The structure of mast cell secretory granules in the blind mole rat (Spalax ehrenbergi).

Eosinophils, basophils, and mast cells produce and secrete active substances whose role is to attack invading parasites and protect the host. In this study we use morphometric methods to study mast cells in the blind mole rat (Spalax ehrenbergi). The subterranean and solitary way of life of this species has led to the evolutionary development of special anatomical, morphological, behavioral, and physiological adaptations. Because of its particular lifestyle, the mole rat is less exposed to parasites than other rodents. This could provide a unique model for research into the pathobiology of mast cells. The paracrystalline structure of the mast cell granule content is composed of parallel plates. Diffraction analysis of electron micrographs of thin sections of araldite-embedded tissues indicated that each crystal line plate is a periodic array of parallelograms. The crystal unit cell volume is approximately 930 nm(3), suggesting that each unit cell is composed of one heparin molecule and one to three additional adsorbed proteins. Morphometric data show that characteristics of the secretory granules of mast cells of the blind mole rat resemble those of other rodents. The mast cell unit granule volume in the present study was calculated to be 0.055 microm(3), similar to that of rat peritoneal mast cells.

Morphometric studies of secretory granule formation in mouse pancreatic acinar cells. Dissecting the early structural changes following pilocarpine injection.

Secretory granule formation in pancreatic acinar cells is known to involve massive membrane flow. In previous studies we have undertaken morphometry of the regranulation mechanism in these cells and in mast cells as a model for cellular membrane movement. In our current work, electron micrographs of pancreatic acinar cells from ICR mice were taken at several time points after extensive degranulation induced by pilocarpine injection in order to investigate the volume changes of rough endoplasmic reticulum (RER), nucleus, mitochondria and autophagosomes. At 2-4 h after stimulation, when the pancreatic cells demonstrated a complete loss of granules, this was accompanied by an increased proportion of autophagosomal activity. This change primarily reflected a greatly increased proportion of profiles retaining autophagic vacuoles containing recognisable cytoplasmic structures such as mitochondria, granule profiles and fragments of RER. The mitochondrial structures reached a significant maximal size 4 h following injection (before degranulation 0.178 +/- 0.028 microm3; at 4 h peak value, 0.535 +/- 0.109 microm3). Nucleus size showed an early volume increase approaching a maximum value 2 h following degranulation. The regranulation span was thus divided into 3 stages. The first was the membrane remodelling stage (0-2 h). During this period the volume of the RER and secretory granules was greatly decreased. At the intermediate stage (2-4 h) a significant increase of the synthesis zone was observed within the nucleus. The volume of the mitochondria was increasing. At the last step, the major finding was a significant granule accumulation in parallel with an active Golgi zone.

Mechanisms leading to cortical reaction in the mammalian egg.

Activation of the mammalian egg results in cortical reaction (CR), which is correlated with an increase in intracellular Ca2+ concentration and PKC activation. The CR is a gradual rather then an "all or none" response, and can be regulated by different concentrations of parthenogenetic activators. To evaluate the biological significance of parthenogenetic induced CR, rat eggs were fertilized or activated by different concentrations of ionomycin and TPA. Cortical granules (CG) were monitored by electron microscopy, while the CG exudate was visualized by Lens culinaris lectin and Texas Red, using light and confocal microscopy. The ability of the CR to trigger a full block to polyspermy was examined in an IVF system. Our study demonstrates the existence of light and dark CG, which differ by number, distribution in the egg cortex, and sensitivity to parthenogenetic activators. Sperm penetration or high concentration of activators, trigger depletion of both light and dark CG, leading to a full CR. Low concentration of activators altered the CG density, the ratio of dark/light CG, and induced partial CR that was sufficient to cause a block to polyspermy. The results imply that Ca2+ rise or PKC activation have different effects on light and dark CG. In recently fertilized or parthenogenetically activated eggs, CG exudate appeared as evenly distributed spots, whereas in more advanced stages of fertilization the exudate was scattered as patchy aggregates. This observation suggests a difference in the dispersion of CG exudate after fertilization as compared to parthenogenetic activation.

Improved hypothermic preservation of rat hearts by furosemide.

The effect of furosemide, a blocker of the Na+/K+/Cl- cotransporter, on hypothermic preservation of rat hearts was studied with use of the Langendorff perfusion system and electron microscopy. Furosemide significantly improved the mechanical recovery and the coronary flow of the hearts preserved for 8 hours in St. Thomas' Hospital cardioplegic solution at a temperature of 4 degrees C. Furosemide at the concentration of 100 mumol/L was found to have an optimal effect, whereas at high concentrations (1000 mumol/L) it was found to have toxic effects. In addition, furosemide reduces the time elapsed between the end of the preservation time and the resumption of myocardial contractions. Ultrastructural evaluations were done in which the presence of swollen mitochondria was chosen as a criterion of hypothermic ischemic damage to the myocardium. Morphometric analysis indicated that the mitochondrial volume of hearts stored for 8 hours in St. Thomas' Hospital cardioplegic solution increased by 72% as compared with the mitochondrial volume of hearts that were not exposed to the hypothermic ischemic conditions (control group). The addition of 100 mumol/L furosemide to the cardioplegic solution resulted in a significant reduction of mitochondrial swelling during the period of 8 hours' storage, which amounted only to 28% as compared with the figure for the control group. The reduction of mitochondrial swelling by furosemide and the improved mechanical and coronary flow recoveries are thought to be related to the blocking of the sarcolemmal Na+/K+/Cl- cotransporter and consequently the reduction of the Na+ influx during hypothermic ischemic storage.

Distribution of lectin receptors sites in the zona pellucida of follicular and ovulated rat oocytes.

Carbohydrates of the zona pellucida (ZP) in mammals are believed to have a role in sperm-egg interaction. We have characterized the biochemical nature and distribution of the carbohydrate residues of rat ZP at the light (LM) and electron microscope (EM) levels, using lectins as probes. Immature female rats were induced to superovulate and cumulus-oocyte complexes were isolated from the oviduct, fixed with glutaraldehyde, and embedded in araldite for LM and LR-Gold for EM histochemistry. For examination of follicular oocytes, rat ovaries were fixed with glutaraldehyde and embedded in paraffin. The araldite or paraffin sections were deresined or deparaffinized, respectively, labeled with biotin-tagged lectins as probes, and avidin-biotin-peroxidase complex as visualant. For EM examination, thin LR-Gold sections were labeled with RCA-I colloidal gold complex (RCA/G) and stained with uranyl acetate. LM analyses indicate that in ovulated oocytes the ZP intensely binds peanut agglutinin (PNA); succinylated wheat germ agglutinin, (S-WGA), Griffonia simplisifolia agglutinin-I (GS-I) and soybean agglutinin (SBA), and to a lesser extent, lectins from Ricinus communis (RCA-I), Concanavaia ensiformis (Con A), Ulex europoeus (UEA-I), and wheat germ agglutinin (WGA). The neighboring cumulus cells are considerably less reactive and exhibit membrane staining only with Con A, WGA, and PNA. EM analysis of RCA/G binding revealed intensive binding to the inner layer region of the ZP and moderate binding to cytoplasmic vesicles of the cumulus cells. The ZP of follicular oocytes exhibits a different lectin binding pattern, expressed in staining strongly with PNA and S-WGA, and in a tendency of the lectin receptors to occur in the outer portion of the ZP.(ABSTRACT TRUNCATED AT 250 WORDS)