Membrane production and yolk degradation in the early fly embryo (Calliphora erythrocephala meig.): An ultrastructural analysis

Formation of nuclear envelopes during the last cleavage mitosis and the formation of the cell membranes during the cellularization of the blastoderm have been studied ultrastructurally in the blowfly egg. Dense bodies arising from yolk granules by budding could contain membrane material destined to be incorporated into the new membranes of the blastoderm. The presence of transitional structures indicates that these bodies can be converted into dark multivesicular bodies. Large amounts of endoplasmic reticulum are found around the mitotic nuclei. Clusters or branched chains of vesicles associated with this are interpreted as evidence for the formation of endoplasmic reticulum by the breakdown of dark multivesicular bodies. Nuclear envelopes of mitotic daughter nuclei probably originate from endoplasmic reticulum. The egg contains both intranuclear and extranuclear annulate lamellae. The main events of cytokinesis are furrow initiation and cell membrane growth during the slow first phase, but probably only cytokinetic movement during the rapid second phase. On the assumption that cell membrane growth occurs by incorporation of complete membrane pieces, the addition of coated vesicles and/or light multivesicular bodies is definitely most probable. Some intermediate profiles indicate that light and dark multivesicular bodies are related. The membrane needed for second phase cytokinesis could well be provided by the unfolding of surface microvilli and protuberances of the furrow canal.     

The Fine Structure of Rhynchocystis pilosa (Sporozoa, Eugregarinida)

SYNOPSIS. The trophozoite of Rhynchocystis pilosa obtained from the seminal vesicles of the earthworm Lumbricus terrestris was studied by light and electron microscopy. The trophozoite's cortical organization is particularly interesting because of its unusual evaginations and associated fibrillar structures. The pellicle is formed by 2 concentric membranes elevated into 60–70 alternating primary and secondary ridges extending posteriad. Numerous long ‘hairs’ or cytopilia originate along the primary ridges and each contains a system of fibrils originating from an underlying longitudinal myoneme. Longitudinal rows of pores lie between adjacent pollicular ridges. Three systems of fibrils lie in the cortex of the trophozoite. A longitudinal myoneme consisting of 12–18 fibrils lies below each primary pellicular ridge. Circular myonemes lie below the pellicle in a parallel array along the length of the organism. Each myoneme consists of 4–8 fibrils structurally similar to those of the longitudinal myonemes. Pairs of fine filaments also lie in the inner pellicular membrane along the apex of each ridge. The trophozoite's anterior end is modified as an attachment organelle consisting of 30–35 delicate pellicular folds which originate at the base of an anterior papilla. The folds extend approximately 15 μ posteriad where they become continuous with the primary pellicular ridges. The nucleus lies in the cytoplasm near the posterior level of the attachment organelle and is surrounded by a double membrane perforated by numerous pores. The cytoplasm contains numerous small vesicles which may be found in dense aggregations. These aggregations often occur in proximity to Golgi complexes and certain membrane-bound bodies. Mitochondria are abundant in the cytoplasm as are large, ovoid paraglycogen bodies. Occasionally layers of granular membranes are arranged parallel to the surface of the paraglycogen bodies but also occur thruout the cytoplasm.     

Microtubules in the cortical region of the egg of Lymnaea during cortical segregation

The cortical region of the 2-cell stage egg of the gastropod Lymnaea palustris was studied by light and electron microscopy. This region includes (1) a vitelline membrane and perivitelline space which contain membrane-limited dense bodies derived from the cell surface, (2) oolemma with surface coat material and microvilli, and (3) a peripheral zone of cytoplasm (0.5-5.0 μm wide) composed of irregular vesicles, electron dense granules, and cytoplasmic microtubules. Microtubules are most abundant in the equatorial region of the egg, where they form arrays that are parallel and oblique to the egg's surface. Microtubular profiles also occur in the cortical region at the animal and vegetal poles of the egg and in the endoplasm. They may play a role in cortical segregation.     

Segregation of Ferritin in Glomerular Protein Absorption Droplets

Ferritin was used as a tracer to study the mechanism by which proteins are segregated into droplets by the visceral epithelium of glomerular capillaries. In glomeruli from both normal and aminonucleoside-nephrotic rats ferritin molecules introduced into the general circulation penetrated the endothelial openings and were seen at various levels in the basement membrane. Striking differences between nephrotic and controls were seen only in the amount of ferritin incorporated into the epithelium. In normal animals, a few ferritin molecules were seen in small invaginations of the cell membrane limiting the foot processes, within minute vesicles in the epithelium, or within occasional large vacuoles and dense bodies. In nephrotics, epithelial pinocytosis was marked, and numerous ferritin molecules were seen within membrane invaginations and in small cytoplasmic vesicles at all time points. After longer intervals, the concentration of ferritin increased in vacuoles and particularly within the dense bodies or within structures with a morphology intermediate between that of vacuoles and dense bodies. In nephrotic animals cleft-like cavities or sinuses were frequently encountered along the epithelial cell surface facing the urinary spaces. Some of these sinuses contained material resembling that filling the dense bodies except that it appeared less compact. The findings suggest that ferritin molecules—and presumably other proteins which penetrate the basement membrane—are picked up by the epithelium in pinocytotic vesicles and transported via the small vesicles to larger vacuoles which are subsequently transformed into dense bodies by progressive condensation. The content of the dense bodies may then undergo partial digestion and be extruded into the urinary spaces where it disperses. The activity of the glomerular epithelium in the incorporation and segregation of protein is similar in normal and nephrotic animals, except that the rate is considerably higher in nephrosis where the permeability of the glomerular basement membrane is greatly increased.     

The cortical actin-membrane cytoskeleton of unfertilized sea urchin eggs: analysis of the spatial organization and relationship of filamentous actin, nonfilamentous actin, and egg spectrin

Whole mounts, cryosections, and isolated cortices of unfertilized sea urchin eggs were probed with fluorescent phalloidin, anti-actin and anti-egg spectrin antibodies to investigate the organizational state of the cortically associated actin-membrane cytoskeleton. Filamentous actin and egg spectrin were localized to the plasma membrane, within microvillar and nonmicrovillar domains. The nonmicrovillar filamentous actin was located immediately subjacent to the microvilli forming an extensive interconnecting network along the inner surface of the plasma membrane. The organization of this filamentous actin network precisely correlated with the positioning of the underlying cortical granules. The cortical cytoplasm did not contain any detectable filamentous actin, but instead contained a sequestered domain of nonfilamentous actin. Spectrin was localized to the cytoplasmic surface of the plasma membrane with concentrated foci co-localized with the filamentous actin present in microvilli. Spectrin was also observed to coat the surfaces of cortical granules as well as other populations of intracellular vesicles. On the basis of light microscopic morphology, intracellular distribution, and co-isolation with the egg cortex, some of these spectrin-coated organelles represent acidic vesicles. Identification of an elaborate organization of inter-related domains of actin (filamentous and nonfilamentous) and spectrin forming the cortical membrane cytoskeleton provides insight into the fundamental mechanisms for early membrane restructuring during embryogenesis. Additionally, the localization of spectrin to the surface of intracellular vesicles is indicative of its newly identified functional roles in membrane trafficking, membrane biogenesis and cellular differentiation.     

Ultrastructural Basis of the Tension Increase in Sea-Urchin Eggs Prior to Cytokinesis

Cortices of sea-urchin eggs were studied by electron microscopy to identify the structure responsible for the rise in tension at the egg surface prior to cleavage. During anaphase the tension increased and fine filaments of 70–90 Å in diameter appeared in the cell cortex forming a thin mesh-work beneath the cell membrane. The meshwork spread all around the egg cortex without reference to the mitotic axis and the number of filaments seemed to increase up to telophase. Immediately before appearance of the cleavage furrow, the meshwork in the anticipated furrow region became dense. As the furrow appeared the tension began to decrease and the meshwork disappeared. In the progressing furrow region fine filaments of the same size as that of the meshwork-filament were oriented in a bundle to form a contractile ring. Treatment with cytochalasin B suppressed both the tension increase and the formation of the filamentous meshwork. These results suggest that the component filament of the meshwork is an actin microfilament, and that the tension increase at anaphase is due to formation of a meshwork of actin microfilaments from which a contractile ring is subsequently derived at late telophase.     

THE FINE STRUCTURE OF THE GALL BLADDER EPITHELIUM OF THE MOUSE

Sections of mouse gall bladder epithelium fixed by perfusion with buffered osmium tetroxide have been studied in the electron microscope as an example of simple columnar epithelium. The free surface presents many microvilli, each presenting a dense tip, the capitulum, and displaying a radiating corona of delicate filaments, the antennulae microvillares. Very small pit-like depressions, representing caveolae intracellulares, are encountered along the cell membrane of the microvilli. The free cell surface between microvilli shows larger cave-like depressions, likewise representing caveolae intracellulares, containing a dense material. The lateral cell borders are extensively folded into pleats, which do not interdigitate extensively with corresponding folds of the adjacent cell membrane. The terminal bars are shown to consist of thickened densities of the cell membrane itself in the region of insertion of the lateral cell wall with the free cell surface. This thickening is associated with an accumulation of dense cytoplasmic material in the immediate vicinity. The terminal bar is thus largely a cytoplasmic and cell membrane structure, rather than being primarily intercellular in nature. The basal cell membrane is relatively straight except for a conical eminence near the center of the cell, projecting slightly into the underlying tunica propria. The basal cell membrane itself is overlain by a delicate limiting membrane, which does not follow the lateral contours of the cell. Unmyelinated intercellular nerve terminals with synaptic vesicles have been encountered between the lateral walls of epithelial cells. A division of the gall bladder epithelial cell into five zones according to Ferner has been found to be convenient for this study. The following cytoplasmic components have been noted, and their distribution and appearance described: dense absorption granules, mitochondria, Golgi or agranular membranes, endoplasmic reticulum or ergastoplasm, ring figures, and irregular dense bodies, perhaps lipoid in nature. The nucleus of these cells is also described.     

Fine structure of the unistellate sperm of the shrimp, Sicyonia ingentis (Natantia)

Sperm of the prawn Sicyonia ingentis were studied cytochemically and ultrastructurally. Striking cytological differences were noted between these natantian sperm and previously studied reptantian sperm. In general, the S. ingentis sperm are composed of a spherical main body that is partially encompassed by a morphologically diverse cap region, from which extends a single appendage or spike. The main body houses an uncondensed, Feulgen-positive nuclear region that is partially surrounded by a cytoplasmic band. A single layer of small, 600 Å, vesicles lines the periphery of the cytoplasmic band. Large membranous vesicles extend from the inner surface of the cytoplasmic band into the nuclear region. The nucleus is separated from the cap or acrosomal complex by a dense plate and a highly organized crystalline lattice, which is composed of geometric squares that are approximately 350 Å in dimension. The cap region also contains convoluted membrane pouches; a central granular core; spherical bodies; an electron-dense, saucer-shaped plate; and a large anterior granule. The convoluted membrane pouches and anterior granule are periodic acid-Schiff (PAS) positive. The anterior granule also demonstrates RNAase-stable red fluorescence with acridine orange staining. A spiralled spike, approximately 6 μm long, extends from the anterior end of the cap. The cap and spike are bound by a double membrane, which results from the fusion of the plasma membrane and the convoluted pouch membrane. The sperm's acrosome is thought to be composed of the two PAS-positive cap components and the spike.     

OBSERVATIONS ON THE FINE STRUCTURE OF PHARYNGEAL MUSCLE IN THE PLANARIAN DUGESIA TIGRINA

Pharyngeal muscle of the planarian Dugesia tigrina was studied by electron microscopy after osmium tetroxide fixation. The muscle cell was observed to contain one myofibril or bundle of myofilaments parallel to its longitudinal axis. The myofilaments were of two types, different in size and distribution. No Z lines or myofilament organization into cross or helical striations were seen. Dense bodies were seen as projections from an invagination of the plasma membrane and as dense lines parallel to the myofilaments. The muscle cells are surrounded by a plasma membrane which is structurally associated with dense body projections, with vesicles and cisternae of sarcoplasmic reticulum, and with synaptic nerve endings. The cell has sarcoplasmic projections perpendicular to its long axis; these projections are seen to contain the nucleus or mitochondria and granules. Mitochondria and granules are also seen in a sarcoplasm rim around the fibril. The dense bodies may serve as attachment for thin myofilaments and function in transmission of stimuli from plasma membrane to the interior of the fibril.     

Cortical differentiation of the animal pole during maturation division in fertilized eggs of Tubifex (Annelida,Oligochaeta): II. Polar body formation

Changes in the cortical organization at the animal pole are examined by scanning and transmission electron microscopy in the Tubifex egg undergoing second polar body formation. At very early anaphase of the second meiosis, the egg surface overlying the meiotic apparatus is undulated, but its neighboring surface appears to be smooth. Although a microfilamentous cortical layer is found in the smooth area, the cortical layer of the undulating area is thin and devoid of filamentous structures except for its central part where some filaments are observed. This local differentiation takes place normally in colchicine-treated eggs where the meiotic apparatus is destroyed. Along with the progression of the anaphase movement, the egg surface of the undulating area is, first, uplifted into a cone-shaped cytoplasmic bulge (presumptive polar body); then the height and surface area of the bulge gradually increase. The distal surface of the growing bulge appears to be undulated whereas the sides of the bulge are relatively smooth. Transmission electron microscopy reveals that a thick microfilamentous cortical layer is always localized at the proximal region of this bulge; other regions of the bulge are characterized by a thin cortical layer which is devoid of filamentous structure except for the apical portion of the bulge. Microfilaments at the base of the bulge are perpendicular or oblique to the egg surface. The cortical layer of the egg which is continuous to that of the proximal region of the bulge comprises microfilaments running parallel to the surface. The attainment of the bulge to its full size is followed by the development of the cleavage furrow along its base. The cleavage furrow appears to bisect the spindle midway between its poles. In cytochalasin B-treated eggs, where some cortical microfilaments are detected at the animal pole, a cytoplasmic bulge lower in height and wider in the diameter of its base than the normal one forms at the animal pole; however, it is subsequently resorbed into the egg. The formation of a cleavage furrow is not observed in these eggs. The mechanism of the polar body formation is discussed in the light of the present observations.     

Furrow formation in Xenopus embryos. Involvement of the golgi body as revealed by ultrastructural localization of thiamine pyrophosphatase activity.

The time-related ultrastructural distribution of thiamine pyrophosphatase (TPPase) activity was studied in first cleavage Xenopus embryos. In the furrow, localisation of the activity varied with the stage of development. The early furrow did not show any activity; however, when the furrow had progressed up to 300 μm into the embryo, activity was present at the furrow tip. With further development, heavy deposits were present in the furrow. In the cytoplasm, the activity was localised within the outermost cisternae of the Golgi body and in some of its associated vesicles. This distribution in the Golgi body prevailed irrespective of the stage of development. Vesicles and membrane cisternae elsewhere in the cytoplasm, especially near the furrow, were also positive. The demonstration of reaction product initially within the vesicles and cisternae of the Golgi body, and subsequently on the furrow surface, indicated that Golgi-derived, TPPase-positive vesicles might be adding membrane and surface material to the developing furrow.     

NEW MEMBRANE FORMATION DURING CYTOKINESIS IN NORMAL AND CYTOCHALASIN B-TREATED EGGS OF XENOPUS LAEVIS : I. Electron Microscope Observations

A method is described for measuring and calculating the preexisting surface in uncleaved Xenopus eggs and the rate of surface growth in cleaving eggs. Surface-marking experiments with cytochalasin B-treated eggs show that the unpigmented surface grows by de novo formation and not by expansion of preexisting pigmented surface. The onset of new surface formation during first cleavage was studied by using transmission electron microscope and scanning electron microscope techniques. At 3–4 min and at 7–8 min after the onset of cleavage the eggs were fixed in the presence of ruthenium red (RR). Evidence is presented that unpigmented surface representing new membrane comes into appearance between four and eight min. This surface has a selective binding capacity for RR. Concomitantly with the appearance of new membrane, endoplasmic reticulum (ER) cisternae are in continuity with, and dense cytoplasmic inclusions coalesce with, the membrane along the furrow. The latter give rise to liposome-like bodies. The possibility is discussed that the ER cisternae transport a surface exudate (a carbohydrate complex), that the dense cytoplasmic inclusions represent pools of membrane precursor, and that membranogenesis takes place by direct insertion of pooled precursors into the cell surface. In a second paper, these findings will be correlated with electrophysiological measurements.     

河鲈锚首吸虫体壁的超微结构观察

寄生鳜鳃部的河鲈锚首吸虫的体壁由表皮合胞体、基板、环肌、纵肌和表皮细胞核周体所组成。合胞体顶部质膜起伏形成表皮的嵴纹,基部质膜折叠形成指状突起伸入到合胞体中。合胞体表面覆盖着一层糖萼。河鲈锚首吸虫的表皮中含有四类分泌体,即电子致密的分泌颗粒、中等电子致密的分泌颗粒、有膜包围的电子稀疏的分泌体和多囊体．可见分泌体和合胞体基质通过胞吐作用排到体外,未见吞饮小泡,推测表皮的主要功能在于分泌和渗透压调节而非营养吸收。在外侧头瓣的乳突状结构所在处,合胞层较薄,基板平滑,在实质组织中的一腔体样结构中可见囊状体、电子致密度各异的颗粒体、泡状体和电子致密的基质团,神经突起分布于腔体周围,这类乳突可能代表一类新的非纤毛感受器类型。     

Membrane protein redistribution during Xenopus first cleavage

A large increase in surface area must accompany formation of the amphibian embryo first cleavage furrow. The additional membrane for this areal expansion has been thought to be provided entirely from cytoplasmic stores during furrowing. We have radioiodinated surface proteins of fertilized, precleavage Xenopus laevis embryos and followed their redistribution during first cleavage by autoradiography. Near the end of first cleavage, membrane of the outer, pigmented surface of the embryo and a short band of membrane at the leading edge of the furrow displayed a high silver grain density, but the remainder of the furrow membrane was lightly labeled. The membrane of the cleavage furrow is thus mosaic in character; the membrane at the leading edge originates in part from the surface of the zygote, but most of the membrane lining the furrow walls is derived from a source inaccessible to surface radioiodination. The furrow membrane adjacent to the outer, pigmented surface consistently showed a very low silver grain density and was underlain by large membranous vesicles, suggesting that new membrane derived from cytoplasmic precursors is inserted primarily in this location, at least during the later phase of cleavage. Radioiodinated membrane proteins and surface-attached carbon particles, which lie in the path of the future furrow, contract toward the animal pole in the initial stages of cleavage while markers in other regions do not. We suggest that the domain of heavily labeled membrane at the leading edge of the definitive furrow contains the labeled elements that are gathered at the animal pole during the initial surface contraction and that they include membrane anchors for the underlying contractile ring of microfilaments.     

The Fine Structure of the Gregarine Lankesteria culicis Parasitic in the Yellow Fever Mosquito Aedes aegypti

SYNOPSIS. The fine structure of the eugregarine Lankesteria culicis from the larvae of the mosquito Aedes aegypti was examined by light and electron microscopy and compared with that of other gregarines. The cell organelles found in L. culicis included a nucleus, mitochondria, Golgi-complexes, endoplasmic reticulum, vesicles, droplets, granules and lipid bodies.
The surface of L. culicis was composed of a highly differentiated membrane-cortex, differing slightly from that of other eugregarines. This complex was limited by a unit membrane, the plasmelemma, and underlying cortical laminae which appeared to be composed of several layers. The homogeneous electrondense layer present in Pyxinoides balani was greatly diminished or absent in L. culicis.
A series of laminar folds supported by ground substance and longitudinal subpellicular fibrils gave the organism's surface a ridge-like appearance. Permanent cytostome-like openings in the surface, which appeared to be supported by a narrow band of thickened cortex, were present as specializations of the surfacemembrane complex. The structural composition of the parasite appeared quite striking in that it was made up almost entirely of vesicles, granules, and droplets which were absent only in the area of the protomerite. The mitochondria were usually found just beneath the surface or near the nucleus. Mitochondria were also seen in the region of demarcation between the protomerite and deutomerite.     

Fine structure of the nephridial muscle layer cells of Phascolosoma granulatum (Sipuncula)

The nephridial muscle layer of Phascolosoma granulatum consists of a network of longitudinal and circular cells separated by connective tissue matrix. The muscle fibers are densely packed with thick and thin myofilaments, among which are scattered cytoplasmic dense bodies. The nucleus and noncontractile cytoplasmic organelles occupy a lateral projection from the contractile portion of the fiber. Cytoplasmic dense bodies are the result of a clustering of an indeterminate number of the thin actin filaments that fill the cytoplasm between thick filaments. Attached to the cytoplasmic face of the cell membrane are membrane-associated electron-dense plaques. These sites are linked to the contractile myofilaments by narrow filamentous bridges. Extracellular narrow filaments extend from these plaques to collagen fibers of the connective tissue matrix. Differences in length of the dense plaques may be related to differences in thick myofilament diameter in three types of muscle fiber, types A, B and C, statistically distinguished by mean fiber size differences. The plaques may serve as connecting links for the transmission of tension from contractile units to the connective tissue of the muscle layer. © 1993 Wiley-Liss, Inc.     

The ultrastructure of the sea urchin egg cortex isolated before and after fertilization

The three-dimensional organization of cortices isolated from unfertilized and fertilized Strongylocentrotus purpuratus eggs has been examined by several techniques of light and electron microscopy. It has been found that when moderate shear forces are used, the isolated unfertilized egg cortex, in addition to cortical granules, contains acidic vesicles and an elaborate network of rough endoplasmic reticulum. This network provides a physical link between the cell surface and several kinds of cytoplasmic organelles (mitochondria, yolk granules, acidic vesicles) which are retained as part of the isolated cortex when gentle shear forces are applied. Furthermore a good visualization of actin in the cortex is provided: it is present as short filaments and mostly within the stubby microvilli of the egg. Finally, it has been noted that plaques exist on the inside face of the plasma membrane ready to assemble into typical clathrin coats that prefigure the burst of coated vesicle endocytosis that takes place after fertilization. The cortex isolated soon after fertilization is shown to contain coated pits and a scaffolding of filaments (mostly actin) in which many acidic vesicles are embedded.     

Some aspects of cleavage in Pomatoceros triqueter eggs

Summary The first cleavage division of Pomatoceros triqueter eggs is described. Time-lapse microcinematographic and electron microscopic studies revealed that, prior to division, the plasma membrane was folded into pleats. These were not present after division. This fact pointed to an unfolding of the plasma membrane which enabled it to cover the increased surface area resulting from cleavage. It is suggested that the pre-cleavage folds are derived from the membranes of the cortical granules, which continue releasing their contents into the perivitelline region following fertilization and first division. Filamentous material in the form of a band was present subjacent to the plasma membrane in the region of the furrow. The individual filaments of this band measure 5 to 7 nm in cross section, the dimensions being similar to those described for other kinds of dividing cells.The authors wish to thank Messrs. P. C. Lloyd, D. Williams and J. Dingley for technical assistance, and Professor B. M. Jones and Dr. G. G. Selman for their helpful criticisms.     

A subcortical, pigment-containing structure in Xenopus eggs with contractile properties

An accumulation of insoluble, finely granular material has been observed under the pigmented surface of Xenopus eggs by a specialized "dry fracture" technique and scanning electron microscopy. Cortical granules and pigment granules can be recognized with the techniques and can be seen to be embedded in the material. Thin sections show that the region also contains mitochondria and membranous vesicles or reticula. Yolk platelets are largely excluded from the heaviest accumulations of the material. The substance is most dense just under the cortex and grades off gradually into the more diffuse, yolk-containing network of the endoplasm. The accumulation of material is much thicker in the animal hemisphere of the egg than in the vegetal hemisphere, and the pigment embedded in it defines the pigmented area of the animal hemisphere. In the pigmented area the material excludes yolk for a thickness of 3-7+ microns from the surface. In the vegetal hemisphere there is no such accumulation and yolk platelets can be found almost touching the plasmalemma. Cortical contractions have been experimentally induced in eggs. Their relative strength correlates with the relative thickness of the finely granular, subcortical material. During contraction the material accumulates to much greater thicknesses, excluding yolk from thicknesses of 15-30+ microns from the surface. The contracting entity is, or is in, the finely granular material. Injection of cytochalasins into the eggs inhibits cleavage furrow operation but does not inhibit the induced cortical contractions. The thus do not seem to be dependent on actin microfilamentogenesis as is the operation of the contractile ring of the cleavage furrow. The differential sensitivity to cytochalasins of the contractile ring and the system responding in the induced cortical contractions, suggests a two-component system for cortical contractions in the egg. A model is presented which accommodates the available data.