Immunohistochemical demonstration of lumbar intervertebral disc innervation in the dog.

Low back pain is a common ailment in dogs, particularly in specific breeds such as the German shepherd dog. A number of structures such as facet joint capsules, ligaments, dorsal root ganglia, periosteum, vertebral endplates and meninges have been associated with this condition. Yet, in spite of all diagnostic efforts, the origin of pain remains obscure in a substantial proportion of all cases. A further structure often being involved in vertebral column disorders is the intervertebral disc. The presence of nerves, however, is a precondition for pain sensation and, consequently, structures lacking innervation can be left out of consideration as a cause for low back pain. Nerve fibres have been demonstrated at the periphery of the intervertebral disc in man, rabbit and rat. With regard to the dog, however, the extent of intervertebral disc innervation is still being disputed. The goal of the present study, therefore, was to substantiate and expand current knowledge of intervertebral disc innervation. Protein gene product (PGP) 9.5 was used for immunohistochemical examination of serial transversal and sagittal paraffin sections of lumbar discs from adult dogs. This general marker revealed nerve fibres to be confined to the periphery of the intervertebral discs. These results indicate that even limited pathological processes affecting the outer layers of the intervertebral disc are prone to cause low back pain.

Exposure of rainbow trout (Oncorhynchus mykiss) to nonylphenol is associated with an increased chloride cell fractional surface area.

Nonylphenol is a biodegradation product of a widely used group of non-ionic detergents. Because of its ubiquitous distribution and persistence, nonylphenol is present in surface waters as a pollutant. Little is known about its biological effects at environmentally relevant concentrations other than its action as a xenoestrogen. The goal of the present paper was to study the trout gill surface epithelium as the major interface between fish and water in view of possible morphological alterations due to exposure to nonylphenol. Rainbow trout were intermittently exposed to 10 micrograms/l nonylphenol and gill samples from experimental and control animals were investigated by scanning electron microscopy. Gill surface epithelium was scrutinised for changes in chloride cell density and their status regarding cell surface modifications. In addition, chloride cell fractional surface area (CCFA) was determined by morphometrical methods. Statistical analysis revealed a highly significant increase of CCFA in animals exposed to nonylphenol as compared to control animals (P = 0.0001). Semi-quantitative assessment of the other parameters suggested a higher chloride cell density and a larger proportion of chloride cells bearing microvilli. Taken together, these results provide evidence that exposure of trout to nonylphenol is associated with a substantial increase in the active interface of chloride cells with water. We interpret these findings as being a means to further the fish's capacity for calcium exchange.

Ultrastructural evidence of transplacental transport of immunoglobulin G in bitches.

In dogs, passive immunity is conferred to fetuses and neonates by the transfer of maternal immunoglobulin G through the placenta during the last trimester of pregnancy and via the mammary gland after parturition, respectively. However, morphological evidence of transplacental transport is still lacking. The aim of the present study was to localize maternal immunoglobulin G in the labyrinthine zone and in the haemophagous zone of the canine placenta by means of immunohistochemistry and immunocytochemistry. In the labyrinthine zone, immunoglobulin G was detected in all the layers of the materno-fetal barrier including the fetal capillaries. Immunoreactivity was particularly prominent in maternal basement membrane material as well as in the syncytiotrophoblast. However, this evidence of transplacental transport of immunoglobulin G originated from a limited number of unevenly distributed maternal vessels only. In the cytotrophoblast of the haemophagous zone, immunoglobulin G was localized to phagolysosomes at various stages but was never detected within fetal vessels. The results indicate that maternal immunoglobulin G is degraded in cytotrophoblast cells of the hemophagous zone and, therefore, that transplacental transport is restricted to a subpopulation of maternal vessels in the labyrinthine zone.

Distinction of gastric Helicobacter spp. in humans and domestic pets by scanning electron microscopy.

BACKGROUND: A number of different Helicobacter spp. can colonize the stomach of humans and domestic pets. Difficulties encountered with primary isolation of these spiral microorganisms and their unusual inertia with respect to biochemical reactions still represent considerable obstacles to their characterization with classic tools. In addition, the high degree of similarity in the 16S rRNA sequence hampers differentiation of Helicobacter spp. using routine molecular biological assays. MATERIALS AND METHODS: Samples from experimentally monoinfected mice, of naturally infected hosts, and of cultured strains were examined by scanning electron microscopy (SEM). In parallel, all samples were analyzed by molecular techniques to ascertain the Helicobacter spp. involved. RESULTS: Using the mouse samples as a reference, microorganisms found in naturally infected hosts were identified by SEM as belonging to H. pylori, H. felis, or a group consisting of H. bizzozeronii and H. heilmannii. A further spiral microorganism with unique morphology was found in a dog that was positive for H. salomonis, but the organism could not be recovered from experimentally infected mice. In culture, most Helicobacter strains lost their ultrastructural characteristics. CONCLUSIONS: When gastric Helicobacter spp. were collected from their natural habitat and examined by SEM, relevant differences could be detected between H. felis, H. bizzozeronii and H. heilmannii, and H. salomonis, respectively. SEM, therefore, seems to be a useful auxillary tool for the distinction of various gastric Helicobacter spp. as based on their ultrastructure.

The glandular chambers of the placenta of the bitch in the second third of pregnancy (day 30-44): an ultrastructural, ultrahistochemical and lectinhistochemical investigation.

The present study represents the first comprehensive investigation of the glandular chambers of the placenta of the bitch. We examined the glandular chambers by SEM, TEM, ultrahistochemical and lectinhistochemical methods. The glandular epithelium is a high columnar epithelium with club-shaped apical protrusions bearing short microvilli. The epithelium forms extensive folds which fill up most of the lumen of the glandular chambers. Proceeding to the placental labyrinth, the glandular chambers are covered by the tips of the chorionic tufts. The trophoblast and the glandular epithelium are separated by a thick layer of secretions. The ultrastructure cytology of the columnar epithelium is characterized by several Golgi complexes and abundant apical-located mucus vesicles with a positive dialysed iron reaction. Lectin histochemistry reveals in general a strong reaction of these mucus vesicles with all lectins used. The mucus in the lumen of the glandular chamber reacts strongly with WGA, NeuWGA, LPA and ConA. The trophoblastic villi projecting into the lumen of the glandular chambers are covered by a pseudostratified epithelium consisting of a flat basal layer and a superficial columnar one. Its cytoplasm is filled with large fusing vacuoles containing longish screw-shaped structures. No morphological equivalent of absorption is found. According to the ultrastructure of the trophoblast, the significance of the mucus in the glandular chambers in stopping the invasive growth of the trophoblast is discussed.

Localization of glucose-6-phosphatase activity and carbohydrates in boar caput epididymal principal cells.

Unidentified tubulovesicular profiles have been reported in the apical cytoplasm of boar caput epididymal principal cells in addition to vesicles considered to be involved in endocytosis and secretion. The main aim of the present study was to clarify the character of these organelles and to differentiate them from the endocytic apparatus. Glucose-6-phosphatase (G6Pase) activity was determined as the reporter enzyme of the endoplasmic reticulum (ER) and phosphotungstic acid was used to visualize carbohydrate moieties in both the proximal and distal caput. Phosphotungstic acid revealed the glycocalyx of the endocytic apparatus, which was similar in both regions studied, and also stained specific granules of the proximal caput. Glucose-6-phosphatase showed the tubulovesicular profiles to be sparsely granulated ER that was poorly developed in the proximal caput and very abundant in the apical cytoplasm of the distal caput principal cells. The function of such large amounts of sparsely granulated ER with corresponding G6Pase activity in caput epididymal principal cells is unknown.

Scanning electron microscopy of the canine placenta.

The zonary placenta of the dog is peculiar in providing three morphologically and functionally distinct areas for transplacental exchange: the endotheliochorial labyrinth of the girdle zone, the hemochorial hemophagous zone of the marginal hematoma and the epitheliochorial free polar zone. Besides, the yolk sac persists throughout pregnancy. Scanning electron microscopy (SEM) was used to study these structures comprehensively with special emphasis on natural surfaces because of their relevance for exchange processes. Unequal cell diameter was typical for trophoblast cells. Surface specializations were observed on trophoblast cells lining the marginal hematoma, the free polar zone and the glandular chambers as well as on uterine epithelial cells of the glandular chambers and of the deep glands. In addition, maternal and fetal endothelial cells in the labyrinth showed major differences. Whereas fetal endothelial cells were extremely thin and polygonal in shape, maternal vessels were lined by thick endothelial cells exhibiting a complex interdigitation pattern of cytoplasmic processes. Thus, most of the natural surfaces could readily be distinguished by SEM due to their typical appearance.

The junctions between efferent ductules and epididymal duct in the boar.

Sperm transit through the male excurrent duct system is dependent on complete luminal patency. Since male excurrent ducts are derived embryologically from originally separate structures, the junctions between mesonephric tubules and the mesonephric duct are of major interest with respect to obstructive disorders. Therefore, we investigated the junctions between efferent ductules and epididymal duct in adult boars by means of serial semithin and ultrathin sections. Based on the anastomosing pattern and on the site of transition from low columnar, ciliated epithelium to high columnar, non-ciliated epithelium, three different types of anastomoses were identified, one of which was associated with luminal stenosis and sperm accumulation. The occurrence of end-to-side junctions with the epithelial transition being localized within the lumen of the tributary may thus impede normal sperm transport.

Ultrastructural validation of an improved culture system for boar efferent duct epithelium.

A tissue culture system in which cells retain defined ultrastructural and functional characteristics was established to provide a basis for functional investigations of the efferent ductules in boars. A currently used culture protocol for rat epididymal epithelium was used as a starting point and was subsequently modified because of unsatisfactory results. Epithelial plaques were isolated by mechanical uncoiling of the ductules and two sequential enzymatic digestion steps. Plaques were seeded onto extracellular matrix-coated permeable membranes and maintained in a two-chamber system. Samples taken before seeding and after 7 days in culture were processed for transmission and scanning electron microscopy. Perfusion-fixed material from earlier studies served as a reference to assess ultrastructural preservation. In addition, endocytotic activity was investigated by adding cationized ferritin to the culture medium on day 8 before fixation. At the end of the disaggregation procedure, cells were cuboidal, while cilia, microvilli and cell organelles were well preserved. After 7 days in culture, three types of cell formation were observed: cysts, pseudotubules and epithelial sheets. Cell sheets were made up of closely juxtaposed cells bearing motile kinocilia and exhibiting well-developed polar differentiation, as judged from the localization of cell junctions and organelles. Although it did not return to the values of native material, cell height was greater than that of cells grown according to the pre-existing protocol. Furthermore, preferential uptake of ferritin by principal cells after 7 days in culture was demonstrated. Preservation of these fundamental characteristics of the in vivo state corroborates that our in vitro system will furnish reliable information.

A scanning electron-microscopic analysis of the morphology of equine lower lip sinus hair.

Sinus hairs, which are tactile organs of most mammals, are differentiated into cavernous and sinus types. The horse has the cavernous type. Horse lower lips were perfused in diluted Karnovsky's fixative and sinus hair processed for scanning electron microscopy. Anastomosing trabeculae of different thickness and shape originate from the internal connective tissue layer and extend to the external layer, thus forming a dense meshwork suspending the hair root like a net. However, many of the distal trabeculae do not reach the external wall of the blood sinus but end in a finger-like knob within the sinus, thus sharing some features of the sinus type. The sinus wall and trabeculae are completely covered by polygonal endothelial cells showing characteristically protruding nuclei, sparse microvilli and distinct cell borders. Endothelial cells on the trabeculae are more elongated. Openings of small blood vessels were found exclusively on the internal wall, indicating that blood exchange occurs via the internal wall only and that turnover of blood may be minimal. The supposed significance and function of the finger-like trabeculae in the distal part of the blood sinus lying close to the majority of nerve terminals is discussed.

Morphological characteristics of boar efferent ductules and epididymal duct.

The aim of the present study was to provide a comprehensive morphological analysis of the porcine epididymis in view of the specific functions being performed in different regions of this organ. Blood supply and microvasculature of efferent ductules and epididymal duct were investigated by means of corrosion casts which were analysed macroscopically and by scanning electron microscopy. This revealed blood supply to the testis and epididymis to be closely related. The capillary pattern was typical for the efferent ductules, the caput, corpus, and distal cauda epididymidis, respectively. Corrosion casts were also used to visualize the course of the efferent ductules themselves. Tissue samples from different regions of the efferent ductules and epididymal duct were examined by light microscopy and both scanning and transmission electron microscopy, with special attention being payed to transitional areas. Morphological criteria allowed the distinction of three segments within the efferent ductules and of the initial segment, proximal caput, distal caput, corpus, proximal cauda, and distal cauda regions of the epididymal duct. Components of the endocytic apparatus of efferent ductule principal cells were identified by ferritin uptake. Ultrastructural evidence of absorption in the epididymal duct was particularly prominent in proximal and distal caput. Extensive cisternae of rough endoplasmic reticulum and a well-developed Golgi apparatus were indicative of active protein synthesis and secretion especially in the distal caput and corpus regions. However, assignment of various organelles in principal cells of the epididymal duct to either absorptive or secretory pathways still remains tentative.

Improved preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM).

Various fixation protocols were used in an attempt to improve preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM). Wash solutions and fixatives of different composition and osmolarity were tested. Paraformaldehyde and glutaraldehyde concentrations were varied between 0.5% and 3%. Ruthenium red was tested as an additive in both primary fixation and postfixation, or in postfixation alone. HR-LVSEM revealed various degrees of ruffing, folding, blebbing, and peeling off of the plasma membrane, as well as holes of different sizes. The plasma membrane overlying the acrosome and the connecting piece proved to be particularly sensitive to varying fixation conditions. Consistent topographical differences were revealed among the different domains over the sperm head. Most of the differences were considered to be artifacts. Their consistency, however, suggests that structural and biochemical differences exist either within the membrane or in the structures subjacent to the membrane. Primary fixation turned out to be less critical than postfixation. Preservation of a smooth plasma membrane without holes could only be achieved when primary fixation in low aldehyde concentrations, with or without ruthenium red, was followed by postfixation with OSO4 and 1,000 ppm ruthenium red. Examination of thin sections of the same material confirmed that even a considerable number of small holes are difficult to detect in transmission electron microscopy. These results show that with the recent increase in resolution of LVSEM there is need for further effort to improve sample processing.

[The glandular compartments of the canine placenta--a scanning electron microscopic study]. / Die Drüsenkammern der Hundeplazenta--eine rasterelektronenmikroskopische Untersuchung.

The glandular chambers in the spongy zone grow out of the superficial endometrial glands during the implantation of the embryo. To our knowledge, no scanning electron microscopic investigations of the spongy layer in the placenta of the bitch have been published so far. We examined the placenta at the 44. day of pregnancy by scanning electron microscopy. The glandular epithelium in the spongy zone forms extensive folds, which fill up most of the lumen of the glandular chambers. They are lined by a simple columnar epithelium with high apical protrusions. The luminal surface of the cells is bordered by short microvilli. Numerous vesicles are stacked in the cytoplasm. The tips of the chorionic tufts project into the lumen of the glandular chambers. The lumen is filled with masses of mucus separating the trophoblast from the glandular epithelium. With respect to its morphology, the trophoblast lining the tips of the chorionic tufts is very different from the trophoblast in the other parts of the placenta. The cells are swollen and club-shaped. The cytoplasm is completely supplanted by big vacuoles. We feel that the trophoblast in the glandular chambers is unable to perform any resorptive activity. The significance of the mucus in the glandular chambers in stopping the invasive growth of the trophoblast is discussed.

Efferent ductules of the boar--a morphological study.

The efferent ductules of the boar were investigated by means of corrosion casts, light microscopy, scanning and transmission electron microscopy. They arise from an extratesticular rete and constitute the major, caudolateral part of the ascending limb of the caput epididymidis. Ductules may be subdivided into three segments: a slightly undulating testicular segment, a highly coiled intermediate segment and a moderately coiled epididymal segment. A decrease in diameter is particularly marked from the intermediate to the epididymal segment. The epithelial transitions from the extratesticular rete to the efferent ductules and from these to the epididymal duct are clearly demarcated. The epithelium of the efferent ducts consists of principal and ciliated cells. Mononuclear leukocytes are found in the basal half. Ultrastructural evidence supports a strong absorptive activity of principal cells. Apical protrusions are not considered to be a proof of apocrine secretion but rather seem to be artifacts. The nature of membrane-bound granules of variable density remains speculative.

Microvasculature of the epididymis in the boar.

Microvasculature of the epididymis was investigated by scanning electron microscopy of vascular corrosion casts. The basic structure of blood supply to the boar epididymis consists of two superimposed vascular networks. Capillaries surrounding the epididymal duct constitute the inner level. They form polygonal meshes around the efferent ductules whereas circular capillaries strongly predominate in the subsequent region of the caput epididymidis. This annulate feature is progressively lost from corpus to cauda, where the capillary network once again has a polygonal appearance. The outer network is composed of feeding and draining vessels. Intertubular arteries pass between the loops of the epididymal duct and give rise to longitudinally oriented vessels attributable to only one adjacent duct segment. They feed the capillary network via circular ramifications debouching in different sectors of its circumference. The sparse veins draining the capillaries encircling the efferent ductules give way to a gradually increasing number of confluent veins up to the cauda.

Cell biology of acrosomal proteins.

Acrosin is a multifunctional enzyme combining several functional properties within a single molecule: the catalytic triad of the proteinase, hydrophobic domains responsible for the special membrane-associating character of the enzyme and the carbohydrate binding sites by which the molecule can bind to the zona pellucida. Acrosin occurs in the sperm acrosome as an inactive precursor, proacrosin, with a molecular mass of 53-55 kDa. Proacrosin is activated by a single proteolytic clip between Arg23 and Val24 generating the high molecular mass acrosin. The activation of proacrosin to the biologically active enzyme which occurs concomitantly with the acrosome reaction appears to be regulated on and by the zona pellucida. It is hypothesized that alternating cycles of binding to the zona, digestion of the zona and release from the zona together with the forward motility of the spermatozoon would be required to achieve penetration.

Boar sperm membranes antigens. I. Topography of a mobile glycoprotein of the sperm cell membrane.

A monoclonal antibody, designated mAb P86/5, was generated by immunization of female Balb/c mice with a membrane vesicle fraction composed of the outer acrosomal membrane and plasma membrane (PM-OAM). As determined by fluorescence microscopy and electron microscopy P86/5 recognizes a sperm plasma membrane antigen that is restricted to the sperm head. In intact spermatozoa the P86/5-antigen is distributed over the surface of the sperm head with the exception of the rostral region. By comparing the antibody binding pattern generated at 4 degrees C and 25 degrees C, it could be shown that the P86/5-antigen is capable to diffuse freely within the cell membrane overlying the acrosome whereas its lateral mobility is restricted to the post-acrosomal region. The P86/5-antigen had a molecular weight of about 78 kDa as revealed by SDS-PAGE and western blotting. The glycoprotein nature of the P86/5-antigen was established by lectin affinity chromatography.

Boar sperm membranes antigens. II. Reorganization of an integral membrane antigen during capacitation and acrosome reaction.

The dynamics of the cell surface during the process of capacitation is impressively shown by means of a monoclonal antibody directed against the P86/5 antigen. This glycoprotein was located in the sperm plasma membrane using the colloidal gold method in combination with specimen preparation in toto. The antigen is absent at the rostral tip of non-capacitated spermatozoa, but forms clusters over the principal segment and the equatorial segment after induction of capacitation. This formation of microdomains with different properties may be a prerequisite for the onset of the acrosome reaction (AR). During AR the diffusion barrier for the P86/5 antigen breakes down and the antigen occupies now the rostral crescent-like area of the sperm head. These observations are discussed with respect to zona binding and induction of the AR in boar spermatozoa.

[The vascularization of the epididymis of the boar with special reference to perfusion fixation]. / Die Vaskularisation des Ebernebenhodens unter besonderer Berücksichtigung der Perfusionsfixation.

Vasculature of the epididymis was investigated by means of corrosion casts. In the boar, epididymal arteries form a complex network around their stem vessel, the testicular artery. Proper perfusion fixation or complete casting therefore require direct injection into one of these branches. To reach the distal cauda, cannulation of the deferential artery is further needed. Connections between all of these feeding vessels occur at the level of the vascular cone. A prominent anastomosis between an epididymal branch and the testicular artery is regularly observed under the caput epididymis. Epididymal veins drain to a large extent into the pampiniform plexus. Unlike the situation in other species, vascularization of testis and epididymis are closely associated in the boar.