THE INFLUENCE OF X-RAYS ON ORGANELLE INDUCTION AND DIFFERENTIATION IN GRASSHOPPER SPERMATOGENESIS

The effect of x-irradiation on grasshopper spermatogenesis was studied with the aid of light and electron microscopy. The insects were irradiated at the second instar prior to the presence of maturation stages and observed at the last instar and imago stages. Dosages of 100 to 600 roentgens were found to retard the differentiation of the nucleus and mitochondrial nebenkern in spermatids. Evidence is presented that irradiation causes a curtailment and disorganization in the differentiation of the nebenkern from mitochondria. The above doses also induced the formation of supernumerary centrioles, flagellar filaments and acrosomes; nuclear disorganization as well as pycnosis and fragmentation also occur. The nucleus appears to be drawn toward each radiation-induced supernumerary acrosome, with consequent multipolarity of the nucleus. Induction of a set of flagellar filaments is seen only where the centriolar structure is in contact with the nucleus. Details are given of an organelle, heretofore not described, that is composed of anastomosed and interwoven cytoplasmic strands.     

An electron microscope study of nebenkern formation and differentiation in spermatids of Murgantia histrionica (Hemiptera, Pentatomidae)

Mitochondria in early spermatids of many insects aggregate and form a round body, the nebenkern. The nebenkern undergoes a structural differentiation and then divides into two separate equal-sized bodies. In the present study, nebenkerns of Murgantia histrionica, a Hemipteran insect, were reconstructed using electron micrographs of serial sections to determine how the mitochondria transform into the two separate bodies. Newly formed nebenkerns are made of one piece, an anastomosis of rod-like segments. Some segments interconnect to join networks of rings. Each network interlocks with another similar network, but networks which interlock are connected with each other by other segments of the nebenkern. Later, the entire nebenkern is made of two unconnected and interlocked networks of rings. The nebenkern appears to remain bipartite during subsequent differentiation. Since the two pieces are interlocked, breaks must occur before the pieces can separate. As breaks occur, each network transforms into a set of curved sheets, producing a nebenkern made of four concentric layers. The three outer layers are each made of two curved sheets which surround a bipartite central core. The surface sheets meet at a furrow in the surface of the nebenkern; segments in each layer are roughly symmetrical with each other about the plane in which the furrow lies. Rod-like segments join alternate segments. The number of layers then decreases to three, and later, to two. These nebenkerns resemble four-layered nebenkerns, but fewer connections between alternate segments are present. The two pieces constituting the nebenkern probably separate after most of the latter connections disappear. Hypotheses to account for the observed changes in nebenkern structure are presented.     

Spermatogenesis in animals as revealed by electron microscopy. VI. Researches on the spermatozoon-dimorphism in a pond snail,Cipango-paludina malleata

This paper reports an electron microscope study of typical and atypical spermatogenesis in the pond snail, Cipangopaludina malteata. In the typical spermatid the nucleus undergoes profound changes as development proceeds, affecting both its form and internal fine structure. A large number of roughly parallel, dense filaments, arranged along the long axis of the nucleus, fuse with each other to form in the end the homogeneous helical body characteristic of the head of the adult spermatozoa. The nebenkern is apparently mitochondrial in nature and, in its early development, is similar to that of insects except that it appears as a double structure from the beginning. As differentiation proceeds, the mitochondria lose their membranes, and the residual, now denuded cristae, reorganize to give a parallel radial arrangement. In the last stages of development, the nebenkern derivations become applied to the sheath of the middle piece in a compact helical fashion. In the development of the atypical spermatozoa, the nucleus fails to differentiate and simply shrinks in volume until only a remnant, devoid of DNA, is left. The cytoplasm shows numerous vesicles containing small Feulgen-positive bodies, 80 to 130 mmicro in diameter. These vesicles plus contents increase in number as spermatogenesis proceeds. The "head" structure of the atypical spermatozoa consists of a bundle (7 to 17) of tail flagella, each with a centriole at its anterior end. The end-piece of the atypical form appears brush-like and is made up of the free ends of the several flagella.     

Spermatogenesis in Animals as Revealed by Electron Microscopy : VI. Researches on the Spermatozoon-Dimorphism in a Pond Snail, Cipangopaludina malleata

This paper reports an electron microscope study of typical and atypical spermatogenesis in the pond snail, Cipangopaludina malteata. In the typical spermatid the nucleus undergoes profound changes as development proceeds, affecting both its form and internal fine structure. A large number of roughly parallel, dense filaments, arranged along the long axis of the nucleus, fuse with each other to form in the end the homogeneous helical body characteristic of the head of the adult spermatozoa. The nebenkern is apparently mitochondrial in nature and, in its early development, is similar to that of insects except that it appears as a double structure from the beginning. As differentiation proceeds, the mitochondria lose their membranes, and the residual, now denuded cristae, reorganize to give a parallel radial arrangement. In the last stages of development, the nebenkern derivations become applied to the sheath of the middle piece in a compact helical fashion. In the development of the atypical spermatozoa, the nucleus fails to differentiate and simply shrinks in volume until only a remnant, devoid of DNA, is left. The cytoplasm shows numerous vesicles containing small Feulgen-positive bodies, 80 to 130 mµ in diameter. These vesicles plus contents increase in number as spermatogenesis proceeds. The "head" structure of the atypical spermatozoa consists of a bundle (7 to 17) of tail flagella, each with a centriole at its anterior end. The end-piece of the atypical form appears brush-like and is made up of the free ends of the several flagella.     

Giant nebenkern produced by colcemid treatment of the spermatocysts of the silkworm,Bombyx mori

Summary The silkworm spermatocysts isolated from testes were cultured for 24 h in a medium containing 1 g of colcemid per ml, which is an inhibitor of tubulin association to microtubules. In the present experiment, the suppression of meiotic division I by this treatment resulted in the formation of a giant nebenkern in the primary spermatocyte. Normally, the nebenkern, an organelle in which all the cell's mitochondria aggregate, is formed in the spermatids of insects and changes into elongated mitochondrial derivatives. After washing with the culture medium without colcemid, the cell bearing the giant nebenkern grew to be a sperm with four flagella. When meiotic division II was blocked, the secondary spermatocytes with a nebenkern appeared and the further culture resulted in sperm with two flagella. These results suggest that the formation of a nebenkern is a necessary step toward the final stages of spermiogenesis.     

Cytostructural dynamics of spreading and translocating cells

Cytostructural changes during fibroblast spreading and translocation and during the transition between the two states have been studied in living cells and in the same cells after fixation and immunofluorescent staining. In time-lapse sequences we observe that birefringent arcs, sometimes circles, concentric with the cell perimeter, form near the periphery of a spreading cell, or that arcs form near the leading edge of a locomoting cell. The arcs move toward the nucleus, where they disappear. In spreading cells, radial stress fibers extend from the region of the cell nucleus to the periphery. The arcs or circles and the stress fibers are visualized in the same cells after fixation and staining with fluorescein-conjugated antiactin antibodies. Stained images of spreading cells show the arcs and stress fibers in the same plane of focus. At points of intersection with arcs, stress fibers are bent toward the substrate on which the cell is moving. During a transitional stage between spreading and translocation the cytostructure undergoes reproducible changes. Arcs and circle cease to form. The radial stress fibers elongate, spiral around the nucleus, and move to the periphery as a band of filaments. We interpret the moving arcs as condensations of a microfilament network that move toward the nucleus as compression waves. As elements of the net are brought close together by the compression wave, contraction may occur and facilitate the condensations.     

Lepidotrachelophyllum fornicis,n. g., n. sp., a Ciliate with an External Layer of Organic Scales (Ciliophora,Litostomatea, Haptoria)1

Lepidotrachelophyllum fornicis n. g., n. sp. was discovered in White Lake, Ontario, Canada, under winter ice. The genus is Trachelophyllum-like, being highly flattened, elongate, and very extensible. The major feature that separates it from other genera in the family Trachelophyllidae is the presence of a dense layer of organic scales which covers the exterior of the cell and through which the cilia emerge. The scales are composed of filamentous material which is organized as an ovoid structure. The “rim” of the baseplate is formed of interwoven filaments. The baseplate is broken by circular or polygonal apertures. The same filaments form an arched superstructure broken by even larger, less regular apertures.     

Spermitin: A Novel Mitochondrial Protein in Drosophila Spermatids

Mitochondria, important energy centers in the cell, also control sperm cell morphogenesis. Drosophila spermatids have a remarkably large mitochondrial formation called the nebenkern. Immediately following meiosis during sperm development, the mitochondria in the spermatid fuse together into two large aggregates which then wrap around one another to produce the spherical nebenkern: a giant mitochondrion about 6 micrometers in diameter. The fused mitochondria play an important role in sperm tail elongation by providing a structural platform to support the elongation of sperm cells. We have identified a novel testis-specific protein, Spermitin (Sprn), a protein with a Pleckstrin homology-like (PH) domain related to Ran-binding protein 1 at its C-terminus. Fluorescence microscopy showed that Sprn localizes at mitochondria in transfected Kc167 cells, and in the nebenkern throughout spermatid morphogenesis. The role of Sprn is unclear, as sprn mutant males are fertile, and have sperm tail length comparable to the wild-type.     

昆虫精细胞内中心粒附体的来源和作用

本研究应用界面铺浮和超薄切片技术,观察了东亚飞蝗(Locusta migratoria manilensis)和七星瓢虫(Coccinella siptempunctata L.)精细胞内中心粒附体(CA)的形成和作用。结果发现,作为电子致密体的CA前体和原顶体颗粒出现在副核和细胞核之间区域。随后,这个主要是由约300 A颗粒组成的CA前体附着在核膜,核内、外膜加厚。在副核分化成两个线粒体衍生物或稍早些时刻,近心中心粒移向CA并嵌入。中心粒镶嵌到校膜上发育成基体,由此生长出轴丝来。随着精细胞的延长, CA的形状也跟着转变和伸长。 250-300A染色质纤维沿精细胞长纵轴连接在CA结构的基部。 当精细胞核向长形转变时,染色质纤维解旋并结合在一起形成缎带结构。因此,可以设想cA是作为暂时性细胞器在组织精细胞内,染色质纤维重新组织排列和指导中心粒移向精细胞核的特定区域中起作用。     

THE FINE STRUCTURE OF THE AXON AND GROWTH CONE OF THE DORSAL ROOT NEUROBLAST OF THE RABBIT EMBRYO

The centrally directed neurite of the dorsal root neuroblast has been described from the period of its initial entrance into the neural tube until a well-defined dorsal root is formed. Large numbers of microtubules, channels of agranular reticulum, and clusters of ribosomes are found throughout the length of the early axons. The filopodia of the growth cone appear as long thin processes or as broad flanges of cytoplasm having a finely filamentous matrix material and occasionally small ovoid or elongate vesicles. At first the varicosity is a small expansion of cytoplasm, usually containing channels of agranular reticulum and a few other organelles. The widely dilated cisternae of agranular reticulum frequently found within the growth cone probably correspond to the pinocytotic vacuoles seen in neurites in tissue culture. The varicosities enlarge to form bulbous masses of cytoplasm, which may measure up to 5 µ in width and 13 µ in length. They contain channels of agranular reticulum, microtubules, neurofilaments, mitochondria, heterogeneous dense bodies, and a few clusters of ribosomes. Large ovoid mitochondria having ribonucleoprotein particles in their matrix are common. Dense membrane specializations are found at the basal surface of the neuro-epithelial cell close to the area where the early neurites first enter the neural tube.     

An electron microscopic study of flight muscle breakdown in an aphid Megoura viciae

Within two days of settling to feed on a host plant degenerative changes can be detected in the flight muscles of aphids at the ultrastructural level. The thick myosin filaments and the M Lines and later the Z bands disappear leaving the thin actin filaments in the cytoplasm. The mitochondria change their configuration and become enveloped in cytosegresomes. As mitochondria become reduced in number the numbers of lysosomes and residual bodies in the cells build up. The cells then appear to become dispersed as finger-like processes protrude from them and are pinched off.     

ORIGIN, DEVELOPMENT, AND NATURE OF INTRANUCLEAR RODLETS AND ASSOCIATED BODIES IN CHICKEN SYMPATHETIC NEURONS

Correlative data are presented here on the developmental history, dynamics, histochemistry, and fine structure of intranuclear rodlets in chicken sympathetic neurons from in vivo material and long-term organized tissue cultures. The rodlets consist of bundles of ~70 ± 10 A proteinaceous filaments closely associated with ~0.4–0.8 µ spheroidal, granulofibrillar (gf) bodies of a related nature. These bodies are already present in the developing embryo a week or more in advance of the rodlets. In early formative stages rodlets consist of small clusters of aligned filaments contiguous with the gf-bodies. As neuronal differentiation progresses these filaments increase in number and become organized into well-ordered polyhedral arrays. Time-lapse cinemicrography reveals transient changes in rodlet contour associated with intrinsic factors, changes in form and position of the nucleolus with respect to the rodlet, and activity of the gf-bodies. With the electron microscope filaments may be seen extending between the nucleolus, gf-bodies, and rodlets; nucleoli display circumscribed regions with fine structural features and staining reactions reminiscent of those of gf-bodies, We suggest that the latter may be derivatives of the nucleolus and that the two may act together in the assemblage and functional dynamics of the rodlet. The egress of rodlet filaments into the cytoplasm raises the possibility that these might represent a source of the cell's filamentous constituents.     

THE ULTRASTRUCTURE OF MAMMALIAN CARDIAC MUSCLE

Papillary muscles of rat and dog hearts were fixed in such a way as to prevent excessive shortening during the procedure. The material was embedded in either araldite or methacrylate and was stained in various ways. The filamentous fine structure of mammalian cardiac muscle is similar to that previously described for striated skeletal muscle. The sarcomeres are composed of a set of thick and thin filaments which interdigitate in the A band proper. The filament ratios and the filamentous array are in accord with those found in skeletal muscle. The functional significance of this twofold array of filaments is not entirely clear. Various other structural aspects of cardiac cells such as surface membranes, mitochondria, nuclei, and cytoplasmic granules are described. The sarcoplasmic reticulum is discussed in detail as are the various structural components forming the intercalated discs. Fairly frequent deep invaginations of the sarcolemma with basement membrane are observed in addition to the intercalated discs. These surface membrane invaginations probably explain the branching appearance of cardiac fibers as seen under the light microscope.     

Life History,Reproductive Morphology and Development of the Antarctic Brown Alga Desmarestia menziesii J. Agardh*

The life history, reproduction and development of Desmarestia menziesii J. Agardh from Antarctica is described. Unilocular sporangia occur singly or in small groups in the outermost cortical layer of the sporophyte. They are formed by periclinal division of cortex cells into a stalk cell and the sporangium initial. Meiospores germinate into dioecious microscopic filamentous gametophytes. As in other perennial Antarctic species of the Desmarestiales, gametangia are formed in culture under short-day conditions or in darkness. In nature, juvenile sporophytes should therefore be formed in winter. They develop only attached to the oogonium. At first they are uniseriate and elongate by means of an intercalary meristem located in their middle part. Laterals are formed predominantly in this region, and they subsequently give rise to secondary laterals. The branching pattern is opposite to alternate in both young and adult plants. Cortication of the main axis is initiated by filaments growing out from the lowermost cells of the primary laterals. In sporophytes of this developmental stage the meristem of the main axis is confined to a small region where cortication starts and above. Lateral branches elongate and become corticated in the same way as the main axis. In mature plants, cells of the inner cortex can become meristematic again and form a meristoderm which contributes to axis thickness by periclinal and anticlinal divisions. The observations are discussed in relation to possible evolutionary relationships in the genus Desmarestia and in the order Desmarestiales.     

The 2G2 Antibody Recognizes an Acidic 110-kDa Human Mitochondrial Protein

Using fluorescence microscopy, the mouse monoclonal antibody 2G2 was found to label mitochondria in human cells, as assessed by double staining with either Rhodamine 123 or a polyclonal antibody to mitochondrial matrix HSP-60 proteins. No reactivity to the 2G2 antibody was detected in cells from mouse, rat and chicken. Immunoblotting analysis demonstrated that the 2G2 antigen corresponds to a human protein with a relative mobility of 110 kDa and an approximate isoelectric point of 6.5 that co-partitions with HSP-60 proteins during isolation of mitochondria from HeLa cells. Close examination of the 2G2 staining pattern in HeLa and Fanconi's anaemia cells revealed differences in the morphology and organization of mitochondria in these two cell types. In HeLa cells, mitochondria appear as individual tubular compartments of variable length and are closely associated with vimentin filaments, particularly at the periphery of the nucleus. In Fanconi's anaemia cells, mitochondria have a filamentous shape and form an interconnected cytoplasmic reticulum running in parallel with both vimentin filaments and microtubules. After stabilization with aldehyde- or alcohol-based fixation protocols that optimize the preservation of cytoskeletal components, the epitope targeted by the 2G2 antibody may serve as a valuable marker in the investigation of relationships between mitochondria and other cellular structures in human cells.     

Some observations on spermatogenesis in Gelastocoris oculatus (Hemiptera) with the aid of the electron microscope

The nuclear cap in the spermatogonial and early spermatocyte cells of Gelastocoris is an aggregate of closely packed mitochondria with their long axes perpendicular to the nuclear membrane. Eventually in the early growth period, the mitochondria move from the cap and appear to become more or less equally distributed in the cytoplasm where they remain until their fusion in the spermatid to form the nebenkern. The Golgi complex consists of clusters of lamellae and vesicles, the Golgi bodies. Granules form within the vesicles, increase in size, move from their place of origin and become distributed at random in the cytoplasm. They are the pro-acrosomal granules and at the end of the growth period fuse to form the proacrosome, about which Golgi bodies collect. The Golgi bodies, however, never fuse into an acroblast. At one end of the oval-shaped pro-acrosome is a small dark body and a less dense vesicle the future of which is uncertain. The dark body eventually occupies a position at the tip of the acrosome. The pro-acrosome, after moving to the side of the nucleus opposite the nebenkern, differentiates into the acrosome which elongates into a tail-like structure. The nuclear membrane of some spermatocytes may appear wave-like in cross section, with the crest and trough different in appearance. Near the membrane and in the troughs of the waves large clusters of granules are frequently present. Similar clusters may be found elsewhere in the cytoplasm. Presumably they had their origin near the membrane but this is not conclusive. Bodies of indeterminate origin and structure may be present in the cytoplasm. They could be lysosomes but evidence is lacking. In late spermatocytes and in spermatids, a group of ten or twelve granules is present. They are smaller than the pro-acrosomal granules, are always closely associated and pass as a group into the tail. Their significance is unknown. The endoplasmic reticulum is typical of cells in general. There are no granule accumulations within the vesicles as in some secretory cells. Vesicles of various shapes and sizes are present within the centrosphere of the first meiotic division. While their location is similar to that of the centriole, the identity of the vesicles is uncertain.     

MITOCHONDRIA IN LIVING CELLS: AN ANALYSIS OF MOVEMENTS

Time-lapse cinephotomicrography of mouse embryonic fibroblasts before and shortly after perfusion of tissue cultures reveals that the elongation of mitochondria caused by coenzyme A results from the terminal association of many shorter rods into a smaller number of long filaments. These are not permanent associations, but they reflect an exaggeration of the cohesive tendency of mitochondria, which in untreated cells is counterbalanced by frequent disjoinings and breakings of the anastomotic network. Our own observations and a survey of the literature suggest that elongate mitochondria with rapid movement and high metabolic activity tend to accompany proliferation in tissue cultures, and that mitotic inhibition of cultured cells may go together with short, slow mitochondria of low metabolic activity. The movement of mitochondria may be both active, reflecting metabolic exchanges with the cytoplasm, and passive, the result of hyaloplasmic currents.     

The dynamics of filamentous structures in the apical band, oral crescent, fission line and the postoral meridional filament in Tetrahymena thermophila revealed by monoclonal antibody 12G9

The ciliate Tetrahymena thermophila possesses a multitude of cytoskeletal structures whose differentiation is related to the basal bodies - the main mediators of the cortical pattern. This investigation deals with immunolocalization using light and electron microscopy of filaments labeled by the monoclonal antibody 12G9, which in other ciliates identifies filaments involved in transmission of cellular polarities and marks cell meridians with the highest morphogenetic potential. In Tetrahymena interphase cells, mAb 12G9 localizes to the sites of basal bodies and to the striated ciliary rootlets, to the apical band of filaments and to the fine fibrillar oral crescent. We followed the sequence of development of these structures during divisional morphogenesis. The labeling of the maternal oral crescent disappears in pre-metaphase cells and reappears during anaphase, concomitantly with differentiation of the new structure in the posterior daughter cell. In the posterior daughter cell, the new apical band originates as small clusters of filaments located at the base of the anterior basal bodies of the apical basal body couplets during early anaphase. The differentiation of the band is completed in the final stages of cytokinesis and in the young post-dividing cell. The maternal band is reorganized earlier, simultaneously with the oral structure. The mAb 12G9 identifies two transient structures present only in dividing cells. One is a medial structure demarcating the two daughter cells during metaphase and anaphase, and defining the new anterior border of the posterior daughter cell. The other is a post-oral meridional filament marking the stomatogenic meridian in postmetaphase cells. Comparative analysis of immunolocalization of transient filaments labeled with mAb12G9 in Tetrahymena and other ciliates indicates that this antibody identifies a protein bound to filamentous structures, which might play a role in relying polarities of cortical domains and could be a part of a mechanism which governs the positioning of cortical organelles in ciliates.     

Nucleation of polar actin filament assembly by a positively charged surface

Polylysine-coated polystyrene beads can nucleate polar assembly of monomeric actin into filamentous form. This nucleation has been demonstrated by a combination of biochemical and structural experiments. The polylysine-coated beads accelerate the rate of actin assembly as detected by two different biochemical assays. Subsequent examination of the beads by electron microscopy reveals numerous actin filaments of similar length radiating from the beads. ATP promotes this bead-induced acceleration of assembly. Decoration of the filaments with the myosin fragment S1 shows that these filaments all have the same polarity, with the arrowhead pattern pointing toward the bead. The relevance of the system to in vitro mechanisms and its usefulness in other studies are discussed.