Werner M. Amselgruber

Expression and Tissue Concentration of Vascular Endothelial Growth Factor, Its Receptors, and Localization in the Bovine Corpus Luteum During Estrous Cycle and Pregnancy

Abstract The presence of vascular endothelial growth factor (VEGF) in the ovary has been reported in a number of species. The objective of the present study was to demonstrate the expression of VEGF, VEGF receptor (R)-1, and VEGFR-2 in detail by different methodological approaches in bovine corpora lutea (CL) obtained from different stages of the estrous cycle and during pregnancy. VEGF and VEGF receptor transcripts were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and ribonuclease protection assay. All components of the VEGF system were found in the bovine CL during the estrous cycle and pregnancy. Analysis of VEGF transcript by RT-PCR shows that CL tissues expressed predominantly the smallest isoforms (VEGF121 and VEGF165). The highest mRNA expression for VEGF and VEGFR-2 mRNA was detected during the early luteal phase, followed by a significant decrease of expression during the mid and late luteal phase and a further decrease of VEGF mRNA after regression. During pregnancy, high levels of expression were always present. In contrast, no significant change in VEGFR-1 mRNA expression during the estrous cycle and pregnancy was found. The VEGF protein concentration in CL tissue was significantly higher (20.9–23.4 ng/g wet weight) during the early luteal phase (Days 1–7), followed by a decrease at the late luteal phase (14.3–18.7 ng/g wet weight) and, especially, after CL regression (2.8 ng/g wet weight). However, relatively high levels were found during pregnancy (10.1 ng/g wet weight). As achieved by immunohistochemistry, VEGF protein was localized predominantly in luteal cells. High VEGF protein and transcript concentrations and increased VEGFR-2 expression during the early luteal phase coincided with luteal vascularization. These results suggest an important role of VEGF in angiogenesis of the newly formed CL. The high VEGF mRNA expression and protein levels during matured vasculature in the mid-stage CL and pregnancy also suggest also a survival function for endothelial cells.

Possible role of growth hormone, IGFs, and IGF-binding proteins in the regulation of ovarian function in large farm animals

The aim of the study and short review was to present evidence that growth hormone (GH), locally produced insulin-like growth factors (IGFs), and IGF-binding proteins (IGFBPs) may have an important role in the control of ovarian function. There is clear evidence for a distinct GH-receptor mRNA expression and protein production in follicles (oocytes and granulosa-cumulus cells) and corpus luteum (CL). In hypophysectomized ewes, GH and LH are necessary for normal CL development. IGF-1 mRNA in the follicles is expressed in theca interstitial cells (TIC) and granulosa cells (GC) with already higher levels in the TIC before follicle selection. In contrast, IGF-2 is mainly expressed in the TIC. The IGFR-1 mRNA is expressed in both the TIC and GC, with increasing levels in GC during the final development of dominant follicles. IGF-1 is a very potent stimulator of progesterone and oxytocin release in GC. IGFBP-1, -2, -3, -4, -5, and -6 have been isolated from follicular fluid or ovarian tissue. Studies indicate that IGFBP expression and production in the developing follicle is dependent on both cell type and follicle size and is regulated by IGF-1 and gonadotropins. The highest expression of IGF-1 and IGFR-1 mRNA was demonstrated during the early luteal phase. Distinct receptors for IGF-1 and IGF-2 were present in CL membrane preparations at all stages investigated. Intense immunostaining for IGF-1 was observed mainly in bovine large and small luteal cells and in a limited number of endothelial cells. In contrast, IGF-2 protein was localized in perivascular fibroblast and pericytes of the capillaries. With the use of a microdialysis system, we found that in vitro and in vivo IGF-1, IGF-2, and GH stimulated the release of progesterone in cultures of luteal cells or intact tissues. In conclusion, there is clear evidence for a central role of the IGFs, IGFBPs, and GH in follicular development and CL function.

Expression and localization of IGF family members in bovine antral follicles during final growth and in luteal tissue during different stages of estrous cycle and pregnancy

The objectives of the study were to monitor the detailed pattern for mRNA expression (RT-PCR and RPA) of IGFs, IGFR-1, IGFBPs, GHR and localization of protein (immunohistochemistry) for IGF-1 and IGFR-1 in bovine follicle classes during final maturation and different corpus luteum (CL) stages during estrous cycle and during pregnancy. A relative high expression of IGF-1 in theca interna (TI) was observed before selection (E<0.5ng/mL). In GC, mRNA expression increased after selection. In contrast, IGF-2 was mainly expressed in the TI. The IGFR-1 mRNA was present in the TI and GC with increasing levels during final development. The expression results were confirmed by localization of IGF-1 and IGFR-1 proteins in GC and TI. There is clear evidence for the local expression of IGFBPs in TI and GC compartment with clear regulatory differences. In CL, the highest mRNA expression of IGF-1, IGF-2 and IGFR-1 was observed during early luteal phase, followed by a decrease, and then by a tendency of an increase during the mid and late luteal phases of the cyclic CL. This level remained low during pregnancy. Intense immunostaining for IGFR-1 in CL was observed mainly in large luteal cells. Evidence for a mRNA for all six IGFBPs were obtained with distinct differences for BP-3, -4 and -5. In conclusion, this comprehensive study gives clear evidence for an important role of the IGFs and IGFBPs in bovine follicular development and CL function. The relative amounts of IGFBPs may ultimately determine ovarian IGF action.

Physiological, haematological and histopathological responses in common carp (Cyprinus carpio L.) fingerlings fed with differently detoxified Jatropha curcas kernel meal.

Protein rich Jatropha curcas kernel meal is toxic. It was detoxified using heat treatment and solvent extraction. Two duration of detoxification process were investigated: shorter (30 min) and longer (60 min) and the detoxified meals so obtained were designated as J(a) and J(b) respectively. Common carp fingerlings (252 fish; 3.2+/-0.07 g) were fed with the following diets: Control containing fishmeal (FM); S(50,) J(a50) and J(b50): 50% of FM protein replaced by soybean meal (SBM), detoxified Jatropha kernel meal (DJ(a)KM and DJ(b)KM); S(75), J(a75) and J(b75): 75% of FM protein replaced by SBM, DJ(a)KM and DJ(b)KM. White blood cells count, mean cell volume and mean cell haemoglobin concentration, calcium and sodium ions and total bilirubin in blood did not differ significantly among the groups. Higher (P>0.05) RBC count was observed in plant protein fed groups compared to control group. Highest alkaline phosphatase and alanine transaminase activities in blood were observed in J(a75), which were not different (P>0.05) from those in J(a50) group, but were higher than in the other groups. No adverse histopathological changes in liver and muscle of any group were observed, but intestinal mucosa of J(a75) groups showed severe pathological lesions. The results demonstrate that Jb was completely detoxified. Since the performance of J(b50) group was similar to control group and better than the other groups, optimum inclusion level of J(b) is 50% replacement of FM protein.

Angiogenesis in the bovine corpus luteum: an immunocytochemical and ultrastructural study.

Immediately after ovulation a neovascular response occurs at the level of the theca interna. Pericytes and endothelial cells of post‐capillary venules locally remodel the surrounding stroma, elongate and migrate into the avascular granulosa folds of the ruptured follicle. In order to examine the composition of the extracellular matrix as well as the growth characteristics of these newly formed vessels, we used immunohistochemical and electron microscopic methods. Initial sprouts were characterized by the appearance of a fibrillary network of fibronectin along the main axis of the sprout. Type IV collagen stained weakly and extracellular deposits of laminin were amorphous and patchy around immature capillary sprouts. In advanced maturational stages of the sprouts the capillaries were surrounded by increased deposits of fibronectin, whereas laminin and type IV collagen displayed a distinct and well‐developed line around endothelial cells and pericytes. These observations indicate that the microvascular extracellular matrix undergoes a series of quantitative rather than qualitative changes during capillary development before achieving final maturation. Ultrastructural analyses showed that early capillary sprouts in the bovine corpus luteum were usually preceded by pericytes migrating at the tips of the sprouts. Endothelial cells comigrated in cohesive cylindrical projections, forming immediately a slit‐like lumen which satisfies the criteria of the intercellular canalization type. Pericytes at the tips of endothelial sprouts exhibited a slender, bipolar morphology and were regularly surrounded by fragmented basal lamina, which is well‐developed around pericytes in a more proximal position of the sprout. The regular association of pericytes with the leading front of the capillary sprouts suggests that these cell types may serve as guiding structures aiding outgrowth of endothelial cells in the bovine corpus luteum.

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).

Cell-specific Localization of the CholecystokininA Receptor in the Porcine Pancreas

Cholecystokinin (CCK) produced in the mucosa of the upper small intestine exerts several biological functions. Its secretion in physiological amounts is modulated by the interaction of extracellular regulators and by binding to intracellular receptors of the target cells. The relative affinity of CCK to its receptor has been characterized in various biological and pharmacological studies and it is now well established that CCK has a higher affinity to the CCKA than to the CCKB receptor. Furthermore CCK influences the secretion of pancreatic enzymes in several species but very little is known about the relationship between CCK and the islet hormone‐producing cells in the pig pancreas. The localization of this receptor at the cellular level showed conflicting results in animal studies and has not been described in pigs. The aim of the present study was to characterize the precise cellular location of the CCKA receptor in the porcine pancreas. Polyclonal antiserum was raised against the N‐terminal epitope of the CCKA receptor molecule and used for localization studies. Using immunohistochemistry on methanol/acetic acid‐fixed, paraffin‐embedded pancreas, the CCKA receptor could successfully be localized in islet cells. Parallel staining of serial sections with antibodies directed against insulin and glucagon revealed colocalization with glucagon in alpha cells. No immunoreaction was found in the exocrine pancreas. Our results support the concept that in the porcine species the stimulation of the exocrine pancreas is mediated by the CCKB rather than the CCKA receptor, as it is known for the rat species.

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.

Localization of the zona glycoproteins ZPB (ZP3α) and ZPC (ZP3β) in the bovine ovary during pre- and postnatal development

Summary The purpose of this study was to investigate the expression of two zona pellucida gene families ZPB (ZP3α) and ZPC (ZP3β. Sections of ovaries from bovine fetuses, calves and cows were labelled with polyclonal antibodies. Immunopositive labelling was found in both the follicle cells and the oocyte. Labelling was dependent on the stage of development. The specific sequence of immuno-positive reactions suggests that in the bovine fetus both the ovary and the follicle cells contribute to the production of the zona pellucida during pre- and postnatal development.