Showing papers by "Fuller W. Bazer 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.

L-Arginine Nutrition and Metabolism in Ruminants.

Abstract: L-Arginine (Arg) plays a central role in the nitrogen metabolism (e.g., syntheses of protein, nitric oxide, polyamines, and creatine), blood flow, nutrient utilization, and health of ruminants. This amino acid is produced by ruminal bacteria and is also synthesized from L-glutamine, L-glutamate, and L-proline via the formation of L-citrulline (Cit) in the enterocytes of young and adult ruminants. In pre-weaning ruminants, most of the Cit formed de novo by the enterocytes is used locally for Arg production. In post-weaning ruminants, the small intestine-derived Cit is converted into Arg primarily in the kidneys and, to a lesser extent, in endothelial cells, macrophages, and other cell types. Under normal feeding conditions, Arg synthesis contributes 65% and 68% of total Arg requirements for nonpregnant and late pregnany ewes fed a diet with ~12% crude protein, respectively, whereas creatine production requires 40% and 36% of Arg utilized by nonpregnant and late pregnant ewes, respectively. Arg has not traditionally been considered a limiting nutrient in diets for post-weaning, gestating, or lactating ruminants because it has been assumed that these animals can synthesize sufficient Arg to meet their nutritional and physiological needs. This lack of a full understanding of Arg nutrition and metabolism has contributed to suboptimal efficiencies for milk production, reproductive performance, and growth in ruminants. There is now considerable evidence that dietary supplementation with rumen-protected Arg (e.g., 0.25-0.5% of dietary dry matter) can improve all these production indices without adverse effects on metabolism or health. Because extracellular Cit is not degraded by microbes in the rumen due to the lack of uptake, Cit can be used without any encapsulation as an effective dietary source for the synthesis of Arg in ruminants, including dairy and beef cows, as well as sheep and goats. Thus, an adequate amount of supplemental rumen-protected Arg or unencapsulated Cit is necessary to support maximum survival, growth, lactation, reproductive performance, and feed efficiency, as well as optimum health and well-being in all ruminants.

Phosphate, Calcium, and Vitamin D: Key Regulators of Fetal and Placental Development in Mammals.

Abstract: Normal calcium and bone homeostasis in the adult is virtually fully explained by the interactions of several key regulatory hormones, including parathyroid hormone, 1,25 dihydroxy vitamin D3, fibroblast growth factor-23, calcitonin, and sex steroids (estradiol and testosterone). In utero, bone and mineral metabolism is regulated differently from the adult. During development, it is the placenta and not the fetal kidneys, intestines, or skeleton that is the primary source of minerals for the fetus. The placenta is able to meet the almost inexhaustible needs of the fetus for minerals by actively driving the transport of calcium and phosphorus from the maternal circulation to the growing fetus. These fundamentally important minerals are maintained in the fetal circulation at higher concentrations than those in maternal blood. Maintenance of these inordinately higher fetal levels is necessary for the developing skeleton to accrue sufficient minerals by term. Importantly, in livestock species, prenatal mineralization of the skeleton is crucial for the high levels of offspring activity soon after birth. Calcium is required for mineralization, as well as a plethora of other physiological functions. Placental calcium and phosphate transport are regulated by several mechanisms that are discussed in this review. It is clear that phosphate and calcium metabolism is intimately interrelated and, therefore, placental transport of these minerals cannot be considered in isolation.

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.

Microarray analysis reveals an important role for dietary L-arginine in regulating global gene expression in porcine placentae during early gestation.

Abstract: BACKGROUND Increasing the dietary provision of L-arginine to pregnant swine beginning at Day 14 of gestation enhances embryonic survival, but the underlying mechanisms are largely unknown. OBJECTIVE This study determined the effects of dietary supplementation with 0.8% L-arginine to gilts between Days 14 and 25 of gestation on the global expression of genes in their placentae. METHODS Between Days 14 and 24 of gestation, gilts were fed 2 kg of a corn- and soybean meal-based diet (containing 12.0% crude protein and 0.70% Arg) supplemented with 0.8% L-arginine or without L-arginine (0.0%; with 1.64% L-alanine as the isonitrogenous control). On Day 25 of gestation, 30 min after the consumption of their top dressing containing 8 g L-arginine or 16.4 g L-alanine, gilts underwent hysterectomy to obtain placentae, which were snap-frozen in liquid nitrogen. Total RNAs were extracted from the frozen tissues and used for microarray analysis based on the 44-K Agilent porcine gene platform. RESULTS L-Arginine supplementation affected placental expression of 575 genes, with 146 genes being up-regulated and 429 genes being down-regulated. These differentially expressed genes play important roles in nutrient metabolism, polyamine production, protein synthesis, proteolysis, angiogenesis, immune development, anti-oxidative responses, and adhesion force between the chorioallantoic membrane and the endometrial epithelium, as well as functions of insulin, transforming growth factor beta, and Notch signaling pathways. CONCLUSION Dietary supplementation with L-arginine plays an important role in regulating placental gene expression in gilts. Our findings help to elucidate mechanisms responsible for the beneficial effect of L-arginine in improving placental growth and embryonic/fetal survival in swine.

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.

Interferon epsilon and preterm birth subtypes; a new piece of the type I interferon puzzle during pregnancy?

Abstract: Interferon epsilon (IFNε) is a unique type I IFN that is expressed in response to sex steroids. Studies suggest that type I IFNs regulate inflammation‐induced preterm birth (PTB), but no study has examined the role of IFNε in human pregnancy.

Elongating porcine conceptuses can utilize glutaminolysis as an anaplerotic pathway to maintain the TCA cycle

Abstract: Abstract During the peri-implantation period of pregnancy, the trophectoderm of pig conceptuses utilize glucose via multiple biosynthetic pathways to support elongation and implantation, resulting in limited availability of pyruvate for metabolism via the TCA cycle. Therefore, we hypothesized that porcine trophectoderm cells replenish tricarboxylic acid (TCA) cycle intermediates via a process known as anaplerosis and that trophectoderm cells convert glutamine to α-ketoglutarate, a TCA cycle intermediate, through glutaminolysis. Results demonstrate: (1) that expression of glutaminase (GLS) increases in trophectoderm and glutamine synthetase (GLUL) increases in extra-embryonic endoderm of conceptuses, suggesting that extra-embryonic endoderm synthesizes glutamine, and trophectoderm converts glutamine into glutamate; and (2) that expression of glutamate dehydrogenase 1 (GLUD1) decreases and expression of aminotransferases including PSAT1 increase in trophectoderm, suggesting that glutaminolysis occurs in the trophectoderm through the GLS-aminotransferase pathway during the peri-implantation period. We then incubated porcine conceptuses with 13C-glutamine in the presence or absence of glucose in the culture media and then monitored the movement of glutamine-derived carbons through metabolic intermediates within glutaminolysis and the TCA cycle. The 13C-labeled carbons were accumulated in glutamate, α-ketoglutarate, succinate, malate, citrate, and aspartate in both the presence and absence of glucose in the media, and the accumulation of 13C-labeled carbons significantly increased in the absence of glucose in the media. Collectively, our results indicate that during the peri-implantation period of pregnancy, the proliferating and migrating trophectoderm cells of elongating porcine conceptuses utilize glutamine via glutaminolysis as an alternate carbon source to maintain TCA cycle flux. Summary Sentence The trophectoderm cells of pigs metabolize glucose through glycolytic biosynthetic pathways and utilize glutamine via glutaminolysis as an alternate carbon source to maintain TCA cycle flux during the peri-implantation period.

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

Abstract: Abstract 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. Summary Sentence Progesterone and interferon tau regulate phosphate, calcium, and vitamin D signaling during the ovine peri-implantation period.

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.

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

Abstract: Abstract 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. Summary Sentence Exogenous progesterone in early pregnancy affects the expression of regulators of calcium, phosphate, and vitamin D in late pregnancy, suggesting previously unappreciated effects on mineral transport at the maternal–conceptus interface of sheep.

Estradiol and zinc-doped nano hydroxyapatite as therapeutic agents in the prevention of osteoporosis; oxidative stress status, inflammation, bone turnover, bone mineral density, and histological alterations in ovariectomized rats

Abstract: Osteoporosis (OP) is a serious health problem, and the most popular therapeutic strategy for OP is hormone replacement (estrogen); however, it increases the risk of reproductive cancers. Hydroxyapatite (HA) nanoparticles have a similar chemical structure to the bone mineral component and can be used as a new remedy for OP. This study was designed to investigate the osteoporosis-protective potential of nano zinc hydroxyapatite (ZnHA-NPs) and/or estradiol (E2) combined therapy. A total of 35 adult female rats were assigned into five groups (n = 7): 1) control group; 2) ovariectomized group (OVX); 3) OVX received oral estradiol replacement therapy (OVX/E2); 4) OVX received ZnHA replacement therapy (OVX/ZnHA); and 5) OVX received both estradiol and ZnHA-NPs combined therapy (OVX/E2+ZnHA). After 3 months of treatment, serum bone markers and estrogen level, oxidative/antioxidant, and inflammatory cytokines were determined. Additionally, femoral expression of estrogen receptors alpha and beta (ESR1; ESR2), receptor activator of nuclear factor-kappa B (RANKL) ligand, osteoprotegerin (OPG), bone mineral density (BMD), histological alterations, and immunohistochemical expression of vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) were assessed. ALP, PINP, Ca, and P concentrations improved significantly (p < 0.05) in all treatment groups, especially in the OVX/E + ZnHA group. MDA and NO were higher in OVX rats, while SOD activity and GSH were lower (p < 0.05). E2 alone or with ZnHA-NPs restored the estimated antioxidant molecules and cytokines toward normal levels in OVX rats (p < 0.05). On the other hand, E2 and ZnHA increased OPG and OC expression in femurs while decreasing ESR1, ESR2, and NF-kB expression (p < 0.05). The combination treatment was superior in the restoration of normal femoral histoarchitecture and both cortical and trabecular BMD (p < 0.05). Overall, the combined therapy of OVX/E2+ZnHA was more effective than the individual treatments in attenuating excessive bone turnover and preventing osteoporosis.

Uptake of Phosphate, Calcium, and Vitamin D by the Pregnant Uterus of Sheep in Late Gestation: Regulation by Chorionic Somatomammotropin Hormone

Abstract: 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.

Sexual dimorphism in placental development and function: Comparative physiology with an emphasis on the pig.

Abstract: Across mammalian species, it has been demonstrated that sex influences birth weight, with males being heavier than females; a characteristic that can be observed from early gestation. Male piglets are more likely to be stillborn and have greater preweaning mortality than their female littermates, despite the additional maternal investment into male fetal growth. Given the conserved nature of the genome between the sexes, it is hypothesized that these developmental differences between males and females are most likely orchestrated by differential placental adaptation. This review summarizes the current understanding of fetal sex-specific differences in placental and endometrial structure and function, with an emphasis on pathways found to be differentially regulated in the pig including angiogenesis, apoptosis, and proliferation. Given the importance of piglet sex in agricultural enterprises, and the potential for skewed litter sex ratios, it is imperative to improve understanding of the relationship between fetal sex and molecular signaling in both the placenta and endometria across gestation.

Editorial: Veterinary Reproductive Immunology

Abstract: EDITORIAL article Front. Vet. Sci., 10 January 2022Sec. Animal Reproduction - Theriogenology Volume 8 - 2021 | https://doi.org/10.3389/fvets.2021.823169

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.

The vaginal and uterine blood flow changes during the ovsynch program and its impact on the pregnancy rates in Holstein dairy cows

Abstract: OvSynch is a hormonal protocol for synchronization of estrus and use of artificial insemination (AI) at an optimal time without adverse effects on the ovaries or uterus. This study investigated the use of noninvasive color Doppler ultrasound to assess changes in uterine and vaginal blood flow during the Ovsynch program for synchronization of estrus and its relation to the pregnancy rates in Holstein cows.The experimental cows received an intramuscular dose of 10 μg of a GnRH analogue (G1), followed 7 days later with an intramuscular injection of synthetic prostaglandin F2α (P: PGF2α) analogue (500 μg cloprostenol sodium), and given a 10 μg, injection of the GnRH analogue (G2) i.m. 48 h after the PGF2α treatment, and the cows were bred 14-16 h after. Uterine and vaginal perfusion were investigated by performing transrectal Doppler ultrasonography of both the uterine and vaginal arteries in Holstein cows at different time points during the Ovsynch program to determine: peak systolic velocity (PSV), time-averaged maximum velocity (TAMV), the volume of blood flow (BFV), pulsatility index (PI), resistance index (RI), resistance impedance (S/D) and diameters of uterine (UA) and vaginal (VA) arteries. Steroid hormones were also assayed. Transrectal ultrasonography (TUS) was performed at 32 and 60 days to confirm the pregnancy per artificial insemination (P/AI).The uterine PSV, TAMV, and PV were greater at the time of the cloprostenol sodium and second GnRH injections (p<0.05) than at the time of the first GnRH injection. The vaginal PSV, PV were greater at the time of the cloprostenol sodium than at the time of the first and second GnRH injections (p<0.05). The receiver operating characteristic curve (ROC curve) indicated a high correlation between the uterine and vaginal blood flow and the rate of the pregnancy (p<0.05). The area under the ROC curve was 0.920 and 0.87 (p<0.05) for vaginal and uterine arteries respectively at time of G2. The serum levels of progesterone, estrogen and cortisol were correlated with the P/AI (p<0.05). The P/AI significantly decreased from 43.9 % at 32 d to 35.37 % at 60 d.These results indicate that noninvasive Doppler ultrasonography is a valid method to evaluate changes in the characteristics of uterine and vaginal blood flow in cows during the Ovsynch protocol. Furthermore, vaginal and uterine blood flow are two determinant factors for the higher conception rates in Holstein dairy cows.

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.

219 Evaluation of Dietary Arginine Supplementation to Increase Placental Nutrient Transporters of Aged Mares

Abstract: Nine pregnant mares (18.2 ± 0.7 y; 493.82 ± 13 kg BW) were used to test the hypothesis that dietary supplementation of arginine would enhance placental vascularity and nutrient transport throughout gestation in aged mares. Mares were balanced by age, BW, and stallion pairing, and randomly assigned to one of two dietary treatments consisting of supplemental L-arginine (50 mg/kg BW; n=5) or L-alanine (100 mg/kg BW; n=4; isonitrogenous control). Mares were individually fed concentrate top-dressed with the respective amino acid treatment plus ad libitum access to Coastal bermudagrass hay. Treatments began on d 14 of gestation and were terminated at parturition. Mare BW, BCS, and rump fat were determined every 28 d and concentrate adjusted accordingly. Parturition was attended with foaling variables and placental measures recorded. Placental tissue from the pregnant horn underwent histological analyses to assess cell-specific localization of vascular endothelial growth factor (VEGF) and cationic amino acid transporter 1 (CAT1) proteins. Semiquantitative analyses were performed using 10 non-overlapping images per sample fixed in a 10X field (Fiji ImageJ v1.2). Data were analyzed using SAS PROC MIXED procedure. BW increased (P < 0.01) in all mares with advancing gestation. BW, BCS, and gestation length of mares were not influenced (P > 0.05) by supplemental arginine. Compared with arginine-supplemented mares, control mares had a thicker rump fat layer (P < 0.01) at parturition. Body length, height, and BW of foals at birth, as well as placental weight, volume, and positive staining for VEGF and CAT1 at parturition were not affected (P > 0.05) by maternal arginine supplementation. These results indicate dietary arginine supplementation (50 mg/kg BW) is safe for gestating mares and promotes mobilization of white adipose tissue. A larger number of mares is required to determine effects of supplemental arginine on embryonic/fetal survival and growth in mares.

Equine enterocytes actively oxidize L-glutamine, but do not synthesize L-citrulline or L-arginine from L-glutamine or L-proline in vitro.

Abstract: In livestock species, the enterocytes of the small intestine are responsible for the synthesis of citrulline and arginine from glutamine and proline. At present, little is known about de novo synthesis of citrulline and arginine in horses. To test the hypothesis that horses of different age groups can utilize glutamine and proline for the de novo synthesis of citrulline and arginine, jejunal enterocytes from 19 horses of three different age groups: neonates (n = 4; 7.54 ± 2.36 d of age), adults (n = 9; 6.4 ± 0.35 yr), and aged (n = 6; 22.9 ± 1.0 yr) with healthy gastrointestinal tracts were used in the present study. Enterocytes were isolated from the jejunum and incubated at 37°C for 30-min in oxygenated (95% O2/5% CO2) Krebs bicarbonate buffer (pH 7.4) containing 5 mM D-glucose and 0 mM, 2-mM L-[U- 14C]glutamine, or 2 mM L-[U- 14C]proline plus 2 mM L-glutamine. Concentrations of arginine, citrulline, and ornithine in cells plus medium were determined using high-performance liquid chromatography. Results indicate that the rate of oxidation of glutamine to CO2 was high in enterocytes from neonatal horses, but low in cells from adult and aged horses. Enterocytes from all age groups of horses did not degrade proline into CO2. Regardless of age, equine enterocytes formed ornithine from glutamine and proline, but failed to convert ornithine into citrulline and arginine. Because arginine is an essential substrate for the synthesis of proteins, but also nitrogenous metabolites (e.g., nitric oxide, polyamines, and creatine), our novel findings have important implications for the nutrition, performance, and health of horses.

Effects of L-carnitine and cryodevices on the vitrification and developmental competence of invitro fertilized buffalo oocytes

Abstract: In the current, study the effect of the addition of L-carnitine (LC) in in vitro maturation (IVM) medium for buffalo oocytes and different cryo-devices on developmental competence. They were matured in IVM medium supplemented with 0.0, 0.3, 0.6 and 1.2 mg/mL of LC and vitrified by using either straw (ST), open pulled-straw (OPS), solid surface vitrification (SSV). The effects of LC during invitro fertilization and invitro culture on the developmental potential were examined. ST showed a higher recovery rate when using 0.6 mg/ml; viability rate (VR) when using 0.3mg/ml and lower zona pellucida (ZP) and cytoplasmic abnormalities when using 1.2 mg/ml LC (P<0.05). OPS showed a higher recovery and viability rates when using 0.3 mg (P<0.05). SSD showed a higher recovery and VR when using 0.6 mg/ml LC (P<0.05). Maturation and fertilization rates were increased when using 0.3 mg LC and OPS (P<0.05). Cleavage was increased in 0.3 (OPS) and 0.6 mg LC (SSD). The blastocyst rate was increased in 0.3 (OPS) and 0.6 mg/ml (SSD) (P< 0.05). In conclusion, 0.3 mg/ml LC and OPS gives a higher recovery and viability rates but 0.6 mg/ml LC and of both SSD and ST gives a higher recovery and viability rates.

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

Abstract: Abstract 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. Summary Sentence Progesterone and interferon tau regulate expression of mRNAs and proteins involved in the metabolism and transport of polyamines in endometria of sheep during the peri-implantation period of pregnancy. Graphical Abstract