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In pigs, the majority of embryonic mortality occurs when free-floating conceptuses (embryos/fetuses and associated placental membranes) elongate, and the uterine-placental interface undergoes folding and develops areolae. Both periods involve proliferation, migration, and changes in morphology of cells that require adenosine triphosphate (ATP). We hypothesize that insufficient ATP in conceptus and uterine tissues contributes to conceptus loss in pigs. Creatine is stored in cells as phosphocreatine for ATP regeneration through the creatine-creatine kinase- phosphocreatine pathway. However, the expression of components of this pathway in pigs has not been examined throughout gestation. Results of qPCR analyses indicated increases in AGAT, GAMT, CKM, CKB, and SLC6A8 mRNAs in elongating porcine conceptuses, and immunofluorescence microscopy localized guanidinoacetate N-methyltransferase, creatine kinase M, and creatine kinase B proteins to the trophectoderm of elongating conceptuses, to the columnar chorionic epithelial cells at the bottom of chorioallantoic troughs, and to endometrial luminal epithelium at the tops of the endometrial ridges of uterine-placental folds on Days 40, 60, and 90 of gestation. Guanidinoacetate N-methyltransferase protein is expressed in endometrial luminal epithelium at the uterine-placental interface, but immunostaining is more intense in luminal epithelium at the bottoms of the endometrial ridges. Results of this study indicate that key elements of the pathway for creatine metabolism are expressed in cells of the conceptus, placenta, and uterus for potential production of ATP during two timepoints in pregnancy with a high demand for energy; elongation of the conceptus for implantation and development of uterine-placental folding during placentation., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Secreted phosphoprotein 1 (SPP1, also known as osteopontin) binds integrins to mediate cell-cell and cell-extracellular matrix communication to promote cell adhesion, migration, and differentiation. Considerable evidence links SPP1 to pregnancy in several species. Current evidence suggests that SPP1 is involved in implantation and placentation in mice, but in vivo localization of SPP1 and in vivo mechanistic studies to substantiate these roles are incomplete and contradictory. We localized Spp1 mRNA and protein in the endometrium and placenta of mice throughout gestation, and utilized delayed implantation of mouse blastocysts to link SPP1 expression to the implantation chamber. Spp1 mRNA and protein localized to the endometrial luminal (LE), but not glandular epithelia (GE) in interimplantation regions of the uterus throughout gestation. Spp1 mRNA and protein also localized to uterine naturel killer (uNK) cells of the decidua. Within the implantation chamber, Spp1 mRNA localized only to intermittent LE cells, and to the inner cell mass. SPP1 protein localized to intermittent trophoblast cells, and to the parietal endoderm. These results suggest that SPP1: (1) is secreted by the LE at interimplantation sites for closure of the uterine lumen to form the implantation chamber; (2) is secreted by LE adjacent to the attaching trophoblast cells for attachment and invasion of the blastocyst; and (3) is not a component of histotroph secreted from the GE, but is secreted from uNK cells in the decidua to increase angiogenesis within the decidua to augment hemotrophic support of embryonic/fetal development of the conceptus., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Our aim was to evaluate whether mechanical forces applied to the placenta of pigs correlate with morphological changes that coordinate the development of placental folds., Methods: We examined changes in the length of placental folds, expression of mechanotransduction-implicated molecules in placental tissues, changes in the size of subepithelial blood vessels within the endometrium, and effects of in vivo supplementation with arginine on fold development., Results: We observed that: 1) the length of folds increased 2) osteopontin, talin and focal adhesion kinase co-localized into aggregates at the maternal placental (uterine)-fetal placental interface; 3) filamin, actin related protein 2, and F-actin were enriched in the tops of maternal placental folds extending into fetal placental tissue; 4) maternal stromal fibroblasts acquired alpha smooth muscle actin; 5) endometrial blood vessels increased in size; and 6) supplementation with arginine increased fold length., Conclusion: Results indicate that lengthening of folds associates with polymerization of actin that coincides with FA assembly, endometrial fibroblasts differentiate into myofibroblasts, and dilation of subepithelial blood vessels correlates with development of folds that is enhanced by arginine. We propose that dilation of subepithelial endometrial blood vessels delivers increased blood flow that pushes upward on the interface between the uterine luminal epithelium (LE) and the placental chorionic epithelium (CE), protrusive forces from growing uterine blood vessels trigger focal adhesion assembly and actin polymerization between the LE and CE, and endometrial fibroblasts differentiate into contractile myofibroblasts that pull connective tissue downward and inward to sculpt folds at the maternal placental-fetal placental interface., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Progesterone (P4) stimulates production and secretion of histotroph, a mixture of hormones, growth factors, nutrients, and other substances required for growth and development of the conceptus (embryo or fetus and placental membranes). Progesterone acts through the progesterone receptor (PGR); however, there is a gap in our understanding of P4 during pregnancy because PGR have not been localized in the uteri and placentae of pigs beyond day 18. Therefore, we determined endometrial expression of PGR messenger RNA (mRNA) and localized PGR protein in uterine and placental tissues throughout the estrous cycle and through day 85 of pregnancy in pigs. Further, 2 components of histotroph, tartrate-resistant acid phosphatase 5 (ACP5; uteroferrin) and secreted phosphoprotein 1 (SPP1; osteopontin) proteins, were localized in relation to PGR during pregnancy. Endometrial expression of PGR mRNA was highest at day 5 of the estrous cycle, decreased between days 5 and 11 of both the estrous cycle and pregnancy, and then increased between days 11 and 17 of the estrous cycle (P < 0.01), but decreased from days 13 to 40 of pregnancy (P < 0.01). Progesterone receptor protein localized to uterine stroma and myometrium throughout all days of the estrous cycle and pregnancy. PGR were expressed by uterine luminal epithelium (LE) between days 5 and 11 of the estrous cycle and pregnancy, then PGR became undetectable in LE through day 85 of pregnancy. During the estrous cycle, PGR were downregulated in LE between days 11 and 15, but expression returned to LE on day 17. All uterine glandular epithelial (GE) cells expressed PGR from days 5 to 11 of the estrous cycle and pregnancy, but expression decreased in the superficial GE by day 12. Expression of PGR in GE continued to decrease between days 25 and 85 of pregnancy; however, a few glands near the myometrium and in close proximity to areolae maintained expression of PGR protein. Acid phosphatase 5 protein was detected in the GE from days 12 to 85 of gestation and in areolae. Secreted phosphoprotein 1 protein was detected in uterine LE in apposition to interareolar, but not areolar areas of the chorioallantois on all days examined, and in uterine GE between days 35 and 85 of gestation. Interestingly, uterine GE cells adjacent to areolae expressed PGR, but not ACP5 or SPP1, suggesting these are excretory ducts involved in the passage, but not secretion, of histotroph into the areolar lumen and highlighting that P4 does not stimulate histotroph production in epithelial cells that express PGR., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The fetal fluids and uterine flushings of pigs contain higher concentrations of fructose than glucose, but fructose is not detected in maternal blood. Fructose can be synthesized from glucose via enzymes of the polyol pathway, aldose reductase (AKR1B1) and sorbitol dehydrogenase (SORD), transported across cell membranes by solute carriers SLC2A5 and SLC2A8, and converted to fructose-1-phosphate by ketohexokinase (KHK). SLC2A8, SLC2A5, AKR1B1, SORD, and KHK mRNAs and proteins were analyzed using quantitative PCR and immunohistochemistry or in situ hybridization in endometria and placentae of cyclic and pregnant gilts, cyclic gilts injected with estrogen, and ovariectomized gilts injected with progesterone. Progesterone up-regulated SLC2A8 protein in uterine luminal (LE) and glandular epithelia during the peri-implantation period, and expression became exclusively placental, chorion and blood vessels, after Day 30. P4 up-regulated SLC2A5 mRNA in uterine LE and glandular epithelia after implantation, and the chorion expressed SLC2A5 between Days 30 and 85. AKR1B1 and SORD proteins localized to uterine LE during the peri-implantation period, but expression switched to chorion by Day 20 and was maintained through Day 85. Uterine expression of AKR1B1 mRNA was down-regulated by estrogen. KHK protein localized to trophectoderm/chorion throughout gestation. These results provide evidence that components for the conversion of glucose to fructose and for fructose transport are present at the uterine-placental interface of pigs. The shift in expression from LE to chorion during pregnancy suggests free-floating conceptuses are supported by fructose synthesized by the uterus, but after implantation, the chorion becomes self-sufficient for fructose synthesis and transport., (© 2016 by the Society for the Study of Reproduction, Inc.)

Interferon tau (IFNT) was discovered as the pregnancy recognition signal in ruminants, but is now known to have a plethora of physiological functions in the mammalian uterus. The mammalian uterus includes, from the outer surface to the lumen, the serosa, myometrium and endometrium. The endometrium consists of the luminal, superficial glandular, and glandular epithelia, each with a unique phenotype, stromal cells, vascular elements, nerves and immune cells. The uterine epithelia secrete or selectively transport molecules into the uterine lumen that are collectively known as histotroph. Histotroph is required for growth and development of the conceptus (embryo and its associated extra-embryonic membranes) and includes nutrients such as amino acids and glucose, enzymes, growth factors, cytokines, lymphokines, transport proteins for vitamins and minerals and extracellular matrix molecules. Interferon tau and progesterone stimulate transport of amino acids in histotroph, particularly arginine. Arginine stimulates the mechanistic target of rapamycin pathway to induce proliferation, migration and protein synthesis by cells of the conceptus, and arginine is the substrate for synthesis of nitric oxide and polyamines required for growth and development of the conceptus. In ruminants, IFNT also acts in concert with progesterone from the corpus luteum to increase expression of genes for transport of nutrients into the uterine lumen, as well as proteases, protease inhibitors, growth factors for hematopoiesis and angiogenesis and other molecules critical for implantation and placentation. Collectively, the pleiotropic effects of IFNT contribute to survival, growth and development of the ruminant conceptus.

The greatest limitation to reproductive performance in most mammals, including humans, is embryonic mortality, which, in general, claims 20%-40% of the embryos during the peri-implantation period of pregnancy. Both arginine and secreted phosphoprotein 1 (SPP1) are multifunctional molecules that increase significantly in ovine uterine histotroph during early pregnancy. However, little is known about the relationship and underlying mechanisms for synergistic effects of arginine and SPP1, if any, on conceptus (embryo/fetus and associated extraembryonic membranes) development. Therefore, we conducted in vitro experiments using our established ovine trophectoderm cell line (oTr1) isolated from Day 15 ovine conceptuses to determine their proliferative response to individual and synergistic effects of arginine and recombinant SPP1 (rSPP1) that contains an RGD binding sequence. At physiological concentrations, arginine (0.2 mM) stimulated oTr1 cell proliferation 1.7-fold (P < 0.05) at 48 h, whereas rSPP1 (10 ng/ml) had no such effect. However, an additive effect on oTr1 cell proliferation was induced by combination of arginine and SPP1 as compared to the control (2.1-fold increase; P < 0.01), arginine alone (1.3-fold increase; P < 0.05), and rSPP1 alone (1.5-fold increase; P < 0.01). This additive effect was mediated through cooperative activation of the PDK1-Akt/PKB-TSC2-MTORC1 cell signaling cascade. Collectively, results suggest that arginine and SPP1 in histotroph act cooperatively to enhance survival, growth, and development of ovine conceptuses., (© 2015 by the Society for the Study of Reproduction, Inc.)

The dialogue between the mammalian conceptus (embryo/fetus and associated membranes) involves signaling for pregnancy recognition and maintenance of pregnancy during the critical peri-implantation period of pregnancy when the stage is set for implantation and placentation that precedes fetal development. Uterine epithelial cells secrete and/or transport a wide range of molecules, including nutrients, collectively referred to as histotroph that are transported into the fetal-placental vascular system to support growth and development of the conceptus. The availability of uterine-derived histotroph has long-term consequences for the health and well-being of the fetus and the prevention of Developmental Origins of Health and Disease (DOHAD). Although mechanisms responsible for differential growth and development of the conceptus resulting in DOHAD phenomena remain unclear, epigenetic events involving methylation of DNA are likely mechanisms. Histotroph includes serine and methionine which can contribute to the one carbon pool, and arginine, lysine and histidine residues which may be targets of methylation. It is also clear that supplementing the diet with arginine enhances fetal-placental development in rodents, swine and humans through mechanisms that remain to be elucidated. However, molecules secreted by conceptuses such as interferon tau in ruminants, estrogens and interferons in pigs and chorionic gonadotrophin, along with progesterone, regulate expression of genes for nutrient transporters. Understanding mechanisms whereby select nutrients regulate expression of genes in cell signaling pathways critical to conceptus development, implantation and placentation is required for improving successful establishment and maintenance of pregnancy in mammals.

The litter-bearing pig is an invaluable model for research in reproductive biology. Spherical pig blastocysts on Day 10 of pregnancy undergo rapid morphological changes to tubular and then filamentous forms by Day 12 and a filamentous conceptus of almost 1m in length by Day 16 of pregnancy. Thus, trophectoderm of each conceptus achieves intimate contact with luminal uterine epithelium (LE) for exchange of nutrients, gases, hormones, growth factors and other key molecules for survival and development. Estrogens secreted between Days 11 and 13 of pregnancy signals pregnancy recognition to ensure that nutrients and prostaglandin F2-alpha (PGF) are secreted into the uterine lumen (exocrine secretion) rather than into the uterine vein (endocrine secretion) which would lead to regression of the corpora lutea (CL) and failure to maintain pregnancy. Pigs have a true epitheliochorial placenta. The fluid filled amnion bouys the embryo so that it develops symmetrically. The allantois fills with allantoic fluid to expand contact of the chorioallantois with uterine LE, and the allanotois supports the vascular system of the placenta. The chorion/trophectoderm in direct contact with uterine LE exchanges gases and nutrients and forms unique structures call areolae that absorb nutrient-rich secretions from uterine glands and transports them directly into fetal blood. The period from Days 20 to 70 of pregnancy is for placental growth in preparation for rapid fetal growth between Days 70 and 114 (term) of gestation. Maturation of the fetal hypothalamic-pituitary-adrenal axis leads to increases in secretion of cortisol from the fetal adrenal glands. Cortisol sets in motion secretion of estrogens, oxytocin, relaxin and prolactin, as well as increases in their receptors, which are required for delivery of piglets and for initiation of lactation and expression of maternal behavior. This review provides details of gestation in the pig with respect to uterine biology, implantation, placentation, fetal development and parturition., (Copyright © 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

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.

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.

Nutrients are primary requirements for development of conceptuses (embryo and extraembryonic membranes), including protein synthesis. We have shown that arginine (Arg), leucine (Leu), and glucose stimulate protein synthesis through phosphorylation of MTOR signaling molecules, thereby increasing proliferation of ovine trophectoderm cells. This study determined whether Arg, Leu, glutamine (Gln), and glucose influence gene expression and protein synthesis in explant cultures of ovine conceptuses recovered from ewes on Day 16 of pregnancy. Conceptuses were deprived of select nutrients and then cultured with either Arg, Leu, Gln, or glucose for 18 h, after which they were analyzed for abundance of MTOR, RPS6K, RPS6, EIF4EBP1 (also known as 4EBP1), IFNT, NOS2, NOS3, GCH1, and ODC1 mRNAs and proteins. Levels of MTOR, RPS6K, RPS6, and EIF4EBP1 mRNAs were not affected by treatment with any of the select nutrients. Similarly, expression of IFNT, NOS2, NOS3, and ODC1 mRNAs were not different. Interestingly, GCH1 mRNA levels increased in response to Arg treatment. Importantly, Arg, Leu, Gln, and glucose increased the abundance of phosphorylated MTOR, RPS6K, RPS6, and EIF4EBP1 proteins as well as NOS and ODC1 proteins, but only Arg increased the abundance of IFNT protein. These findings indicate that Arg, Leu, Gln, and glucose stimulate translation of mRNAs to increase synthesis of proteins through phosphorylation and activation of components of the MTOR signaling pathway. Increases in abundance of IFNT protein (the pregnancy recognition signal), NOS2, NOS3 and GCH1 for conversion of Arg to nitric oxide, and ODC1 for synthesis of polyamines are all important for growth and development of the ovine conceptus during pregnancy.

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.

Reproduction in mammals is a highly complex biological process. The critical importance of reproduction to propagation of species required the natural evolution of various strategies that vary considerably across species. Regardless of species, a dialogue between the developing conceptus (embryo-fetus and associated placental membranes) and maternal uterus must be established during the peri-implantation period. The uterus must provide a microenvironment that supports growth and development of the conceptus and is receptive to implantation. During the same period, the conceptus must provide its pregnancy recognition signaling to sustain the functional life of corpora lutea for production of progesterone which is essential for implantation and placentation; critical events for successful pregnancy. However, it is within the peri-implantation period that most embryonic deaths occur due to deficiencies attributed to uterine functions or to the failure of the conceptus to develop appropriately, signal pregnancy recognition and/or undergo implantation and placentation. The challenge is to understand the complexity of key mechanisms that are characteristic of successful reproduction in humans and animals and to use that knowledge to enhance fertility and reproductive health or to establish acceptable methods for control of fertility.

In pigs, endometrial functions are regulated primarily by progesterone and placental factors including estrogen. Progesterone levels are high throughout pregnancy to stimulate and maintain secretion of histotroph from uterine epithelia necessary for growth, implantation, placentation, and development of the conceptus (embryo and its extra-embryonic membranes). This study determined effects of long-term progesterone on development and histoarchitecture of endometrial luminal epithelium (LE), glandular epithelium (GE), and vasculature in pigs. Pigs were ovariectomized during diestrus (day 12), and then received daily injections of either corn oil or progesterone for 28 days. Prolonged progesterone treatment resulted in increased weight and length of the uterine horns, and thickness of the endometrium and myometrium. Hyperplasia and hypertrophy of GE were not evident, but LE cell height increased, suggesting elevated secretory activity. Although GE development was deficient, progesterone supported increased endometrial angiogenesis comparable to that of pregnancy. Progesterone also supported alterations to the apical and basolateral domains of LE and GE. Dolichos biflorus agglutinin lectin binding and α(v) integrin were downregulated at the apical surfaces of LE and GE. Claudin-4, α(2)β(1) integrin, and vimentin were increased at basolateral surfaces, whereas occludins-1 and -2, claudin-3, and E-cadherin were unaffected by progesterone treatment indicating structurally competent trans-epithelial adhesion and tight junctional complexes. Collectively, the results suggest that progesterone affects LE, GE, and vascular development and histoarchitecture, but in the absence of ovarian or placental factors, it does not support development of GE comparable to pregnancy. Furthermore, LE and vascular development are highly responsive to the effects of progesterone.

Pigs experience significant conceptus loss near mid-gestation, correlating with increasing glandular epithelial (GE) development and secretory activity. Secreted phosphoprotein 1 (SPP1, osteopontin) increases in GE between days 30 and 40 of pregnancy and is expressed in the GE of day 90 pseudopregnant pigs, suggesting that progesterone (P(4)) from corpora lutea is responsible for induction of SPP1 in GE. In this study, pigs were ovariectomized and treated daily with P(4) to assess effects of 40 days of P(4) exposure on SPP1, P(4) receptor (PGR), uteroferrin (ACP5), and fibroblast growth factor 7 (FGF7) expression in porcine endometria. PGR mRNA decreased in pigs injected with P(4) compared with pigs injected with corn oil (CO), and PGRs were downregulated in the luminal epithelium (LE) and GE. ACP5 mRNA increased in pigs injected with P(4) compared with pigs injected with CO, and ACP5 was induced in the GE of P(4)-treated pigs. FGF7 mRNA increased in pigs injected with P(4) compared with pigs injected with CO, and FGF7 was induced in the LE and GE of P(4)-treated pigs. SPP1 mRNA was not different between pigs injected with P(4) compared with pigs injected with CO, and SPP1 was not present in the GE of P(4)-treated pigs. Therefore, long-term P(4), in the absence of ovarian and/or conceptus factors, does not induce SPP1 expression in GE. We hypothesize that a servomechanism involving sequential effects of multiple hormones and cytokines, similar to those for sheep and humans, is required for GE differentiation and function, including the synthesis and secretion of SPP1.

Attachment and migration of trophectoderm (Tr) cells, hallmarks of blastocyst implantation in mammals, are unique uterine events. Secreted phosphoprotein 1 (SPP1) in the uterus binds integrins on conceptus Tr and uterine luminal epithelium (LE), affecting cell-cell and cell-matrix interactions. The signal transduction pathways activated by SPP1 and integrins in conceptuses have not been elucidated. Results of this study demonstrate that SPP1 binds alphavbeta3 and alpha5beta1 integrins to induce focal adhesion assembly, a prerequisite for adhesion and migration of Tr, through activation of: 1) P70S6K via crosstalk between FRAP1/mTOR and MAPK pathways; 2) mTOR, PI3K, MAPK3/MAPK1 (Erk1/2) and MAPK14 (p38) signaling to stimulate Tr cell migration; and 3) focal adhesion assembly and myosin II motor activity to induce migration of Tr cells. These cell signaling pathways, acting in concert, mediate adhesion, migration and cytoskeletal remodeling of Tr cells essential for expansion and elongation of conceptuses and attachment to uterine LE for implantation., (Copyright (c) 2010 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.)

The process of implantation is mediated by a complex network of signaling and adhesive factors. In the pig, latent and active transforming growth factor beta (TGFB), TGFB receptors (TGFBR), and integrins (ITGs) are present during the peri-implantation period. TGFB signals via TGFBR and activates downstream effector SMAD proteins 2 and 3 (p-SMAD2/3). Latency-associated peptide (LAP), part of the latent TGFB complex, is known to bind to ITG heterodimers and activate TGFB. We hypothesize that active TGFBs and TGFBRs along with LAP and ITGs functionally interact at the conceptus-maternal interface to mediate events essential for conceptus development and attachment in pigs. Uteri and conceptuses from days 10, 12, 16, 20, and 24 pregnant gilts were immunostained for TGFB, LAP, and ITG subunits (ITGAV, ITGB1, ITGB3, ITGB5, ITGB6, and ITGB8). Activation of TGFBRs was evaluated by the presence of phosphorylated downstream effector SMAD2/3. Binding of LAP to ITGs was also evaluated using porcine trophectoderm cells. Abundant active TGFB was detected at the apical surfaces of epithelia at the conceptus-maternal interface, and p-SMAD2/3 was detected at both conceptus attachment and nonattachment sites during implantation. Separate aggregates of LAP, ITGB1, ITGB5, and later ITGB3 were detected at the porcine conceptus-maternal interface, and binding of LAP to ITGs on apical surfaces was demonstrated. Results suggest that functional LAP-ITG adhesion complexes support conceptus attachment and promote TGFB activation leading to TGFB interaction with TGFBR supporting events of porcine implantation.

Establishment of pregnancy in ruminants requires conceptus elongation and production of interferon-tau (IFNT), the pregnancy recognition signal that maintains ovarian progesterone (P4) production. These studies determined temporal and spatial alterations in IGF binding protein (IGFBP)-1 and IGFBP3 in the ovine and bovine uterus; effects of P4 and IFNT on their expression in the ovine uterus; and effects of IGFBP1 on ovine trophectoderm cell proliferation, migration, and attachment. IGFBP1 and IGFBP3 were studied because they are the only IGFBPs specifically expressed by the endometrial luminal epithelia in sheep. In sheep, IGFBP1 and IGFBP3 expression was coordinate with the period of conceptus elongation, whereas only IGFBP1 expression was coordinate with conceptus elongation in cattle. IGFBP1 mRNA in the ovine endometria was between 5- and 29-fold more abundant between d 12 and 16 of pregnancy compared with the estrous cycle and greater on d 16 of pregnancy than nonpregnancy in the bovine uterus. In sheep, P4 induced and IFNT stimulated expression of IGFBP1 but not IGFBP3; however, the effect of IFNT did not mimic the abundant increase observed in pregnant ewes. Therefore, IGFBP1 expression in the endometrium is regulated by another factor from the conceptus. IGFBP1 did not affect the proliferation of ovine trophectoderm cells in vitro but did stimulate their migration and mediate their attachment. These studies reveal that IGFBP1 is a common endometrial marker of conceptus elongation in sheep and cattle and most likely regulates conceptus elongation by stimulating migration and attachment of the trophectoderm.

FRAP1 (FK506-binding protein 12-rapamycin complex-associated protein 1), a component of the nutrient-sensing cell signaling pathway, is critical for cell growth and metabolism. The present study determined expression of FRAP1 and associated members of the mTORC1 and mTORC2 cell signaling pathways in uteri of cyclic and pregnant ewes and conceptuses, as well as effects of pregnancy, progesterone (P4), and interferon tau (IFNT) on their expression. The mRNAs for FRAP1, LST8, MAPKAP1, RAPTOR, RICTOR, TSC1, TSC2, RHEB, and EIF4EBP1 were localized to luminal, superficial glandular, and glandular epithelia and stromal cells of uteri from cyclic and pregnant ewes, as well as trophectoderm and endoderm of conceptuses between Days 13 and 18 of pregnancy. The abundance of FRAP1, RAPTOR, RICTOR, TSC1, and TSC2 mRNAs in endometria was unaffected by pregnancy status or by day of the estrous cycle or pregnancy; however, levels of LST8, MAPKAP1, RHEB, and EIF4EBP1 mRNA increased in endometria during early pregnancy. In ovariectomized ewes, P4 and IFNT stimulated expression of RHEB and EIF4EBP1 in uterine endometria. Total endometrial FRAP1 protein and phosphorylated FRAP1 protein levels were affected by pregnancy status and by day after onset of estrus, and phosphorylated FRAP1 protein was detected in nuclei of uterine epithelia and conceptuses. In endometria of pregnant ewes, increases in abundance of mRNAs for RICTOR, RHEB, and EIF4EBP1, as well as RHEB protein, correlated with rapid conceptus growth and development during the peri-implantation period. These results suggest that the FRAP1 cell signaling pathway mediates interactions between the maternal uterus and peri-implantation conceptuses and that P4 and IFNT affect this pathway by regulating expression of RHEB and EIF4EBP1.

Nitric oxide (NO) and polyamines are critical for implantation and development of conceptuses (embryo and extraembryonic membranes), but mechanisms regulating their biosynthesis in uteri and conceptuses are largely unknown. This study determined the effects of the estrous cycle, pregnancy, progesterone, and interferon tau (IFNT) on expression of NO synthases (NOS1, NOS2, and NOS3), guanosine triphosphate (GTP) cyclohydrolase (GCH1, the key enzyme in de novo synthesis of tetrahydrobiopterin, a cofactor for NO production), and ornithine decarboxylase (ODC1) in uterine endometria in cyclic ewes (Days 10-16) and pregnant ewes (Days 10-20). The mRNAs and proteins for NOS1 and ODC1 were most abundant in uterine luminal (LE) and superficial glandular (sGE) epithelia, and abundance was affected by day of estrous cycle and early pregnancy. NOS2, GCH1, and NOS3 mRNAs were detected in very low abundance in uterine epithelia and stromal cells in both cyclic and pregnant ewes. NOS1 mRNA also was expressed very weakly in conceptuses, whereas NOS3 mRNA was abundant in the trophectoderm and endoderm of conceptuses, as were total NOS1 and NOS3 proteins, inhibitory p-NOS1 protein, and stimulatory p-NOS3 protein. GCH1 mRNA was abundant in the trophectoderm and endoderm of conceptuses between Days 13 and 15 of pregnancy and then decreased thereafter, whereas ODC1 mRNA abundance increased in conceptuses between Days 13 and 18 of pregnancy. GCH1 protein was localized primarily in the nuclei of trophectoderm and endoderm, and its abundance decreased after Day 14 of pregnancy, whereas ODC1 protein was more abundant in the trophectoderm than in the endoderm between Days 13 and 18 of pregnancy. Progesterone stimulated NOS1 and GCH1 expression in LE/sGE and glandular epithelia, whereas IFNT inhibited NOS1 expression in these cell types. Thus, biosynthesis of NO and polyamines in ovine uterine endometria and conceptuses is potentially regulated at transcriptional, translational, and posttranslational levels to favor conceptus development and implantation.

Wnt genes are involved in critical developmental and growth processes. The present study comprehensively analyzed temporal and spatial alterations in Wnt and Fzd gene expression in the mouse uterus during peri-implantation of pregnancy. Expression of Wnt4, Wnt5a, Wnt7a, Wnt7b, Wnt11, Wnt16, Fzd2, Fzd4, and Fzd6 was detected in the uterus during implantation. Wnt4 mRNA was most abundant in the decidua, whereas Wnt5a mRNA was restricted to the mesometrial decidua during decidualization. Wnt7a, Wnt7b, and Wnt11 mRNAs were abundantly detected in the endometrial epithelia. The expression of Wnt7b was robust in the luminal epithelium (LE) at the implantation site on Gestational Day 5, whereas Wnt11 mRNA disappeared in the LE adjacent to the embryo in the antimesometrial implantation chamber but remained abundant in the LE. Wnt16 mRNA was localized to the stroma surrounding the LE on Day 4 and remained in the stroma adjacent to the LE but not in areas undergoing the decidual reaction. Fzd2 mRNA was detected in the decidua, Fzd4 mRNA was in the vessels and stroma surrounding the embryo, and Fzd6 mRNA was observed in the endometrial epithelia, stroma, and some blood vessels during implantation. Ovarian steroid hormone treatment was found to regulate Wnt genes and Fzd receptors in ovariectomized mice. Especially, single injections of progesterone stimulated Wnt11 mRNA, and estrogen stimulated Wnt4 and Wnt7b. The temporal and spatial alterations in Wnt genes likely play a critical role during implantation and decidualization in mice.

The integrity of the fetal-maternal interface is critical for proper fetal nourishment during pregnancy. Integrins are important adhesion molecules present at the interface during implantation; however, in vivo evidence for integrin activation and focal adhesion formation at the maternal-conceptus interface is limited. We hypothesized that focal adhesion assembly in uterine luminal epithelium (LE) and conceptus trophectoderm (Tr) results from integrin binding of extracellular matrix (ECM) at this interface to provide increased tensile forces and signaling to coordinate utero-placental development. An ovine model of unilateral pregnancy was used to evaluate mechanotransduction events leading to focal adhesion assembly at the maternal-conceptus interface and within the uterine wall. Animals were hysterectomized on days 40, 80, or 120 of pregnancy, and uteri immunostained for integrins (ITGAV, ITGA4, ITGA5, ITGB1, ITGB3, and ITGB5), ECM proteins (SPP1, LGALS15, fibronectin (FN), and vitronectin (VTN)), cytoskeletal molecules (ACTN and TLN1), and a signal generator (PTK2). Focal adhesion assembly in myometrium and stroma was also studied to provide a frame of reference for mechanical stretch of the uterine wall. Large focal adhesions containing aggregates of ITGAV, ITGA4, ITGA5, ITGB1, ITGB5, ACTN, and PTK2 were detected in interplacentomal uterine LE and Tr of gravid but not non-gravid uterine horns and increased during pregnancy. SPP1 and LGALS15, but not FN or VTN, were present along LE and Tr interfaces in both uterine horns. These data support the idea that focal adhesion assembly at the maternal-conceptus interface reflects adaptation to increasing forces caused by the growing fetus. Cooperative binding of multiple integrins to SPP1 deposited at the maternal-conceptus interface forms an adhesive mosaic to maintain a tight connection between uterine and placental surfaces along regions of epitheliochorial placentation in sheep.

Arginine is an essential amino acid for conceptus (embryo/fetus and trophoblast/placenta) growth and development; however, the mechanisms for arginine transport into the uterine lumen and uptake by conceptuses are largely unknown. In this study, expression of System y(+) (SLC7A1, SLC7A2, and SLC7A3) cationic amino acid transporters in uteri of cyclic and pregnant ewes and conceptuses was studied, and effects of pregnancy, progesterone (P4), and interferon tau (IFNT) on their expression were investigated. SLC7A1 mRNA was most abundant in endometrial luminal (LE) and superficial glandular (sGE) epithelia on Day 16 of the estrous cycle and on Days 16-20 of pregnancy, whereas SLC7A2 mRNA was most abundant in LE and mid to deep glandular (GE) epithelia on Days 14-20 of gestation. Expression of SLC7A1 and SLC7A2 was enhanced in pregnant ewes in a cell-specific manner, but abundance of SLC7A3 was not affected by day of the estrous cycle or by pregnancy status. SLC7A1, SLC7A2, and SLC7A3 mRNAs were expressed in trophectoderm and endoderm of conceptuses. In ovariectomized ewes, short-term treatment of ewes with P4 and IFNT did not affect endometrial SLC7A1 mRNA, while long-term treatment with P4 stimulated SLC7A1 in LE and GE, and IFNT tended to increase SLC7A1 abundance in LE. SLC7A2 mRNA abundance increased 4.1-fold in response to short-term P4 treatment and an additional 1.7-fold by IFNT primarily in endometrial LE/sGE, and these effects were ablated by a P4 receptor antagonist. These results indicate that coordinate changes in SLC7A1, SLC7A2, and SLC7A3 expression in uterine endometria and conceptuses are likely important in transport of arginine that is critical to conceptus growth, development, and survival.

This article focuses on the potential roles of interferons (IFNs) in establishing uterine receptivity to implantation. A common feature of the peri-implantation period of pregnancy in most mammals is production of type I and/or type II IFNs by trophoblasts that induce and/or stimulate expression of an array of IFN-stimulate genes (ISGs). These effects range from pregnancy recognition signaling in ruminants through IFN tau to effects on cellular functions of the uterus and uterine vasculature. For actions of IFNs, progesterone (P4) is permissive to the expression of many effects and to the expression of ISGs that are induced directly by an IFN or induced by P4 and stimulated by an IFN in a temporal and/or cell-specific manner. Uterine receptivity to implantation is P4 dependent; however, implantation events are preceded by loss of expression of progesterone (PGR) and estrogen (ESR1) receptors by uterine epithelia. Therefore, P4 likely acts via PGR-positive stromal cells to induce expression of fibroblast growth factors-7 and -10 and/or hepatocyte growth factor (progestamedins) that then act via their respective receptors on uterine epithelia and trophectoderm to affect expression of ISGs. The permissive effects of P4 on the expression of ISGs and the effects of P4 to induce and IFNs to stimulate gene expression raise the question of whether uterine receptivity to implantation requires P4 and IFN to activate unique, but complementary, cell signaling pathways. Uterine receptivity to implantation, depending on species, involves changes in the expression of genes for the attachment of trophectoderm to the uterine lumenal epithelium (LE) and superficial glandular epithelium (sGE), modification of the phenotype of uterine stromal cells, the silencing of PGR and ESR1 genes, the suppression of genes for immune recognition, alterations in membrane permeability to enhance conceptus-maternal exchange of factors, increased vascularity of the endometrium, activation of genes for transport of nutrients into the uterine lumen, and enhanced signaling for pregnancy recognition. Differential expression of genes by uterine LE/sGE, mid- to deep-glandular epithelia (GE), and stromal cells in response to P4 and IFNs is likely to influence uterine receptivity to implantation in most mammals. Understanding the roles of IFNs in uterine receptivity for implantation is necessary to develop approaches to enhance reproductive health and fertility in humans and domestic animals.

Total glucose in ovine uterine lumenal fluid increases 6-fold between Days 10 and 15 of gestation, but not the estrous cycle; however, mechanisms for glucose transport into the uterine lumen and uptake by conceptuses (embryo/fetus and associated membranes) are not established. This study determined the effects of the estrous cycle, pregnancy, progesterone (P4), and interferon tau (IFNT) on expression of both facilitative (SLC2A1, SLC2A3, and SLC2A4) and sodium-dependent (SLC5A1 and SLC5A11) glucose transporters in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy, as well as in conceptuses from Days 10 to 20 of pregnancy. The SLC2A1 and SLC5A1 mRNAs and proteins were most abundant in uterine luminal epithelia and superficial glandular epithelia (LE/sGE), whereas SLC2A4 was present in stromal cells and glandular epithelia (GE). SLC5A11 mRNA was most abundant in endometrial GE, whereas SLC2A3 mRNA was not detectable in endometria. SLC2A1, SLC2A3, SLC2A4, SLC5A1, and SLC5A11 were expressed in the trophectoderm and endoderm of conceptuses. Steady-state levels of SLC2A1, SLC5A1, and SLC5A11 mRNAs, but not SLC2A4 mRNA, were greater in endometria from pregnant than from cyclic ewes. Progesterone increased SLC2A1, SLC5A11, and SLC2A4 mRNAs in the LE/sGE and SLC5A1 in the GE of ovariectomized ewes. Expression of SLC5A1 was inhibited by ZK136,317 (progesterone receptor antagonist), and the combination of ZK136,317 and IFNT further decreased expression in GE. In constrast, P4 induced and IFNT stimulated expression of SLC2A1 and SLC5A11, and these effects were blocked by ZK136,317. Results of this study indicate differential expression of facilitative and sodium-dependent glucose transporters in ovine uteri and conceptuses for transport and uptake of glucose, and that P4 or P4 and IFNT regulate their expression during the peri-implantation period of pregnancy.

Nutrients in uterine secretions are essential for development and survival of conceptuses (embryo and associated extraembryonic membranes) during pregnancy; however, little is known about changes in the amounts of specific nutrients in the uterine fluids of cyclic and pregnant ruminants. This study determined quantities of glucose, amino acids, glutathione, calcium, sodium, and potassium in uterine lumenal fluid from cyclic (Days 3-16) and pregnant (Days 10-16) ewes. Total recoverable glucose, Arg, Gln, Leu, Asp, Glu, Asn, His, beta-Ala, Tyr, Trp, Met, Val, Phe, Ile, Lys, Cys, Pro, glutathione, calcium, and sodium were greater in the uterine fluid of pregnant compared with cyclic ewes between Days 10 and 16. In cyclic ewes, only modest changes in the total amounts of glucose, Asn, Cit, Tyr, Trp, Met, Val, Cys, glutathione, calcium, and potassium were detected between Days 3 and 16. However, in pregnant ewes, amounts of glucose, Arg, Gln, Glu, Gly, Cys, Leu, Pro, glutathione, calcium, and potassium in uterine fluids increased 3- to 23-fold between Days 10 and 14 and remained high to Day 16. Of particular interest were increases in glucose, Arg, Leu, and Gln in uterine flushings of pregnant ewes between Days 10 and 16 of pregnancy. Total amounts of His, ornithine, Lys, Ser, Thr, Ile, Phe, Trp, Met, and Cit in uterine fluids also increased, but to a lesser extent during early pregnancy. These novel results indicate activation of pregnancy-associated mechanisms for transport of nutrients into the uterine lumen, and they provide a framework for future studies of nutrients, including glucose, amino acids, and glutathione, required to activate nutrient-sensing cell signaling pathways for growth, development, and survival of conceptuses, as well as for optimization of culture media for in vitro studies of conceptus development.

Secreted phosphoprotein one (SPP1, osteopontin) may regulate conceptus implantation and placentation. We investigated effects of progesterone (P(4)) and the conceptus on expression and localization of SPP1 in the ovine uterus. Steady-state levels of SPP1 mRNA in the endometrium of unilaterally pregnant ewes did not differ significantly between nongravid and gravid horns within their respective days of pregnancy; however, levels did increase as pregnancy progressed. SPP1 mRNA was detectable in the glandular epithelium (GE) of both nongravid and gravid horns via in situ hybridization. SPP1 protein was localized to the apical surface of the luminal epithelium of both nongravid and gravid uterine horns. Gravid horns exhibited extensive stromal SPP1 on Days 40 through 120, whereas SPP1 was markedly lower in the stroma of nongravid uterine horns through Day 80 of pregnancy. By Day 120, stromal expression of SPP1 between nongravid and gravid horns was similar. Long-term P(4) treatment of ovariectomized ewes induced SPP1 in the uterine stroma and GE. A bioactive 45-kDa SPP1 fragment was purified from uterine secretions and promoted ovine trophectoderm cell attachment in vitro. Interestingly, increased stromal cell expression of SPP1 was positively associated with vascularization as assessed by von Willebrand factor staining. Finally, ovine uterine artery endothelial cells produced SPP1 during outgrowth into three-dimensional collagen matrices in an in vitro model system that recapitulates angiogenesis. Collectively, P(4) induces and the conceptus further stimulates SPP1 in uterine GE and stroma, where SPP1 likely influences histotrophic and hematotrophic support of conceptus development.

MHC class I molecules and beta(2)-microglobulin (beta(2)m) are membrane glycoproteins that present peptide Ags to TCRs, and bind to inhibitory and activating receptors on NK cells and other leukocytes. They are involved in the discrimination of self from non-self. Modification of these molecules in the placenta benefits pregnancy, but little is known about their genes in the uterus. We examined the classical class I swine leukocyte Ags (SLA) genes SLA-1, SLA-2, and SLA-3, the nonclassical SLA-6, SLA-7, and SLA-8 genes, and the beta(2)m gene in pig uterus during pregnancy. Uterine SLA and beta(2)m increased in luminal epithelium between days 5 and 9, then decreased between days 15 and 20. By day 15 of pregnancy, SLA and beta(2)m increased in stroma and remained detectable through day 40. To determine effects of estrogens, which are secreted by conceptuses to prevent corpus luteum regression, nonpregnant pigs were treated with estradiol benzoate, which did not affect the SLA or beta(2)m genes. In contrast, progesterone, which is secreted by corpora lutea, increased SLA and beta(2)m in luminal epithelium, whereas a progesterone receptor antagonist (ZK137,316) ablated this up-regulation. To determine effects of conceptus secretory proteins (CSP) containing IFN-delta and IFN-gamma, nonpregnant pigs were implanted with mini-osmotic pumps that delivered CSP to uterine horns. CSP increased SLA and beta(2)m in stroma. Cell-type specific regulation of SLA and beta(2)m genes by progesterone and IFNs suggests that placental secretions control expression of immune regulatory molecules on uterine cells to provide an immunologically favorable environment for survival of the fetal-placental semiallograft.

IGF-II, a potent stimulator of cellular proliferation, differentiation, and development, regulates uterine function and conceptus growth in several species. In situ hybridization analyses found that IGF-II mRNA was most abundant in the caruncular endometrial stroma of both cyclical and pregnant ewes. In the intercaruncular endometrium, IGF-II mRNA transitioned from stroma to luminal epithelium between d 14 and 20 of pregnancy. IGF-II mRNA was present in all cells of the conceptus but was particularly abundant in the yolk sac. Immunohistochemical analyses revealed that phosphorylated (p)-protooncogenic protein kinase 1, p-ribosomal protein S6 kinase, p-ERK1/2, and p-P38 MAPK proteins were present at low levels in a majority of endometrial cells but were most abundant in the nuclei of endometrial luminal epithelium and conceptus trophectoderm of pregnant ewes. In mononuclear trophectoderm cells isolated from d-15 conceptuses, IGF-II increased the abundance of p-pyruvate dehydrogenase kinase 1, p-protooncogenic protein kinase 1, p-glycogen synthase kinase 3B, p-FK506 binding protein 12-rapamycin associated protein 1, and p-ribosomal protein S6 kinase protein within 15 min, and the increase was maintained for 90 min. IGF-II also elicited a rapid increase in p-ERK1/2 and p-P38 MAPK proteins that was maximal at 15 or 30 min posttreatment. Moreover, IGF-II increased migration of trophectoderm cells. Collectively, these results support the hypothesis that IGF-II coordinately activates multiple cell signaling pathways critical to survival, growth, and differentiation of the ovine conceptus during early pregnancy.

Conceptus trophectoderm and uterine luminal epithelial cells interact via endocrine, paracrine, and autocrine modulators to mediate pregnancy recognition and implantation. Pig conceptuses not only release estrogens for pregnancy recognition but also secrete interferons during implantation. Because interferon-stimulated genes are increased by interferons secreted for pregnancy recognition in ruminants, we asked whether the interferon-stimulated gene, STAT1, is up-regulated in pig endometrium by conceptus estrogens and/or interferons. STAT1 expression in response to day of pregnancy, estrogen injection, and intrauterine infusion of conceptus secretory proteins in pigs indicated 1) estrogen increases STAT1 in luminal epithelial cells, 2) conceptus secretory proteins that contain interferons increase STAT1 in stroma, 3) STAT1 increases in close proximity to the conceptus, and 4) early estrogen results in conceptus death and no STAT1 in stroma. The interactions of estrogen and interferons to regulate cell-type-specific expression of STAT1 highlight the complex interplay between endometrium and conceptus for pregnancy recognition and implantation.

Pig conceptuses secrete estrogen for pregnancy recognition, and they secrete interferons (IFNs) gamma and delta during the peri-implantation period. The uterine effects of pig IFNs are not known, although ruminant conceptuses secrete IFN tau for pregnancy recognition, and this increases the expression of IFN-stimulated genes (ISGs) in the endometrium. In sheep, the transcriptional repressor interferon-regulatory factor 2 (IRF2) is expressed in the endometrial luminal epithelium (LE) and appears to restrict IFN tau induction of most ISGs, including IRF1, to the stroma and glands. Interestingly, MX1, which is an ISG in sheep, is also expressed in the endometrial stroma of pregnant pigs. The objective of the present study was to determine if estrogen and/or conceptus secretory proteins (CSPs) that contain IFNs regulate IRF1 and IRF2 in pig endometria. The endometrial levels of IRF1 and IRF2 were low throughout the estrus cycle. After Day 12 of pregnancy, the levels of the classical ISGs, which include IRF1, STAT2, MIC, and B2M, increased in the overall endometrium, with expression of IRF1 and STAT2 being specifically localized to the stroma. IRF2 increased in the LE after Day 12. To determine the effects of estrogen, pigs were treated with 17 beta-estradiol benzoate (E2). To determine the CSP effects, pigs were treated with E2 and implanted with mini-osmotic pumps that delivered control serum proteins (CX) to one ligated uterine horn and CSP to the other horn. Estrogen increased the level of IRF2 in the endometrial LE. The administration of E2 and infusion of CSP increased the level of IRF1 in the stroma. These results suggest that conceptus estrogen induces IRF2 in the LE and limits the induction of IRF1 by conceptus IFNs to the stroma. The cell-specific expression of IRF1 and IRF2 in the pig endometrium highlights the complex and overlapping events that are associated with gene expression during the peri-implantation period, when pregnancy recognition signaling and uterine remodeling for implantation and placentation are necessary for successful pregnancy.

Fibroblast growth factor 7 (FGF7) stimulates cell proliferation, differentiation, migration and angiogenesis. The consensus is that FGF7, expressed by mesenchymal cells, binds FGF receptor 2IIIb (FGFR2) on epithelia, thereby mediating epithelial-mesenchymal interactions. The pig uterus is unique in that FGF7 is expressed by the luminal epithelium (LE) and FGFR2 is expressed by the LE, glandular epithelium (GE), and trophectoderm to effect proliferation and differentiated cell functions during conceptus development and implantation. FGF7 expression by the uterine LE of pigs increases between Days 9 and 12 of the estrus cycle and pregnancy, as circulating concentrations of progesterone increase, progesterone receptors (PGR) in the uterine epithelia decrease, and the conceptuses secrete estradiol-17beta (E(2)), for pregnancy recognition. Furthermore, E(2) increases the expression of FGF7 in pig uterine explants. The present study investigates the relationships between progesterone, E(2), and their receptors and the expression of FGF7 in the pig uterus in vivo. Pigs were ovariectomized on Day 4 of the estrus cycle and injected i.m. daily from Day 4 to Day 12 with either corn oil (CO), progesterone (P4), P4 and ZK317,316 (PZK), E(2), P4 and E(2) (PE), or P4 and ZK and E(2) (PZKE). All gilts (n = 5/treatment) were hysterectomized on Day 12. The results suggest that: 1) P4 is permissive to FGF7 expression by down-regulating PGR in LE; 2) P4 stimulates PGR-positive uterine stromal cells to release an unidentified progestamedin that induces FGF7 expression by LE; 3) E(2) and P4 can induce FGF7 when PGR are rendered nonfunctional by ZK; and 4) E(2) from conceptuses interacts via estrogen receptor alpha, but not estrogen receptor beta in LE to induce maximal expression of FGF7 in LE on Day 12 of pregnancy in pigs.

Secreted phosphoprotein 1 (SPP1, commonly referred to as osteopontin and formerly known as bone sialoprotein 1, early T-lymphocyte activation 1) is an extracellular matrix/adhesion molecule that is upregulated in the pregnant uterus of all mammals examined to date. This study focused on the pig, which has true epitheliochorial placentation and exhibits induction of SPP1 mRNA in luminal epithelium (LE) just before conceptus attachment and in glandular epithelium (GE) after Day 30 of pregnancy. The objective of this study was to determine steroid regulation of SPP1 mRNA and protein in porcine uterine epithelium. To examine the effect of estrogen, cyclic gilts were treated daily (Days 11-14) with 5 mg estradiol benzoate (i.m.) and hysterectomized on Day 15. To evaluate the long-term effect of pseudopregnancy, cyclic gilts were given daily injections (Days 11-15) with steroid as above and hysterectomized on Day 90. In situ hybridization showed high expression of SPP1 mRNA only in LE contiguous with apposing conceptus tissue on Day 15 of pregnancy. In contrast, estrogen injection resulted in moderate but uniform SPP1 mRNA in all LE of Day 15 nonpregnant gilts, with expression maintained through Day 90 of pseudopregnancy. SPP1 mRNA also localized to the GE of Day 90 pseudopregnant gilts, similar to expression in late gestation. Consistent with in situ hybridization results, SPP1 protein localized to the apical surface of LE in all estrogen-treated gilts and in the GE on Day 90 of pseudopregnancy. We conclude that, in pregnant pigs, SPP1 is induced by conceptus estrogen in uterine LE and is regulated in GE in a manner coincident with CL/placental progesterone production.

Osteopontin (OPN) is the most highly up-regulated extracellular matrix/adhesion molecule in the uterus of humans and domestic animals as it becomes receptive to implantation. Studies in sheep and pigs have shown that OPN is a component of ovine and porcine histotroph characterized by a complex temporal and spatial pattern of uterine and conceptus expression involving immune, epithelial, and stromal cells. It is proposed that these expression events are orchestrated to contribute to conceptus attachment and placentation. However, differences in OPN expression between sheep and pigs have been detected that relate to differences in placentation. Therefore, this study examined OPN expression in the caprine uterus and conceptus to gain insight into mechanisms underlying OPN function(s) during pregnancy through comparative analysis of differences in placentation between pigs, sheep, and goats. Goats were hysterectomized (n = 5/day) on Days 5, 11, 13, 15, 17 or 19 of the estrous cycle, and Days 5, 11, 13, 15, 17, 19 or 25 of pregnancy. Slot-blot hybridization showed increases in endometrial OPN mRNA beginning on Day 17 of the estrous cycle and Day 19 of pregnancy. In situ hybridization localized OPN mRNA to endometrial glandular epithelium (GE), Day 25 myometrium, and cells scattered within the placenta hypothesized to be immune. Immunofluorescence microscopy detected OPN protein on the apical surface of endometrial lumenal epithelium (LE), in GE, and on conceptus (Tr). Western blot analysis detected primarily the native 70-kDa OPN protein in endometrial extracts from the estrous cycle and pregnancy, as well as in uterine flushings from pregnant goats. Co-induction of OPN and alpha-smooth muscle actin, but not desmin proteins, was observed in uterine stroma by Day 25 of pregnancy. OPN in cyclic GE, Day 25 myometrium, and desmin-negative endometrial stroma is unique and reflects subtle differences among superficial implanting species that correlate with the depth of Tr invasion., ((c) Elsevier Ltd. All rights reserved.)

Progesterone is unequivocally required for maternal support of conceptus (embryo/fetus and associated extraembryonic membranes) survival and development. In cyclic sheep, progesterone is paradoxically involved in suppressing and then initiating development of the endometrial luteolytic mechanism. In cyclic and pregnant sheep, progesterone negatively autoregulates progesterone receptor (PR) gene expression in the endometrial luminal (LE) and superficial glandular epithelium (GE). In cyclic sheep, PR loss is closely followed by increases in epithelial estrogen receptor (ERalpha) and then oxytocin receptor (OTR), allowing oxytocin to induce uterine release of luteolytic prostaglandin F2alpha pulses. In pregnant sheep, the conceptus produces interferon tau (IFNtau) that acts on the endometrium to inhibit transcription of the ERalpha gene and thus development of the endometrial luteolytic mechanism. After Day 13 of pregnancy, the endometrial epithelia do not express the PR, whereas the stroma and myometrium remain PR positive. The absence of PR in the endometrial GE is required for onset of differentiated function of the glands during pregnancy. The sequential, overlapping actions of progesterone, IFNtau, placental lactogen (PL), and growth hormone (GH) comprise a hormonal servomechanism that regulates endometrial gland morphogenesis and terminal differentiated function during gestation. In pigs, estrogen, the pregnancy-recognition signal, increases fibroblast growth factor 7 (FGF-7) expression in the endometrial LE that, in turn, stimulates proliferation and differentiated functions of the trophectoderm, which expresses the receptor for FGF-7. Strategic manipulation of these physiological mechanisms may offer therapeutic schemes to improve uterine capacity, conceptus survival, and reproductive health of domestic animals and humans.

Osteopontin (OPN) is a phosphorylated and glycosylated, secreted protein that is present in various epithelial cells and biological fluids. On freezing and thawing or treatment with proteases, the native 70-kDa protein gives rise to 45- and 24-kDa fragments. Secreted OPN functions as an extracellular matrix (ECM) protein that binds cell surface receptors to mediate cell-cell adhesion, cell-ECM communication, and cell migration. In sheep and humans, OPN is proposed to be a secretory product of uterine glandular epithelium (GE) that binds to uterine luminal epithelium (LE) and conceptus trophectoderm to mediate conceptus attachment, which is essential to maintain pregnancy through the peri-implantation period. Cell-cell adhesion, communication, and migration likely are important at the interface between uterus and placenta throughout pregnancy, but to our knowledge, endometrial and/or placental expression of OPN beyond the peri-implantation period has not been documented in sheep. Therefore, the present study determined temporal and spatial alterations in OPN mRNA and protein expression in the ovine uterus between Days 25 and 120 of pregnancy. The OPN mRNA in total ovine endometrium increased 30-fold between Days 40 and 80 of gestation. In situ hybridization and immunofluorescence analyses revealed that the predominant source of OPN mRNA and protein throughout pregnancy was the uterine GE. Interestingly, the 45-kDa form of OPN was detected exclusively, continuously, and abundantly along the apical surface of LE, on conceptus trophectoderm, and along the uterine-placental interface of both interplacentomal and placentomal regions through Day 120 of pregnancy. The 45-kDa OPN is a proteolytic cleavage fragment of the native 70-kDa OPN, and it is the most abundant form in uterine flushes during early pregnancy. The 45-kDa OPN is more stimulatory to cell attachment and cell migration than the native 70-kDa protein. Collectively, the present results support the hypothesis that ovine OPN is a component of histotroph secreted by the uterine GE that accumulates at the uterine-placental interface to influence maternal-fetal interactions throughout gestation in sheep.

Osteopontin (OPN) is a component of the extracellular matrix that interacts with cell surface receptors, including integrins, to mediate cell adhesion, migration, differentiation, survival, and immune function. In pregnant mice and primates, OPN has been detected in decidualized stroma and is considered to be a gene marker for decidualization. Decidualization involves transformation of spindle-like fibroblasts into polygonal epithelial-like cells that are hypothesized to limit conceptus trophoblast invasion through the uterine wall during invasive implantation. Decidualization is not considered characteristic of species with noninvasive implantation, such as domestic animals. However, the extent of trophoblast invasion between sheep and pigs differs, with sheep exhibiting erosion of the uterine luminal epithelium (LE) and fusion of trophectoderm with LE to form syncytia, and pigs maintaining an intact LE throughout pregnancy. Therefore, the present study measured changes in the decidualization marker genes OPN, desmin, and alpha smooth muscle actin (alphaSMA) in ovine and porcine uterine stroma throughout pregnancy. The morphology of endometrial stromal cells in pregnant ewes changes following conceptus attachment, with cells increasing in size and becoming polyhedral in shape by Day 35 of pregnancy. Expression of OPN mRNA and protein, as well as desmin and alphaSMA proteins, was observed in this same uterine stromal compartment. In contrast, no morphological changes in uterine stroma nor induction of OPN mRNA and protein, or desmin protein, were detected during porcine pregnancy. Interestingly, alphaSMA protein was absent on Day 20, but prominent in uterine stroma of pregnant pigs on Day 45. Collectively, these results indicate that the uterine stroma of sheep undergoes a program of differentiation similar to decidualization in invasive implanting species, whereas porcine stroma exhibits differentiation that is more limited than that in sheep, rodents, or primates. Results suggest that uterine stromal decidualization is common to species with different types of placentation, but the extent is variable and correlates with the depth of trophoblast invasion during implantation.

Pregnancy and interferon-tau (IFN tau) upregulate uterine Mx gene expression in ewes; however, the only known role for Mx is in the immune response to viral infection. We hypothesize that Mx functions as a conceptus-induced component of the anti-luteolytic mechanism and/or regulator of endometrial secretion or uterine remodeling during early pregnancy. This study was conducted to determine the effects of early pregnancy on uterine Mx expression in domestic farm species with varied mechanisms of pregnancy recognition. Endometrium from cows, gilts, and mares was collected during the first 20 d of the estrous cycle or pregnancy, and total messenger RNA (mRNA) and protein were analyzed for steady-state levels of Mx mRNA and protein. Northern blot analysis of Mx mRNA detected an approximately 2.5 Kb of mRNA in endometrium from each species. In pregnant cows, steady-state levels of Mx mRNA increased 10-fold (P < 0.05) above levels observed in cyclic cows by d 15 to 18. In cyclic gilts, slot blot analysis indicated that endometrial Mx mRNA levels did not change between d 5 and 18 of the cycle. However, in pregnant gilts, Mx levels tended (P = 0.06) to be elevated two-fold on d 16 only, and in situ hybridization indicated that this increase occurred in the stroma. In mares, Mx mRNA was low, but detectable, and did not change between ovulation (d 0) and d 20, regardless of reproductive status. Western blot analysis revealed multiple immunoreactive Mx protein bands in each species. One band was specific to pregnancy in cows. As in ewes, in situ hybridization analysis indicated that Mx mRNA was strongly expressed in the luminal epithelium, stroma, and myometrium by d 18 in cows. However, on d 14 in gilts, Mx was expressed primarily in the stroma, and on d 14 in mares, low levels of Mx expression were confined largely to the luminal epithelium. The uteruses of cows, gilts, and mares express Mx, and expression is upregulated during pregnancy in cows and gilts--animals whose conceptuses secrete interferons during early pregnancy, but that possess different mechanisms for pregnancy recognition.

Major histocompatibility complex (MHC) class I molecules, consisting of an alpha chain and beta2-microglobulin (beta2MG), play an important role in immune rejection responses by discriminating self and nonself and are increased by type I interferons during antiviral responses. Interferon tau (IFNtau), the pregnancy-recognition signal in ruminants, is a type I interferon produced by the ovine conceptus between Days 11 and 21 of gestation. In study 1, expression of MHC class I alpha chain and beta2MG mRNA and protein was detected primarily in endometrial luminal epithelium (LE) and glandular epithelium (GE) on Days 10 and 12 of the estrous cycle and pregnancy. On Days 14-20 of pregnancy, MHC class I and beta2MG expression increased only in endometrial stroma and GE and, concurrently, was absent in LE and superficial ductal GE (sGE). Although neither MHC class I nor beta2MG proteins were detected in Day 20 trophectoderm, beta2MG mRNA was detected in conceptus trophectoderm. In study 2, cyclic ewes were ovariectomized on Day 5, treated daily with progesterone to Day 16, received intrauterine infusions between Days 11 and 16 of either control serum proteins or recombinant ovine IFNtau, and were hysterectomized on Day 17. The IFNtau increased MHC class I and beta2MG expression only in endometrial stroma and GE. During pregnancy, MHC class I and beta2MG gene expression is inhibited in endometrial LE and sGE but, paradoxically, is stimulated by IFNtau in the stroma and GE. The silencing of MHC class I alpha chain and beta2MG genes in the endometrial LE and sGE during pregnancy recognition and establishment may be a critical mechanism preventing immune rejection of the conceptus allograft.

After parturition, the uterus undergoes marked remodelling during involution; however, little is known of the hormonal, cellular and molecular mechanisms that regulate this process. The working hypothesis used in this study is that return of the ovine uterus to a non-pregnant state involves termination of a hormonal servomechanism that regulates endometrial gland morphogenesis and function during pregnancy. Suffolk ewes were ovariohysterectomized on postpartum days 1, 7, 14 or 28. Serum concentrations of oestradiol were high at parturition, declined to postpartum day 4, peaked on postpartum day 6, and then declined and remained low thereafter. Progesterone was undetectable in plasma from ewes post partum. Uterine wet mass and horn length decreased after postpartum day 1, but ovarian mass did not change. Residual placental cotyledons were present in the maternal caruncles on postpartum days 1 and 7 and were extruded by postpartum day 14 as plaques that were resorbed by postpartum day 28. The width of the total endometrium, stratum compactum, stratum spongiosum and myometrium, as well as endometrial gland density, decreased after parturition. Most apoptotic cells in the involuting uterus were large, vacuolated and located between the endometrial glandular epithelial cells on postpartum days 1 and 7. Immunofluorescence analyses identified both T and B cells within the glandular epithelium on postpartum day 1. Cell proliferation was detected in the luminal epithelium and glandular epithelium on postpartum days 1 and 7. On postpartum day 1, expression of oestrogen receptor alpha (ERalpha) was not detected in luminal epithelium and was low in glandular epithelium, but ERalpha was present in epithelia thereafter. Progesterone receptor (PR) protein was not detected in endometrial epithelia on postpartum day 1, but was detected in the glandular epithelium thereafter. Between postpartum days 1 and 7, ERalpha and PR protein increased substantially in the endometrial glandular epithelium. On postpartum days 1-28, abundant expression of oxytocin receptor mRNA was detected in endometrial luminal epithelium and superficial to the middle glandular epithelium. Prolactin receptor (PRLR) mRNA was detected in glandular epithelium on all postpartum days, whereas mRNA for uterine milk protein (UTMP), an index of secretory capacity of glandular epithelium, was present only on postpartum day 1. Collectively, these results indicate that uterine involution in ewes involves remodelling of both caruncular and intercaruncular areas of the uterine wall and termination of differentiated uterine gland functions characteristic of pregnancy.

Interferon stimulated gene 17 (ISG17) and Mx are up-regulated in the ruminant uterus in response to interferon-tau (IFNtau) during early pregnancy. Recent evidence strongly indicates that expression of ISGs occur only in stroma (ST) and glandular epithelium (GE) during this time as a result of transcriptional repression by interferon regulatory factor two (IRF-2) expression in the LE. The present report tested this hypothesis by examining mRNA and protein expression of ISG17 and Mx in serial uterine cross-sections obtained from cyclic and early pregnant ewes. In situ and immunocytochemical analysis revealed that ISG17 mRNA and protein were low to undetectable, whereas Mx mRNA was expressed in the lumenal (LE) and superficial GE at all days of the estrous cycle examined. Both ISG17 and Mx mRNA increased in the stratum compactum ST between Days 11 and 13, and expression extended into the deep GE and stratum spongiosum ST on Days 15 through 17 in pregnant ewes. Interestingly the Mx gene continued to be strongly expressed in LE and superficial GE through Day 17 of pregnancy, whereas ISG17 remained low to undetectable in these cells. Collectively, this study highlights the complexity of the uterine environment by unequivocally illustrating differential temporal and spatial expression of the IFN-responsive genes ISG17 and Mx.

All mammalian uteri contain endometrial glands that synthesize or transport and secrete substances essential for survival and development of the conceptus (embryo/fetus and associated extraembryonic membranes). In rodents, uterine secretory products of the endometrial glands are unequivocally required for establishment of uterine receptivity and conceptus implantation. Analyses of the ovine uterine gland knockout model support a primary role for endometrial glands and, by default, their secretions in peri-implantation conceptus survival and development. Uterine adenogenesis is the process whereby endometrial glands develop. In humans, this process begins in the fetus, continues postnatally, and is completed during puberty. In contrast, endometrial adenogenesis is primarily a postnatal event in sheep, pigs, and rodents. Typically, endometrial adenogenesis involves differentiation and budding of glandular epithelium from luminal epithelium, followed by invagination and extensive tubular coiling and branching morphogenesis throughout the uterine stroma to the myometrium. This process requires site-specific alterations in cell proliferation and extracellular matrix (ECM) remodeling as well as paracrine cell-cell and cell-ECM interactions that support the actions of specific hormones and growth factors. Studies of uterine development in neonatal ungulates implicate prolactin, estradiol-17 beta, and their receptors in mechanisms regulating endometrial adenogenesis. These same hormones appear to regulate endometrial gland morphogenesis in menstruating primates and humans during reconstruction of the functionalis from the basalis endometrium after menses. In sheep and pigs, extensive endometrial gland hyperplasia and hypertrophy occur during gestation, presumably to provide increasing histotrophic support for conceptus growth and development. In the rabbit, sheep, and pig, a servomechanism is proposed to regulate endometrial gland development and differentiated function during pregnancy that involves sequential actions of ovarian steroid hormones, pregnancy recognition signals, and lactogenic hormones from the pituitary or placenta. That disruption of uterine development during critical organizational periods can alter the functional capacity and embryotrophic potential of the adult uterus reinforces the importance of understanding the developmental biology of uterine glands. Unexplained high rates of peri-implantation embryonic loss in humans and livestock may reflect defects in endometrial gland morphogenesis due to genetic errors, epigenetic influences of endocrine disruptors, and pathological lesions.

Interferon tau (IFNtau) is the signal for maternal recognition of pregnancy in ruminants. The positive effects of IFNtau on IFN-stimulated gene (ISG) expression are mediated by ISG factor 3 (ISGF3), which is composed of signal transducer and activator of transcription (Stat) 1, Stat 2, and IFN regulatory factor-9 (IRF-9), and by gamma-activated factor (GAF), which is a Stat 1 homodimer. Induction of ISGs, such as ISG17 and 2',5'-oligoadenylate synthetase, by IFNtau during pregnancy is limited to the endometrial stroma (S) and glandular epithelium (GE) of the ovine uterus. The IRF-2, a potent transcriptional repressor of ISG expression, is expressed in the luminal epithelium (LE). This study determined effects of the estrous cycle, pregnancy, and IFNtau on expression of Stat 1, Stat 2, IRF-9, IRF-1, and IRF-2 genes in the ovine endometrium. In cyclic ewes, Stat 1, Stat 2, IRF-1, and IRF-9 mRNA and protein were detected at low levels in the S and GE. During pregnancy, expression of these genes increased only in the S and GE. Expression of IRF-2 was detected only in the LE and superficial GE (sGE) of both cyclic and pregnant ewes. In cyclic ewes, intrauterine administration of IFNtau stimulated Stat 1, Stat 2, IRF-9, and IRF-1 expression in the endometrium. Ovine IRF-2 repressed transcriptional activity driven by IFN-stimulated response elements that bind ISGF3, but not by gamma-activation sequences that bind GAF. These results suggest that IRF-2 in the LE and sGE restricts IFNtau induction of ISGs to the S and GE. In the S and GE, IFNtau hyperactivation of ISG expression likely involves formation and actions of the transcription factors ISGF3 and, perhaps, IRF-1.

The enzymes which comprise the 2',5'-oligoadenylate synthetase (OAS) family are interferon (IFN) stimulated genes which regulate ribonuclease L antiviral responses and may play additional roles in control of cellular growth and differentiation. This study characterized OAS expression in the endometrium of cyclic and pregnant ewes as well as determined effects of IFNtau and progesterone on OAS expression in cyclic or ovariectomized ewes and in endometrial epithelial and stromal cell lines. In cyclic ewes, low levels of OAS protein were detected in the endometrial stroma (S) and glandular epithelium (GE). In early pregnant ewes, OAS expression increased in the S and GE on Day 15. OAS expression in the lumenal epithelium (LE) was not detected in uteri from either cyclic or pregnant ewes. Intrauterine administration of IFNtau stimulated OAS expression in the S and GE, and this effect of IFNtau was dependent on progesterone. Ovine endometrial LE, GE, and S cell lines responded to IFNtau with induction of OAS proteins. In all three cell lines, the 40/46-kDa OAS forms were induced by IFNtau, whereas the 100-kDa OAS form appeared to be constitutively expressed and not affected by IFNtau. The 69/71-kDa OAS forms were induced by IFNtau in the S and GE cell lines, but not in the LE. Collectively, these results indicate that OAS expression in the endometrial S and GE of the early pregnant ovine uterus is directly regulated by IFNtau from conceptus and requires the presence of progesterone.

The complexity of implantation necessitates intimate dialogue between conceptus and maternal cells, and precise coordination of maternal and conceptus signalling events. Maternal and conceptus-derived steroid hormones, growth factors and cytokines, as well as integrins and their ligands, have important and inter-related roles in mediating adhesion between apical aspects of conceptus trophectoderm and maternal uterine luminal epithelium that leads to formation of an epitheliochorial placenta. Integrin receptors appear to play fundamental roles in the implantation cascade and may interact with extracellular matrix molecules and other ligands to transduce cellular signals through autocrine and paracrine mechanisms. Functional in vitro analyses can be used to monitor individual contributions of specific integrin receptors and ligands to the signalling cascades of the maternal-conceptus interface. Integrative studies of implantation in pigs, using in vivo and in vitro approaches, are required to understand conceptus attachment and implantation in this species, and provide valuable opportunities to understand the fundamental mechanisms of implantation in all species.

The aim of this study was to develop immortalized cell lines from porcine uterus. Endometrial cells including luminal epithelium (LE), glandular epithelium (GE), stroma (ST), and myometrium (MYO) were enzymatically isolated from the uterus of a day 12 pregnant gilt. Primary cultures were immortalized by transduction with a retroviral vector containing the E6 and E7 open reading frames of human papillomavirus type 16 (LXSN-16E6E7) packaged by the amphotropic fibroblast line PA-317. Cells having integrated the vector were selected by resistance to the neomycin analog G418 (0.4-1.5 mg/ml). Surviving cells were maintained in complete culture medium containing G418 (0.1 mg/ml) and subcultured for 1 yr. Expression of the E7 protein was confirmed in all cell lines by Western blotting. Phase contrast microscopy revealed that LE and GE cells exhibited cobblestone morphology, whereas ST and MYO cells exhibited spindle-shaped morphology. The epithelial origin of LE and GE was confirmed by positive immunostaining for cytokeratin. Stromal and MYO cells were vimentin-positive, but cytokeratin-negative. The MYO cell lines were positive for smooth muscle alpha-actin staining, whereas LE, GE, and ST cell lines were negative for alpha-actin. Western blotting indicated that all cell lines expressed both estrogen and progesterone receptors, but only GE cells secreted uteroferrin (UF). Collectively, these porcine uterine cell lines provide an in vitro model for studying cell type-specific actions of hormones and cytokines, signal transduction pathways, cell-cell interactions, and gene expression.

Osteopontin (OPN) is an acidic phosphorylated glycoprotein component of the extracellular matrix that binds to integrins at the cell surface to promote cell-cell attachment and cell spreading. This matrix constituent is a ligand that could potentially bind integrins on trophectoderm and endometrium to facilitate superficial implantation and placentation. OPN mRNA increases in the endometrial glandular epithelium (GE) of early-pregnant ewes, and OPN protein is secreted into the uterine lumen. Therefore, progesterone and/or interferon-tau (IFNtau) may regulate OPN expression in the uterine GE. Cyclic ewes were ovariectomized and fitted with intrauterine (i. u.) catheters on Day 5 and treated daily with steroids (i.m.) and protein (i.u.) as follows: 1) progesterone (P, Days 5-24) and control serum proteins (CX, Days 11-24); 2) P and ZK 136.317 (ZK; progesterone receptor [PR] antagonist, Days 11-24) and CX proteins; 3) P and recombinant ovine IFNtau (roIFNtau, Days 11-24); or 4) P and ZK and roIFNtau. All ewes were hysterectomized on Day 25. Progesterone induced the expression of endometrial OPN mRNA in the GE and increased secretion of a 45-kDa OPN protein from endometrial explants maintained in culture for 24 h. Administration of ZK ablated progesterone effects. Intrauterine infusion of roIFNtau did not affect OPN gene expression or secretion in any of the steroid treatments. Interestingly, OPN mRNA-positive GE cells lacked detectable PR expression, although PR were detected in the stroma. Results indicate that progesterone regulates OPN expression in GE through a complex mechanism that includes PR down-regulation, and we suggest the possible involvement of a progesterone-induced stromal cell-derived growth factor(s) that acts as a progestamedin.

Ubiquitin cross-reactive protein (UCRP) is a functional ubiquitin homolog synthesized by the ruminant endometrium in response to conceptus-derived interferon-tau (IFNtau). Progesterone is required for IFNtau to exert antiluteolytic actions on the endometrium. Therefore, this study was designed to determine whether progesterone is requisite for IFNtau induction of UCRP expression within the ovine uterus. Cyclic ewes were ovariectomized and fitted with intrauterine (i.u.) catheters on Day 5 and treated daily with steroids (i.m.) and protein (i.u.) as follows: 1) progesterone (P, Days 5-24) and control serum proteins (CX, Days 11-24); 2) P and ZK 137.316 (ZK; progesterone receptor antagonist, Days 11-24) and CX proteins; 3) P and recombinant ovine IFNtau (roIFNtau, Days 11-24); or 4) P and ZK and roIFNtau. All ewes were hysterectomized on Day 25. In P-treated ewes, roIFNtau increased endometrial UCRP mRNA and protein levels. However, administration of ZK to ewes ablated roIFNtau induction of UCRP. Recombinant ovine IFNtau induced expression of UCRP mRNA in progestinized endometrial luminal (LE) and glandular (GE) epithelium as well as in both stratum compactum and spongiosum layers of the stroma (ST). Progesterone receptor protein was located in endometrial ST, but not in LE and GE from these ewes. Results support the hypothesis that progesterone is required for IFNtau induction of type I IFN-responsive genes, such as UCRP, in the ruminant uterus.