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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., (© 2015 American College of Veterinary Ophthalmologists.)

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.

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.

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.

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.

A 3.5-year-old male Labrador retriever dog showed a short history of illness characterized by vomiting, apathy, and fever. Ultrasonographically, large nodular liver masses of high echogenicity were noted in both left and right liver lobes. Cytological and bacteriological examinations revealed a neutrophilic hepatitis without detectable agents. During treatment with doxycycline a four-fold decrease of serum titers to Leptospira (L.) icterohaemorrhagiae and L. sejroe was detected in paired serum samples by use of the complement-fixation test. The dog remained without clinical signs and no significant biochemical changes were recorded. However, ultrasonsographic examinations showed a progression of the hepatic lesions, presenting now as nodular parts with high echogenicity and cavernous parts with lower echogenicity. Diagnostic laparotomy was performed and the dog was euthanized due to severity of hepatic lesions. Histopathologically, a severe chronic granulomatous hepatitis with numerous parasitic structures was diagnosed. Morphology of the parasitic structures was comparable to the metacestode stage of Echinococcus multilocularis.

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., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Squamous metaplasia of the oviduct epithelium is a rare disorder of reproductive organs. We noted squamous metaplasia of the oviduct epithelium in a sow routinely slaughtered at day 2 of the oestrous cycle. Expression of transforming growth factor beta3 (TGF beta3) in the metaplastic epithelia was evaluated by immunohistochemistry, because TGF beta3 appears to play a key role as regulator of a variety of tissue remodelling events. Our results show that TGF beta3 immunostaining was specifically localized to foci of squamous metaplasia of the epithelial linings. Non-metaplastic epithelial cells of the oviduct were not immunostained with anti-TGF beta3 antibody. At the subcellular level, TGF beta3-labelled cells occasionally showed signs of apoptotic cell death. It is concluded that signals produced by TGF beta3 in metaplastic lesions of the oviduct are potentially involved in pathophysiological processes.

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.

Transforming growth factor-beta (TGF-beta) proteins are growth factors that have been shown to be involved in regulation of ovarian follicular development. Ovarian expression, activity and functional significance of TGF-beta1 and TGF-beta2 isoforms were extensively studied in most species. However, little is known about the biological role of TGF-beta3 previously shown to be expressed independently of the other two isoforms. Therefore, expression of TGF-beta3 mRNA and protein was evaluated by RT-PCR and immunohistochemistry, respectively, in porcine ovaries collected during different phases of the oestrus cycle. Results of RT-PCR analysis showed that TGF-beta3 mRNA is expressed throughout the oestrus cycle. The level of TGF-beta3 mRNA expression was found to be higher at metoestrus and dioestrus. Weak TGF-beta3 immunoreactivity was present in follicular epithelial cells and oocytes of preantral follicles in all stages examined. TGF-beta3 protein expression was exclusively present in theca interna cell layer of antral follicles, and was particularly prominent in large antral follicles. Immediately after ovulation, almost all theca cells outside of the granulosa cell layer were intensively stained with anti-TGF-beta3. Immunostaining of TGF-beta3 in theca lutein cells rapidly decreased during corpus luteum development. It is suggested that TGF-beta3 may play an important role in modulating theca cell function of pre- and postovulatory follicles of the pig.

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.

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.

Clusterin is a well-known glycoprotein expressed by many cell types involved in multiple physiological functions. In rat pancreatic tissue it is expressed along with islet cell development and found to be involved in regeneration of pancreatic endocrine cells after various types of tissue injuries. These results led us to propose that clusterin might play a crucial role in organization and assembling processes of islet cells during pre- and postnatal development. Therefore, the aim of this study was to find out whether and in which cell type clusterin is expressed during islet cell organization in the porcine species which could play a future role in the field of xenotransplantation. For this purpose we examined the expression pattern of clusterin at different developing stages in the porcine pancreas by double-immunostaining with antibodies against chromogranin A and clusterin, and clarified whether distinct islet hormones were coexpressed with clusterin. Further, we checked by RT-PCR whether clusterin was locally expressed or possibly locally bound to the corresponding receptor. In newborn and up to 3-month-old animals clusterin was found to be expressed in a special cell type which is closely associated and intermingled with other endocrine cells. In fully developed adult islets clusterin-cells then reorganize and were found to be mainly localized in the mantle area of Langerhans islets. Double-immunostaining with antibodies against clusterin and different islet hormones such as insulin, glucagon, and somatostatin clearly demonstrate that clusterin expression was found in an own special cell type and it is also present in a subset of glucagon producing A-cells. Taken together, our results show that clusterin expression in porcine species is found in an own, as yet unidentified cell type during postnatal developmental stages, and probably labels immature precursor cells in adult animals, which finally have the potential to differentiate into glucagon-expressing cells.

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 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.

The cellular form of the prion protein (PrP(c)) is thought to be a substrate for an abnormal isoform of the prion protein (PrP(sc)). One emerging hypothesis is that the proposed conversion phenomenon takes place at the site at which the infectious agent meets PrP(c). PrP(c) is abundant in the central nervous system, but little is known about the cell-type-specific distribution of PrP(c) in non-neuronal tissues of cattle. We have studied whether PrP(c), a protein found predominantly in neurons, also exists in bovine podocytes, since neurons and podocytes share a large number of similarities. We have therefore examined the expression of PrP(c) by immunohistochemistry, reverse transcription/polymerase chain reaction and enzyme-linked immunosorbent analysis. Immunostained serial sections and specific antibodies against PrP(c) have revealed that PrP(c) is selectively localized in podocytes and is particularly strongly expressed in extraglomerular mesangial cells but not in endothelial or intraglomerular mesangial cells. The selective expression of PrP(c) in podocytes is of special importance, as it suggests that these cells represent possible targets for peripheral infection with prions and demonstrates that PrP(c) can be added to the list of neuronal factors expressed in mammalian podocytes.

Expression of the cellular prion protein (PrP(c)) has been shown to be crucial for the development of transmissible spongiform encephalopathies and for the accumulation of the disease-associated conformer (PrP(sc)) 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 PrP(c). In this work we have studied whether PrP(c), 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 PrP(c) in a series of fetal and postnatal bovine pancreatic tissue by immunohistochemistry and RT-PCR. Using immunostained serial sections and specific antibodies against bovine PrP(c), insulin, glucagon, somatostatin, chromogranin A and chromogranin B we found that PrP(c) 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 PrP(c) gene-neighbour chromogranin B as well as chromogranin A are coexpressed together with PrP(c). The selective expression of PrP(c) 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.

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.

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.

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.

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., (Copyright 2004 Springer-Verlag)

The inositol 1,4,5-trisphosphate receptor type 2 (IP(3)R-2) is an intracellular Ca(2+) release channel responsible for mobilizing of Ca(2+) from intracellular storage sites and plays a key role in biological processes such as fertilization, cell differentiation, and growth. To study the cell-type-specific IP(3)R-2 expression in porcine ovaries during different phases of the oestrous cycle, we used reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. A total of 24 ovaries from gilts were collected in early luteal, mid-luteal, and follicular phases of the oestrous cycle. When amplified with the primers common to IP(3)R-2, a RT-PCR product of the expected size (approximately 388 bp) was clearly detected in the follicular and early luteal phase of the oestrous cycle, but there was no detectable PCR product in the corpus luteum of the mid-luteal phase of the oestrous cycle. Immunohistochemical studies showed that IP(3)R-2 protein is expressed in granulosa cells and theca cells of growing follicles. IP(3)R-2 immunostaining was first detected during the late pre-antral stage in granulosa and theca cells. Granulosa cell IP(3)R-2 expression increased from the pre-antral to mid-antral stage, but was strongly reduced in pre-ovulatory follicles. In the developing corpus luteum, intense IP(3)R-2 immunostaining was also present in luteal cells, but undetectable in mid-luteal corpora lutea. Furthermore, oocytes, atretic follicles and regressed corpora lutea were negative for IP(3)R-2. Our results indicate that the expression of the IP(3)R-2 protein was downregulated in terminally differentiated granulosa cells of pre-ovulatory follicles when granulosa cells lose follicle-stimulating hormone responsiveness. Therefore, we strongly suggest that IP(3)R-2 may play an important role in the initiation and propagation of intracellular Ca(2+) signals during follicular development of the pig.

Objective: The third eyelid of domestic animals is important for the production and distribution of tears, in removing ocular debris and in protection of the globe, and has significant immunologic functions. Although it is known that tears contain antibodies of the immunoglobulin A (IgA) isotype which are produced mainly by plasma cells of the lacrimal gland, very little is known about the antibody repertoires in the third eyelid of domestic animals. To assess whether IgA is derived from local synthesis, we analyzed the location of IgA-producing cells and the cellular distribution of secretory component (SC) in the third eyelid of domestic animals in a comparative study., Animal Studied: A total of 83 third eyelids of dogs, cats, pigs, cows, sheep, goats and horses were investigated in the course of this study., Procedures: Third eyelids were obtained immediately after death, cut length-wise, fixed overnight and processed for immunohistochemical detection of IgA and SC by the ABC technique., Results: The results show that IgA-producing plasma cells are densely populated in subepithelial spaces of the surface epithelium as well as in the nictitating gland in a species-specific manner. In contrast, the SC could be demonstrated exclusively in glandular acinar and ductal epithelial cells and in different cell types of the surface epithelium, preferentially located on the bulbar side of the nictitating membrane., Conclusion: It is suggested that most of the SC is locally produced by resident plasma cells and subsequently transferred through the surface epithelium and glandular duct cells by transcytosis. This indicates that the third eyelid is an important member of the secretory immune system in domestic animals.

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.

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.

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 against 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. In the early pseudoglandular stage of development (day 70 of gestation) all the columnar epithelial cells lining the tubular endbuds strongly expressed CK 18 predominantly in the apical cell compartment. A modest staining was found in the more cuboidal cells of the canalicular stage (day 80 of gestation) where the labelling occurred as a distinct positive rim at the apical cell membrane in most of the cells lining the canaliculi. In 96- and 100-day-old foetuses, parts of the gas exchanging area were formed as terminal sacs by extreme attenuation of the epithelium. In this stage, CK 18 was clearly detectable in the flat type I as well as in the cuboidal type II alveolar epithelial cells. A marked change of the CK 18 expression pattern occurred during formation of the alveoli by septal outgrowth and maturation of the epithelium in 105- and 111-day-old foetuses. Differentiated type I cells no longer expressed CK 18, whereas type II cells were still labelled. Moreover, a specific change in the subcellular distribution pattern from the luminal periphery in immature porcine type II cells to a cytoplasmic localization in differentiated type II cells could be observed. Our investigation additionally demonstrated that the epithelium of bronchi, bronchioli and terminal bronchioli expressed CK 18 in all pre- and post-natal developmental stages. From the 96 days of gestation onwards the epithelial cells of developing bronchial glands were also labelled. Our results clearly show that during porcine lung development profound changes in the cellular expression pattern of CK 18 occur and that CK 18 can be regarded as a selective marker for differentiated porcine alveolar type II cells from the 105th day of gestation onwards. We also assume that the intermediate filament CK 18 could be of significance in the maturation process of the type II alveolar cells.

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.

The newly formed corpus luteum (CL) rapidly develops after ovulation and has the features of active vascularisation and mitosis of steroidogenic cells. These stage-specific mechanisms also may contribute to gain the function of prostaglandin F2 alpha (PGF2 alpha)-resistant CL at this stage. Recent studies suggest that the vasoactive peptide angiotensin II (Ang II) regulates luteal function. Thus, this study aimed to investigate (i) the expression of angiotensin-converting enzyme (ACE) mRNA by RT-PCR and the ACE protein expression by immunohistochemistry, (ii) the effects of angiogenic growth factors, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), on the secretion of Ang II, PGF2 alpha, progesterone and oxytocin (OT), and (iii) the effects of luteal vasoactive peptides (Ang II and endothelin-1 (ET-1)) or OT on the secretion of PGF2 alpha, progesterone and OT from bovine early CL (days 3--4 of the oestrous cycle), and evaluate a possible interaction of these substances with PGF2 alpha. The expression of mRNA for ACE was found in theca interna of mature follicle, early CL and endothelial cells from developing CL as well as pituitary and kidney, but granulosa cells of mature follicle were negative. The immunohistochemical analysis revealed that blood capillaries (endothelial cells) were stained for ACE, but luteal cells were negative in early CL. To examine the effects of substances on the secretory function of the CL, an in vitro microdialysis system was used as a model. The infusion of bFGF and VEGF stimulated Ang II and PGF2 alpha secretion as well as progesterone, but not OT secretion in early CL. The infusion of Ang II after PGF2 alpha infusion continued the stimulatory effect on progesterone and OT release within early CL until 3 h thereafter. However, the infusion of ET-1 alone had no effect on progesterone or OT release. The infusion of luteal peptides such as Ang II and OT stimulated PGF2 alpha secretion, whereas the infusion of ET-1 did not. In conclusion, the overall results of this study indicate that a functional angiotensin system exists on the endothelial cells of early CL, and that angiogenic factors bFGF and VEGF upregulate luteal Ang II and PGF2 alpha secretion, which fundamentally supports the mechanism of progesterone secretion in bovine early CL. This idea supports the concept that the local regulatory mechanism involved in active angiogenesis ensures the progesterone secretion in the developing CL in vivo.

The purpose of this comparative study was to evaluate morphological differences between the cartilages of the third eyelid in dogs, cats, pigs, cows, small ruminants and horses. For that reason a total of 83 third eyelids were investigated. By the aid of a modified maceration technique, the three-dimensional form of the cartilage could be demonstrated for the first time. Generally, the cartilage consists of a long narrow appendix which is followed by a variable crossbar. In dogs the appendix is cone shaped in the basal end and extends to form a triangular plate. The former is crescent-like in shape and has a marked bulge. The cartilage of the cat consists of an appendix which is enlarged in the proximal end as compared to the dog. The crossbar resembles a reverse s-form with ends tapering off to a point. In contrast pig and cow cartilage possess a typical anchorform whereas the cartilage of small ruminants starts with a thin rod which extends in a slightly curved form ending in an oval plate. The crossbar is crescent-like in these animals. In the horse the base of the cartilage is surrounded by a massive fatty tissue and the crossbar has a characteristic hook-form. Moreover, there are significant differences in regard to the quality of the cartilage, especially concerning the presence and distribution of elastic fibres. In cats and horses the elastic fibres of the adjacent connective tissue penetrate the perichondrium. Additionally, the centre of the cartilage shows a very dense network consisting of fine elastic fibres. In dogs, pigs, cows and small ruminants the cartilage consists of hyaline quality and only in the neighbouring connective tissue are some elastic fibres detectable.

Vascular endothelial growth factor (VEGF) is the most important factor in the regulation of angiogenesis. Associated with luteinisation and formation of corpus luteum (CL) are alterations in luteal vascularity. The aim of the study was to test under in vitro conditions the stimulation of VEGF and progesterone (P) secretion of bovine granulosa cells by LH, IGF1 (insulin like growth factor) or by factors known to be produced by luteinised granulosa cells or in the early CL. Localisation of VEGF protein in preovulatory follicle and early CL were achieved by immunohistochemistry. LH and IGF1 stimulated dose dependently and significantly P and VEGF when tested alone. Both hormones added simultaneously had clear additive and even more interesting far greater (synergistic) effects on P with LH (0.1 ng/ml) plus 5 or 10 ng IGF1. In contrast, VEGF was stimulated only additively with 0.1 ng/ml of LH plus 5 or 10 ng IGF1. But with the higher dose of LH (1 ng/ml) additionally to the additive effect a tendency for a synergistic action (which was significant with 1 ng LH plus 5 ng IGF1/ml) was observed. Endothelin, oxytocin, progesterone, atrial natiuretic peptide, angiotensin II, prostaglandin F2 alpha alpha, prostaglandin E2, cortisol, fibroblast growth factor 1 and 2 and growth hormone showed no effect neither on P nor on VEGF. Tumour necrosis factor alpha (TNF alpha) stimulated (P < 0.05) VEGF with 10 or 100 ng/ml but not P. TPA (12-0 tetra decaenoyl-phorbol-13-acetate) or Ca2+ ionophore did not show a stimulatory effect in contrast to forskolin which increased P and VEGF secretion dose dependently. The VEGF protein was localised in follicle (granulosa cells, theca cells and some endothelial cells) and early (about 24 h after ovulation) CL (granulosa-lutein cells and endothelial cells). The same signalling pathway by stimulation of cAMP production and proteinkinase A activation for luteinisation and neo-vascularisation demonstrates a close temporal and spatial relationship of these normal physiological processes.

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.

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.

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.

alpha-Galactosyl residues in the carbohydrate part of cellular glycoconjugates can serve as cell type-associated markers and are implicated in intercellular adhesion and biosignaling. This biological significance explains the interest to characterize probes with respective specificity as the Griffonia simplicifolia I-isolectin B4. Due to the documented occurrence of an alpha-galactoside-binding immunoglobulin G fraction in human serum we compared the extent of binding and its pattern for the lectin and the antibody using surface-immobilized extract proteins and fixed sections of bovine testicular tissue with known lectin reactivity. The antibody fractions were obtained either solely from affinity chromatography isolation on immobilized melibiose or after an additional step to deplete this fraction of galactoside-binding activities without pronounced specificity to the alpha-anomeric linkage. They yielded a rather indistinguishable reactivity in comparison to that of the lectin, when an indirect approach was used. Labeling of the antibodies with a hydrazide derivative of biotin did not affect the pattern of binding. However, significant differences were noted, when conjugation of label was targeted to amino groups via N-hydroxy-succinimide esters of biotin and digoxigenin despite performance of the modification under activity-preserving conditions. Notably, the apparent strong staining of Leydig cells and nuclei of primary spermatocytes, respectively, was not inhibitable by sugar. These differences were corroborated by a nonidentical response of the various probes in solid-phase assays with extract proteins. Thus, care should be exercised in the interpretation of histochemical data, obtained with this type of modified antibody. When these precautions are fulfilled, this immunoglobulin fraction from human serum has the potential as an alpha-galactosyl-specific histochemical tool.

Galactosyltransferase (GalTase) has been discovered on a variety of cells where it is believed to be involved in cell-cell adhesion and cell-substratum adhesion as well as in metastasis of carcinoma cells. This immunohistochemical study was undertaken to identify the topography and the cellular distribution of GalTase in normal prostatic tissue, benign prostatic hyperplasia (11 cases), and prostatic carcinoma (26 cases). Immunoreactive GalTase was found to be exclusively associated with carcinoma cells and with premalignant epithelial cells in prostatic hyperplasia. In highly differentiated carcinomas, most of the carcinoma cells are positive for GalTase, whereas in poorly differentiated tumors, GalTase immunoreactivity was restricted to a subset of carcinoma cells with obviously invasive behavior. At the cellular level, GalTase was localized in the cytoplasm and at the cell membrane. In sections of normal prostatic tissue, as well as in unaltered acini of prostatic hyperplasic tissue, GalTase was not expressed. Fibroblasts, smooth muscle cells, and endothelial cells of the prostatic stroma were also consistently negative. With the use of immunoblots, we could confirm the presence of GalTase with a molecular mass of 45 kDa in the extracts of benign prostatic hyperplasic tissue and in prostatic carcinoma tissue but not in normal prostatic tissue. The results of our immunohistochemical study suggest that GalTase is a valuable marker to diagnose neoplastic transformation in prostatic tissue.

The present study deals with the immunohistochemical localization of S-100 protein in the testes of seven mammalian species including rat, cat, dog, pig, sheep, cattle and horse. Significant differences are demonstrated in the cellular distribution and intensity of immunoreaction for the protein. In bull, ram, boar and cat testes S-100 protein was localized in the cytoplasm and nuclei of Sertoli cells. A particularly intense staining was seen in the modified Sertoli cells of the terminal tubular segment. With the exception of the cat and horse S-100 protein immunoreactivity was additionally found in epithelial cells of the straight testicular tubules and in the epithelial cells of the rete testis. Endothelial cells of capillaries, veins and lymphatic vessels are regularly S-100 immunoreactive in ruminants. Leydig cells were found to be strongly positive for S-100 protein in the cat and rat testes and to a lower degree in pig and horse testes. Finally a distinct immunostaining of peritubular cells was restricted to the testis of dogs and rats. The remarkable species-specific variations of immunoreactivity for S-100 protein in different cell types of the testis support the hypothesis that S-100 protein is multifunctional protein and may have a different function in testicular physiology.

To date, reliable and sensitive methods to localize the estrogen receptor (ER) in rat tissues and human breast cancers have required the use of frozen sections. This not only incurs poor tissue structure but also precludes the study of small breast lesions that are usually paraffin embedded for histological evaluation. We have developed and optimized a dinitrophenyl hapten sandwich staining (DHSS) immunocytochemical procedure to demonstrate ER in paraffin-embedded, hormone-sensitive tissues of the rat and in human breast cancers. The method was applicable to formalin- and Bouins-fixed material, with trypsinization of sections being essential. The immunocytochemical system utilized a dinitrophenyl (DNP) hapten-labeled monoclonal antibody to the receptor. Mouse IgM anti-DNP was used secondarily, followed by a DNP/peroxidase complex, diaminobenzidine/hydrogen peroxide chromogen, and silver intensification. This highly sensitive method localized the ER within paraffin-embedded rat uterus, fallopian tube, vagina, and normal and cancerous mammary gland. Furthermore, excellent staining was generated in human breast cancers in accordance with their ER-ICA status. Control sections involving simultaneous incubation with DNP-labeled and unlabeled H222 were background free, while uteri from castrated rats demonstrated reduced receptor immunostaining. Staining was also absent in ER-negative human breast tumors.

We have developed an immunocytochemical method to demonstrate estrogen receptor in hormone-sensitive tissues of the rat using a dinitrophenyl (DNP) hapten-labeled rat antihuman estrogen receptor monoclonal antibody (MAb), H222. Mouse IgM anti-DNP was used secondarily, followed by a DNP/peroxidase conjugate, diaminobenzidine/hydrogen peroxide chromogen, and silver intensification. This method was applied to tissues from intact female rats and showed that estrogen receptor was localized in the nuclei of the stromal and glandular components of the uterine endometrium. Reduced receptor staining was observed in the luminal epithelium, with minimal myometrial staining. Anterior pituitary glands showed heterogeneous immunostaining and ovaries expressed the receptor predominantly in the interstitial cells; fallopian tubes demonstrated substantial epithelial staining. Uteri from chemically castrated rats showed reduced estrogen receptor immunostaining in both stromal and luminal cells, whereas staining was enhanced in the glandular elements. Classical estrogen-unresponsive tissues (heart, lung, and spleen) were unstained. Antibody controls involved pre-blocking antibody recognition sites on the receptor with unlabeled antibodies to estrogen receptor (H222, H226, and D547), as well as use of an inappropriate DNP-labeled antibody to metallothionein. These controls illustrated the specific nature of the DNP-H222 binding.

A colorimetric tetrazolium (MTT) cleavage test was modified and established as a bioassay for the cytotoxicity of mycotoxins. Using the human erythroleukemia cell line K562 and porcine white blood cells (lymphocytes and granulocytes) we evaluated the influence of deoxynivalenol, ochratoxin A, and zearalenone on cellular MTT cleavage activity. The yellow MTT is reduced by mitochondrial enzymes of metabolically active cells into a dark blue formazan product, the optical density (OD) of which can be measured by an ELISA reader. After an exposure time of 24hours, concentrations of deoxynivalenol and ochratoxin A as low as 0.4 \gmg/mL were found to inhibit significantly the cleavage activity in K562 cells. Cytotoxicity in lymphocytes and granulocytes was observed at concentrations of 0.8 up to 0.4 \gmg/mL for deoxynivalenol and 3.1 and 0.8 \gmg/mL for ochratoxin A, respectively. Zearalenone concentrations of 25.0 to 12.5 \gm/mL inhibited the mitochondrial cleavage activity of lymphocytes and of K562 cells significantly, whereas in granulocytes none of the concentrations tested was proved to be toxic. Morphological findings on the ultrastractural level showed that toxin incubation (28 hours) resulted in massive cell damage. Similar alterations were observed in about 15% of control cells. This indicates, that the massive cytotoxic effect of the mycotoxin deoxynivalenol is more likely to be an unspecific than a specific one. The modified MTT cleavage assay was found to be a quick (28 hours) and efficient colorimetric test for examining the cytotoxicity of three mycotoxins. The simplicity and speed of the procedure, which allows the simultaneous testing of various parameters and the possibility of objective data analysis could establish this test as an additional bioassay for the evaluation of cytotoxicity of mycotoxins.

The application of a modified colorimetric assay for the evaluation of mycotoxin-derived cytotoxicity is reported. Using four mammalian monolayer cell types (swine kidney, African green monkey kidney, Madin Darbin canine kidney and bovine embryonic lung cells), the influence of the mycotoxins zearalenone and deoxynivalenol on the cellular methylthiazoltetrazolium (MTT)-cleavage activity was evaluated after different toxin exposure times. The yellow tetrazolium salt is converted by mitochondrial enzymes of metabolically active cells into purple formazan products. These crystals were dissolved in dimethylsulphoxide to obtain a homogeneous solution, the optical density of which was suitable for precise spectrophotometric measurement by a plate reader. Zearalenone proved to be less cytotoxic than deoxynivalenol and even enhanced the cleavage activity of the cell types tested. At concentrations of 10 and 100 mug/ml, deoxynivalenol showed a marked suppressive effect on the activity of all cell types, depending on the length of the incubation time. The results indicate that monolayers are useful targets in the MTT assay for investigating the action of mycotoxins and for performing comparative studies in mammalian cells.