Útero/metabolismoRESUMO
Given recent reports of expression of postnatal mineral transport regulators at the maternal-conceptus interface during the peri-implantation period, this study tested the hypothesis that progesterone (P4) and interferon tau (IFNT) regulate phosphate, calcium, and vitamin D signaling in the ovine endometrium. Mature Rambouillet ewes (n = 24) were surgically fitted with intrauterine catheters on day 7 of the estrous cycle. Ewes received daily intramuscular injections of 50 mg of P4 in corn oil vehicle and 75 mg of progesterone receptor antagonist (RU486) in corn oil from days 8 to 15, and twice-daily intrauterine injections of either control proteins (CX) or IFNT (25 µg/uterine horn/day) from days 11 to 15 resulting in four treatment groups: P4 + CX; P4 + IFNT; RU486 + P4 + CX; and RU486 + P4 + IFNT. On day 16, ewes were hysterectomized. RU486 + P4 + CX treated ewes had lower concentrations of 25 (OH) D in plasma than P4 + CX treated ewes (P < 0.05). Endometria from ewes treated with IFNT had greater expression of FGF23 (P < 0.01), S100A9 (P < 0.05), and S100A12 (P = 0.05) mRNAs and lower expression of ADAM10 mRNA (P < 0.01) than of ewes treated with CX proteins. Expression of FGF23 mRNA was greater in endometria of ewes that received RU486 + P4 + IFNT than in ewes that received RU486 + P4 + CX (hormone × protein interaction, P < 0.05). The expression of S100G mRNA was greater in endometria of ewes that received P4 + IFNT compared to ewes that received RU486 + P4 + IFNT (P < 0.05; hormone × protein interaction, P < 0.01). These data implicate P4 and IFNT in the regulation of phosphate, calcium, and vitamin D signaling during the peri-implantation period of pregnancy and provide a platform for continued mechanistic investigations.
Assuntos
Interferon Tipo I , Progesterona , Animais , Cálcio/metabolismo , Óleo de Milho/metabolismo , Óleo de Milho/farmacologia , Endométrio/metabolismo , Feminino , Interferon Tipo I/metabolismo , Mifepristona/farmacologia , Fosfatos/metabolismo , Fosfatos/farmacologia , Gravidez , Proteínas da Gravidez , Progesterona/metabolismo , Progesterona/farmacologia , Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ovinos , Carneiro Doméstico , Vitamina D/farmacologiaRESUMO
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.
Assuntos
Frutose-Bifosfato Aldolase , Frutose , Animais , Feminino , Frutoquinases , Glucose , Lactatos , Lipídeos , Pentoses , Gravidez , Piruvatos , Ovinos , Carneiro DomésticoRESUMO
This study aimed to determine whether the acceleration of conceptus development induced by the administration of exogenous progesterone (P4) during the preimplantation period of pregnancy alters calcium, phosphate, and vitamin D signaling at the maternal-conceptus interface. Suffolk ewes (n = 48) were mated to fertile rams and received daily intramuscular injections of either corn oil (CO) vehicle or 25 mg of progesterone in CO (P4) for the first 8 days of pregnancy and hysterectomized on either Day 9 (CO, n = 5; P4, n = 6), 12 (CO, n = 9; P4, n = 4) or 125 (CO, n = 14; P4, n = 10) of gestation. The expression of S100A12 (P < 0.05) and fibroblast growth factor receptor (FGFR2) (P < 0.01) messenger RNAs (mRNAs) was lower in endometria from P4-treated ewes on Day 12. The expression of ADAM10 (P < 0.05) mRNA was greater in endometria from P4-treated ewes on Day 125. The expression of ADAM10 (P < 0.01), FGFR2 (P < 0.05), solute carrier (SLC)20A1 (P < 0.05), TRPV5 (P < 0.05), and TRPV6 (P < 0.01) mRNAs was greater, but KL mRNA expression was lower (P < 0.05) in placentomes from P4-treated ewes at Day 125. There was lower endometrial and greater placentomal expression of mRNAs involved in mineral metabolism and transport in twin compared to singleton pregnancies. Further, the expression of mRNAs involved in mineral metabolism and transport was greater in P4-treated twin placentomes. KL, FGF23, vitamin D receptor (VDR), S100A9, S100A12, S100G, and CYP27B1 proteins were immunolocalized in endometria and placentomes. Exogenous P4 in early pregnancy altered the expression of regulators of calcium, phosphate, and vitamin D on Day 125 of pregnancy indicating a novel effect of P4 on mineral transport at the maternal-conceptus interface.
Assuntos
Cálcio , Progesterona , Animais , Cálcio/metabolismo , Endométrio/metabolismo , Feminino , Masculino , Minerais/metabolismo , Minerais/farmacologia , Fosfatos/metabolismo , Fosfatos/farmacologia , Placenta/metabolismo , Gravidez , Progesterona/metabolismo , RNA Mensageiro/metabolismo , Proteína S100A12/metabolismo , Proteína S100A12/farmacologia , Ovinos , Carneiro Doméstico , Vitamina D/farmacologiaRESUMO
Minerals are required for the establishment and maintenance of pregnancy and regulation of fetal growth in mammals. Lentiviral-mediated RNA interference (RNAi) of chorionic somatomammotropin hormone (CSH) results in both an intrauterine growth restriction (IUGR) and a non-IUGR phenotype in sheep. This study determined the effects of CSH RNAi on the concentration and uptake of calcium, phosphate, and vitamin D, and the expression of candidate mRNAs known to mediate mineral signaling in caruncles (maternal component of placentome) and cotyledons (fetal component of placentome) on gestational day 132. CSH RNAi Non-IUGR pregnancies had a lower umbilical vein−umbilical artery calcium gradient (p < 0.05) and less cotyledonary calcium (p < 0.05) and phosphate (p < 0.05) compared to Control RNAi pregnancies. CSH RNAi IUGR pregnancies had less umbilical calcium uptake (p < 0.05), lower uterine arterial and venous concentrations of 25(OH)D (p < 0.05), and trends for lower umbilical 25(OH)D uptake (p = 0.059) compared to Control RNAi pregnancies. Furthermore, CSH RNAi IUGR pregnancies had decreased umbilical uptake of calcium (p < 0.05), less uterine venous 25(OH)D (vitamin D metabolite; p = 0.055), lower caruncular expression of SLC20A2 (sodium-dependent phosphate transporter; p < 0.05) mRNA, and lower cotyledonary expression of KL (klotho; p < 0.01), FGFR1 (fibroblast growth factor receptor 1; p < 0.05), FGFR2 (p < 0.05), and TRPV6 (transient receptor potential vanilloid member 6; p < 0.05) mRNAs compared to CSH RNAi Non-IUGR pregnancies. This study has provided novel insights into the regulatory role of CSH for calcium, phosphate, and vitamin D utilization in late gestation.
Assuntos
Cálcio , Lactogênio Placentário , Animais , Cálcio/metabolismo , Cálcio da Dieta , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/metabolismo , Humanos , Mamíferos/metabolismo , Fosfatos/metabolismo , Placenta/metabolismo , Lactogênio Placentário/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ovinos/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Útero/metabolismo , Vitamina D/metabolismoRESUMO
Appropriate mineralization of the fetal skeleton requires an excess of phosphate in the fetus compared to the mother. However, mechanisms for placental phosphate transport are poorly understood. This study aimed to identify phosphate regulatory pathways in ovine endometria and placentae throughout gestation. Suffolk ewes were bred with fertile rams upon visual detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n = 3-14/Day), ewes were euthanized and hysterectomized. Phosphate abundance varied across gestational days in uterine flushings, allantoic fluid, and homogenized endometria and placentae (P < 0.05). The expression of mRNAs for sodium-dependent phosphate transporters (SLC20A1 and SLC20A2) and klotho signaling mediators (FGF7, FGF21, FGF23, FGFR1-4, KL, KLB, ADAM10, and ADAM17) were quantified by qPCR. Day 17 conceptus tissue expressed SLC20A1, SLC20A2, KLB, FGF7, FGF21, FGF23, FGFR1, and FGFR2 mRNAs. Both sodium-dependent phosphate transporters and klotho signaling mediators were expressed in endometria and placentae throughout gestation. Gestational day influenced the expression of SLC20A1, ADAM10, ADAM17, FGF21, FGFR1, and FGFR3 mRNAs in both endometria and placentae (P < 0.05). Gestational day influenced endometrial expression of FGF7 (P < 0.001), and placental expression of FGF23 (P < 0.05). Immunohistochemistry confirmed that both FGF23 and KL proteins were expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile of KL-FGF signaling suggests a potential role in the establishment of pregnancy and regulation of fetal growth. This study provides a platform for further mechanistic investigation into the role for KL-FGF signaling in the regulation of phosphate transport at the ovine maternal-conceptus interface.
Assuntos
Proteínas Klotho/genética , Redes e Vias Metabólicas , Minerais/metabolismo , Fosfatos/metabolismo , Carneiro Doméstico/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato/genética , Animais , Feminino , Gravidez , Prenhez , Transdução de Sinais , Proteínas Cotransportadoras de Sódio-Fosfato/metabolismoRESUMO
Mineralization of the fetal mammalian skeleton requires a hypercalcemic gradient across the placenta from mother to fetus. However, the mechanisms responsible for maintaining the placental transport of calcium remain poorly understood. This study aimed to identify calcium and vitamin D regulatory pathway components in ovine endometria and placentae across gestation. Suffolk ewes were bred with fertile rams upon detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n=3-14/Day), ewes were euthanized and hysterectomized. Calcium abundance was influenced by gestational day in uterine flushings and allantoic fluid (P<0.05). The expression of S100G, S100A9, S100A12, ATP2B3, ATP2B4, TRPV5, TRPV6, CYP11A1, CYP2R1, CYP24, and VDR mRNAs known to be involved in calcium binding, calcium transport, and vitamin D metabolism were quantified by qPCR. Mediators of calcium and vitamin D signaling were expressed by Day 17 conceptus tissue, and endometria and placentae across gestation. Gestational day influenced the expression of S100G, S100A9, S100A12, TRPV6, VDR, and CYP24 mRNAs in endometria and placentae (P<0.05). Gestational day influenced endometrial expression of ATP2B3, and placental expression of TRPV5, ATP2B4, and CYP11A1 (P<0.05). VDR protein localized to the endoderm and trophectoderm (Day 17 conceptus) and was expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile suggests a potential role of calcium and vitamin D in the establishment of pregnancy and regulation of fetal and placental growth, providing a platform for further mechanistic investigation.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Cálcio/metabolismo , Carneiro Doméstico/fisiologia , Transdução de Sinais , Vitamina D/metabolismo , Animais , Feminino , Gravidez , Prenhez , Vitaminas/metabolismoRESUMO
Arginine is a key amino acid in pregnant females as it is the precursor for nitric oxide (NO) via nitric oxide synthase and for polyamines (putrescine, spermidine, and spermine) by either arginase II and ornithine decarboxylase to putrescine or via arginine decarboxylase to agmatine and agmatine to putrescine via agmatinase. Polyamines are critical for placental growth and vascularization. Polyamines stabilize DNA and mRNA for gene transcription and mRNA translation, stimulate proliferation of trophectoderm, and formation of multinucleated trophectoderm cells that give rise to giant cells in the placentae of species such as mice. Polyamines activate MTOR cell signaling to stimulate protein synthesis and they are important for motility through modification of beta-catenin phosphorylation, integrin signaling via focal adhesion kinases, cytoskeletal organization, and invasiveness or superficial implantation of blastocysts. Physiological levels of arginine, agmatine, and polyamines are critical to the secretion of interferon tau for pregnancy recognition in ruminants. Arginine, polyamines, and agmatine are very abundant in fetal fluids, fetal blood, and tissues of the conceptus during gestation. The polyamines are thus available to influence a multitude of events including activation of development of blastocysts, implantation, placentation, fetal growth, and development required for the successful establishment and maintenance of pregnancy in mammals.
Assuntos
Agmatina , Poliaminas , Animais , Arginina , Feminino , Mamíferos , Camundongos , Placenta , GravidezRESUMO
PURPOSE: Through their feeding practices, adult caregivers play an important role in shaping children's eating behaviors. However, the feeding practices of child care teachers have received little attention. The purpose of this study was to compare child care teachers' self-reported feeding practices and observed feeding practices during a preschool meal. METHODS: Rhode Island Head Start teachers (n = 85) were observed during breakfast and lunch where feeding practices were coded using a tool adapted from the Environmental Policy Assessment and Observation (EPAO) tool. Teachers completed a questionnaire adapted from the EPAO Self-Report to capture self-reported feeding practices. Agreement between reported and observed was compared by percent agreement. RESULTS: Teachers were predominantly White (89%) and female (98%). There was a higher level of agreement among self-reported and observed controlling feeding practices (78.8-97.6% agreement) compared to healthful feeding practices (11.8-20.0% agreement). CONCLUSIONS: Although self-report measures are typically used to capture feeding practices, there are inconsistencies between self-report and observation measures. The inconsistencies found among healthful self-reported and observed feeding practices have implications for future research protocols, measurement refinement, and training of child care teachers.
Assuntos
Dieta Saudável/psicologia , Comportamento Alimentar/psicologia , Professores Escolares/psicologia , Adulto , Pré-Escolar , Feminino , Humanos , Masculino , Refeições , Pessoa de Meia-Idade , Rhode Island , AutorrelatoRESUMO
Large chromosomal aberrations occur commonly during development, resulting in complex and multisystem diseases. In spite of this high frequency, there are currently no means for correcting these disorders due to their complexity and involvement of multiple genes. Recently, several new approaches have been devised that target whole chromosomes in vitro, which are collectively referred to as "Chromosome Therapies." These include silencing and selection for loss of the extra chromosome in trisomies, promotion of euploidy in an aneuploid culture, and forced loss and replacement of a chromosome. Here, we provide a review of Chromosome Therapy, and discuss potential directions for these methods clinically, as well as research applications and cellular models that can be made using these technologies. © 2016 Wiley Periodicals, Inc.
Assuntos
Aberrações Cromossômicas , Transtornos Cromossômicos/terapia , Terapia Genética/métodos , Cromossomos/genética , Terapia Genética/tendências , HumanosRESUMO
Hyperandrogenic disorders, such as polycystic ovary syndrome, are often associated with metabolic disruptions such as insulin resistance and hyperinsulinemia. Studies in sheep, a precocial model of translational relevance, provide evidence that in utero exposure to excess testosterone during days 30-90 of gestation (the sexually dimorphic window where males naturally experience elevated androgens) programs insulin resistance and hyperinsulinemia in female offspring. Extending earlier findings that adverse effects of testosterone excess are evident in fetal day 90 pancreas, the end of testosterone treatment, the present study provides evidence that transcriptomic and phenotypic effects of in utero testosterone excess on female pancreas persist after cessation of treatment, suggesting lasting organizational changes, and induce a male-like phenotype in female pancreas. These findings demonstrate that the female pancreas is susceptible to programmed masculinization during the sexually dimorphic window of fetal development and shed light on underlying connections between hyperandrogenism and metabolic homeostasis.
Assuntos
Pâncreas , Testosterona , Transcriptoma , Animais , Feminino , Ovinos , Transcriptoma/efeitos dos fármacos , Transcriptoma/genética , Gravidez , Pâncreas/metabolismo , Pâncreas/efeitos dos fármacos , Masculino , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Resistência à Insulina , Hiperandrogenismo/metabolismo , Hiperandrogenismo/genética , Desenvolvimento Fetal/efeitos dos fármacos , Caracteres SexuaisRESUMO
BACKGROUND: Tissue non-specific alkaline phosphatase (TNSALP; encoded by the ALPL gene) has a critical role in the postnatal regulation of phosphate homeostasis, yet how TNSALP activity and expression are regulated during pregnancy remain largely unknown. This study tested the hypothesis that progesterone (P4) and/or interferon tau (IFNT) regulate TNSALP activity during pregnancy in sheep. METHODS: In Exp. 1, ewes were bred and received daily intramuscular injections of either corn oil vehicle (CO) or 25 mg progesterone in CO (P4) for the first 8 days of pregnancy and were hysterectomized on either Day 9, 12, or 125 of gestation. In Exp. 2, ewes were fitted with intrauterine catheters on Day 7 of the estrous cycle and received daily intramuscular injections of 50 mg P4 in CO and/or 75 mg progesterone receptor antagonist (RU486) in CO from Days 8 to 15, and twice daily intrauterine injections of either control proteins (CX) or IFNT (25 µg/uterine horn/d) from Days 11 to 15 (treatment groups: P4 + CX; P4 + IFNT; RU486 + P4 + CX; and RU486 + P4 + IFNT) and were hysterectomized on Day 16. RESULTS: In Exp. 1, endometria from ewes administered P4 had greater expression of ALPL mRNA than ewes administered CO on Day 12. TNSALP activity appeared greater in the epithelia, stratum compactum stroma, and endothelium of the blood vessels in the endometrium and myometrium from ewes administered P4 than ewes administered CO on Day 12. On Day 125, TNSALP activity localized to uterine epithelial and endothelial cells, independent of P4 treatment. TNSALP activity in placentomes appeared greater in P4 treated ewes and was detected in endothelial cells and caruncular tissue in P4 treated but not CO treated ewes. In Exp. 2, endometrial homogenates from ewes administered RU486 + P4 + CX had lower TNSALP activity those for P4 + CX and P4 + IFNT ewes. Immunoreactive TNSALP protein appeared greater in the mid- and deep-glandular epithelia in RU486 + P4 + CX treated ewes as compared to the other treatment groups. Enzymatic activity appeared greater on the apical surface of the deep glandular epithelia in endometria from ewes treated with RU486 + P4 + CX compared to the other treatment groups. CONCLUSIONS: These results suggest that P4, but not IFNT, regulates the expression and activity of TNSALP in utero-placental tissues and has the potential to contribute to the regulation of phosphate availability that is critical for conceptus development during pregnancy.