Calcium oscillations in fertilized pig oocytes are associated with repetitive interactions between STIM1 and ORAI1.

The Ca2+ entry mechanism that sustains the Ca2+ oscillations in fertilized pig oocytes was investigated. Stromal interaction molecule 1 (STIM1) and ORAI1 proteins tagged with various fluorophores were expressed in the oocytes. In some cells, the Ca2+ stores were depleted using cyclopiazonic acid (CPA); others were inseminated. Changes in the oocytes' cytosolic free Ca2+ concentration were monitored, while interaction between the expressed fusion proteins was investigated using fluorescence resonance energy transfer (FRET). Store depletion led to an increase of the FRET signal in oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, indicating that Ca2+ release was followed by an interaction between these proteins. A similar FRET increase in response to CPA was also detected in oocytes co-expressing mVenus-STIM1 and mTurquoise2-STIM1, which is consistent with STIM1 forming punctae after store depletion. ML-9, an inhibitor that can interfere with STIM1 puncta formation, blocked store-operated Ca2+ entry (SOCE) induced by Ca2+ add-back after a CPA treatment; it also disrupted the Ca2+ oscillations in fertilized oocytes. In addition, oocytes overexpressing mVenus-STIM1 showed high-frequency Ca2+ oscillations when fertilized, arguing for an active role of the protein. High-frequency Ca2+ oscillations were also detected in fertilized oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, and both of these high-frequency Ca2+ oscillations could be stopped by inhibitors of SOCE. Importantly, in oocytes co-expressing mVenus-STIM1 and mTurquoise2-ORAI1, we were also able to detect cyclic increases of the FRET signal indicating repetitive interactions between STIM1 and ORAI1. The results confirm the notion that in pig oocytes, SOCE is involved in the maintenance of the repetitive Ca2+ transients at fertilization.

Store-Operated Ca2+ Entry Sustains the Fertilization Ca2+ Signal in Pig Eggs.

The role of store-operated Ca(2+) entry (SOCE) in the maintenance of sperm-induced Ca(2+) oscillations was investigated in porcine eggs. We found that 10 µM gadolinium (Gd(3+)), which is known to inhibit SOCE, blocked Ca(2+) entry that was triggered by thapsigargin-induced store depletion and also caused an abrupt cessation of the fertilization Ca(2+) signal. In a similar manner 3,5-bis(trifluoromethyl)pyrazole 2 (20 µM), and tetrapandin-2 (10 µM), potent SOCE inhibitors, also blocked thapsigargin-stimulated Ca(2+) entry and disrupted the Ca(2+) oscillations after sperm-egg fusion. The downregulation of Stim1 or Orai1 in the eggs did not alter the Ca(2+) content of the intracellular stores, whereas co-overexpression of these proteins led to the generation of irregular Ca(2+) transients after fertilization that stopped prematurely. We also found that thapsigargin completely emptied the endoplasmic reticulum, and that the series of Ca(2+) transients stopped abruptly after the addition of thapsigargin to the fertilized eggs, indicating that the proper reloading of the intracellular stores is a prerequisite for the maintenance of the Ca(2+) oscillations. These data strengthen our previous findings that in porcine eggs SOCE is a major signaling cascade that is responsible for sustaining the repetitive Ca(2+) signal at fertilization.

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.

Intrauterine infusion of latency-associated peptide (LAP) during early porcine pregnancy affects conceptus elongation and placental size.

In the pig, transforming growth factor beta (TGFB), TGFB receptors (TGFBRs), and integrins are present during the peri-implantation period. Latency-associated peptide (LAP), a part of latent TGFB, can bind to integrin heterodimers via its Arg-Gly-Asp (RGD) sequence; therefore, ligand-receptor interactions between TGFB and TGFBRs, along with LAP and integrin heterodimers, may be functional in mediating events supporting conceptus elongation and attachment. With the use of surgically implantable osmotic pumps, we were able to maintain pregnancy with the aim of mechanistically altering in vivo receptor-ligand interactions involving TGFB with TGFBRs and LAP with integrins during porcine pregnancy. Day 9 pregnant gilts received intrauterine infusions of LAP-RGD, a recombinant mutant of LAP (LAP-RGE), or vehicle control and were ovariohysterectomized on Day 13 or 24 of pregnancy. We hypothesized that intrauterine infusion of LAP-RGD would decrease downstream signaling of TGFB while increasing LAP-integrin interactions and that net effect would enhance conceptus survival and attachment early in the peri-implantation period but possibly increase the chance of abnormal placentation later in pregnancy. Additionally, we hypothesized that infusion of LAP-RGE would disrupt TGFB signals but not alter integrin signaling, and thus the net result would be decreased conceptus survival and abnormal development. Unexpectedly, LAP-RGD intrauterine infusions resulted in a reduction of conceptus elongation, whereas infusions of LAP-RGE permitted implantation and placentation but resulted in larger fetal weight, allantois length, and allantoic fluid volume. Results suggest TGFB and integrins are contributing factors in the regulation of conceptus elongation and placental and fetal size.

Transforming growth factor beta (TGFB) signaling is activated during porcine implantation: proposed role for latency-associated peptide interactions with integrins at the conceptus-maternal interface.

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.

Regulation of ornithine aminotransferase gene expression and activity by all-transretinoic acid in Caco-2 intestinal epithelial cells.

Ornithine aminotransferase (OAT) is a crucial enzyme in the synthesis of citrulline and arginine from glutamine/glutamate and proline by enterocytes of the small intestine. However, a role for OAT in intestinal polyamine synthesis and cell growth is not known. All-transretinoic acid (RA), an active metabolite of vitamin A, regulates the activity of several metabolic enzymes related to OAT, including ornithine decarboxylase and arginase, which may influence the function of OAT through effects on substrate (ornithine) availability. The objective of the present study was to test the hypothesis that RA regulates OAT mRNA expression and enzymatic activity in intestinal epithelial cells. Caco-2 cells were cultured for 12-72 h in the presence of 0, 0.01 and 1 microM RA and then used for measurements of OAT mRNA levels and enzyme activity as well as ornithine and polyamines. Treatment with RA induced increases in OAT gene expression and enzymatic activity, which resulted in decreased intracellular concentrations of ornithine and polyamines (putrescine, spermidine and spermine) in a dose-dependent manner. These changes occurred concomitantly with a decrease in the total number of cells, and the increase in OAT activity was due to increased OAT mRNA expression. In cells treated with 1 microM RA, addition of 10 microM putrescine to culture medium restored both cellular levels of polyamines and cell numbers to the values for the control group (without addition of RA). We conclude that exposure of Caco-2 cells to RA induces OAT expression for increasing ornithine catabolism. This leads to a reduced availability of intracellular ornithine for polyamine synthesis, thereby decreasing cell proliferation. These novel findings indicate a functional role for OAT in regulating intestinal polyamine synthesis and growth.

Estradiol up-regulates AUF1p45 binding to stabilizing regions within the 3'-untranslated region of estrogen receptor alpha mRNA.

Estradiol up-regulates expression of the estrogen receptor alpha gene in the uterus by stabilizing estrogen receptor alpha mRNA. Previously, we defined two discrete minimal estradiol-modulated stability sequences (MEMSS) within the extensive 3'-untranslated region of estrogen receptor alpha mRNA with an in vitro stability assay using cytosolic extracts from sheep uterus. We report here that excess MEMSS RNA inhibited the enhanced stability of estrogen receptor alpha mRNA in extracts from estradiol-treated ewes compared with those from control ewes. Several estradiol-induced MEMSS-binding proteins were characterized by UV cross-linking in uterine extracts from ewes in a time course study (0, 8, 16, and 24 h after estradiol injection). The pattern of binding proteins changed at 16 h post-injection, concurrent with enhanced estrogen receptor alpha mRNA stability and the highest rate of accumulation of estrogen receptor alpha mRNA. The predominant MEMSS-binding protein induced by estradiol treatment was identified as AUF1 (A + U-rich RNA-binding factor 1) protein isoform p45 (a product of the heterogeneous nuclear ribonucleoprotein D gene). Immunoblot analysis indicated that only two of four AUF1 protein isoforms were present in the uterine cytosolic extracts and that estradiol treatment strongly increased the ratio of AUF1 isoforms p45 to p37. Nonphosphorylated recombinant AUF1p45 protected estrogen receptor alpha mRNA in vitro in a dose-dependent manner. These studies describe estrogenic induction of AUF1p45 binding to the estrogen receptor alpha mRNA as a molecular mechanism for post-transcriptional up-regulation of gene expression.

Pig conceptuses increase uterine interferon-regulatory factor 1 (IRF1), but restrict expression to stroma through estrogen-induced IRF2 in luminal epithelium.

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.

Regulation of expression of fibroblast growth factor 7 in the pig uterus by progesterone and estradiol.

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.

Functional effects of transforming growth factor beta on adhesive properties of porcine trophectoderm.

In pigs, expression and amounts of biologically active TGFbetas at the conceptus-maternal interface increase significantly as conceptuses elongate and begin the implantation process. Before their activation, secreted TGFbetas are noncovalently associated with their respective, isoform-specific latency-associated peptides (LAPs), which contain the Arg-Gly-Asp (RGD) amino acid sequence that serves as a ligand for numerous integrins. Objectives of this study were to determine whether TGFbeta1 increases production of fibronectin by porcine trophectoderm, whether porcine trophectoderm adheres specifically to fibronectin and LAP, and whether functional interactions between porcine trophectoderm and the two TGFbeta-associated proteins, fibronectin and LAP, are integrin mediated. Porcine trophectoderm cells (pTr2) were cultured in presence of TGFbeta1, LAP, or pan-neutralizing anti-TGFbeta antibody; TGFbeta specifically increased (P < 0.05) fibronectin mRNA levels, as determined by Northern and slot blot analyses. Immunofluorescence microscopy demonstrated a TGFbeta-induced increase in fibronectin in pTr2 cells. In dispersed cell adhesion assays, adhesion of pTr2 cells to fibronectin was inhibited by an RGD-containing peptide (P < 0.05) and pTr2 cells attached to recombinant LAP but not to an LAP mutant, which contained an RGE sequence rather than the RGD site (P < 0.05). Fibronectin- and LAP-coated microbeads induced integrin activation at apical surfaces of both trophectoderm and uterine luminal epithelial cells, as indicated by aggregation and transmembrane accumulation of talin detected with immunofluorescence microscopy. Cell surface biotinylation and immunoprecipitation revealed integrin subunits alphav and beta1 on apical membranes of pTr2 cells. These results suggest multiple effects of TGFbeta at the porcine conceptus-maternal interface, including integrin-mediated conceptus-maternal communication through LAP.

Arginine deficiency in preterm infants: biochemical mechanisms and nutritional implications.

Arginine, an amino acid that is nutritionally essential for the fetus and neonate, is crucial for ammonia detoxification and the synthesis of molecules with enormous importance (including creatine, nitric oxide, and polyamines). A significant nutritional problem in preterm infants is a severe deficiency of arginine (hypoargininemia), which results in hyperammonemia, as well as cardiovascular, pulmonary, neurological, and intestinal dysfunction. Arginine deficiency may contribute to the high rate of infant morbidity and mortality associated with premature births. Although hypoargininemia in preterm infants has been recognized for more than 30 years, it continues to occur in neonatal intensive care units in the United States and worldwide. On the basis of recent findings, we propose that intestinal citrulline and arginine synthesis (the major endogenous source of arginine) is limited in preterm neonates owing to the limited expression of the genes for key enzymes (e.g., pyrroline-5-carboxylate synthase, argininosuccinate synthase and lyase), thereby contributing to hypoargininemia. Because premature births in humans occur before the normal perinatal surge of cortisol (an inducer of the expression of key arginine-synthetic enzymes), its administration may be a useful tool to advance the maturation of intestinal arginine synthesis in preterm neonates. Additional benefits of cortisol treatment may include the following: 1) allowing early introduction of enteral feeding to preterm infants, which is critical for intestinal synthesis of citrulline, arginine, and polyamines as well as for intestinal motility, integrity, and growth; and 2) shortening the expensive stay of preterm infants in hospitals as a result of accelerated organ maturation and the restoration of full enteral feeding. Further studies of fetal and neonatal arginine metabolism will continue to advance our understanding of the mechanisms responsible for the survival and growth of preterm infants. This new knowledge will be beneficial for designing the next generation of enteral and parenteral amino acid solutions to optimize nutrition and health in this compromised population.

Psittacid herpesviruses associated with mucosal papillomas in neotropical parrots.

Mucosal papillomas are relatively common lesions in several species of captive neotropical parrots. They cause considerable morbidity and in some cases, result in mortality. Previous efforts to identify papillomavirus DNA and proteins in these lesions have been largely unsuccessful. In contrast, increasing evidence suggests that mucosal papillomas may contain psittacid herpesviruses (PsHVs). In this study, 41 papillomas from 30 neotropical parrots were examined by PCR with PsHV-specific primers. All 41 papillomas were found to contain PsHV DNA. This 100% prevalence of PsHV infection in the papilloma population was found to be significantly higher than PsHV infection prevalence observed in other surveys of captive parrots. PsHV genotypes 1, 2, and 3, but not 4 were found in these lesions. Psittacus erithacus papillomavirus DNA and finch papillomavirus DNA were not found in the papillomas. A papilloma from a hyacinth macaw (Anodorhynchus hyacinthinus) was found to contain cells that had immunoreactivity to antiserum made to the common antigenic region of human papillomavirus (HPV) L1 major capsid protein. However, four other mucosal papillomas were negative for this immunoreactivity, and negative control tissues from a parrot embryo showed a similar staining pattern to that seen in the cloaca papilloma of the hyacinth macaw, strongly suggesting that the staining seen in hyacinth macaw papilloma was nonspecific. Based on these findings, it was concluded that specific genotypes of PsHV play a direct role in the development of mucosal papillomas of neotropical parrots and there is no evidence to suggest the concurrent presence of a papillomavirus in these lesions.

Gene expression and activity of enzymes in the arginine biosynthetic pathway in porcine fetal small intestine.

We recently reported the presence of ornithine aminotransferase (OAT) enzymatic activity and mRNA expression in the intestine of fetal pigs from 30 to 110 d of gestation. Here we describe the activities and mRNA expression patterns of other key enzymes in the arginine biosynthetic pathway, specifically carbamoyl phosphate synthase I (CPS-I), ornithine carbamoyl transferase (OCT), and pyrroline-5-carboxylate reductase (P5CR), in the fetal porcine small intestine from 30 to 110 d of gestation. The activities of all three enzymes increased from d 30 to d 110 of gestation, and in situ hybridization demonstrates that 1) CPS-I and OCT genes are expressed in distinct patterns and are confined to the mucosal epithelium and 2) P5CR mRNA is present in mucosal epithelium and lamina propria of the fetal porcine small intestine from d 30 to d 110 of gestation. The presence of CPS-I and OCT in conjunction with the presence of OAT suggests that the fetal porcine small intestine is capable of synthesizing citrulline from P5C. In addition, the presence of P5CR suggests that the fetal porcine small intestine is able to synthesize proline from ornithine via OAT. This ability of the fetal small intestine to synthesize amino acids may be important for development and metabolic activity of the intestine during somatic growth of the fetus.

Integrins and extracellular matrix proteins at the maternal-fetal interface in domestic animals.

Establishment of pregnancy in mammals requires coordinated conceptus-maternal interactions involving numerous hormones, growth factors and cytokines acting via specific receptors in the uterus. Uterine secretions play an important role in establishing synchrony between development of the conceptus and uterine receptivity, as well as in conceptus remodeling, adhesion, implantation and placentation in domestic species. Studies of non-invasive implantation in domestic livestock provide valuable opportunities to investigate fundamental processes of the initial events of apposition, attachment and adhesive interactions that are shared among species. In pigs and sheep, it appears that integrins play a dominant role in these fundamental processes via interactions with extracellular matrix molecules and other ligands to transduce cellular signals in uterine epithelial cells and conceptus trophectoderm. This review considers several of the potential integrin-binding ligands involved in the complex implantation adhesion cascade in pigs and sheep along with in vitro evidence for the transduction of cytoplasmic signals that may be required to sustain fetal and maternal contributions to the formation of the epitheliochorial placenta.

Analysis of osteopontin at the maternal-placental interface in pigs.

Noninvasive, epitheliochorial placentation in the pig follows a prolonged preimplantation period characterized by migration, spacing and elongation of conceptuses, and secretion of estrogen for maternal recognition of pregnancy. Osteopontin (OPN) is an extracellular matrix protein that binds integrins to promote cell-cell attachment and communication. OPN appears to play a key role in conceptus implantation and maintenance of pregnancy in sheep; however, a role for OPN in the porcine uterus has not been established. Therefore, this study examined OPN expression and function in the porcine uterus and conceptus (embryo/fetus and associated extraembryonic membranes). Northern and slot blot hybridization detected an increase in endometrial OPN expression between Days 25 and 30, and levels remained elevated through Day 85 of pregnancy. In situ hybridization localized OPN mRNA to discrete regions of the uterine luminal epithelium (LE) on Day 15 of pregnancy and to the entire LE thereafter. Glandular epithelial (GE) expression of OPN mRNA was first detected on Day 35 of pregnancy and increased through Day 85. Both 70- and 45-kDa forms of OPN protein were detected in cyclic and pregnant endometrium by Western blotting. OPN protein was localized to the LE and GE by immunofluorescence; however, only the 70-kDa OPN was detected in uterine flushings. OPN protein was present along the entire uterine-placental interface after Day 30 of pregnancy. In addition, OPN mRNA and protein were localized to immune-like cells within the stratum compactum of the endometrium in both Day 9 cyclic and pregnant gilts. Incubation of OPN-coated microbeads with porcine trophectoderm and uterine luminal epithelial cells induced Arg-Gly-Asp (RGD)-dependent integrin activation and transmembrane accumulation of cytoskeletal molecules at the apical cell surface as assessed by immunofluorescence detection of talin or alpha-actinin as markers for focal adhesions. These results suggest that OPN, expressed by uterine epithelium and immune cells, may interact with receptors (i.e., integrins) on conceptus and uterus to promote conceptus development and signaling between these tissues as key contributors to attachment and placentation in the pig.