Keratinocyte growth factor is up-regulated by estrogen in the porcine uterine endometrium and functions in trophectoderm cell proliferation and differentiation.

Keratinocyte growth factor (KGF) is expressed by uterine endometrial epithelial cells during the estrous cycle and during pregnancy in pigs, whereas KGF receptor is expressed in conceptus trophectoderm and endometrial epithelia. In particular, KGF expression in the endometrium is highest on day 12 of pregnancy. This corresponds to the period of maternal recognition of pregnancy in pigs, which is signaled by large amounts of estrogen secreted by conceptus trophectoderm acting on the endometrium. Our hypothesis is that estrogens of conceptus origin stimulate endometrial epithelial KGF expression, and, in turn, secreted KGF stimulates proliferation and differentiation of conceptus trophectoderm. To determine the factors affecting KGF expression in the uterus, endometrial explants from gilts on day 9 of the estrous cycle were cultured in the presence of 17beta-estradiol, catechol estrogens, or progesterone. 17beta-Estradiol stimulated the expression of KGF (P < 0.05), whereas catechol estrogens had no effect (P > 0.05). Between days 9 and 15 of pregnancy, proliferating cell nuclear antigen was abundant in conceptuses, but was barely detectable in uterine endometrial epithelia. To determine the effects of KGF on conceptus trophectoderm, porcine trophectoderm (pTr) cells were treated with recombinant rat KGF (rKGF). rKGF increased the proliferation of pTr cells (P < 0.01) as measured by [(3)H]thymidine incorporation. rKGF elicited phosphorylation of KGF receptor and activated the mitogen-activated protein kinase (ERK1/2) cascade in pTr cells. pTr cell differentiation was affected by rKGF, because it increased expression of urokinase-type plasminogen activator, a marker for differentiation in pTr cells. Collectively, these results indicate that estrogen, the pregnancy recognition signal from the conceptus in pigs, increases uterine epithelial KGF expression, and, in turn, KGF stimulates the proliferation and differentiation of conceptus trophectoderm.

Effect of prolonged administration of dietary capsaicin on broiler growth and Salmonella enteritidis susceptibility.

The effect of continuous (42 days) dietary administration of 5 or 20 ppm capsaicin to broiler chickens on Salmonella enteritidis susceptibility, body weight, and feed efficiency was investigated. Chickens were weighed at 1, 21, and 42 days of age. No significant differences in body weight or feed efficiency were observed. Chickens were challenged with 1 x 10(8) colony-forming units of S. enteritidis at 21, 28, or 42 days of age. The S. enteritidispositive culture rate for cecal tonsils was significantly lower (P < 0.05) in the treatment groups receiving 5 ppm or 20 ppm dietary capsaicin than in the untreated control group at all challenge times. Dietary capsaicin (5 and 20 ppm) resulted in protection against S. enteritidis organ invasion at 28 days in one experiment and at both 21 and 42 days in the other. These results indicate that continual dietary capsaicin administration increases resistance to S. enteritidis colonization and organ invasion throughout the normal growth period without detrimental effects on growth in broiler chickens.

Lead transport in IEC-6 intestinal epithelial cells.

This study evaluated the use of IEC-6 cells as a model for studying lead (Pb) transport by intestinal epithelial cells (IECs) and examined potential transport mechanisms for Pb uptake and extrusion. Pb accumulation in IEC-6 cells exposed to 5 and 10 microM Pb for up to 60 min was time- and dose-dependent. Reduction of incubation temperature significantly reduced the total cellular Pb content of IEC-6 cells. Simultaneous exposed of cells to zinc (Zn) and Pb resulted in decreased total cellular Pb contents compared to total cellular Pb contents of cells exposed to Pb only. IEC-6 cells treated with ouabain (1 mM) or sodium azide (1 mM) and 5 microM Pb accumulated more Pb than cells exposed to Pb only. Cells treated with p-chloromercuribenzensulfonic acid (50 microM), p-chloromercuribenzoic acid (50 microM), or iodoacetimide (50 microM) accumulated less Pb than cells treated with Pb only. We conclude that Pb uptake by IEC-6 cells depends on the extracellular Pb concentration. Our data suggest that the mechanism of Pb uptake by IECs is complex, and that Pb transport in IEC-6 cells is time- and temperature-dependent, involves sulfhydryl groups, and is decreased by the presence of Zn. Extrusion of Pb is at least partially dependent on metabolic energy.

Immunolocalization of epidermal growth factor (EGF) and EGF receptors in the porcine upper gastrointestinal tract.

The factors controlling growth and maturation in the porcine gastrointestinal tract are not well understood. Epidermal growth factor (EGF) is a polypeptide that has been implicated in the control of gastrointestinal tract growth, maturation, and protection in other species. Immunoreactive EGF (IR-EGF) and EGF receptors (EGF-R) were histochemically identified in formalin-fixed tissues of the upper digestive tract of 1-, 3-, 7-, 14-, 21-, and 28-day-old pigs. The ductal epithelium consistently contained IR-EGF in the parotid salivary gland of pigs of all ages and in the mandibular salivary gland in pigs greater than or equal to 7 days old. Immunoreactive EGF was detected in the mucosal epithelium of the esophagus and nonglandular portion of the stomach, and in the pancreas and liver in all pigs. Gastric gland IR-EGF was inconsistently detected in pigs less than 14 days old and was consistently observed in all older pigs. Enterocyte EGF immunoreactivity was usually weak and was variably detected in the duodenum of pigs less than or equal to 7 days old and in the jejunum of pigs less than or equal to 14 days old, but was consistently observed in older pigs. Ileal immunoreactivity was erratic. Immunoreactive EGF-R were identified in the esophageal epithelium of all pigs, and in the nonglandular gastric and glandular gastric mucosa of all pigs, except for two 7-day-old pigs and one 7-day-old pig, respectively. Immunoreactive EGF-R were detected in the duodenal, jejunal, and ileal enterocytes of pigs of all ages examined.

Lectin binding to small intestinal goblet cells of newborn, suckling, and weaned pigs.

Lectin binding of small intestinal goblet cells was examined in newborn, suckling, and weaned pigs. Sections of duodenum, proximal portion of the jejunum, distal portion of the jejunum, and ileum were embedded in a hydrophilic acrylic resin and treated with each of the following lectins: Canavalia ensiformis, Ricinus communis I, Glycine max, Ulex europaeus I, and Triticum vulgaris. Percentages of goblet cells binding each lectin were calculated within intestinal regions. Differences in lectin-binding affinity were detected among pigs of various ages and among various intestinal regions within pig age groups.

Effect of orally administered epidermal growth factor on the jejunal mucosa of weaned pigs.

The effect of ingested epidermal growth factor (EGF) on the small intestinal mucosa of conventionally weaned pigs was determined. At 21 days of age, 39 pigs were randomly distributed into suckling and weaned treatment groups that were administered 124 micrograms of EGF, 372 micrograms of EGF, or the dosing compound daily. Fecal water content was determined daily. On postweaning days 0, 3, 6, and 9, representative pigs from each group were euthanatized, and jejunal mucosa samples were collected for determination of villus-to-crypt ratio, total protein content, disaccharidase activities, and microbiological populations. At postweaning day 3, the 372-micrograms dose of EGF significantly (P less than or equal to 0.05) increased jejunal lactase and sucrase activities in the weaned pigs. Increased lactase activity was not greater than that of the suckling pig controls, whereas sucrase activity was significantly (P less than or equal to 0.05) higher than that of the suckling pig controls. Significant changes were not observed in villus-to-crypt ratio, mucosal protein content, or disaccharidase activities on other collection days.

Growth-stimulating substances in porcine milk.

Colostral and milk samples were collected from 6 primiparous sows on postpartum days 0, 9, 18, and 27. Insulin content was measured and epidermal growth factor (EGF) concentration was determined by use of radioimmunoassay and radioreceptor assay, respectively. Total milk protein concentration was measured colorimetrically. Concentrations of insulin, EGF, and protein in the colostral samples were significantly (P less than 0.01) higher than those found in the milk. Milk samples obtained from postpartum days 9, 18, and 27 did not differ significantly in insulin, EGF, or total protein content.

Immunohistochemical localization of transforming growth factor-alpha in suckling porcine intestine.

The distribution of transforming growth factor-alpha (TGF alpha), a member of the epidermal growth factor (EGF) family, was studied immunohistochemically in small intestinal tissues of suckling pigs which were 7, 14 and 21 days of age. TGF alpha immunostaining was similar in villous epithelium in all intestinal regions of all ages examined, and was diffuse throughout the epithelial cytoplasm and more prominent in the apical and brush-border regions of the cells. Cytoplasmic immunoreactive TGF alpha was also identified in crypt epithelial cells; the pattern and extent of immunostaining differed among intestinal regions and ages. In all cases, the relative number of TGF alpha-immunopositive crypt epithelial cells was fewer than the number of immunonegative cells within the compartment. In duodenal and jejunal segments, relative numbers of immunopositive crypt epithelial cells were greater in 14- and 21- than in 7-day-old pigs. In all 7-day-old pigs, a relatively greater number of immunopositive epithelial cells was present in the jejunal crypts than in the duodenal crypts. Intracellular TGF alpha immunostaining was present within Peyer's patches of distal jejunum and ileum, and is a previously unreported finding. The presence of TGF alpha immunoreactive protein within small intestinal crypt epithelium has not been previously described in any species, and may be unique to suckling animals or, alternatively, the porcine species. These data suggest that TGF alpha may be an endogenous ligand for the EGF receptor in suckling porcine intestine. Increased expression of TGF alpha in suckling pigs during the period in which marked structural and functional changes occur within the small intestinal mucosa suggests a role for this growth factor in remodelling and maturation of the neonatal mucosa.

Differential expression of beta transforming growth factors (TGF beta 1, TGF beta 2, and TGF beta 3) and their receptors (type I and type II) in peri-implantation porcine conceptuses.

Beta transforming growth factors (TGF beta 1, TGF beta 2, and TGF beta 3) and type I and II TGF beta receptors were immunohistochemically localized in peri-implantation porcine conceptuses (embryos and associated membranes) collected on Day 10 through Day 14 of gestation. Our results indicate specific immunolocalization of TGF beta isoforms and their receptors in conceptuses during these gestational days. In parietal endoderm, TGF beta 1 immunoreactions were weak to undetectable, TGF beta 2 immunoreactions were intense, and TGF beta 3 immunoreactions were intermediate in intensity to TGF beta 2 and TGF beta 1. In contrast to immunoreactions in endoderm, TGF beta 1 and TGF beta 3 immunostaining in trophectoderm (Tr) was intense. Differences in TGF beta 2 immunostaining of Tr were observed from Days 10 to 14 of gestation. A drastic decrease in cytoplasmic immunostaining of ectoderm and mesoderm was detected from Days 12 to 14 for all TGF beta isoforms and type II receptor; however, type I receptor immunoreactions were consistently detected between Days 10 and 14. Concurrent expression of both type I and type II receptors in the peri-implantation conceptuses suggests that porcine conceptuses are capable of binding and responding to TGF beta s during this period. Differential expression of the three TGF beta isoforms suggests different roles for TGF beta s 1, 2, and 3 in conceptus development. Our results suggest possible roles for TGF beta s in early growth and differentiation of the embryo, differentiation of the Tr, and implantation.

Immunolocalization of acidic and basic fibroblast growth factors in porcine uterine and conceptus tissues.

Immunohistochemical techniques were used to localize acidic and basic fibroblast growth factor (FGF) polypeptides in porcine uterine and conceptus tissues collected on Days 10 through 14 of gestation, which is the peri-implantation period, and in uterine tissues collected on the same days of the estrous cycle. Our results demonstrate differential expression of acidic and basic FGF (aFGF, bFGF) in porcine uterine and conceptus tissues. Localization of these peptides in the uterus of cycling and pregnant pigs was different from that reported for other species, suggesting species-specific roles for FGFs in early pregnancy. Increases in both cytoplasmic and nuclear bFGF immunostaining were detected in uterine luminal and glandular epithelial cells from Days 10 to 14 of gestation but not in the uterine epithelium of cycling pigs. Acidic FGF immunostaining was not detected in luminal or glandular epithelium of either cycling or pregnant uterine tissue; however, differential stromal staining was observed. Uterine tissues collected from pigs on Days 10-14 of the estrous cycle had diffuse aFGF immunostaining throughout the stroma. During early pregnancy, however, intense aFGF immunostaining was concentrated around the glandular epithelial and below the luminal epithelial cells as gestation progressed. Basic FGF, but not aFGF, was detected in porcine conceptuses collected during the peri-implantation period (Days 10-14 of gestation). Although both acidic and basic FGF belong to the same family of proteins, results of the present study indicate that they likely play different roles in uterine function and conceptus development in pigs.

Ornithine aminotransferase messenger RNA expression and enzymatic activity in fetal porcine intestine.

In most neonatal animals, the small intestinal epithelium is responsible for endogenous arginine production. The ability of neonatal enterocytes to synthesize arginine immediately after birth suggests that the enzymes involved are present prenatally. Pyrroline-5-carboxylate is the common intermediate in the intestinal pathways for the synthesis of citrulline and arginine from both glutamine and proline and is interconverted into ornithine by ornithine aminotransferase (OAT). In this study, OAT enzymatic activity and mRNA expression in the intestine of fetal pigs from 30 to 110 d of gestation were determined. Enzymatic activity (nanomoles per minute per milligram of protein) peaked at d 45 of gestation and increased again between d 60 and 110 of gestation. At 30 and 35 d of gestation, OAT mRNA expression was detected throughout the mucosal epithelium of the small intestine. Throughout the remainder of gestation, OAT expression was notably higher in the villus epithelium than in the crypt epithelium. The presence of OAT in the small intestinal epithelium throughout gestation suggests that the porcine small intestine is capable of interconverting ornithine and pyrroline-5-carboxylate during fetal development. This capability may be important for synthesis of arginine, proline, ornithine, and polyamines for development and metabolic activity of the intestine during gestation or for somatic growth of the fetus.

Mucosal morphogenesis and cytodifferentiation in fetal porcine small intestine.

BACKGROUND: Development of the small intestine is essential for proper nutrition of the fetus and the neonate. This investigation examines the morphogenesis and cytodifferentiation of developing fetal porcine small intestinal mucosa. METHODS: Fetuses were collected from gilts after hysterectomy. Small intestinal segments were removed and processed for light and electron microscopy. RESULTS: Fetal porcine small intestine developed from a simple tube of stratified epithelium to a tube containing villus and intervillus regions of simple columnar epithelium. This development occurred in a proximal to distal direction. By Day 40 of gestation, cytodifferentiation was evident with the presence of goblet cells and enteroendocrine cells in the duodenum. As development progressed, microvilli lengthened and components of the apical endocytic complex (AEC) were observed. By Day 110 of gestation, tubular and vesicular components of the AEC were confined to the jejunum, whereas large lysosomal vacuoles were observed in the distal jejunum. Duodenal epithelium at Day 110 was similar to postnatal epithelium. CONCLUSIONS: The pattern of fetal porcine small intestinal development is similar to that reported for fetal human small intestine. Villus development and cytodifferentiation occur at similar relative times in gestation when compared to the human. These observations support the use of the fetal pig as a model for investigations of human small intestinal development.

A cortisol surge mediates the enhanced polyamine synthesis in porcine enterocytes during weaning.

This study was conducted to determine whether a cortisol surge mediates the enhanced expression of intestinal ornithine decarboxylase (ODC) in weanling pigs. Piglets were nursed by sows until 21 days of age, when 40 pigs were randomly assigned into one of four groups (10 animals/group). Group 1 continued to be fed by sows, whereas groups 2-4 were weaned to a corn and soybean meal-based diet. Weanling pigs received intramuscular injections of vehicle solvent (sesame oil), RU-486 (a potent blocker of glucocorticoid receptors; 10 mg/kg body wt), and metyrapone (an inhibitor of adrenal cortisol synthesis; 5 mg/kg body wt), respectively, 5 min before weaning and 24 and 72 h later. At 29 days of age, pigs were used to prepare jejunal enterocytes for ODC assay and metabolic studies. To determine polyamine (putrescine, spermidine, and spermine) synthesis, enterocytes were incubated for 45 min at 37 degrees C in 2 ml Krebs-bicarbonate buffer containing 1 mM [U-(14)C]arginine, 1 mM [U-(14)C]ornithine, 1 mM [U-(14)C]glutamine, or 1 mM [U-(14)C]proline plus 1 mM glutamine. Weaning increased intestinal ODC activity by 230% and polyamine synthesis from ornithine, arginine, and proline by 72-157%. Arginine was a quantitatively more important substrate than proline for intestinal polyamine synthesis in weaned pigs. Administration of RU-486 or metyrapone to weanling pigs prevented the increases in intestinal ODC activity and polyamine synthesis, reduced intracellular polyamine concentrations, and decreased villus heights and intestinal growth. Our results demonstrate an essential role for a cortisol surge in enhancing intestinal polyamine synthesis during weaning, which may be of physiological importance for intestinal adaptation and remodeling.

Beta transforming growth factors (TGFss) at the porcine conceptus-maternal interface. Part I: expression of TGFbeta1, TGFbeta2, and TGFbeta3 messenger ribonucleic acids.

Spatial and temporal mRNA expression of beta transforming growth factors (TGFbeta1, TGFbeta2, and TGFbeta3) in porcine uterus and conceptuses was determined during the peri-implantation period (Days 10-14 of gestation). Northern blotting identified a major 3. 5-kilobase (kb) and a minor 2.5-kb transcript for TGFbeta1 mRNA. TGFbeta2 transcripts were 6.2 kb, 5.4 kb, and 2.7 kb, and a single 3. 5-kb transcript was detected for TGFbeta3. With semiquantitative in situ hybridization analysis, progressive increases were detected in TGFbetas 1, 2, and 3 mRNA expression in uterine luminal epithelium (ULE), uterine glands (UGs), and underlying stroma (stroma spongiosum, SS) from Days 10 through 14 of gestation (p < 0.05). In myometrium, TGFbeta mRNA expression did not differ between Days 10 through 14 of gestation. In porcine conceptuses, TGFbetas 1, 2, and 3 mRNA expression was detected in trophectoderm, endoderm, embryonic ectoderm, and mesoderm. For the three TGFbeta isoforms examined, mRNA expression increased 2- to 4-fold in trophectoderm and endoderm between Days 10 and 14 of gestation. TGFbeta1 mRNA levels increase significantly in embryonic ectoderm, but not mesoderm, between Days 12 and 14 of gestation; during that same time, TGFbeta2 mRNA levels increased, but no change was detected in TGFbeta3 mRNA levels, in embryonic ectoderm and mesoderm. Progressive increases in TGFbeta mRNA expression in conceptus trophectoderm, endoderm, ULE, UGs, and SS suggest important roles for these growth factors in porcine conceptus development during the peri-implantation period.

Beta transforming growth factors (TGFbeta) at the porcine conceptus-maternal interface. Part II: uterine TGFbeta bioactivity and expression of immunoreactive TGFbetas (TGFbeta1, TGFbeta2, and TGFbeta3) and their receptors (type I and type II).

Porcine uterine tissues were collected from Days 10 to 14 of gestation (peri-implantation period) or corresponding days of the estrous cycle. Results indicated a marked increase in beta transforming growth factors (TGFbeta1, TGFbeta2, and TGFbeta3) and TGFbeta receptor (type I and type II) immunostaining in uterine luminal epithelium (ULE) between Days 10 and 14 of gestation, but there was no increase in ULE immunostaining on the corresponding days of the estrous cycle. Uterine glands and stroma were intensely immunopositive in pregnant gilts for TGFbeta isoforms and their receptors, but immunostaining was weak to undetectable in cycling gilts. No differences were detected in myometrium, in which immunostaining was moderate in both cycling and pregnant gilts. Additionally, TGFbeta2 and TGFbeta receptor (type I and type II) immunostaining was detected in uterine monocyte/macrophage-like cells. Western blotting detected the presence of all three TGFbeta isoforms in uterine luminal flushings. The CCL64 cell TGFbeta bioassay detected bioactive TGFbetas++ in uterine luminal flushings on Days 12, 13, an 14 of gestation. These results strongly indicate that uterine expression of TGFbetas and their receptors is pregnancy specific and that bioactive TGFbetas are present at the conceptus-maternal interface in the peri-implantation period in pigs. Thus TGFbetas are likely to be involved in autocrine-paracrine interactions between the maternal uterus and the conceptus.

Muc-1, integrin, and osteopontin expression during the implantation cascade in sheep.

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 alpha(v), alpha(4), alpha(5), beta(1), beta(3), and beta(5) 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 alpha(v)beta(3), alpha(v)beta(1), alpha(v)beta(5), alpha(4)beta(1), and alpha(5)beta(1) 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 alpha-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.

Functional analysis of autocrine and paracrine signalling at the uterine-conceptus interface in pigs.

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.