Laurie A. Jaeger

Muc-1, Integrin, and Osteopontin Expression During the Implantation Cascade in Sheep

Abstract The extracellular matrix protein osteopontin (OPN) is a component of histotroph that increases in uterine flushings from pregnant ewes during the peri-implantation period and is localized on the apical surfaces of the uterine luminal epithelium (LE) and conceptus trophectoderm (Tr). The potential involvement of OPN in the implantation adhesion cascade in sheep was investigated by examining temporal, spatial, and potential functional relationships between OPN, Muc-1, and integrin subunits during the estrous cycle and early pregnancy. Immunoreactive Muc-1 was highly expressed at the apical surfaces of uterine luminal (LE) and glandular epithelium (GE) in both cycling and pregnant ewes but was decreased dramatically on LE by Day 9 and was nearly undetectable by Day 17 of pregnancy when intimate contact between LE and Tr begins. In contrast, integrin subunits αv, α4, α5, β1, β3, and β5 were constitutively expressed on conceptus Tr and at the apical surface of uterine LE and GE in both cyclic and early pregnant ewes. The apical expression of these subunits could contribute to the apical assembly of several OPN receptors including the αvβ3, αvβ1, αvβ5, α4β1, and α5β1 heterodimers on endometrial LE and GE, and conceptus Tr in sheep. Functional analysis of potential OPN interactions with conceptus and endometrial integrins was performed on LE and Tr cells in vitro using beads coated with OPN, poly-l-lysine, or recombinant OPN in which the Arg-Gly-Asp sequence was replaced with RGE or RAD. Transmembrane accumulation of talin or α-actinin at the apical surface of uterine LE and conceptus Tr cells in contact with OPN-coated beads revealed functional integrin activation and cytoskeletal reorganization in response to OPN binding. These results provide a physiological framework for the role of OPN, a potential mediator of implantation in sheep, as a bridge between integrin heterodimers expressed by Tr and uterine LE responsible for adhesion for initial conceptus attachment.

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

Abstract 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 α-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.

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 Dulbeccos modified Eagles 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.

Regulation of Expression of Fibroblast Growth Factor 7 in the Pig Uterus by Progesterone and Estradiol

Abstract 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 (E2), for pregnancy recognition. Furthermore, E2 increases the expression of FGF7 in pig uterine explants. The present study investigates the relationships between progesterone, E2, 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), E2, P4 and E2 (PE), or P4 and ZK and E2 (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) E2 and P4 can induce FGF7 when PGR are rendered nonfunctional by ZK; and 4) E2 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.

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.

Effect of Prolonged Administration of Dietary Capsaicin on Salmonella enteritidis Infection in Leghorn Chicks

The effect of 14 or 19 days of dietary capsaicin (18 ppm) on Salmonella enteritidis infection and histological, morphometric, and pH changes of the ceca was investigated. At day 13 or day 18, chicks were challenged with 10(8) colony-forming units of S. enteritidis. Chicks were killed and cultured 24 hours later. The total number of S. enteritidis-organ-culture-positive chicks was significantly lower among chicks fed capsaicin for either 14 or 19 days than among controls (P < 0.05). Subjective histological examination revealed a mild to moderate infiltration of mononuclear cells and heterophils in lamina propria of ceca, as well as epithelial cell proliferation in chicks following either 14 or 19 days of capsaicin administration. Using morphometric analysis, the mean lamina propria thickness and mean epithelial cell thickness in chickens fed capsaicin for 14 or 19 days were significantly greater than in controls (P < 0.05). Capsaicin significantly decreased luminal pH in both trials (P < 0.05). These data indicate that the observed capsaicin-induced resistance to S. enteritidis organ invasion is associated with measurable pH and morphological changes of the cecal mucosa.

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.

Pig Conceptuses Increase Uterine Interferon-Regulatory Factor 1 (IRF1), but Restrict Expression to Stroma Through Estrogen-Induced IRF2 in Luminal Epithelium

Abstract 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.

Estradiol Up-regulates AUF1p45 Binding to Stabilizing Regions within the 3′-Untranslated Region of Estrogen Receptor α mRNA

Estradiol up-regulates expression of the estrogen receptor α gene in the uterus by stabilizing estrogen receptor α mRNA. Previously, we defined two discrete minimal estradiol-modulated stability sequences (MEMSS) within the extensive 3′-untranslated region of estrogen receptor α 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 α 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 α mRNA stability and the highest rate of accumulation of estrogen receptor α 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 α mRNA in vitro in a dose-dependent manner. These studies describe estrogenic induction of AUF1p45 binding to the estrogen receptor α mRNA as a molecular mechanism for post-transcriptional up-regulation of gene expression.