Distribution of N- and O-linked oligosaccharides on surface of spermatozoa from normal and infertile subjects

Sperm glycocalyx modifications are very important for gamete recognition and fertilisation in mammals. These processes may be associated with specific changes in the content and distribution of surface carbohydrates. The purpose of this study was to determine the distribution of surface carbohydrates in human spermatozoa from normal and oligospermic subjects. Fifteen ejaculates each from normal fertile and oligospermic individuals were analysed. N-linked and O-linked surface carbohydrates were detected by fluorescence microscopy using fluorescein isothiocynate-conjugated lectins. Triticum vulgaris agglutinin (WGA)-binding sites were found to be decreased on acrosomal domain in spermatozoa from oligospermic individuals, while no changes were observed in the binding sites of Concanavalin ensiformis, Peanut agglutinin and Lens clunaris agglutinin. A reduction in binding sites for soybean agglutinin and Ricinus communis agglutinin was observed on the acrosomal domains in spermatozoa from oligospermic individuals. Changes in sperm glycocalyx observed in this study provide new insights into molecular rearrangement of sperm membrane in infertility.     

Characterizing the glycocalyx of poultry spermatozoa: I. Identification and distribution of carbohydrate residues using flow cytometry and epifluorescence microscopy

The aim of the present work was to use a battery of lectins to 1) delineate the carbohydrate content of sperm glycocalyx in the turkey and chicken using flow cytometry analysis, and 2) evaluate the distribution of existing sugars over the sperm plasma membrane surface with epifluorescent microscopy. Carbohydrate groups (corresponding lectins) that were investigated included galactose (GS-I, Jacalin, RCA-I, PNA), glucose and/or mannose (Con A, PSA, GNA), N-acetyl-glucosamine (GS-II, s-WGA, STA), N-acetyl-galactosamine (SBA, WFA), fucose (Lotus, UEA-I), sialic acid (LFA, LPA), and N-acetyl-lactosamine (ECA). Spermatozoa were assessed before and after treatment with neuraminidase to remove sialic acid. Mean fluorescence intensity (MnFI) was used as indicator of lectin binding for flow cytometry analysis. Nontreated spermatozoa from both species showed high MnFI when incubated with RCA-I, Con A, LFA, and LPA, as did chicken spermatozoa incubated with s-WGA. Neuraminidase treatment increased the MnFI for most lectins except LFA and LPA, as expected. Differences in MnFI between species included higher values for s-WGA and ECA in chicken spermatozoa and for WFA in turkey spermatozoa. Microscopy revealed segregation of some sugar residues into membrane-specific domains; however, the 2 staining techniques (cell suspension vs fixed preparation) differed in identifying lectin binding patterns, with fixed preparations yielding a high degree of nonspecific binding. We conclude that 1) the glycocalyx of turkey and chicken spermatozoa contains a diversity of carbohydrate groups, 2) these residues are extensively masked by sialic acid, 3) the glycocalyx composition is species-specific, and 4) some glycoconjugates appear to be segregated into membrane-specific domains. Characterization of the poultry sperm glycocalyx is the first step in identifying the physiological impact of semen storage on sperm function.     

利用氩气凝血器行不阻断肝门的肝叶切除术的临床研究

自１９９０年１０月－１９９２年７月共行肝叶切除术４１例，其中２１例用氩气凝血器止血，切肝时不阻断肝门。对照组２０例行常规的肝切除方法。研究结果表明：利用ＡＢＣ行肝叶切除术可以不阻断入肝血流，且具有减少术中出血量，术后不阻断入肝血流，且具有减少术中出血量，术后并发症少，残肝断面可不缝合，不附加任何覆盖物等优点。     

ConA-binding proteins of the sperm surface are conserved through evolution and in sperm maturation

Summary.  Lectin-binding glycoconjugates present on the surface of spermatozoa are believed to play a crucial role in sperm maturation, capacitation, acrosome reaction, or sperm-egg interaction. We have studied ConA-binding surface proteins on spermatozoa from different mammalian species. First, ConA-binding proteins were isolated from boar spermatozoa by affinity chromatography. ConA-binding ability was confirmed by Enzyme-linked Lectin assay (ELLA). Monoclonal (MAb436/10) and polyclonal antibodies were raised against chromatography fractions containing purified ConA-binding proteins of boar spermatozoa. MAb436/10 (IgG_2a) recognizes a 40 kD ConA-binding antigen. Indirect immunofluorescence on fixed and unfixed boar spermatozoa with MAb436/10 indicated a plasma membrane localization of antigen 436/10 in the acrosomal macrodomain. Interspecies cross-reactivity with MAb436/10 was found by whole cell ELISA and immunocytochemistry. MAb436/10 cross-reacted with human, horse, guinea-pig, bull, and ram spermatozoa in both assays. Expression of ConA-binding antigen 436/10 on guinea pig sperm surface was detectable during spermiogenesis and in early stages of sperm maturation. Change of regionalization of the antigen did not occur during the epididymal passage. ConA-binding antigen 436/10 was also detectable in testis and caudal segments of the epididymis. These findings suggest that ConA-binding proteins located in the acrosomal region are highly conserved through evolution as well as in sperm maturation indicating an important role for the physiology of spermatozoa.     

Electron microscopic investigation of the bladder urothelium and glycocalyx in patients with interstitial cystitis

The electron microscopic appearance of the bladder urothelium and glycocalyx was investigated in ten patients with well defined interstitial cystitis and compared to the findings in ten control patients with stress incontinence as the only symptom. Ruthenium red, a polycationic dye which binds specifically to cell surface acid polysaccharides, was used to demonstrate the glycocalyx. In cases of interstitial cystitis two types of luminal cell were observed, each possessing a distinct surface glycocalyx. One type of cell possessed numerous plaques of asymmetric unit membrane associated with a relatively thin glycocalyx. The second type of cell was characterised by numerous microvilli and a relatively thick glycocalyx. In control material each type of cell and its associated glycocalyx was identified with similar frequency. Our study concludes that there are no differences in the morphologic appearances of the glycocalyx and of urothelial cells in patients with interstitial cystitis when compared with controls. Hence, the hypothesis that an important pathogenic factor in interstitial cystitis is a defective glycocalyx associated with a permeable urothelium, has not been supported.     

Escherichia coli-induced alterations of human spermatozoa. An electron microscopy analysis

This study evaluated if the negative influence of Escherichia coli on the motility of human spermatozoa is a consequence of E. coli-induced ultrastructural alterations. Suspensions of spermatozoa were artificially infected with E. coli from a serotyped, pathogenic strain and incubated at 37 degrees C for 6 h. After incubation, spermatozoa were fixed in glutaraldehyde, stained with osmium tetroxide and ruthenium red and embedded in Spurr(R)-resin followed by ultramicrotomy. The sections were analysed subsequently by use of transmission electron microscopy. Uninfected suspensions of spermatozoa in medium and bacterial suspensions served as controls. Negative contrast technique was performed to facilitate visualization of ultrastructural details of the bacterial capsule after experimental exposure to spermatozoa. Electron microscopic evaluation revealed multiple and profound alterations in the ultrastructure of spermatozoa such as membrane defects and cytoplasmic vacuoles exclusively in spermatozoa of infected samples (> 90%). Morphological alterations involved all superficial structures of spermatozoa, in particular the plasma membrane of the mid-piece and neck as well as the inner and outer acrosomal membrane of the acrosome, indicating that morphological defects account for the immobilization of spermatozoa by E. coli. The results suggest that E. coli infection of ejaculates results in immobilization and impaired acrosomal function in human spermatozoa, findings that support the indication for antimicrobial chemotherapy in symptomatic and silent infections that affect the ejaculate.     

Capacitated and acrosome reacted spermatozoa of goat (Capra indicus): a fluorescence and electron microscopic study

Summary. Plasma membrane alterations accompanying in vitro capacitation and acrosome reaction of goat spermatozoa were studied using lectin labelling, scanning electron microscopy, and freeze-fracture methods. Fluorescein isothi-ocyanate linked lectins namely; Canavalia ensiformis (ConA), Maclura pomifera (MPA), Arachis hypogaea (PNA), Glycine max (SBA) and Triticum vulgaris (WGA) agglutinin were used to examine the distribution of surface carbohydrates during these two events. The head and the sperm tail reveal altered lectin labelling features after capacitation and acrosome reaction. After capacitation the surface coat components for MPA, SBA, and WGA are shed from the spermatozoon head. ConA receptors on the head are retained after capacitation but are partially shed in the acrosome reacted spermatozoa. SBA receptor sites appear on the sperm tail of the capacitated spermatozoa. Unusual morphological changes attending capacitation involve the sperm tail-end which develops a novel entity, which we have termed 'spatula'. The 'spatula' shows strong binding with ConA and WGA only. In the acrosome reacted spermatozoa the spatulated tail-end unwinds with a concomitant loss of lectin labelling. Highly ordered membrane particles, 'ladders' of the middle piece of the epididymal sperm tail, disappear and IMP clearings appear on the middle piece and in the spatulated ends of the capacitated spermatozoa. But in the acrosome reacted sperm IMPs reappear and are randomly disposed on the middle-piece and are clustered in small patches on the principal-piece. IMP free areas appear on the plasma membrane covering the acrosome and the outer acrosomal membrane (OAM) of the capacitated spermatozoa. The plasma membrane and OAM fuse at multiple foci and appear as acrosomal 'ghosts' which remain associated with the sperm head even after acrosome reaction.     

Motility initiation of quiescent spermatozoa from rat caudal epididymis: effects of pH, viscosity, osmolality and inhibitors

Non-motile spermatozoa freshly extruded from the rat caudal epididymis can be initiated to full motility immediately after diluting with 0.9% NaCl. The motility initiation was dependent on the pH, viscosity and osmolality of the diluent. Diluent with pH 4 to 8 could optimally initiate the motility. Osmolality of most diluents suitable for the initiation was between 130 to 600 mOsm/kg. The motility initiation was inhibited by Hg²⁺ > Cu²⁺ > Cd²⁺, chlorpromazine, Triton X-100 and SDS. The following compounds showed essentially no inhibitory effect: EGTA, chlortetracycline, calcein, ruthenium red, phloridzin, myo-inositol and carnitine. The findings suggested that spermatozoa were kept in quiescence in the cauda epididymis not by the pH, osmolality, viscosity, myo-inositol, carnitine, Ca²⁺ or K⁺ of the caudal epididymal fluid. It was also suggested that motility initiation did not involve Ca²⁺. calmodulin and transport of Ca²⁺ or glucose across sperm membrane.     

Hydrocortisone preserves the vascular barrier by protecting the endothelial glycocalyx

BACKGROUND: Hydrocortisone protects against ischemia-reperfusion injury, reduces paracellular permeability for macromolecules, and is routinely applied in the prevention of interstitial edema. Healthy vascular endothelium is coated by the endothelial glycocalyx, diminution of which increases capillary permeability, suggesting that the glycocalyx is a target for hydrocortisone action. METHODS: Isolated guinea pig hearts were perfused with Krebs-Henseleit buffer. Hydrocortisone was applied in a stress dose (10 microg/ml) before inducing 20 min of ischemia (37 degrees C). Hearts were reperfused for 20 min at constant flow (baseline perfusion pressure, 70 cm H2O) with Krebs-Henseleit buffer or Krebs-Henseleit buffer plus 2 g% hydroxyethyl starch (130 kd). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Hearts were perfusion fixed to visualize the glycocalyx. RESULTS: Ischemia-induced degradation of the glycocalyx enhanced coronary perfusion pressure (118.8 +/- 17.3 cm H2O) and increased vascular permeability (8 +/- 0.2 microl x min(-1) x cm H2O(-1) at baseline vs. 34 +/- 3.3 microl x min(-1) x cm H2O(-1) after reperfusion). Enzymatic digestion of the glycocalyx (heparinase) elicited similar effects. Hydrocortisone reduced postischemic oxidative stress, perfusion pressure (86.3 +/- 6.4 cm H2O), and transudate formation (11 +/- 0.6 microl x min(-1) x cm H2O(-1)). Applying colloid augmented this (70.6 +/- 5.6 cm H2O and 9 +/- 0.5 microl x min(-1) x cm H2O(-1)). Postischemic shedding of syndecan-1, heparan sulfate, and hyaluronan was inhibited by hydrocortisone, as was release of histamine from resident mast cells. Electron microscopy revealed a mostly intact glycocalyx after hydrocortisone treatment, but not after heparinase treatment. CONCLUSIONS: Hydrocortisone preserves the endothelial glycocalyx, sustaining the vascular barrier and reducing interstitial edema. The effect of colloids suggests that prevention of postischemic rise in coronary resistance by hydrocortisone could also be based on alleviation of endothelial swelling. Stabilization of myocardial mast cells by hydrocortisone may account for the mitigated inflammatory affect of ischemia-reperfusion.     

Cytochalasin D inhibits penetration of hamster eggs by guinea pig and human spermatozoa

Fertilization experiments using zona-free hamster eggs and spermatozoa from both guinea pig and human were conducted in the presence of cytochalasin D to evaluate the possible role of actin filaments in fertilization processes. When the actin filament inhibitor, cytochalasin D, was added to fertilization media at concentrations of 10 to 30 microM, penetration of eggs was significantly inhibited. Preincubation of the eggs with cytochalasin D and washing prior to addition of spermatozoa had no effect on penetration as quantitated by the number of swollen heads in the egg cytoplasm. However, spermatozoa preincubated with cytochalasin D and subsequently washed prior to egg addition showed reduced penetration of the same magnitude as when spermatozoa and eggs were coincubated with cytochalasin D. Both the percentage of zona-free eggs showing decondensed sperm heads and the penetration indices (total decondensed spermatozoa/total eggs) were significantly affected when spermatozoa were exposed to cytochalasin D. The DMSO vehicle used to dissolve cytochalasin D had little effect on the number of decondensed heads. When the concentration of cytochalasin D was increased (DMSO remaining constant) in human sperm experiments, percent penetration decreased and progressively fewer decondensed spermatozoa were recorded, indicating dose-responsiveness to cytochalasin D. Motility parameters of human spermatozoa were not altered at any of the concentrations of cytochalasin D tested. Neither guinea pig sperm motility nor acrosome reaction was altered significantly by cytochalasin D or the DMSO vehicle. These experiments suggest that cytochalasin D may be an inhibitor of some fertilization processes such as sperm penetration or sperm head decondensation.     

Glycocalyx damage estimated using colloidal iron staining

Anionic constituents in the peritubular capillary basement membranes and the glomerular endothelial cells have been demonstrated to function as a size- and charge-selective barrier. Cationic colloidal iron staining of human biopsy specimens revealed a glycocalyx on the surface of the glomerular basement membrane (GBM), peritubular capillary (PTC) endothelial cells, and brush border of the tubular epithelial cells of normal kidney. However, the glycocalyx was abolished in the PTC wall of C4d-positive acute humoral rejected kidney, and in the GBM as well as the PTC wall of a chronic, allograft, nephropathy kidney. In addition, cyclosporine eliminated the glycocalyx in the PTC wall, while treatment with heparin inhibited the elimination of the PTC glycocalyx. In conclusion, the glycocalyx on the surface of the GBM and PTC is an important component in the endothelial cell barrier.     

Hydrocortisone Preserves the Vascular Barrier by Protecting the Endothelial Glycocalyx

Background: Hydrocortisone protects against ischemia-reperfusion injury, reduces paracellular permeability for macromolecules, and is routinely applied in the prevention of interstitial edema. Healthy vascular endothelium is coated by the endothelial glycocalyx, diminution of which increases capillary permeability, suggesting that the glycocalyx is a target for hydrocortisone action.

Methods: Isolated guinea pig hearts were perfused with Krebs-Henseleit buffer. Hydrocortisone was applied in a stress dose (10 [mu]g/ml) before inducing 20 min of ischemia (37[degrees]C). Hearts were reperfused for 20 min at constant flow (baseline perfusion pressure, 70 cm H2O) with Krebs-Henseleit buffer or Krebs-Henseleit buffer plus 2 g% hydroxyethyl starch (130 kd). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Hearts were perfusion fixed to visualize the glycocalyx.

Results: Ischemia-induced degradation of the glycocalyx enhanced coronary perfusion pressure (118.8 +/- 17.3 cm H2O) and increased vascular permeability (8 +/- 0.2 [mu]l [middle dot] min-1 [middle dot] cm H2O-1 at baseline vs. 34 +/- 3.3 [mu]l [middle dot] min-1 [middle dot] cm H2O-1 after reperfusion). Enzymatic digestion of the glycocalyx (heparinase) elicited similar effects. Hydrocortisone reduced postischemic oxidative stress, perfusion pressure (86.3 +/- 6.4 cm H2O), and transudate formation (11 +/- 0.6 [mu]l [middle dot] min-1 [middle dot] cm H2O-1). Applying colloid augmented this (70.6 +/- 5.6 cm H2O and 9 +/- 0.5 [mu]l [middle dot] min-1 [middle dot] cm H2O-1). Postischemic shedding of syndecan-1, heparan sulfate, and hyaluronan was inhibited by hydrocortisone, as was release of histamine from resident mast cells. Electron microscopy revealed a mostly intact glycocalyx after hydrocortisone treatment, but not after heparinase treatment.     

Sperm washing method alters the ability of seminal plasma proteins to revert the cold-shock damage on ram sperm membrane

Prolonged exposure of spermatozoa to seminal plasma (SP) has been found to have adverse effects on sperm function. Therefore, the separation of mammalian spermatozoa from SP is a practice routinely used in the laboratory and in assisted reproductive technology application. We have previously shown that adsorption of seminal plasma proteins (SPP) to the sperm cell surface partially restores the functional characteristics of damaged spermatozoa, reproducing those of live cells. In the present report, we have compared the influence of two different semen washing methods, a dextran/swim-up or a filtration procedure, on the SPP adsorption to the sperm surface, and on their ability to recover membrane integrity of cold-shocked sperm. Seminal plasma proteins were added to cold-shocked sperm samples obtained by both washing procedures. Adsorption of proteins to the sperm membrane surface was assessed by centrifugal counter-current distribution (CCCD) in an aqueous two-phase system, as well as membrane integrity being determined by fluorescence markers. The percentage of reversion of cold-shock effect in the sample containing plasma proteins with respect to the control sample was determined by assessing the percentage of membrane-intact spermatozoa, i.e. propidium iodide-negative. The addition of 700 microg of SPP to the swim-up damaged samples caused a 32% reversion, whereas the reversal percentage found with the same amount of SPP on filtered damaged samples was 10% (p < 0.05). Likewise, the loss of heterogeneity and the decrease in viability after the cold-shock revealed by CCCD analysis were greatly reversed by the addition of SPP to sperm samples obtained by swim-up. However, this restorative effect was again much lower when SPP were added to the cold-shocked filtered sample. These results strongly suggest that the washing method influences the ability of SPP to recover membrane integrity of cold-shocked sperm. Separation of spermatozoa by swim-up accounts for higher viability recovery than that obtained by filtration washing.     

Anti‐oxidative Status and Semen Quality during Cooled Storage in Stallions

Activity of the anti‐oxidative enzymes glutathione peroxidase (GSH‐Px), superoxide dismutase (SOD) and catalase (CAT), content of thiobarbituric acid reactive substances (TBARS) and SH‐groups were determined in native stallion semen (n = 8 stallions). Semen was then diluted in Kenney extender, EquiPro^® extender either with or without addition of N‐acetyl cysteine or phosphate‐buffered saline (PBS) and stored for 72 h at 5°C. Correlations between initial activity of enzymes and development of semen motility and membrane integrity were calculated. Activities of GSH‐Px, SOD and CAT immediately after semen collections were 10.0 ± 0.6 picokatals, 0.40 ± 0.03 SOD units and 0.70 ± 0.05 nanokatals/10⁶ spermatozoa respectively. TBARS content was 0.06 ± 0.01 nmol and SH‐group content 1.7 ± 0.5 mmol/10⁶ spermatozoa. The loss of motile spermatozoa during storage did not differ between extenders. N‐acetyl cysteine had no effect on semen motility and membrane integrity. The loss in membrane‐intact spermatozoa was highest (P < 0.05) in semen diluted in PBS. Motility and membrane integrity after addition of extender were positively correlated with GSH‐Px and CAT, indicating that anti‐oxidative mechanisms contribute to the initial high percentage of motile and membrane‐intact spermatozoa. However, in these samples the decrease in semen quality was most pronounced. No correlations existed between initial activity of anti‐oxidative enzymes, peroxidation products and semen quality during storage. This indicates that once extender has been added, peroxidative damage to sperm membranes is not the predominant cause of losses in semen quality.     

Microvascular effects of chondroitinase ABC and chymopapain. An in vivo experimental study on hamsters and rabbits

Immediate and long-term microvascular effects of chondroitinase ABC, 200 unit/ml, were analyzed in ten hamsters. The immediate effects on the microcirculation were studied by vital microscopy following local injection in the cheek pouch. There were no detectable effects on the microvascular blood flow during the 60 minutes of observation for chondroitinase ABC or the control. A therapeutic concentration (2000 pKat/ml) of chymopapain stopped the microcirculation in the injected area immediately, with numerous microbleedings at the border zone. Long-term effects were studied after subcutaneous injections in the ears of six rabbits. Chondroitinase ABC and the control did not cause any macroscopic or microangiographic effects. However, light microscopy showed a moderate inflammatory reaction in the subcutaneous layer for both chondroitinase ABC and the control. Chymopapain induced severe effects on the cartilage and surrounding tissues. Microangiography revealed a vessel-free zone at the injection site. Since 200 units/ml of chondroitinase ABC is four to eight times higher than the concentration that might be used for chemonucleolysis, i.e., dissolution of intervertebral discs by local enzyme injection, the present investigation suggests a wide margin of safety regarding the potential effects on blood vessels in tissues surrounding the disc.     

Mitochondrial permeability transition and cytochrome c release in ischemia-reperfusion injury of the rat liver

BACKGROUND: We investigated whether ischemia-reperfusion causes activation of caspases and whether this activation is related to cytochrome c release from the mitochondria into the cytosol as a result of the mitochondrial inner membrane permeability transition. MATERIALS AND METHODS: Rats were subjected to 30 min to 120 min of hepatic ischemia followed by 6 h of reperfusion. Cyclosporin A or ruthenium red (inhibitors of the mitochondrial inner membrane permeability transition) was given intravenously at 60 and 30 min before ischemia, respectively. RESULTS: Reperfusion after ischemia caused the release of liver enzymes accompanied by mitochondrial membrane depolarization, DNA fragmentation, and translocation of cytochrome c from the mitochondria into the cytosol. Accumulation of cytochrome c in the cytosol and activation of caspase-3-like protease was already detected during ischemia and before reperfusion. Pretreatment with cyclosporin A or ruthenium red significantly ameliorated the loss of the mitochondrial membrane potential, the increase of plasma membrane permeability, the cytosolic accumulation of cytochrome c, DNA fragmentation, and caspase-3-like protease activation. CONCLUSIONS: The mitochondrial inner membrane permeability transition occurs during ischemia and/or after reperfusion, resulting in translocation of cytochrome c and activation of caspases.     

The Effect of Caffeine on Guinea Pig Epididymal Spermatozoa: Motility and Fertilizing Capacity

Guinea pig epididymal spermatozoa, when taken from different segments of the epididymis, are known to differ markedly in their in vitro motility, metabolism and morphology. In this report, the fertilizing capacity of epididymal spermatozoa isolated from proximal and distal segments and treated with caffeine was tested. Twenty six guinea pig sows were inseminated 16–26 h after parturition with isolated epididymal spermatozoa. In 15 cases the spermatozoa were treated with caffeine (10 mM), and 11 cases served as controls. Sperm characteristics (mean ± SE) were: proximal sperm; concentration 48 ± 3.9 millions/ml, vitality 94 ± 0.9%, motility 42 ± 5.3%. Distal sperm: concentration 161 ± 21 million/ml, vitality 94 ± 0.9% and motility 86 ± 3.0%. Caffeine increased significantly the motility of proximal spermatozoa (by 59.5%). These suspensions of spermatozoa were used for intraperitoneal artificial insemination.
The impregnation rate of untreated proximal sperm was zero (0 out of 4), compared with 83% (5 out of 6) for the caffeine treated sperm. The impregnation rate of untreated distal segment sperm was 42.8% (3 out of 7) compared with 100% (9 out of 9) for caffeine treated sperm.     

Alterations of anionic charge and/or sites of the glomerular basement membrane in the heterologous phase of passive Heymann nephritis.

Alterations of the anionic charge and/or sites of the glomerular basement membrane (GBM) in the heterologous phase of passive Heymann nephritis (PHN) were studied. Rats with PHN induced by a single injection of anti-Fx1A IgG were examined at days 1, 2, 3 and 4. The left kidney was perfused with ruthenium red (RR) solution as a cationic probe. The RR particles (= anionic sites) in the GBM were counted and expressed as the number of RR particles per unit length of GBM. For quantitative determination of the total anionic charge of the GBM, the GBM-bound ruthenium (= anionic charge) was measured with an atomic absorption spectrophotometer (AAS). Abnormal proteinuria corresponding to a decrease in anionic charge was detected at days 3 and 4. The anionic sites in the lamina rara externa (LRE) adjacent to immune complex (IC) deposits were found to have diminished earlier from day 1 onwards. This diminution was largely confined to areas adjacent to the IC deposits and was significantly correlated with the amount of urinary albumin excretion. Proteinuria in the heterologous phase of PHN would thus appear to be causally related to a decrease in the number of anionic sites in the LRE adjacent to IC deposits.     

Overtime expression of plasma membrane and mitochondrial function markers associated with cell death in human spermatozoa exposed to nonphysiological levels of reactive oxygen species

In many cell types, the potential of reactive oxygen species to induce death processes has been largely demonstrated. Studies in spermatozoa have associated the imbalance of reactive oxygen species and phosphatidylserine externalisation as an apoptosis marker. However, the lack of consensus about time effect in the joint expression of these and other death markers has made it difficult to understand the set of mechanisms influenced beyond the concentration effect of reactive oxygen species to stimulate cell death. Here, the plasma membrane permeability and integrity, phosphatidylserine externalisation and mitochondrial membrane potential were jointly evaluated as death markers in human spermatozoa stimulated with H₂O₂. The results showed a profound and sustained effect of dissipation in the mitochondrial membrane potential and an increased phosphatidylserine externalisation in human spermatozoa exposed to 3 mmol⁻¹ of H₂O₂ at 30 min. This was followed by an increased membrane permeability after 45 min. The last observed event was the loss of cell membrane integrity at 60 min. In conclusion, mitochondria are rapidly affected in human spermatozoa exposed to reactive oxygen species, with the barely detectable mitochondrial membrane potential coexisting with the high phosphatidylserine externalisation in cells with normal membrane permeability.     

The anionic sites at luminal surface of peritubular capillaries in rats

Anionic sites have been demonstrated in the basement membranes of peritubular capillaries. The anionic barrier function of peritubular capillary wall has been ascribed to these sites. Fenestrated capillaries in other organs have anionic sites in the endothelial cell glycocalyx and at the luminal surface of the fenestral diaphragms. The purpose of this study was to map anionic sites at the luminal surface of peritubular capillaries and to assess whether a concentration gradient for albumin exists across the endothelium. Partial chemical characterization of these anionic sites was done by in vivo enzymatic degradation. The difference in distribution of albumin following enzyme digestion was also studied. The binding of cationized ferritin to the luminal surface indicated that the rat peritubular capillaries have anionic sites along the entire luminal surface of the endothelial cell, including the fenestral diaphragms. Partial biochemical characterization of these sites shows that the sites in the glycocalyx are mainly from neuraminic acid, while the fenestral diaphragms have mainly heparan sulfate proteoglycans. Intravascular albumin extended to the endothelial luminal plasmalemma and to the luminal surface of fenestral diaphragms. Digestion with heparitinase was associated with the leakage of albumin outside the capillary wall. These findings suggest that the anionic surface of fenestrae constitutes a charge barrier of the peritubular capillaries.