Metabolic pathways for glucose and fructose: I synthesis and metabolism of fructose by ovine conceptuses.

Fructose, the most abundant hexose sugar in fetal fluids and 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: KHK-A protein was more abundant in trophectoderm and cotyledons of placentomes, while KHK-C protein was more abundant in endoderm of Day 16 conceptuses and 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 TCA cycle, as well as substrates for the pentose cycle, hexosamine biosynthesis pathway and one-carbon metabolism required for conceptus development throughout pregnancy.

Metabolic pathways of glucose and fructose: II spatiotemporal expression of genes involved in synthesis and transport of lactate in ovine conceptuses.

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 (LDH) 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.

Characterization of TNSALP expression, localization, and activity in ovine utero-placental tissues†.

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.

Regulation of synthesis of polyamines by progesterone, estradiol, and their receptors in uteri of cyclic ewes†.

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.

Creatine metabolism at the uterine-placental interface throughout gestation in sheep†.

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.

Phosphate, calcium, and vitamin D signaling, transport, and metabolism in the endometria of cyclic ewes.

BACKGROUND: Recent evidence suggests important roles for progesterone (P4) and interferon tau in the regulation of calcium, phosphate, and vitamin D signaling in the uteri of pregnant sheep. However, the effects of P4 and estradiol (E2), with respect to the expression of their receptors PGR and ESR1, respectively, in uterine epithelia on mineral signaling during the estrous cycle has not been investigated. Estrous cycles of mature Suffolk ewes were synchronized, prostaglandin F2α was administered, and ewes were observed for estrus (designated as Day 0) in the presence of vasectomized rams. On Days 1, 9, or 14 of the estrous cycle, hysterectomies were performed. RESULTS: 25-hydroxyvitamin D was more abundant in plasma from ewes on Day 14 than Day 1 (P < 0.05). Expression of fibroblast growth factor receptor 2 (FGFR2), a disintegrin and metalloprotease 17 (ADAM17), and parathyroid hormone-related protein (PTHrP) mRNAs was greater in endometria on Day 9 compared to Days 1 and 14 (P < 0.01). Similarly, expression of transient receptor potential cation channel subfamily V member 6 (TRPV6) mRNA was greater in endometria on Day 9 than Day 1 (P < 0.05). ATPase plasma membrane Ca2+ transporting 4 (ATP2B4) and S100 calcium binding protein G (S100G) mRNA expression was greater in endometria on Day 14 than on Days 1 and 9 (P < 0.01). In contrast, endometrial expression of vitamin D receptor (VDR) mRNA was lower on Days 9 and 14 than Day 1 (P < 0.01). Expression of klotho (KL) (P < 0.05) and cytochrome P450 family 24 subfamily A member 1 (CYP24) (P < 0.01) mRNAs was lower on Day 14 than Days 1 and 9. ADAM17, FGF23, CYP2R1, CYP27B1, KL, and VDR proteins immunolocalized to the uterine myometrium, blood vessels, and uterine luminal (LE), superficial glandular (sGE), and glandular (GE) epithelia. S100A9 protein was weakly expressed in the uterine myometrium, LE, sGE, and GE. Immunoreactivity of CYP2R1 and KL proteins in uterine LE and sGE was less on Day 1 than on Days 9 and 14. In contrast, S100G protein was expressed exclusively by GE, and immunoreactive S100G protein was less on Day 9. S100A12 protein localized to stromal cells of the uterine stratum spongiosum and blood vessels, but not by uterine epithelial cells. CONCLUSION: Collectively, these results implicate E2, P4, and PGR in the regulation of phosphate, calcium, and vitamin D signaling in cyclic ewes.

The ovine conceptus utilizes extracellular serine, glucose, and fructose to generate formate via the one carbon metabolism pathway.

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.

Creatine metabolism at the uterine-conceptus interface during early gestation in sheep†.

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.

Inhibition of SHMT2 mRNA translation increases embryonic mortality in sheep†.

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.

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

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.

Effects of progesterone and interferon tau on ovine endometrial phosphate, calcium, and vitamin D signaling†.

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.

Effects of exogenous progesterone on the expression of mineral regulatory molecules by ovine endometrium and placentomes†.

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.

Progesterone and interferon tau regulate expression of polyamine enzymes during the ovine peri-implantation period†.

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.

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.

Pre-implantation exogenous progesterone and pregnancy in sheep. II. Effects on fetal-placental development and nutrient transporters in late pregnancy.

BACKGROUND: Administration of progesterone (P4) to ewes during the first 9 to 12 days of pregnancy accelerates blastocyst development by day 12 of pregnancy, likely due to P4-induced up-regulation of key genes in uterine epithelia responsible for secretion and transport of components of histotroph into the uterine lumen. This study determined if acceleration of blastocyst development induced by exogenous P4 during the pre-implantation period affects fetal-placental development on day 125 of pregnancy. Suffolk ewes (n = 35) were mated to fertile rams and assigned randomly to receive daily intramuscular injections of either corn oil vehicle (CO, n = 18) or 25 mg progesterone in CO (P4, n = 17) for the first 8 days of pregnancy. All ewes were hysterectomized on day 125 of pregnancy and: 1) fetal and placental weights and measurements were recorded; 2) endometrial and placental tissues were analyzed for the expression of candidate mRNAs involved in nutrient transport and arginine metabolism; and 3) maternal plasma, fetal plasma, allantoic fluid, and amniotic fluid were analyzed for amino acids, agmatine, polyamines, glucose, and fructose. RESULTS: Treatment of ewes with exogenous P4 did not alter fetal or placental growth, but increased amounts of aspartate and arginine in allantoic fluid and amniotic fluid, respectively. Ewes that received exogenous P4 had greater expression of mRNAs for SLC7A1, SLC7A2, SLC2A1, AGMAT, and ODC1 in endometria, as well as SLC1A4, SLC2A5, SLC2A8 and ODC1 in placentomes. In addition, AZIN2 protein was immunolocalized to uterine luminal and glandular epithelia in P4-treated ewes, whereas AZIN2 localized only to uterine luminal epithelia in CO-treated ewes. CONCLUSIONS: This study revealed that exogenous P4 administered in early pregnancy influenced expression of selected genes for nutrient transporters and the expression of a protein involved in polyamine synthesis on day 125 of pregnancy, suggesting a 'programming' effect of P4 on gene expression that affected the composition of nutrients in fetal-placental fluids.

Pre-implantation exogenous progesterone and pregnancy in sheep: I. polyamines, nutrient transport, and progestamedins.

BACKGROUND: Administration of exogenous progesterone (P4) to ewes during the pre-implantation period advances conceptus development and implantation. This study determined effects of exogenous P4 on transport of select nutrients and pathways that enhance conceptus development. Pregnant ewes (n = 38) were treated with either 25 mg P4 in 1 mL corn oil (P4, n = 18) or 1 mL corn oil alone (CO, n = 20) from day 1.5 through day 8 of pregnancy and hysterectomized on either day 9 or day 12 of pregnancy. Endometrial expression of genes encoding enzymes for synthesis of polyamines, transporters of glucose, arginine, and glycine, as well as progestamedins was determined by RT-qPCR. RESULTS: On day 12 of pregnancy, conceptuses from P4-treated ewes had elongated while those from CO-treated ewes were spherical. The mRNA expression of AZIN2, an arginine decarboxylase, was lower in endometria of P4-treated than CO-treated ewes on day 9 of pregnancy. Expression of FGF10, a progestamedin, was greater in endometria of CO and P4-treated ewes on day 12 of gestation in addition to P4-treated ewes necropsied on day 9 of gestation. Treatment with P4 down-regulated endometrial expression of amino acid transporter SLC1A4 on day 12 of pregnancy. CONCLUSIONS: Results indicated that administration of exogenous P4 during the pre-implantation period advanced the expression of FGF10, which may accelerate proliferation of trophectoderm cells, but also was correlated with decreased expression of glycine and serine transporters and polyamine synthesis enzyme AZIN2. Further research with increased sample sizes may determine how differential expression affects endometrial functions and potentially embryonic loss.