EXTRUSION OF NUCLEOLI FROM PRONUCLEI OF THE RAT

Electron microscope observations of osmium tetroxide-fixed rat eggs indicate that small nucleoli are extruded from pronuclei in a sharply demarcated time period after sperm penetration. Approximately 4½ hours after sperm penetration, fine fibrous material aggregated in distinct loci along the inner surface of the nuclear envelope and condensed into small, dense bodies. The term tertiary nucleolus or extrusion body is used to designate the forming bodies. The small tertiary nucleoli form distinct protrusions from the pronuclei during the following developmental period and finally bud off into the cytoplasm, carrying with them a small portion of the double nuclear envelope. The extrusion bodies can be observed only in the vicinity of the pronuclei and have not been seen near the cell membrane. The fate of the tertiary nucleoli is not known; apparently they transform or disappear after they have passed into the cytoplasm. Eleven hours after sperm penetration, tertiary nucleoli are not present near the nuclear membrane and the extrusion activity has apparently ceased. Large and small nucleoli react similarly to cytochemical reagents: they are Feulgen negative; they are positive to the Millon, Sakaguchi, brom-phenol blue, and PAS reactions. Azure B stain combined with nuclease extraction indicates the presence of small amounts of RNA in the nucleoli.     

Fine structure of germinatingPenicillium megasporum conidia

Summary Penicillium megasporum conidia have spore walls consisting of several layers. There is no visible change in the outer wall layers during spore germination, but the inner layers increases in thickness on only one side of the spore, resulting in a rupture of the outer wall layers and subsequently in germ tube formation. Invaginations in the plasma membrane disappear as the germ tube forms and emerges, and the nucleus migrates into the developing germ tube. Mitochondria gather at the base of the germ tube during its formation. During germination, the amount of lipid in the spore decreases and portions migrate into the germ tube. Membrane-bound, electron dense bodies are present in resting spores. These bodies decrease in size as germination proceeds, and the cytoplasm in the developing germ tube appears much more electron dense than the cytoplasm within the spore.     

Two RING Finger Proteins, the Oncoprotein PML and the Arenavirus Z Protein, Colocalize with the Nuclear Fraction of the Ribosomal P Proteins

The promyelocytic leukemia (PML) protein forms nuclear bodies which are relocated to the cytoplasm by the RNA virus lymphocytic choriomeningitis virus (LCMV). The viral Z protein directly binds to PML and can relocate the nuclear bodies. Others have observed that LCMV virions may contain ribosomes; hence, we investigated the effects of infection on the distribution of ribosomal P proteins (P0, P1, and P2) with PML as a reference point. We demonstrate an association of PML bodies with P proteins by indirect immunofluorescence and coimmunoprecipitation experiments, providing the first evidence of nucleic acid-binding proteins associated with PML bodies. We show that unlike PML, the P proteins are not redistributed upon infection. Immunofluorescence and coimmunoprecipitation studies indicate that the viral Z protein binds the nuclear, but not the cytoplasmic, fraction of P0. The nuclear fraction of P0 has been associated with translationally coupled DNA excision repair and with nonspecific endonuclease activity; thus, P0 may be involved in nucleic acid processing activities necessary for LCMV replication. During the infection process, PML, P1, and P2 are downregulated but P0 remains unchanged. Further, P0 is present in virions while PML is not, indicating some selectivity in the assembly of LCMV.     

Ribosomal bodies in early oogenetic stages of the lizard Lacerta sicula Raf.

During hibernation, ribosomal bodies are present in the germ cells of Lacerta sicula, which have undergone meiotic prophase, but neither in oogonia nor in stroma cells. These bodies are similar in structure, although smaller in size, to those which have been described in the cytoplasm of growing oocytes; in the germ cells at zygopachytene, they are frequently associated with the nuclear envelope.     

Precocious formation of synaptonemal-like polycomplexes and their subsequent fate in female Ascaris lumbricoides var. suum

During meiotic interphase, before leptotene, synaptonemal-like polycomplexes are seen in the cytoplasm of the Ascaris lumbricoides oocytes and in the communal anucleate rachis. In some females short intranuclear synaptonemal complexes are present briefly at that early stage. The number of extranuclear complexes increases just before leptotene, some are attached to the pores of the nuclear membrane. During zygotene most polycomplexes disappear. At late pachytene they reappear in some females but not in others. The morphology, when first seen, is that of disorganized filamentous bodies, later lateral elements appear among the filaments. The dimensions of the lateral elements of the polycomplexes are variable. In the male the distribution of polycomplexes among the rachis, the cell cytoplasm, and at the nuclear envelope is similar to that observed in the female.These observations confirm the precocious occurrence of synaptonemal-like polycomplexes reported by Bogdanov (1977). Ascaris lumbricoides thus, uniquely, appears to manufacture synaptonemal complex-like material in the communal cytoplasm of the germ cells prior to the time that the full complement of synaptonemal complexes appears in the nucleus.     

Unusual structure of nuclei in a B–mutant strain of Schizophyllum commune with intercellular nuclear migration

Serial sections of multinucleate hyphae of a B–mutant strain of Schizophyllum commune , in which intercellular nuclear migration takes place, revealed appendages attached to nuclei. The appendages were strand–like organelles surrounded by a membrane with an electron–dense content. In addition, the outer membranes of the envelopes of adjacent nuclei were often joined and surface sections of these nuclei showed fibrous material with only a few nuclear pores. These ultrastructural features have not been reported before in the vegetative hyphae of S. commune , and they are suggested to be necessary for intercellular nuclear movement. The appendages may create the force or may result from the force necessary for the movement of the nucleus, while the joining of the outer membranes of the nuclear envelopes makes possible simultaneous movement of several nuclei, although only one has an appendage. The fibrous material in the nuclear envelope may facilitate sliding of the nuclei through the cytoplasm.     

Immunoelectron Microscopy of RNA Combined with Nucleic Acid Cytochemistry in Plant Nucleoli

The immunoelectron microscopy detection of RNA using anti-RNA monoclonal antibodies has been performed for the first time over different plant cells. The use of the methylation-acetylation (MA) method permits clear distinction among the nuclear and nucleolar compartments and can be combined with the immunogold approach. Cytochemical methods for nucleic acids were performed together with the immunoassays, providing additional data about the different composition of the various nucleolar components. Anti-RNA antibodies highly labeled the ribosome-rich areas of the cytoplasm and the nucleolus. The interchromatin region also is labeled. The labeling was intense in the granular component, lower in the dense fibrillar component, and very scarce in the fibrillar centers. The MA method made possible the statistical evaluation of the labeling density in the various nuclear compartments by permitting the clear assignment of the particles to precise nuclear structures.     

An electron microscope study on the cytoplasmic and nuclear components of rat primary oocytes

Summary This paper reports on the structure of rat primary oocytes, as observed with the electron microscope. Four main components are described in the cytoplasm: Golgi apparatus, centrioles, mitochondria and multivesicular bodies.The components of the Golgi apparatus are forming a single mass confined to a limited region of the cytoplasm and the centrioles were found located in a clear zone sited in the middle of this mass. Mitochondria are scattered at random in the cytoplasm. Multivesicular bodies are elements integrated by an enveloping membrane containing a varied number of tiny vesicles. They are generally found associated with a short number of small free vesicles. Only one two groups of this kind are found per oocyte. This contrast with what has been observed previously in full-grown rat oocytes, where the groups are numerous and constituted by many units.Two components were described for the oocyte nucleus: nucleoli and chromosomes. Nucleoli are constituted by a tangled thread whose elemental component is a fine fibrous material of high electron density.At the age studied on this paper, primary oocytes are undergoing meiotic prophase, chromosomes have at this time the same components observed by different authors in primary spermatocytes. These are two thick ribbon-like threads helically twisted around a thinner medial filament. Each tripartite group is attached by one end to the nuclear membrane. It was actually seen tripartite groups incompletely organized; the images recorded of such groups suggest that the medial filament is the first to appear in the nucleoplasm. The possible significance of these filaments in respect to the meiotic phase called chromosome pairing is discussed.     

Ultrastructure of fat body cells of the velvetbean caterpillar infected with a nuclear polyhedrosis virus

During a study of the ultrastructure of a nuclear polyhedrosis virus of the velvetbean caterpillar, Anticarsia gemmatalis, various types of nuclear and cytoplasmic inclusions were found in fat body tissue heavily infected with the virus. Virogenic stroma was present in the nuclei of most infected cells. Bundles of fibrous material were observed in the nuclei and cytoplasm of cells containing polyhedral bodies. Other nuclear inclusions included concentric multilayered material, vacuoles, and membrane structures.     

Fate of sperm tail components after incorporation into the hamster egg

The changes in the cytoplasmic organelles of sperm tail in golden hamster eggs fertilized in vivo were observed by electron microscopy. Eggs were obtained from oviducts of hamsters that had been superovulated and inseminated by injection of cauda epididymal spermatozoa into the uteri. In the egg cytoplasm 10 hours after insemination, some of the mitochondria of the spermatozoon midpiece had begun to swell, and a number of multivesicular bodies were observed surrounding the midpiece. The fibrous sheath of the principal piece quickly disappeared prior to the first cleavage, whereas the axoneme and outer dense fibers were unaltered. During the two-cell stage, numerous multivesicular bodies gathered around the midpiece and fused with the mitochondria. The heterophagic vacuoles thus formed then gradually separated from the axial fibers. The outer dense fibers were disarranged and partially torn into small segments; then they seemed to dissociate into substructural granular components. The axonemal microtubules had begun to swell but were still present in the two blastomeres. It is indicated from these observations that at least the mitochondria of the tail constituents carried into the oocyte are digested into small molecular elements by the multivesicular bodies and are possibly distributed as nutrients for the blastomeres during the early stage of development.     

On the ultrastructure of the ovary of the liver fluke (Fasciola hepatica L.)

Summary The ovary of the liver fluke has been studied with light and electron microscopy. The organ consisted of germ cells and a layer of peripheral cells suggested to be nurse cells, and was surrounded by a capsule containing muscular tissue. The peripheral cells rested on a thick basement membrane and were irregular in outline. Their nuclei were of irregular shape, the mitochondria were dark with few cristae and the endoplasmic reticulum was tubular or vesicular and partly studded with ribosomes. The germ cells were rounded or polyhedral except in the outer part of the ovary where some of them showed irregular processes. The germ cells of the outer region (oogonia) were relatively small and in close contact with the cells suggested to be nurse cells. The inner germ cells (oocytes) were large and loosely packed. Their nuclei were irregular and contained round distinct nucleoli. The nuclear envelopes showed numerous pores. The endoplasmic reticulum was very sparse, but free ribosomes were abundant in the cytoplasm. This corresponded with a strong basophilia removable with RNase. In addition round basophilic bodies formed by densely packed ribosomes and membraneous material occurred in close spatial relation to mitochondria. The latter contained dense granules and few cristae. Groups of vesicles and membraneous lamellae were found in the cytoplasm, but they were considerably smaller than vertebrate Golgi complexes. Numerous dense spherical granules were found mainly in the periphery of the large germ cells. The granules were strongly osmiophilic except in the terminal part of the ovary. They were PAS-positive, but negative to Sudan dyes.Supported by a grant from Jordbrukets Forskningsråd, Stockholm.     

Fine structure of the epineural connective tissue sheath of the subesophageal ganglion in Helix aspersa

Summary The epineural connective tissue sheath investing the subesophageal ganglion of Helix aspersa consists of a superficial region and a deeper region. The superficial region contains masses of globular cells intermingled with smooth muscle cells and nerve fibers all embedded in a connective tissue matrix. The histochemical and fine structural features of the globular cells show seasonal changes. During autumn to winter glycogen accumulates in their cytoplasm; this accumulation is accompanied by the appearance of dense, cytoplasmic globules which fuse together and ultimately form large pools of granular material. All the organelles and cytoplasm are displaced towards the cell periphery. Various cell-membrane invaginations containing dense material are prominent but there is no direct evidence to link these structures with the uptake of metabolites for glycogenesis. In winter there is a concentration of homogeneous, membrane-bound inclusions in the vicinity of the Golgi bodies. It is suggested that these inclusions constitute a lipid store. They decrease in number during summer. The globular cells do not bear any intimate relation to neurons and there is no reason to include these cells in the neuroglia. The muscle cells often weave around the globular cells but there is no direct contact. Nerve fibers innervate at least some of the muscle cells. The connective tissue consists of large and small diameter fibers suggesting that maturation of the fibrous components of the intercellular matrix is taking place in the superficial regions of the epineural sheath.This work has been supported by the Australian Research Grants Committee.     

FORMATION OF GERM CELL-LIKE CELLS IN THE PERITONEAL EPITHELIUM OF THE TESTES OF ESTROGEN-TREATED ADULT MALES OF THE JAPANESE RED-BELLIED NEWT, CYNOPS PYRRHOGASTER PYRRHOGASTER

Columnar cells of the peritoneal epithelium in slender cords of the testes were examined in normal and estradiol benzoate-treated Japanese red-bellied newt, Cynops pyrrhogaster pyrrhogaster, by light and electron microscopy. In normal newts, the peritoneal epithelium covering the slender cord consists of columnar cells, which contain extraordinarily large, oval or spindle-shaped nuclei with conspicuous indentations. The nucleus contains chromatin granules and the cytoplasm is filled with numerous tonofilaments. The primordial germ cells are scattered throughout the slender cord, and each cell is surrounded by a few follicle cells. Between the germ cells and follicle cells there are microvilli-like processes. The nucleus of primordial germ cells is multilobate and has electron lucent areas, dispersed chromatin and several electron-dense nucleoli. In the lighter cytoplasm, the nuage material is found very near to nuclear pores, and is frequently seen among the mitochondria. The nucleolus-like body is not associated with other organelles. The primary spermatogonia have bilobate nuclei. It is remarkable that most of the cytoplasmic organelles are found in the deep nuclear indentations. The nuage material and nucleolus-like body are well developed in the cytoplasm. After treatment of newts with estradiol benzoate for one year, four types of cells can be distinguished in the peritoneal epithelium. One type is quite different from the columnar cells. These newly appeared cells are large and light in appearance. Their nucleus is highly lobate, and contains dispersed chromatin and several nucleoli with compact electron dense material in its periphery. The cells are characterized by the presence of nuage material and nucleolus-like bodies in the cytoplasm. There are microvilli-like processes between these cells and adjacent elongated cells. These ultrastructural characteristics of the light cells are very similar to those of primordial germ cells and/or primary spermatogonia in normal testes. These findings suggest that the light cells which appear in the peritoneal epithelium of the testes on administration of estrogen may be germ line cells.     

Unique virus morphogenesis and cytopathology of a baculovirus (hypertrophy strain) in larva of the armyworm,Pseudaletia unipuncta

Nuclear polyhedrosis in tracheal cells caused by the hypertrophy strain of a nuclear polyhedrosis virus (HNPV) has many morphological and developmental features that distinguish it from that caused by the typical strain (TNPV). The most obvious difference is the morphogenic sequence due to the relatively slow virogenic development in HNPV-infected cells, in which are found extensive membranous profiles similar to viral envelopes, electron dense granules, large fibrous bodies, and microtubules. These structures also occur in TNPV-infected cells but are far less abundant and conspicuous. Fibrous bodies found in the cytoplasm and nucleus appear to be morphologically identical. Cellular distortion in a hypertrophied tracheal cell is seen as tearing of mestracheon folds between cells, separation of septate desmosomes, and attenuation in the cellular sheath.     

Intracellular distribution of the U1A protein depends on active transport and nuclear binding to U1 snRNA.

Nuclear transport of the U1 snRNP-specific protein U1A has been examined. U1A moves to the nucleus by an active process which is independent of interaction with U1 snRNA. Nuclear localization requires an unusually large sequence element situated between amino acids 94 and 204 of the protein. U1A transport is not unidirectional. The protein shuttles between nucleus and cytoplasm. At equilibrium, the concentration of the protein in the nucleus and cytoplasm is not, however, determined solely by transport rates, but can be perturbed by introducing RNA sequences that can specifically bind U1A in either the nuclear or cytoplasmic compartment. Thus, U1A represents a novel class of protein which shuttles between cytoplasm and nucleus and whose intracellular distribution can be altered by the number of free binding sites for the protein present in the cytoplasm or the nucleus.     

Ultrastructural study on nuclear-cytoplasmic relationships in oocytes of the African lungfish,Protopterus aethiopicus

Summary Growing oocytes of Protopterus, like those of some amphibians and teleosts, show an impressive development of the nucleolar apparatus. Numerous nucleolus-like bodies establish close spatial relationships with the nuclear envelope by extending pedicels and streams of finely dispersed material towards the inner membrane.At such contact points, gaps in the perinuclear cistern are more frequent than elsewhere along the nuclear boundary. Expansion of the outer nuclear membrane gives rise to blebs, with or without visible content, and these become pinched off to form small vesicles in the perinuclear cytoplasm.Small, electron dense aggregates, indistinguishable from nucleolar material occur on both sides of the nuclear envelope opposite to each other, some being connected by a slender portion of the same material within a nuclear pore. Such accumulations are interpreted as detached parts of nucleolar bodies in transit to cytoplasmic sites where they presumably participate in the biogenesis of ribosomes. At the height of nucleolar emission, nucleoplasm and perinuclear cytoplasm are so rich in small electron dense particles that they are almost indistinguishable from each other.At this stage of massive transport, the route provided by the nuclear pores seems to be insufficient and another, more spacious, gateway may be in operation. The latter involves direct passage of material across the nuclear membranes preferentially where these form blebs.This view is supported not only by the overt spatial relationships between nucleolar pedicels and blebs, but by the occurrence within perinuclear lacunae and blebs of particles that seem to be derived from nucleolar bodies. Furthermore, frequent interruptions in the nuclear membranes preferentially located where they expand into outpocketings suggest that at these sites temporary gateways may exist in the living cell that permit easy access of intranuclear components to the cytoplasm.Supported by grants AM-3984, NB-00840, and NB-05219 from the U.S.P.H.S.     

ELECTRON MICROSCOPIC EXAMINATION OF THE SITES OF NUCLEAR RNA SYNTHESIS DURING AMPHIBIAN EMBRYOGENESIS

The site of H³-uridine incorporation and the fate of labeled RNA during early embryo-genesis of the newt Triturus pyrrhogaster were studied with electron microscopic autoradiography. Isolated ectodermal and mesodermal tissues from the embryos were treated in H³-uridine for 3 hours and cultured in cold solution for various periods before fixation with OsO₄ and embedding in Epon. At the blastula stage, the only structural component of the nucleus seen in electron micrographs is a mass of chromatin fibrils. At the early gastrula stage, the primary nucleoli originate as small dense fibrous bodies within the chromatin material. These dense fibrous nucleoli enlarge during successive developmental stages by the acquisition of granular components 150 A in diameter, which form a layer around them. Simultaneously larger granules (300 to 500 A) appear in the chromatin, and they fill the interchromatin spaces by the tail bud stage. Autoradiographic examination has demonstrated that nuclear RNA synthesis takes place in both the nucleolus and the chromatin, with the former consistently showing more label per unit area than the latter. When changes in the distribution pattern of radioactivity were studied 3 to 24 hours after immersion in isotope at each developmental stage, the following results were obtained. Labeled RNA is first localized in the fibrous region of the nucleolus and in the peripheral region of chromatin material. After longer culture in non-radioactive medium, labeled materials also appear in the granular region of the nucleolus and in the interchromatin areas. Further incubation gives labeling in cytoplasm.