Spermatogenesis in animals as revealed by electron microscopy

Summary The early spermatid nuclei of the grasshopper, Acrida lata, have been observed electron microscopically. The irregularly compact chromatin mass appears closely attached to the nuclear envelope. This mass migrates subsequently into a more central portion. It seems to participate in the formation of the nucleolus as a nucleolar organizer. At the time when the chromatin mass and frequently the nucleolus undergo involution, clusters of peculiar granular bodies 130 m in average diameter and 200 A wide filamentous elements among the bodies make their appearance in the nucleoplasm. The particles constituting the granular bodies are composed of DNA, but their matrix consists of RNA. The term microkaryosome is proposed for such granular body, because it is similar in chemical components to karyosome, but the former is smaller in size than the latter. It is suggested that the microkaryosome may be related with the paracrystalline formation of nucleoprotein.     

Spermatogenesis in animals as revealed by electron microscopy

Summary The first indication of differentiation of the Jensen's ring has been detected in an early stage of spermiogenesis of Felis catus Linné when the pair of centrioles takes up a position immediately beneath the plasma membrane. The chromatoid bodies appear in the early spermatid cytoplasm through the nuclear pore complex. In a more advanced stage, such bodies have been found in association with the striated columns, the distal centriole or the proximal part of flagellum and the Jensen's ring. As the spermiogenesis proceeds, the bodies have decreased their size and density, and finally disappear in mature spermatozoa. The chromatoid bodies seem, therefore, to share with the centriole the capacity to form the connecting piece. As a consequence of disorganization of triplet microtubules of the centriole, a noticeable material appears in the center of lumen of the centriole to be identifiable as a distinct precursor of the central pair of axonemal complex. Microtubules are first developed as the sheath of principal piece of the sperm flagellum, originating from the plasma membrane surrounding the axonemal complex.     

Spermatogenesis in animals as revealed by electron microscopy

Summary Testes of Bombyx mori Linné were fixed in buffered (pH 8.2) 1% OsO₄ or 3 % KMnO₄ and thin sections of the tissue, embedded in methacrylate or epoxy Epon resin, were studied under the electron or light microscope.At the late stage of differentiation of the spermatid, the nucleus shows an elongated conical contour, being composed of fine fibrillar elements. These fibrillar elements fixed in OsO₄ measure 100 to 130 Å in diameter, while those fixed with KMnO₄ are approximately 70 Å in diameter.It has been found for the first time in the spermiogenesis of the silkworm that two bands and a tubular structure develop in close proximity to one another and attached to the plasma membrane of the spermatid. The two bands fixed in OsO₄ are electron dense, but in the material fixed with KMnO₄, one of them, situated within the cell body, is as dense as that fixed in OsO₄, while the other, outside the cell body, is much less dense. These apparently novel apparatuses develop from the caudal nuclear region along the elongating spermatid, but the dense band intertwines with the acrosome in the apical region of the nucleus along the major axis of spermatid, while the tubular structure and the clear band reach far into the nutritive cell where the dense band and acre-some are not visible.A possible relationship between the tubular structure and the nutritive cell has been discussed.This study was supported by Grant GM-8327-03 from the United States Public Health Service.     

Spermatogenesis in animals as revealed by electron microscopy

Summary The fine structure of the developing middle piece of the silkworm spermatid was studied in the electron microscope.The formation of Nebenkern and its metamorphosis are characteristic, for a given species of animal, and different from those of other species.Microtubules, with a wall approximately 50 Å thick and a lumen 110 Å in diameter, have been found in the developing middle piece; these are in close relationship with the differentiation of the axial tail filaments and of the Nebenkern derivatives.When the middle piece has reached a certain degree of maturity, a peripheral cytoplasmic layer of the middle piece is separated by the outer membrane of the endoplasmic reticulum, and later sloughed off from its trunk. The naked middle piece is subsequently covered by a mantle provided with 12–18 projections which develop descending from the proximal head part of the late spermatid.The clear band developing along the surface of the middle piece is a characteristic, complex structure which seems to represent an apparatus for pinocytosis or exosmosis.This study was supported by Grant GM-8327-04 from the United States Public Health Service.     

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.     

Spermatogenesis in animals as revealed by electron microscopy

Summary The development of nuclei and cytoplasmic microtubules was studied in the maturing spermatids of the grasshopper, Acrida lata, fixed with glutaraldehyde-potassium bichromate-osmium tetroxide and embedded in epoxy Epon-resin. Utilization of microkaryosomes for the formation of paracrystalline nucleoprotein is suggested by the fact that they are no longer visible in the advanced spermatid nuclei showing the paracrystalline structure. The cytoplasmic microtubules approximately 220 Å in diameter develop in close association with a linear material similar in density to the nuclear envelope. Only a single layer of the double-layered nuclear envelope is visible during the development of microtubules. Although cytoplasmic microtubules are assumed to have several physiological functions, such apparatus seem to be related to the polymerization of nucleoproteins as well, since the depolymerization of nucleoproteins occurs simultaneously along with the disappearance of cytoplasmic microtubules.     

Spermatogenesis in animals as revealed by electron microscopy

Summary The fine structure of the spermatids in late stages of the differentiation, which appeared in the testis of early pupa of the silkworm Bombyx mori Linné, was studied in the electron imcroscope, being fixed in buffered (pH 8.2) 2.5% osmium tetroxide or 3% potassium permanganate.The clear band differentiates into elaborated elements consisting of an array of at least 12 membranes which run loosely winding along the major axis of the spermatid. The elaborated clear band, i. e., clear band derivatives, may be an apparatus to facilitate the activity of spermatozoa, since they are present along the full length of the remarkably elongated premature spermatozoa.It has been revealed that the clear band derivatives possess a highly ordered, fine structure which is seen to be of a paracrystalline nature. The periodic pattern has first occurred along the long axis of the clear band derivatives. After such structure is decomposed into an apparently homogeneous material, a characteristic periodic pattern occurs again crossed the major axis of the clear band derivatives, the significance of such ultrastructural changes remaining obscure.The tubular structure appears through the head part of the developing spermatids, revealing even in an apical region where the nucleus is not visible, and it appears enlarged at the base of the nucleus, but no more visible in the tail piece. In the stages when the clear band becomes progressively specialized, the tubular structure appears attached to the nucleus, although it situated at the peripheral part of the cell in a more early stage of the differentiation. The tubule is incompletely separated into two layers by a dense septum projected from the tubular wall, suggesting that such structure provides the tubule with a relatively large interior surface for metabolic reactions.This study was supported by Grant GM-8327-04 from the United States Public Health Service.     

Spermatogenesis in animals as revealed by electron microscopy

Summary The dense bodies appearing in the cytoplasm of spermatids during early spermiogenesis of the grasshopper, Acrida lata, correspond to the chromatoid bodies of light microscopy, since they are composed of RNP. So far as the present material is concerned, the chromatoid bodies contribute to the formation of the centriole adjunct, because both structures consist of similar components and the former appear attached closely to the latter until the latter is completely formed. It has been tentatively suggested that the function of the centriole adjunct is to provide nutritive materials for the developing axial tail filament bundle.     

Spermatogenesis in animals as revealed by electron microscopy

Summary Testes of the pond snail, Cipangopaludina malleata Reeve, were fixed in 1% osmium tetroxide, 3% permanganate, or 4% formaldehyde followed by 1% osmium tetroxide, each being buffered to pH 7.2 with Veronal-acetate or Sörensen's phosphate buffer. On the other hand, testes fixed with 4% formaldehyde adjusted to pH 7.2 with 0.075 M Na-cacodylate were incubated in Novikoff-Goldfischer medium for demonstrating thiamine pyrophosphatase, uridine or inosine diphosphatase, uridine monophosphatase or adenosine triphosphatase. The specimens incubated were postfixed in 1% osmium tetroxide buffered to pH 7.2 with Veronal-acetate buffer. Thin sections of the epoxy Epon resin-embedded tissue were stained either singly with saturated aqueous uranyl acetate or doubly with saturated aqueous uranyl acetate followed by lead citrate.In a concentric lamellar structure consisting of the granular endoplasmic reticulum in the cytoplasm of early atypical spermatids, disappearance of ribosomes attached to the outer surface of cisternae seems to have initiated at the central part of the structure, and the cisterna-attached ribosomes seem to participate in the formation of dense granules appearing in the vesicles representing the endoplasmic reticulum of atypical spermatids.The Golgi apparatus of the atypical spermatids in the advanced stages of development is composed of at least three different layers, the central part consisting of an amorphous material, the following lamellar and vesicular elements, and the peripheral fine vesicles.It has been assumed that the mechanism by which the nucleic acid, especially DNA is converted into the polysaccharide might be attributed to the function of the Golgi apparatus, because the transformation of dense granules into less dense granules as well as diphosphatase activities have been detected within the Golgi apparatus.This study was supported by Grant GM-8327-06 from the United States Public Health Service.     

Structure of trichomonads as revealed by scanning electron microscopy.

A scanning electron microscope (SEM) study of Hypotrichomonas acosta (Moskowitz), Trichomonas vaginalis Donné, Pentatrichomonas hominis (Davaine), and Tritrichomonas foetus (Riedmüller) provided new information about the structure of the periflagellar canal; emergence of the flagella from the cell body; structure of the undulating membrane; and position, shape, and size of the pelta. Of special interest were the spatial relationships of the attached part of the recurrent flagellum and the accessory filament in Hypotrichomonas and in the members of Trichomonadinae, i.e. Trichomonas and Pentatrichomonas.     

Structural association of Bax with nuclear matrix and cytomatrix revealed by embedment-free immunogold electron microscopy

Bax is a cellular protein functioning as a promoter of apoptosis. It is ultrastructurally associated with mitochondrial membranes, where it participates in permeability transition pore formation. By employing embedment-free electron microscopy (EFEM), we present evidence that Bax is also associated with the nuclear matrix and cytomatrix of cultured human tumour cells (COLO 205, PA-1, U-373 MG). Extracted cellular scaffolds were probed with anti-Bax antibody using the immunogold electron microscopy technique. Bax immunoreactivity was found on 10-15 nm intermediate filaments of karyo- and cytoskeleton, stretched between the nucleus, nuclear lamina and cell periphery. Bax immunoreactivity was preferentially localized to certain areas of filaments (spot-like). The target molecules for Bax binding in the cellular matrix and their physiological significance remain to be established.     

Mitosis in the fungusBasidiobolus ranarum as revealed by electron microscopy

Summary Mitosis of nuclei in vegetative hyphae of the fungusBasidiobolus ranarum has been studied by electron microscopy. Cells fixed with glutaraldehyde and OsO⁴ were embedded in Vestopal. Sections were obtained of single cells whose mitotic status was known. Attention was paid to the behaviour of the microtubules, the nuclear envelope and the nucleolus. Nuclear division begins with the dilution and rearrangement of nucleolar material and the gradual breakdown of the nuclear envelope. At this stage the nucleus is surrounded by a sheet of closely packed microtubules. Some of these penetrate into the nucleus through gaps in the envelope. Dissolution of the envelope is followed or accompanied by the development of an extensive labyrinth of membranous cisternae which persists at the periphery of the division site through mitosis and probably contributes material to the envelopes of the daughter nuclei. The drum-shaped spindle of metaphase is composed of large numbers of microtubules aligned parallel to each other. Many of them are associated with chromosomes. Metaphase is soon followed by the movement of dense masses of nucleolar material and chromosomes to the poles of the division figure to form the socalled end plates. Microtubules extend into the end plates but not beyond. Neither centrioles nor centriolar plaques have been seen.     

Expression of BAX in cell nucleus after experimentally induced apoptosis revealed by immunogold and embedment-free electron microscopy

The unique combination of immunocytochemistry with embedment-free electron microscopy was applied for precise and specific localisation of BAX in the human colon adenocarcinoma COLO 205 cell line stimulated to undergo apoptosis by camptothecin (DNA topoisomerase I inhibitor). Camptothecin-induced apoptosis was associated with redistribution of BAX from cytosol to organelle membranes: mitochondria, Golgi apparatus, endoplasmic reticulum and via nuclear envelope pores to the nucleus, occurring within 60-180 min of cell exposure to the drug. An increase in BAX immunoreactivity on fine filaments and the lamina-pore complex of the nuclear matrix was also observed. The increase in BAX expression in the nuclear area of camptothecin-treated COLO 205 cells was confirmed by quantitative analysis using laser scanning cytometry. The subcellular translocations of BAX preceded the appearance of any morphological symptoms of apoptosis.