Martin Steffl

Expression and localisation of extracellular matrix degrading proteases and their inhibitors during the oestrous cycle and after induced luteolysis in the bovine corpus luteum.

The corpus luteum (CL) offers the opportunity to study high proliferative processes during its development and degradation processes during its regression. We examined the mRNA expression of matrix metalloproteases (MMP)-1, MMP-2, MMP-9, MMP-14, MMP-19, tissue inhibitor of MMP (TIMP)-1, TIMP-2, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), uPA-receptor (uPAR), PA-inhibitors (PAI)-1, PAI-2 in follicles 20 h after GnRH application, CLs during days 1-2, 3-4, 5-7 and 8-12 of the oestrous cycle as well as after induced luteolysis. Cows in the mid-luteal phase were injected with Cloprostenol and the CLs were collected at 0.5, 2, 4, 12, 24, 48 and 64 h after PGF2alpha injection. Real-time RT-PCR determined mRNA expressions. Expression from 20 h after GnRH to day 12: MMP-1, MMP-2, MMP-14 and tPA showed a clear expression, but no regulation. TIMP-1 and uPAR mRNA increased when compared with the follicular phase. TIMP-2, MMP-9, MMP-19 and uPA increased from the follicular phase to days 8-12. PAI-1 and PAI-2 expression increased from days 1-7 and decreased to days 8-12. Induced luteolysis: MMP-1, MMP-2, MMP-9, MMP-14, MMP-19 and TIMP-1 all increased at different time points and intensities, whereas TIMP-2 was constantly decreased from 24 to 64 h. The plasminogen activator system and their inhibitors were up-regulated from 2 to 64 h, tPA was already increased after 0.5 h. Immunohistochemistry for MMP-1, MMP-2, MMP-14: an increased staining for MMP-1 and MMP-14 was seen in large luteal cells beginning 24 h after PGF2alpha application. MMP-2 showed a strong increase in staining in endothelial cells at 48 h.

Serum IgA-Promoting Effects Induced by Feed Loads Containing Isolated Deoxynivalenol (DON) in Growing Piglets

Deoxynivalenol (DON), a Fusarium toxin belonging to the trichothecene group, has been reported to produce a variety of adverse health effects in farm animals, such as inhibition of protein synthesis, reduction of feed intake, and alteration of the immune system. In pigs, the effects of increasing levels of chemically pure DON in a semisynthetic diet on performance, health, and serum immunglobulin A (IgA) levels were examined. A diet, without grain components and trichothecene free (8 main trichothecenes), with doses of 0, 300, 600, and 1200 μg pure DON/kg was fed to 34 female pigs for a period of 8 wk after weaning under standardized conditions. Body weight gain and biochemical and hematological values in the blood and serum, including concentrations of IgA, blood glucose, cortisol, and insulinlike growth factor 1 (IGF-1), were determined. Increasing levels of DON in the feed induced a significant depression of glucose levels. Cortisol and IGF-1 levels were not significantly affected but differed between groups at the end of the experiment. A significant increase of IgA concentration in the serum even at a dosage level of 600 μg DON/kg feed was observed. This is the first report demonstrating in vivo that limited dosages of DON are able to stimulate IgA levels in the serum of growing piglets.

Effect of the luteinising hormone surge on regulation of vascular endothelial growth factor and extracellular matrix-degrading proteinases and their inhibitors in bovine follicles

The aim of the present study was to evaluate the pattern of regulation of vascular endothelial growth factor (VEGF)-A (isoforms 121, 165, 189), VEGF receptor tyrosine kinases (VEGF-R1 and VEGF-R2), matrix metalloproteinase (MMP)-1, MMP-2, MMP-14, MMP-19, tissue-specific inhibitor of metalloproteinases (TIMP)-1, TIMP-2, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) in time-defined follicle classes before (0 h) and after the application of gonadotrophin-releasing hormone (GnRH). Bovine ovaries containing periovulatory follicles or new corpora lutea (CL; Days 1-2) were collected 0, 4, 10, 20 and 25 h (follicles) or 60 h (CL) after the injection of GnRH. Transcripts of VEGF isoforms (VEGF(121), VEGF(165), VEGF(189)) were upregulated 4 h after GnRH injection (during the luteinising hormone (LH) surge) and decreased thereafter to lowest levels around ovulation. All VEGF isoforms and their receptors were upregulated again after ovulation. The VEGF peptide concentration in follicular fluid decreased 20 h after GnRH injection, followed by an increase in follicles 25 h after GnRH. Expression of MMP-1 mRNA increased rapidly 4 h after GnRH injection and remained high during the entire experimental period. In contrast, MMP-19 mRNA increased significantly only after ovulation. Expression of TIMP-1 mRNA increased 4 h after GnRH and again after ovulation. Expression of tPA mRNA increased 4 h after GnRH and remained high during the entire experimental period, whereas expression of uPA transcripts increased significantly only after ovulation. Both uPAR and PAI-1 mRNA levels increased in follicles 4 h after GnRH and again after ovulation. The amount of MMP-1 protein (immunolocalisation) increased in follicles 10 h after GnRH: additional staining was observed in the granulosa cell layer. In conclusion, the temporal and spatial pattern of regulation of VEGF and extracellular matrix-degrading proteinases during periovulation suggests they are important mediators of the LH-dependent rupture of bovine follicles and for early CL formation (angiogenesis).

Expression and possible role of fibroblast growth factor family members in porcine antral follicles during final maturation.

The aim of this study was to investigate the possible participation of fibroblast growth factor (FGF) family members (FGF1, FGF2 and FGF7 and their receptors) in porcine follicles (polyovulatory species) under special consideration for FGF2 during final growth. A classification of follicles was done by size and follicular fluid content of oestradiol-17beta, progesterone and prostaglandin F2alpha. The mRNA expression of examined factors was analysed by real-time PCR. The hormone concentration was estimated by enzyme immunoassay, protein characterisation by western blotting and localisation by immunohistochemistry. Follicle tissue separated in theca interna and granulosa cells was extracted and tested for mRNA of FGF1, FGF2, FGF7 and receptors (FGFR1IIIc, FGFRIIIb and FGFR2IIIc). Additionally, the mRNA expression of FSHR, LHR and aromatase cytochrome P450 for further characterisation of follicles was analysed. Significantly, higher FGF2 protein levels were measured in stroma when compared with total follicle or corpus luteum tissue. This result was confirmed by western blot with two strong bands. Immunological localisation of FGF2 only in stroma (fibroblasts) confirms the protein measurements. The results show a clear difference for FGF2 protein expression during final growth of follicles if monovulatory (bovine) and polyovulatory (porcine) species are compared. FGF2 protein in porcine ovary may be (due to localisation and concentration in stroma) important for support of angiogenesis of more follicles (polyovulatory species) and not of a single follicle like in cows.

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.

Expression of Cytokeratin 18 During Pre- and Post-Natal Porcine Lung Development

The expression pattern of the intermediate filament protein cytokeratin 18 (CK 18) is described during pre‐ and post‐natal development of the porcine lung using a monoclonal antibody aginst human CK 18. Lungs from 16 foetuses in pseudoglandular, canalicular, saccular and alveolar stages of lung development and lungs from 12 pigs ranging in age from birth to 49 days after birth were studied by immunohistochemistry.

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.

Cellular prion protein in the bovine mammary gland is selectively expressed in active lactocytes

The cellular prion protein (PrPc) is a highly conserved glycoprotein with a still enigmatic physiological function. It is mainly expressed in the central nervous system but accumulating data suggest that PrPc is also found in a broad spectrum of non-neuronal tissue. Here we investigated the cell-type-related PrPc expression in the bovine mammary gland by using immunohistochemistry (IHC), ELISA, Western blot, and real-time RT-PCR. Specific immunostaining of serial sections revealed that PrPc is selectively localized in mammary gland epithelial cells. Particularly strong expression was found at the basolateral surface of those cells showing active secretion. Results obtained by RT-PCR and ELISA complemented IHC findings. No correlation was found between the level of PrPc expression and other parameters such as age of the animals under study or stage of lactation.