Armin E. Friess

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

SummaryThe 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.

Density and distribution of anionic sites on boar ejaculated and epididymal spermatozoa

Abstract. Current knowledge implies that spermatozoa successively acquire negative surface charges as they migrate through the epididymis. Until recently, however, techniques used were not amenable to statistical analysis. In the present study, a novel approach allowing numerical assessment of negative charge labelling was used in order to determine the density and distribution of anionic sites on ejaculated and maturing spermatozoa collected from six regions of the boar epididymis. Labelling was assessed quantitatively for the three morphologically distinct membrane domains on the sperm head. Statistical analysis revealed that labelling density was highest on efferent duct spermatozoa, declined up to the proximal corpus and then increased again. Densities of anionic sites on distal corpus, proximal cauda and ejaculated sperm cells were similar but significantly below the values obtained for efferent duct spermatozoa. All three sperm membrane domains underwent parallel changes. However, the overall density of negative charges on the postacrosomal segment was significantly higher as compared to the acrosomal plasma membrane. These alterations reflect sperm surface modifications through removal and addition of anionic groups. Since charge interactions are considered to play a pivotal role in sperm–egg interactions, these processes should be viewed as an integral part of sperm maturation.

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.

Electron microscopic localization of a fucose-binding protein in acrosome reacted boar spermatozoa by the fucosyl-peroxydase-gold method

SummaryIn this study we have examined the behaviour and the localization of the fucose-binding protein (FBP) in boar spermatozoa during ionophore induced acrosome reaction (AR) by means of normal TEM and specimen preparation in toto. During early stages of AR the FBP is first localized at the border between equatorial segment and anterior acrosome. With the propagation of the AR the FBP is dramatically expressed and visible over the entire surface of the acrosome and equatorial segment. TEM pictures of this stages show that the FBP is associated with the OAM. At later stages of AR, when acrosomal ghost formation occur, the FBP is associated with the acrosomal ghost, and equatorial segment and to a very low degree also with the IAM. It is concluded from this data that the FBP is responsible for the specific binding of the ghost-sperm unit to the zone pellucida.

Blood-tissue barriers in the male reproductive tract of the dog: a morphological study using lanthanum nitrate as an electron-opaque tracer.

Blood-tissue barriers preventing an uncontrolled exchange of larger molecules between adjacent but metabolically separate compartments have been demonstrated in various organs. One prominent example is the blood-testis barrier which has been investigated in a number of species. A key function of this barrier is to shield developing germ cells from the immune system in order to avoid autoimmune reactions. This requirement also applies to the male excurrent duct system. Yet, very few investigations have addressed the morphology of the blood-epididymal barrier. The goal of the present study, therefore, was to revisit the blood-testis barrier in the dog and to identify the structures constituting the blood-epididymal barrier in this species. Lanthanum nitrate was used as a tracer for electron microscopy. In the testis, lanthanum had free access to the intercellular space of the seminiferous epithelium up to the Sertoli cell junctions. Similarly, epithelial tight junctions were found to represent the permeability barrier in the epididymis. The present study highlights species differences with respect to the blood-testis barrier and extends the knowledge of the blood-epididymal barrier by providing morphofunctional data in this domestic species.

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

SummaryA 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° C and 25° 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.

Immunocytological characteriziation of the outer acrosomal membrane (OAM) during acrosome reaction in boar

SummaryIn order to study the acrosome reaction in boar, spermatozoa were incubated in a calcium-containing medium in the presence of the calcium ionophore A23187. The time course of the acrosome reaction was assessed by phasecontrast microscopy and correlated with the movement characteristics of the spermatozoa determined by means of multiple-exposure photography (MEP). Different stages of the acrosome reaction could be observed by indirect immunofluorescence using an antibody fraction raised in rabbits against the isolated outer acrosomal membrane (OAM). At the start of the acrosome reaction, a bright fluorescence located exclusively at the acrosomal cap of the sperm head could be observed, whereas after 60–120 min, the fluorescence vanished, indicating the complete loss of the OAM. However, to gain more insight into the stages of the plasma membrane and OAM during the acrosome reaction, immunoelectron-microscopical studies were performed using anti-OAM antibodies detected by the protein-A gold method. Ultrathin sections and total preparations in combination with transmission electron microscopy (TEM) confirmed, that boar spermatozoa start their acrosome reaction by a vesiculation of the plasma membrane, thus exposing the heavily labelled OAM, which is then lost as sheets or large vesicles. The newly exposed inner acrosomal membrane did not show any labelling with gold, thereby indicating clear differences in the antigenicity of both acrosomal membranes.

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.

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.

SummaryMicrovasculature 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.