Evidence that ADH-stimulated intramembrane particle aggregates are transferred from cytoplasmic to luminal membranes in toad bladder epithelial cells

In freeze-fracture (FF) preparations of ADH-stimulated toad urinary bladder, characteristic intramembrane particle (IMP) aggregates are seen on the protoplasmic (P) face of the luminal membrane of granular cells while complementary parallel grooves are found on the exoplasmic (E) face. These IMP aggregates specifically correlate with ADH-induced changes in water permeability. Tubular cytoplasmic structures whose membranes contain IMP aggregates which look identical to the IMP aggregates in the luminal membrane have also been described in granular cells from unstimulated and ADH-stimulated bladders. The diameter of these cytoplasmic structures (0.11 +/- 0.004 micrometers) corresponds to that of tubular invaginations of the luminal membrane seen in thin sections of ADH-treated bladders (0.13 +/- 0.005 micrometers). Continuity between the membranes of these cytoplasmic structures (which are not granules) and the luminal membrane has been directly observed in favorable cross-fractures. In FF preparations of the luminal membrane, these apparent fusion events are seen as round, ice-filled invaginations (0.13 +/- 0.01 micrometer Diam), of which about half have the characteristic ADH-associated aggregates near the point of membrane fusion. They are less numerous than, but linearly related to, the number of aggregates counted in the same preparations (n = 78, r = 0.71, P less than 0.01). These observations suggest that the IMP aggregates seen in luminal membrane after ADH stimulation are transferred preformed by fusion of cytoplasmic with luminal membrane.     

Dynamics of the mammalian sperm plasma membrane in the process of fertilization

Sexual reproduction requires the fusion of sperm cell and oocyte during fertilization to produce the diploid zygote. In mammals complex changes in the plasma membrane of the sperm cell are involved in this process. Sperm cells have unusual membranes compared to those of somatic cells. After leaving the testes, sperm cells cease plasma membrane lipid and protein synthesis, and vesicle mediated transport. Biophysical studies reveal that lipids and proteins are organized into lateral regions of the sperm head surface. A delicate reorientation and modification of plasma membrane molecules take place in the female tract when sperm cells are activated by so-called capacitation factors. These surface changes enable the sperm cell to bind to the extra cellular matrix of the egg (zona pellucida, ZP). The ZP primes the sperm cell to initiate the acrosome reaction, which is an exocytotic process that makes available the enzymatic machinery required for sperm penetration through the ZP. After complete penetration the sperm cell meets the plasma membrane of the egg cell (oolemma). A specific set of molecules is involved in a disintegrin-integrin type of anchoring of the two gametes which is completed by fusion of the two gamete plasma membranes. The fertilized egg is activated and zygote formation preludes the development of a new living organism. In this review we focus on the involvement of processes that occur at the sperm plasma membrane in the sequence of events that lead to successful fertilization. For this purpose, dynamics in adhesive and fusion properties, molecular composition and architecture of the sperm plasma membrane, as well as membrane derived signalling are reviewed.     

Intramembrane protein distribution in cell cultures is affected by 50 Hz pulsed magnetic fields

Intramembrane proteins (IMP) represent a class of proteins located in the lipid bilayer of the cell membrane which function as ion channels, enzymes or receptors. Since it has been argued that biological effects of extremely low frequency (ELF) electromagnetic fields are mediated by plasma membrane, this work was designed to study the possible effects of 50 Hz pulsed magnetic fields (PMF) of the type used to stimulate bone repair, on the distribution of IMP in the plasma membrane of Swiss NIH 3T3 fibroblasts. Evaluations were based on the calculation of a distribution factor, which allows discrimination between random, regular and clustered distribution of IMP, in electron microscope images of freeze-fractured membranes. The results indicate that cells exposed to PMF for more than two hours have a significant clustering of the IMP distribution compared to control unexposed cells. Bioelectromagnetics 18:463–469, 1997. © 1997 Wiley-Liss, Inc.     

Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin- phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.     

Quantitative analysis of modulations in numerical and lateral distribution of intramembrane particles during the cell cycle of neuroblastoma cells

Modulations in the internal structure of the plasma membrane during the cell cycle of mouse C1300 neuroblastoma cells (clone Neuro-2A) have been studied by freeze-fracture electron microscopy. Both the numerical and lateral distributions of the intramembrane particles (IMP) of the P face of the medium-exposed plasma membrane were determined as a function of the IMP diameter. The lateral IMP-distribution was quantified by a differential density distribution analysis, that could distinguish between random, aggregated, and dispersed distributions of IMP-subpopulations at various levels of spatial organization. Nonrandom lateral IMP-distribution was considered to indicate significant directional constraints on the lateral mobility of the represented molecules. The analysis demonstrated that the density, the size distribution, and the lateral distribution of the IMP are modulated during the cell cycle, such that characteristic structural and dynamic membrane properties can be attributed to the various cell cycle phases (M, G1, S, and G2). The results are interpreted in terms of asynchronous assembly of different membrane components and dynamic reorganizations within the plasma membrane during the cell cycle. Furthermore, they provide a structural manifestation of earlier observed changes in the dynamic properties of membrane proteins and lipids, and functional membrane transport properties in these neuroblastoma cells.     

Sperm head membrane reorganisation during capacitation

The sperm cell has a characteristic polarized morphology and its surface is also highly differentiated into different membrane domains. Junctional protein ring structures seal the surface of the mid-piece from the head and the tail respectively and probably prevent random diffusion of membrane molecules over the protein rings. Despite the absence of such lateral diffusion-preventing structures, the sperm head surface is also highly heterogeneous. Furthermore, lipid and membrane protein ordering is subjected to changes when sperm become capacitated. The forces that maintain the lateral polarity of membrane molecules over the sperm surface, as well as those that cause their dynamic redistribution, are only poorly understood. Nevertheless, it is known that each of the sperm head surface regions has specific roles to allow sperm to fertilize the oocyte: a specific region is devoted to zona pellucida binding, a larger area of the sperm head surface is involved in the acrosome reaction (intracellular fusion), while yet another region is involved in egg plasma membrane binding and fertilization fusion (intercellular membrane fusion). All three events occur in the area of the sperm head where the plasma membrane covers the acrosome. Recently, lipid ordered microdomains (lipid rafts) were discovered in membranes of many biological specimens including sperm. In this review, we cover the latest insights about sperm lipid raft research and discuss how sperm lipid raft dynamics may relate to sperm-zona binding and the zona-induced acrosome reaction.     

The development of regionalized lipid diffusibility in the germ cell plasma membrane during spermatogenesis in the mouse

The lipids and proteins of sperm cells are highly regionalized in their lateral distribution. Fluorescence recovery after photobleaching studies of sperm membrane component lateral diffusibility have shown that the sperm plasma membrane is also highly regionalized in the extents and rates of diffusion of its surface components. These studies have also shown that regionalized changes in lateral diffusibility occur during the differentiative processes of epididymal maturation and capacitation. Unlike mammalian somatic cells, sperm cells exhibit large nondiffusing lipid fractions. In this paper, we will show that both regionalized lipid diffusibility and nondiffusing lipid fractions develop with the morphogenesis of cell shape during spermatogenesis in the mouse. Pachytene spermatocytes and round spermatids show diffusion rates and the nearly complete recoveries (80-90%) typical of mammalian somatic cells. In contrast, stage 10-11 condensing spermatids, testicular spermatozoa, cauda epididymal spermatozoa, as well as the anucleate structures associated with these later stages of spermatogenesis (residual bodies and the cytoplasmic droplets of condensing spermatids and testicular spermatozoa), exhibit large nondiffusing fractions. Both the diffusion rates and diffusing fractions observed on the anterior and posterior regions of the head of stage 10-11 condensing spermatids are the same as the values obtained for these regions on testicular spermatozoa. Possible mechanisms of lipid immobilization and possible physiological implications of this nondiffusing lipid are discussed.     

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

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.     

A freeze-etch study of the effects of extracellular freezing on cellular membranes of wheat

Seedlings of Triticum aestivum L. cv. Lennox were grown in different environments to obtain different hardiness. Pieces of laminae and leaf bases were slowly cooled to sub-zero temperatures and the damage caused was assessed by an ion-leakage method. Comparable pieces of tissue were slowly cooled to temperatures between 2° and-14°C and were then freeze-fixed and freeze-etched. Membranes generally retained their lamellar structures indicated by the abundance of typical membrane fracture faces in all treatments, and some membrane fracture faces had patches which lacked the usual scattering of intramembranous particles (IMP). These IMP-free areas were present in the plasma membrane of tissues given a damaging freezing treatment, but were absent from the plasma membrane of room-temperature controls, of supercooled tissues, and of tissues given a non-damaging freezing treatment. The frequency of IMP-free areas and the proportion of the plasma membrane affected increased with increasing damage. In the most damaged tissue (79% damage; leaf bases exposed to-8°C), 20% of the plasma membrane was IMP-free. The frequencies of IMP at a distance from the IMP-free areas were unaffected by freezing treatments. There was a patchy distribution of IMP in other membranes (nuclear envelope, tonoplast, thylakoids, chloroplast envelope), but only in the nuclear envelope did it appear possible that their occurrence coincided with damage. The IMP-free areas of several membranes were sometimes associated together in stacks. Such membranes lay both to the outside and inside of the plasma membrane, indicating that at least some of the adjacent membrane fragments arose as a result of membrane reorganization induced by the damaging treatment. Occasional views of folded IMP-free plasma membrane tended to confirm this conclusion. The following hypothesis is advanced to explain the damage induced by extracellular freezing. Areas of plasma membrane become free of IMP, probably as a result of the freezing-induced cellular dehydration. The lipids in these IMP-free patches may be in the fluid rather than the gel phase. The formation of these IMP-free patches, especially in the plasma membrane, initiates or involves proliferation and possibly fusion of membranes, and during or following this process, the cells become leaky.Abbreviations EF exoplasmatic fracture face - IMP intramembranous particles - PF protoplasmatic fracture face     

Electron microscopic research on the mitochondria-rich bladder cells of the frog

The ultrastructural peculiarities of mitochondria-rich cells of the frog urinary bladder are analysed using three electron microscopic methods: ultrathin sections, scanning electron microscopy, freeze fracture. The mitochondria and tubular and vesicular structures are most abundant in the apical region of cytoplasm. The P-face (PF) of the apical plasma membrane is characterized by the presence of rod-shaped intramembrane particles (IMP), whereas the E-face (EF) possesses complementary pits. Depending on the distribution density of the rod-shaped IMP, three types of cells are described. The apical plasma membrane has an invert distribution of the globular IMP: a great quantity of IMP on the EF and a few particles on the PF. This structure of the apical plasma membrane is supposed to correlate with its very low water permeability. Using filipin as a marker of cholesterol localization, it has been shown that the mitochondria-rich cell apical membrane contains more cholesterol than that of the granular cells. The nature of the rod-shaped IMP and their role in the transmembrane ion transport have been discussed.     

Phagocytosis of bacteria by polymorphonuclear leukocytes: a freeze-fracture, scanning electron microscope, and thin-section investigation of membrane structure

The changes in membrane structure of rabbit polymorphonuclear (PMN) leukocytes during bacterial phagocytosis was investigated with scanning electron microscope (SEM), thin-section, and freeze-fracture techniques. SEM observations of bacterial attachment sites showed the involvement of limited areas of PMN membrane surface (0.01-0.25μm(2)). Frequently, these areas of attachment were located on membrane extensions. The membrane extensions were present before, during, and after the engulfment of bacteria, but were diminished in size after bacterial engulfment. In general, the results obtained with SEM and thin-section techniques aided in the interpretation of the three-dimensional freeze-fracture replicas. Freeze-fracture results revealed the PMN leukocytes had two fracture faces as determined by the relative density of intramembranous particles (IMP). Membranous extensions of the plasma membrane, lysosomes, and phagocytic vacuoles contained IMP's with a distribution and density similar to those of the plasma membrane. During phagocytosis, IMPs within the plasma membrane did not undergo a massive aggregation. In fact, structural changes within the membranes were infrequent and localized to regions such as the attachment sites of bacteria, the fusion sites on the plasma membrane, and small scale changes in the phagocytic vacuole membrane during membrane fusion. During the formation of the phagocytic vacuole, the IMPs of the plasma membrane appeared to move in with the lipid bilayer while maintaining a distribution and density of IMPs similar to those of the plasma membranes. Occasionally, IMPs were aligned to linear arrays within phagocytic vacuole membranes. This alignment might be due to an interaction with linearly arranged motile structures on the side of the phagocytic vacuole membranes. IMP-free regions were observed after fusion of lysosomes with the phagocytic vacuoles or plasma membrane. These IMP-free areas probably represent sites where membrane fusion occurred between lysosomal membrane and phagocytic vacuole membrane or plasma membrane. Highly symmetrical patterns of IMPs were not observed during lysosomal membrane fusion.     

Freeze-fracture studies on isolated sperm cells ofSpinacia oleracea L.

Summary With freeze-fracturing sperm cells appear to be fractured preferentially through the plasma membranes. Only few fracture planes through the cytoplasm are found. Both the PF as well as the EF side of the sperm cell plasma membranes show a slightly undulating surface and contain intramembrane particles. The particle distribution is irregular and does not show any clustering. The EF side of the plasmamembrane contains approximately 3 times more particles per m² than the PF side.Abbreviations EF extraplasmatic fracture face - IMP intramembrane particles - FDA fluorescein diacetate - PF plasmatic fracture face     

Structural differentiation of membranes involved in the secretion of polysaccharide slime by root cap cells of cress (Lepidium sativum L.)

The peripheral secretion tissue of the root cap of Lepidium sativum L. was investigated by electronmicroscopy and freeze-fracturing in order to study structural changes of membranes involved in the secretion process of polysaccharide slime. Exocytosis of slime-transporting vesicles occurs chiefly in the distal region of the anticlinal cell walls. The protoplasmic fracture face (PF) of the plasmalemma of this region is characterized by a high number of homogenously distributed intramembranous particles (IMPs) interrupted by areas nearly free of IMPs. Near such areas slime-transporting vesicles are found to be underlying the plasma membrane. It can be concluded that areas poor in particles are prospective sites for membrane fusion. During the formation of slime-transporting vesicles, the number of IMPs undergoes a striking change in the PF of dictyosome membranes and their derivatives. It is high in dictyosome cisternae and remarkably lower in the budding region at the periphery of the cisternae. Slime-transporting vesicles are as poor in IMPs as the areas of the plasmalemma. Microvesicles rich in IMPs are observed in the surroundings of dictyosomes. The results indicate that in the plasmalemma and in membranes of the Golgi apparatus special classes of proteins — recognizable as IMPs — are displaced laterally into adjacent membrane regions. Since the exoplasmic fracture face (EF) of these membranes is principally poor in particles, it can be concluded that membrane fusion occurs in areas characterized by a high quantity of lipid molecules. It is obvious that the Golgi apparatus regulates the molecular composition of the plasma membrane by selection of specific membrane components. The drastic membrane transformation during the formation of slime-transporting vesicles in the Golgi apparatus causes the enrichment of dictyosome membranes by IMPs, whereas the plasma membrane probably is enriched by lipids. The structural differentiations in both the plasma membrane and in Golgi membranes are discussed in relation to membrane transformation, membrane flow, membrane fusion, and recycling of membrane constituents.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face - IMP intramembranous particle     

A designed probe for acidic phospholipids reveals the unique enriched anionic character of the cytosolic face of the mammalian plasma membrane

It is generally accepted that the cytosolic face of the plasma membrane of mammalian cells is enriched in acidic phospholipids due to an asymmetric distribution of neutral and anionic phospholipids in the two bilayer leaflets. However, the phospholipid asymmetry across intracellular membranes is not known. Two models have been proposed for the selective targeting of K-Ras4B, which contains a C-terminal farnesyl cysteine methyl ester adjacent to a polybasic peptide segment, to the cytosolic face of the plasma membrane. One involves electrostatic interaction of the lipidated polybasic domain with anionic phospholipids in the plasma membrane, and the other involves binding of K-Ras4B to a specific protein receptor. To address this issue, we prepared by semi-synthesis a green fluorescent protein variant that is linked to a farnesylated, polybasic peptide corresponding to the K-Ras4B C terminus as well as a variant that contains an all-d amino acid version of the K-Ras4B peptide. As expected based on electrostatics, both constructs showed preferential in vitro binding to anionic phospholipid vesicles versus those composed only of zwitterionic phospholipid. Both constructs fully targeted to the plasma membrane when microinjected into live Chinese hamster ovary and Madin-Darby canine kidney cells. Because the all-d amino acid peptide should be devoid of binding affinity to a putative highly specific K-Ras membrane receptor, these results support an electrostatic basis for the targeting of K-Ras4B to the plasma membrane, and they support an intracellular landscape of phospholipids in which the cytosolic face of the plasma membrane is the most enriched in acidic phospholipids.     

The surface membrane of Leishmania mexicana mexicana: comparison of amastigote and promastigote using freeze-fracture cytochemistry

The freeze fracture replica technique has been used to compare the plasma membranes of amastigote and promastigote stages of Leishmania mexicana mexicana with respect to intramembranous particle (integral protein) distribution and to beta-hydroxysterols content as revealed by the distribution of lesions induced by the polyene antibiotic filipin. Intramembranous particle (IMP) density was greater in promastigote than in amastigote plasma membranes. Intramembranous particles were more abundant in the protoplasmic face (PF) than in the exoplasmic face (EF) of promastigotes, but this situation was found to be reversed in amastigotes. Filipin-induced lesions in glutaraldehyde-fixed parasites indicated higher levels of beta-hydroxysterols in the amastigote than in the promastigote plasma membrane, and in the promastigote flagellar membrane than in the body membrane. Amphotericin B (a related polyene antibiotic used in chemotherapy of leishmaniasis) induced IMP aggregation in the PF of unfixed amastigotes but did not appear to influence sterol distribution as demonstrated by freeze-fracture of subsequently-fixed and filipin-treated organisms.     

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as 'markers' for the sequential events of synthesis and assembly of zonulae occludentes: development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: reshaping the cell surface to align future junction-containing regions with those of adjacent cells, delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

Membrane fusion correlates with surface charge in exocytic vesicles

Stimulation of gastric parietal cells results in exocytic recruitment of the proton pump (H(+),K(+)-ATPase) from a pool of intracellular membranes (tubulovesicles) to the apical plasma membrane. We have previously reconstituted a step in this process, the homotypic fusion of tubulovesicles, and shown that they also fuse with liposomes in a protein-dependent manner [Duman, J. G., Singh, G., Lee, G. Y., Machen, T. E., and Forte, J. G. (2002) Traffic 3, 203-17]. Further, the lipid composition of the liposomes affects their ability to undergo fusion with tubulovesicles. In the present study, we investigated the lipid requirements for tubulovesicular membrane fusion using a fluorescent probe relaxation assay as well as transfer of protein between tubulovesicles and liposomes of defined composition. Initially, we tested the ability of tubulovesicles to undergo fusion with a panel of synthetic phosphatidylcholine-based liposomes containing a variety of common membrane lipids of various shapes and charges. We found that anionic lipids such as phosphatidylserine, phosphatidic acid, and phosphoinositides were best able to enhance tubulovesicle-liposome fusion and that they did it in a dose-dependent, apparently saturable manner. Next, we altered the lipid compositions of actual tubulovesicles and observed that addition of anionic lipids was able to enhance tubulovesicle-tubulovesicle fusion in vitro; thus, we hypothesized that the charge imparted by the lipids, per se, was responsible for the enhancement of membrane fusion. Accordingly, addition of negative charges to one of two pools of tubulovesicles in a fusion assay using anionic detergents increased membrane fusion; whereas, addition of positively charged cationic detergent decreased membrane fusion and could be used to back-titrate the anionic effects. Surprisingly, when both pools of fusing membranes were loaded with anionic detergents, fusion was markedly increased. The ability of anionic charges to enhance fusion was diminished as the ionic strength of the fusion medium was increased, suggesting that the mechanism of fusion enhancement depends on the surface charge of the membranes. Finally, the fusion reaction was highly dependent on temperature, and anionic charge appears to lower the activation energy of the fusion reaction. Taken together, these data suggest that (1) tubulovesicular fusion is enhanced by an increase in membrane surface negative charge associated with a lower activation energy and (2) neutralization or reversal of the surface charge prevents tubulovesicular fusion.     

Topographical relations of intramembrane particle distribution patterns in human sperm membranes

The distribution of intramembrane particles in human sperm membranes has been explored with particular reference to the topographical region of the sperm cell and the membranes' fracture face. Conspicuous differences in the size, arrangement, density, and lateral mobility of intramembrane particles between some topographically distinct membrane domains are demonstrated. The greatest regionality is exhibited by the plasma membrane. In sperm head regions, it shows a significant variability and changes its particle distribution during culture in capacitating medium. In contrast, little variability and no changes during the incubation are seen in the acrosomal and nuclear membranes. Striking is the difference in particle distribution on the E face of the outer acrosomal membrane between the acrosomal and equatorial regions. It is suggested that the invariable regional difference in the organization of the outer acrosomal membrane may bear on the different behavior of its two main domains during sperm capacitation and acrosome reaction.     

Electron microscopic study of colonic epithelial cells from the grass frog Rana temporaria under different intensity of water absorption

Three cell types have been revealed in the epithelium of the frog large intestine: granular, mitochondria-rich, and mucosal cells. Under a low water permeability (0.12 +/- 0.10 mkl/(min.cm2)) the distribution of intramembrane particles (IMP) in the apical cell membrane was the same as in the most cell plasma membranes studied with freeze-fracture method. Under rising osmotic permeability and water absorption (0.43 +/- 0.05 mkl/(min.cm2)) the IMP distribution did not change. In these conditions, the quantity of fusion sites between granule membranes and the apical membrane increased, and the intercellular spaces in basolateral epithelial region were diluted. A a low water permeability, in addition to usual microtubules, bundles of noncentrosomal microtubules with associated osmiophilic globules were revealed. A comparative analysis has been made of the present evidence and previously obtained data on the frog urinary bladder epithelium.     

Lipids of Xenopus laevis Spermatozoa

Xenopus laevis sperm lipid composition has been studied. The cholesterol content of Xenopus spermatozoa is 194 μ/mg DNA. Their content of glycolipids and phospholipids (measured as inorganic phosphorus) is respectively 40 and 27 μ/mg DNA. The phospholipid pattern is quite homogeneous and all the principal molecular species are present. In all the examined samples, a glycolipid with low mobility, not yet structurally identified, is present. Finally, using as a probe filipin, we have observed cholesterol distribution on the Xenopus sperm plasma membrane by freeze-fracture. In agreement with the chemical data here presented, Xenopus spermatozoa are heavily labelled by filipin. The filipin-cholesterol complexes seem to be distributed on the entire sperm plasma membrane and appear as protuberances on the P face, suggesting that most of the cholesterol reside in the inner leaflet of the membrane.