Lamellar body ultrastructure revisited: high-pressure freezing and cryo-electron microscopy of vitreous sections

Lamellar bodies are the storage sites for lung surfactant within type II alveolar epithelial cells. The structure-function models of lamellar bodies are based on microscopic analyses of chemically fixed tissue. Despite available alternative fixation methods that are less prone to artifacts, such as cryofixation by high-pressure freezing, the nature of the lung, being mostly air filled, makes it difficult to take advantage of these improved methods. In this paper, we propose a new approach and show for the first time the ultrastructure of intracellular lamellar bodies based on cryo-electron microscopy of vitreous sections in the range of nanometer resolution. Thus, unspoiled by chemical fixation, dehydration and contrasting agents, a close to native structure is revealed. Our approach uses perfluorocarbon to substitute the air in the alveoli. Lung tissue was subsequently high-pressure frozen, cryosectioned and observed in a cryo-electron microscope. The lamellar bodies clearly show a tight lamellar morphology. The periodicity of these lamellae was 7.3 nm. Lamellar bifurcations were observed in our cryosections. The technical approach described in this paper allows the examination of the native cellular ultrastructure of the surfactant system under near in vivo conditions, and therefore opens up prospectives for scrutinizing various theories of lamellar body biogenesis, exocytosis and recycling.

Structural alterations in adult rat carotid bodies exposed to hyperbaric oxygenation

Inhibition of carotid body (CB) function is the main mechanism involved in the attenuation of respiratory drive observed during hyperoxia However, only a few studies at 5 0 atmospheres absolutes (ATA) have analyzed carotid body structure or function in hyperbaric oxygenation (HBO(2)) situations We hypothesized that rats will present CB structural alterations when exposed to different lower hyperbaric oxygen doses enough to alter their chemosensory response to hypoxia Methods - Twenty-one adult male Wistar rats, divided into three groups, were maintained in room air or exposed to O(2) at 2 4 or 3 0 ATA for six hours Histological, ultrastructural and immunohistochemical analyses for neuronal nitric oxide synthase (nNOS) and F2-isoprostane were performed in the excised CBs Results - Histological analyses revealed signs of intracellular edema in animals exposed to both conditions, but this was more marked in the 3 0 ATA group, which showed ultrastructural alterations at the mitochondrial level There was a significant increase in the volume density of intraglomic-congested capillaries in the 3 0 ATA group associated with an arteriolar vasoconstriction In the 2 4 ATA group, there was a relative increase of glomic light cells and a decrease of glomic progenitor cells Additionally, there was a stronger immunoreactivity for F2-isoprostane in the 3 0 ATA O(2)-exposed carotid bodies The glomic cells stained positive for nNOS, but no difference was observed between the groups Our results show that high 02 exposures may induce structural alterations in glomic cells with signs of lipid peroxidation We further suggest that deviation of blood flow toward intraglomic capillaries occurs in hyperbaric hyperoxia

Ultrastructure of last larval instar fat body cells of Pachycondyla (= Neoponera) villosa (Formicidae : Ponerinae): cytochemical and chemical analysis

The ultrastructure of the fat body cells (trophocytes) of the last larval instar of Pachycondyla (= Neoponera) villosa is presented. The cytoplasm is restricted to the cell periphery and to the smaller strips among the vacuoles, protein granules, lipid droplets, and around the nucleus. Cytochemically, the presence of basic amino acids in the protein granules and in the nuclei was observed by using the ethanolic phosphotungstic acid technique (EPTA). The lipid droplets stained for unsaturated lipids. This result was further confirmed by gas chromatography and mass spectrometry, where the unsaturated fatty acids were identified as oleic and linoleic acids together with saturated fatty acids such as palmitic and stearic acid. Carbohydrates (glycogen) were also detected in the fat body. The glycogen is present as beta particles distributed among the lipid droplets and sometimes attached to them.

Spermatozoal ultrastructure of spiny oysters (Spondylidae, Bivalvia) including a comparison with other bivalves

Sperm ultrastructure in three representative species of the marine bivalve family Spondylidae (spiny or thorny oysters) is examined and compared with available data on other bivalves, especially other families of the subclass Pteriomorphia. Spondylid spermatozoa are of the externally fertilizing aquasperm. type (ect-aquasperm). The acrosomal vesicle is conical with a deep basal invagination extending almost the full length of the vesicle. Vesicle contents are divisible into an inner, highly electron-dense anterior layer and a less dense posterior layer. The anterior layer is folded back on itself posteriorly and exhibits radiating plates (best developed peripherally). The vesicle rests on, and is partially embedded in, an extensive granular deposit of subacrosomal. material at the nuclear apex. This deposit extends partly into acrosomal vesicle invagination and also fills a broad depression in the anterior of the nucleus. No pre-formed axial rod (perforatorium) is present. The nucleus is round-pyriform and its contents coarsely fibrogranular. At the base of the nucleus, four broad depressions partially accommodate the midpiece mitochondria. The midpiece consists the four spherical mitochondria and the proximal and distal centrioles. The centrioles are arranged at approximately 90degrees to each other, and each consists of nine, angularly-oriented, microtubular triplets embedded in a granular matrix. A short, periodically banded rootlet connects the proximal centriole to the nuclear fossa, whereas the distal centriole, which forms the basal body to the flagellar axoneme, is anchored to the plasma membrane by nine terminally forked satellite fibres. Extensive deposits of putative glycogen rosettes surround the centrioles and mitochondria. The flagellum consists of a 9+2 axoneme sheathed by the plasma membrane. Spondylid spermatozoa strongly resemble those of the Pectinidae, further confirming the traditional view (based on comparative anatomy and shell morphology) of a close relationship between the Spondylidae and the Pectinidae. Differences in acrosomal shape and dimensions were noted between the three species examined, indicating potential taxonomic utility for comparative sperm ultrastructure within the Spondylidae.

Comparative sperm ultrastructure in five genera of the nudibranch family Chromodorididae (Gastropoda : Opisthobranchia)

Sperm ultrastructure is examined in representatives of five genera of the nudibranch gastropod family Chromodorididae: (Chromodoris, Hypselodoris, Glossodoris, Risbecia and Pectenodoris) and the results compared with previous work on other gastropods, especially other nudibranchs. As chromodoridid phylogeny is still incompletely understood, this study partly focuses on the search for new and as yet untapped sources of informative characters. Like spermatozoa of most other heterobranch gastropods, those of the Chromodorididae are elongate, complex cells composed of an acrosomal complex (small, rounded acrosomal vesicle, and columnar acrosomal pedestal), a condensed nucleus, sub-nuclear ring, a highly modified mid-piece (axoneme + coarse fibres surrounded by a glycogen-containing, helically-coiled mitochondrial derivative) and terminally a glycogen piece (or homologue thereof). The finely striated acrosomal pedestal is a synapomorphy of all genera examined here, but interestingly also occurs in at least one dorid (Rostanga arbutus). Substantial and potentially taxonomically informative differences were also observed between genera in the morphology of the nucleus, the neck region of the mid-piece, and also the terminal glycogen piece. The subnuclear ring is shown for the first time to be a segmented, rather than a continuous structure; similarly, the annular complex is shown to consist of two structures, the annulus proper and the herein-termed annular accessory body.

Is the coiled body involved in nucleolar functions?

Coiled bodies (CBs) are structural constituents observed in nuclei of most eukaryotic cells. They usually occur in the nucleoplasm as well as in contact with the nucleolar surface. In this work we studied the hepatocyte nuclei of hibernating dormice in order to investigate possible modifications of CBs along the seasonal cycle. CBs were abundant during hibernation and rapidly disappeared upon arousal from hibernation. Moreover, CBs were frequently found to be integrated into the nucleolar body. Immunocytochemical analyses showed that CBs contain nucleoplasmic as well as nucleolar RNA-processing factors, suggesting an "ambiguous" role for this organelle in the nuclear functions.

Ultrastructure of Endogenous Stages of Eimeria ninakohlyakimovae Yakimoff & Rastegaieff, 1930 Emend. Levine, 1961 in Experimentally Infected Goat

The ultrastructure of endogenous stages of Eimeria ninakohlyakimovae was observed in epithelial cells of cecum and colon crypts from a goat experimentally infected with 2.0 x 105 oocysts/kg. The secondary meronts developed above the nucleus of the host cell. The nucleus first divides and merozoites then form on the surface of multinucleated meronts. Free merozoites in the parasitophorous vacuole present a conoid, double membrane, one pair of rhoptries, micronemes, micropore, anterior and posterior polar ring, a nucleus with a nucleolus and peripheral chromatin. The microgamonts are located below the nucleus of the host cell and contain several nuclei at the periphery of the parasite. The microgametes consist of a body, a nucleus, three flagella and mitochondria. The macrogamonts develop below the nucleus of the host cell and have a large nucleus with a prominent nucleolus. The macrogametes contain a nucleus, wall-forming bodies of type I and type II. The young oocysts present a wall containing two layers and a sporont

Ultrastructure of spermatogenesis and sperm development in Saccocoelioides godoyi Kohn & Froes, 1986 (Digenea, Haploporidae)

Ultrastructural observations of spermatogenesis and sperm development of Saccocoelioides godoyi, an intestinal parasite of Leporinus friderici (Bloch, 1794) are described. The irregular-shaped spermatogonia form a peripheral layer, and show a prominent nucleus. Spermatocytes are larger than spermatogonia, and in the early stage present synaptonemal complex. Spermatids show nuclei smaller than the spermatocytes. Spermiogenesis is characterized by outgrowth of the zone of differentiation, presenting basal bodies, separated by an intercentriolar body. At the end of this process, the spermatozoa are released into the residual cytoplasmic mass. The spermatozoa of S. godoyi are elongate, similar to the pattern described for other Digenea, showing nuclei, mitochondria and two axonemes with the 9+1 configuration. The peripheral cortical microtubules on the dorsal and ventral faces are laterally interrupted.

Ultrastructure and chaetotaxy of sensory eeceptors in the cercaria of a species of Allopodocotyle Pritchard, 1966 (Digenea: Opecoelidae)

Previous investigations of sensory systems in opecoelid cercariae have focused on chaetotaxy and ultrastructure of sensory receptors. They revealed chaetotaxic patterns within family, genus, and species as well as different receptors. Chaetotaxic and ultrastructural observations have rarely been combined. We investigated the ultrastructure of cercarial sensory receptors in conjunction with chaetotaxy and neuromorphology in a species of Allopodocotyle. Cercariae were treated with acetylthiocholine iodide and silver nitrate, and some were processed for light, scanning (SEM), and transmission (TEM) electron microscopy. Five nerve regions were distinguished. Chaetotaxy was consistent with that of other opecoelids. Five types of receptors were distinguished with SEM. Types differed in number of cilium-like structures (one or more), length of cilium-like structure (short, moderately long, or long), presence or absence of a tegumentary collar, and length of tegumentary collar (low, moderately low, or very high). Internal ultrastructure of some types revealed unsheathed cilium-like structures, basal body, and thickened nerve collars. Possible subtegumentary and sheathed receptors are introduced. Some receptor types were site-specific. For example, receptors with multiple cilium-like structures were concentrated on cephalic region whereas receptors with short cilium-like structure were widespread throughout most regions. Ultrastructure and site-specificity observations suggest that most receptors are mechanoreceptors.

Histophatology and ultrastructure of Henneguya caudalongula sp. n. infecting Prochilodus lineatus (Pisces: Prochilodontidae) cultivated in the state of São Paulo, Brazil

The histological and ultrastructural characteristics of a new species of Henneguya and the host reactions to infection by this species are reported. Henneguya caudalongula sp. n. was found in the inter and intralamellar regions of the gills of Prochilodus lineatus (Valenciennes, 1836) cultivated at Center for the Research and Management of Continental Fishing Resources located in the municipality of Pirassununga, state of São Paulo, Brazil. The plasmodia were white and round or ellipsoidal and measured 0.2 to 1 mm in length. The development of the parasite was asynchronous and the mature spores were fusiform, with a total length 71 ± 1.4 µm, body length of 16.6 ± 0.54 µm and width 4.6 ± 0.2 µm. The caudal process was 52.6 ± 1.5 µm long. The polar capsules were elongate (length 6.1 ± 0.19 µm, width 1.6 ± 0.15 µm) and of equal size. The polar filament was coiled in 10-11 turns. The prevalence of the parasite was 48.3% and did not vary significantly with the season or host size.

Spermiogenesis and spermatozoon ultrastructure of the bothriocephalidean cestode Clestobothrium crassiceps (Rudolphi, 1819), a parasite of the teleost fish Merluccius merluccius (Gadiformes: Merlucciidae)

Spermiogenesis and the ultrastructure of the spermatozoon of the bothriocephalidean cestode Clestobothrium crassiceps (Rudolphi, 1819), a parasite of the teleost fish Merluccius merluccius (Linnaeus, 1758), have been studied by means of transmission electron microscopy. Spermiogenesis involves firstly the formation of a differentiation zone. It is characterized by the presence of two centrioles associated with striated rootlets, an intercentriolar body and an electron-dense material in the apical region of this zone. Later, two flagella develop from the centrioles, growing orthogonally in relation to the median cytoplasmic process. Flagella then undergo a rotation of 90° until they become parallel to the median cytoplasmic process, followed by the proximodistal fusion of the flagella with the median cytoplasmic process. The nucleus elongates and afterwards it migrates along the spermatid body. Spermiogenesis finishes with the appearance of the apical cone surrounded by the single helical crested body at the base of the spermatid. Finally, the narrowing of the ring of arched membranes detaches the fully formed spermatozoon. The mature spermatozoon of C. crassiceps is filiform and contains two axonemes of the 9 + '1' trepaxonematan pattern, a parallel nucleus, parallel cortical microtubules, and electron-dense granules of glycogen. The anterior extremity of the gamete exhibits a short electron-dense apical cone and one crested body, which turns once around the sperm cell. The first axoneme is surrounded by a ring of thick cortical microtubules that persist until the appearance of the second axoneme. Later, these thick cortical microtubules disappear and thus, the mature spermatozoon exhibits two bundles of thin cortical microtubules. The posterior extremity of the male gamete presents only the nucleus. Results are discussed and compared particularly with the available ultrastructural data on the former 'pseudophyllideans'. Two differences can be established between spermatozoa of Bothriocephalidea and Diphyllobothriidea, the type of spermatozoon (II vs I) and the presence/absence of the ring of cortical microtubules.

Spermiogenesis and spermatozoon ultrastructure of the bothriocephalidean cestode Clestobothrium crassiceps (Rudolphi, 1819), a parasite of the teleost fish Merluccius merluccius (Gadiformes: Merlucciidae)

Spermiogenesis and the ultrastructure of the spermatozoon of the bothriocephalidean cestode Clestobothrium crassiceps (Rudolphi, 1819), a parasite of the teleost fish Merluccius merluccius (Linnaeus, 1758), have been studied by means of transmission electron microscopy. Spermiogenesis involves firstly the formation of a differentiation zone. It is characterized by the presence of two centrioles associated with striated rootlets, an intercentriolar body and an electron-dense material in the apical region of this zone. Later, two flagella develop from the centrioles, growing orthogonally in relation to the median cytoplasmic process. Flagella then undergo a rotation of 90° until they become parallel to the median cytoplasmic process, followed by the proximodistal fusion of the flagella with the median cytoplasmic process. The nucleus elongates and afterwards it migrates along the spermatid body. Spermiogenesis finishes with the appearance of the apical cone surrounded by the single helical crested body at the base of the spermatid. Finally, the narrowing of the ring of arched membranes detaches the fully formed spermatozoon. The mature spermatozoon of C. crassiceps is filiform and contains two axonemes of the 9 + '1' trepaxonematan pattern, a parallel nucleus, parallel cortical microtubules, and electron-dense granules of glycogen. The anterior extremity of the gamete exhibits a short electron-dense apical cone and one crested body, which turns once around the sperm cell. The first axoneme is surrounded by a ring of thick cortical microtubules that persist until the appearance of the second axoneme. Later, these thick cortical microtubules disappear and thus, the mature spermatozoon exhibits two bundles of thin cortical microtubules. The posterior extremity of the male gamete presents only the nucleus. Results are discussed and compared particularly with the available ultrastructural data on the former 'pseudophyllideans'. Two differences can be established between spermatozoa of Bothriocephalidea and Diphyllobothriidea, the type of spermatozoon (II vs I) and the presence/absence of the ring of cortical microtubules.

Spermiogenesis and the spermatozoon ultrastructure of Robphildollfusium fractum (Digenea: Gyliauchenidae), an intestinal parasite of Sarpa salpa (Pisces: Teleostei)

Spermiogenesis in Robphildollfusium fractum begins with the formation of a differentiation zone containing: two centrioles, each bearing striated rootlets, nucleus, several mitochondria and an intercentriolar body constituted by seven electron-dense layers. The two centrioles originate two free flagella growing orthogonally to the median cytoplasmic process. Later, the free flagella rotate and undergo proximodistal fusion with the median cytoplasmic process. Nuclear and mitochondrial migrations occur before this proximodistal fusion. Finally, the young spermatozoon detaches from the residual cytoplasm after the constriction of the ring of arched membranes. The spermatozoon of R. fractum exhibits two axonemes of different length of the 9 +"1" trepaxonematan pattern, nucleus, two mitochondria, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. Additionally, a shorter axoneme, which does not reach the nuclear region, the presence of an electron-dense material in the anterior spermatozoon extremity and the morphologies of both spermatozoon extremities characterize the mature sperm of R. fractum.

Spermiogenesis and ultrastructure of the spermatozoon of Wardula capitellata (Digenea, Mesometridae), an intestinal parasite of the sparid teleost Sarpa salpa in Senegal

The spermiogenesis process in Wardula capitellata begins with the formation of a differentiation zone containing two centrioles associated with striated rootlets and an intercentriolar body. Each centriole develops into a free flagellum orthogonal to a median cytoplasmic process. Later these flagella rotate and become parallel to the median cytoplasmic process, which already exhibits two electron-dense areas and spinelike bodies before its proximodistal fusion with the flagella. The final stage of the spermiogenesis is characterized by the constriction of the ring of arched membranes, giving rise to the young spermatozoon, which detaches from the residual cytoplasm. The mature spermatozoon of W. capitellata presents most of the classical characters reported in digenean spermatozoa such as two axonemes of different lengths of the 9 + '1' trepaxonematan pattern, nucleus, mitochondrion, two bundles of parallel cortical microtubules and granules of glycogen. However, some peculiarities such as two lateral expansions accompanied by external ornamentation of the plasma membrane and spinelike bodies characterize the mature sperm. Moreover, a new spermatological character is described for the first time, the so-called cytoplasmic ornamented buttons.

Spermiogenesis and the spermatozoon ultrastructure of Robphildollfusium fractum (Digenea: Gyliauchenidae), an intestinal parasite of Sarpa salpa (Pisces: Teleostei)

Spermiogenesis in Robphildollfusium fractum begins with the formation of a differentiation zone containing: two centrioles, each bearing striated rootlets, nucleus, several mitochondria and an intercentriolar body constituted by seven electron-dense layers. The two centrioles originate two free flagella growing orthogonally to the median cytoplasmic process. Later, the free flagella rotate and undergo proximodistal fusion with the median cytoplasmic process. Nuclear and mitochondrial migrations occur before this proximodistal fusion. Finally, the young spermatozoon detaches from the residual cytoplasm after the constriction of the ring of arched membranes. The spermatozoon of R. fractum exhibits two axonemes of different length of the 9 +"1" trepaxonematan pattern, nucleus, two mitochondria, two bundles of parallel cortical microtubules, external ornamentation of the plasma membrane, spine-like bodies and granules of glycogen. Additionally, a shorter axoneme, which does not reach the nuclear region, the presence of an electron-dense material in the anterior spermatozoon extremity and the morphologies of both spermatozoon extremities characterize the mature sperm of R. fractum.