Licht- und elektronenmikroskopische Untersuchungen an den Eihüllen der Elritze (Phoxinus phoxinus [L.];Teleostei,Cyprinidae)

Zusammenfassung Die Oocyten der ElritzePhoxinus phoxinus wurden licht- und elektronenmikroskopisch untersucht. Sie sind im Stadium I nur von der primären Oocytenmembran umgeben, die Mikrovilli zum Follikelepithel hin aussendet. Die Bildung der eigentlichen Eihülle, des Cortex radiatus, beginnt im frühen Stadium II. Der Cortex radiatus differenziert sich bald in den Cortex radiatus externus und den Cortex radiatus internus, wobei der Externus vor dem Internus angelegt wird. Die Differenzierung der Cortex-Schichten ist gegen Ende des Stadiums III abgeschlossen. Aussehen und Aufbau des Cortex radiatus externus der Elritze weichen stark von dem bisher bei anderen Cypriniden-Eizellen gefundenen Schema ab. DiePhoxinus-Eihüllen gleichen mehr denen von Salmoniden. Die Bildung der Rindenvakuolen erfolgt in einfacher Schicht überall gleichzeitig in den Eizellen unterhalb der primären Oocytenmembran. Sie breiten sich zentripetal aus. Die Rindenvakuolen entstehen beiPhoxinus phoxinus wahrscheinlich größtenteils durch Verschmelzung von Vesikeln.

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Light and electron microscopic studies of the envelopes in egg cells of the minnow (Phoxinus phoxinus [L.];Teleostei, Cyprinidae)

Summary The oocytes of the minnowPhoxinus phoxinus were examined by light and electron microscopy. In stage I they are only enveloped by the primary oocyte membrane which extends microvilli towards the follicle epithelium. The development of the actual egg envelope, the cortex radiatus, starts early in stage II. The cortex radiatus differentiates soon into the cortex radiatus externus and the cortex radiatus internus. In the course of this development the externus is formed earlier than the internus. The differentiation of the cortex layers is finished about the end of stage III. The exterior and the structur of the cortex radiatus externus of the minnow differs widely from the scheme found at other cyprinid oocytes. ThePhoxinus egg envelopes resemble more those of salmonids. The development of the cortical alveoli takes place simultaneously in the oocytes beyond the primary oocyte membrane. They expand centripetally. The cortical alveoli ofPhoxinus phoxinus probably derive for the most part from fusion of vesicles.

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Herrn Prof. Dr. Dr. h. c. W. E.Ankel zu seinem 80. Geburtstag gewidmet.     

Licht- und elektronenmikroskopische Untersuchungen an den Eihüllen des marinen TeleosteersLutjanus synagris

Zusammenfassung 1. Die Struktur der Eier und Eihüllen reifender Oocyten vonLutjanus synagris (L.) wurde licht- und elektronenmikroskopisch untersucht.2. Die Bildung der Kortikalschicht beginnt im frühen Stadium II. Eine elektronenoptisch dichte Substanz wird zwischen den Mikrovilli der Oocyte abgelagert.3. Im frühen Stadium III differenziert sich die Kortikalschicht in einen Cortex radiatus externus und einen Cortex radiatus internus.4. Im Stadium IV wird der Cortex radiatus externus weiter differenziert: Er besteht aus drei deutlich unterscheidbaren Lagen, einer sehr dunklen Außenlage und zwei darunterliegenden Schichten unterschiedlicher Granulierung.5. Die Kortikalschicht wird von Poren durchbrochen, in denen die Mikrovilli der Oocyte und die Fortsätze des Follikelepithels verlaufen.6. Der Stofftransport von der Eizelle zum Follikel und umgekehrt erfolgt mit großer Wahrscheinlichkeit auf dem Wege der Pinocytose.7. Cytoplasmatische Verbindungen an den Kontaktstellen zwischen den Mikrovilli der Eizelle und den Fortsätzen des Follikelepithels konnten nicht beobachtet werden.

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Light and electron microscopic studies of the envelopes in egg cells of the marine teleost fishLutjanus synagris

The cortical layer development of the oocytes of the oviparous teleost fishLutjanus synagris (Linnaeus) were investigated by light and electron microscopy. In stage I the young oocytes and the follicle cells are in contact with each other. During the early growth period, microvilli cover the surface of the oocytes. In the early stage II, patches of electron dense homogeneous material for the cortex are deposited between the microvilli. In stage III the cortical layer differentiates into a cortex radiatus externus and a cortex radiatus internus. Just before ovulation in stage IV the cortex radiatus externus is strengthened by further differentiations. The transport of material from the egg to the follicle cells and vice versa is probably mediated by pinocytosis. Cytoplasmatic links at the points of contact between microvilli and the branches of the follicular cells could not be observed.

     

Studies on the formation of germ cells in a compound ascidian Botryllus,primigenus oka

Gametogenesis of a compound ascidian Botryllus primigenus was studied histologically. On either side of the zooid (stage 9), in the gonadal space between the epidermis and the atrial epithelium, either a single testis or a complex of an egg follicle and a testis can be formed. The egg follicle consists of a single ovum (occasionally two ova) and its accessory cells and is connected with the atrial epithelium by the follicle stalk. The egg follicle is always accompanied by the brood pouch, a diverticulum of the atrial cavity. The testis is equipped with a vestigial spermiduct and is attached to the atrial epithelium. Buds of stage 8 comprise, besides the developing testes and, egg follicles, loose aggregations of hemoblasts and oocytes of early developmental stages, which are already accompanied by primary follicular cells. Both the oocytes and the primary follicular cells seem to arise from the hemoblasts. The young oocytes are isolated in the gonadal space of the buds nnd are transferred to buds of the succeeding generations until they finally mature. In the bud of stage 3, a compact mass of cells appears, attaching to tbe inner vesicle on either side of the body. It is derived from the hemoblasts lodged there in the preceding generation and presumably also from the circulating hemoblasts. When the cell mass receives a large oocyte derived from the preceding generation, part of the cell mass differentiates into egg envelopes, forming an egg follicle, and a follicle stalk and the remainder into a testis. When the cell mass receives no oocyte, it differentiate as a whole into a testis. In the egg follicle thus formed the outer and inner follicular cells increase in number by mitotic division. Subsequently, initial test cells are derived from the inner follicle by migration across the developing chorion; then they increas2 in number by mitosis. In the testis, meiosis and spermiogenesis take place.     

ULTRASTRUCTURAL STUDY ON THE ATTACHING FILAMENTS AND VILLI OF THE OOCYTE OF ORYZIAS LATIPES DURING OOGENESIS

The formation of the attaching filaments and villi on the surface of the oocyte of Oryzias latipes were studied electron-microscopically. The oocyte at the early stage has almost smooth surface with a few tufts of microvilli. Some parts of the surface of the oocyte are in contact with the follicle cell, and these parts subsequently become protrusions. As maturation proceeds, a mass of fine granules appears in the space between the protrusion and the follicle cell. Similar granules begin to appear also in the space between the microvilli. These granules later become the outer layer of the chorion. The protrusions are reduced in height, and consequently become almost flat. At the same time, there appears some amorphous material of high electron density on the above-mentioned granules on the flat part. A bundle of parallel microtubules is formed in the material. The tubule is 180–200 A in diameter, and its wall consists of 12 or 13 subunits. The bundle increases in volume, and becomes the attaching filament or villus.     

Dynamics of apoptosis in the ovarian follicle cells during the late stages of Drosophila oogenesis

In the present study, we demonstrate the apoptotic events of the ovarian follicle cells during the late stages of oogenesis in Drosophila melanogaster. Follicle cell morphology appears normal from stage 10 up to stage 14, exhibiting a euchromatic nucleus and a well-organized cytoplasm. First signs of apoptosis appear at the anterior pole of the egg chamber at stage 14A. They are characterized by loss of microvilli at the apical cell membrane, alterations in nuclear morphology, such as chromatin condensation and convolution of the nuclear membrane, and also by condensation and vacuolization of the cytoplasm. During the following stage 14B, the follicle cell nuclei contain fragmented DNA as is demonstrated by acridine orange staining and TUNEL (TdT-mediated dUTP nick end-labeling) assay. Finally, the apoptotic follicle cells seem to detach from the eggshell when the mature egg chamber exits the ovariole. The detached follicle cells exhibit condensed nuclear chromatin, a disorganized cytoplasm with crowded organelles and are surrounded by epithelial cells. The above results seem to be associated with the abundant phagocytosis that we observed at the entry of the lateral oviducts, where the epithelial cells contain apoptotic cell bodies. Additionally, we tested the effect of etoposide treatment in the follicular epithelium and found that it induces apoptosis in a stage- and site-specific manner. These observations suggest a possible method of absorption of the apoptotic follicle cells that prevents the blockage of the ovarioles and helps the regular production of mature eggs.     

Transitional epithelium of urinary tract in normal and dehydrated rats

Summary This report is a light microscopic histochemical and fine structural study of transitional epithelium of the urinary tract of normal and dehydrated rats. Four types of cells were recognized: basal, intermediate, squamous or luminal and bundle cells. The transitional epithelium of normal rat ureter and bladder shows distinct cytoplasmic staining of the squamous cells layer by PAS. The luminal free border stains more intensely with PAS. With the electron microscope, abundant cytoplasmic tonofilaments, free ribosomes and the characteristic thick-walled fusiform and round vesicles are observed, which were in greater number in the squamous cells. Lysosomes are identified with PAS, and Toluidine Blue 0, by their content of acid phosphatase and non-specific carboxylic esterase, and by their ultrastructural appearance. The bundle cell (Hicks, 1965) is characterized by histochemical technics. These cells form about 2.5% of the total cell population of normal transitional epithelium. The bundle cell contains basophilic metachromatic granules, which indicates the presence of a weakly acid mucosubstance. It is suggested that bundle cell granules are released in the intercellular spaces of transitional epithelium and that the mucosubstance may regulate flow of ions and metabolites in the epithelial intercellular channels.Several ultrastructural changes occur in the transitional epithelium of dehydrated rats: marked increase in number of thick-walled vesicles, development of polysomes, relative increase of cytoplasmic filaments and greater number of enlarged lysosomes. Bundle cells decrease in number. These ultrastructural changes promptly regressed by allowing the animal to drink water.It is suggested that the rate of formation of the characteristic vesicles of transitional epithelium, a function of membrane synthesis, may be under the control of the antidiuretic hormone.This investigation was supported in part by the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina, through a travel grant to Dr. Monis, who would like to thank Dr. E. de Robertis for the use of the electron microscope facilities of the Instituto de Anatomía General y Embriología, Facultad de Medicina, Universidad de Buenos Aires.     

Surface specializations of the olfactory epithelium of rainbow trout, Salmo gairdneri

Receptors for olfactory stimulus molecules appear to be located at the surface of olfactory receptor cells. The ultrastructure of the distal region of rainbow trout (Salmo gairdneri) olfactory epithelium was examined by transmission electron microscopy. On the sensory olfactory epithelium, which occurs in the depressions of secondary folds of the lamellae of the rosettes, five cell types were present. Type I cells have a knob-like apical projection which is unique in this species because it frequently contains cilia axonemes within its cytoplasm in addition to being surrounded by cilia. Type II cells bear many cilia oriented unidirectionally on a wide, flat surface. Type III cells have microvilli on a constricted apical surface and centrioles in the subapical cytoplasm. Type IV cells contain a rod-like apical projection filled with a bundle of filaments, and type V cells are supporting cells. Cilia on the sensory epithelium contain the 9 + 2 microtubule fiber pattern. Dynein arms are clearly present on the outer doublet fibers, which suggests that the cilia in the olfactory region are motile. Their presence in olfactory cilia of vertebrates has been controversial. The cilia membrane in this species is unusual in often showing outfoldings, within which are included small, irregular vesicles or channels. In addition, cilia on type II cells frequently contain dense-staining bodies closely apposed to the membranes, along with a densely stained crown at the cilia tip. Previous biochemical evidence indicates that odorant receptors are associated with the cilia.     

Ultrastructure of the indifferent gonad in male and female pig embryos.

Pig embryos aged 24 days were obtained from artifically inseminated sows for ultrastructural study of the indifferent gonads. Sex was identified by chromosome analysis. The gonads are composed in both sexes of three different tissues: the surface epithelium, the gonadal blastema and the mesenchyme. The surface epithelial cells contained elongate mitochondria, granular endoplasmic reticulum, free polysomes, the Golgi complex, fine filaments and coated vesicles. The primitive cords were continuous with the surface epithelium and the interior of the gonad was occupied by blastema cells. They had prominent nucleoli, elongate mitochondria, granular endoplasmic reticulum, the Golgi complex, free polysomes, some lipid droplets and occasionally circular smooth membrane profiles resembling the agranular endoplasmic reticulum. Individual primordial germ cells were seen in all parts of the gonad. They were roundish with prominent nucleoli, globular mitochondria, granular endoplasmic reticulum, free polysomes, the Golgi complex, coated vesicles, lipid droplets and dense bodies. Degenerating cells and cells having pseudopods were also encountered. In comparison to the gonad at the age of 22 days, the primordium had grown into a longitudinal roundish protrusion and the number of primoridal germ cells had increased. Histological and ultrastructural observations showed that the pig gonads at the age of 24 days were similar in both sexes.     

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.     

Ultrastructure of the thread cells in the slime gland of Japanese hagfishes,Paramyxine atami and Eptatretus burgeri

The thread cells in the slime gland of Japanese hagfishes, Paramyxine atami and Eptatretus burgeri were studied by light and electron microscopy. The mature thread cells are large elements (180 times 80 mu) filled with an intricately coiled thread, approximately 2 mu in diameter. The protein nature of the thread has been confirmed by histochemical examination. In the initial stage of growth, the thread consists of a bundle of distinctly parallel filaments approximately 90-120 A in diameter and a centrally located tubular component approximately 230-260 A in diameter which occurs singly or occasionally as a double and triple structure. The developing thread displays thin filaments, approximately 30-60 A in diameter. The thin filaments are composed of fine fibrous structures, subfilaments, approximately 10-30 A in diameter. On the outer surface of the thread a coating is apparent, giving it a fluffy appearance. Polysomal clusters consisting of five or six ribosomes are predominant. Fine fibrous structures are also found among the threads; they seem to have a spatial relationship with the polysomes and resemble the subfilament constituents of the thin filaments. From these results, it may be suggested that the fine fibrous structures synthesized by polysomes, twist together and coalesce into a thread. The problem of the polysome size and the molecular weight of the fibrous protein synthesized is discussed.     

The follicle cells of styela plicata (ascidiacea, tunicata): a sem study

The morphological aspect of the follicle cells of Styela plicata eggs is described by means of scanning electron microscope investigations. The follicular layer is made of spaced, cylindrical box-like cells which are arranged hexagonally. They adhere to the egg through a complex network of membrane extensions making an overall thin layer on the vitelline coat. The walls of the follicle cells are plentifully provided with microvilli, filopodia and lamellipodia, which allow a connection among the cells. At their apical end lies a large vacuole containing a granule, probably involved in secretion. At insemination the majority of spermatozoa is distributed on the apical membrane of the follicle cells. The membrane often breaks after sperm-egg impact and the granule is therefore displaced. By means of the present investigations it is once again suggested a role played by follicle cells in ascidian eggs at fertilization.     

Follicular placenta of the viviparous fish,Heterandria formosa: II. Ultrastructure and development of the follicular epithelium

Embryos of the viviparous poeciliid fish, Heterandria formosa, develop to term in the ovarian follicle where they undergo a 3,900% increase in embryonic dry weight. Maternal-embryonic nutrient transfer occurs across a follicular placenta that is formed by close apposition of the embryonic surface (i.e., the entire body surface during early gestation and the pericardial amnionserosa during mid-late gestation) to the follicular epithelium. To complement our recent study of the embryonic component of the follicular placenta, we now describe the development and fine structure of the maternal component of the follicular placenta. Transmission electron microscopy reveals that the ultrastructure of the egg envelope and the follicular epithelium that invests vitellogenic oocytes is typical of that described for teleosts. The egg envelope is a dense matrix, penetrated by microvilli of the oocyte. The follicular epithelium consists of a single layer of cuboidal cells that lack apical microvilli, basal surface specializations, and junctional complexes. Follicle cells investing the youngest embryonic stage examined (Tavolga's and Rugh's stage 5–7 for Xiphophorus maculatus) also lack apical microvilli and basal specializations, but possess junctional complexes. In contrast, follicle cells that invest embryos at stage 10 and later display ultrastructural features characteristic of transporting epithelial cells. Apical microvilli and surface invaginations are present. The basal surface is extensively folded. Apical and basal coated pits are present. The cytoplasm contains a rough endoplasmic reticulum, Golgi complexes, and dense staining vesicles that appear to be lysosomes. The presence of numerous apically located electron-lucent vesicles that appear to be derived from the apical surface further suggests that these follicle cells may absorb and process follicular fluid. The egg envelope, which remains intact throughout gestation and lacks perforations, becomes progressively thinner and less dense as gestation proceeds. We postulate that these ultrastructural features, which are not present in the follicles of the lecithotrophic poeciliid, Poecilia reticulata, are specializations for maternal-embryonic nutrient transfer and that the egg envelope, follicular epithelium, and underlying capillary network form the maternal component of the follicular placenta. © 1994 Wiley-Liss, Inc.     

The role of the follicular epithelium in growing eggs of a dipteran insect during late oogenesis and cleavage

The gall midge Heteropeza pygmaea can reproduce by means of paedogenesis (i.e., larval parthenogenesis). In that process, follicles are produced that develop while floating in the hemocoele of the mother larva. A chorion is not formed at the end of oogenesis, and the growing embryos remain enveloped by the follicular epithelium. To investigate possible adaptations of the follicular epithelium to this unusual egg development, its ultrastructure has been studied during late oogenesis and cleavage. Earlier investigations had shown that the follicle cells are provided with a specifically arranged microtubular frame, which may be responsible for the anisometric growth of the egg. The present work shows that the follicle cells are always joined by desmosomes and septate junctions. During development, the septate junctions increase their surface and change their orientation to become parallel to the longitudinal egg axis, thus increasing the resistance of the follicle cells to being torn apart by growth tensions. The total surface of the follicular epithelium increases during development. Well-developed nucleoli in the nuclei and numerous ribosomes in the cytoplasm of follicle cells indicate a high level of synthetic activity. This activity may be required to support the increase in the membrane surface and the establishment of the microtubular frame. Lipid droplets, glycogen, and different inclusions in the follicle cells may represent nutrient and energy reserves. Structures indicating a quantitative significant transfer of nutrients from the follicle cells to the egg were not found.     

Development of the follicle complex and oocyte staging in red drum,sciaenops ocellatus linnaeus, 1776 (perciformes,sciaenidae)

Pelagic egg development in red drum, Sciaenops ocellatus, is described using tiered staging. Based on mitosis and meiosis, there are five periods: Mitosis of Oogonia, Active Meiosis I, Arrested Meiosis I, Active Meiosis II, and Arrested Meiosis II. The Periods are divided into six stages: Mitotic Division of Oogonia, Chromatin Nucleolus, Primary Growth, Secondary Growth, Oocyte Maturation and Ovulation. The Chromatin Nucleolus Stage is divided into four steps: Leptotene, Zygotene, Pachytene, and Early Diplotene. Oocytes in the last step possess one nucleolus, dispersed chromatin with forming lampbrush chromosomes and lack basophilic ooplasm. The Primary Growth Stage, characterized by basophilic ooplasm and absence of yolk in oocytes, is divided into five steps: One‐Nucleolus, Multiple Nucleoli, Perinucleolar, Oil Droplets, and Cortical Alveolar. During primary growth, the Balbiani body develops from nuage, enlarges and disperses throughout the ooplasm as both endoplasmic reticulum and Golgi develop within it. Secondary growth or vitellogenesis has three steps: Early Secondary Growth, Late Secondary Growth and Full‐Grown. The Oocyte Maturation Stage, including ooplasmic and germinal vesicle maturation, has four steps: Eccentric Germinal Vesicle, Germinal Vesicle Migration, Germinal Vesicle Breakdown and Resumption of Meiosis when complete yolk hydration occurs. The period is Arrested Meiosis II. When folliculogenesis is completed, the ovarian follicle, an oocyte and encompassing follicle cells, is surrounded by a basement membrane and developing theca, all forming a follicle complex. After ovulation, a newly defined postovulatory follicle complex remains attached to the germinal epithelium. It is composed of a basement membrane that separates the postovulatory follicle from the postovulatory theca. Arrested Meiosis I encompasses primary and secondary growth (vitellogenesis) and includes most of oocyte maturation until the resumption of meiosis (Active Meiosis II). The last stage, Ovulation, is the emergence of the oocyte from the follicle when it becomes an egg or ovum. J. Morphol. 2012. © 2012 Wiley Periodicals, Inc.     

The receptor-like tyrosine phosphatase lar is required for epithelial planar polarity and for axis determination within drosophila ovarian follicles.

The follicle cell monolayer that encircles each developing Drosophila oocyte contributes actively to egg development and patterning, and also represents a model stem cell-derived epithelium. We have identified mutations in the receptor-like transmembrane tyrosine phosphatase Lar that disorganize follicle formation, block egg chamber elongation and disrupt Oskar localization, which is an indicator of oocyte anterior-posterior polarity. Alterations in actin filament organization correlate with these defects. Actin filaments in the basal follicle cell domain normally become polarized during stage 6 around the anterior-posterior axis defined by the polar cells, but mutations in Lar frequently disrupt polar cell differentiation and actin polarization. Lar function is only needed in somatic cells, and (for Oskar localization) its action is autonomous to posterior follicle cells. Polarity signals may be laid down by these cells within the extracellular matrix (ECM), possibly in the distribution of the candidate Lar ligand Laminin A, and read out at the time Oskar is localized in a Lar-dependent manner. Lar is not required autonomously to polarize somatic cell actin during stages 6. We show that Lar acts somatically early in oogenesis, during follicle formation, and postulate that it functions in germarium intercyst cells that are required for polar cell specification and differentiation. Our studies suggest that positional information can be stored transiently in the ECM. A major function of Lar may be to transduce such signals.     

The structure of the chorion and associated surface filaments in Oryzias--evidence for the presence of extracellular tubules

The structure of the chorion with its associated surface filaments has been examined in Oryzias latipes using several techniques, including scanning and transmission electron microscopy, enzymatic digestion, and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The chorion of the recently fertilized egg was found to be organized into three zones: an outer, fuzzy electron-lucent zone that was continuous over the surface of filaments, a middle, homogeneous electron-dense zone, and an inner zone of ten to 12 horizontal, fibrous lamellae. Two topographically distinct types of filaments were found on the chorionic surface: nonattaching and attaching. Nonattaching filaments showed a regular spatial distribution over the chorion with an interfilament distance of about 60-70 microns. Attaching filaments originated from a localized portion of the chorion and united with those of neighboring eggs to anchor the egg cluster to the gonoduct of the female. Both nonattaching and attaching filaments were morphologically regionalized into basal and distal segments. Internally, nonattaching and attaching filaments were constructed of unbranched, packed tubules with an average outside diameter of approximately 19.5 and 18.8 nm, respectively. Using the attaching filament for further study, it was determined by rotational analysis (Markham et al., '63) that the wall of each tubule was a cylinder composed of 14 globular subunits. Two structural types of attaching filaments were identified. The type I attaching filament was similar in internal organization to the nonattaching filament and consisted of only tubules. The type II attaching filament, however, showed a highly osmiophilic, electron-dense bar surrounded by packed tubules. Tubules of attaching filaments of the adult were resistant to the action of Triton X-100 and colchicine, but sensitive to a 0.1% protease solution. However, colchicine-treated ovary tissue showed an absence and pattern of disorganization of tubules at the periphery of developing filaments. Solubilized attaching filament samples electrophoresed on 7.5% polyacrylamide-SDS gels were resolved into a pair of Coomassie-blue-positive bands that comigrated with purified porcine brain tubulin. The apparent molecular weight of the attaching filament polypeptide was determined to be approximately 55,000 daltons. These data suggest that the extracellular, tubular components of attaching filaments (as well as nonattaching filaments) are proteinaceous and show properties similar to those of cytoplasmic microtubules. Tubular precursor material was electron-dense and appeared to originate in the cisternae of the rough endoplasmic reticulum of ovarian foll     

Colocalization of heparin and histamine in the intracellular granules of test cells from the invertebrate Styela plicata (Chordata-Tunicata)

In most ascidian species the oocytes are surrounded by two types of accessory cells called follicle cells and test cells. Test cells are located on the periphery of oocytes and remain in the perivitelline space during egg development until hatching. Heparin and histamine were previously described in the test cells of the ascidian Styela plicata. In the present study, electron microscopy techniques were used to characterize the ultrastructure of the S. plicata test cells and to localize heparin and histamine in these cells. Test cells contain several intracellular granules with unique ultrastructural features. They are formed by elongated filaments composed of serial globules with an electron-lucent circle, containing a central electron-dense spot. Immunocytochemistry showed that heparin and histamine colocalize at the border of granule filaments in the test cell. Compound 48/80, a potent secretagogue of heparin-containing mast cells, also induced degranulation of test cells. According to these results, we suggest that test cells represent ancient effector cells of the innate immunity in primitive chordates.     

Cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor cluster in cell cultures

Whole-mount stereo electron microscopy has been used to examine the cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor (AChR) clusters in cultures of Xenopus nerve and muscle cells. The cells were grown on Formvar-coated gold electron microscope (EM) finder grids. AChR clusters were identified in live cultures by fluorescence microscopy after labeling with tetramethylrhodamine-conjugated alpha-bungarotoxin. After chemical fixation and critical-point drying, the cytoplasmic specializations of identified cells were examined in whole mount under an electron microscope. In the presynaptic nerve terminal opposite to the AChR cluster, synaptic vesicles were clearly suspended in a lattice of 5-12- nm filaments. Stereo microscopy showed that these filaments directly contacted the vesicles. This lattice was also contiguous with the filament bundle that formed the core of the axon. At the AChR cluster, an increased cytoplasmic density differentiated this area from the rest of the cytoplasm. This density was composed of a meshwork of filaments with a mean diameter of 6 nm and irregularly shaped membrane cisternae 0.1-0.5 micron in width, which resembled the smooth endoplasmic reticulum. These membrane structures were interconnected via the filaments. Organelles that were characteristic of the bulk of the sarcoplasm such as the rough endoplasmic reticulum and the polysomes, were absent from the cytoplasm associated with the AChR cluster. These results indicate that the cytoskeleton may play an important role in the development and/or the maintenance of the neuromuscular synapse, including the release of transmitter in the nerve terminal and the clustering of AChRs in the postsynaptic membrane.     

Synthesis and function of the fibrous layers covering the eggs of Siphlonurus lacustris (Ephemeroptera, Siphlonuridae)

Ultrastructural analysis (transmission and electron scanning microscopy) of the eggs of the mayfly Siphlonurus lacustris (Eaton) showed that they are wrapped in a thick coat composed of a network of tightly entwined filaments. Groups of twisted filaments form slightly uplifted buttons that are scattered on the coat surface. After experimentally induced egg deposition, egg–water interaction promotes marked cohesion of the eggs and their firm adhesion to the substrate. Egg masses include numerous gametes; the covering of those located close to the substrate greatly extends to anchor the whole mass. Eggs removed from the coat reveal a slightly punctuated smooth chorion and tagenoform micropyles (three to five). The coat increases egg size by about 20%. The lack of female reproductive accessory glands in Ephemeroptera transfers the synthesis of the adhesive coats to the follicle cells, which are typically competent for insect egg shell deposition (vitelline envelope and chorionic layers). This covering results from electron‐dense granules that give rise to filaments progressively organized to form superimposed layers variously orientated around the egg. In addition to egg adhesion to the substrate, a trophic function and protection from shear stress are postulated for this covering.     

On the infiltration of Golgi bodies from the follicular epithelium to the egg

Summary 1. In a well advanced oocyte of the tortoise the egg membranes besides the theca and the single-layered epithelium consist of a zona pellucida often differentiated into zona striata and a homogeneous layer; underlying these two layers is a layer of cortical fibrillae or fibrillar layer, Next to this layer, is the limiting membrane of the egg which is not present in all stages and generally disappears in a well developed oocyte. In certain animals either the homogeneous layer or fibrillar layer is absent. 2. In certain animals,Golgi bodies seem to be extruded into the follicle cells from the theca cells. 3. At a particular stage of development the follicle cells become very active and produce a large number of smallGolgi bodies. TheseGolgi granules filter through canalicular passages of the zona radiata into either the homogeneous layer and from thence into the fibrillar layer or where a homogeneous layer is not present directly to the fibrillar layer. Where a fibrillar layer is not present they are transferred directly to the limiting membrane and from thence to the egg. 4. In certain cases e. g. in Fowl, Calotes and Uromastix, fairly large lumps ofGolgi bodies are extruded from the follicle cells through the zona pellucida into the egg. Here the fine canilicular passages do not seem to form a vehicle for the passage of these comparatively larger bodies. 5. The fine canalicular passages in the zona radiata ofTestudo graeca andKachuga smithii and the fibrillar prolongation of the cytoplasm which we have called the fibrillar layer are marked features of the egg membranes at certain stages of development of the egg. During the period when infiltration ofGolgi bodies through these passages takes place slides prepared by silver nitrate and osmic methods show black beaded chains ofGolgi granules in various stages of descent. 6. It is claimed that the extrusion and infiltration ofGolgi bodies from the follicular epithelium to the egg are established phenomena at least in the Vertebrates.