L-Arginine Nutrition and Metabolism in Ruminants.

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.

Adverse organogenesis and predisposed long-term metabolic syndrome from prenatal exposure to fine particulate matter.

Exposure to fine particulate matter (PM) during pregnancy is associated with high risks of birth defects/fatality and adverse long-term postnatal health. However, limited mechanistic data are available to assess the detailed impacts of prenatal PM exposure. Here we evaluate fine PM exposure during pregnancy on prenatal/postnatal organogenesis in offspring and in predisposing metabolic syndrome for adult life. Between days 0 and 18 of gestation, two groups of adult female rats (n = 10 for each) were placed in a dual-exposure chamber device, one with clean ambient air (∼3 µg·m-3) and the other with ambient air in the presence of 100 to 200 µg·m-3 of ultrafine aerosols of ammonium sulfate. At birth (postnatal day 0, PND0), four males and four females were selected randomly from each litter to be nursed by dams, whereas tissues were collected from the remaining pups. At PND21, tissues were collected from two males and two females, whereas the remaining pups were fed either a high- or low-fat diet until PND105, when tissues were obtained for biochemical and physiological analyses. Maternal exposure to fine PM increased stillbirths; reduced gestation length and birth weight; increased concentrations of glucose and free fatty acids in plasma; enhanced lipid accumulation in the liver; and decreased endothelium-dependent relaxation of aorta. This lead to altered organogenesis and predisposed progeny to long-term metabolic defects in an age-, organ-, and sex-specific manner. Our results highlight the necessity to develop therapeutic strategies to remedy adverse health effects of maternal PM exposure on conceptus/postnatal growth and development.

Lipid metabolism is altered in maternal, placental, and fetal tissues of ewes with small for gestational age fetuses†.

Nutrient restriction (NR) has the potential to negatively impact birthweight, an indicator of neonatal survival and lifelong health. Those fetuses are termed as small for gestational age (SGA). Interestingly, there is a spectral phenotype of fetal growth rates in response to NR associated with changes in placental development, nutrient and waste transport, and lipid metabolism. A sheep model with a maternal diet, starting at Day 35, of 100% National Research Council (NRC) nutrient requirements (n = 8) or 50% NRC (n = 28) was used to assess alterations in fetuses designated NR SGA (n = 7) or NR NonSGA (n = 7) based on fetal weight at Day 135 of pregnancy. Allantoic fluid concentrations of triglycerides were greater in NR SGA fetuses than 100% NRC and NR NonSGA fetuses at Day 70 (P < 0.05). There was a negative correlation between allantoic fluid concentrations of triglycerides (R2 = 0.207) and bile acids (R2 = 0.179) on Day 70 and fetal weight at Day 135 for NR ewes (P < 0.05). Bile acids were more abundant in maternal and fetal blood for NR SGA compared to 100% NRC and NR NonSGA ewes (P < 0.05). Maternal blood concentrations of NEFAs increased in late pregnancy in NR NonSGA compared to NR SGA ewes (P < 0.05). Protein expression of fatty acid transporter SLC27A6 localized to placentomal maternal and fetal epithelia and decreased in Day 70 NR SGA compared to 100% NRC and NR NonSGA placentomes (P < 0.05). These results identify novel factors associated with an ability of placentae and fetuses in NR NonSGA ewes to adapt to, and overcome, nutritional hardship during pregnancy.

Placental adaptation to maternal malnutrition.

Maternal malnutrition gives rise to both short- and long-term consequences for the survival and health of the offspring. As the intermediary between mother and fetus, the placenta has the potential to interpret environmental signals, such as nutrient availability, and adapt to support fetal growth and development. While this potential is present, it is clear that at times placental adaptation fails to occur resulting in poor pregnancy outcomes. This review will focus on placental responses to maternal undernutrition related to changes in placental vascularization and hemodynamics and placental nutrient transport systems across species. While much of the available literature describes placental responses that result in poor fetal outcomes, novel models have been developed to utilize the inherent variation in fetal weight when dams are nutrient restricted to identify placental adaptations that result in normal-weight offspring. Detailed analyses of the spectrum of placental responses to maternal malnutrition point to alternations in placental histoarchitectural and vascular development, amino acid and lipid transport mechanisms, and modulation of immune-related factors. Dietary supplementation with selected nutrients, such as arginine, has the potential to improve placental growth and function through a variety of mechanisms including stimulating cell proliferation, protein synthesis, angiogenesis, vasodilation, and gene regulation. Improved understanding of placental responses to environmental cues is necessary to develop diagnostic and intervention strategies to improve pregnancy outcomes.

Amino Acid Nutrition and Reproductive Performance in Ruminants.

Amino acids (AAs) are essential for the survival, growth and development of ruminant conceptuses. Most of the dietary AAs (including L-arginine, L-lysine, L-methionine and L-glutamine) are extensively catabolized by the ruminal microbes of ruminants to synthesize AAs and microbial proteins (the major source of AAs utilized by cells in ruminant species) in the presence of sufficient carbohydrates (mainly cellulose and hemicellulose), nitrogen, and sulfur. Results of recent studies indicate that the ruminal microbes of adult steers and sheep do not degrade extracellular L-citrulline and have a limited ability to metabolize extracellular L-glutamate due to little or no uptake by the cells. Although traditional research in ruminant protein nutrition has focused on AAs (e.g., lysine and methionine for lactating cows) that are not synthesized by eukaryotic cells, there is growing interest in the nutritional and physiological roles of AAs (e.g., L-arginine, L-citrulline, L-glutamine and L-glutamate) in gestating ruminants (e.g., cattle, sheep and goats) and lactating dairy cows. Results of recent studies show that intravenous administration of L-arginine to underfed, overweight or prolific ewes enhances fetal growth, the development of brown fat in fetuses, and the survival of neonatal lambs. Likewise, dietary supplementation with either rumen-protected L-arginine or unprotected L-citrulline to gestating sheep or beef cattle improved embryonic survival. Because dietary L-citrulline and L-glutamate are not degraded by ruminal microbes, addition of these two amino acids may be a new useful, cost-effective method for improving the reproductive efficiency of ruminants.

Maternal Nutrient Restriction and Skeletal Muscle Development: Consequences for Postnatal Health.

Severe undernutrition and famine continue to be a worldwide concern, as cases have been increasing in the past 5 years, particularly in developing countries. The occurrence of nutrient restriction (NR) during pregnancy affects fetal growth, leading to small for gestational age (SGA) or intrauterine growth restricted (IUGR) offspring. During adulthood, SGA and IUGR offspring are at a higher risk for the development of metabolic syndrome. Skeletal muscle is particularly sensitive to prenatal NR. This tissue plays an essential role in oxidation and glucose metabolism because roughly 80% of insulin-mediated glucose uptake occurs in muscle, and it represents around 40% of body weight. Alterations in myofiber number, hypertrophy and myofiber type composition, decreased protein synthesis, lower mitochondrial content and activity of oxidative enzymes, and increased accumulation of intramuscular triglycerides are among the described programming effects of maternal NR on skeletal muscle. Together, these features would add to a phenotype that is prone to insulin resistance, type 2 diabetes, obesity, and metabolic syndrome. Insights from diverse animal models (i.e. ovine, swine, and rodent) have provided valuable information regarding the molecular mechanisms behind those altered developmental pathways. Understanding those molecular signatures supports the development of efficient treatments to counteract the effects of maternal NR on skeletal muscle, and its negative implications for postnatal health.

Functional role of arginine during the peri-implantation period of pregnancy. I. Consequences of loss of function of arginine transporter SLC7A1 mRNA in ovine conceptus trophectoderm.

Arginine, the common substrate for production of nitric oxide (NO) and polyamines in mammals, increases in the uterine lumen during the peri-implantation period of pregnancy. However, functional roles of arginine within the uterine lumen for conceptus (embryo and extraembryonic membranes) development have not been elucidated in vivo. To assess roles of arginine in reproductive tissue for survival and development of the conceptus, we conducted an in vivo morpholino antisense oligonucleotide (MAO)-mediated knockdown of SLC7A1 mRNA, the arginine transporter in ovine conceptus trophectoderm (Tr). Translational knockdown of SLC7A1 mRNA resulted in retarded conceptus development and abnormal function compared to MAO control. Use of MAO-SLC7A1 knockdown in conceptuses decreased arginine transport (73%, P<0.01), the abundance of ornithine decarboxylase, and nitric oxide synthase (NOS3) proteins, arginine-related amino acids [citrulline (76%, P<0.05) and ornithine (40%, P<0.05)], and polyamines, which likely accounts for their retarded development. Also, no alternative arginine precursors (glutamine and glutamate), isoforms of nitric oxide synthase (NOS1 and NOS2), or alternative pathways for polyamine biosynthesis via arginine decarboxylase and agmatinase were activated to rescue conceptus development. Collectively, SLC7A1 is the key transporter of arginine by conceptus Tr, and arginine is essential for conceptus survival and development.-Wang, X., Frank, J. W., Little, D. R., Dunlap, K. A., Satterfield, M. C., Burghardt, R. C., Hansen, T. R., Wu, G., and Bazer, F. W. Functional role of arginine during the peri-implantation period of pregnancy. I. Consequences of loss of function of arginine transporter SLC7A1 mRNA in ovine conceptus trophectoderm.

Arginine decarboxylase and agmatinase: an alternative pathway for de novo biosynthesis of polyamines for development of mammalian conceptuses.

Ornithine decarboxylase (ODC1) is considered the rate-controlling enzyme for the classical de novo biosynthesis of polyamines (putrescine, spermidine, and spermine) in mammals. However, metabolism of arginine to agmatine via arginine decarboxylase (ADC) and conversion of agmatine to polyamines via agmatinase (AGMAT) is an alternative pathway long recognized in lower organisms, but only recently suggested for neurons and liver cells of mammals. We now provide evidence for a functional ADC/AGMAT pathway for the synthesis of polyamines in mammalian reproductive tissue for embryonic survival and development. We first investigated cellular functions of polyamines by in vivo knockdown of translation of mRNA for ODC1 in ovine conceptus trophectoderm using morpholino antisense oligonucleotides (MAOs) and found that one-half of the conceptuses were morphologically and functionally either normal or abnormal. Furthermore, we found that increases in ADC/AGMAT mRNA levels and in the translation of AGMAT mRNA among conceptuses in MAO-ODC1 knockdown compensated for the loss of ODC1, supporting polyamine synthesis from arginine and accounting for the normal and abnormal phenotypes of conceptuses. We conclude that the majority of polyamine synthesis is by the conventional ODC1-dependent pathway (arginine-ornithine-putrescine) and that deficiencies in ODC1 result in increased activity of the rescue ADC/AGMAT-dependent pathway (arginine-agmatine-putrescine) for production of polyamines. The presence of an alternative ADC/AGMAT pathway for converting arginine into putrescine is functionally important for supporting survival and development of mammalian conceptuses.

Functional role of arginine during the peri-implantation period of pregnancy. II. Consequences of loss of function of nitric oxide synthase NOS3 mRNA in ovine conceptus trophectoderm.

Nitric oxide (NO) is a gaseous molecule that regulates angiogenesis and vasodilation via activation of the cGMP pathway. However, functional roles of NO during embryonic development from spherical blastocysts to elongated filamentous conceptuses (embryo and extraembryonic membrane) during the peri-implantation period of pregnancy have not been elucidated in vivo. In order to assess roles of NO production in survival and development of the ovine conceptus, we conducted an in vivo morpholino antisense oligonucleotide (MAO)-mediated knockdown trial of nitric oxide synthase-3 (NOS3) mRNA, the major isoform of NO synthase, in ovine conceptus trophectoderm (Tr). Translational knockdown of NOS3 mRNA results in small, thin, and underdeveloped conceptuses, but normal production of interferon-tau, the pregnancy recognition signal in sheep. MAO-NOS3 knockdown in conceptuses decreased the abundance of NOS3 (72%, P < 0.05) and the arginine transporter SLC7A1 proteins in conceptus Tr. Furthermore, the amounts of ornithine and polyamines were less (P < 0.01) in uterine fluid, whereas the amounts of arginine (58%, P < 0.01), citrulline (68%, P < 0.05), ornithine (68%, P < 0.001), glutamine (78%, P < 0.001), glutamate (68%, P < 0.05), and polyamines (P < 0.01) were less in conceptuses, which likely accounts for the failure of MAO-NOS3 conceptuses to develop normally. For MAO-NOS3 conceptuses, there were no compensatory increases in the expression levels of either nitric oxide synthase-1 (NOS1) or nitric oxide synthase-2 (NOS2) or in expression of enzymes for synthesis of polyamines (ornithine decarboxylase, arginine decarboxylase, agmatinase) from arginine or ornithine with which to rescue development of MAO-NOS3 conceptuses. Thus, the adverse effect of MAO-NOS3 to reduce NO generation and the transport of arginine and ornithine into conceptuses is central to an explanation for failure of normal development of MAO-NOS3, compared to control conceptuses. The study, for the first time, created an NO-deficient mammalian conceptus model in vivo and provided new insights into the orchestrated events of conceptus development during the peri-implantation period of pregnancy. Our data suggest that NOS3 is the key enzyme for NO production by conceptus Tr and that this protein also regulates the availability of arginine in conceptus tissues for synthesis of polyamines that are essential for conceptus survival and development.

Metabolism and Nutrition of L-Glutamate and L-Glutamine in Ruminants.

Although both L-glutamate (Glu) and L-glutamine (Gln) have long been considered nutritionally nonessential in ruminants, these two amino acids have enormous nutritional and physiological importance. Results of recent studies revealed that extracellular Gln is extensively degraded by ruminal microbes, but extracellular Glu undergoes little catabolism by these cells due to the near absence of its uptake. Ruminal bacteria hydrolyze Gln to Glu plus ammonia and, intracellularly, use both amino acids for protein synthesis. Microbial proteins and dietary Glu enter the small intestine in ruminants. Both Glu and Gln are the major metabolic fuels and building blocks of proteins, as well as substrates for the syntheses of glutathione and amino acids (alanine, ornithine, citrulline, arginine, proline, and aspartate) in the intestinal mucosa. In addition, Gln and aspartate are essential for purine and pyrimidine syntheses, whereas arginine and proline are necessary for the production of nitric oxide (a major vasodilator) and collagen (the most abundant protein in the body), respectively. Under normal feeding conditions, all diet- and rumen-derived Glu and Gln are extensively utilized by the small intestine and do not enter the portal circulation. Thus, de novo synthesis (e.g., from branched-chain amino acids and α-ketoglutarate) plays a crucial role in the homeostasis of Glu and Gln in the whole body but may be insufficient for maximal growth performance, production (e.g., lactation and pregnancy), and optimal health (particularly intestinal health) in ruminants. This applies to all types of feeding systems used around the world (e.g., rearing on a milk replacer before weaning, pasture-based production, and total mixed rations). Dietary supplementation with the appropriate doses of Glu or Gln [e.g., 0.5 or 1 g/kg body weight (BW)/day, respectively] can safely improve the digestive, endocrine, and reproduction functions of ruminants to enhance their productivity. Both Glu and Gln are truly functional amino acids in the nutrition of ruminants and hold great promise for improving their health and productivity.

Arginine nutrition and fetal brown adipose tissue development in nutrient-restricted sheep.

Intrauterine growth restriction is a significant problem worldwide, resulting in increased rates of neonatal morbidity and mortality, as well as increased risks for metabolic and cardiovascular disease. The present study investigated the role of maternal undernutrition and L-arginine administration on fetal growth and development. Embryo transfer was utilized to generate genetically similar singleton pregnancies. On Day 35 of gestation, ewes were assigned to receive either 50 or 100% of their nutritional requirements. Ewes received i.v. injections of either saline or L-arginine three times daily from Day 100 to Day 125. Fetal growth was assessed at necropsy on Day 125. Maternal dietary manipulation altered circulating concentrations of leptin, progesterone, and amino acids in maternal plasma. Fetal weight was reduced in nutrient-restricted ewes on Day 125 compared with 100% fed ewes. Compared with saline-treated underfed ewes, maternal L-arginine administration did not affect fetal weight but increased weight of the fetal pancreas by 32% and fetal peri-renal brown adipose tissue mass by 48%. These results indicate that L-arginine administration enhanced fetal pancreatic and brown adipose tissue development. The postnatal effects of increased pancreatic and brown adipose tissue growth warrant further study.

Impacts of arginine nutrition on embryonic and fetal development in mammals.

Embryonic loss and intrauterine growth restriction (IUGR) are significant problems in humans and other animals. Results from studies involving pigs and sheep have indicated that limited uterine capacity and placental insufficiency are major factors contributing to suboptimal reproduction in mammals. Our discovery of the unusual abundance of the arginine family of amino acids in porcine and ovine allantoic fluids during early gestation led to the novel hypothesis that arginine plays an important role in conceptus (embryo and extra-embryonic membranes) development. Arginine is metabolized to ornithine, proline, and nitric oxide, with each having important physiological functions. Nitric oxide is a vasodilator and angiogenic factor, whereas ornithine and proline are substrates for uterine and placental synthesis of polyamines that are key regulators of gene expression, protein synthesis, and angiogenesis. Additionally, arginine activates the mechanistic (mammalian) target of rapamycin cell signaling pathway to stimulate protein synthesis in the placenta, uterus, and fetus. Thus, dietary supplementation with 0.83 % L-arginine to gilts consuming 2 kg of a typical gestation diet between either days 14 and 28 or between days 30 and 114 of pregnancy increases the number of live-born piglets and litter birth weight. Similar results have been reported for gestating rats and ewes. In sheep, arginine also stimulates development of fetal brown adipose tissue. Furthermore, oral administration of arginine to women with IUGR has been reported to enhance fetal growth. Collectively, enhancement of uterine as well as placental growth and function through dietary arginine supplementation provides an effective solution to improving embryonic and fetal survival and growth.

Viral particles of endogenous betaretroviruses are released in the sheep uterus and infect the conceptus trophectoderm in a transspecies embryo transfer model.

The sheep genome contains multiple copies of endogenous betaretroviruses highly related to the exogenous and oncogenic jaagsiekte sheep retrovirus (JSRV). The endogenous JSRVs (enJSRVs) are abundantly expressed in the uterine luminal and glandular epithelia as well as in the conceptus trophectoderm and are essential for conceptus elongation and trophectoderm growth and development. Of note, enJSRVs are present in sheep and goats but not cattle. At least 5 of the 27 enJSRV loci cloned to date possess an intact genomic organization and are able to produce viral particles in vitro. In this study, we found that enJSRVs form viral particles that are released into the uterine lumen of sheep. In order to test the infectious potential of enJSRV particles in the uterus, we transferred bovine blastocysts into synchronized ovine recipients and allowed them to develop for 13 days. Analysis of microdissected trophectoderm of the bovine conceptuses revealed the presence of enJSRV RNA and, in some cases, DNA. Interestingly, we found that RNAs belonging to only the most recently integrated enJSRV loci were packaged into viral particles and transmitted to the trophectoderm. Collectively, these results support the hypothesis that intact enJSRV loci expressed in the uterine endometrial epithelia are shed into the uterine lumen and could potentially transduce the conceptus trophectoderm. The essential role played by enJSRVs in sheep reproductive biology could also be played by endometrium-derived viral particles that influence development and differentiation of the trophectoderm.

Parenteral administration of L-arginine enhances fetal survival and growth in sheep carrying multiple fetuses.

The frequency of multiple fetuses has increased in human pregnancies due to assisted reproductive technologies. This translates into a greater proportion of premature and low-birth weight infants in the United States and worldwide. In addition, improvements in sheep breeding have resulted in new breeds with increased litter size but reduced fetal survival and birth weight. Currently, there are no treatments for preventing fetal growth restriction in humans or sheep (an established model for studying human fetal physiology) carrying multiple fetuses. In this work, Booroola Rambouillet ewes (FecB+/-) with 2-4 fetuses were fed a diet providing 100% of NRC-recommended nutrient requirements. Between d 100 and 121 of gestation, ewes received an i.v. bolus injection of either saline solution or 345 µmol arginine-HCl/kg body weight 3 times daily. The arginine treatment reduced (P < 0.05) the percentage of lambs born dead by 23% while increasing (P = 0.05) the percentage of lambs born alive by 59%. The i.v. administration of arginine enhanced (P < 0.05) the birth weights of quadruplets by 23% without affecting maternal body weight. The improved pregnancy outcome was associated with an increase in maternal plasma concentrations of arginine, ornithine, cysteine, and proline, as well as a decrease in circulating levels of ammonia and ß-hydroxybutyrate. These novel results indicate that parenteral administration of arginine to prolific ewes ameliorated fetal mortality and growth retardation. Our findings provide support for experiments to assess the clinical use of arginine to enhance fetal growth and survival in women gestating multiple fetuses.

Proline and hydroxyproline metabolism: implications for animal and human nutrition.

Proline plays important roles in protein synthesis and structure, metabolism (particularly the synthesis of arginine, polyamines, and glutamate via pyrroline-5-carboxylate), and nutrition, as well as wound healing, antioxidative reactions, and immune responses. On a per-gram basis, proline plus hydroxyproline are most abundant in collagen and milk proteins, and requirements of proline for whole-body protein synthesis are the greatest among all amino acids. Therefore, physiological needs for proline are particularly high during the life cycle. While most mammals (including humans and pigs) can synthesize proline from arginine and glutamine/glutamate, rates of endogenous synthesis are inadequate for neonates, birds, and fish. Thus, work with young pigs (a widely used animal model for studying infant nutrition) has shown that supplementing 0.0, 0.35, 0.7, 1.05, 1.4, and 2.1% proline to a proline-free chemically defined diet containing 0.48% arginine and 2% glutamate dose dependently improved daily growth rate and feed efficiency while reducing concentrations of urea in plasma. Additionally, maximal growth performance of chickens depended on at least 0.8% proline in the diet. Likewise, dietary supplementation with 0.07, 0.14, and 0.28% hydroxyproline (a metabolite of proline) to a plant protein-based diet enhanced weight gains of salmon. Based on its regulatory roles in cellular biochemistry, proline can be considered as a functional amino acid for mammalian, avian, and aquatic species. Further research is warranted to develop effective strategies of dietary supplementation with proline or hydroxyproline to benefit health, growth, and development of animals and humans.

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.

HSD11B1, HSD11B2, PTGS2, and NR3C1 expression in the peri-implantation ovine uterus: effects of pregnancy, progesterone, and interferon tau.

Establishment of pregnancy in ruminants requires conceptus elongation and production of interferon tau (IFNT), the pregnancy recognition signal that maintains the corpus luteum and progesterone (P4) secretion. The enzymes hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1) and HSD11B2 catalyze the interconversion of inactive cortisone and active cortisol, which is a biologically active glucorticoid and ligand for the receptor subfamily 3, group C, member 1 (glucocorticoid receptor) (NR3C1). The activity of HSD11B1 is stimulated by P4, prostaglandins, and cortisol. These studies determined the effects of pregnancy, P4, and IFNT on HSD11B1, HSD11B2, prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase) (PTGS2), and nuclear NR3C1 in the ovine uterus. Endometrial HSD11B1 mRNA levels were more abundant between Days 12 and 16 of pregnancy than the estrous cycle, and HSD11B1 and PTGS2 expression in the endometrial luminal and superficial glandular epithelia was coincident with conceptus elongation. HSD11B1 mRNA was very low in the conceptus, whereas HSD11B2 mRNA was abundant in the conceptus but not in the uterus. Treatment of ewes with P4 induced, and intrauterine infusions of IFNT modestly stimulated, HSD11B1 expression in the endometrial luminal and superficial glandular epithelia. In all of the studies, HSD11B1 and PTGS2 expression was coincident in the endometrial epithelia, and NR3C1 was present in all endometrial cell types. Collectively, these results support hypotheses that endometrial epithelial HSD11B1 expression is induced by P4 as well as stimulated by IFNT and PTGS2-derived prostaglandins and that HSD11B1-regenerated cortisol acts via NR3C1 to regulate ovine endometrial functions during early pregnancy.

Select nutrients and their associated transporters are increased in the ovine uterus following early progesterone administration.

The intrauterine milieu is a complex mixture of substances originating from serum and endometrium that support blastocyst growth and development. The present study identified alterations in glucose and amino acids in response to an early rise in progesterone (P4), which accelerates blastocyst growth and development. Bred ewes received daily injections of either corn oil (CO) vehicle or P4 from 36 h postmating (Day 0) to either Day 9 or Day 12. Another group of ewes received P4 to Day 8 and the antiprogestin mifepristone (RU486) from Day 8 to Day 12. The total amount of glucose, aspartate (acidic amino acid), arginine and lysine (basic amino acids), and citrulline, asparagine, serine, glutamine, beta-alanine, and alanine (neutral amino acids) was greater in uterine flushings from early P4- than CO-treated ewes on Day 9. On Day 12, only arginine and lysine were higher in uterine flushings from P4-treated ewes, whereas citrulline was reduced. Glucose transporters, SLC2A1 and SLC5A1, were increased in uterine luminal (LE) and superficial glandular (sGE) epithelia of early P4-treated ewes on Days 9 and 12 but were reduced in endometria from ewes treated with both P4 and RU486 (P4+RU). SLC7A2B, a transporter of basic amino acids, increased in LE/sGE of P4- versus CO-treated ewes on Day 12 but was reduced in P4+RU-treated ewes. Thus, select nutrients are increased in the uterine lumen by P4 concomitant with the upregulation of epithelial transporters for glucose and basic amino acids, suggesting that these nutrients stimulate blastocyst growth and development.

Sildenafil citrate treatment enhances amino acid availability in the conceptus and fetal growth in an ovine model of intrauterine growth restriction.

Adequate placental blood flow is essential for the optimal delivery of nutrients from mother to fetus for conceptus growth. Restricted fetal development results from pathophysiological and environmental factors that alter utero-placental blood flow, placental function, and, therefore, nutrient availability in the fetus. To test this hypothesis, 0, 75, or 150 mg/d sildenafil citrate (Viagra) was administered subcutaneously from d 28 to 115 of gestation to either nutrient-restricted [50% of NRC requirements) or adequately-fed ewes (100% of NRC requirements). On d 115, maternal, fetal, and placental tissues and fluids were collected. Concentrations of total amino acids and polyamines in uterine venous and arterial sera, amniotic and allantoic fluids, and fetal umbilical venous serum were lower (P < 0.05) in nutrient-restricted ewes than in adequately fed ewes, as were the ratios of total amino acids in fetal umbilical venous serum to uterine arterial serum. Sildenafil citrate dose-dependently increased (P < 0.05) total amino acids and polyamines in amniotic fluid, allantoic fluid, and fetal serum without affecting values in maternal serum. Fetal weight was lower (P < 0.05) in nutrient-restricted ewes on d 115. Sildenafil citrate treatment dose-dependently increased (P < 0.05) fetal weight in both nutrient-restricted and adequately fed ewes. This study supports the hypothesis that long-term sildenafil citrate treatment enhances fetal growth, at least in part, by increasing the availability of amino acids in the conceptus. These findings may lead to the clinical use of sildenafil citrate in human pregnancies suspected to be at risk for intrauterine fetal growth retardation.

Parenteral administration of L-arginine prevents fetal growth restriction in undernourished ewes.

Intrauterine growth restriction (IUGR) is a major health problem worldwide that currently lacks an effective therapeutic solution. This study was conducted with an ovine IUGR model to test the hypothesis that parenteral administration of l-arginine (Arg) is effective in enhancing fetal growth. Beginning on d 28 of gestation, ewes were fed a diet providing 100% (control-fed) or 50% (underfed) of NRC-recommended nutrient requirements. Between d 60 of gestation and parturition, underfed ewes received i.v. infusions of saline or 155 micromol Arg-HCl/kg body weight 3 times daily, whereas control-fed ewes received only saline. The birth weights of lambs from saline-infused underfed ewes were 23% lower (P < 0.01) than those of lambs from control-fed dams. Administration of Arg to underfed ewes increased (P < 0.01) concentrations of Arg (69%), ornithine (55%), proline (29%), methionine (37%), leucine (36%), isoleucine (35%), cysteine (19%), and FFA (43%) in maternal serum, decreased maternal circulating levels of ammonia (18%) and triglycerides (32%), and enhanced birth weights of lambs by 21% compared with saline-infused underfed ewes. There was no difference in birth weights of lambs between the control-fed and the Arg-infused underfed ewes. These novel results indicate that parenteral administration of Arg to underfed ewes prevented fetal growth restriction and provide support for its clinical use to ameliorate IUGR in humans. The findings also lay a new framework for studying cellular and molecular mechanisms responsible for the beneficial effects of Arg in regulating conceptus growth and development.