ELECTRON MICROSCOPY OF BASOPHILIC STRUCTURES OF SOME INVERTEBRATE OOCYTES : II. FINE STRUCTURE OF THE YOLK NUCLEI

RNA containing yolk nuclei from the surf clam Spisula solidissima have been studied with the light microscope and with the electron microscope. A variety of structures can be seen with both and a correlation can be made between bodies studied with the electron microscope and those studied with the light microscope. The electron microscope shows many of these structures to be composed of double walled lamellae arranged in space, in various ways. The variety of patterns seen with the electron microscope can be satisfactorily explained on the basis of a hypothetical model. The presence of yolk platelets within the yolk nuclei lends support to classical observations on the synthesis of yolk within yolk nuclei or yolk nuclei derivatives. Small granules (about 100 A) are included within the double walled lamellae and their presence probably accounts for the basophilic nature of the entire body. The presence of small granules attached to electron-dense layers relates the yolk nuclei described here to ergastoplasm discussed by others.     

Intranuclear and cytoplasmic annulate lamellae in grasshopper spermatocytes (Genus Melanoplus)

Intranuclear and cytoplasmic annulate lamellae were studied in grasshopper spermatocytes (Melanoplus) with the electron microscope. Although cytoplasmic annulate lamellae were observed in all three species examined, intranuclear annulate lamellae were found in only one species. The intranuclear annulate lamellae encompass certain nuclear material adjacent to the nuclear envelope forming a vesicle that is extruded into the spermatocyte cytoplasm. In this same species, cytoplasmic annulate lamellae are seen contiguous with granular masses of varying size. These structures were noted as being morphologically indistinguishable from the "yolk nuclei" of dragonfly oocytes (Kessel and Beams, 1969; Kessel, 1973).     

Degradation of yolk in the brine shrimp Artemia. Biochemical and morphological studies on the involvement of the lysosomal system

The degradation of yolk granules during the development of Artemia was studied. The results obtained suggest that lysosomes are involved in the process. In homogenates of embryos and larvae at different stages of development, the distribution of 2 lysosomal markers, acid phosphatase and cathepsin B, was studied by sucrose isopycnic gradient centrifugation. Three peaks of enzyme activity of densities > 1.3 and around 1.25 and 1.18 were observed. As revealed by electron microscope analysis, the 3 peaks were found to be associated with increasingly degraded yolk structures which stained for acid phosphatase. The process can be mimicked in vitro by incubating isolated yolk granules and lysosomes. The enzyme activity levels of the 3 peaks observed during development presented an oscillatory pattern, suggesting that degradation of yolk is cyclic. Five cycles of degradation were observed during the initial 60 hr of development.     

Vasopressinergic axon collaterals and axon terminals in the magnocellular neurosecretory nuclei of the rat hypothalamus

Axon collaterals emerging from the vasopressinergic neurons of the supraoptic (SON) and paraventricular (PVN) nuclei and recurving back towards their respective nuclei have been previously reported. Since such axon collaterals can play a role in the neuromodulation of SON and PVN, these nuclei have been further investigated immunohistochemically under the light and electron microscope. The PAP technique, using a commercial antibody, was employed. Vasopressin-positive axon collaterals were seen to recurve towards their nuclei of origin. In the latter, vasopressinergic intrinsic neurons were also observed. Under the electron microscope, axon terminals containing vasopressin-immunoreactive neurosecretory granules were noted. Such terminals presumably arise from the vasopressin-positive recurrent axon collaterals or from the intrinsic neurons for the purpose of neuromodulation within the SON and PVN.     

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.     

Avian Trypanosome Division; A Light and Electron Microscope Study1

SYNOPSIS. A magpie isolate of Trypanosoma avium was studied using cultured epimastigotes and trypomastigotes from experimentally infected canaries. Apparent signs of division (double nuclei, flagella, and kinetoplasts) were observed in trypomastigote forms with the light microscope. Three or 4 flagella were seen within a single flagellar pocket in epimastigotes by electron microscopy. It is suggested that apparently dividing trypomastigotes are actually blocked in the process of division.     

The formation of intracellular crystals in midgut glands of Limnoria lignorum

Certain morphological features of intracellular crystal formation within the midgut glands of Limnoria lignorum (Rathke) have been studied with the electron microscope and cytochemical methods. A correlation has been established between Golgi membranes and formation of the crystals. The Prussian blue reaction reveals quantities of iron localized in the intracellular crystals and in small granular structures seen in the apical region of the cells. These granules can be identified as accumulations of Golgi membranes, with which iron-containing particles are associated. When these membrane configurations are studied with the electron microscope, they can be classified and arranged in an assumed sequence which is thought to represent successive stages in the development of crystals. As the membrane systems become progressively specialized, increasing accumulations of dense granular material appear within their interstices. This material is rich in iron and probably represents the component responsible for the positive Prussian blue reaction. This material also appears to be a precursor substance for iron-containing protein molecules which are synthesized and arranged to make up the crystals. These iron-containing molecules are first deposited in orderly array as double rows of dense particles on certain internal membranes of the specialized Golgi complexes. The membranes later disappear and the particles form definitive crystals by rearrangement into a hexagonal close-packed pattern.     

The formation of “Accessory Nuclei” and annulate lamellae in the oöcytes of the flour moth anagasta kühniella

Summary Oöcytes in various stages of maturation were studied with the electron microscope. Stacks of annulate lamellae were found attached to large ribosome studded vesicles. The outer nuclear membrane was found to form blebs and it is considered that these produce the large vesicles. It is suggested that mRNA enters the cytoplasm in membraneous vesicles which collapse, liberating the mRNA, and thus produce annulate lamellae which may later form E.R. Oöcyte nuclei were examined in the E.M. as a potential source of accessory nuclei. Membrane bound organelles containing granules were found in the periphery of the nucleus. The granules were shown to be extruded from the oöcyte nucleolus. The timing of the disappearance of these organelles from the nucleus, together with their structure, suggests that they are precursors of accessory nuclei.The author is indebted to Miss Audrey Taylor, Mr. A. W. Morison, and Mr. M. Craig for technical assistance. The Science Research Council provided a grant for the purchase of equipment with the author gratefully acknowledges.     

The Formation of Intracellular Crystals in Midgut Glands of Limnoria lignorum

Certain morphological features of intracellular crystal formation within the midgut glands of Limnoria lignorum (Rathke) have been studied with the electron microscope and cytochemical methods. A correlation has been established between Golgi membranes and formation of the crystals. The Prussian blue reaction reveals quantities of iron localized in the intracellular crystals and in small granular structures seen in the apical region of the cells. These granules can be identified as accumulations of Golgi membranes, with which iron-containing particles are associated. When these membrane configurations are studied with the electron microscope, they can be classified and arranged in an assumed sequence which is thought to represent successive stages in the development of crystals. As the membrane systems become progressively specialized, increasing accumulations of dense granular material appear within their interstices. This material is rich in iron and probably represents the component responsible for the positive Prussian blue reaction. This material also appears to be a precursor substance for iron-containing protein molecules which are synthesized and arranged to make up the crystals. These iron-containing molecules are first deposited in orderly array as double rows of dense particles on certain internal membranes of the specialized Golgi complexes. The membranes later disappear and the particles form definitive crystals by rearrangement into a hexagonal close-packed pattern.     

Ultrastructural cytochemical localizations by back-scattered electron imaging of white blood cells

White blood cells have been studied in the back-scattered electron imaging (BEI) mode of scanning electron microscopy (SEM) with cytochemical methods for endogenous peroxidase, acid phosphatase, and a silver-staining method for nuclei. Peroxidase-positive granules were seen with good contrast and resolution in myeloid precursor cells and acid phosphatase activity was easily detected in macrophages and monoblasts. Silver staining permitted recognition of the shapes and location of the nuclei. In spite of the cytochemical procedures, cell surface structures were reasonably well-preserved in all methods, making direct correlation of BEI and secondary electron imaging (SEI) images an attractive feature in cell research with the scanning electron microscope.     

STUDIES ON SMALL INTESTINAL CRYPT EPITHELIUM : I. The Fine Structure of the Crypt Epithelium of the Proximal Small Intestine of Fasting Humans

Small intestinal crypt epithelium obtained from normal fasting humans by peroral biopsy of the mucosa was studied with the electron microscope. Paneth cells were identified at the base of the crypts by their elaborate highly organized endoplasmic reticulum, large secretory granules, and small lysosome-like dense bodies within the cytoplasm. Undifferentiated cells were characterized by smaller cytoplasmic membrane-bounded granules which were presumed to be secretory in nature, a less elaborate endoplasmic reticulum, many unattached ribosomes and, in some cells, the presence of glycogen. Some undifferentiated cells at the base of the crypts contained lobulated nuclei and striking paranuclear accumulations of mitochondria. Membrane-bounded cytoplasmic fragments, probably originating from undifferentiated and Paneth cells, were frequently apparent within crypt lumina. Of the goblet cells, some were seen actively secreting mucus. In these, apical mucus appeared to exude into the crypt lumen between gaps in the microvilli. The membrane formerly surrounding the apical mucus appeared to fuse with and become part of the plasma membrane of the cell, suggesting a merocrine secretory mechanism. Enterochromaffin cells were identified by their location between the basal regions of other crypt cells and by their unique intracytoplasmic granules.     

Annulate lamellae and crystalline inclusions in granular endoplasmic reticulum of the islet organ and associated tissues of a cyclostome,Myxine glutinosa

Cytoplasmic annylate lamellae were found in the islet organ of a cyclostome, the hagfish (Myxine glutinosa), predominantly in cells interpreted as young proliferating beta-cells, and also in endocrine cells and enterocytes of the bile duct and gut and in the endothelial cells of small blood vessels. A close association was observed annulate lamellae and granular endoplasmic reticulum. Both in cells with and in those without annulate lamellae, crystalline inclusions of proteinaceous nature were seen in granular endoplasmic reticulum. These inclusions were occasionally closely associated to annulate lamellae, and a direct continuity could be seen between granular endoplasmic reticulum and the outer nuclear membrane surrounding an inclusion partially situated in the perinuclear cisterna. Rod-shaped structures and rounded electron dense bodies were seen in the nuclei of some islet parenchymal cells. The presence of annulate lamellae in the islet organ and associated tissues of Myxine glutinosa is believed to be related to the very high phylogenetic age of this species. The close association observed between annulate lamellae, granular endoplasmic reticulum, crystalline inclusions, and sometimes also nuclear membranes, may be of functional significance.     

The fine structure of the adipose cell of the leech Glossiphonia complanata

The two distinct types of cytoplasm seen with the light microscope in the adipose cell of the leech Glossiphonia complanata have been identified in the electron microscope image of this cell. One of these, the basophil cytoplasm, contains many well oriented, paired membranes which are much more clearly evident when calcium ions are added to the fixative. The membranes sometimes appear as concentric arrays of lamellae and are thought to represent sections through a phospholipide-containing body. The paired membranes and the concentric lamellae have granules attached to them and resemble in size and structure the membranes of the endoplasmic reticulum encountered in many mammalian cells. Small dense cytoplasmic particles are present throughout the cell; they may be ferritin molecules, derived from the breakdown of haemoglobin taken in as food. On the basis of a previous histochemical study and the present electron microscope investigation, it is suggested that these paired membranes are similar to the organized type of mammalian ER and the results seem to confirm the belief that these membranes are composed of layers of phospholipoprotein together with attached particles of ribonucleoprotein.     

Oogenesls in the terrestrial hermit crab,coenobita clypeatus (decapoda,anomura): II. Vitellogenesis

Oocytes from the land hermit crab, Coenobita clypeatus, in various stages of vitellogenesis were examined by light and electron microscopy. Early vitellogenic oocytes are characterized by accumulations of discrete vesicles of endoplasmic reticulum in the perinuclear cytoplasm. As oocytes develop, the endoplasmic reticulum becomes abundant, and numerous Golgi complexes are seen. There is a well developed Golgi-endoplasmic reticulum interaction. Within the confines of the reticulum are discrete intracisternal granules, which can be seen coalescing into electron-dense yolk bodies. Lipid accumulation is seen throughout the cytoplasm. Coincident with the burst of intra-oocytic metabolism are oolemma modifications and micropinocytosis, which provide ultrastructural evidence for extra-oocytic yolk production. The mature oocyte contains numerous yolk and lipid vesicles of varying electron density that comprise both intra- and extra-oocytic substrates.     

Annulate lamellae in adenomas of human pituitary glands.

Twelve nontumorous adenohypophyses and 36 various pituitary adenomas, removed by surgery, have been investigated by electron microscopy in order to shed some light on annulate lamellae, primarily on their ultrastructural features, incidence, origin, fate and functional significance. No annulate lamellae were found in the nontumorous adenohypophyses and in 33 pituitary adenomas. They were, however, detected in two adenomas consisting of undifferentiated cells and one adenoma composed of sparsely granulated prolactin cells indicating that these unique membrane configurations cannot be regarded as an exceedingly rare finding and, furthermore, that they may be disclosed not only in undifferentiated but occasionally in highly differentiated cells. Annulate lamellae may arise from endoplasmic reticulum and/or nuclear envelope and consist of arrays of smooth walled double membrane sheets exhibiting regularly spaced interruptions as well as continuities with the endoplasmic reticulum. No relationship was established between annulate lamellae and adenohypophysial secretory activity. Our findings seem to be consistent with the view that annulate lamellae are present in those cells which have the tendency to proliferate.     

Electron microscope studies on developing oocytes of a coelenterate medusa with special reference to vitellogenesis

In a hydrozoan jellyfish, the female gonad is differentiated from a specialized region of the epidermis near the manubrium. Changes in the oocytes during growth and vitellogenesis are described as observed with electron microscopic and cytochemical techniques. Three major types of yolk are formed; these include lipid, glycogen, and membrane-bound granules consisting of both protein and carbohydrate. The latter first appear evident within vesicular and cisternal elements of the numerous Golgi complexes. The orientation and structural variations noted between the endoplasmic reticulum and forming face of the Golgi complexes suggest that the protein component of the yolk granules may be transferred from the cisternae of the endoplasmic reticulum to the Golgi complex where it is joined to carbohydrate perhaps synthesized by the Golgi complexes. Stages in the release of the precursor yolk material sequestered in cisternal elements of the Golgi complexes are illustrated. The presence of coated and uncoated vesicles in the Golgi regions and their possible role in intracellular transport are described and discussed. The presence and possible method of morphogenesis of vesiculate yolk bodies are also described. What appear to represent invaginations of the oolemma extend into the ooplasm and display a special orientation with respect to lamellae of the rough-surfaced endoplasmic reticulum. Intraooplasmic synthesis appears to constitute the major pathway for protein-carbohydrate yolk deposition.     

THE ORIGIN OF PROTEIN AND FATTY YOLK IN RANA PIPIENS : II. Electron Microscopical and Cytochemical Observations of Young and Mature Oocytes

Electron microscope studies of young oocytes have demonstrated that the plate-like, hexagonally shaped yolk bodies previously observed in living cells are wholly within the substance of oocyte mitochondria and that they remain within these mitochondria while increasing in size. These bodies possess a crystalline structure consisting of what appear to be lines, with a spacing of 70 to 85 A, and appear very dense in the electron microscope. After formalin fixation such bodies give an intense positive test for protein, and when viewed in the electron microscope are only slightly less dense than after OsO₄ fixation. Evidence is presented for the origin of these crystals within a single crista. The clusters of yolk globules previously studied in living cells are seen to consist of several types of bodies, but an irregular dense droplet predominates. This dense material is apparently secreted by small spherical bodies which, the evidence suggests, originate from the breaking up of filamentous mitochondria and which possess an outer double membrane and sometimes internal cristalike membranes. When thin sections of young oocytes are immersed in xylol the dense globules of the clusters are dissolved, but the hexagonal bodies are unaffected, indicating that the globules are of a predominantly fatty nature, while the hexagonal bodies are of a predominantly protein nature. Examination of mature or almost mature oocytes has revealed that the main body of the yolk platelets is crystalline in nature and is surrounded by a thick matrix which, in light microscope study, masks the fact that the face view of the main body of the platelets is often hexagonal. The spacing within the main body is found to be 70 to 85 A. The crystal laminae of this material can be resolved quite clearly into rows of particles. Dense globules of varying sizes are found in the cytoplasm between the platelets. When thin sections of these OsO₄-fixed oocytes are immersed in xylol, the material of the globules is extracted and the crystalline material of the platelets remains unaffected, indicating the fatty nature of the globules and the protein nature of the platelets. The platelets of the mature egg resemble the hexagon bodies, previously described in young oocytes, in their protein nature, their crystalline spacing, and their hexagonal outline. This is given as strong evidence for the origin of the mature platelets by the growth of the intramitochondrial hexagon bodies. The biochemical implications of this study are discussed.     

Intranuclear and cytoplasmic annulate lamellae in the polyploid giant cells of the trophoblast

Intranuclear and cytoplasmic annulate lamellae in polyploid giant cells of the trophoblast have been studied in rat placenta on days 12--17 of development. The annulate lamellae are present in the cytoplasm within a limited time, being visible on day 12 only. These are arranged in bundles near the nucleus to be moving then to the cytoplasm. The end parts of annulate lamellae are broadened to make cisterns of rough endoplasmic reticulum. Unlike the cytoplasmic annulate lamellae, those found within the nucleus are seen in part of the nuclei investigated throughout the whole period examined to look as single structures (not gathered in bundles), they can be branching, separating closed spaces within the nucleus (making local swellings in the loci of branching; the latter having electron dense or transparent vesicles). Association with nuclear chromatin in some regions is a peculiar feature of the intranuclear annulate lamellae. This association is especially obvious at endoprophase in the cycle ofthe polytene nucleus during the somatic conjugation--chromonemes unite in a bundle and condense. Ultrastructural changes of the annulate lamellae is noted throughout the polytene nucleus cycle and during the cell differentiation. It is supposed that in the case of temporary labile chromosome polyteny in the nuclear cycle, which is characteristic of mammalian trophoblasts, annulate lamellae can well compare, in their function, with the synaptonemal complex--these prevent from too tight associations of homologues in the course of somatic conjugation of chromosomes.     

染色质在非洲爪蟾卵的无细胞系统中形成组装核的研究

从大鼠肝、鸡肝提取了染色质并从受精未孵育鸡蛋胚下表层卵黄颗粒提取了染色质,在荧光显微镜和电子显微镜下观察了这些染色质在无细胞系统中形成的组装核;研究二阶钙镁离子对形成组装核的影响。在有ＡＴＰ再生系统存在的无细胞系统中,围绕染色质能形成与间期细胞核相似的组装核;二价钙离子不利于形成组装核,而二价镁离子是形成组装核所必须的,但过高浓度的镁离子对形成组装核有抑制作用。