Cell differentiation during early development of Nassarius reticulatus L. (Gastropoda Prosobranchia)

Summary The ultrastructure of the zygote and of early segmentation stages, up to the 16-cell stage, was investigated in normal Nassarius reticulatus from Roscoff (France). This study deals predominantly with structures that remain morphologically unchanged throughout this period, such as yolk granules, lipid droplets, and multimembranous vesicles. These organelles do not change in position, fine structure, or quantity from the egg to the 16-cell stage. The cortex and the vitelline layer also remain almost unchanged until the 16-cell stage. Cortical granules could not be observed and thus the thin vitelline layer is not transformed to a thick fertilization membrane. This phenomenon seems to be related to the facts that the eggs are protected by a tough capsule and that the cells of the embryo have to incorporate extraembryonic nutrient substances. From the 2-cell stage until the 16-cell stage micropinocytotic vesicles 500–2000 Å in diameter are associated with the plasmalemma. Perhaps they help to incorporate the extraembryonic nutrient reserves. Desmosomes first appear at the 4-cell stage and are common at the 8-cell stage, when a small transitory blastocoel appears. Normally the cell borders are in close proximity during interphase. Syncytial connections between the blastomeres were not observed. Beginning at the trefoil stage the mitochondria increase in number and many apparent division stages are observed. The increase in mitochondria occurs in the perinuclear region throughout the embryo and does not result in a polar lobe especially rich or poor in mitochondria. From the 4-cell stage onwards, the number of ribosomes increases differentially in different blastomeres.Dedicated to Prof. B. Rensch on his 75th birthday. We wish to thank Mrs. C. Mehlis for her valuable technical assistance and Professor J. Bergérard for the excellent working conditions at the Station Biologique at Roscoff (France).     

The ultrastructure of the yolk nucleus during early cleavage of Nassarius reticulatus L. (Gastropoda,Prosobranchia)

Summary In all blastomeres of Nassarius from 4- to 16-cell stage yolk nuclei occur. Most of them are spherical bodies, lying juxtanuclearly between the nucleus and the apical plasmalemma. Strangely they are not ultrastructurally uniform but fall into two categories (Fig. 5): Type I is a massive spherical accumulation of mitochondria embedded in a dense intermitochondrial substance, which appears to contain both granules and filaments. Type II is a ball of radially arranged small Golgi stacks clustered around a centre of Golgi vesicles and other organelles embedded in a ground cytoplasm structurally similar to the intermitochondrial substance of type I. The function of both types of yolk nuclei is unknown. These segmentation yolk nuclei have nothing to do with yolk synthesis any more. On the other hand there are no indications that yolk nuclei occurrence is correlated with the break-down of yolk because neither lipid droplets nor protein yolk granules are observed in or beside the yolk nuclei.We wish to thank Mrs. Chr. Mehlis for her valuable technical assistance as well as Professor J. Bergérard for the excellent working conditions on the Station biologique at Roscoff (France).     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary The ultrastructure of the intermediate lobe of the hypophysis was studied in Anolis carolinensis with the use of a threefold aldehyde fixative. Lizards with a brown skin were selected. The possibility of two types of secretory cells is discussed; neither cell type is innervated. Type I cells are rarely found and contain dense granules approximately 0.3 m in diameter; Type II cells vary widely in secretory activity. Most of the Type II cells contain a large number of dense secretory granules (up to about 1.3 m in diameter) almost filling the cytoplasm. Rough endoplasmic reticulum (RER), Golgi apparatus and mitochondria are poorly developed. Only some of these cells show signs suggesting a high secretory activity, namely a well developed RER, Golgi apparatus and numerous mitochondria. In these cells the RER sometimes forms large intracisternal droplets (up to 7 m in diameter). Two of the animals exhibited a more uniform, high secretory activity. Large (about 2 m in diameter), pale vacuoles, probably of extracellular character, were found mostly in the vicinity of the perivascular septum. Their role in the release of MSH is discussed. The present data, which are discussed with reference to earlier findings (Forbes, 1972), form the morphological basis for an experimental study on regulation of MSH release (Larsson et al., 1979).Supported by grants from the Swedish Natural Science Research Council (to P. Meurling) and the Royal Physiographic Society of LundThe authors are indebted to Mrs. Ingrid Hallberg, Mrs. Kirsten Thörneby and Mrs. Lena Sandell for valuable technical assistance and to Miss Inger Norling for photographic aid     

Mitochondrial degeneration after organic phosphate poisoning in prosimian primates

Summary The degenerative reaction of mitochondria to tricresylphosphate (TCP) poisoning in spinal ganglion cells of Slow Loris (Nycticebus coucang coucang) were studied with the electron microscope. In neurones of animals treated with TCP, mitochondria display various stages of alterations which confirm mitochondrial involvement in TCP poisoning. The role of degenerated mitochondria in the formation of neuronal lipofuscin is discussed. It is suggested that the lipofuscin granule is a metabolic product inherently related to mitochondrial degeneration, irrespective of the primary cause: ageing or intoxication.Fellow of the Deutscher Akademischer Austauschdienst on study leave from the Medical Faculty, University of SingaporeA grant from the Deutsche Forschungsgemeinschaft (Gl. 28/20) is gratefully acknowledgedThe skilfull technical assistance of Mr. Tajuddin b.M. Ali, Mr. P. Gopal, Mr. R. Dungan and Mrs. C. Weinrichter is gratefully acknowledged     

Release of neurosecretory material by protrusions of bounding membranes extending through the axolemma,in Diphyllobothrium dendriticum (Cestoda)

Summary The release of neurosecretory material was studied in the cestode tapeworm Diphyllobothrium dendriticum. In plerocercoid larvae cultivated in vitro the neurosecretory vesicles release their content by way of protrusions extending through the axolemma into the intercellular space. The released material is directed predominantly towards the muscles. No omega figures were detected. The significance of this new type of release is discussed.The authors are greatly indebted to Mrs. Annukka Kärkkäinen and Mr. Esa Nummelin for technical assistance     

The ultrastructure of the eyes of<Emphasis Type="Italic"> Paravortex karlingi</Emphasis> (Plathelminthes,Rhabdocoela)

Both eyes of Paravortex karlingi belong to the rhabdomeric type. Each eye consists of a single pigment cup cell and three sensory cells. Modified mitochondria lie in three protrusions of the cup cell, and several of these mitochondria form giant mitochondrial derivates. The centres of the derivates include electron-dense substances. These modified structures may have lenticular functions. Furthermore, it is shown that giant mitochondrial derivates develop by fusion of smaller ones. Embryos have many small mitochondria in the developing eyes whereas the adult possesses less numerous, but giant forms. This circumstance leads to the assumption that all such lenticular structures within the rhabdocoels result from identical processes.     

Use of mitochondrial DNA as a sensitive and specific marker for localization of mitochondria in fractionated plant extracts

Upon subfractionation of certain plant seed homogenates on sucrose density gradients, we encountered problems in defining the location and amount of mitochondria using marker enzymes. In order to overcome the inherent limitations of enzyme assays, we utilized a heterologous DNA probe specific foratp6 in maize orBrassica tournefortii to detect mitochondria. The samples were treated with SDS, proteinase K, and RNase A followed by agarose gel electrophoresis, and blotting. The immobilized DNA was detected with [³²P]-labelled probes, and quantified using a phosphor imager. The assay is specific, sensitive, and independent of species, cell type, and developmental stage, thus circumventing the need for expressed protein to assay enzyme activity.     

The ultrastructural basis of steroid production in the Y-organ and the mandibular organ of the crabs Hemigrapsus nudus (Dana) and Carcinus maenas L.

Summary The ultrastructure of the steroid producing Y-organ and the mandibular organ of the crustaceans Hemigrapsus nudus and Carcinus maenas has been studied with reference to the well investigated steroid secreting cells (SSC) of mammals. In accordance with the most important characteristic of mammalian SSC, abundant SER could be shown in the Y-organ, where it is unevenly distributed. The amount of SER seems to vary in correlation with the secretion of moulting hormone during the moult cycle. Most Y-organ cells contain a great number of mitochondria of the tubular type, another important characteristic of mammalian SSC. The ultrastructure of the mandibular organ of C. maenas differs considerably from that of the Y-organ. Some SER was found, mitochondria of unusual shape and size were conspicuous. No definite conclusion as to the function of the mandibular organ is yet to be drawn.Dedicated to Prof. Dr. Peter Karlson on the occasion of his 60th birthdaySupported by the Deutsche Forschungsgemeinschaft, grant Ad 24/4We wish to thank Dr. A. Owczarzak, Oregon State University, Corvallis, Oregon, for providing the facilities for our work with H. nudus and Thomas Gallenstein for many helpful discussions of technical problems     

Ultrastructure of the sternal glands inThrips validus uzel (Thysanoptera,terebrantia)

Summary The ultrastructure of the sternal glands of the males ofThrips validus is consistent with the hypothesis that they are involved in pheromone production. The secretory cells, which are strongly modified epidermal cells, are characterized by the abundance of mitochondria and the presence of agranular, tubular endoplasmic reticulum (ATER) and microbodies. Each of the numerous apical microvilli encloses one or several tubular extensions of ATER. Some mitochondria are in close contact with ATER tubules. The mitochondrial matrix is probably involved in the elaboration of the secretory product, which is extruded and possibly modified by the agranular reticulum. The secretion is sudanophilic, but not osmiophilic. No lipid droplets are found in the cytoplasm. After storage in the strongly dilated subcuticular space, the secretion is released by epicuticular ductules with a diameter of about 120 å.I should like to thank the staff of the EM laboratory of the Zoological Institute for technical assistance, especially the photographer, Mrs. M. Ullmann     

Continuity of the membrane systems in the cells of imaginal discs

Summary Imaginal discs from mature larvae of the fly Calliphora erythrocephala fixed in permanganate and embedded in Epon 812 were observed by electron microscopy. Irrespective of the type of the disc, the endoplasmic reticulum in the cells was found to be in unbroken contact with the outer nuclear membrane, Golgi areas, the mitochondria and the cell membrane. The imaginai discs are syncytial in that all cells are in cytoplasmic communication through secondary breaks in the cell membranes.The investigation was facilitated by grants from the Nordic Insulin Foundation.I am indebted to Mrs. Mariann Carleson and Miss Brita Nilsson for technical aid.     

Isolation and Characterization of Tightly Coupled Mitochondria from Wild Type and nap Mutant Neurospora crassa

A fast and reproducible procedure was elaborated for isolation of tightly coupled mitochondria from wild type and nap mutant Neurospora crassa cells harvested at different growth stages. The isolated mitochondrial preparations had controlled metabolic states and were tightly coupled, i.e., displayed good respiratory control and had close to the theoretically expected maximal ADP/O ratios upon oxidation of Krebs cycle intermediates and exogenous NADH. They contained the fully competent respiratory chain with all three points of energy conservation. Oxidation of all examined substrates by mitochondria from both wild type and mutant cells was mediated by two alternative terminal oxidative systems, albeit to varying extent, with the more pronounced engagement of the alternative oxidase in the stationary growth phase and with a minor contribution of this non-phosphorylating pathway in the substrate oxidation by mutant mitochondria. Oxidation of NAD-dependent substrates by mitochondria from the two cell types was accommodated via both rotenone-sensitive and rotenone-insensitive pathways, while the level of rotenone-insensitive pathway in mutant cells was lower than in wild type cells. It is suggested that a more limited contribution of alternative non-phosphorylating oxidative pathways to the total respiration in mutant cells, as compared with wild type cells, could, at least partially, explain an elevated ATP level in these cells. However, the absence of principal differences in the arrangement of the respiratory chain in mitochondria of wild type and mutant cells implies that the elevated ATP level in the nap mutant is largely related to reduced ATP expenses for transport processes in these cells.     

Ultrastructure and phylogeny of the spermatozoid ofChara vulgaris (Charophyceae)

At maturity, spermatozoids of the green algaChara vulgaris are biflagellated, contain little cytoplasm, and coil for approximately 2 1/2 gyres within the mother cell wall. The anterior of the cell contains an ovoid headpiece anchoring two slightly staggered basal bodies that are positioned above and directly in front of approximately 30 linearly arranged mitochondria. An elongated stellate pattern occupies the transition zone between the BBs and axonemes. Flagella emerge from the cell just in front of the nucleus and encircle the full length of the spermatozoid. The spline comprises a maximum of 38 microtubules surrounding the anterior mitochondria and gradually decreases posteriorly to a minimum of 11. The dense nucleus is narrow, cylindrical, and occupies the central revolution of the cell. Six starch-laden plastids and associated mitochondria are linearly arranged at the cell posterior. Phylogenetic analyses of charalean taxa and archegoniates based on spermatogenesis strongly support the orderCharales, withNitella as the sister group toChara. Diagnostic features ofChara spermatozoids include absence of a lamellar strip and axonemes embedded in the cell for almost the entire length of the anterior mitochondria. Potential relationships amongCharales, Coleochaetales and archegoniates are evaluated in regards to the probable course of evolution of streamlined biflagellated gametes.     

Movement of mitochondria in the ovarian trophic cord of Dysdercus intermedius (Heteroptera) resembles nerve axonal transport

Summary The motile behaviour of mitochondria in the ovarian trophic cord of the red cotton bug, Dysdercus intermedius, was observed optically using video-enhanced differential interference contrast (AVEC-DIC) microscopy. The motion of 258 video-recorded mitochondria was analysed of which 10%–30% were found to move during the observation periods. Of the moving mitochondria 76% travelled towards the oocyte with an average velocity of 3.37 m/ min, and 24% towards the tropharium with 2.84 m/min. The movement was found to be basically of the saltatory type I as known from nerve axons characterized by the absence of directional reversal. In some cases short periods of interrupted motion of type II, i.e. with local oscillations, were observed. Individual mitochondria often showed velocity variations during the excursions. The hemipteran trophic cords are known to contain numerous parallel microtubules. As the observed type of mitochondrial motility resembles axonal transport, a modified transport hypothesis is presented for the microtubule-based motility of organelles in the nurse strands of telotrophic insect ovarioles.     

Gonadotropic cells in the Atlantic salmon,Salmo salar

Summary The gonadotropin-producing cells (GTH-cells) in the Atlantic salmon were studied light and electron microscopically before, during and after spawning, and after injections of luteinizing hormone-releasing hormone (LH-RH). The double immunofluorescent technique was applied using rabbit anti-carp GTH as the first antibody. Numerous immunofluorescent cells were observed throughout the pars distalis, but very few in the pars intermedia. These cells are basophilic and PAS-positive, and ultrastructurally classified as globular gonadotropes. Only one gonadotropic cell type could be identified; its size, morphology and fine structure vary considerably. In the same specimen the GTH-cells can be predominantly globular or vesicular in appearance, depending on the reproductive phase of the fish. At spawning and after LH-RH injection, many GTH-cells reach a vacuolar stage; the content of the vacuoles is not immunofluorescent. Another cell type, which resembles GTH-cells in semithin sections, did not show gonadotropic properties; its nature and functional significance are unknown. In addition, the present study revealed an increase in the synthetic and exocytotic activity of prolactin cells after LH-RH injections. It is suggested that LH-RH mediates this effect via LH and eventually via estradiol.The authors are indebted to Dr. N. Johansson and superintendent N. Steffner for generously providing facilities at their institutes in Älvkarleby. They also wish to thank Mrs. Y. Lilliemarck and Mrs. V. Lundin for their skillful technical assistance. The grants made available by the Swedish Natural Science Research Council (No. 2124-037) are also gratefully acknowledged. Thanks are also due to Mrs. S.M. McNab for linguistic assistance     

Fine structural analysis of the basal epidermal receptor cells in the earthworm (Lumbricus terrestris L.)

Summary A fine structural investigation was performed on receptor cells lying at the base of the epidermis in the earthworm, Lumbricus terrestris. Two types of receptor cells with many similarities, but also with major differences, were discriminated.One receptor is of the microvillar receptor type, that appears to be identical with the photoreceptor cell described earlier by Röhlich et al. (1970). Proximal to the nucleus is a large vacuole (phaosome, Binnenkörper) with many daughter cavities containing a large number of microvilli and several cilia with the 9 × 2 + 0 microtubular pattern. The intracellular cavity has no connection with the surface membrane, in contrast to that in hirudineans (White and Walther, 1969) and pogonophores (Nørrevang, 1974).The other receptor is the ciliated receptor type, that is presently described for the first time. This receptor also has a comparatively large uniform cavity, few microvilli and about 20 cilia with the 9 × 2 + 2 microtubular pattern. The cilia leave the cell in the proximal part through a wide opening, make a turn of 180 °, and proceed toward the epidermal surface. Receptors of a similar type have been described by Golding and Whittle (1975) in the cerebral ganglion of four limicole oligochaete annelids; they presumed that these cells have an osmoreceptor function. The new epidermal receptor type described in the present investigation probably has a chemoreceptor function of hitherto unknown kind.This investigation was supported by the Royal Physiographic Society at Lund, Sweden. The author would like to express his thanks to Mrs. Lena Sandell for skilful technical assistance     

Wall projections in the vegetative cell ofParietaria officinalis L. Pollen

Summary Ultrastructural aspects of pore development inParietaria officinalis L. pollen are described. The main feature is the presence of intine wall projections formed in correspondence to the pores during the late microspore stage and persisting in the shedding pollen. This structure coexists with the tubules occurring in the pore and first appearing as evaginations of the plasma membrane.The morphological evidence and other features such as the occurrence of mitochondria next or in contact with wall projections lead us to compare the pollen vegetative cell to a transfer-cell. The possible functions of these transfer-cell type structures are discussed.     

Development of the olfactory organ in the rainbow fish Nematocentris maccullochi (Atheriniformes,Melanotaeniidae)

Summary The development of the olfactory organ in the rainbow fish, Nematocentris maccullochi, was studied using scanning and transmission electron microscopy; it was compared with the developmental process in other teleosts, especially in the closely related atherinids and cyprinodonts. The formation of the nares parallels that in atherinids, salmonids, cyprinids and heterosomats, but differs from that found in cyprinodonts. Another ontogenetic feature in which the olfactory organs of the rainbow fish and also of atherinids differ from those of cyprinodonts, is the occurrence of transitory kinociliary cells which disappear during the postlarval period. The divergent evolutionary pathways are discussed with reference to experimental investigations. During development, ciliated and microvillous receptor cell types occur. At the primary larval stage ciliated receptor neurons are exclusively present. At a later stage the microvillous type develops and becomes equal in frequency. Thus, the microvillous receptor represents a separate type of olfactory neuron and is not a progenitor of the ciliated receptor cell.This work was supported by the Deutsche ForschungsgemeinschaftThe authors would like to acknowledge Prof. G. Hartmann (Hamburg) and Prof. V. Storch (Kiel) for use of the SEM, Prof. K. Seifert (Neumünster) for valuable discussion. For skillful technical assistance we are indebted to Miss C. Heim, Mrs. K. Hoffmann, and Mr. H.P. Dreyer     

Heart FoF1-ATPase changes during the acute phase of Trypanosoma cruzi infection in rats

The kinetic properties of ATP hydrolysis and synthesis by FoF1-ATPase of heart mitochondria were evaluated during the acute phase of T. cruzi infection in rats. Mitochondria and submitochondrial particles were isolated 7 days (early stage) and 25 days (late stage) following infection of rats with 2 × 10⁵ trypomastigote forms of the Y strain of T. cruzi. The kinetic properties for ATP hydrolysis were altered for the early but not the late stage, showing a changed pH profile, increased K_0.5 values, and a decreased total V_max. The Arrhenius' plot for membrane-associated enzyme showed a higher transition temperature with a lower value for the activation energy in body temperature. For the Triton X-100 - solubilized enzyme, the plot was similar to the control. A decrease in the efficiency of ADP phosphorylation by mitochondria, measured by the firefly-luciferase luminescence, was observed only during the late stage and appeared to be correlated with a decrease in the affinity of the FoF1-ATPase for ADP. It is proposed that in the early stage, during the acute phase of T. cruzi infection in rats, heart FoF1-ATPase undergoes a membrane-dependent conformational change in order to maintain the phosphorylation potential of mitochondria, which would compensate for the uncoupling of mitochondrial function. Also, during both the early and late stages, the enzyme seems to be under the regulation of the endogenous inhibitor protein for the preservation of cellular ATP levels.     

Cell differentiation during early development of Nassarius reticulatus L. (Gastropoda,prosobranchia)

Summary In Nassarius reticulatus the nuclei and nucleoli undergo important morphological changes from the zygote to the 16-cell stage. In the zygote and in the trefoil (2-cell) and 4-cell stage, several agranular, fibrillar nucleolus-like bodies 1 m diameter are present in the interphase nucleus. Granular nucleoli first appear at the 8-cell stage. These nucleoli have fibrillar and granular regions. The granular regions are made up predominantly of ribosome-like osmiophilic granules. From the 16 and 32-cell stage onwards, the one or two spherical nucleoli of each nucleus measure 2.5 m in diameter and show a concentric organization with a very dense central region surrounded by a broad peripheral zone containing numerous granules, possibly of ribonucleoprotein. At the same time the number of ribosome-like particles increase in the karyoplasm and that of ribosomes in the cytoplasm. These findings are surprising because in eggs with radial cleavage, which have been subjected to more detailed analysis, the first granular nucleoli appear after the end of the cleavage, at the blastula or gastrula stage. The early appearance of granular nucleoli which seem to be characteristic of several eggs with spiral cleavage is discussed in connection with biochemical data on RNA-synthesis.This investigation was supported by the Deutsche Forschungsgemeinschaft and the Stiftung VolkswagenwerkWe wish to thank Mrs. C. Mehlis for valuable technical assistance and Prof. J. Bergerard for the excellent working conditions at the Station biologique at Roscoff (France)     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary Morphological changes in the disconnected neuro-intermediate lobe were studied in the lizard, Anolis carolinensis from the 2nd to the 14th post-operative day using a threefold aldehyde fixative (Rodríguez, 1969). Two phases of colour change capacity were exhibited: Phase I started immediately after the transection, lasted for 6 days (mean) and was characterised by an excessive MSH release (brown skin). This phase proceeded gradually into Phase II, designated by an interruption of the MSH release (green skin).The degenerative processes and final elimination of neurons in the disconnected neural lobe propagate in a rostro-caudal direction from the transected area. The aminergic fibres (Type II) disappear within 2 days postoperatively, whereas the degeneration continues for more than 10 days in the peptidergic fibres (Type III, IV and V). The glia cells (ependyma and pituicytes) serve as very active macrophages, engulfing fragments of axons already affected by autolysis and transferring them into glial lysosomes. No apparent morphological changes occur in the shift from Phase I to II.The great majority of the secretory cells of the intermediate lobe are not affected by degenerative processes and appear to be markedly activated by the stalk transection. They exhibit numerous mitochondria, well-developed Golgi complexes forming numerous Golgi vesicles and extensive parallel cisternae of the rough endoplasmic reticulum, sometimes forming large intracisternal droplets (7 m in diameter). Numerous pale vacuoles are seen, especially toward the intact capillaries, suggesting their coupling to the MSH release by extension of the active membrane area toward the perivascular septum. The number of these vacuoles is very markedly reduced in Phase II (no release), whereas the formation of new granules seems to proceed in early stages. The interruption of the MSH release implies a successive refilling of gradually growing secretory granules and a concomitant reduction in the development of the synthetic apparatus. Mechanisms probably involved in the control of the synthesis and release of MSH are discussed.Supported by grants from the Swedish Natural Science Research Council (to P. Meurling) and the Royal Physiographic Society of LundThe authors are indebted to Mrs. Lena Sandell, Mrs. Ingrid Hallberg and Mrs. Kirsten Thörneby for technical assistance and skillful attention of the animals and to Miss Inger Norling for photographic aid.