The olfactory organ of the trout Salmo trutta fario: A novel localization for a progestin receptor

A progestin receptor (PR) has been detected in the olfactory organ of the trout Salmo trutta fario. The specificity of this receptor was high for 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17α,20β‐DP), but it also bound 17α‐hydroxy‐progesterone (17α‐OHP) and 21‐hydroxyprogesterone (21‐OHP), even when present at low concentrations (10‐fold in relative binding affinity assay). Progesterone (P) competed effectively at much higher concentrations (1,000‐fold in relative binding affinity assay). Immunohistochemical studies carried out with three different monoclonal antibodies against human progesterone receptor (hPR), chicken progesterone receptor hinge region (cPR), and chicken progesterone receptor A/B domain (PR22), revealed that immunoreactivity was present in the epithelium of the olfactory organ of females and males of the trout Salmo trutta fario only against hPR. Western blotting showed two hPR immunoreactive bands of about 62 and 66 kDa. Finally, a portion of the cDNA of about 300 nucleotides extending over the DNA binding domain and the ligand binding domain was cloned and sequenced, revealing a high degree of sequence homology of the PR in Salmo trutta fario with the PR in other teleosts. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

The hepatocytes of the brown trout (Salmo trutta fario): A stereological study of some cytoplasmic components with the breeding cycle

Sex differences exist in fish hepatocytes, but studies for characterizing their cytology throughout the breeding cycle are still scarce; suggesting changes, but most lacking quantitative data. To address this limitation, to complement baseline data generated from the brown trout model, and to prove that sex‐specific seasonal changes exist, we made an unbiased stereological evaluation of the hepatocytic cytoplasm. Unprecedentedly for fish liver, the stereological design was exempt from model (biased) assumptions. Five (3 years old) animals per sex were studied in endogenous vitellogenesis, exogenous vitellogenesis, and spawning season end. Liver pieces for analysis were systematically sampled. Stereology was done in transmission electron microscopy (TEM) micrographs. Primary data generated relative volume estimates of the major cytoplasmic components. Such values were used for deriving absolute volumes (per cell and per liver). Lipid droplets did not show changes. As to other targets, trends at cell and liver levels were not always equal. If the hepatocyte was the reference space, the contents in mitochondria, dense bodies, glycogen, and cytosol changed seasonally, in both sexes. If taking the liver as the reference, changes attained the Golgi apparatus and rough endoplasmic reticulum (RER), besides dense bodies, glycogen (in females), and cytosol. The components volumes (namely per liver) were often positively (negatively for glycogen) correlated with the ovary weight, disclosing new associations and implications in fish. While also offering gold‐standard data for backing morphofunctional correlations and pathology, we revealed a new process by which females increase the amount of RER and Golgi throughout vitellogenesis, breaking from the idea on how this event happens in fish. Microsc. Res. Tech. 73:766–778, 2010. © 2010 Wiley‐Liss, Inc.     

Ultrastructural study on the body surface of the acanthocephalan parasite Dentitruncus truttae in brown trout

Scanning electron microscope (SEM) investigations on the holdfast elements, proboscis hooks, and trunk spines of Dentitruncus truttae (Acanthocephala, Palaeacanthocephala), an endoparasite of Salmo trutta (brown trout), provide more data about the surface of these taxonomic relevant structures. In both acanthocephalan sexes, the fully everted cylindrical proboscis possessed 18 longitudinal rows of hooks with 18 hooks per row (rarely 19-20). Hook length varied according to position on the proboscis; apical hooks were 40-52 microm long, middle hooks were 31.7-36.6 microm, and basal hooks were 38.1-40 microm. Starting from the anterior end of the metasoma, numerous cuticular spines (26.7-30 microm in length) were visible and their number progressively decreased posteriorly. SEM observations of D. truttae hooks and spines revealed the presence of many surface striations on each proboscis hook. These surface striations were absent from trunk spines. From the base of the hook, the striations ran parallel toward the point of convergence. Additionally, survey of longitudinal and transversal sections of the hook using transmission electron microscope confirmed that the hook surface was not smooth. SEM comparison with the hooks of several palaeacanthocephalan species, as well as with the hooks of species belonging to Eoacanthocephala and Polyacanthocephala, indicated that the striations are currently exclusive to D. truttae proboscis hooks.     

Ultrastructural histopathology of human olfactory dysfunction.

This paper presents electron-microscopic observations on biopsies of the olfactory mucosae of several classes of patients with smell disorders: 1) patients with loss of smell function following head injury (post-traumatic anosmics or hyposmics); 2) patients with loss of smell function following severe head colds and/or sinus infections (post-viral olfactory dysfunction, or PVOD); and 3) patients that have lacked smell function since birth (congenital anosmics). Of these, the traumatic anosmics' olfactory epithelia were quite disorganized; the orderly arrangement of supporting cells, ciliated olfactory receptor neurons, microvillar cells, and basal cells was disrupted. Although many somata of ciliated olfactory receptors were present, few of their dendrites reached the epithelial surface. The few olfactory vesicles present usually lacked olfactory cilia. The post-viral anosmics, too, had a greatly reduced number of intact ciliated olfactory receptor neurons, and most of those present were aciliate. The post-viral hyposmics had a larger population of intact, ciliated olfactory receptor cells. In the seven cases of congenital anosmia studied, no biopsies of olfactory epithelium were obtained, indicating the olfactory epithelium is either absent--or greatly reduced in area--in these individuals.     

Ultrastructural changes of the olfactory bulb in manganesetreated mice

The effect of manganese toxicity on the ultrastructure of the olfactory bulb was evaluated. Male albino mice were injected intraperitoneally with MnCl₂ (5 mg/Kg/day) five days per week during nine weeks. The control group received NaCl (0.9%). The olfactory bulbs of five mice from each group were processed for transmission electron microscopy after 2, 4, 6 and 9 weeks of manganese treatment. On week 2, some disorganization of the myelin sheaths was observed. After 4 weeks, degenerated neurons with dilated cisternae of rough endoplasmic reticulum and swollen mitochondria appeared. A certain degree of gliosis with a predominance of astrocytes with swollen mitochondria, disorganization of the endomembrane system, dilation of the perinuclear cisternae and irregularly shaped nuclei with abnormal chromatin distribution were observed after 6 weeks. Some glial cells showed disorganization of the Golgi apparatus. On week 9, an increase in the number of astrocytes, whose mitochondrial cristae were partially or totally erased, and a dilation of the rough endoplasmic reticulum were found. Neurons appear degenerated, with swollen mitochondria and a vacuolated, electron dense cytoplasm. These changes seem to indicate that the olfactory bulb is sensitive to the toxic effects of manganese.     

Fine structural aspects of secretion and extrinsic innervation in the olfactory mucosa.

The mucus at the surface of the olfactory mucosa constitutes the milieu in which perireceptor events associated with olfactory transduction occur. In this review, the ultrastructure of olfactory mucus and of the secretory cells that synthesize and secrete olfactory mucus in the vertebrate olfactory mucosa is described. Bowman's glands are present in the olfactory mucosa of all vertebrates except fish. They consist of acini, which may contain mucous or serous cells or both, and ducts that traverse the olfactory epithelium to deliver secretions to the epithelial surface. Sustentacular cells are present in the olfactory epithelium of all vertebrates. In fish, amphibia, reptiles, and birds, they are secretory; in mammals, they generally are considered to be "non-secretory," although they may participate in the regulation of the mucous composition through micropinocytotic secretion and uptake. Goblet cells occur in the olfactory epithelium of fish and secrete a mucous product. Secretion from Bowman's glands and vasomotor activity in the olfactory mucosa are regulated by neural elements extrinsic to the primary olfactory neurons. Nerve fibers described in early anatomical studies and characterized by immunohistochemical studies contain a variety of neuroactive peptides and have several targets within the olfactory mucosa. Ultrastructural studies of nerve terminals in the olfactory mucosa have demonstrated the presence of adrenergic, cholinergic and peptidergic input to glands, blood vessels, and melanocytes in the lamina propria and of peptidergic terminals in the olfactory epithelium. The neural origins of the extrinsic nerve fibers and terminals are the trigeminal, terminal, and autonomic systems.     

Ultrastructural studies of microtubules and microtubule organizing centers of the vertebrate olfactory neuron.

The olfactory neuron is specialized along its length into highly determined morphological regions. These regions include the dendritic cilia, dendritic vesicle, dendritic shaft proper, perikaryon, axon, zone of transition where the axon widens as it approaches its termination, and the axon terminal. Except for the zone of transition and the terminal, characteristic populations of microtubules occur in these compartments. In the olfactory vesicle, three discrete microtubule organizing centers (MTOCs) nucleate microtubules: the basal body, the lateral foot associated with the body, and dense masses of nearby material. Little is known about MTOCs elsewhere in the neuron, although the polarity of the axonal microtubules indicate that they originate at or near the perikaryon. An attempt is made to summarize what is known of the origin, structure, distribution, and function of microtubules in vertebrate olfactory neurons, which are useful model systems in which to study microtubules. Information about olfactory neuron microtubules may be applicable to neurons in general (e.g., the discovery that axons contain microtubules of uniform polarity was first made in the olfactory neuron) or to microtubules in other eukaryotic cells.     

Ciliated and microvillar receptor cells degenerate and then differentiate in the olfactory epithelium of rainbow trout following olfactory nerve section.

We used scanning (SEM) and transmission (TEM) electron microscopy to examine ultrastructural changes in the olfactory epithelium (OE) of rainbow trout following unilateral olfactory nerve section. Both ciliated receptor cells (CRC) and microvillar receptor cells (MRC) degenerated and subsequently differentiated from unidentified precursor cells. The following changes took place in fish that were held at 10 degrees C at the stated period following olfactory nerve section: on day 7, MRC and CRC contained intracellular vacuoles; on day 12, the olfactory knobs appeared disrupted; by day 26, olfactory receptor cells were absent from the OE; on day 42, there were receptor cell bodies and a few CRC with short cilia at the apical surface; and on day 55, a small number of both CRC and MRC had differentiated. By day 76, both CRC and MRC repopulated the OE. Degenerative changes in the cytoplasm of the sustentacular cells (SC) and ciliated nonsensory cells (CNC) were observed in the first 26 days following olfactory nerve section, but these cells remained intact throughout the experiment. The degeneration and subsequent differentiation of CRC and MRC supports and extends previous observations that both cell types are olfactory receptor neurons with axons that extend along the olfactory nerve to the olfactory bulb.     

Changes in the amount of kidney pigmented macrophage aggregates throughout the breeding cycle of female Ohrid trout, Salmo letnica Kar. (Teleostei, Salmonidae)

Changes in fish macrophages (Macs) are useful indicators of environmental pressures, but responses due to chemical and nonchemical stresses may be confounded by natural sources of variability. These may include sex and gonadal stage. In this study, we start addressing the following question: is the seasonally dependent ovary stage a factor to consider when using kidney Macs as biomarkers? To tackle this problem, the relative amount of pigmented Macs in kidney (head, trunk, and tail portions) was stereologically estimated in Ohrid trout, and related with the breeding status. The amount of Macs significantly increased from pre vitellogenesis to late vitellogenesis and showed a decreasing trend then after, with lower values noted after spawning in the head (1.9% versus 7.5% versus 2.0%), trunk (1.8% versus 7.5% versus 2.5%), and tail (2.5% versus 6.7% versus 2.9%) kidney. The decrease seen at spawning was significant in head and trunk kidney, and at post spawning it was significant for all kidney portions. The amounts of Macs were positively correlated with the ovary relative weights and plasma estradiol levels. We proved for the first time that fish kidney pigmented Macs can vary in amount during the breeding cycle. Our data, combined with literature, strongly support that the sex‐steroid profile and kidney status‐seasonal remodeling both influence the Macs pool; likely not only in female trout. So, while increases in Macs may warn of ecosystem problems, we show that using kidney Macs for biomonitoring should also take into account seasonally, particularly that related with ovary maturation. Microsc. Res. Tech. 2011. © 2011 Wiley Periodicals, Inc.     

A qualitative and quantitative study of the hepatic pigmented macrophage aggregates during the breeding cycle of Ohrid trout, Salmo letnica Kar. (Teloestei, Salmonidae)

It is well known that factors such as aging and water quality may influence pigmented macrophages (PMacs) in fishes, but it is not established yet if PMacs undergo seasonal and breeding dependent variations. This study explored this caveat and reports qualitative histological and stereological data on liver PMacs from wild female Ohrid trout, Salmo letnica, during the annual breeding cycle. Data showed that a minority of PMacs contained melanin and that the vast majority of them contained only hemosiderin or hemosiderin and lipofuscin/ceroid pigment. It was suggested that this is the normal scenario for the species. One remarkable result was the demonstration of a striking increase, after spawning, of the relative and total volumes of the hepatic macrophages, both parenchyma and stroma located. Because the melano PMacs did not vary, those changes were due to fluctuations in the hemosiderin-laden PMacs. We concluded that Ohrid trout presented a pigment composition in liver macrophages that differed from other fish, including salmonids, where most liver phagocytes essentially display melanin. Our quantitative data support interspecies differences in the amount of liver macrophages and also that after spawning expansion of the macrophage pool is crucial and most likely connected with the needs of liver remodeling (leading to a decrease in hepatic mass). So, we suggested that the hormonal (sex steroidal) constellation influenced the liver macrophage pool. Additionally, we proved that the use of fish liver macrophages for biomonitoring should take into account the considerable natural breeding/seasonal dependent variations that are expected to occur.     

Ultrastructural effects of the high molecular weight cryoprotectants Dextran and polyvinyl pyrrolidone on liver and brown adipose tissue in vitro

The ultrastructural changes caused by incubation of rat liver and brown fat in buffered solutions of high molecular weight cryoprotectives (Dextran and polyvinyl pyrrolidone) at high concentrations (up to 25% w/v) have been examined. Under appropriate conditions of incubation rather small qualitative changes were found. Hepatocytes showed some signs of plasmolysis. Evidence for the endocytosis of Dextran and polyvinyl pyrrolidone was obtained. Cryoultramicrotomy of unfixed, quench-frozen specimens after the same incubation procedures showed good cutting properties and only slight ice-crystal damage. Several of the larger tissue compartments were recognizable in such sections. Although high molecular weight cryoprotectants do penetrate cells by endocytosis (and these effects require further functional evaluation), the present observations provide further support for the idea that such compounds significantly improve the quench-freezing of biological specimens and offer a practical way forward for the preparation of material for X-ray microanalysis of diffusible elements.     

Recent progress in the neurobiology of the vomeronasal organ

In many terrestrial tetrapodes, a pair of vomeronasal organs (VNOs), which are chemosensory apparatuses, are situated at the base of the nasal septum in the anterior nasal cavity. The purposes of this review are to summarize comparative neuroanatomy and to introduce recent progress in neurobiological studies of the VNO. Five types of VNOs can be identifiable in terms of anatomical organization; snakes possess the most complex one. Sensory cells in the VNO, vomeronasal receptor neurons (VRNs), are located in its neuroepithelium, vomeronassal sensory epithelium. The VRNs retain the characteristic of epithelial cells in that they are born continuously from progenitor cells. They contain two prominent subcellular structures: microvilli and extraordinarily large amounts of smooth endoplasmic reticulum and a few unique glycoconjugates. The VRNs express two types of G-protein -subunits: Gi(alpha2) and Go(alpha) and each of them is coupled with putative pheromone receptors, V1Rs and V2Rs, respectively. Recent physiological and biochemical studies have demonstrated that pheromones depolarize the V1R-Gi(alpha2) and V2R-Go(alpha) VRNs via IP(3)-mediated mechanisms. The VRNs do not show adaptation and are ultrasensitive to putative pheromones. Other than being a chemosensory organ, the VNO and its primordium might play important roles for brain development; hypothalamic neurons that produce gonadotropin-releasing hormone are born in the VNO primordium and a few other neuron-like cells may be born in the VNO primordium and VNO. In human fetuses, anatomical findings strongly suggest that their VNOs contain a neuroepithelium. By contrast, it is unlikely that adult human VNO serves as a chemosensory organ.     

Drosophila as a focus in olfactory research: mapping of olfactory sensilla by fine structure, odor specificity, odorant receptor expression, and central connectivity.

This review intends to integrate recent data from the Drosophila olfactory system into an up-to-date account of the neuronal basis of olfaction. It focuses on (1) an electron microscopic study that mapped a large proportion of fruitfly olfactory sensilla, (2) large-scale electrophysiological recordings that allowed the classification of the odor response spectra of a complete set of sensilla, (3) the identification and expression patterns of candidate odorant receptors in the olfactory tissues, (4) central projections of neurons expressing a given odorant receptor, (5) an improved glomerular map of the olfactory center, and (6) attempts to exploit the larval olfactory system as a model of reduced cellular complexity. These studies find surprising parallels between the olfactory systems of flies and mammals, and thus underline the usefulness of the fruitfly as an olfactory model system. Both in Drosophila and in mammals, odorant receptor neurons appear to express only one type of receptor. Neurons expressing a given receptor are scattered in the olfactory tissues but their afferents converge onto a few target glomeruli only. This suggests that in both phyla, the periphery is represented in the brain as a chemotopic map. The major difference between mammals and fruitflies refers to the numbers of receptors, neurons, and glomeruli, which are largely reduced in the latter, and particularly in larvae. Yet, if activated in a combinatorial fashion, even this small set of elements could allow discrimination between a vast array of odorants.     

Ultrastructural studies on membrane, cytoskeletal, mucous, and protective compartments in olfaction.

There is a great variety in the morphological appearance of olfactory structures across the metazoan animal kingdom. Despite this variety the receptive structures themselves have a strikingly similar architecture, namely some type of elongated cellular extension that is spanned by a membrane and surrounded by mucus. These cellular extensions can either be modified primary or secondary cilia, or microvilli. There are more similarities between membranes of these extensions than between the cytoskeletal elements immediately underneath the membranes. One might infer that the cytoskeletal elements of the cellular extensions merely serve as a scaffold for the membranes, whereas the similarity in membrane ultrastructure provides morphological evidence supporting the concept that these membranes are responsible for the initial olfactory transduction process. The transduced message is transported to the brain, where it is decoded to initiate the cascade of events resulting in the organisms' appropriate behavioral response to the initial odorous stimulus. The varying appearance of olfactory structures across the animal kingdom is probably produced by evolutionary pressure to adapt the olfactory system to the animal's environment. This review deals with the ultrastructural aspects of these facets of olfaction.     

Development,growth, and plasticity in the crayfish olfactory system

Decapod crustaceans have a well-defined olfactory system characterised by a set of chemosensitive sensilla grouped together in an array (the olfactory organ) on their antennules. Olfactory receptor neurons in the olfactory organ project exclusively to, and terminate in, cone-shaped olfactory glomeruli in a discrete neuropil in the brain, the olfactory lobe. The olfactory organ appears to be the only afferent input to the olfactory lobe, making the system convenient for the study of its development and growth. The progression of development of the olfactory system is a continuum and can be traced from the first appearance of peripheral receptor cells and central stem cells through to the construction of the tracts and neuropils that constitute the adult system. Cell proliferation leading to the production of peripheral and central olfactory neurons can be observed with mitotic markers in both embryonic stages and in postembryonic growth. Cell proliferation in the olfactory system in crayfish persists throughout the lives of the animals and can be modulated by manipulating the living conditions imposed on growing animals. Large serotonergic neurons that are associated with the olfactory system may play a role in the regulation of cell proliferation.     

Ultrastructural diagnosis of human pituitary adenomas.

Electron microscopy, which has been instrumental in the characterization of normal pituitary cell types, has also played a crucial role in the morphologic classification of pituitary adenomas arising in the presently known 5 cell types, and in the recognition of 3 adenoma types with yet undisclosed cell derivation. This review deals with the application of electron microscopy for study of pituitary adenomas in order to provide specific pathological diagnosis and aid the clinician in selecting appropriate postoperative treatment. In addition to the ultrastructural appearance and diagnostic features of 15 adenoma types, the morphology of hyperplastic proliferations and that of known normal counterparts of various adenoma types are also discussed. Specific morphologic diagnosis of pituitary lesions is important not only for adequate postoperative management of patient, but is also a prerequisite for study of the natural history and biological behaviour of various adenoma types.     

Seasonal changes in hepatocytic lipid droplets,glycogen deposits,and rough endoplasmic reticulum along the natural breeding cycle of female ohrid trout (Salmo letnica Kar.)—A semiquantitative ultrastructural study

This study on wild female Ohrid trout was primarily designed to provide a general overview of the breeding cycle influence upon selected aspects of hepatocytes. According with a semiquantitatively evaluation, some of these cell's structural compartments change during the breeding cycle. Structural modifications were disclosed in the relative occurrence of lipid, glycogen, and RER content during breeding cycle. The relative amount of lipid deposits in the hepatocytes was much greater in previtellogenesis, and decreased postspawning. So, while the seasonal changes in RER were positively related with the ovary maturation status, those of the lipid droplets followed an opposite trend. The hepatocytic glycogen occurred rarely, mainly in late‐vitellogenesis and spawning, suggesting that in this species such kind of energy storage is comparatively unimportant. Lipid accumulation and later usage is, probably, the relevant biochemical pathway for Ohrid trout in the wild. While glycogen and RER contents were positively correlated with the gonadosomatic index, lipids were negatively correlated. Additionally, glycogen inclusions were positively correlated with the plasma estradiol levels. When comparing seasonal patterns from wild Ohrid trout with those from well‐studied rainbow and brown trout (specimens studied were from aquaculture), there are contradicting results as to lipid and glycogen reserves, and also as to RER loads. The differences among the mentioned trout can result from intrinsic interspecies differences or may be associated with natural feeding conditions versus feeding with commercially prepared diets, or other factors. This study offers new data useful as standard to access liver pathology in wild and aquacultured Ohrid trout. Microsc. Res. Tech. 79:700–706, 2016. © 2016 Wiley Periodicals, Inc.     

Fine structure of olfactory epithelia of gastropod molluscs.

Among gastropod molluscs the chemical senses are most important for location of distant objects. They are used in food finding, locating mates, avoiding predators, trail following, and homing. Chemoreceptors are commonly associated with the oral area, the tentacles, and the osphradium, which lies in the mantle cavity. Most chemosensory neurons are primary sensory neurons, although secondary sensory cells have been reported in the osphradium of some prosobranch gastropods. Most chemosensory organs contain sensory cells with ciliated sensory endings that are in contact with the external environment. Some sensory endings have only microvilli or have no surface elaborations. Cilia on sensory endings are commonly of the conventional type, but some species have modified cilia; some lack rootlets, some have an abnormal microtubular content, and some have paddle-shaped endings. The perikarya of sensory neurons may be within the sensory epithelium, below it, or in ganglia near the sensory surface. In some groups of gastropods there are peripheral ganglia in the olfactory pathway; in others chemosensory axons appear to pass directly to the CNS. Olfactory epithelia of terrestrial pulmonates have modified brush borders with long branching plasmatic processes and a spongy layer of cytoplasmic tubules which extend from the epithelial cells. Sensory endings of the olfactory receptors are entirely within this spongy layer. Aquatic pulmonates may have a similar spongy layer in their olfactory epithelia, but the cilia of sensory endings, as well as motile cilia of epithelial cells, extend well beyond the spongy layer.     

Ultrastructural imaging of freeze-fractured plant cells in the scanning electron microscope.

The application of the freeze-fracture and cytoplasmic maceration technique in ultrastructural studies of plant cells is described. A major advantage of the technique is, that by extracting mobile cytoplasmic components from the freeze-fractured cells, surface relief is introduced and three-dimensional information is obtainable. The details of specimen preparation are described and the results obtained are reviewed. The use of chitosan embedding for very small or fragile specimens is described.