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 ß-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.

Comparative morphological evaluation of domestic animal cornea.

OBJECTIVE: This study described corneal morphology in different domestic animals using histological and immunohistochemical methods. Specifically, we evaluated the species-specific distribution pattern of cytokeratins (CKs) and aquaporins (AQPs) to assess their contribution to the strength and transparency of the cornea, respectively. PROCEDURES: Corneal sections (2 µm thick) were obtained from 28 pigs, 11 cows, two goats, six horses, four dogs, and five cats and stained with hematoxylin-eosin and periodic acid-Schiff (PAS) reaction. Immunohistochemistry was also performed using CK1 to 3 and AQP1 and 5 antibodies. RESULTS: Domestic animal corneas were composed of corneal epithelium, stroma, Descemet's membrane, and corneal endothelium. Bowman's layer was not detected using the PAS reaction. The three-layered epithelium was thinnest in carnivores and thickest in equines and bovines. CKs were demonstrated immunohistochemically in all species examined, especially in the most superficial layer of the corneal epithelium. CKs were more widely distributed in canine and feline corneal epithelial cell layers compared to other species. The corneal monolayer endothelium was immunostained with AQP1 in all species examined, and it was also present in stromal keratocytes in a species-specific manner. In contrast, AQP5 was exclusively localized to the corneal epithelium. Epithelial staining patterns varied markedly between species, and the widest distribution of AQP5 was demonstrated in feline epithelial cell layers. CONCLUSIONS: Differences in the distribution of CKs and AQPs in various species suggest species differences in the maintenance of structural integrity and fluid balance.

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

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.

Mycoplasma suis infection results endothelial cell damage and activation: new insight into the cell tropism and pathogenicity of hemotrophic mycoplasma.

Hemotrophic mycoplasmas (HM) are highly specialized red blood cell parasites that cause infectious anemia in a variety of mammals, including humans. To date, no in vitro cultivation systems for HM have been available, resulting in relatively little information about the pathogenesis of HM infection. In pigs, Mycoplasma suis-induced infectious anemia is associated with hemorrhagic diathesis, and coagulation dysfunction. However, intravasal coagulation and subsequent consumption coagulopathy can only partly explain the sequence of events leading to hemorrhagic diathesis manifesting as cyanosis, petechial bleeding, and ecchymosis, and to disseminated coagulation. The involvement of endothelial activation and damage in M. suis-associated pathogenesis was investigated using light and electron microscopy, immunohistochemistry, and cell sorting. M. suis interacted directly with endothelial cells in vitro and in vivo. Endothelial activation, widespread endothelial damage, and adherence of red blood cells to the endothelium were evident in M. suis-infected pigs. These alterations of the endothelium were accompanied by hemorrhage, intravascular coagulation, vascular occlusion, and massive morphological changes within the parenchyma. M. suis biofilm-like microcolonies formed on the surface of endothelial cells, and may represent a putative persistence mechanism of M. suis. In vitro analysis demonstrated that M. suis interacted with the endothelial cytoskeletal protein actin, and induced actin condensation and activation of endothelial cells, as determined by the up-regulation of ICAM, PECAM, E-selectin, and P-selectin. These findings demonstrate an additional cell tropism of HM for endothelial cells and suggest that M. suis interferes with the protective function of the endothelium, resulting in hemorrhagic diathesis.

Immunohistochemical localization of low density lipoprotein (LDL) receptor-related protein-1 (LRP1) in the endometrium of cyclic and pregnant pigs.

The low density lipoprotein (LDL) receptor-related protein-1 (LRP1), also known as α2macroglobulin receptor or CD 91, is a multifunctional cell surface receptor that plays an important role in the endocytosis of several ligands and regulation of signalling pathways. In human endometrium, LRP1 was shown to be involved in the endocytic clearance of specific matrix metalloproteinases (MMPs) from the stroma during different phases of the cycle. However, in the pig, it is currently not known whether LRP1 is actually expressed in the endometrium and functions in a similar manner, respectively. For that reason, we examined the localization of LRP1 in the porcine endometrium at different stages of the estrous cycle and pregnancy by immunohistochemistry. Our results showed that LRP1 immunostaining is found in all endometrial specimens examined of both cyclic and pregnant animals. Especially in metestrus and estrus, immunoreactivity (IR) of LRP1 was strongly detected in stromal cells underlying the luminal epithelium (LE). Endometrial glands were mostly surrounded by LRP1-positive cells, which showed some concomitant staining with an antibody against porcine macrophages. In pregnant animals, the number of LRP1-positively stained cells was comparable high within the subepithelial stroma of early pregnant pigs. During apposition and implantation, IR of LRP1 remained high in stromal cells of the endometrium and declined markedly during the ongoing pregnancy stages examined. Our data show, that endometrial LRP1 protein expression was specifically high in such cyclic and pregnancy stages which have a high tissue remodelling activity in dependence of differing steroid hormone concentrations.

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.

Glucose intolerance and reduced proliferation of pancreatic beta-cells in transgenic pigs with impaired glucose-dependent insulinotropic polypeptide function.

OBJECTIVE: The insulinotropic action of the incretin glucose-dependent insulinotropic polypeptide (GIP) is impaired in type 2 diabetes, while the effect of glucagon-like peptide-1 (GLP-1) is preserved. To evaluate the role of impaired GIP function in glucose homeostasis and development of the endocrine pancreas in a large animal model, we generated transgenic pigs expressing a dominant-negative GIP receptor (GIPR(dn)) in pancreatic islets. RESEARCH DESIGN AND METHODS: GIPR(dn) transgenic pigs were generated using lentiviral transgenesis. Metabolic tests and quantitative stereological analyses of the different endocrine islet cell populations were performed, and beta-cell proliferation and apoptosis were quantified to characterize this novel animal model. RESULTS: Eleven-week-old GIPR(dn) transgenic pigs exhibited significantly reduced oral glucose tolerance due to delayed insulin secretion, whereas intravenous glucose tolerance and pancreatic beta-cell mass were not different from controls. The insulinotropic effect of GIP was significantly reduced, whereas insulin secretion in response to the GLP-1 receptor agonist exendin-4 was enhanced in GIPR(dn) transgenic versus control pigs. With increasing age, glucose control deteriorated in GIPR(dn) transgenic pigs, as shown by reduced oral and intravenous glucose tolerance due to impaired insulin secretion. Importantly, beta-cell proliferation was reduced by 60% in 11-week-old GIPR(dn) transgenic pigs, leading to a reduction of beta-cell mass by 35% and 58% in 5-month-old and 1- to 1.4-year-old transgenic pigs compared with age-matched controls, respectively. CONCLUSIONS: The first large animal model with impaired incretin function demonstrates an essential role of GIP for insulin secretion, proliferation of beta-cells, and physiological expansion of beta-cell mass.

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.

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).

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

The cellular prion protein (PrP(c)) 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 PrP(c) is also found in a broad spectrum of non-neuronal tissue. Here we investigated the cell-type-related PrP(c) 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 PrP(c) 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 PrP(c) expression and other parameters such as age of the animals under study or stage of lactation.

Changes in fibroblast growth factor 2 and its receptors in bovine follicles before and after GnRH application and after ovulation.

The aim of this study was to evaluate the expression pattern of fibroblast growth factor 2 (FGF2), its receptor variants (FGFR1IIIc, FGFR2IIIc) and nucleolin in time-defined follicle classes before and after GnRH application and after ovulation in the cow. Ovaries containing preovulatory follicles or new corpora lutea (CL) were collected at approximately 0, 4, 10, 20 and 25 h (follicles) and 60 h (new CL) relative to injection of GnRH to induce an LH surge (n = 5 animals per group). The expressions of FGF2 and FGFR1IIIc mRNA were significantly up-regulated only in the follicle group 4 h after GnRH (during the LH surge) with a significant down-regulation immediately afterwards. Western blot analyses showed two protein bands with at 22 and 18 kDa with apparent up-regulation beginning with the LH surge (4 h) and maximum levels 20 h after GnRH. FGF2 protein in follicles collected at 0 h (before LH surge) was localised in theca tissue (endothelial and pericytes of blood vessels) but not in granulosa cells (GCs). The FGF2 staining (by immunohistochemistry) pattern changed dramatically after the LH surge for a short period (about 2 days) and FGF2 protein was localised dominantly in the nucleus of many GCs, while most capillary endothelial cells were FGF2 immunonegative. In conclusion, the novel observation of FGF2 up-regulation and the distinct change in FGF2 localisation from theca (cytoplasm of endothelial cells) to the nucleus of GCs after the LH surge may be important for survival of GCs or for the transition of the GCs to luteal cells.

Estrous cycle specific immunolocalization of different domains of the epidermal growth factor receptor in the porcine oviduct.

Although porcine uterus is known to contain active and inactive forms of epidermal growth factor receptor (EGF-R), the latter consist of the extracellular domain only; it is currently unknown whether different EGF-R isoforms are expressed in the porcine oviduct during estrous cycle. Therefore, we used two different monoclonal antibodies, one against the extracellular and the other against the cytoplasmic domain of the EGF-R, to investigate cycle-dependent and cell-type-specific expression of full-size and truncated receptor forms. At metestrus, the majority of epithelial cells of the oviduct were strongly immunopositive for both antibodies, indicating the presence of the full-size receptor. In diestrous and proestrous stages, we found a low level of cytoplasmic but no extracellular EGF-R staining in epithelial cells. While the staining intensity of cytoplasmic domain of the EGF-R was only faint or absent in muscular tissue and blood vessels throughout the estrous cycle, extracellular domain of the EGF-R exhibited a strong immunostaining of smooth muscle cells and vascular smooth muscle cells, especially in diestrous and proestrous stages. There was no significant difference between the oviductal ampulla and isthmus in either the intensity or the pattern of both cytoplasmic and extracellular EGF-R immunostaining. We conclude that the restricted presence of the functional full-size receptor to the epithelial layer indicates a specific role during early embryonic development, whereas truncated EGF-R forms may potentially regulate contractions and blood flow in the oviduct.

Determination of target cells for cholecystokinin in the porcine pancreas.

The cholecystokinin (CCK) family of peptides and receptors is present throughout the brain and gastrointestinal tract including pancreatic tissue. The expression of these molecules in the gut and pancreas is species-specifically regulated and the role of CCK in porcine pancreatic islet hormone secretion is still a matter of discussion. Therefore, in this study we have determined the cell-type specific localization of and its high affinity CCKA-receptor in islet cells using immunohistochemical techniques. Serial sectioning followed by double-immunostaining of methanol/acetic acid-fixed, paraffin-embedded pancreatic tissues were performed with antibodies against CCK, CCKA-receptor, glucagon and somatostatin. To determine whether CCK specific mRNA is locally expressed, total RNA was isolated, transcribed to cDNA and analysed with specific primer for CCK gene expression. Our results clearly show that CCK and the CCKA-receptor coexist in glucagon--but not in somatostatin-producing cells. Moreover our RT-PCR experiments demonstrate that there is no local gene expression of CCK in the porcine pancreas. Our results provide evidence that, in the porcine species, blood-borne CCK binds specifically to the CCKA-receptor and may thereby modulate the glucose homeostasis via a direct action on A-cells.

Cell-type specific expression of IGF-1R in porcine islet cells.

Insulin-like growth factor-1 (IGF-1) functions as a growth factor regarding physiological regulations of cellular metabolism, regeneration and growth. In pancreas islets their potential function is unclear and only little information is available on occurrence and distribution of the corresponding insulin-like growth factor-1 receptor (IGF-1R) in islet cells. Therefore, we investigated the localization of IGF-1R by immunohistochemical techniques and its possible co-localization with other islet hormones. Further, we applied molecular biology techniques to determine the present of local gene expression of IGF-1R and IGF-1. Immunostaining on serial sections with anti-insulin, anti-glucagon and anti-somatostatin antibodies shows, IGF-1R was selectively expressed in insulin-producing B-cells and additionally more pronounced in somatostatin-containing D-cells, which are located in the periphery of porcine pancreatic islets. Furthermore, the RT-PCR experiment demonstrates clearly that IGF-1 and IGF-1R was expressed together in the porcine pancreas. The high expression of IGF-1R in porcine D-cells indicates that mammalian IGF-1R genes are regulated in a different manner since it was shown that in all other species IGF-1R was expressed in B- and A-cells but not in D-cells.

Immunophenotype of porcine oviduct epithelial cells during the oestrous cycle: a double-labelling immunohistochemical study.

The luminal epithelium of the porcine oviduct is composed of ciliated cells and secretory cells, but it is assumed for several species that under the control of steroid hormones secretory cells are able to be transformed into ciliated cells. In order to better understand such physiological changes during the different stages of the oestrous cycle, we evaluated epithelial cell proliferation together with oestrogen receptor alpha (ERalpha) expression of porcine ampullary oviducts. To identify the immunophenotype of proliferating cells, double immunohistochemistry was performed using anti-chromogranin A antibody (anti-CgA) as the second primary antibody. Anti-CgA, recently shown to be an immunocytochemical marker of ciliated cells of the cow, also labelled specifically the luminal surface of ciliated cells of the pig. Double labelling of sections with the monoclonal antibody MIB-1 against the proliferation-associated nuclear epitope Ki-67 and anti-CgA clearly demonstrates that MIB-1 was selectively localised in the nuclei of secretory cells. Proliferative activity was not observed in CgA-positive ciliated cells in all examined stages of the oestrous cycle. The percentage of Ki-67-positive epithelial cells was higher at pro-oestrus, compared with the other stages of the oestrous cycle. Furthermore, ERalpha immunoreactivity was exclusively detected in the nuclei of the epithelial cells, which were negative for CgA. We conclude, therefore, that oestrogen may induce the initial proliferation of secretory cells and promote the differentiation into ciliated cells.

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 Brunner's 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.

Real-time changes of the local vasoactive peptide systems (angiotensin, endothelin) in the bovine corpus luteum after induced luteal regression.

There is evidence that angiotensin II (Ang II) and endothelin-1 (ET-1) may interact in an additive or synergistic way during luteal regression. The aim of the study was to investigate real time changes in luteal tissue of angiotensin and endothelin system members in mRNA expression, tissue concentrations, tissue localization, and ACE (angiotensin converting enzyme) antagonist application after prostaglandin F(2alpha) (PG) induced (days 8-12) luteal regression in cow. Corpora lutea (CL) were collected by transvaginal ovaryectomy before and 2, 4, 12, 24, 48, and 64 hr (n = 5/time point) after PG injection. ACE mRNA expression (RT-PCR) increased continuously and peaked at 12, 24 hr; ECE-1 (endothelin converting enzyme) peaked at 12 hr, and both peptides in tissue (Ang II and ET-1) increased significantly and peaked at 24 hr. The expression of receptors for Ang II (AT1R and AT2R) did not change in contrast to ET receptors (ETR-A and ETR-B), which were up-regulated. Localization in tissue revealed very weak staining for Ang II and ET-1 before PG application followed by a clear increase of staining predominantly in large luteal cells, but also in endothelial cells. In two experiments, the attempt was made to block ACE by the antagonist captopril with two different doses. In both experiments with captopril, progesterone levels were not significantly different from controls. Ang II alone seems to be not essential for functional luteolysis in bovine system. In conclusion, the results suggest that both Ang II and ET-1 are in parallel up-regulated during luteal regression and may act as vasoconstrictors during functional luteolysis, but also as apoptosis inducer during functional/structural luteolysis.

Characterization of POMT2, a novel member of the PMT protein O-mannosyltransferase family specifically localized to the acrosome of mammalian spermatids.

Over the past few years it has emerged that O-mannosyl glycans are not restricted to yeasts and fungi but are also present in higher eukaryotes, including humans. They play a substantial role in the onset of muscular dystrophy and neuronal migration disorders, like muscle-eye-brain disease. Protein O-mannosyltransferase genes (PMTs) are evolutionarily conserved from yeast to human; however, little is known about these enzymes in higher eukaryotes. In this study, we cloned the first PMT2 subfamily members from human (hPOMT2), mouse (mPomt2), and Drosophila (DmPOMT2). A detailed characterization of the mammalian POMT2, with emphasis on mouse Pomt2, shows that mammalian POMT2 is predominantly expressed in testis tissue. Due to differential transcription initiation of the mPomt2 gene, two distinct mRNA species that vary in length are formed. The shorter transcript is present in all somatic tissues examined. Expression of the corresponding hPOMT2 cDNA in mammalian cells identified POMT2 as an integral membrane protein of the endoplasmic reticulum with an apparent molecular weight of 83 kDa. The longer mPomt2 transcript is restricted to testis and encodes a testis-specific mPOMT2 protein isoform. Using in situ hybridization and immunolocalization, we demonstrate that in testis tissue mPOMT2 localizes to maturing spermatids and is abundant within the acrosome, a sperm-specific organelle essential for fertilization. Our data suggest a novel and specific role for the putative protein O-mannosyltransferase POMT2 in the maturation and/or function of sperm in mammals.