Elsevier

Theriogenology

Volume 150, 1 July 2020, Pages 75-83
Theriogenology

Interferon tau: Influences on growth and development of the conceptus

https://doi.org/10.1016/j.theriogenology.2020.01.069Get rights and content

Highlights for Review

  • Interferon tau (IFNT) abrogates the uterine luteolytic mechanism to ensure maintenance of functional corpora lutea for production of progesterone (P4).

  • IFNT, in concert with P4, induces expression of genes in uterine luminal (LE) and superficial glandular (sGE) epithelia for transport and/or secretion of histotroph into the uterine lumen.

  • IFNT and P4 induce transporters responsible for transport of arginine into the uterine lumen to activate the mechanistic target of rapamycin cell signaling pathway essential for growth and development of the conceptus.

  • IFNT and P4 induce transporters for glucose that the conceptus converts to fructose and those two hexose sugars are metabolized via the hexosamine biosynthesis pathway, one-carbon metabolism, and pentose phosphate pathway.

Abstract

Interferon tau (IFNT), the pregnancy recognition signal secreted from trophectoderm cells of ruminant conceptuses abrogates the uterine luteolytic mechanism to ensure maintenance of functional corpora lutea for production of progesterone (P4). Importantly, IFNT, in concert with P4, also induces expression of genes in uterine luminal (LE) and superficial glandular (sGE) epithelia for transport and/or secretion of histotroph into the uterine lumen to support growth and development of the conceptus. For example, IFNT and P4 induce transporters responsible foer transport of glucose and arginine into the uterine lumen during the peri-implantation period of pregnancy. Arginine activates the mechanistic target of rapamycin (MTOR) nutrient sensing cell signaling pathway to stimulate proliferation, migration, differentiation and translation of mRNAs essential for growth and development of the conceptus. Glucose not utilized by the conceptus is converted to fructose and those two hexose sugars are metabolized via aerobic glycolysis to produce metabolites used in the hexosamine biosynthesis pathway, pathways for one-carbon metabolism, and pentose phosphate pathway for synthesis of ribose sugars and NADPH. Arginine is metabolized to nitric oxide (NO) that stimulates angiogenesis in uterine and placental tissues, and to polyamines required for many cellular functions critical for growth and development of the conceptus. In summary, IFNT and P4 regulate expression of genes for transport of select nutrients into the pregnant uterus during the peri-implantation period of pregnancy. Those nutrients are then metabolized via multiple metabolic pathways to not only provide ATP, but also substrates for synthesis of nucleotides, amino acids, co-factors required for growth, development, and survival of conceptuses during the peri-implantation period of pregnancy.

Introduction

Interferon tau (IFNT), a Type 1 interferon is secreted by mononuclear trophectoderm cells of conceptuses (embryo and extra-embryonic membranes) of ruminants. IFNT has antiviral, antiproliferative and immunomodulatory properties as do other Type 1 interferons such as IFN alpha; however, IFNT is the antiluteolyic pregnancy recognition signal unique to ruminants. IFNT silences expression of estrogen receptor alpha (ESR1) and, in turn, oxytocin receptors in uterine epithelia to abrogate the mechanism that otherwise results in pulsatile release of luteolytic prostaglandin F (PGF) that would induce regression of corpora lutea and prevent their production of progesterone (P4) [1]. Of equal importance to establishment and maintenance of pregnancy, P4 and IFNT act cooperatively to silence expression of classical interferon stimulated genes specifically in ovine uterine luminal (LE) and superficial glandular (sGE) epithelia while simultaneously, via an alternate cell signaling mechanism, stimulating expression of genes required for growth and development of the conceptus. Those genes expressed by uterine LE/sGE include transporters of glucose and amino acids [2]. The IFNT-induced cell signaling mechanism unique to uterine LE/sGE is not known. Further, undefined mechanisms of action of IFNT are linked to a servomechanism in which IFNT is permissive to actions of ovine placental lactogen (CSH1) and placental growth hormone (GH) to affect development of uterine glands and their expression of genes throughout gestation [3]. IFNT may also acts systemically to induce expression of interferon stimulated genes that influence secretion of progesterone by the corpus luteum. This review is based primarily on results from studies with sheep which have been studied more extensively than other ruminan species to understand the molecular, cellular and physiological mechanisms whereby IFNT, in concert with P4, effects changes in uterine biology required for growth, development and survival of conceptuses (see Fig. 1).

Spherical ovine blastocysts “hatch” from the zona pellucida on Days 6 and 7 of pregnancy and expand to 0.4 mm diameter on Day 10. The blastocysts then elongate to filamentous forms between Days 12 (1.0 by 33 mm) and 15 (1 by 150–190 mm), and conceptus growth extends through the uterine body and into the contralateral uterine horn by Days 16–18 of pregnancy [4]. Elongation of ovine conceptuses is a prerequisite for central implantation that initially involves apposition and then adhesion between trophectoderm and uterine LE/sGE. As conceptuses elongate, they metabolize and are responsive to a complex mixture of molecules in the uterine lumen referred to as histotroph. Histotroph includes hormones, enzymes, growth factors, cytokines, transport proteins, adhesion factors, nutrients and other molecules required for development, implantation and placentation of the conceptus (Fig. 1). Although not studied in sheep or other ruminants, in vitro studies with mouse embryos revealed that leucine or arginine in culture medium and presumably uterine fluid is required as early as the developmental transition of morulae to blastocysts [5,6].

Elongation of ovine conceptuses precedes attachment of trophectoderm to uterine LE/sGE and formation of a mosaic of interactions between integrins and extracellular matrix (ECM) proteins that contribute to stable adhesion during implantation and placentation (Fig. 2). Elongation and implantation are complex events that require significant sources of energy and histotroph, for example amino acids, in the uterine lumen [7]. Glucose, fructose, arginine, leucine, glutamine, glycine and serine are major components of histotroph. Embryonic mortality during this period of peri-implantation period of development of conceptuses of livestock and other species is a major constraint to improving reproductive efficiency and profitability in livestock enterprises and fertility in humans and other animals. Therefore, our research has focused on hormonal, cellular and molecular mechanisms whereby secretions from uterine epithelia and/or nutrients transported into the uterine lumen enhance growth, differentiation, development and survival of the conceptus using the ovineexperimental model.

Uterine receptivity to implantation is dependent on P4 that is permissive to actions of IFNT, placental GH and CSH1. The paradox is that cessation of expression of receptors for progesterone(PGR) and estrogen (ESR1) by uterine epithelia is a prerequisite for uterine receptivity to implantation, expression of genes for secretory proteins, and expression of genes for transporters of nutrients into the uterine lumen that support conceptus development. Down-regulation of PGR precedes loss of expression of proteins on uterine LE, such as mucin1 (MUC1), that interfere with implantation. Loss of expression of PGR in uterine epithelia also restricts actions of P4 to PGR-positive uterine stromal cells that express progestamedins that include fibroblast growth factor-7 (FGF7), FGF10, and hepatocyte growth factor (HGF). The progestamedins then exert paracrine effects on uterine epithelia and conceptus trophectoderm that express receptors for FGF7 and FGF10 (FGFR2IIIb) and HGF (MET; protooncogene MET). A fundamental unanswered question is whether the actions of progestamedins and IFNT on uterine epithelia or other uterine cell types involve unique cell-specific cell signaling pathways (Fig. 3). This is of particular interest for uterine LE/sGE which do not express interferon stimulated genes (ISG) including signal transducer and activator of transcription (STAT1) and STAT2, and interferon regulatory factor 9 (IRF9) required for classical cell signaling in response to Type 1 IFNs. Cell-specific gene expression in the ovine uterine LE/sGE is due, at least in part, to the fact that IFNT induces expression of IRF2, a potent inhibitor of transcription, including transcription of STAT1, STAT2, IRF9 and ESR1 genes. However, classical ISGs are expressed in ovine uterine GE and stromal cells, and ISGs are expressed in immune cells in blood of ruminants including interferon-stimulated gene 15(ISG15), myxovirus resistance 1, mouse, homolog of (MX1, MX2) and 2′,5′oligoadenylate synthetase (OAS1,OAS2,OAS3).

In ewes, the abundances of glucose, all amino acids except for taurine, and glutathione increase significantly in the uterine lumen between Days 13 and 16 of pregnancy and are more abundant in the uterine lumen of pregnant than cyclic ewes [14,15]. Further, P4 and IFNT act in concert to increase expression of transporters for the glucose solute carrier family (SLC) members SLC2A1 and SLC5A11 and cationic amino acids such as those for arginine, SLC7A2 and SLC7A2B, and neutral amino acids such as glutamine, SLC1A5. P4 alone induces expression of glucose transporters SLC2A4 and SLC5A1, and amino acid transporter SLC7A1; however, expression of transporters for the large neutral amino acids (SLC7A5, SLC7A8 and SLC43A2) are constitutive in ovine uterine endometrium as neither P4, IFNT nor their combination affects their expression. Abundances of amino acids in the uterine lumen and expression of their transporters during the peri-implantation period of pregnancy in sheep have been published [12,13].

Leu, Arg and Gln are of particular interest because outgrowth and expansion of the trophectoderm of mouse blastocysts in preparation for implantation require Leu or Arg [5,6]. Leucine and Arg activate the serine-threonine kinase and mechanistic target of rapamycin (MTOR) cell signaling pathways to regulate protein synthesis and catabolism, and induce expression of insulin-like growth factor 2 (IGF2), nitric oxide synthases (NOS1, NOS2, and NOS3), and ornithine decarboxylase (ODC1) that likely coordinate differentiation of trophectoderm and development of uterine epithelia in preparation for implantation [11,13]. There are also differential effects of Leu, Arg and Gln on hypertrophy and hyperplasia of cells important for conceptus development during the peri-implantation period of pregnancy [[16], [17], [18]]. Physiological levels of Leu, Arg and Gln stimulate MTOR and ribosomal protein S6 (RPS6) kinase required for proliferation of trophectoderm cells. Actions of Gln require the presence of physiological concentrations of glucose or fructose (a precursor of fructose-6-phosphate and glucosamine-6-phosphate) suggesting that hexosamines influence growth and development of the conceptus [19]. Cellular events associated with elongation of ovine conceptuses during the peri-implantation period of pregnancy involve both cellular hyperplasia and hypertrophy, as well as cytoskeletal reorganization during the transition of spherical blastocysts to tubular and filamentous conceptuses [20].

The human placenta transports amino acids into the fetal circulation against concentration gradients utilizing both sodium dependent and sodium independent transporters including System A amino acid transporters that primarily transport small and neutral amino acids [21]. In women with intra-uterine growth restriction there is an inverse relationship between placental size and System A amino acid transporters [22]. Those results indicate that amino acid transporters are critical components of the nutrient sensing system and that amino acids stimulate MTORC1 which in turn enhances expression of transporters linking maternal nutrient availability and development of the conceptus [23].

Nitric oxide (NO) and polyamines (putrescine, spermidine, and spermine) are products of Arg catabolism critical for placental growth [11]. Arginine stimulates placental production of NO by enhancing expression of GTP cyclohydrolase I (GCH1), the first and rate-controlling enzyme for synthesis of tetrahydrobiopterin (BH4), an essential cofactor for all isoforms of NO synthase. Glutathione, synthesized from glutamate, glycine and cysteine, is the major antioxidant in the conceptus. Fetal intra-uterine growth restriction (IUGR) is associated with impaired transport of basic, neutral and acidic amino acids by the placenta; therefore, maternal protein/amino acid nutrition impacts embryonic/fetal survival [11]. Along with insulin-like growth factors, vascular endothelial growth factors and other growth factors, NO and polyamines are crucial for angiogenesis, embryogenesis, placental growth, utero-placental blood flows, and the transfer of nutrients from mother to fetuses [11].

The Arg family of amino acids is very abundant in ovine uterine fluid and allantoic fluid (e.g., 10 mM citrulline and 25 mM Gln on Day 60 of gestation) [25]. The ovine placenta expresses arginase; therefore, citrulline is “stored” in abundant amounts (approximately 10 mmol/L on Day 60 of gestation) in ovine allantoic fluid. Accordingly, rates of NO and polyamine synthesis in ovine placentae are greatest during early gestation when placental growth is most rapid [26]. Impaired placental growth (including vascular growth) or function results from reduced placental synthesis of NO and polyamines, thereby contributing to IUGR in both underfed and overfed dams based on results of studies with sheep, pigs and rats [27].

NO generated from conversion of Arg to NO by eNOS and/or iNOS in trophoblast cells activates guanyl cyclase to produce cGMP, stimulates trophectoderm motility perhaps by modifying the extra-cellular matrix (ECM), induces vasodilation of maternal blood vessels and regulates cellular energy metabolism. During elongation and implantation of ovine conceptuses, there is a significant increase in expression of secreted phosphoprotein1 (SPP1; also known as osteopontin, OPN) by uterine GE that may be influenced by NO to increase cell adhesion and invasion [28]. In addition, HGF-induced motility of human trophoblast cells is activated by NO signaling through phosphatidylinositol bisphosphate-3 kinase (PI3K), serine/threonine kinase (AKT) and MTOR cell signaling [26]. Increases in eNOS and iNOS activities in ovine placentomes between Days 30 and 60 of gestation are sustained to Day 140 of gestation and those increases in placental NO synthesis parallel increases in growth of the placental vasculature and utero-placental blood flows in ewes [29].

Changes in migration of trophectoderm cells may result from increases in expression of ODC1, the rate-limiting enzyme in the synthesis of polyamines from Arg, proline and ornithine [30]. Polyamines associate with DNA and nuclear proteins to produce normal chromatin required for gene transcription, proliferation of trophectoderm and formation of multinucleated trophectoderm cells that give rise to giant cells in the placentae [see 26]. Polyamine-induced cell signaling pathways include tyrosine and mitogen activated protein kinases (MAPK), proto-oncogenes (c-myc, c-jun, c-fos) and MTOR to stimulate protein synthesis in trophectoderm cells. ODC1 stimulates motility, integrin signaling via focal adhesion kinases, cytoskeletal organization, and invasiveness of mouse blastocysts through modification of beta-catenin phosphorylation and changes in uterine epithelial cells required for blastocysts to adhere to uterine LE and undergo implantation [see 26].

Synthesis of polyamines is greatest in rapidly developing ovine placentomes, both in the caruncular and cotyledonary components, and endometria between Days 30 and 60 of gestation and high levels of polyamines in placental and endometrial tissues in the second half of pregnancy likely contribute to continued development of the placental vascular bed for increased uterine blood flow to support fetal growth [26]. Knockout of the Odc1 gene in mice is not lethal until the gastrulation stage of embryogenesis, but Odc1 null blastocyst do not survive due to apoptosis of inner cell mass cells. This condition can be rescued by providing putrescine (a precursor of spermidine and spermine) in drinking water of the dam up to the early implantation stage, but not beyond that stage of pregnancy [31].

The abundance of Arg increases approximately 8-fold in the ovine uterine lumen between Days 10 and 15 of pregnancy and is involved in numerous metabolic pathways including biosynthesis of NO and polyamines. In order to assess pathways for which Arg is critical, we used in utero morpholino anti-sense oligonucleotide (MAO) loss-of-function studies to block translation of mRNAs for: 1) SLC7A1, the primary transporter for Arg into conceptus trophectoderm; 2) ODC1, the rate limiting enzyme in conversion of ornithine to polyamines (Arg to ornithine by arginase (ARGI/II) and ornithine to polyamines by ODC1); and 3) nitric oxide synthase 3 (NOS3) required for production of NO and citrulline. The use of MAO in vivo for knockdown of mRNA translation in conceptuses is unique in that trophectoderm cells of conceptuses, but not uterine epithelial cells, take up the MAO.

MAO knockdown of translation of SLC7A1 mRNA in ovine conceptus trophectoderm did not affect expression of SLC7A1 mRNA or protein in the uterus or the amount of Arg in the uterine lumen of ewes, but decreased abundances of Arg, citrulline, ornithine, ODC1 and NOS3 in conceptuses. Further, the total amount of IFNT in uterine flushings from ewes with MAO-SLC7A1 conceptuses was significantly less than for control conceptuses. Thus, SLC7A1 is critical for transport of Arg into conceptus trophectoderm for synthesis of polyamines for proliferation and migration of conceptus trophectoderm required for elongation and secretion of IFNT.

NOS3 converts Arg to NO and NOS3 mRNA and protein are abundant in the trophectoderm and endoderm of peri-implantation ovine conceptuses, whereas NOS1 mRNA and protein are expressed very weakly [39]. Following knock-down of translation of NOS3 mRNA in ovine conceptuses, they conceptuses elongated, but they were smaller, thinner and disorganized compared to MAO-control conceptuses. However, there was no effect of loss of function of NOS3 protein on IFNT production. Interestingly, SLC7A1 protein was less abundant in MAO-NOS3 conceptuses, while ODC1 protein was similar for MAO control and MAO-NOS3 conceptuses suggesting cross-talk between the Arg transporter SLC7A1 and NOS3 at the protein level. In addition, Arg, ornithine, Gln and Glu were less abundant in MAO-NOS3 conceptuses suggesting disruption of pathways for synthesis or transport of those amino acids in the absence of NO. There were no significant differences in NOS1, NOS2, ADC or AGMAT proteins between MAO-NOS3 and MAO control ewes; therefore, alternative pathways for production of NO and polyamines were not affected. An increase in agmatine in the uterine lumen and conceptuses of MAO-NOS3 conceptuses may account for the smaller and thinner, but elongated phenotype that produced IFNT since agmatine is a precursor for synthesis of polyamines. Results from studies of MAO-NOS3 conceptuses revealed the importance of NO for normal growth and development of ovine conceptuses.

ODC1 is the rate-controlling enzyme for classical de novo biosynthesis of polyamines. However, we discovered that knockdown of translation of ODC1 mRNA in ovine conceptuses resulted in upregulation of expression of arginine decarboxylase (ADC) and agmatinase (AGMAT). ADC converts Arg to agmatine and agmatinase converts agmatine to putrescine. The ADC/AGMAT pathway for synthesis of polyamines compensated for loss of ODC1 activity to allow for one-half of the ODC1-MAO conceptuses being morphologically and functionally normal and the other one-half failing to develop normally. MAO-ODC1 conceptuses with a normal phenotype expressed significantly more ADC/AGMAT mRNA and protein than did the abnormal conceptuses. Although the ODC1-pathway is the primary pathway for synthesis of polyamines, the ADC/AGMAT pathway is a complimentary pathway for production of polyamines in ovine conceptuses.

Agmatine is also a neurotransmitter that, in the brain, is released from synaptic vesicles during membrane depolarization. Agmatine is inactivated by conversion to putrescine by AGMAT. Agmatine binds α2-adrenergic receptors (ADRA2A) and imidazoline receptors (IRAS) and blocks N-methyl-d-aspartate receptor (NMDR) and other cation ligand-gated channels. Agmatine also inhibits NOS and induces secretion of some peptide hormones by cells in the hypothalamus such as oxytocin and vasopressin [40]. Exogenous agmatine has neuroprotective effects in animal models of ischemia and neurotrauma and it acts via IRAS to stimulate synthesis of eicosanoids (prostaglandins, leukotrienes, thromboxanes, prostacyclins) that enhance cell migration. Effects of agmatine on synthesis of eicosanoids may be important during pregnancy, as prostaglandins from ovine endometrium affects expression of genes critical to elongation and implantation of the ovine conceptus [41].

The synthesis, metabolism and function of agmatine in animal tissues has received little attention, but agmatine is formed in mitochondria, and arginase II and mitochondrial NOS (mtNOS) differ from cytosolic forms [see 36]. The NO formed in mitochondria may modulate the respiratory rate and ATP synthesis by inhibiting cytochrome c oxidase that would lead to formation of peroxynitrite-induced apoptosis [see 36].

The concentration of glutamine increases in the uterine lumen during the peri-implantation period of pregnancy in sheep and pigs, and glutamine affects proliferation of porcine Tr cells in vitro [14,42]. Glutamine is involved in the biosynthesis of nucleotides and can be rate-limiting for cell cycle progression. Many cancer cells undergo metabolic reprogramming that makes them highly dependent on glutamine for survival and proliferation. In the absence of glutamine, those cells stop growing and die [43,44]. Glutamine-dependent cell lines consume glutamine as the preferred anaplerotic substrate, as evident from their oxaloacetate pools, 90% of which are derived from glutaminolysis [44]. Glutaminolysis is the process by which cells convert glutamine into TCA cycle metabolites through the activities of multiple enzymes (Fig. 5). Glutamine is converted into glutamate via glutaminase (GLS/GLS2) and glutamate is converted into alpha-ketoglutarate (α-KG) via two divergent pathways. The first is through the activity of glutamate dehydrogenase (GLUD). The second is through the activity of transaminases, including glutamate–oxaloacetate transaminase (GOT), glutamate–pyruvate transaminase (GPT), and phosphoserine transaminase (PSAT). The α-KG serves as an anaplerotic substrate for the TCA cycle. We hypothesize that ovine and porcine conceptuses utilize glucose and fructose within the uterine lumen via the glycolytic biosynthetic pathway and that glycolytic intermediates are shunted into pathways for the de novo synthesis of nucleotides and amino acids, and that glutamine within the uterine lumen is an alternate carbon source to maintain TCA cycle flux (Fig. 5). Thus, glutamine supports conceptus development by directly contributing carbon and nitrogen with the carbon contributing to fatty acid and amino acid synthesis and nitrogen contributing to the biosynthesis of nucleotides and some amino acids.

Total glucose in ovine uterine lumenal fluid increases 6-fold between Days 10 and 15 of gestation, but not during the estrous cycle [14]. In the ovine uterus, SLC2A1 and SLC5A1 mRNAs and proteins are most abundant in uterine LE/sGE, whereas SLC2A4 is expressed by stromal cells and GE [45]. SLC5A11 mRNA is also abundant in endometrial GE. SLC2A1, SLC2A3 and SLC2A4, SLC5A1 and SLC5A11 are expressed in the trophectoderm and endoderm of sheep conceptuses and steady-state levels of mRNAs for SLC2A1, SLC5A1 and SLC5A11 mRNAs, but not SLC2A4 mRNA are greater in endometria from pregnant than cyclic ewes. P4 increased SLC2A1, SLC5A11 and SLC2A4 mRNAs in LE/sGE, and SLC5A1 in GE of ovariectomized ewes, while P4-induced and IFNT further stimulated expression of SLC2A1 and SLC5A11 in ovine uterine LE/sGE. Thus, there is differential expression of facilitative and sodium-dependent glucose transporters in ovine uteri and conceptuses.

The placentae of sheep and other livestock species are fructogenic. That is, glucose not metabolized by the placenta is converted into fructose by trophectoderm and it is abundant in fetal blood and allantoic fluid [19]. Although, concentrations of fructose are 11.1–33.3 mM as compared to only 2.2 mM for glucose in allantoic fluid, the roles of fructose during pregnancy in sheep and other livestock species are not established.

McGowan et al. [46] examined the rate of production of [14C]fructose and [14C]lactate from [U–14C]glucose by the ovine placenta and the contribution of 14CO2 from fetal oxidation of these metabolic products to the calculation of glucose oxidation rate in fetal sheep. Eighty percent of total fetal CO2 production was from direct oxidation of carbon atoms in glucose, while CO2 production from fructose was 21, 20, and 30% and CO2 production from lactate was 16, 13, and 11% in hypoglycemic, normoglycemic and hyperglycemic lambs, respectively. The conclusion was that fetal oxidation of substrates derived primarily from glucose metabolism occurs in the placenta.

Cell metabolism through the TCA cycle and oxidative phosphorylation in mitochondria produces ATPs. However, proliferating cells, such as trophectoderm cells, have characteristics of cancer cells and activated lymphocytes [47] that, even in the presence of oxygen, utilize aerobic glycolysis (Warburg effect) as the major metabolic pathway [48,49]. The concept is that aerobic glycolysis yields glycolytic intermediates for metabolism via branching pathways of glycolysis that lead to de novo synthesis of nucleotides, amino acids, and fatty acids to meet metabolic demands of rapidly proliferating cells [15].

Proliferating trophectoderm cells of sheep and pig conceptuses switch from reliance on oxidative phosphorylation to activation of aerobic glycolysis using glucose and fructose as metabolic substrates (Fig. 5). The trophectoderm cells utilize glucose for glycolytic branching pathways including the pentose phosphate pathway, serine biosynthesis, one-carbon metabolism, and hexosamine biosynthesis [19,50]. For example, ovine conceptus trophectoderm cells express key enzymes required for and active pentose phosphate pathway [see 15]. The Warburg effect and aerobic glycolysis appears to be operational in proliferating sheep and pig conceptuses.

There are reports [19] that: 1) fructose can be utilized for synthesis of nucleic acids and generation of NADPH + H+ in fetal pigs and in HeLa cells; neither fructose nor glucose was metabolized via the pentose phosphate pathway in the ovine placenta; and fructose and glucose are equivalent for synthesis of neutral lipids and phospholipids in heart, liver, kidney, brain and adipose tissue of fetal lambs. These findings indicate that fructose metabolism in fetal-placental tissues is not understood. It is clear that the abundance of fructose in blood, allantoic fluid and amniotic fluid of the fetal pig decreases as glucose increases between Days 82 and 112 of the 114 day period of gestation in pigs and that fructose is cleared rapidly from blood into urine of neonatal piglets by 24 h post-partum. Thus, the neonatal piglet and probably the neonatal lamb do not utilize fructose as an energy source. In fact, piglets die within 30 h post-partum without a source of glucose.

Hyaluronic acid (HA), a glycosaminoglycan synthesized via the hexosamine biosynthesis pathway, is critical for angiogenesis and other cellular functions, particularly in the placenta during pregnancy [see 19, 50]. Pig and sheep trophectoderm cells metabolize both glucose and fructose via the hexosamine biosynthesis pathway to synthesize glycosaminoglycans (e.g., hyaluronic acid), uridine diphosphate-N-acetyl glucosamine, a cell signaling molecule, and uridine diphosphate-N-acetyl galactosamine that is involved in synthesis of glycolipids, glycosaminoglycans and proteoglycans. Gln, another substrate for hexosamine biosynthesis, is particularly abundant in the allanotic fluid of sheep (e.g., 25 mM on Day 60 of gestation). Hyaluronic acid, also known as Wharton’s Jelly, accumulates in the placentae of most mammals and localizes to the umbilical cord primarily, and, to a lesser extent around placental blood vessels. Hyaluronic acid in the ECM: 1) provides for hydration and hydrodynamics; 2) stimulates proliferation, migration and adhesion of cells; 3) supports blood vessels and fragments of hyaluronic acid stimulate angiogenesis; 4) influences innate immunity; 5) supports mesenchymal cells; and 6) provides a niche for stem cells such as hematopoietic stem cells [51].

Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) is involved in intracellular signaling as a substrate for O-linked N-actetylglucosamine transferases, nuclear pore formation and nuclear signaling, glucose sensing mechanisms, and insulin sensitivity of cells [50]. Uridine diphosphate-N-acetylgalactosamine (UDP-GalNAc) is another sugar donor that, with its addition to serine or threonine residues, represents the first step in biosynthesis of mucins such as O-glycans that provide structural features to mucin glycoproteins and membrane receptors, and resistance of cells to thermal change and proteolytic attack. The O-linked carbohydrate side chains function as ligands for receptors, lymphocyte and leukocyte homing, and as signals for protein sorting.

Glucose and fructose-induce proliferation of ovine trophectoderm (oTr1) cells involving UDP-GlcNAc. After 48 and 96 h of culture, UDP-GlcNAc increases proliferation of oTr1 cells in the absence of 1 mM glucose, with 10 mM UDP-GlcNAc being optimal for maximum stimulation of cell proliferation. UDP-GlcNAc increases the abundance of total and phosphorylated MTOR, Tuberous Sclerosis Complex 2 (TSC2), and protein kinase B (AKT) in oTr1 cells. After 96 h of incubation, p-MTOR increased 2.3-fold, p-TSC2 increased 4.0-fold and p-AKT increased 1.9-fold in UDP-GlcNAc-treated cells compared to control cells. These effects were inhibited when oTr cells were incubated in medium containing alloxan, an inhibitor of O-linked N-acetylglucosamine transferase (OGT). In addition, knockdown of OGT mRNA translation inhibited proliferation and O-GlcNAcylation-mediated phosphorylation of components of the MTOR cell signaling cascade. Thus, fructose and glucose metabolized via the nonoxidative hexosamine biosynthesis pathway to GlcN-6-P by glutamine-fructose-6-phosphate transaminase 1 (GFPT1) increases O-GlcNAcylation of cellular proteins/enzymes by OGT, and then activates the Akt-TSC2-MTOR cell signaling cascade to stimulate proliferation of ovine trophectoderm cells.

Research focused on IUGR and adult onset of metabolic disease in humans, use sheep as a model, but ignore fructose which is the major hexose sugar in the ovine conceptus [see 19, 50]. In ewes, the maximum concentrations of glucose in allantoic fluid are about 1.1 mM between Days 35 and 140 of pregnancy, while concentrations of fructose are between 11.1 and 33 mM [32]. Therefore, fructose is exerting maximum effects on proliferation of ovine trophectoderm cells at molar concentrations well below those in allantoic fluid [see 22]. Thus, fructose promotes embryonic/fetal growth and development during pregnancy which should be taken into account when assessing effects of nutrition and other environmental factors on sheep as a model for research on IUGR.

The uterine environment during the peri-implantation period of pregnancy is hypoxic and that influences utilization of specific metabolic pathways that favor growth and survival of organisms, including conceptuses of sheep, pigs and other domestic animals. The African naked mole-rat tolerates hours of extreme hypoxia and survives 18 min of total oxygen deprivation (anoxia) during which time they switch to anaerobic metabolism fueled by fructose which was actively accumulated and metabolized to lactate in the brain [52]. Global expression of GLUT5 fructose transporter and high levels of expression of ketohexokinase in tissues of naked mole rats under anoxia resulted in fructose-driven glycolytic respiration that circumvented the normal feedback inhibition of glycolysis via phosphofructokinase. This was considered key to prolonged viability of the naked mole rats under hypoxic or anoxic conditions. Ketohexokinase converts fructose to fructose-1-PO4 that is metabolized to glyceraldehyde, dihydroxyacetone phosphate and glyceraldehyde 3 phosphate, but the ketohexokinase pathway is not inhibited by pH, citrate or ATP as occurs when glucose is phosphorylated to glucose-6-PO4 by hexokinase and glucose-6-PO4 is converted to fructose-6-PO4 and then to fructose-1,6 bisphosphate by phosphofructokinase. The hexokinase pathway is subject to feedback inhibition by pH, citrate and ATP at the phosphofructokinase level of metabolism.

As noted previously (Fig. 5), trophectoderm cells of sheep and pigs in their hypoxic environment are metabolically distinct from cells of resting tissues, and reflect characteristics of cancer cells and activated lymphocytes in their ability to enhance glycolysis, even in the presence of oxygen by implementing the Warburg effect or aerobic glycolysis [15,53]. There is evidence that, at least in pig trophectoderm cells, the ketohexokinase enzyme is abundantly expressed [54]; therefore, aerobic glycolysis via fructose-1-PO4 is not inhibited by ATP, acidic pH, or citrate. Therefore, the ketohexose pathway used to prolong survival of naked mole rats under hypoxia/anoxia may be the key the key metabolic pathway in trophectoderm cells of sheep and pigs under hypoxic conditions during the peri-implantation period of pregnancy and, perhaps, into later stages of gestation because a major metabolic product of aerobic glycolysis is lactic acid that creates an acidic environment for the trophectoderm cells [55].

Lactate was a major metabolite of the Naked Mole rat under hypoxic conditions and it may also play a role in survival of those rats [52]. In mice, aerobic glycolysis also provides for a high carbon flux to fulfil biosynthetic demands and to increase concentrations of lactate and low pH around the conceptus [55]. Lactate may activate cell signaling under hypoxic conditions at implantation sites to: 1) increase expression of hypoxia inducible factor 1 alpha (HIF1A) and downstream growth factors such as bioactive VEGF to increase angiogenesis; 2) modulate local immune responses to favor immune tolerance; and 3) modulate expression of enzymes that modify the extracellular matrix of the endometrium in preparation for implantation [55]. The conversion of pyruvate to lactate via lactate dehydrdogenase also regenerates NAD+ required for glycolysis as maintenance of the NAD+/NADH redox balance is necessary for conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate and NADH is a cofactor for the transcriptional regulator C-terminal-binding protein involved in cell growth, differentiation, and transformation [56].

Section snippets

Concluding remarks

In addition to its role as the pregnancy recognition signal in ruminants, IFNT, in concert with P4, induces expression of genes in uterine LE and sGE for transports of glucose and amino acids such as arginine and glutamine. Glucose can then be converted to fructose and both of those hexose sugars can be metabolized via aerobic glycolysis to provide intermediates for hexosamine biosynthesis for glycosaminoglycans such as hyaluronic acid, one-carbon metabolism for biosynthesis of purines,

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgements

We wish to acknowledge from the National Research Initiative Competitive Grants no. 2011-67015-20028, 2015-67015-23276, 2016-67015-24958, 2018-67015-28093 from the USDA National Institute of Food and Agriculture.

References (55)

  • C. Yang et al.

    Glutamine oxidation maintains the TCA cycle and cell survival during impaired mitochondrial pyruvate transport

    Mol Cell

    (2014)
  • G. Andrejeva et al.

    Similarities and distinctions of cancer and immune metabolism in inflammation and tumors

    Cell Metabol

    (2017)
  • L. Yang et al.

    Hypoxia-induced miR-214 expression promotes tumour cell proliferation and migration by enhancing the Warburg effect in gastric carcinoma cells

    Canc Lett

    (2018)
  • X. Wang et al.

    Functional roles of fructose: crosstalk between O-linked glycosylation and phosphorylation of Akt-TSC2-MTOR cell signaling cascade

    Biol Reprod

    (2016)
  • F.W. Bazer et al.

    Historical Aspects: chronicling the discovery of IFNT

    Reproduction

    (2017)
  • F.W. Bazer et al.

    Mechanisms for the establishment and maintenance of pregnancy: synergies from scientific collaborations

    Biol Reprod

    (2018)
  • T.E. Spencer et al.

    Progesterone and placental hormone actions on the uterus: insights from domestic animals

    Biol Reprod

    (2004)
  • T.E. Spencer et al.

    Implantation mechanisms: insights from the sheep

    Reproduction

    (2004)
  • R.B. Gwatkin

    Nutritional requirements for post-blastocyst development in the mouse. Amino acids and protein in the uterus during implantation

    Int J Fertil

    (1969)
  • F.W. Bazer et al.

    Interferons and uterine receptivity

    Semin Reprod Med

    (2009)
  • F.W. Bazer et al.

    Select nutrients in the uterine lumen of sheep and pigs affect conceptus development

    J Reprod Dev

    (2012)
  • G. Wu et al.

    Impacts of arginine nutrition on embryonic and fetal development in mammals

    Amino Acids

    (2013)
  • F.W. Bazer et al.

    Amino acids and conceptus development during the peri-implantation period of pregnancy

    Adv Exp Med Biol

    (2015)
  • H. Gao et al.

    Select nutrients in the ovine uterine lumen: I. Amino acids, glucose and ions in uterine lumenal fluid of cyclic and pregnant ewes

    Biol Reprod

    (2009)
  • G.A. Johnson et al.

    Cellular events during ovine implantation and impact for gestation

    Anim Reprod

    (2018)
  • J. Kim et al.

    Select Nutrients in the ovine uterine lumen: VII. Effects of arginine, leucine, glutamine and glucose on trophectodem cell signaling, proliferation and migration

    Biol Reprod

    (2011)
  • J. Kim et al.

    Select Nutrients in the ovine uterine lumen: VIII. Arginine stimulates proliferation of ovine trophectoderm cells through mTOR-RPS6K-RPS6 signaling cascade and synthesis of nitric oxide and polyamines

    Biol Reprod

    (2011)
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      IFN-τ acts on the endometrial lumen and glandular epithelium in a paracrine way, and plays an anti-luteinolytic role by inhibiting the expression of endometrial estrogen receptor and oxytocin receptor genes, thus maintaining the normal progress of pregnancy [22]. IFN-τ also induces or enhances the expression of endometrial interferon-stimulating genes (ISGs) during early pregnancy in sheep and cows by binding to interferon receptors (IFNAR, two subunits: IFNAR1 and IFNAR2), and plays a role in regulating endometrial receptivity, gestational elongation and implantation [23,24]. It has been suggested that IFN-τ plays a role in endometrium-induced classical interferon-stimulated gene expression, which promotes gestational implantation, establishment of pregnancy, and regulation of immune cells in mother-to-pregnancy interaction [25].

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      This is part of a cascade of events that start even before the implantation events, with a downregulation of receptors for progesterone (PGR) and estrogen in the uterine epithelia [29]. This loss of PGR expression makes the action of progesterone to be restricted to PGR-positive uterine stromal cells, which express prostamedins such as FGF7 and FGF10 [12]. Then, the prostamedins act on uterine epithelia and trophectoderm to regulate expression of interferons-stimulated genes, with a following process that is important for establishing uterine receptivity to implantation and maintenance of conception in mammals [12,30].

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      The failure to make meaningful progress in improving embryo survival in livestock can be explained by insufficient knowledge regarding the cellular and molecular events during the pre-implantation period [34]. Recent reviews on conceptus development, attachment, and implantation have summarized the role of cell-cell adhesion molecules [11,35], interferon tau (IFN-τ) [12,35–40], kisspeptin [13] and exosomes/extracellular vesicles [41–49]. It is abundantly clear that ongoing communication between the conceptus and uterus is fundamental to implantation and pregnancy [34,38,50].

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