Organization and activity in the pre- and postovulatory follicle of Necturus maculosus.

The established follicle envelope of Necturus maculosus consists of a layer of follicle cells (granulosa) surrounding the developing oocyte, a layer of theca comprised of connective tissue cells, fibers, and matrix, and a layer of serosal cells. The changes in shape and fine structure of these layers during differentiation accompanying oogenesis are described. The cells and capillaries of the follicle envelope are engaged in an extensive pinocytotic activity, the details of which are described. We used cytochemical techniques to analyze the activity of the follicle envelope with respect to lipid accumulation and alkaline phosphatase activity. Radioautographic results indicate that cells of the follicle envelope are capable of incorporating tritium-labeled uridine and amino acids at certain times during oocyte growth. A comparative analysis was made of the soluble proteins in follicle envelopes isolated from immature oocytes and of those in follicle envelopes isolated from nearly mature oocytes and in postovulatory follicles. After the oocyte is ovulated, the cells of the follicle envelope are converted into a postovulatory follicle. The cells of the postovulatory follicle undergo further differentiation resulting in their becoming actively engaged in the formation of a secretion, the details of which are described at the electron microscope level. Analysis of the postovulatory follicle by thin-layer chromatography and cytochemistry demonstrated the presence of a wide variety of lipid substances and the possible presence of steroid. That the postovulatory follicle may be engaged in steroid biosynthesis is also suggested by studies involving the demonstration of 3 beta-hydroxysteroid dehydrogenase activity with cytochemical techniques applied to frozen sections and to soluble proteins separated by gel electrophoresis.

Annulate lamellae and "yolk nuclei" in oocytes of the dragonfly, Libellula pulchella.

Annulated membranes in the form of single and short lamellae are present adjacent to and parallel to the nuclear envelope in oogonia and early oocyte (synaptene) stages of the dragonfly, Libellula pulchella. These solitary and short annulate lamellae are usually continuous with long, part rough- and part smooth-surfaced cisternae which extend into more distal areas of the oogonial ooplasm. These particular annulate lamellae then either disappear or decrease in number to be replaced by a much more extensive system of annulate lamellae in the cortical ooplasm of previtellogenic oocytes. The differentiation of extensive stacks of annulate lamellae is consistently observed to be restricted to large cytoplasmic areas of considerable electron density. These cytoplasmic regions consist of material which stains basophilic and contains RNA but differs structurally from the large number of ribosomes which surround the dense masses. The cytoplasmic dense masses, in terms of their formation and staining reactions, are comparable to the "yolk nuclei" or "Balbiani bodies" described in insect oocytes in earlier studies. The results of the present study thus provide evidence that the appearance of cortical ooplasmic stacks of annulate lamellae in the dragonfly oocyte is specifically limited to cytoplasmic areas of high electron density which contain RNA but which do not have a ribosomal morphology.

Intracellular synthesis, transport, and packaging of proteinaceous yolk in oocytes of Orconectes immunis.

The incorporation of leucine-(3)H into either ovarian or oocyte proteins occurs throughout vitellogenesis, but is at a maximum during early phases of this process. The labeling of ovarian and oocyte proteins is inhibited with cycloheximide. Oocytes are permeable to actinomycin D, and this drug does not affect the incorporation of amino acids into oocyte proteins but does block oocyte RNA synthesis. By means of both light microscope and high resolution radioautography, it has been demonstrated that the initial incorporation of leucine-(3)H under both in vitro and in vivo conditions occurs in elements of the rough-surfaced endoplasmic reticulum in the oocyte. Under pulse-chase conditions, the label subsequently becomes associated with intracisternal (precursor yolk) granules now aggregated within the cisternae of the connected smooth-surfaced endoplasmic reticulum. By 7 days, mature yolk globules are extensively labeled. The results of experiments designed to assess the possible contribution of maternal blood proteins to yolk deposition indicate that such a contribution is minimal. It is concluded that the crayfish oocyte is programmed for and capable of synthesizing the massive store of proteinaceous yolk present in the egg at the end of oogenesis.

Electron microscopic and autoradiographic studies on vitellogenesis in Necturus maculosus.

Electron microscope studies on Necturus maculosus oocytes ranging in size from 1.1-1.5 mm in diameter indicate the primary proteinaceous yolk to arise within structures referred to in other amphibian oocytes as yolk precursor sacs or bodies. The origin of these yolk precursor sacs appears to result from the activity of the Golgi complexes which form multivesicular and granular-vesicular bodies, the limiting membrane of which is at times incomplete. During differentiation, the yolk precursor sacs contain small vesicles similar in size to Golgi vesicles, larger vesicles similar to vesicular elements of the agranular endoplasmic reticulum and, on occasion, a portion of a mitochondrion. The interior of these sacs becomes granular, perhaps by a dissolution of the components just described, and soon becomes organized into a crystalline configuration. In oocytes 2.0-2.5 mm in diameter, an extensive micropinocytotic activity begins, continues throughout vitellogenesis, and constitutes the primary mechanism for the formation of secondary yolk protein. Numerous coated and smooth-surfaced vesicles, as well as electron-dense and electronlucent ones, fuse in the cortical ooplasm to form progressively larger yolk platelets.

Effects of prolonged antitubulin culture on annulate lamellae in mouse alpha L929 fibroblasts.

The fine structure of alpha L929 fibroblasts cultured in colchicine or vinblastine sulfate for periods as long as 48 hr was compared to control cells not exposed to antitubulins . In response to prolonged antitubulin culture, several changes in cell ultrastructure were noted: Control fibroblasts contain cytoplasmic annulate lamellae (AL), but prolonged exposure to either vinblastine sulfate or colchicine results in enhanced development of AL. Single pore complexes are present in the rough-surfaced endoplasmic reticulum (rER) in both control and antitubulin-treated cells, but stacked porous cytomembranes also occur under both conditions. Polyribosomes often are closely associated or continuous with the pore complexes. Many antitubulin-treated cells become multinucleate. Some nuclei in both control and antitubulin-treated cells contain large and multiple nucleoli. The large and multiple nucleoli are either attached directly to the inner membrane of the nuclear envelope or to infoldings of the nuclear envelope. Antitubulin-treated cells, after 48-hr exposure, appear also to contain enhanced quantities of smooth-surfaced endoplasmic reticulum (sER) and cytoplasmic filaments (and in some cells, lysosomes and rER as well) when compared to untreated cells. In both control and colchicine-treated cells, AL can exhibit continuity with either rER or sER. Further, all three membrane systems may at times be continuous, but the quantity of these membranes appears to be greater in colchicine-treated cells than in control cells. The results are discussed with respect to possible functional significance.

Effects of prolonged antitubulin culture on the ultrastructure of anterior limb bud cells of the chick embryo.

The anterior limb bud mesenchyme cells of stage 24 chick embryos were dissociated by trypsinization followed by gentle pipetting, and placed in a tissue culture medium of F12 containing 10% fetal calf serum and antibiotics. As the cells became nearly confluent, some of them were exposed to colchicine or vinblastine sulfate for durations as long as 48 hr. The control and antitubulin-treated cells were processed for transmission electron microscopy and the ultrastructure of the cells was compared. Annulate lamellae (AL) were observed in small amounts in both control and antitubulin-treated cells. The amount of AL did not markedly differ in the control versus antitubulin-treated cells. Furthermore, few multinucleated cells were observed in antitubulin-treated cultures. These results indicate that prolonged culture of cells in antitubulins need not, in itself, lead to a condition of enhanced AL development as reported in several other studies using various cell types.

Intracisternal tubules and intramitochondrial filaments in cells of a snail, Limnaea.

Epithelial and peritubular cells associated with the reproductive tract of the snail, Limnaea stagnalis, contain an extensive system of endoplasmic reticulum that is often dilated with many closely packed intracisternal tubules. The intracisternal tubules are approximately 24-28 nm in diameter and they are often hexagonally packed. They have a two-layered wall, possess fine interconnections, and extend linearly for considerable distances, but angular bends in the tubules also occur. Mitochondria in the peritubular cells contain solid, filamentous structures 9-12 nm in diameter and triangular-shaped structures when sectioned in the mitochondrial matrix.

Freeze fracture and scanning electron microscope studies on the nuclear envelope and perinuclear cytomembranes (parabasal apparatus) in the protozoan, Lophomonas blattarum.

Phase contrast, scanning electron microscope (SEM), and freeze fracture studies on the parasitic flagellate, Lophomonas blattarum, have demonstrated that the endomembrane system (parabasal apparatus) is highly ordered, restricted in position to a perinuclear zone at the anterior end of the organism, and is supported in this localized cytoplasmic region by overlapping sheets or plates of microtubules, previously called the calyx and axial filament, which may participate in supporting the nucleus-endomembrane system in a restricted region of the cell. Light microscope observations, SEM and freeze fracture data provide support to previous views that the rough- and smooth-surfaced endoplasmic reticulum are interconnected and attached to the outer layer of the nuclear envelope. The continuity of these membrane systems provides an orderly and restricted packing in the perinuclear cytoplasm since other areas of the cell may become filled with yeast. These flagellates are especially adept at phagocytosis of entire yeast. In yeast-laden cells, the flagella-nucleus-parabasal body-calyx-axial filament complex may separate from the remainder of the cell and assume a motile existence for a time. The significance of the described relationships, in addition to providing efficiency in endomembrane localization, may also reflect synthesis of enzymes and proteins by the RER and packaging in the SER, both of which are continuous. Granules characteristic of glycogen are concentrated around the SER which may be involved in glycogen metabolism. Although critical information is lacking, the endomembrane system may also be involved in the synthesis and morphogenesis of lysosomes, and perhaps peroxisomes. Lophomonas thus amplifies a highly ordered spatial relationship between the nuclear envelope and the ER.

Intercellular junctions in the follicular envelope of the teleost, Brachydanio rerio.

The nature and distribution of intercellular junctions in the outer ovarian epithelium (serosa, mesothelium), endothelium, and follicle cells of the teleost oocyte-follicle complex were investigated by freeze-fracture electron microscopy. Tight-junctions were common between outer squamous epithelial cells, sometimes closely associated with intercalated foci of communicating junctions. The tight junctions consisted of one to several sealing strands which possessed focal discontinuities. In addition, the strands existed as loops or as short, free-ending elements; a condition that could indicate lability in their assembly or disassembly. The presence of free-ending strands could also mean that the structure serves for attachment as well as involved in the formation of occluding zonules. The free ends of some bars comprising the tight junctional strands are enlarged slightly. In outer ovarian epithelial or serosal cells, as is the case for mammalian mesothelium described by others, clusters of particles comprising communicating (gap) junctions are often intercalated within or are located in close proximity to tight junctional strands. In freeze-cleaved replicas, the outer squamous epithelial (serosal) cells contained a multitude of micropinocytotic pits (caveolae) and vesicles. Capillary endothelium also contains tight junctional components which are often closely associated with communicating junctions. Tight junctions also exist between follicle cells, but their structure changes during oocyte growth. Communicating junctions between follicle cells tend to be focal in distribution and not closely associated with tight junctions.

The annulate lamellae--from obscurity to spotlight.

This review aims to provide a comprehensive and in-depth survey of a cell organelle, the annulate lamellae, that is widely distributed and especially prevalent in both female and male sex cells as well as tumor and cancer cells. The organelle is also present in many somatic cells and plant cells. Emphasis is placed on the contributions that electron microscopy and associated experimental approaches have made in providing information about the distribution, ultrastructure, morphogenesis and relationships of annulate lamellae to other cellular organelles, especially the nuclear envelope and endoplasmic reticulum, as well as cell product. An increasing number of experimental manipulations have recently been shown to alter, either increase or decrease, the amount of annulate lamellae and these studies are explored in depth. Information about the origin and morphogenesis of annulate lamellae in different cells is summarized and extensive coverage is given to several hypotheses about possible annulate lamellae function. A detailed bibliography provides a thorough compilation of research dealing with annulate lamellae. A major goal of this extensive review is to generate increased awareness of, and interest in, this cell organelle for students and investigators of the cell who, by bringing current techniques in cell and molecular biology to bear, might focus and intensify studies on the function of an organelle whose precise role in the cell is presently enigmatic.

Intranuclear membranes (vesicles, lamellae, annulate lamellae) in oocytes of the ascidian, Styela partita.

Transmission electron microscope studies on developing oocytes of Styela partita that were preserved with a primary fixative of buffered glutaraldehyde and a secondary fixative of buffered osmium tetroxide have dealt with the origin, distribution, and associations of intranuclear annulate lamellae in both previtellogenic and vitellogenic oocytes. These studies (1) substantiate that, developmentally, intranuclear vesicles appear to precede differentiated intranuclear annulate lamellae although intranuclear vesicles and intranuclear annulate lamellae may coexist in the same nucleus (germinal vesicle), (2) substantiate an earlier demonstration that the inner membrane of the nuclear envelope forms blebs which could, after detachment, become intranuclear vesicles, (3) confirm that the pore-associated material of the pores in intranuclear annulate lamellae appears to be structurally identical to that of the pores in the nuclear envelope, (4) demonstrate that at certain times during oogenesis electron-dense material inside the nucleus, thought to represent nucleolar-derived material, is continuous with the pore-associated material of pores in both intranuclear annulate lamellae and the nuclear envelope, (5) propose a function for intranuclear annulate lamellae that considers the pores to play a role in processing or assembling various RNA's and proteins to become cytoplasmic polyribosomes as has recently been suggested also for the pores in the nuclear envelope as well as pores in cytoplasmic or extranuclear annulate lamellae, and (6) provide additional evidence that intranuclear annulate lamellae are not remnants of the nuclear envelope persisting after mitosis as has been described to be the case in certain other cells.