Seminal Phagocytes Detection at Light-Microscopy Level using Three Different Techniques

The phagocytes are normally present in the conective tissue at different organs, and their number increased in immunological or inflammatory processes. In these cases, they are present too in corporal fluids.The presence of phagocytes in semen was referred by several authors, but that phenomenon has not been suficiently analysed. At smear Papanicolaou stained analysis, the seminal phagocytes can be inadverted thus, their relationship to the male infertility or another reproductive tract pathology can not be established.In this study we implemented three techniques to detect the presence of phagocytes in semen of 28 infertile patients: Myeloperoxidase for polymorphonuclears (leukocyte), Neutral red for macrophages and the semithin sections analysis at light microscopical level of the seminal pellet obtained by centrifugation and included in epoxyresin.

Ultrastructural study of Trichinella spiralis with emphasis on adult female reproductive organs

The ultrastructure of the reproductive organs of adult female T. spiralis was described on the basis of extensive observation under an electron microscope. The organisms were recovered from the host intestine 7 days after oral infection. The organs consisted of a single ovary, seminal receptacle, uterus and vagina, confirming the reports described by previous authors at light microscopical level. All the organs were surrounded by a basal lamina and epithelial cells, and were bathed in haemolymph. Ova were formed in a germinal zone on the ovary wall exhibiting a half-moon shape, and spread to other sides of the ovary Mature ova exhibited a smooth cell surface, cuboidal shape, prominent cytoplasmic polysomes, a clear nucleus and a well-developed nucleolus. Embryos in early stage of development consisted of numerous small cells and were surrounded by a sheath.As they matured they shed the sheath and left a pool of sheaths in the uterus. During oogenesis and embryogenesis of adult worms in normal development, there occurred lipid droplets and the degradation of embryos; reportedly signs of worm damage.

Immunoelectron characterisation of the inter-endothelial junctions of human term placenta

The molecular constituents of the paracellular clefts in human placental microvessels were investigated using antibodies against PECAM-1, pan-cadherin, A-CAM (N-cadherin), cadherin-5 and two types of integrins (those recognised by antibodies to the beta 1 chain and alpha v beta 3). Ultrastructural localisation of these molecules in ultrathin frozen sections of human term placentae was attempted using colloidal gold immunocytochemistry, after establishing their presence by indirect immunofluorescence. At the light microscopical level, the endothelial paracellular clefts were found to be immunoreactive to the antibodies against PECAM-1, cadherin-5 and pan-cadherin, but not the integrins. The latter showed diffuse distribution in the endothelium and in the abluminal interstitial space. PECAM-1 and pan-cadherin were also seen in the cytoplasm and luminal surface of the endothelium. Immunoelectron studies revealed that the cadherins and PECAM-1 were present in the wide regions of the paracellular clefts, but not in tight junctional regions. Using immunocytochemistry, these wide junctional areas were found to be associated with the cytoskeletal linking molecules vinculin and alpha-actinin. These regions may therefore contain adherens-type junctions. Cadherin-5, localised by two different monoclonal antibodies, 7B4 and TEA, was the only antigen which was cleft-specific, the others also being seen in the cytoplasm of the microvascular endothelium. Cadherin-5 and pan-cadherin were co-localised in the same wide junction, but were usually seen to occupy different microdomains of, and different wide zones of, the same cleft. The cell adhesion molecules localised in the paracellular wide junctions of the human placental microvessels may play a role in maintaining the intercellular spacing between endothelial cells, and may be part of a paracellular “fibre matrix” with permeability-restricting properties.

Vitronectin receptor has a role in bone resorption but does not mediate tight sealing zone attachment of osteoclasts to the bone surface.

During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycine-arginine-glycine-aspartic acid-serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts. When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed intense staining of the cell membrane with occasional small unstained areas, probably corresponding to the sealing zones. Immunoelectron microscopy confirmed the results obtained by light microscopy showing specific labeling only at the ruffled borders and basolateral membranes (0.82 and 2.43 gold particles/microns of membrane, respectively), but not at the sealing zone areas (0.06 gold particles/microns of membrane). Both alpha v and beta 3 subunits of the vitronectin receptor were similarly localized. These results strongly suggest that, although the vitronectin receptor is important in the function of osteoclasts, it is not mediating the final sealing zone attachment of the osteoclasts to the mineralized bone surface.

Multiple subtypes of glycine-immunoreactive neurons in the goldfish retina: Single- and double-label studies

The glycinergic system in goldfish retina was studied by immunocytochemical localization of glycine antiserum at the light-microscopical level. Numerous amacrine cells, a type of interplexiform cell, interstitial cell, and displaced amacrine cell were glycine-immunoreactive (IR). Amacrine cells, accounting for 97% of the glycine-IR neurons, were of four types based solely on their level of dendritic stratification: stratified amacrine cells of the first, third, and fifth sublayers and bistratified amacrine cells of the first and fifth sublayers. Double-labeling experiments were carried out to determine possible co-localization of glycine-IR with GABA-IR, serotonin-IR, substance P-IR and somatostatin-IR. No evidence for co-localization of glycine-IR with these other transmitter substances was found, despite reports of co-localization of these substances in retinas of other species. Glycinergic neurons in goldfish retina appear to consist of a heterogeneous population of at least seven morphologically distinct subtypes that are also neurochemically distinct in regard to GABA, serotonin, substance P, and somatostatin. Since dendritic stratification in the inner plexiform layer is correlated with ON-, OFF-response types, we suggest that the subtypes of glycine-IR amacrine cells play different roles in the encoding of visual information.

Immunolabelling of glomerular deposits in kidney biopsies routinely fixed in glutaraldehyde

Various kidney diseases are characterized by the presence of dense deposits in the glomeruli. The type(s) of immunoglobulins (Igs) present in the dense deposits are characteristic of the disease. The accurate Identification of the deposits is therefore of utmost diagnostic and prognostic importance. Immunofluorescence (IF) used routinely at the light microscopical level is unable to detect and characterize small deposits found in early stages of glomerulonephritis. Although conventional TEM is able to localize such deposits, it is not capable of determining their nature. It was therefore attempted to immunolabel at EM level IgG, IgA IgM, C3, fibrinogen and kappa and lambda Ig light chains commonly found in glomerular deposits on routinely fixed ( 2% glutaraldehyde (GA) in 0.1M cacodylate buffer) kidney biopsies.The unosmicated tissue was embedded in LR White resin polymerized by UV light at -10°C. A postembedding immunogold technique was employed

Some ultrastructural aspects of Crithidia guilhermei n.sp. isolated from Phaenicia cuprina (Diptera: Calliphoridae)

A flagellate trypanosomatid was isolated from the fly Phaenicia cuprina captured in Rio de Janeiro, Brazil. It grows well in liver infusion – trypticase medium, in the form of choanomastigotes, typical of the genus Crithidia. Morphometrical data obtained at the light microscopical level indicated that the new isolated Crithidia is smaller than Crithidia luciliae, a parasite isolated from Phaenicia sericata. Transmission electron microscopy of thin sections revealed that this trypanosomatid has a flagellar pocket divided into two compartments, one basal and the other apical, separated by a region of attachment of the flagellum to the cell body. The attachment region was characterized in freeze-fracture replicas. The flagellate has a compact kinetoplast DNA network. As in endosymbiote-containing trypanosomatids previously described, no subpellicular microtubules were seen in the regions where the mitochondria touched the plasma membrane, although no endosymbiotes were found in this flagellate. Electrophoretic mobility of six enzymes showed that the parasite could not be grouped in any of the isoenzymic pattern groups of other Crithidia spp. These observations indicate that the trypanosomatid isolated from P. cuprina is a new species of Crithidia. The flagellate is described as Crithidia guilhermei n.sp.

Secondary and backscatter electron imaging of silver stained ciliated protozoa

The use of secondary electron (SE) signals in the scanning electron microscope provides a wealth of information on the cortical topography of the ciliated protozoa. Surface manifestations of such morphogenetic events as cell division and regeneration can easily be visualized in the SE mode of the SEM, but concomitant alterations occurring below the surface of the cell can not be visualized. Ciliatologists have used the "Protargol" (silver protein) method to study the cortical features of the ciliates at the light microscopical level. The protein silver stain clearly reveals cilia, microtubules, kinetosomes and nuclei. Small et al. have demonstrated the usefulness of this technique in conjunction with backscatter electron (BSE) detection in the SEM. A strong BSE signal can be obtained from argentophilic structures within the specimen due to the atomic number contrast between the deposited silver and the low atomic number elements normally found in cell cytoplasm. Therefore, the use of the SE and BSE signals with good spatial resolution should clearly demonstrate the relationship between surface and subsurface features.