Plasma membrane structure of bat spermatozoa: observations on epididymal and uterine spermatozoa in Myotis lucifugus

Plasma membrane structure of bat spermatozoa was examined utilizing electron microscopy of thin sections and freeze-fracture replicas. Notable membrane features observed in replicas from cauda epididymal spermatozoa included specialized particle aggregates at the junction between the acrosomal and postacrosomal region of the head (a membrane structure not previously described in mammalian spermatozoa) and another row of rod-like particles just anterior to the posterior ring. Both of these specializations in fractured plasma membranes correspond with regions where the membrane is closely apposed to underlying structures when viewed in thin sections. The postacrosomal sheath appears to be composed of an array of longitudinally oriented filamentous components. Characteristic ordering of intramembranous particles was also noted in replicas from the midpiece region and the annulus. Major changes in plasma membrane structure were not seen in spermatozoa stored in the female reproductive tract; however, the appearance of linear particle aggregations in the principal piece membrane was noted. No evidence was obtained to suggest that an acrosome reaction had occurred in spermatozoa stored in females.     

Firm structural associations between migratory pigment granules and microtubules in crayfish retinula cells

The morphology of associations between mobile pigment granules and microtubules of the crayfish retinula cells was examined with transmission electron microscopy. Many pigment granules were found associated with microtubules through linkages of fuzzy appearance in thin sections. The linkages were revealed as discrete strands of variable shape in rotary-shadowed replicas of freeze-fractured and deep- etched specimens. The only feature of constant morphology among these connections consisted of 2-4-nm filaments projecting laterally from the microtubules. The firmness of the pigment granule-microtubule associations was judged by their ability to hold up during cell disruption procedures of increasing disaggregation effects in a low- Ca++ stabilization buffer. The results of these tests were inspected with scanning electron microscopy and with transmission electron microscopy of negatively stained preparations. Numerous pigment granules remained associated with a stable microtubule framework after the plasma membrane had been stripped away. Moreover, granule- microtubule attachments survived breakdown of this framework into free fascicles of microtubules. The pigment granules were associated with the free microtubules either individually or as clusters entangled in a fibrous material interwoven with 10-nm filaments. These findings attest that many pigment granules are bound to microtubules through linkages that constitute effective attachments. Further, it is demonstrated that a highly cohesive substance associates the pigment granules with one another. These conclusions are discussed in terms of a pigment transport mechanism in which a network of interconnected granules would establish firm transient interactions with a supporting skeleton of microtubules.     

The centriolar rim. The structure that maintains the configuration of centrioles and basal bodies in the absence of their microtubules

Preparations of centrioles from bovine spleen were incubated in solutions of NaCl, MgCl2, HCl, NaOH, EDTA and heparin. Their effects on the centrioles were studied by electron microscopy of ultrathin sections. It was found that the microtubules of centriolar cylinders gradually disintegrate at a higher than physiological ionic strength and at a pH value lower than 3.5 and higher than 8.5. After microtubule extraction, a closely apposed rim or sheath of dense centriolar matrix remains which has the same dimensions of length and width as the original centriole. Some other centriolar structures, including the pericentriolar satellites and certain structures in the cylinders (hub) are also preserved. The basal bodies of fish spermatozoa revealed similar structures, including the centriolar rim and hub, after microtubule extraction. Thus, the microtubule triplets are not involved in maintaining the structure of the centriolar cylinder; this role is rather carried out by amorphous material--the matrix, surrounding the microtubules.     

Ultrastructural Observations on the Spermatozoa of Two Temnocephalids (Platyhelminthes)

The spermatozoa of two Temnocephalidae collected in Uruguay, Temnocephala iheringi Haswell, 1893 (Host: Pomacea canaliculata) and Temnocephala axenos Monticelli, 1899 (Host: Parastacus varicosus), were studied with a transmission electron microscope. In both species the spermatozoon is made up of a long sperm body which bears at one extremity two free flagella of the 9+‘1’ flatworm pattern. The sperm body contains the nucleus, mitochondria, dense bodies and parallel, cortical, longitudinal singlet microtubules. Along a part of the sperm body the palissade of the microtubules displays a spiral pattern in transverse sections. A part of the perimeter of the cell is thus lined by two overlapping rows of microtubules. This spiral pattern of the singlets is considered as a synapomorphy of the family Temnocephalidae. The singlet microtubules are interconnected by two kinds of links: tangential links between neighbouring singlets in the same row and radial links between singlets belonging to two rows. The presence of these links suggests that this structure could be a motile system of singlets.     

Structural organization of the "zipper line" in Drosophila species with giant spermatozoa

The "zipper line" of Drosophila melanogaster and of Drosophila species characterized by giant spermatozoa (D. hydei, D. kanekoi and D. bifurca) was studied by electron microscopy using conventional thin-sections, lectin labeling and freeze-fracture replicas. In cross sections the membrane specializations are located either at the level of the short cistern close to the large mitochondrial derivative where a small tuft of glycocalyx is visible or, in species characterized by long spermatozoa, along a cistern beneath the plasma membrane. In correspondence of such cistern, the plasma membrane exhibits a thick and extended glycocalyx. At this level, as well as at the short tuft of D. melanogaster, alpha-mannose residues were detected. The "zipper" of D. melanogaster consists of rows of intramembrane particles longitudinally disposed along the sperm tail and associated with the external face of the plasma membrane. On the protoplasmatic face a narrow ribbon of transversal grooves is visible. Freeze-fracture replicas have revealed, in the region characterized by extended glycocalyx, the presence of a large ribbon of intramembrane particles disposed in parallel transversal rows, associated with the protoplasmatic membrane face. On the complementary external face a ribbon of parallel transversal grooves was observed. It is suggested that membrane specializations are mechanical devices to protect spermatozoa from torsion and bending in the seminal vesicles and then in the female storage organ.     

Cholesteric organization of DNA in the stallion sperm head

The fine structure of chromatin in sperm heads was investigated by different microscopic techniques: in vivo examinations in the polarizing microscope, thin sections and freeze-fracture replicas observed by transmission electron microscopy. The freeze-fractured chromatin appears to be formed of superimposed lamellae, each one 330 A thick. These lamellae are parallel to the flattening plane of the sperm head. This situation was already described in other mammal spermatozoa and in particular in the bull and the rabbit. This work presents a new interpretation of this lamellated aspect. The chromatin structure of these spermatozoa is that of a cholesteric liquid crystal. This structure resembles that of a plywood, made of superimposed layers of parallel filaments, but instead of having a right angle between two successive layers, there is a progressive rotation and similar orientation occurs at each 180 degrees rotation. The apparent lamellae result from cleavages due to freeze-fracture between levels of parallel filament orientation. The thickness of lamellae corresponds therefore to the half helicoidal pitch of the cholesteric liquid crystal. This model is consistent with our observations by polarizing microscopy. The lamellation is not visible in thin sections of stallion spermatozoa. There are however biochemical methods to decondense chromatin and we are able to observe this lamellation in sections normal to the flattening plane of sperm heads. The methods used classically to decondense the sperm chromatin lead to extremely varied aspects which are discussed, some of them being closely related to the structure of cholesteric liquid crystals.     

Surface Features of Bacillus polymyxa Spores as Revealed by Scanning Electron Microscopy

The surface features of Bacillus polymyxa spores were compared by use of thin sections, carbon replicas, and the scanning electron microscope. Some features of the characteristic ridges, previously reported in ultrathin sections and carbon replicas of spores of this species, were more clearly revealed with the scanning electron microscope. A three-dimensional image is provided because of the greater depth of focus possible with this instrument. End-on views of B. polymyxa spores readily illustrate the polygonal porelike structure present.     

Acoel spermatozoa: ultrastructure and immunocytochemistry of tubulin

Acoel spermatozoa are filiform and contain two parallel axonemes, which do not show the trepaxonematan 9 + ‘1’ pattern, but instead, another kind of 9 + ‘1’ pattern, or a 9 + 0 or 9 + 2 pattern. Spermatozoa have either cortical singlet microtubules or central microtubules. Identification of these groups of microtubules and recognition of homologies between species is difficult with electron microscopy. In addition to conventional electron microscopy, indirect immunofluorescence of tubulin was performed on three species (Symsagittifera schultzei, Symsagittifera psammophila, and Actinoposthia beklemischevi). This technique facilitated understanding of the general morphology of the filiform spermatozoon and of the arrangement of the microtubular organelles along its length. We have found that different monoclonal antibodies (anti-alpha-, anti-alpha-acetylated- and anti-beta-tubulin) can distinguish distinct subcellular populations of microtubules. The axonemes were labelled by the three antibodies in all species. The cortical microtubules (in Actinoposthia beklemischevi) were labelled by the three antibodies. The central microtubules (in Symsagittifera schultzei and S. psammophila) were labelled with the anti-beta-tubulin antibody and not labelled by the anti-alpha- and anti-alpha-acetylated-tubulin. Similar experiments were performed on other Platyhelminthes and indicated that immunocytochemistry of spermatozoa may provide new characters for phylogenetic studies. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Comparative electron microscopic study of cytoplasmic microtubules and axial unit tubules in a spermatozoon and a protozoan

Cytoplasmic microtubules and axial unit tubules were studied in both sectioned and negatively-stained material. Walls of microtubules of frog lung-fluke (Haematoloechus medioplexus) spermatozoa have a helical substructure, while those of the flagellate, Trypanosoma lewisi, are composed of ten longitudinally-oriented filaments. Cross-bridges occur between some filaments of trypanosome microtubules. Doublet tubules of axial units in both cell types are structurally similar to the trypanosome microtubules, which may indicate similarity of function. Microtubules of fluke spermatozoa appear to be somewhat rigid, are resistant to sonication, and are considered to be mainly supportive. Circular profiles of wall subunits are seen in transverse sections of microtubules of both cell types and in doublet tubules of the trypanosome. Comparisons are made between sectioned and negatively-stained material; while negative-staining better reveals the fundamental substructure of microtubular elements, some distortion appears to occur. In connection with this research, a brief preliminary article demonstrated the presence of subunits in the walls of cytoplasmic microtubules of fluke spermatozoa (Burton, '66). Also, it was shown that the wall of these tubular elements possesses a helical structure, and a diagrammatic representation of the wall structure was set forth.     

MICROTUBULAR PATTERNS IN SPERMATOZOA OF COCCID INSECTS IN RELATION TO BENDING

Flagella-like motion occurs in filamentous spermatozoa of coccid insects, which have diameters (0.16–0.65 µ) and lengths (150–300 µ) similar to those of long flagella, but have no doublets or 9 + 2-like arrangements of microtubules. Light and electron microscope investigations of spermatozoa from 10 species reveal many bizarre patterns of microtubules and suggest some basic similarities to flagella. Detailed analyses of spermatozoa which are naturally bent in definable planes during their elongation in the male and their storage in the female provide evidence that a constant topographical relationship is maintained between their unorthodox patterns of microtubules, as viewed in transections, and the direction of bending. The configuration common to most coccid spermatozoa consists of an acentrically positioned crescent of microtubules surrounded by one to several concentric rings. A line drawn to connect the two ends of the crescent appears to remain perpendicular to the plane of bending, and it defines a plane in which bisection of the spermatozoon produces halves with unequal numbers of microtubules. Bisection of the 9 + 2 motile apparatus in a plane perpendicular to that of bending also appears to produce halves with unequal numbers of microtubules. Therefore, the indispensable elements for flagellar and flagella-like motion may be microtubules arranged in "asymmetric" patterns.     

Post-testicular changes in the density and distribution of intramembrane particles of stallion sperm surface domains

Freeze-fracture replicas of stallion spermatozoa, collected from the proximal caput, corpus and cauda epididymides regions, were analyzed by electron microscopy to explore the distribution and density of intramembrane particles (IMP). Conspicuous differences in density and arrangement of the IMP were observed in the different topographical domains of mature and immature spermatozoa. A reduction of IMP, especially remarkable in the post-acrosomal domain, was observed in mature epididymal spermatozoa when compared with samples collected from ductuli efferentes. Some structural species-specific differences were also observed. The significance of these changes has not been determined, but remodeling of membrane components during developmental processes constitutes a fine control mechanism to ensure that key molecules are in the correct membrane position and during an appropriate timeframe to mediate fertilization.     

Membrane differentiations in spermatozoa of the squid,Loligo pealeii

Regional differences in the structure of the plasma membrane and acrosome membrane of squid spermatozoa were studied by freeze-fracture and thin section electron microscopy. In regions of close apposition the plasma membrane and acrosome membrane are adjoined to one another by regularly spaced linkages. These linkage sites, overlie a set of fibers located at the inner face of the acrosomal membrane. The acrosomal fibers terminate in a layer of granular material located at the base of the acrosome. Detergent treatment of sperm releases the fibers and granular material as an interconnected complex. Freeze-fracture replicas reveal a random arrangement of intramembranous particles in the plasma membrane over the sperm head and linear aggregates of intramembranous particles in the acrosomal membrane. Several regional differences in the structure of the flagellar plasma membrane are present. The thickness of the glycocalyx is progressively reduced distally along the flagellum. Freeze-fracture replicas show evenly spaced linear arrays of intramembranous particles which extend parallel t o the flagellar long axis. Examination of spermatozoa extracted to disrupt flagellar geometry suggest that the dense fiber-doublet microtubule complexes are attached to the plasma membrane. The possible functional role of these membrane differentiations and their relationship t o membrane structures in mammalian spermatozoa are discussed.     

Spermatogenesis in animals as revealed by electron microscopy

Summary Testes of the Japanese crayfish, Cambaroides japonicus, were fixed in buffered (pH 7.4) 4% formaldehyde followed by buffered (pH 7.4) 1% osmium tetroxide, and thin sections of the epoxy Epon resin-embedded tissue were studied with the electron microscope. Spermatozoa from vasa deferentia and spermatids from the testis were examined in smear preparations and thick sections by an ordinary light microscope, employing the Feulgen nuclear technique, fast green or periodic acid-Schiff reagent. On the other hand, testes fixed with buffered (pH 7.4) 4% formaldehyde were incubated in Novikoff and Goldfischer's medium or in Mölbert and coworkers' mixture for demonstrating thiamine pyrophosphatase (TPPase) or alkaline phosphatase, and observed in the electron microscope.The microtubules 220 Å to 310 Å in diameter appearing in the nuclear process seem to represent some unit structure of chromosomes in this species. The microtubules are composed of the tubular subunits which are disposed twisted along the peripheral part of major axis of the microtubules. Such tubular subunits are approximately 20 Å thick in wall and 10 Å wide in lumen. The acrosome in a helmet-like shape has been found to have a hornlike process at its proximal part, though the function of such process remains unsettled in the present study. With incubation in disodiumphenylphosphate, no final product is deposited in any part of the premature spermatozoa. The convoluted membrane as well as the invagination of nuclear envelope are revealed to be specific sites for TPPase activity, and such finding suggests that TPPase may act as an intermediary in formation of nuclear processes of the crayfish sperm.This study was supported by Grant GM-8327-05 from the United States Public Health Service.Scientist from the Laboratory of Electron Microscopy, Department of Biology, Kyung Pook National University, Taegu, Korea.     

Tektin 4 is located on outer dense fibers, not associated with axonemal tubulins of flagella in rodent spermatozoa

Tektins, which are thought to be the constitutive proteins of microtubules in cilia, flagella, basal bodies, and centrioles, have been reported to be involved in the stability and structural complexity of axonemal microtubules. Four types of mammalian Tektins have been reported, and at least two types of Tektins, Tektin 2 and Tektin 4, have been verified to be present in sperm flagella. To elucidate the molecular localization of Tektin 4 in flagella of rodent spermatozoa, we performed immunocytochemistry, fractionation study followed by immunoblot analysis, and immunogold electron microscopy. Confocal laser scanning microscopy and immunogold electron microscopy indicated that Tektin 4 was associated with outer dense fibers (ODFs) in both the middle and principal piece of flagella in rat and mouse spermatozoa. Tektin 4 in rat spermatozoa is completely released by 6 M urea treatment, but not extracted by 1% Triton X-100 and 0.6 M potassium thiocyanate. Pre-embedding immunoelectron microscopy demonstrated that Tektin 4 located on the abaxial (convex) surface of ODFs in flagella, not associate with axonemal microtubules. Our data strongly suggested that Tektin 4 is not associated with axonemal tubulins but an ODFs-affiliated molecule in rodent spermatozoa.     

Association between coated vesicles and microtubules

In this study, a possible functional association between microtubules and coated vesicles is described. We have found that our preparations of microtubules contained coated vesicles in quantities of usually above 10%. These coated vesicles were identified both by immunological methods using anticoat antibodies and by electron microscopy of negatively stained specimens. In the immune replica, two components of coated vesicles, i.e., heavy (clathrin) and light chains, were recognized as constituents of the preparations. In the electron microscope, it was found that coated vesicles were attached predominantly along the length of microtubules. Furthermore, projections from the microtubules to the triskelion centers of the clathrin lattice were identified and thus seem to serve as linkers between the cytoskeletal structure of the organelle. A similar type of association was detected in tissue culture cells; bridges between coated vesicles and microtubules were clearly identified by electron microscopy of thin sections.     

Stereo views and immunogold labeling of the pellicular microtubules at the inner surface of the plasma membrane of Leishmania as revealed by fracture-flip.

We used a modification of fracture-flip to reveal the nanoanatomy of the inner surface of the plasma membrane in promastigotes of Leishmania. After freeze-fracture, lightly fixed promastigotes were coated with a stabilizing layer of carbon evaporated from an electron gun, thawed, and washed. Fractured promastigotes attached to the carbon casts by the protoplasmic (i.e., inner) halves of their plasma membranes were treated with Triton X-100, followed by exposure to low concentrations of trypsin and thorough washing. This was followed by picking up and flipping of the replicas, followed by air-drying. The actual inner surfaces of the plasma membrane were then imaged by platinum shadowing. Extended, three-dimensional, high-resolution views of the inner surface of the plasma membrane showed parallel arrays of microtubules (average spacing 47 nm) closely apposed to the inner surface. Cytochemical labeling confirmed the morphological identification of both subpellicular and flagellar microtubules, as determined by treatment with mouse monoclonal anti-alpha- or anti-beta-tubulin, followed by labeling with goat anti-mouse IgG adsorbed to colloidal gold. Removal of the microtubules revealed parallel arrays of particles (average diameter 17 nm). We hypothesize that these particles represent the cytoplasmic portion of proteins that link the microtubules to the plasma membrane.     

Microtrabecular structure of the axoplasmic matrix: visualization of cross-linking structures and their distribution

Axoplasmic transport is a dramatic example of cytoplasmic motility. Constituents of axoplasm migrate as far as 400 mm/d or at approximately 5 micron/s. Thin-section studies have identified the major morphological elements within the axoplasm as being microtubules, neurofilaments (100-A filaments), an interconnected and elongated varicose component of smooth endoplasmic reticulum (SER), more dilated and vesicular organelles resembling portions of SER, multivesicular bodies, mitochondria, and, finally, a matrix of ground substance in which the tubules, filaments, and vesicles are suspended. In the ordinary thin-section image, the ground substance is comprised of wispy fragments which, in not being noticeably tied together, do not give the impression of representing more than a condensation of what might be a homogeneous solution of proteins. With the high-voltage microscope on thick (0.5-micron) sections, we have noticed, however, that the so- called wispy fragments are part of a three-dimensional lattice. We contend that this lattice is not an artifact of aldehyde fixation, and our contention is supported by its visability after rapid-freezing and freeze-substitution. This lattice or microtrabecular matrix of axoplasm was found to consist of an organized system of cross-bridges between microtubules, neurofilaments, cisternae of the SER, and the plasma membrane. We propose that formation and deformation of this system are involved in rapid axonal transport. To facilitate electron microscope visualization of the trabecular connections between elements of axoplasm, the following three techniques were used: first, the addition of tannic acid to the primary fixative, OsO4 postfixation, then en bloc staining in uranyl acetate for conventional transmission electron microscope (TEM); second, embedding tissue in polyethylene glycol for thin sectioning, dissolving out the embedding medium from the sections and blocks, critical-point-drying (J. J. Wolosewick, 1980, J. Cell Biol., 86:675-681.), and then observing the matrix-free sections with TEM or the blocks with a scanning electron microscope; and third, rapid freezing of fixed tissue followed by freeze-etching and rotary- shadowing with replicas observed by TEM. All of these procedures yielded images of cross-linking elements between neurofilaments and organelles of the axoplasm. These improvements in visualization should enable us to examine the distribution of trabecular links on motile axonal organelles.     

Spermatozoa of an Old World Ricinulei (Ricinoides karschii, Ricinoidae) with notes about the relationships of Ricinulei within the Arachnida

The ultrastructure of spermatozoa is a valuable tool for phylogenetic and systematic studies. Ricinulei are enigmatic and poorly studied arachnids. So far, spermatozoa are only known from New World ricinuleids. The goals were to study, by means of light and transmission electron microcopy, the spermatozoa of an Old World species with regard to their phylogenetic implications, e.g., does the sperm structure contribute to the debated sister-group relationship of Acari and Ricinulei. The spermatozoa are coiled-flagellate and characterized by a cap-like acrosomal vacuole covered by electron-dense material, an elongated nucleus covered by a manchette of microtubules during spermiogenesis, an axoneme with a 9+2 microtubular pattern, a nuclear tube and axonemal basis which both originate underneath the acrosomal vacuole and cleistospermia as transfer form equipped with three intracellular plates. The data of the present study did not support a close relationship of Ricinulei and Acari which have aflagellate sperm with various synapomorphies as e.g., lacking nuclear envelopes/membranes in Actinotrichida (very similar to Solifugae) or vacuolated spermatozoa in Anactinotrichida. Affinities of Ricinulei are discussed in the light of the ultrastructure of arachnid spermatozoa.     

Fibroblast spreading in the presence of cytochalasin D: immunoelectron microscopy of the cytoskeleton

Spreading of mouse embryo fibroblasts in the presence of cytochalasin D (1 microgram/ml) was studied using scanning electron microscopy, immunofluorescence, and electron microscopy of platinum replicas. Whereas circular lamellae were formed around the cell body during normal spreading, separate processes appeared at the cell periphery during spreading in cytochalasin-containing medium. The processes gradually elongated and branched. Cytoskeletons of fibroblasts spreading in the cytochalasin-containing medium were obtained by Triton X-100 extraction. They contained microtubules, intermediate filaments, actin "paracrystals" looking like short microfilament bundles, and patches of a meshwork-granular material. Immunogold coating of the cytoskeletons with anti-actin antibody showed that some meshwork-granular patches were decorated with gold particles, whereas the others were not. Non-actin patches were usually located on the distal ends of the processes, thus leaving behind the actin cytoskeletal components during the process growth. Another characteristic feature of this unidentified material is its usual association with the substratum and microtubules. These results suggest that the process protrusion during cell spreading in cytochalasin-containing medium may occur not due to actin polymerization as in the control cells, but due to involvement of some other non-actin cytoskeletal components. These components seem to be able to move along microtubules and to bind to the substratum.     

Fine structure of spermatozoa of Chondrostoma nasus and Rutilus meidingerii (Teleostei, Cyprinidae), as revealed by scanning and transmission electron microscopy

Fürböck, S., Patzner, R.A. and Lahnsteiner, F. 2008. Fine structure of spermatozoa of Chondrostoma nasus and Rutilus meidingerii (Teleostei, Cyprinidae), as revealed by scanning and transmission electron microscopy. — Acta Zoologica (Stockholm) 91 : 88–95
The fine structure of spermatozoa of sneep or nase, Chondrostoma nasus , and lake chub, Rutilus meidingerii , was investigated by means of scanning and transmission electron microscopy. The uniflagellate spermatozoa of C. nasus lacked an acrosome. The flagellum contained the conventional nine peripheral doublets and one central pair of microtubules (9 + 2 pattern) and lacked lateral fins. The uniflagellate spermatozoa of R. meidingerii were made up of a head, also without an acrosome. For both species the sperm tail was covered by a plasma membrane. The midpiece of C. nasus contained five or six mitochondria on average, vesicles and glycogen granules, whereas the midpiece of R. meidingerii had seven mitochondria of a spherical or ovoid shape. The centriolar complex was located caudolaterally with respect to the nucleus. In C. nasus , the centrioles were orientated at an angle of 125° to each other, whereas the centrioles of R. meidingerii were at an angle of 110°. The fine structure of C. nasus and R. meidingerii spermatozoa showed species-specific differences in the position of the proximal centriole relative to the distal centriole, the position and number of mitochondria, size of the head and the length of the flagellum. (Correction added on 11 June 2009, after first online publication: The word 'axoneme' was deleted from the sentence 'The flagellum contained the conventional nine peripheral doublets and one central pair of microtubules (9 + 2 pattern) axoneme and lacked lateral fins.')