Conceptus Trophectoderm Research Articles

A Potential Autocrine Role for Interferon Tau in Ovine Trophectoderm

Interferon tau (IFNT), a type I IFN produced by the conceptus trophectoderm, is the signal for maternal pregnancy recognition in ruminants. The purpose of this study was to investigate whether IFNT effected on the proliferation of ovine trophectoderm cells in an autocrine manner. Elongated ovine conceptuses (Days 15, Day 0=day of mating) were collected for isolation of mononuclear ovine trophectoderm (oTr-1) cells, and conceptuses (Days 15 and 20, n=4 and 3, respectively) were collected for RNA extraction. We demonstrated that the IFNT receptor, IFNAR1, was expressed in trophectoderm of day 15 and 20 conceptuses. Interestingly, the ovine trophectoderm cell line oTr-1 cultured in the presence of recombinant bovine IFNT (rbIFNT) displayed increased expressions of IFN-stimulated genes (ISGs), such as IFN-stimulated gene 15 (ISG15), 2-5-oligoadenylate synthetase 1 (OAS1) and bone marrow stromal cell antigen 2 (BST2). Meanwhile, the presence of rbIFNT in the culture media could promote the cell proliferation in a dose-dependent manner. Furthermore, the connective tissue growth factor, which has diverse functions in cell proliferation and is involved in conceptus elongation, was upregulated in oTr-1 cell by rbIFNT treatment in vitro. These data indicated that IFNT could act as an autocrine factor to regulate trophectoderm cell proliferation.

Contributions of an animal scientist to understanding the biology of the uterus and pregnancy

I developed a passion for reproductive biology when taking a course in Physiology of Reproduction at Louisiana State University while preparing to apply for Veterinary School at Texas A&M University. My career path changed. I entered graduate school, obtained a Ph.D. and have enjoyed an academic career conducting research in uterine biology and pregnancy in animal science departments at the University of Florida and at Texas A&M University. My contributions to science include: (1) identification of molecules secreted by or transported by uterine epithelia into the uterine lumen that are critical to successful establishment and maintenance of pregnancy, (2) discovery of steroids and proteins required for pregnancy-recognition signalling and their mechanisms of action in pigs and ruminants, (3) patterns of fetal-placental development and placental transport of nutrients, (4) identification of links between nutrients and components of histotroph that affect fetal-placental development, (5) characterising aspects of the endocrinology of pregnancy and (6) contributing to efforts to exploit the therapeutic value of interferon tau, particularly for treatment of autoimmune and inflammatory diseases. Current research focuses on select nutrients in the uterine lumen, specifically amino acids, glucose and fructose, that affect conceptus development, the therapeutic potential for interferon tau, stromal-epithelial cell signalling whereby progesterone and oestrogen act via steroid receptors in uterine stromal cells to stimulate secretion of growth factors (e.g. fibroblast growth factors and hepatocyte growth factor) that regulate uterine epithelial cells and conceptus trophectoderm, and roles of toll-like receptors expressed by uterine epithelia and conceptus trophectoderm in pregnancy.

Select nutrients, progesterone, and interferon tau affect conceptus metabolism and development.

Interferon tau (IFNT), a novel multifunctional type I interferon secreted by trophectoderm, is the pregnancy recognition signal in ruminants that also has antiviral, antiproliferative, and immunomodulatory bioactivities. IFNT, with progesterone, affects availability of the metabolic substrate in the uterine lumen by inducing expression of genes for transport of select nutrients into the uterine lumen that activate mammalian target of rapamycin (mTOR) cell signaling responsible for proliferation, migration, and protein synthesis by conceptus trophectoderm. As an immunomodulatory protein, IFNT induces an anti-inflammatory state affecting metabolic events that decrease adiposity and glutamine:fructose-6-phosphate amidotransferase 1 activity, while increasing insulin sensitivity, nitric oxide production by endothelial cells, and brown adipose tissue in rats. This short review focuses on effects of IFNT and progesterone affecting transport of select nutrients into the uterine lumen to stimulate mTOR cell signaling required for conceptus development, as well as effects of IFNT on the immune system and adiposity in rats with respect to its potential therapeutic value in reducing obesity.

Application of next generation sequencing in mammalian embryogenomics: Lessons learned from endogenous betaretroviruses of sheep

Endogenous retroviruses (ERVs) are present in the genome of all vertebrates and are remnants of ancient exogenous retroviral infections of the host germline transmitted vertically from generation to generation. The sheep genome contains 27 JSRV-related endogenous betaretroviruses (enJSRVs) related to the pathogenic Jaagsiekte sheep retrovirus (JSRV) that have been integrating in the host genome for the last 5–7 million years. The exogenous JSRV is a causative agent of a transmissible lung cancer in sheep, and enJSRVs are able to protect the host against JSRV infection. In sheep, the enJSRVs are most abundantly expressed in the uterine epithelia as well as in the conceptus (embryo and associated extraembryonic membranes) trophectoderm. Sixteen of the 27 enJSRV loci contain an envelope (env) gene with an intact open reading frame, and in utero loss-of-function experiments found the enJSRVs Env to be essential for trophoblast outgrowth and conceptus elongation. Collectively, available evidence supports the ideas that genes captured from ancestral retroviruses were pivotal in the acquisition of new, important functions in mammalian evolution and were positively selected for biological roles in genome plasticity, protection of the host against infection of related pathogenic and exogenous retroviruses, and a convergent physiological role in placental morphogenesis and thus mammalian reproduction. The discovery of ERVs in mammals was initially based on molecular cloning discovery techniques and will be boosted forward by next generation sequencing technologies and in silico discovery techniques.

Effects of In Vivo Knockdown of Translation of Arginine-Associated Gene Transcripts in Conceptus Trophectoderm on Growth, Development, and Gene Expression by Ovine Conceptuses.

Arginine (Arg), a nutritionally essential amino acid for conceptus development, can be metabolized to nitric oxide (NO) and polyamines. Arg increases 13-fold in the ovine uterine lumen between Days 10 and 15 of pregnancy and activates mechanistic target of rapamycin cell signaling. This study tested the hypothesis that knockdown of translation of Arg-associated gene transcripts inhibits ovine conceptus development. Morpholino antisense oligonucleotides (MAOs) were designed to inhibit translation of mRNAs for: 1) SLC7A1, Arg transporter; 2) NOS3, an enzyme for NO synthesis; 3) ODC1, an enzyme for polyamine synthesis; and 4) mismatch morpholino as MAO-control. On Day 8 post-mating, the uterine horn of ewes (n=7 or 8 /MAO type) ipsilateral to the CL was ligated and MAO injected into the uterine lumen. On Day 16, ewes were hysterectomized to obtain conceptuses, uterine flushings, and uterine tissues from the treatment groups: 1) MAO-SLC7A1 (n=8); 2) MAO-NOS3 (n=7); 3) MAO-ODC1 (n=8); and 4) MAO-control (n=7). Conceptuses in the control uterine horn were healthy, elongated and intact. Conceptuses exposed to the SLC7A1-MAO were small, thin, fragile and fragmented. Conceptuses exposed to the NOS3-MAO were thin and small, but elongated and healthy. In the ODC1-MAO group, half of the conceptus were small, fragile and fragmented, but half were healthy and elongated. Compared to protein concentration (2.1 + 0.5 mg/ml), uterine flush volume recovered (8.2 + 0.8 ml) and total protein in uterine flushing (15.7±3.5 mg) for MAO-control ewes, values did not differ (P>0.05) for treatment MAOs. Slot blot analysis did not detect effects of treatment (P>0.05) on interferon-tau (IFNT) in uterine flushings. However, radioimmunoassay data revealed an increase (P<0.05) in IFNT in MAO-NOS3 (15,057±2,538 ng) compared to MAO-control (9,353±1,768 ng) ewes. Pregnancy rates were not affected by treatment (P>0.2). Thus, knockdown of SLC7A1 protein attenuated conceptus development. Knockdown of NOS3 and ODC1 proteins partially suppressed conceptus development likely due to compensatory effects of NOS1/NOS2, other cationic amino acid transporters and production of putrescine via the arginine decarboxylase and agmatinase pathway. This research was supported by Agriculture and Food Research Initiative Competitive Grant no. 2011-67015-20028 from the USDA National Institute of Food and Agriculture.

Functional roles of fructose

During the periimplantation period of pregnancy, pig blastocysts undergo morphological changes and differentiation requiring secretion and transport of nutrients (histotroph) into the uterine lumen. Of these nutrients, glucose is converted to fructose, an isomer of glucose, by conceptus trophectoderm. Although glucose is an energy source for proliferation and growth of mammalian cells, the role of fructose in uterine histotroph is unclear although it is the most abundant hexose sugar in fetal blood and fluids of ungulate mammals (e.g., cows, sheep, and pigs). In this study, we used porcine trophectoderm cells to determine that fructose increased cell proliferation, as did glucose. Western blot analyses of porcine trophectoderm cell extracts revealed that fructose increased the abundance of phosphorylated-RPS6K, -EIF4EBP1, and -RPS6 over basal levels within 30 min, and those levels remained elevated to 120 min. Phosphorylation of both RPS6K and EIF4EBP1 proteins in response to fructose was inhibited by inhibitors of both PI3K and MTOR. Further, when we investigated the inhibition of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) by azaserine (an inhibitor of GFPT1) and GFPT1 siRNA, we found that MTOR-RPS6K and MTOR-EIF4EBP1 signaling in response to fructose is mediated via GFPT1 activation and the hexosamine pathway. We further demonstrated that fructose stimulates the production of hyaluronic acid via GFPT1 and the hexosamine biosynthesis pathway. Collectively, these results demonstrate critical roles for fructose that are mediated via the hexosamine biosynthesis pathway to stimulate MTOR cell signaling, proliferation of porcine trophectoderm cells, and synthesis of hyaluronic acid, a significant glycosaminoglycan in the pregnant uterus.

Interaction of the conceptus and endometrium to establish pregnancy in mammals: role of interleukin 1β

Implantation and the establishment of pregnancy in mammals involves an intricate interplay of hormones, cytokines, growth factors, proteins, lipids, ions and the extracellular matrix between the uterine epithelium, stroma, immune cells and the conceptus trophectoderm. The divergent nature of implantation in the mouse, human and pig provides not only an interesting contrast in the establishment of pregnancy and early embryonic development but also intriguing similarities with regard to early endometrial-conceptus signaling. An interesting pro-inflammatory cytokine expressed in a number of mammalian species during the period of implantation is interleukin-1β (IL1B). The presence of IL1B might be involved with immunotolerance at the maternal-placental interface and has been proposed as one of the mediators in placental viviparity. The production of IL1B and other proinflammatory cytokines might play a role in establishing pregnancy through modulation of the nuclear factor kappa-B (NFKB) system in a number of species. A model for the regulation of cellular progesterone receptor expression and NFKB activation for endometrial receptivity and conceptus attachment is continuing to evolve and is discussed in the present review.

Swine Leukocyte Antigen-DQ Expression and Its Regulation by Interferon-Gamma at the Maternal-Fetal Interface in Pigs1

Successful pregnancy requires an appropriate intrauterine immune response to the conceptus, which is a semiallograft within the uterus. We reported that swine leukocyte antigen-DQA (SLA-DQA), a major histocompatibility complex (MHC) class II gene, is expressed in the uterine endometrium at the time of conceptus implantation in pigs. Because MHC molecules play critical roles in the immune system, SLA-DQ was hypothesized to be involved in immune regulation during pregnancy. Therefore, we examined expression of SLA-DQ in uterine endometrial tissues obtained during the estrous cycle and pregnancy. SLA-DQA and SLA-DQB mRNAs were detected as 1.3-kb and 1.2-kb bands, respectively. Real-time RT-PCR analysis indicated that SLA-DQA and SLA-DQB mRNA expression was affected by day and pregnancy status, with the highest expression on Day 15 of pregnancy. SLA-DQ was localized primarily to subepithelial stromal cells and endothelial cells of the uterus. Using endometrial explant cultures from Day 12 of the estrous cycle, we determined that expression of SLA-DQA and SLA-DQB mRNAs increased in response to interferon-gamma (IFNG), which is produced by pig conceptus trophectoderm between Days 14 and 18 of pregnancy. The abundance of SLA-DQ protein was less in endometria from gilts with conceptuses resulting from somatic cell nuclear transfer compared with endometria from gilts with conceptuses resulting from natural mating. These results support our hypothesis that SLA-DQ is expressed in response to IFNG from the conceptus, and likely regulates immune response at the maternal-fetal interface to support the maintenance of pregnancy in pigs.

Select Nutrients in the Uterine Lumen of Sheep and Pigs Affect Conceptus Development

Interferon tau (IFNT) is the pregnancy recognition signal from ruminant conceptuses. IFNT also acts with P4 to induce expression of genes for transport of nutrients, such as glucose (Gluc) and arginine (Arg) into the uterine lumen to activate mechanistic mammalian target of rapamycin (MTOR) cell signaling that stimulates proliferation, migration, gene transcription and mRNA translation by conceptus trophectoderm (Tr). In ewes, Arg and Gluc increase significantly in the uterine lumen between Days 10 and 15 of pregnancy due to increased expression of transporters for Gluc (SLC2A1 and SLC5A1) and Arg (SLC7A2B) by uterine epithelia. Arg and Gluc stimulate proliferation, migration and mRNA translation by Tr. Arg increases expression of GTP cyclohydrolase 1 (GCH1) and IFNT mRNAs while Arg and Gluc increase ornithine decarboxylase, nitric oxide synthase 2, and GCH1 mRNAs and proteins by Tr cells. GCH1 is required for synthesis of tetrahydrobiopterin, an essential cofactor for all NOS isoforms. Arg is metabolized to nitric oxide and polyamines that increase proliferation and migration of Tr cells. In pigs, Gluc, Arg, leucine (Leu) and glutamine (Gln) increase in the uterine lumen between Days 12 and 15 of pregnancy due to enhanced expression of transporters for Gluc and amino acids. Transporters for Gluc in porcine uterine LE (SLC2A1) and conceptus trophectoderm (SLC2A2) are abundant. Transporters for glutamate and neutral (SLC1A1, SLC1A4) and cationic (SLC7A1, SLC7A2, SLC7A7, SLC7A9) amino acids are expressed in uterine LE and SLC7A3 mRNA is expressed in conceptus Tr. Arg and Leu increase MTOR cell signaling and proliferation of pig Tr, as do Gluc and fructose. Azaserine, an inhibitor of hexosamine biosynthesis, inhibits effects of Gluc and fructose. Thus, select nutrients in the uterine lumen affect gene transcription and mRNA translation to affect conceptus development.

Endogenous Retroviruses of Sheep: A Model System for Understanding Physiological Adaptation to an Evolving Ruminant Genome

Endogenous retroviruses (ERVs) are present in the genome of all vertebrates and are remnants of ancient exogenous retroviral infections of the host germline transmitted vertically from generation to generation. Sheep betaretroviruses offer a unique model system to study the complex interaction between retroviruses and their host. The sheep genome contains 27 endogenous betaretroviruses (enJSRVs) related to the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV), the causative agent of a transmissible lung cancer in sheep. The enJSRVs can protect their host against JSRV infection by blocking early and late steps of the JSRV replication cycle. In the female reproductive tract, enJSRVs are specifically expressed in the uterine luminal and glandular epithelia as well as in the conceptus (embryo and associated extraembryonic membranes) trophectoderm and in utero loss-of-function experiments found the enJSRVs envelope (env) to be essential for conceptus elongation and trophectoderm growth and development. Collectively, available evidence in sheep and other mammals indicate that ERVs coevolved with their hosts for millions of years and were positively selected for biological roles in genome plasticity and evolution, protection of the host against infection of related pathogenic and exogenous retroviruses, and placental development.

L-Arginine stimulates the mTOR signaling pathway and protein synthesis in porcine trophectoderm cells

Impairment of placental growth is a major factor contributing to intrauterine growth retardation (IUGR) in both human pregnancy and animal production. Results of recent studies indicate that administration of l-arginine (Arg) to gestating pigs or sheep with IUGR fetuses can enhance fetal growth. However, the underlying mechanisms are largely unknown. The present study tested the hypothesis that Arg stimulates the mammalian target of rapamycin (mTOR) signaling pathway and protein synthesis in porcine conceptus trophectoderm (pTr2) cells. The cells were cultured for 4 days in Arg-free Dulbecco's modified Eagle's Ham medium containing 10, 50, 100, 200, 350 or 500 μM Arg. Cell numbers, protein synthesis and degradation, as well as total and phosphorylated levels of mTOR, ribosomal protein S6 kinase 1 (p70S6K) and eukaryotic initiation factor 4E-binding protein-1 (4EBP1), were determined. The pTr2 cells exhibited time (0–6 days)- and Arg concentration (10–350 μM)-dependent increases in proliferation. Addition of 100 and 350 μM Arg to culture medium dose-dependently increased (a) protein synthesis and decreased protein degradation and (b) the abundance of total and phosphorylated mTOR, p70S6K and 4EBP1 proteins. Effects of 350 μM Arg on intracellular protein turnover were only modestly affected when nitric oxide synthesis was inhibited. Collectively, these results indicate a novel and important role for Arg in promoting growth of porcine placental cells largely via a nitric-oxide-independent pathway. Additionally, these findings help to explain beneficial effects of Arg supplementation on improving survival and growth of embryos/fetuses in mammals.

Uterine Histotroph and Conceptus Development: Select Nutrients and Secreted Phosphoprotein 1 Affect Mechanistic Target of Rapamycin Cell Signaling in Ewes1

Interferon tau (IFNT), the pregnancy recognition signal in ruminants, abrogates the uterine luteolytic mechanism to ensure maintenance of function for the corpora lutea to produce progesterone (P4). IFNT also suppresses expression of classical IFN-stimulated genes by uterine lumenal epithelium (LE) and superficial glandular (sGE) epithelium but, acting in concert with progesterone, affects expression of a multitude of genes critical to growth and development of the conceptus. The LE and sGE secrete proteins and transport nutrients into the uterine lumen necessary for conceptus development, pregnancy recognition signaling, and implantation. Secretions include arginine and secreted phosphoprotein 1 (SPP1). Arginine can be metabolized to nitric oxide and to polyamines or act directly to activate the mechanistic target of rapamycin cell signaling pathway to stimulate proliferation, migration, and mRNA translation in trophectoderm cells. SPP1 binds alphavbeta3 and alpha5beta1 integrins to induce focal adhesion assembly, adhesion, and migration of conceptus trophectoderm cells during implantation. Thus, arginine and SPP1 mediate growth, migration, cytoskeletal remodeling, and adhesion of trophectoderm essential for pregnancy recognition signaling and implantation. This minireview focuses on components of histotroph that affect conceptus development in the ewe.

Expression of Toll-Like Receptors in Uteri and Conceptuses of Ewes.

Toll-like Receptors (TLRs) are type I transmembrane proteins involved in the innate immune response to pathogen-associated molecular pattern (PAMPs) molecules. They are pattern recognition receptors which, after binding to their ligands, initiate signaling cascades by means of inflammation, phagocytosis, activation of complement, release of inflammatory cytokines, apoptosis and activation of transcription factors. TLRs are expressed primarily in antigen processing and presenting cells, although they have been detected in endothelial cells, fibroblasts and epithelium of multiple tissues including the ovary and uterus. Influences of the conceptus on the innate immune system have been described for humans and mice; therefore, we investigated differences in expression of TLRs in cyclic and pregnant ewes. Expression of TLR was analyzed in uterine endometria from ewes on Days 10, 12, 14 and 16 of the estrous cycle and endometria and conceptuses from ewes on Days 10, 12, 14, 16, 18 and 20 of pregnancy (n=4 or 5 ewes/day; Day 0 is the day of onset of estrus). Expression of mRNAs for TLR1-TLR9 was quantified by real-time quantitative PCR. Relative quantification of gene expression was evaluated using a comparative CT method by subtracting the expression of alpha tubulin (TUBA1) CT value of each sample from the target gene CT value and data were analyzed to detect differences due to Day, Pregnancy Status and Day X Pregnancy Status interaction using PROC GLM of SAS. Expression of all TLR were affected by Day (P<0.01) except for TLR4 and TLR8 (P>0.05). Expression of TLR2, TLR3, TLR6, TLR7 and TLR8 was different between cyclic and pregnant (P<0.01) ewes and there were Day X Pregnancy Status effects for TLR2, TLR6, TLR7 (P<0.01) and TLR8 and TLR9 (P<0.05). Immunoreactive TLR7- and TLR8-like proteins were detected in uterine luminal epithelium, superficial glandular epithelium and stroma between Days 10 and 16 of the estrous cycle. Conversely, in pregnancy, endometrial expression of TLR7- and TLR8-like proteins decreased between Days 10 to Day 16, but then increased between Days 18 and 20 with abundant expression in the conceptus trophectoderm. Additionally, intraepithelial lymphocytes present mainly in the deep uterine glands expressed TLR7 on all days studied. Results of this study indicate that TLRs are differentially expressed in most uterine cell types as well as resident immune cells during the estrous cycle, and that the pattern of expression is modified during the peri-implantation period of pregnancy in ewes. (poster)

Activated Leukocyte Cell Adhesion Molecule: Expression in the Uterine Endometrium during the Estrous Cycle and Pregnancy in Pigs

The pig exhibits true epitheliochorial placentation, where the fetal membrane maintains attachment throughout pregnancy but does not invade into the maternal uterine endometrium. Accordingly, the expression and function of cell adhesion molecules are very important for embryo implantation and the establishment of pregnancy. In our recent microarray analysis, we found that activated leukocyte cell adhesion molecule (ALCAM) was expressed in the uterine endometrium during pregnancy in pigs. To better understand the roles of ALCAM in the establishment and maintenance of pregnancy, we examined ALCAM expression in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that ALCAM was differentially expressed in the uterine endometrium during the estrous cycle and pregnancy, with the highest levels on D12 of pregnancy. ALCAM mRNA was localized to the luminal and glandular epithelial cells and to the trophectoderm of conceptuses during early pregnancy. The steroid hormones estrogen and progesterone had no effect on ALCAM expression in an endometrial explant culture study. Further, we found that ALCAM expression in the uterine endometrium from gilts with somatic cell nuclear transfer-derived embryos was not different from that in gilts with embryos from natural mating. ALCAM was expressed in a pregnancy stage- and cell type-specific manner in the uterine endometrium and conceptuses during pregnancy. These findings suggest that ALCAM may play a role in the establishment of pregnancy. Further analysis of ALCAM will provide insight into the implantation process and establishment of pregnancy in pigs.

Contributions of an Animal Scientist to Reproductive Biology

I became interested in biology as an undergraduate in a premedical curriculum but developed a passion for the field of reproductive biology because of a course in physiology of reproduction taken to meet requirements for admission to veterinary school. My career path changed, and I entered graduate school, obtained the Ph.D., and have enjoyed an academic career as a reproductive biologist conducting research in uterine biology and pregnancy in animal science departments at the University of Florida and at Texas A&M University. However, I have never allowed academic boundaries to interfere with research and graduate education as that is contrary to collegiality, the cornerstone of great universities. I consider that my major contributions to science include 1) identification of proteins secreted by cells of the uterine endometrium that are critical to successful establishment and maintenance of pregnancy; 2) discovery of steroids and proteins required for pregnancy recognition signaling and their mechanisms of action in pigs and ruminant species; 3) investigation of fetal-placental development and placental transport of nutrients, including water and electrolytes; 4) identification of linkages between nutrition and fetal-placental development; 5) defining aspects of the endocrinology of pregnancy; and 6) contributing to efforts to exploit the therapeutic value of interferon tau, particularly for treatment of autoimmune diseases. My current studies are focused on the role of select nutrients in the uterine lumen, specifically amino acids and glucose, that affect development and survival of the conceptus and translation of mRNAs and, with colleagues at Seoul National University, gene expression by the avian reproductive tract at key periods postovulation. Another goal is to understand stromal-epithelial cell signaling, whereby progesterone and estrogen act via uterine stromal cells that express receptors for sex steroids to stimulate secretion of growth factors (e.g., fibroblast growth factors and hepatocyte growth factor) that, in turn, regulate functions of uterine epithelial cells and conceptus trophectoderm.

Prostaglandins Regulate Conceptus Elongation and Mediate Effects of Interferon Tau on the Ovine Uterine Endometrium1

In ruminants, both the endometrium and the conceptus (embryo and associated extraembryonic membranes) trophectoderm synthesizes and secretes prostaglandins (PG) during early pregnancy. In mice and humans, PGs regulate endometrial function and conceptus implantation. In Study One, bred ewes received intrauterine infusions of vehicle as a control (CX) or meloxicam (MEL), a PG synthase (PTGS) inhibitor from Days 8-14 postmating, and the uterine lumen was flushed on Day 14 to recover conceptuses and assess their morphology. Elongating and filamentous conceptuses (12 cm to >14 cm in length) were recovered from all CX-treated ewes. In contrast, MEL-treated ewes contained mostly ovoid or tubular conceptuses. PTGS activity in the uterine endometrium and amounts of PGs were substantially lower in uterine flushings from MEL-treated ewes. Receptors for PGE2 and PGF2 alpha were present in both the conceptus and the endometrium, particularly the epithelia. In Study Two, cyclic ewes received intrauterine infusions of CX, MEL, recombinant ovine interferon tau (IFNT), or IFNT and MEL from Days 10-14 postestrus. Infusion of MEL decreased PGs in the uterine lumen and expression of a number of progesterone-induced endometrial genes, particularly IGFBP1 and HSD11B1. IFNT increased endometrial PTGS activity and the amount of PGs in the uterine lumen. Interestingly, IFNT stimulation of many genes (FGF2, ISG15, RSAD2, CST3, CTSL, GRP, LGALS15, IGFBP1, SLC2A1, SLC5A1, SLC7A2) was reduced by co-infusion with MEL. Thus, PGs are important regulators of conceptus elongation and mediators of endometrial responses to progesterone and IFNT in the ovine uterus.

Endogenous retroviruses of sheep: a model system for understanding physiological adaptation to an evolving ruminant genome

Endogenous retroviruses (ERVs) are present in the genome of all vertebrates and are remnants of ancient exogenous retroviral infections of the host germline transmitted vertically from generation to generation. Sheep betaretroviruses offer a unique model system to study the complex interaction between retroviruses and their host. The sheep genome contains 27 endogenous betaretroviruses (enJSRVs) related to the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV), the causative agent of a transmissible lung cancer in sheep. The enJSRVs can protect their host against JSRV infection by blocking early and late steps of the JSRV replication cycle. In the female reproductive tract, enJSRVs are specifically expressed in the uterine luminal and glandular epithelia as well as in the conceptus (embryo and associated extraembryonic membranes) trophectoderm and in utero loss-of-function experiments found the enJSRVs envelope (env) to be essential for conceptus elongation and trophectoderm growth and development. Collectively, available evidence in sheep and other mammals indicate that ERVs coevolved with their hosts for millions of years and were positively selected for biological roles in genome plasticity and evolution, protection of the host against infection of related pathogenic and exogenous retroviruses, and placental development.

Endogenous Retroviruses: From Infectious Elements to Essential Genes.

Endogenous retroviruses (ERVs) are present in the genome of all vertebrates and are remnants of ancient exogenous retroviral infections of the host germline transmitted vertically from generation to generation. Syncytins are envelope (env) genes of retroviral origin that have been co-opted by the host to mediate placental development. Two syncytin genes have been identified in primates (HERV-W (ERVWE1) termed syncytin-1 and HERV-FRD termed syncytin-2), as well as distinct, nonorthologous genes in rodents (syncytin-A and -B) and rabbits (syncytin-Ory1). The syncytins can induce cell-cell fusion, and placental development in those mammals requires the fusion of trophoblast cells into a highly specialized, multinucleated syncytiotrophoblast layer, which is essential for absorption of nutrients from the mother. All of the syncytins are expressed in the syncytiotrophoblasts, and null mutation of the syncytin-A gene in mice results in disruption of the syncytiotrophoblast-containing labyrinth placenta. The null placentae exhibit decreased vascularization and poor transport, resulting in fetal growth retardation and ultimately embryo loss between 11.5 and 13.5 days of gestation. In both humans and mice, one of the two syncytins (human syncytin-2 and mouse syncytin-B) is also immunosuppressive, suggesting they may be also involved in immunotolerance of the semiallogeneic fetus. The sheep genome contains 27 JSRV-related endogenous betaretroviruses (enJSRVs) related to the pathogenic Jaagsiekte sheep retrovirus (JSRV) that have been integrating in the host genome for the last 5-7 million years. The exogenous JSRV is a causative agent of a transmissible lung cancer in sheep, and enJSRVs are able to protect their host against JSRV infection by blocking different steps of the viral replication cycle via receptor interference and late replication steps. Indeed, endogenization and positive selection of ERVs acting as restriction factors is a mechanism used by the host to fight exogenous retroviral infections. In sheep, the enJSRVs are most abundantly expressed in the uterine luminal and glandular epithelia as well as in the conceptus (embryo and associated extraembryonic membranes) trophectoderm. Sixteen of the 27 enJSRV loci contain an env gene with an intact open reading frame, and in utero loss-of-function experiments found the enJSRVs envelope (env) to be essential for conceptus elongation and trophoblast growth and differentiation. Collectively, available evidence supports the ideas that genes captured from ancestral retroviruses have been pivotal in the acquisition of new, important functions in mammalian evolution and were positively selected for biological roles in genome plasticity, protection of the host against infection of related pathogenic and exogenous retroviruses, and placental morphogenesis. This work supported by NIH Grant HD052745 and by a Strategic Research Developmental Grant by the Scottish Funding Council. (platform)

Pregnancy and interferon tau regulate N-myc interactor in the ovine uterus

In ruminants, interferon tau (IFNT) is synthesized and secreted by the mononuclear trophectoderm cells of the conceptus and maintains the corpus luteum and its secretion of progesterone for successful implantation and maintenance of pregnancy. In this study, we examined regulation of the expression of N-myc interactor ( NMI) gene by IFNT in the ovine uterus based on results of microarray data from a study that compared gene expression by human 2fTGH and U3A (STAT1-null 2fTGH) cell lines in response to treatment with IFNT or vehicle. In the present study, semiquantitative reverse transcription-polymerase chain reaction analyses verified that IFNT stimulated expression of NMI mRNA in 2fTGH (ie, in a STAT1-dependent manner), but not in U3A (STAT1-null) cells. Furthermore, results of western blot analyses indicated that immunoreactive NMI proteins in 2fTGH and U3A cell lines increased in a time-dependent manner only in response to IFNT. In ovine endometria, steady-state levels of NMI mRNA increased between days 14 and 16 of pregnancy and then decreased slightly by day 20, but there was no effect of day of the estrous cycle. Expression of NMI mRNA was most abundant in endometrial stromal cells, glandular epithelium, and conceptus trophectoderm. Intrauterine infusion of IFNT in cyclic ewes increased expression of NMI in the endometrium. Expression of NMI in ovine and bovine uterine cell lines increased in response to IFNT. Collectively, the results of the present study indicate that IFNT regulates expression of NMI mRNA and protein in ovine endometria during pregnancy via a STAT1-dependent cell signaling pathway.

Select Nutrients in the Ovine Uterine Lumen. VII. Effects of Arginine, Leucine, Glutamine, and Glucose on Trophectoderm Cell Signaling, Proliferation, and Migration1

Histotroph is required for survival and development of ovine conceptuses (embryo and extraembryonic membranes). Results from our laboratory indicate that arginine (Arg), leucine (Leu), glutamine (Gln), and glucose increase in the uterine lumen between Days 10 and 15 of pregnancy, coincident with increases in expression of amino acid and glucose transporters by uterine epithelia as well as trophectoderm and yolk sac of conceptuses and elongation of the conceptus trophectoderm. Therefore, we hypothesized that Arg, Leu, Gln, and glucose have differential effects on hypertrophy, hyperplasia, and differentiated functions of trophectoderm cells that are critical to conceptus development. Primary ovine trophectoderm (oTr) cells isolated from Day 15 conceptuses were serum-starved for 24 h in a customized medium, deprived of select nutrients, and then treated with either Arg, Leu, Gln, or glucose. Western blot analyses of whole oTr cell extracts revealed that Arg, Leu, and glucose, but not Gln, increased phosphorylated AKT1 by 2.8-, 2.5-, and 1.8-fold, respectively, within 15 min, and the increase was maintained to 60 min. Arg, Leu, and glucose also stimulated increases in phosphorylated ribosomal protein S6K (pRPS6K) by 4.2-, 4.7-, and 2.3-fold, respectively, within 15 min, as well as increases in phosphorylated ribosomal protein S6 (pRPS6) between 0 and 30 min posttreatment, that were sustained to 60 min. When oTr cells were treated with Arg, pRPS6K protein increased in nuclei, but this was not observed in nuclei of oTr cells treated with Leu and glucose. Immunocytochemical analyses also revealed abundant amounts of pRPS6 protein in the cytoplasm of oTr cells treated with Arg, Leu, and glucose. Furthermore, Arg and Leu increased proliferation and migration of oTr cells. Collectively, these results indicate that Arg, Leu, and glucose, but not Gln, in histotroph coordinately activate AKT1-mechanistic target of rapamycin and RPS6K-RPS6 cell signaling pathways to stimulate hypertrophy, hyperplasia, and migration of oTr cells.