Markus Schweiger

Differential expression of EGF receptor in the pig duodenum during the transition phase from maternal milk to solid food.

Background. The aim of this investigation was to study the cell type-specific expression of epidermal growth factor receptor (EGF-R) and to evaluate changes of the EGF-R distribution during transition from maternal milk to solid food in the gastrointestinal tract of young piglets. Methods. Duodenal tissue probes from six pigs were taken 2 days before (−2d) and 2 days (+2d) and 14 days (+14d) after transition from milk to solid food. The specimens were fixed in methanol/glacial acetic acid (2 : 1). A monoclonal antibody against EGF-R was used to examine the pattern and topographical shift of EGF-R. To assess a possible correlation between EGR-R-positive cells and mitotic activity, the mitotic index (MI) were evaluated based on expression of the Ki-67 antigen. Results. A significant change in the topographical and cellular distribution of the EGF-R could be successfully determined during the transition period. The highest immunoreactivity for EGF-R was found in enterocytes 2 days before transition from maternal milk, predominantly around the villous tips. Two days after transition consistent staining along the villi and crypts could be demonstrated. Fourteen days later the expression was significant lower around the villous tips and was more concentrated in Brunners glands. Additionally, distinct expression of the receptor is selectively found in stimulated goblet cells. The analysis of the mitotic activity during the transition period shows that cells that highly express the EGF-R have a rather low proliferation rate. Conclusions. Our findings suggest that EGF plays an important role in cell differentiation (rather than cell proliferation) in young animals, and it may be involved in stimulating mucus secretion.

Review of apoptotic and non-apoptotic events in non-ciliated cells of the mammalian oviduct.

Reproductive organs are known to undergo dynamic changes during the oestrus cycle and pregnancy. Cell growth and regeneration of the reproductive tissues are closely correlated with ovarian steroid hormone levels. This review focuses on apoptotic and non-apoptotic degenerative events within oviduct epithelium that occur in a species-, cycle-, and segment-specific manner. Epithelial extrusion of larger cell fragments including nuclei and whole cells is the characteristic feature of non-apoptotic cell loss of non-ciliated cells in large (pig, sheep, goat, cattle) and small animals (dog). This mechanism of epithelial cell loss is most frequently observed in the luteal phase of the oestrus cycle and after progesterone treatment, respectively. Using light- and electron-microscopic techniques, typical apoptotic epithelial cells characterized by extensive nuclear and cytoplasmic fragmentation are found very sporadically in most species. In contrast, oviduct epithelial cells of subhuman primates and cats in part show marked signs of apoptosis, which could be explained by their respective cycle-specific characteristics. Recent investigations using histochemical markers of apoptosis and our own findings in the porcine oviduct suggest that the degenerative process in the mammalian oviduct includes the death of numerous epithelial cells by apoptosis. Advancement in the knowledge of elimination of oviduct epithelial cells is necessary to understand the physiological process of epithelial renewal and pathological processes caused by imbalances between cell renewal and elimination.

Estrous cycle-dependent activity of neutrophils in the porcine endometrium: possible involvement of heat shock protein 27 and lactoferrin.

Neutrophil infiltration into the porcine endometrium is thought to be a specific feature during the follicular phase of the estrous cycle. To specify the localization and distribution of neutrophil granulocytes at different stages of the estrous cycle, porcine uterine samples were evaluated by immunohistochemical methods using anti-bovine lactoferrin (LF) antibody. Additionally, blood samples were collected from 30 pigs at different stages of the estrous cycle with a special focus on peri-estrous phase. Manual 100-cell differential counts were performed on routinely stained blood smears and evaluated statistically. Finally, the expression of granulocyte-colony-stimulating factor (G-CSF) and heat shock protein 27 (HSP 27), which are known to influence activation of the neutrophilic granulocytic lineage, was analyzed in porcine uteri using immunohistochemistry. Results show that LF is expressed regularly in the cytoplasm of neutrophil granulocytes. An increasing infiltration of subepithelial neutrophils was detected in the follicular phase. The highest number of intra- and subepithelial LF-positive cells was found on d 2 of the estrous cycle. Maximum level was followed by a strong decrease on d 3. Blood analysis revealed that the percentage of neutrophil granulocytes was significantly lower at d 2 (26.2+/-11.1%) than d 1 (42.1+/-11%) of the estrous cycle. HSP 27 staining was predominantly localized to luminal epithelium (LE) and glandular epithelium (GE) depending on stage of the estrous cycle. Strong immunostaining of HSP 27 is only found in LE during estrus. At d 2 of the estrous cycle, HSP 27 immunoreactivity in LE and superficial GE is reduced but moderate staining is found in deep GE. G-CSF immunostaining is uniformly not detected in endometrial cells of cyclic pigs. In conclusion, there is a clinically relevant relationship between neutrophil count in the blood and neutrophil infiltrate in the endometrium of the pig during the estrous cycle. This association may reflect the possibility of translocation of neutrophils from the blood to the endometrium up to d 2 of the estrous cycle. Additionally, HSP 27 could be a good candidate involved in migration and/or function of neutrophils within the porcine endometrium.

Non-neuronal acetylcholine and choline acetyltransferase in oviductal epithelial cells of cyclic and pregnant pigs

Certain female reproductive tissues are known to express the non-neuronal cholinergic system. Using different experimental approaches, we tested the hypothesis that acetylcholine (ACh) in the porcine oviduct may also be derived from non-neuronal structures. Immunohistochemistry was performed to detect acetylcholine synthesizing enzyme choline acetyltransferase (ChAT) in different segments of the oviduct of cyclic and pregnant sows. Immunohistochemical experiments revealed strong immunoexpression of ChAT in the entire oviductal epithelium at metoestrus. Thereby, a particular pronounced staining was found in the supranuclear region of almost all epithelial cells. Immunostaining of ChAT decreased markedly during dioestrus and prooestrus stages, respectively. At prooestrus, ChAT immunoreactivity was confined to ciliated cells. Furthermore, we found elevated level of staining intensity of ChAT in the pregnant oviduct at day 13. Using the same ChAT antibody for Western blot analyses, we detected immunoreactive bands of MW 69,000 and 46,000 mainly in ampulla, while MW 58,000 and 30,000 forms were present mainly in infundibulum and isthmus. Furthermore ACh was detected by HPLC and fluorimetric methods in oviductal epithelium. In conclusion, we show expression of ChAT in oviductal epithelial cells at different stages of the oestrus cycle and pregnancy, indicating that these cells can synthesize ACh in a cycle-dependent manner. These results suggest as yet unexplored roles of epithelial ACh in the oviduct.

The normal cellular prion protein (PrPc) is strongly expressed in bovine endocrine pancreas

SummaryExpression of the cellular prion protein (PrPc) has been shown to be crucial for the development of transmissible spongiform encephalopathies and for the accumulation of the disease-associated conformer (PrPsc) in the brain and other tissues. One of the emerging hypotheses is that the conversion phenomenon could take place at the site where the infectious agent meets PrPc. In this work we have studied whether PrPc, a protein found predominantly in neurons, could also exist in pancreatic endocrine cells since neuroectoderm-derived cells and pancreatic islet cells share a large number of similarities. For this purpose we have examined the expression of PrPc in a series of fetal and postnatal bovine pancreatic tissue by immunohistochemistry and RT-PCR. Using immunostained serial sections and specific antibodies against bovine PrPc, insulin, glucagon, somatostatin, chromogranin A and chromogranin B we found that PrPc is highly expressed in all endocrine cells of fetal and adult pancreatic islets with a particular strong expression in A-cells. Moreover it became evident that the PrPc gene-neighbour chromogranin B as well as chromogranin A are coexpressed together with PrPc. The selective expression of PrPc in the bovine endocrine pancreas is of particular importance regarding possible iatrogenic transmission routes and demonstrates also that bovine pancreatic islet cells could represent an interesting model to study the control of PrP-gene expression.

Cellular prion protein in mammary gland and milk fractions of domestic ruminants.

The present study shows that PrP(c) is expressed in the mammary gland and milk fractions of domestic ruminants in a species-specific manner. By applying immunohistochemistry, Western blot and ELISA, clear expression differences between bovine, ovine and caprine mammary gland, skimmed milk, acid whey and cream could be demonstrated, the highest relative PrP(c) levels being associated with the cream fraction. In the bovine gland PrP(c) was preferentially detectable at the basolateral surface of mammary gland epithelial cells, whereas in ovine and caprine samples the prion protein was more homogeneously distributed. Moreover, in ovine and caprine bovine mammary gland epithelial cells, apocrine secretory vesicles were strongly stained. Ovine and caprine milk proved to contain PrP(c) in all fractions with an additional truncated form at 12kDa in Western blot. This truncated isoform is the predominate one in caprine acid whey. These results support the hypothesis that the apocrine secretion mode of milk fat globules is a major way of PrP(c) transport into the milk.

The zinc transporter ZnT8 (slc30A8) is expressed exclusively in beta cells in porcine islets

Abstract Diabetes represents a major endemic disease throughout the world, and different therapeutic methods are used to treat the disease. Xenotransplantation of pig islet cells is a potential treatment for type 1 diabetes, but studies of protein expression in distinct islet cells are rare. ZnT8, a member of the slc30A gene family, is involved in islet endocrine hormone release and is a diabetes auto-antigen, raising the question of whether ZnT8 expression is regulated similarly in pig and human pancreas. We used nested RT-PCR to detect ZnT8 expression in pig pancreas and polyclonal antibody to examine possible co-localization with other islet hormones. Immunohistochemistry of sequential serial sections as well as double immunostaining of pancreatic tissues with antibodies against ZnT8, insulin, glucagon, and somatostatin shows that pig ZnT8 is exclusively co-expressed in insulin-producing, but not in glucagon- or somatostatin-producing cells. The absence of ZnT8 in glucagon-producing cells in pig islets indicates that zinc homeostasis is mediated by a different cellular mechanism compared with human islet cells. Our findings provide important information about the cell-type-specific expression of ZnT8 in porcine islet cells, which should be taken into account when evaluating different xenotransplantation approaches.

Colocalization of Chromogranin A and Inositol 1,4,5-Trisphosphate receptor in ciliated cells of the bovine oviduct

Previous investigations of the expression of chromogranin A (CgA) have been performed primarily in neuroendocrine tissues containing amine and peptide secretory vesicles. More recently it has been shown that CgA, as a high capacity Ca2+ storage protein, interacts with the inositol 1,4,5-trisphosphate receptor/Ca2+ channel (InsP3R) which has been found to be selectively localized in oviductal cells of the mouse. To examine a possible role of this coupling in the Ca2+-dependent ciliary movement, we investigated the topographical and cellular distribution of cells positive for CgA and inositol 1,4,5-trisphosphate receptor type 2 (InsP3R2) in the bovine oviduct at different stages of the oestrous cycle. Using immunohistochemical techniques on paraffin-embedded tissue we have successfully shown that CgA is selectively expressed in ciliated cells of the bovine oviduct. The labelled cells show intense positive staining in the apical surface area in close vicinity to the ciliary apparatus. CgA-positive ciliated cells are most frequently observed at dioestrous while a lower number appears at oestrous. Additionally, secretory and intraepithelial neuroendocrine cells consistently do not stain with the CgA-antiserum. We then investigated whether the reported expression of the InsP3R in oviductal cells of the mouse corresponds to the expression of the InsP3R in bovine oviductal cells. Using a polyclonal antibody to the type 2 InsP3R, we found that the receptor is also selectively expressed in a similar matter to CgA in the apical cytoplasm of ciliated cells. This is the first morphological demonstration of the colocalization of CgA and InsP3R in epithelial ciliated cells of the bovine oviduct. Our results suggest that CgA and InsP3R could be involved in controlling the ciliary activity of oviductal epithelial cells.

Expression and localization of growth hormone receptor in the oviduct of cyclic and pregnant pigs and mid-implantation conceptuses

Previously it was shown that growth hormone (GH) and its receptor (GH-R) are involved in growth-promoting events during early embryonic development. However, it is still unknown if GH-induced GH-R signalling may support other functions within the oviduct. The purpose of our study was to analyse GH-R expression and localization in the porcine oviduct during different stages of the oestrus cycle and pregnancy (days 2–3 post inseminationem to days 65–71). As shown by reverse transcription polymerase chain reaction (RT-PCR), GH-R is expressed in the porcine oviduct during all stages of the oestrus cycle and pregnancy, respectively. Additionally, GH-R mRNA was detected in porcine conceptuses collected at day 18 of pregnancy. Using immunohistochemistry, GH-R was exclusively localized to the epithelium of the porcine oviduct throughout all segments examined. Localization of GH-R was mainly observed in the cytoplasm of ciliated epithelial cells. Generally, the number of GH-R-positive cells was elevated in the periovulatory phase of the oestrus cycle. Except for the isthmic epithelium, staining intensity of GH-R-positive cells was highest at oestrus and markedly reduced at met- and dioestrous stages. In infundibular and ampullar segments, percentage of GH-R-positive cells was significantly higher at days 2–3 post inseminationem compared to days 65–71 of pregnancy. Furthermore, in porcine conceptuses on day 18 of pregnancy GH-R protein expression was almost exclusively localized to trophectoderm. Our data suggest that GH-R mRNA and protein expression in the porcine oviduct throughout the oestrus cycle and pregnancy may suggest other activities of GH not described previously. Specifically, autocrine or paracrine GH-induced GH-R signalling may be linked to ciliated cell homeostasis of the porcine oviduct. Additionally, our results indicate that GH-R expression in the pig trophectoderm may be responsible for trophoblastic elongation.

Co-localization of the zinc transporter ZnT8 (slc30A8) with ghrelin and motilin in the gastrointestinal tract of pigs.

Zinc is an important co-factor for insulin storage in pancreatic β-cells of different species and the uptake of this ion into insulin containing secretory vesicles is managed by the zinc transporter, ZnT8, a member of the slc30A gene family. Recent studies indicate that this protein is a major autoimmune target in human type 1A diabetes and has also been implicated by genome-wide association studies in type 2 diabetes. Since individuals suffering from type 1 diabetes often develop gastrointestinal motility disorders, we investigated the expression of ZnT8 in the porcine gastrointestinal tract. For this purpose, we studied the cell-type specific expression of ZnT8 in the gut and its co-expression with endocrine hormones that are closely linked to intestinal motility regulation. Nested RT-PCR and immunostaining of sequential serial sections, as well as double-immunostaining using antibodies directed against ZnT8, ghrelin, motilin, neurotensin, serotonin and glucagon-like peptide 1, indicated that ZnT8 is co-localized with ghrelin and motilin. Our findings provide important information about the cell-type specific expression of ZnT8 in the porcine gastrointestinal system. The selective and exclusive expression of ZnT8 in two endocrine cell-types that are engaged in motility functions may be of particular interest for further investigations into type I diabetes-associated gastrointestinal dysfunctions.