Negatively stained punctated cell contacts between luteal cells in rabbit and rat.

Cell contacts from rat and rabbit luteal cells have been spread on phosphotungstic acid solution after tissue fractionation or spreading was accomplished by a direct technique avoiding homogenization. Sectioned material was also examined. It was shown by negative staining that the cell contacts were composed of a multitude of bridges. These bridges were either filamentous (condensed form) or clearly showed a larger (expanded form) central particle. The corresponding image in top view showed respectively small and larger granules without any particular arrangement. Large granular structures were also seen on single plasma membranes. Evidence is presented that the condensed and the expanded form are two aspects of the same junctional structure. It is also suggested that both aspects are related to the tightness of the contact. Up to now this type of cell contract has been called "septate-like". However because of the dotted appearance of these contacts after colloidal lanthanum and after negative staining the denomination "punctated cell contact" may be better adapted.

The p185erbB2 protein is localized on cell organelles involved in cell motility.

In a previous paper, we have shown that the c-erbB-2-encoded protein p185erbB2 is localized on the brush border of the proximal tubule kidney cells. In invasive duct cell carcinomas, the labeling was most obvious on the villous plasma membrane protrusions. From these observations the hypothesis was raised that p185erbB2 could play a role in motility. To test this hypothesis, we quantified its distribution on the microvilli and plasma membrane protrusions and on the straight parts of the cell membrane after immunoelectron microscopy. These findings were compared with the localization on p185erbB2 overexpressing SK-BR-3 human breast cancer cells before and after stimulation of motility by treatment with conditioned medium from COLO-16 cells (CM), which is also able to induce chemotaxis of these cells in a Boyden chamber assay. In the invasive duct cell carcinomas, the number of gold particles was nine times higher at the plasma membrane protrusions than at the straight parts of the cell membrane. In untreated SK-BR-3 cells, p185erbB2 was similarly concentrated on the membrane of small microvilli and plasma membrane protrusions. Treatment of SK-BR-3 cells with CM leads to cell spreading, enlargement of the microvilli, formation of pseudopodia and chemotaxis. Aggregation of p185erbB2 at the plasma membrane protrusions and pseudopodia is observed on immunofluorescence light microscopy. The concentration of p185erbB2 is several times higher on these membrane extensions than on the straight parts after immunogold labeling. It is concluded that p185erbB2 is localized on cell organelles involved in motility, and it is suggested that the molecule plays a role in cell movement, providing the capacity of tumor cells to spread and metastasize.

Detection of sialic acid residues in the axonal reticulum of rat superior cervical ganglion cells by lectin-gold cytochemistry.

Highly glycosylated compounds have been demonstrated in the axonal reticulum elements of the superior cervical ganglion cells of the rat, and this is considered to suggest a connection of the reticulum with the trans Golgi side. In the present study, the axonal reticulum and the Golgi elements were further characterized by post-embedding methods of lectin-gold cytochemistry to determine their carbohydrate residues and to see, more specifically, if sialic acid residues could be detected in the axonal reticulum elements. Therefore, the affinity of neuronal cell structures for Limax flavus agglutinin (LFA), wheat germ agglutinin (WGA), and Ricinus communis agglutinin I (RCA-I) was tested in ultra-thin sections of glycolmethacrylate-embedded material, counterstained with phosphotungstic acid (PTA) at low pH. The trans Golgi network, the Golgi-associated axonal reticulum, the reticulum within axons, the large dense-cored vesicles, and the plasma membranes were reactive for all three lectins used. We conclude that the axonal reticulum elements carry sialic acid residues, relating them to the trans Golgi network. The present results support the concept that the axonal reticulum is an extension of the trans network of the Golgi apparatus specialized for neurosecretion.

Immunocytochemical demonstration of dopamine-beta-hydroxylase and cytochrome B561 on the axonal reticulum in bovine sympathetic neurons.

In sympathetic neurons the axonal reticulum can be considered an extension of the secretory pole of the Golgi apparatus. If this tubular system indeed represents the neurosecretory apparatus, it would likely contain on its membranes the enzymes involved in catecholamine synthesis. To test this hypothesis, we investigated the distribution of dopamine-beta-hydroxylase and cytochrome b561 in bovine splenic nerve and nerve terminals in the vas deferens with an immunogold procedure after glycolmethacrylate embedding. Counterstaining with phosphotungstic acid at low pH selectively revealed the axonal reticulum elements. With antibodies against both enzymes, gold labeling was observed over the large dense-cored vesicles, the Golgi-associated axonal reticulum, the reticulum within axons, and the tubular complex at the nerve terminal. From our results it can be concluded that in sympathetic neurons the axonal reticulum represents a tubular neurosecretory system, extending from the Golgi apparatus in the cell soma to the nerve terminal. This concept emphasizes the local production of neurosecretory vesicles and may be of importance in the interpretation of neuronal transmission in normal and diseased states.

Ultrastructural evidence for a separate, small synaptic vesicle (SSV) pathway in ligated bovine splenic nerves, incubated in vitro.

In sympathetic nerves the tubules of the axonal reticulum make up the immature elements of the neurosecretory apparatus. The formation of the mature large dense granules occurs via a less dense tubular intermediate, representing the maturing part. At a terminal small synaptophysin-positive vesicles are found intermingled with the dense granules. The biogenesis of these clear, small synaptic vesicles and their relationship with dense granules remains to be determined. In search for the small synaptic vesicles we undertook a careful ultrastructural examination of the axons proximal to a ligation in bovine splenic nerve incubated in vitro for 3 h. The distended axons were crowded with tubules, granulo-tubular elements and dense granules. Occasionally homogeneous clusters of small, uniform vesicles were detected. They were shown to be positive for synaptophysin and were negative for dopamine-beta-hydroxylase, a marker for the granular pathway. The clusters of small vesicles could be found in close spatial relationship with the maturing and mature elements of granular secretion. Our findings argue for the presence of two separate neurosecretory pathways in sympathetic nerves and favour the idea that both small synaptic vesicles and dense granules are a differentiation product of the axonal reticulum. This configuration can explain the biogenesis of small synaptic vesicles and dense granules both in the cell body and at the nerve terminal.

Differential ultrastructural distribution of synapsin and synaptophysin proximal to a ligation in bovine splenic nerve.

Synaptophysin and synapsin, closely correlated on synaptic vesicles in terminals, may show a differential distribution at synapse formation and maturation. In order to disclose the fine structural details of these differences, synapsin and synaptophysin distribution was studied by immunocytochemistry on ligated bovine splenic axons in vitro and compared with terminals in the vas deferens. In the synaptic differentiations taking place proximally synapsin could only be detected on the accumulating elements of the axonal reticulum. Large dense granules and clusters of small synaptic vesicles were negative. Synaptophysin was restricted to these clusters. In the vas deferens, co-localization of synapsin and synaptophysin could be seen on small vesicles. From their formation small synaptic vesicles carry synaptophysin. Synapsin may be involved in the dynamic membrane changes taking place at the ligation. At a functional terminal, synapsin shifts to small synaptic vesicles.

Postganglionic, direct axo-axonal contacts on the splenic nerve.

The splenic nerve is made up almost exclusively of adrenergic fibers. Consequently it was used as a model system in the study of autonomic sympathetic neurotransmission. The splenic nerve regulates the vasoconstriction and volume reduction of the spleen. Brain-immune interactions via modulation of the splenic nerve activity may regulate peripheral cellular immunity. An inhibition of noradrenaline release by alpha(2)-adrenoceptor activation has been reported. As we were interested in a structurally detailed distribution of synaptophysin, immunocytochemical methods were applied to splenic nerve axons. In 1 microm plastic sections a network of synaptophysin-positive varicosities could be observed all along the splenic nerve. They were also positive for dopamine-beta-hydroxylase and cytochrome b561. At the ultrastructural level the varicosities were seen to establish direct contact with the splenic axons. In normal morphology the varicosities revealed small synaptic vesicles and several dense granules. It is demonstrated that a network of direct symmetric contacts of adrenergic nature is present all along the nerve. These terminals may have an inhibitory effect on the splenic nerve activity via axonal receptors. This finding opens new perspectives for the study of the splenic nerve in general and more particularly for its role in the regulation of peripheral cellular immunity.

The organisation of the axonal reticulum at a ligation, in in vitro incubated bovine splenic nerves.

From previous studies we concluded that in noradrenergic neurons the axonal reticulum can be considered to be an extension of the Golgi apparatus, directly involved in the condensation and packaging of neurosecretion. But the precise ultrastructure of the organisation of the axonal reticulum in relation to neurosecretory granule formation remained to be elucidaded. This conversion was studied in ligated bovine splenic nerve incubated in vitro for three hours. The ultrastructure of the material accumulating proximally and distally was examined and its nature was determined by phosphotungstic acid staining and immunocytochemistry on glycolmethacrylate sections. Proximal to the ligation predominantly electron-lucent vesicles and tubules were found. Tubules of intermediate electron density appeared in between. The latter, especially in thicker sections, were seen to form complexes with tubules and granules of high electron density. All those elements were shown to be positive for dopamine-beta-hydroxylase and cytochrome b561. In the distal part multivesicular bodies accumulated and they were also positive for both enzymes. From these findings it is concluded that the different types of structures accumulating proximally belong to a neurosecretory axonal reticulum. At a block the axonal reticulum is triggered to generate a reticular differentiation, in which granular densities of different size are found. This configuration compares well with that in nerve terminals and strongly suggests that granule formation is basically a local process.

Ultrastructural localization of neuropeptide Y-immunoreactivity in the axonal reticulum elements, accumulating anterogradely in transected rat sciatic nerve.

The detection of dopamine-beta-hydroxylase and cytochrome B561 on the membranes of the axonal reticulum demonstrated that in sympathetic neurons the different compartments of the axonal reticulum participate in the formation of neurosecretory vesicles. In the present study we tried to reveal that the components of the vesicular content are also channeled along the axonal reticulum, by examining whether neuropeptide Y could be localized in elements of the axonal reticulum. Therefore 6 h transected rat sciatic nerve was embedded in glycolmethacrylate and an immunogold labeling was performed. Counterstaining with phosphotungstic acid at low pH selectively contrasted the accumulated axonal reticulum elements and associated granules. In the non-myelinated axons gold labeling was localized on granules and on tubular and granular profiles, demonstrating the presence of neuropeptide Y in the accumulated axonal reticulum elements. This indicates that neuropeptides are indeed transported via the axonal reticulum to the nerve ending and suggests that the accumulation of large dense-cored vesicles at a block is mainly due to local new formation rather than down transport.

Fertilization and pregnancy after assisted oocyte activation and intracytoplasmic sperm injection in a case of round-headed sperm associated with deficient oocyte activation capacity.

OBJECTIVE: To investigate a method of assisted activation of human oocytes for the treatment of infertility resulting from globozoospermia associated with deficient oocyte activation capacity. DESIGN: The mouse oocyte activation test was used to analyze the oocyte activation capacity of the sperm cells of a patient with globozoospermia. Fresh donor human oocytes were used for determining the most appropriate procedure for oocyte activation. SETTING: Infertility Center, University Hospital of Ghent. PATIENT(S): A couple with infertility resulting from globozoospermia. INTERVENTION(S): Intracytoplasmic sperm injection, assisted oocyte activation, and embryo transfer. MAIN OUTCOME MEASURE(S): Oocyte activation and fertilization rates, implantation, and pregnancy. RESULT(S): Deficiency in oocyte activation capacity was found in the sperm of a patient with globozoospermia. This deficiency was proven by the mouse oocyte activation test and was confirmed further by lack of activation of human oocytes after simple sperm injection. Only human oocytes that were injected with sperm and subjected to calcium chloride and ionophore treatment underwent activation. Transfer of embryos obtained by this procedure of assisted oocyte activation resulted in an ongoing pregnancy. CONCLUSION(S): Assisted oocyte activation of human oocytes is useful when globozoospermia is associated with absence of oocyte activation capacity in the sperm. These cases can be identified by the mouse oocyte activation test.

Influence of aging on fluidity and coupling between beta-receptors and G-proteins in rat lung membranes.

We tested the hypothesis that in rat lung membranes, the age-related decline in the percentage of beta-receptors coupled with high affinity to G-proteins, is due to limitation of the diffusion caused by a decrease in membrane fluidity. We measured both parameters simultaneously in a crude membrane preparation from lungs of rat of different age. In contrast to what is found in crude membrane preparations from rat liver and brain, in rat lung fluidity was increased upon aging. We conclude that the age-related alteration in coupling between receptor and G-protein is difficult to explain by alterations of membrane fluidity.

Ultrastructural alterations in the sialic acid distribution in minimal change disease and membranous glomerulonephritis.

Kidney biopsy specimens from patients with minimal change disease and membranous glomerulonephritis were embedded in glycolmethacrylate and stained with phosphotungstic acid (PTA) at low pH. Biopsy specimens from patients without proteinuria served as a control. The PTA staining at low pH on glycolmethacrylate sections was used to study the changes in the sialic acid content of the lamina rara externa of the glomerular basement membrane. This method also gives a clear picture of the changes occurring at the epithelial cell coat and these alterations have implications on the distribution of the negative charges. In minimal change disease no alterations could be observed in the sialic acid content of the lamina rara externa. But the luminal epithelial cell coat showed obvious changes in conjunction with extensive foot process widening. In membranous glomerulonephritis with heavy deposits the staining of the lamina rara externa became almost completely negative and the foot process architecture was strongly affected. Obvious defects at the luminal epithelial cell coat, as observed in minimal change disease, were also found regularly. The alterations at the epithelial cell coat are tentatively related to the selective proteinuria reported in minimal change disease. In addition the non-selective proteinuria observed in non-minimal glomerulopathies, may find its origin in the absence of sialic acid molecules from the lamina rara externa.

M cells are damaged and increased in number in inflamed human ileal mucosa.

Ileocolonoscopy and biopsies of patients with spondylarthropathy revealed gut inflammation in 62% of the cases. In order to better understand the pathogenetic mechanisms of spondylarthropathy related gut inflammation the follicle associated epithelium was examined. Biopsies from 9 controls and 18 patients with spondylarthropathy were studied by electron microscopy. Membranous (M) cells were investigated in normal and inflamed ileum. In normal mucosa M cells were scarce whereas in inflamed mucosa their number was increased (up to 24% of follicle associated epithelial cells). They showed a thin rim of cytoplasm covering groups of lymphocytes. In chronic ileitis necrotic M cells, ruptures of M cell cytoplasm and lymphocytes entering the gut lumen were observed. The bursts of M cells at the top of the lymphoid follicles lead to interruption of the gut epithelial lining and give luminal content access to the lymphoid tissue. This pathogenetic mechanism may cause aphthoid ulcers.

Spondylarthropathy and gut inflammation: immunopathogenetic mechanisms.

In 62% of patients with spondylarthropathy (SpA) we found clinically unsuspected gut inflammation to feature acute or chronic enterocolitis on ileocolonoscopy. A proportion of patients of the latter group had subclinical Crohn's disease as demonstrated clinically, endoscopically, genetically, histologically and immunopathologically. There is a relation between the activity of the gut inflammation and clinical rheumatic symptoms. A pathogenetic mechanism is increased handling of luminal (bacterial or nutritional) antigen, hence inducing T-cell activation and intestinal inflammation. In ileal mucosal biopsies we showed two different cellular events: increased enterocytic expression of class II molecules in active inflammation and an increase of membranous (M) cells overlying lymphocytes in inflamed mucosa. Although the number of intraepithelial lymphocytes was not increased in inflammation, intraepithelial T lymphocytes had protruding processes in close apposition to the tips of epithelial plasma membranes. Lymphocytes crossing the basement membrane were a frequent finding. These features indicate that the intestinal immune response caused by increased antigen processing in the terminal ileum induces an inflammatory immunocascade responsible for the clinical picture of SpA.

The Hermansky-Pudlak syndrome.

The Hermansky-Pudlak syndrome (HPS) associates oculocutaneous albinism with a haemorrhagic diathesis and the accumulation of ceroid-like material in different tissues. HPS is not an uncommon type of albinism as it was diagnosed in 13.5% (8/59) of our autosomal recessive albinos. These eight patients were evaluated ophthalmologically and haematologically. Apart from the symptoms caused by the albinism, accompanying signs vary from ecchymoses to life threatening haemorrhages and death by associated restrictive lung disease. Recognition of this syndrome by the ophthalmologist can be of major importance in this serious and eventually fatal disorder.

Endocytosis and multivesicular body formation in rabbit luteal cells during pseudopregnancy.

Horseradish peroxidase (HRP) was injected directly into the corpora lutea of rabbits on different days of pseudopregnancy. Young luteal cells have also been incubated "in vitro" in a medium containing horseradish peroxidase. In the "in vivo" experiments the 5 to 9 days old luteal cells take up far more horseradish peroxidase than the older ones (14-20 days). Different types of endocytic vacuoles, MvB's and also some DB's are already peroxidase positive shortly (15-20 min) after injection of the tracer. In the "in vitro" experiments the cells are more heavily loaded. The normal morphology of pale, dense and terminal MvB's is also described and staining with PTA at low pH provides further information on changes occurring in the MvB's. The various findings on multivesicular bodies are compared and two possible pathways for endocytic vacuoles are proposed: one to DB's the other to MvB's. The related phenomena of the uptake of material and the internalization of plasma membrane are discussed in the light of the possible function of endocytosis in luteal cells.

The axonal reticulum in the neurons of the superior cervical ganglion of the rat as a direct extension of the Golgi apparatus.

Adrenergic neurons from the superior cervical ganglion of the rat have been studied with the chromaffin reaction and the zinc iodide-osmium tetroxide method. Phosphotungstic acid staining at a low pH and a combined acid phosphatase reaction and phosphotungstic acid staining have also been performed on glycolmethacrylate-embedded tissue. The results indicate that phosphotungstic acid-positive elements lacking acid phosphatase activity are present at the inner side of the Golgi apparatus. These elements give rise directly to reticular differentiations, carrying catecholamines, or to tubular extensions, representing the origin of the axonal reticulum. On these tubules, reticular differentiations can again be formed at any level. In the cell body, the differentiations are mainly found close to the neurolemma. In the axons, they are especially abundant at the axon terminals. Large granules may be associated with the reticular differentiations and small and large granules may detach from them. It is concluded that the whole catecholamine-producing and/or -storing system in sympathetic neurons can be considered as a direct extension of the Golgi apparatus, set up for local catecholamine synthesis. The relative importance of small and large granules along this system may reflect th functional status of the nerve cell.

Decrease in the sialoglycoprotein content of the glomerular basement membrane in experimental nephrosis.

Phosphotungstic acid at low pH on glycolmethacrylate sections allows the detection of sialic acid groups in the lamina rara externa of the glomerular basement membrane. This staining procedure was used to study the alterations in puromycin aminonucleoside nephrosis and adriamycin nephrosis. In both model systems defects were detected in the sialic acid content of the lamina rara externa and the epithelial cell coat was also affected. The possible role of sialic acid in glomerular filtration is discussed.

Demonstration of sialic acid groups in the glomerular basement membrane of the rat with phosphotungstic acid at low pH.

This paper reports an unrecognized aspect of phosphotungstic acid staining at low pH. It provides an on-section staining method in which sialic acid-containing molecules can be demonstrated in the laminae rarae of the rat glomerular basement membrane. The staining in the basement membrane became negative after perfusion with the following cations: protamine sulphate, hexadimethrine, Alcian Blue, Ruthenium Red and Toluidine Blue. Blocking was not achieved with Alcian Blue at about pH 1. The staining was also abolished after mild methylation and demethylation restored the contrast. This is suggestive of the involvement of carboxyl groups. Prior digestion with pronase, trypsin and neuraminidase rendered the laminae rarae negative, whereas hyaluronidase, chondroitinase ABC and crude heparinase were without effect. This indicates that sialic acid groups are detected by this method and that heparan sulphate does not interfere. The staining of the epithelial plasma membrane, also carrying sialic acid groups, remained positive after neuraminidase treatment. It is presumed that this method can be applied successfully for detecting changes in the sialic acid content of the laminae rarae in rat glomerular basement membranes under normal and pathological conditions.