Effect of the local anesthetic dibucaine on the surface membrane in sterol-manipulated Tetrahymena pyriformis studied by freeze-fracture electron microscopy

The effect of the local anesthetic dibucaine on the membrane ultrastructure of sterol-manipulated Tetrahymena pyriformis (NT-1 strain) was studied by freeze-fracture electron microscopy. Dibucaine-treated, ergosterol-replaced Tetrahymena cells had marked alterations in their plasma membranes. IMP-free small depressions (exoplasmic fracture face) and protrusions (protoplasmic fracture face) were formed on the plasma membranes which was in contact with the outer alveolar membrane. In addition, large IMP-free surface "blebs" covered with hexagonally-arranged depressions and protrusions appeared on both the plasma and outer alveolar membranes. These "blebs" were pinched off when the membranes were severely affected. Our previous study (28) demonstrated that the plasma membrane of dibucaine-treated native Tetrahymena cells that contain tetrahymanol showed vertical displacement of its intramembranous particles and that subsequently a smooth, flat surface appeared. Therefore, the structural changes in ergosterol-replaced membranes produced by dibucaine differ strikingly from changes in the native membranes. The remarkable difference in the ultrastructural deformation of the plasma membrane probably is due to a difference in the membrane lipid composition induced by sterol-manipulation.     

Density and size distributions of the intramembranous particles in the cytoplasmic membrane of human peripheral blood lymphocytes. II. Ultrastructural differences between T and B cells

Separated T and B lymphocytes from human peripheral blood were studied using the freeze-fracture technique. Quantitative analysis performed on density and size of intramembranous particles (IMPs) present on both fracture faces of the plasma membrane has revealed remarkable differences between cells belonging to the two main lymphocyte populations. In particular: (a) both fracture faces of the cytoplasmic membrane of B lymphocytes exhibit larger particles than T lymphocytes; (b) the mean densities, on both protoplasmic (PF) and external (EF) fracture faces, in B lymphocytes are lower than in T lymphocytes; (c) in B cells the partition ratio of particles between PF and EF is reversed with respect to T cells; (d) on both fracture faces of B lymphocytes, the IMP densities present a normal distribution while on T cells, density values show bimodal distributions indicating the existence of two cell subsets differing in particle density.     

Freeze-fracture analysis of phloem structure in plant tissue cultures. II. The sieve element plasma membrane

Sieve element plasma membranes reveal a unique distribution of intramembrane particles (IMPs) in tissue cultures fixed and cyroprotected prior to freeze-fracturing. Sieve element IMPs are smaller than those found in the plasma membranes of callus parenchyma cells from these same cultures. The PF/EF ratio of plasma membrane IMPs is 9.6 for parenchyma cells and 1.21 for sieve elements. The increased binding of IMPs to the sieve element E face may be related to the role of membrane proteins in the loading of sucrose and other molecules by these cells. The enlargement of the cell wall at the site of sieve area pores creates complementary ridges and depressions in the E and P fracture faces of sieve element plasma membranes. No alteration of IMP density is seen at the sieve area pore site.     

Freeze-fracture study of rat liver peroxisomes: evidence for an induction of intramembrane particles by agents stimulating peroxisomal proliferation

The membrane ultrastructure of isolated rat liver peroxisomes has been observed by rapid freezing and freeze-fracture techniques. Unidirectional and rotary shadowing allows a clear visualization of the intramembrane particles (IMPs) on both the protoplasmic fracture (PF) leaflet and the endoplasmic fracture (EF) leaflet and reveals an asymmetric distribution of IMPs. Both fracture faces were uniformly studded by IMPs, and the frequency was about seven times higher on the P face (2322 per 1.0 micron2) than on the E face (322 per 1.0 micron2). Administration of the peroxisomal proliferator clofibrate (ethyl-p-chlorophenoxyisobutyrate) induced a marked increase in the frequency of IMPs on both the P face (2.2-fold) and the E face (1.7-fold). The average size decreased (P less than 0.001) from 45.7 +/- 16.5 nm2 to 35.2 +/- 10.8 nm2 on the P face. A similar increase in the frequency of IMPs was observed on the P face (1.8-fold) and the E face (1.8-fold) of peroxisomes from rats fed a semisynthetic diet containing 20% (w/w) of partially hydrogenated fish oil. The average size increased (P less than 0.001) from 36.6 +/- 19.7 to 50.0 +/- 23.5 nm2 on the E face. This study demonstrates alterations both in frequency and size distribution of IMPs in liver peroxisomal membranes on exposure of rats to agents known to induce peroxisomal proliferation. The increase in frequency of IMPs was as expected from the observed increase in one of the major integral membrane polypeptides, with apparent molecular mass of 69 (or 70) kDa, in proliferating rat liver peroxisomes.     

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     

Morphological detection of filipin-sterol complexes in the cytoplasmic membrane of staphylococcal L-form

Filipin, a sterol-specific antibiotic, and freeze-fracture electron microscopy were used to study the presence and distribution of sterol in the cytoplasmic membrane of stable staphylococcal L-form cells. Fixed cells were treated with filipin, and then observed by freeze-fracture electron microscopy. Freeze-fractured profiles of the L-form cells treated with filipin demonstrated irregular distribution of protuberances or pits of 25-30 nm, representing filipin-sterol complexes, on the proto-plasmic fracture face (PF) and exoplasmic fracture face (EF) of the cytoplasmic membrane. In contrast, no such structure was detected in the filipin-treated parent cells or protoplasts. The results suggest that some sterol molecules, which are usually not found in staphylococcal or other bacterial cells, emerged on the cytoplasmic membrane after the cells were converted to the stable L-form.     

Effect of hyperthermia on the plasma membrane structure of Chinese hamster V79 fibroblasts: a quantitative freeze-fracture study

Hyperthermia in the range 41-45 degrees C can induce wide biochemical, physiological, and morphological changes in mammalian cells both in vivo and in vitro. In general, its effects on membranes, particularly on the plasma membrane, are still poorly understood. To investigate the effects of heat on this cell structure, Chinese hamster V79 fibroblasts were exposed to 43 degrees C hyperthermia for 1 h, immediately fixed with glutaraldehyde after treatment, and freeze-fractured for electron microscopic examination. Particular attention was given to the density and size of intramembranous particles (IMPs) on both protoplasmic (PF) and external (EF) fracture faces of the plasma membrane. The quantitative study performed by an interactive image analyzer on the IMPs, generally reported as plasma membrane proteins, showed in heat-treated cells a statistically significant increase in their density and size on both fracture faces. The differences observed demonstrate that in our experimental conditions, hyperthermia in plasma membranes produces structural changes whose biological significance has to be clarified. Moreover, our findings seem to support recent data indicating an involvement of membrane proteins in the cell response to hyperthermia.     

Formation of B type gap junctions between PHA-stimulated rabbit lymphocytes.

Contact areas of PHA-stimulated and consequently agglutinated rabbit peripheral blood and spleen lymphocytes were studied with ultrathin-section and freeze-fracture techniques. Broad contact zones (BCZ) between adjacent cells were characterized in freeze-fracture replicas as plasma membrane areas in which at the protoplasmic fracture face (PF) a heterogeneous population of redistributed intramembranous particles (IMP) appear to assemble. In addition homogeneous particles of 11 nm diameter, found to be concentrated at the external fracture face (EF) at the site of the BCZ, aggregate to clusters and after longer culture periods appear to participate in the formation of gap junctional complexes. Evidence is provided that the BCZ—probably an area of concentrated PHA-binding sites—may well serve as a formation plaque for gap junction constitution in the system studied.     

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.     

The plasma membrane of growing root hairs is composed of zones of local differentiation

Growing root hairs of cress (Lepidium sativum L.) were investigated using freeze-fracture and electron-microscopic techniques. Three zones of differentiation could be detected: the tip zone, the zone of vacuolation and the foot zone. Corresponding to these zones, the plasmatic fracture face of the plasma membrane showed areas of pronounced differentiation with respect to the distribution and frequency of intramembranous particles (IMPs). The tip zone was characterized by an irregular fracture plane caused by a large number of blisters which were more or less free of IMPs. These blisters coincided in size and shape with Golgi vesicles accumulated in the ground cytoplasm near the very tip. Outside these blisters, IMPs were randomly distributed. The surrounding cell wall was very thin and mainly composed of amorphous material. The plasma membrane of the vacuolation zone often revealed areas of hexagonally ordered particles (HOPS). Such patterns of particles were observed in chemically fixed and unfixed root hairs with a maximum surface density of 1200 HOPS per area. Mostly, however, 15–50 HOPS per area were found. The number of such areas increased with increasing distance from the tip up to five areas per m². Additionally, imprints of large cellulose microfibrils could be detected in unfixed material; they were mainly parallel to the root-hair axis and sometimes ended in areas of HOPS. However, HOPS were observed only in approximately 60% of the root hairs. Otherwise, large areas free of IMPs were interspersed between areas of randomly distributed IMPs. The particle frequency was relatively low and varied greatly in the tip as well as in the vacuolation zone, that is, from 1200 to 2000 IMPs m^-2. Finally, the plasma membrane of the foot zone showed a very constant number of approx. 2000 IMPs m^-2. These particles were mainly distinct and randomly distributed. In this zone, HOPS were never observed in spite of the fact that the cell wall was composed of numerous parallel-running cellulose microfibrils. Since membrane material is mainly incorporated in the tip zone where IMPs are statistically distributed, the results indicate that the plasma membrane of the outgrowing part of the root-hair cells is characterized by a high lateral mobility of its components. Furthermore, they indicate that specifically arranged particles are involved in the synthesis of cellulose microfibrils. These areas of HOPS seem to be locally restricted and — or limited with respect to their lifetime.Abbreviations cmf(s) cellulose microfibril(s) - EF extraplasmatic fracture face - HOPS hexagonally ordered particles - IMP intramembranous particle - PF plasmatic fracture face - pm plasma membrane Dedicated to Professor Dr. Kurt Mühlethaler, Zürich, on the occasion of his 65th birthday     

The eyespot of the flagellateTetraselmis cordiformis stein (Chlorophyceae): Structural spezialization of the outer chloroplast membrane and its possible significance in phototaxis of green algae

Summary The eyespot region of the flagellateTetraselmis cordiformis Stein (Chlorophyceae) was investigated with the freeze-fracture technique. The only fracture faces observed in this region were the two complementary fracture faces (PF and EF) of the outer chloroplast envelope membrane. Intramembranous particle numbers on both fracture faces of this membrane were significantly higher in the eyespot region as compared to regions outside the eye-spot. Higher numbers of particles on the PF face in the eyespot region were mainly caused by an increase in particle numbers of the size class 6–8 mm, while on the EF face particle size distribution was not significantly different between eyespot and other regions. Functional implications are discussed and evidence is presented that the outer chloroplast envelope membrane may be the site of photoreceptor location in green algal phototaxis.     

Intercellular junctions of antennal gland epithelial cells in the crayfish,Orconectes virilis

Summary Labyrinth and nephridial canal cells of the crayfish (Orconectes virilis) antennal gland possess two types of intercellular junctions revealed by freeze-fracture studies. Apical margins of the cells are connected by long septate junctions. In replicas, these junctions consist of many parallel rows of 80–140 Å intramembrane particles situated on the PF membrane face (EF and PF fracture faces of Branton et al., 1975). Rows of pits are found on the EF fracture face and are deemed complementary to the rows of particles. Moreover, lateral margins of basal regions of the epithelial cells are attached by many intercellular junctions. These contacts are characterized in thin plastic sections by a narrow dense cytoplasmic plaque located subjacent to the plasma membrane at sites of adjoined cells, and 5 to 12 fine strands of dense material that extend across the intercellular gap between adjoined cells. In freeze-fracture replicas, EF intramembrane faces basal to the region of the plasma membrane containing septate junctions exhibit numerous discoid clusters of particles. The particle aggregates, assumed to represent freeze-cleave images of adhering junctions, range from 900 to 3,700 Å in diameter, with individual particles about 185 Å in diameter. These junctions appear to connect epithelial cell processes formed by basal infoldings of the plasma-lemma, and occur between adjacent cells as well as adjacent processes of a single cell. The discrete aggregates of particles resemble replicated desmosomes (Shienvold and Kelly, 1974) and hemi-desmosomes (Shivers, 1976); therefore, they probably do not constitute a basis for electrical coupling between antennal gland epithelial cells.Supported by the National Research Council of Canada     

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.     

A freeze-fracture electron microscope study of Trichomonas vaginalis Donné and Tritrichomonas foetus (Riedmüller)

Two strains of Trichomonas vaginalis, JH162A , with low pathogenicity, and Balt 44, with high pathogenicity, as well as one highly pathogenic strain, KV-1, of Tritrichomonas foetus were studied by freeze-fracture electron microscopy. The protoplasmic faces ( PFs ) of the cell membranes of all three strains of both species had similar numbers of intramembranous particles (IMPs); however, the particles in the external faces (EFs) of these membranes were least abundant in Trichomonas vaginalis strain Balt 44 and most numerous in those of strain JH162A of this species. In Tritrichomonas foetus strain KV-1 the number of IMPs in the EF was close to but somewhat lower than that in the mild strain of the human urogenital trichomonad . In both species, the anterior, but not the recurrent, flagella had rosette-like formations, consisting of approximately 9 to 12 IMPs on both the PFs and EFs. The numbers and distribution of the rosettes appeared to vary among different flagella and in different areas of individual flagella of a single organism belonging to either species. The freeze-fracture electron micrographs provided a more complete understanding of the fine structure of undulating membranes of Trichomonadinae , as represented by Trichomonas vaginalis, and of Tritrichomonadinae (the Tritrichomonas augusta -type), as exemplified by Tritrichomonas foetus, than was gained from previous transmission and scanning electron microscope studies. Typically three longitudinal rows of IMPs on the PF of the recurrent flagellum of Trichomonas vaginalis were noted in the area of attachment of this flagellum to the undulating membrane. The functional aspects of the various structures and differences between certain organelles revealed in the two trichomonad species by the freeze-fracture method are discussed.     

Freeze-Fracture Analysis of Plasma Membranes of CHO Cells Stably Expressing Aquaporins 1-5

Several studies suggest that aquaporin water channels can be identified in membranes by freeze-fracture electron microscopy. For this report, Chinese Hamster ovary cells were stably transfected with cDNAs encoding aquaporins 1–5. Measurement of the osmotic water permeability of the cells confirmed that functional protein was expressed and delivered to the plasma membrane. By freeze-fracture electron microscopy, a 20% increase in intramembrane particle (IMP) density was found in plasma membranes of cells expressing AQP2, 3 and 5, and a 100% increase was measured in AQP1-expressing cells, when compared to mock-transfected cells. On membranes of cells expressing AQP4, large aggregates of IMPs were organized into orthogonal arrays, which occupied 10–20% of the membrane surface. IMP aggregates were never seen in AQP2-transfected cells. Hexagonally packed IMP clusters were detected in ∼5% of the membranes from AQP3-expressing cells. Particle size-distribution analysis of rotary shadowed IMPs showed a significant shift from 13.5 (control cells) to 8.5 nm or less in AQP-expressing cells; size distribution analysis of unidirectionally shadowed IMPs also showed a significant change when compared to control. Some IMPs in AQP expressing cells had features consistent with the idea that aquaporins are assembled as tetramers. The results demonstrate that in transfected CHO cells, AQP transfection modifies the general appearance and number of IMPs on the plasma membrane, and show that only AQP4 assembles into well-defined IMP arrays. Received: 17 March 1998/Revised: 19 June 1998     

Plasma-membrane rosettes in root hairs of Equisetum hyemale

Particle arrangement in the plasma membrane during cell wall formation was investigated by means of the double-replica technique in root hairs of Equisetum hyemale. Particle density in the protoplasmic fracture face of the plasma membrane was higher than in the extraplasmic fracture face. Apart from randomly distributed particles, particle rosettes were visible in the PF face of the plasma membrane. The rosettes consisted of six particles arranged in a circle and had an outer diameter of approx. 26 nm. No gradient in the number of rosettes was found, which agrees with micrifibril deposition taking place over the whole hair. The particle rosettes were found individually, which might indicate that they spin out thin microfibrils as found in higher-plant cell walls. Indeed microfibril width in these walls, measured in shadowed preparations, is 8.5±1.5 nm. It is suggested that the rosettes are involved in microfibril synthesis. Non-turgid cells lacked microfibril imprints in the plasma membrane and no particle rosettes were present on their PF face. Fixation with glutaraldehyde caused, probably as a result of plasmolysis, the microfibril imprints to disappear together with the particle rosettes. The PF face of the plasma membrane of non-turgid hairs sometimes showed domains in which the intramembrane particles were aggregated in a hexagonal pattern. Microfibril orientation during deposition will be discussed.Abbreviations EF extraplasmic fracture face - PF protoplasmic fracture face     

Changes in plasmalemma organization in cowpea radicle during imbibition in water and NaCl solutions

Abstract Freeze-fracture electron microscopy of the plasmalemma of dry cowpea radicle cells disclosed a normal-appearing membrane with a high area density of intramembraneous particles (IMPs). Seeds imbibed in either water or salt solution exhibited decreased area density of IMPs, but water-imbibed tissue showed the greatest decline. Mean particle size increased with hydration but not enough to suggest aggregation as the cause for this density decrease. Calculations of plasmalemma area expansion during imbibition show that such expansion can account for the decrease in IMPs per unit area in the cytoplasmic side (PF) of the membrane in water-imbibed, but not in salt-imbibed, tissue. During imbibition, there is a change in the ratio of IMPs per unit area of the PF versus EF (external) membrane faces, suggesting a relative increase in the number of EF particles. These changes in membrane structure are probably not related to any decrease in membrane permeability during the early phases of imbibition.     

FREEZE-FRACTURE STUDY OF THE SINGLE MEMBRANE BETWEEN HOST CELL AND ENDOCYTOBIONT IN THE DINOFLAGELLATES GLENODINIUM FOLIACEUM AND PERIDINIUM BALTICUM1

The dinoflagellates Glenodinium foliaceum Stein and Peridinium balticum (Levander) Lemmermann harbor a chrysophytic endocytobiont which is bounded by only a single membrane. This unique membrane is of particular interest because it could correspond to an intermediate stage in the evolution of “complex” plastids found in many Plastids of this type are surrounded by three or membranes instead of the usual two. With freeze-fracture techniques, we show that the single membrane in P. balticum has a pronounced polarity with respect to the distribution of intramembrane particles (IMPs) on the two corresponding fracture faces. The inner face exhibited more IMPs than the outer. We suggest that this stdedness identifies the separating membrane as the plasma membrane of the endocytobiont. A symbiontophoric vacuole with a separate membrane apparently is lacking. In the endocytobiosis of G. foliaccum, the single membrane separating host and endocylobiont exhibits a symmetrical particle partition. Nevertheless, from the size distribution of the IMPs it appears likely that this membrane, too, corresponds to the plasma membrane of the symbiont.     

Ultrastructural study of cholestasis induced by longterm treatment with estradiol valerate. II. Gap junctional analysis

Treatment of male Wistar rats with estradiol valerate induced alterations in hepatic gap junctions as visualized by the freeze-fracture technique. The alterations involved the spacing, and regularity of packing of the membrane particles of the P face (PF) and complementary pits on the E face (EF), as well as internalization and changes in the number, size and shape of the junctional domains. In approximately 20% of the PF's of the lateral membrane of treated animals the nonjunctional IMPs were aggregated, while the bile canalicular membrane was never involved, maintaining its random distribution of particles. It is proposed that the changes in junctional area and the more general arrangement of the junctional particles may indicate a decrease in coupling between hepatocytes. The invaginations of gap junctions may represent a means for removing gap junctional membrane from the surface or may be an expression of a higher turnover of gap junctions. We assume that the alterations observed here are due to the specific effects of estrogen. This study addresses in detail a number of possible sites of activity and modes of action for estrogen.     

Distribution of intramembranous particles and filipin-sterol complexes in mouse sperm membranes: polyene antibiotic filipin treatment

The distribution of intramembranous particles (IMPs) and membrane filipin-sterol complexes (FSC) was examined ultrastructurally in mouse spermatozoa from the male reproductive tract and ejaculates. IMPs were qualitatively analyzed on freeze-fracture replicas of glutaraldehyde-fixed tissue, while membrane FSC were quantitatively analyzed on replicas of filipin-treated cells. The distribution pattern of IMPs of mouse spermatozoa was fundamentally similar to that of other mammalian spermatozoa. 1) In the head, the plasma membrane had a heterogeneous population density, e.g., few IMPs on the acrosomal region, particularly few on the marginal segment, and somewhat regularly arranged IMPs on the postacrosomal region. The acrosomal membrane had many IMPs in hexagonal arrays. The nuclear membrane had many IMPs on the P-face, few IMPs on the variegated E-face, and an intense population density on the P-face of the basal plate. 2) In the neck, the plasma membrane had many IMPs with square arrangements of small IMPs in some areas on the P-face; the redundant nuclear membrane had a few IMPs on both P- and E-faces. 3) In the tail, the plasma membrane had diagonal rows of IMPs in some areas amongst larger IMPs on the middle piece, while it had "zippers" composed of IMPs running parallel to the axis on the principal piece. The distribution of sperm membrane FSC may be summarized as follows: 1) In the head, the acrosomal plasma membrane, which was heavily labeled with filipin, had much more FSC in the equatorial segment than in the marginal segment throughout the study. The postacrosomal plasma membrane generally had no FSC, but some sperm in ejaculates were slightly positive to filipin. The acrosomal membranes (both outer and inner) had no FSC. The nuclear membrane in the main part of the head had less FSC in vas deferens and ejaculated sperm than in the epididymal sperm. The nuclear membrane on the basal plate had no FSC. 2) In the neck, the plasma membrane had little FSC. The redundant nuclear envelope had scattered FSC with a higher incidence in the epididymal sperm than in those from the vas deferens and ejaculates. The membrane scroll, which was elongated from the extreme caudal end of the redundant nuclear envelope, had abundant FSC in the vas deferens and ejaculated sperm. 3) The tail plasma membrane (both middle and principal piece), which was weakly labeled with filipin, had less FSC in sperm from the vas deferens and ejaculates than in those from the epididymis. The limiting membrane covering the mitochondria had no FSC.