Histochemical Approaches Research Articles

Histochemical analyses and quantum dot imaging of microvascular blood flow with pulmonary edema in living mouse lungs by “in vivo cryotechnique”

Light microscopic imaging of blood vessels and distribution of serum proteins is essential to analyze hemodynamics in living animal lungs under normal respiration or respiratory diseases. In this study, to demonstrate dynamically changing morphology and immunohistochemical images of their living states, "in vivo cryotechnique" (IVCT) combined with freeze-substitution fixation was applied to anesthetized mouse lungs. By hematoxylin-eosin staining, morphological features, such as shapes of alveolar septum and sizes of alveolar lumen, reflected their respiratory conditions in vivo, and alveolar capillaries were filled with variously shaped erythrocytes. Albumin was usually immunolocalized in the capillaries, which was confirmed by double-immunostaining for aquaporin-1 of endothelium. To capture accurate time-courses of blood flow in peripheral pulmonary alveoli, glutathione-coated quantum dots (QDs) were injected into right ventricles, and then IVCT was performed at different time-points after the QD injection. QDs were localized in most arterioles and some alveolar capillaries at 1s, and later in venules at 2s, reflecting a typical blood flow direction in vivo. Three-dimensional QD images of microvascular networks were reconstructed by confocal laser scanning microscopy. It was also applied to lungs of acute pulmonary hypertension mouse model. Erythrocytes were crammed in blood vessels, and some serum components leaked into alveolar lumens, as confirmed by mouse albumin immunostaining. Some separated collagen fibers and connecting elastic fibers were still detected in edematous tunica adventitia near terminal bronchioles. Thus, IVCT combined with histochemical approaches enabled us to capture native images of dynamically changing structures and microvascular hemodynamics of living mouse lungs.

Evaluation of myelin sheath and collagen reorganization pattern in a model of peripheral nerve regeneration using an integrated histochemical approach

Peripheral nerves are complex histological structures that can be affected by a variety of conditions with different degree of axonal degeneration and demyelination. For the study of peripheral nerve regeneration in pathology and tissue engineering, it is necessary to evaluate the regeneration, remyelination and extracellular matrix reorganization of the neural tissue. Currently, different histochemical techniques must be used in parallel, and a correlation among their findings should be further performed. In this work, we describe a new histochemical method for myelin and collagen fibers based on luxol fast blue and picrosirius methods, for the evaluation of the morphology, the myelin sheath and the collagen fiber reorganization using a model of peripheral nerve regeneration. Whole brain, normal sciatic nerve and regenerating peripheral nerve samples were fixed in 10% neutral buffered formalin and paraffin-embedded, for the performance of the hematoxylin-eosin stain, the Luxol fast blue method and the new histochemical method for myelin and collagen. The results of this technique revealed that this new histochemical method allowed us to properly evaluate histological patterns, and simultaneously observe the histochemical reaction for myelin sheath and collagen fibers in normal tissue, and during the regeneration process. In conclusion, this new method combines morphological and histochemical properties that allowed us to determine with high accuracy the degree of remyelination and collagen fibers reorganization. For all these reasons, we hypothesize that this new histochemical method could be useful in pathology and tissue engineering.

Abstract 2928: Regulation of MST1 and MST2 by mTOR and androgen receptor signaling in prostate cancer

Abstract The serine/threonine kinases MST1 and MST2 (hippo in Drosophila) were originally identified as pro-apoptotic proteins. In addition to their pro-apoptotic functions, evidence suggests that deficiency in MST1 and MST2 and their downstream effectors has been linked to developmental defects, tissue overgrowth, and cancer, including prostate cancer (PCa). However, the molecular mechanism that may modulate MST1 or MST2 signaling in PCa cells remains unknown. Here we investigate the impact of androgen receptor (AR) and PI3K-AKT-mTOR signaling on MST1 or MST2. We used biochemical, mutagenesis, histochemical, and pharmacological approaches to carry out this investigation. First, we demonstrate that AR and MST1 co-localize and form protein complexes in cell nuclei. Androgen further stimulates the interaction between the two proteins and reduces MST1 kinase activity in LNCaP PCa cells, suggesting that AR may sequester MST1 in an inactive form. Second, we showed that MST1 at Threonine120 (T120) was exclusively phosphorylated in cell nuclei, whereas MST2 at Threonine117 (T117), corresponding to MST1-T120, was phosphorylated only in the cytoplasm, although MST1 and MST2 were found in both locations. Similar phosphorylation patterns were also observed in prostate cancer tissue samples. Third, we have found that the T120 is not a physiologic target of AKT kinase in vivo, because the inhibition of AKT activity by a specific PI3K inhibitor, LY294002, had no effect on the phosphorylation of MST1 at the T120 site, but it did inhibit MST1-T117 phosphorylation. Furthermore, attenuation of mTOR activity by Ku0063794, a specific mTOR1 and mTOR2 inhibitor, abolished MST2-T117 phosphorylation, but without having an effect on the levels of phospho-MST1-Thr120. Lastly, the use of combined LY294002 and Ku0063794 compounds synergistically inhibited PCa cell growth, relative to a single compound. Our data suggest that AR and mTOR pathway signaling may oppose MST1 and MST2 in discrete cell locations. Thus, understanding the functional relationship between these signal networks in the context of cell proliferation and cell death may have important therapeutic implications in prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2928. doi:10.1158/1538-7445.AM2011-2928

Evolution of the localisation and composition of phenolics in grape skin between veraison and maturity in relation to water availability and some climatic conditions

Several studies have investigated the composition of phenolics in grape skin during grape maturation under various conditions of light exposure, water stress, nitrogen supply and mineral nutrition, but their localisation during berry development is not well known. In this study the composition and localisation of proanthocyanidins were monitored for three years on four plots known to induce a distinctive behaviour of the vine (Cabernet Franc). The composition of phenolics was determined by spectrophotometry; also, in one year, proanthocyanidins were determined by high-performance liquid chromatography. Further information was obtained histochemically by means of toluidine blue O staining and image analysis. The results indicated that clear differences in phenolic quantification existed between the biochemical and histochemical approaches; the proportion of cells without phenolics was not linked with the quantity determined by the analytical methods used. The histochemical method showed the evolution of the localisation and typology of cells with and without phenolics during ripening. The number of cells without any phenolic compounds appeared to be very dependent on the mesoclimatic conditions and only slightly dependent on the site water status. Clear differences in phenolic quantification existed between the biochemical and histochemical approaches; the proportion of cells with phenolics was not linked with the quantity determined by biochemistry. The histochemical method showed an evolution of the localisation and typology of cells with and without phenolics in which mesoclimatic conditions were the most influential factor. Finally, the study showed some advantages of the histochemical approach: it gives information about the anatomy of the tissue as well as the nature and distribution of some of the large macromolecules and allows reconstruction of the three-dimensional plant structure.

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

Histochemistry through the years, browsing a long-established journal: novelties in traditional subjects

Histochemical journals represent a traditional forum where methodological and technological improvements can be presented and validated in view of their applications to investigate not only cytology and histology in normal and diseased conditions but to test as well hypotheses on more basic issues for life sciences, such as comparative and evolutionary biology. The earliest scientific journals on histochemistry began their publication in the first half of the ‘50s of the last century, and their readership did not probably change over the years; rather, the authors’ interests may have progressively been changing as well as the main topics of their articles. This hypothesis is discussed, based on the subjects of the article published in the first and last ten years in the European Journal of Histochemistry, as an example of old journal which started publication in 1954, being since then the official organ of the Italian Society of Histochemistry. This survey confirmed that histochemistry has provided and still offers unique opportunities for studying the structure, chemical composition and function of cells and tissues in a wide variety of living organisms, especially when the topological distribution of specific molecular components has diagnostic or predictive significance, as it occurs in human and veterinary biology and pathology. Some subjects (e.g. histochemistry applied to muscle cells or to mineralized tissues) have recently become rather popular, whereas a wider application of the histochemical approach may be envisaged for plant cells and tissues.

Histochemical and morpho­metrical study of mouse intestine epithelium after a long term diet containing genetically modified soybean

Diet can influence the structural characteristics of both small and large intestine. In this study, we investigated the duodenum and colon of mice fed on genetically modified (GM) soybean during their whole life span (1–24 months) by focusing our attention on the histological and ultrastructural characteristics of the epithelium, the histochemical pattern of goblet cell mucins, and the growth profile of the coliform population. Our results demonstrate that controls and GM-soybean fed mice are similarly affected by ageing. Moreover, the GM soybean-containing diet does not induce structural alterations in duodenal and colonic epithelium or in coliform population, even after a long term intake. On the other hand, the histochemical approach revealed significant diet-related changes in mucin amounts in the duodenum. In particular, the percentage of villous area occupied by acidic and sulpho-mucin granules decreased from controls to GM-fed animals, whereas neutral mucins did not change.

Cytomorphological Analysis of Keratinocytes in Oral Smears from Tobacco Users and Oral Squamous Cell Carcinoma Lesions — A Histochemical Approach

AimTo analyse the cytomorphological features of keratinocytes in smears obtained from the oral mucosa of tobacco users and from oral squamous cell carcinoma lesions.MethodologyOral smears were obtained from clinically, normal appearing mucosa of oral squamous cell carcinoma patients (n=20) and from the mucosa of smokers (n=20), and apparently healthy individuals (n=20) were used as controls. The smears were histochemically stained and cytomorphological assessment of the keratinocytes was carried out. One-way ANOVA (Analysis of Variance) was used for comparing the parameters among multiple groups and Tukey-HSD test was used to compare the mean values between groups.ResultsThe mean nuclear area of keratinocytes from the mucosa of tobacco users was 46 ± 2.57 and that of the oral squamous cell carcinoma lesion was 81.54 ± 4.31. While there was a significant (P=0.001) reduction in the cellular area of keratinocytes from oral squamous cell carcinoma lesion when compared with those from oral smears of tobacco users.ConclusionCytomorphometric analysis of keratinocytes can serve as a useful adjunct in the early diagnosis of oral squamous cell carcinomas.

新しいアプローチから見た食品機能性成分の動態研究

Dietary habits are related to many problems including food, energy, environment and health. Thus, the researches in dietary habits must be conducted in the paradigm of complex system. But, it is important to accumulate the experimental observations by the reductionistic approarch. In this review, I focus on the new approach to the materials and methods. First, I examined the function of oleuropein, a major polyphenol in olive leaf using the rats fed on high cholesterol diets. As the result, the values of total bile acids and total lipids in the feces decreased significantly in the rat fed on the olive leaf powder and oleuropein. The values of total cholesterol in the feces and triglyceride in the liver decreased significantly in the group fed on the olive leaf extract. These results indicate that the oleuropein facilitates the lipid absorption and the bile acid reabsorption in the ileum. Second, in order to reveal the relationship between renal reabsorptional systems of vitamin B12 (B12) and vitamin A, I analized the effect of B12 deficient status on the localization of megalin, the receptor of both vitamins, and retinol binding protein (RBP). As the result, in B12 deficient rats, megalin was accumulated on the membrane in the proximal tubules and a few tubules containing RBP were identified, which suggested inhibition of megalin-mediated endocytosis and renal vitamin A uptake. Third, I produced D-histidine specific antibody to trace biokinetics. Histochemical approach may be a useful tool to understand the nutrition and dietary habits.

Identification of distinct cellular pools of interleukin-1β during the evolution of the neuroinflammatory response induced by transient middle cerebral artery occlusion in the brain of rat

The proinflammatory cytokine interleukin(IL)-1β plays a crucial role in ischemic pathophysiology, since pharmacologic inhibition of its biological effects provides neuroprotection after stroke. However, there is evidence suggesting that under certain circumstances the cytokine may also exert beneficial functions on brain injury. We have investigated the regional and cellular expression of IL-1β after ischemia–reperfusion injury in the brain of rat, and correlated cytokine expression with the activation/recruitment of glial cells in the damaged tissue. By using a double immunofluorescence histochemical approach, we observed an increased cytokine immunoreactivity in the ischemic core, as early as 1 h after middle cerebral artery occlusion, in few activated OX-42-positive microglial cells and in perivascular GFAP-positive astrocytes, suggesting that the cytokine may participate in the early response of the neurovascular unit to reduced blood supply. After 2 h ischemia, followed by 2 h reperfusion, cytokine staining was evident in the astrocytes of the penumbra and in activated microglial cells of the ischemic core. Microglial activation increases with the progression of damage and, after 22 h reperfusion, OX-42-immunopositive cells were strongly labelled for IL-1β in the core and, even more intensely, in the penumbra. At this later stage, GFAP-positive cells, appearing hypertrophic and distributed in a ring-like pattern around the ischemic core, do no longer express IL-1β. Thus, a specific cellular and regional pattern of IL-1β expression characterises the progression of ischemia–reperfusion injury. Depending on the stage and intensity of the insult, the different cellular origin of the cytokine may suggest a distinct role of this neuroinflammatory mediator in ischemic pathophysiology.

Early undernutrition increases glycogen content and reduces the activated forms of GSK3, AMPK, p38 MAPK, and JNK in the cerebral cortex of suckling rats

Exposure to maternal undernutrition during development increases the risk for neurological and cognitive defects. However, little is known about the underlying mechanisms involved. Peripheral responses to insulin are increased following food-restriction, thus the possibility arises that brain insulin actions are affected by undernutrition, causing damages to the higher cerebral functions. In this study, we examined the effects of early undernutriton on molecular targets of insulin actions such as glucose transporters, glycogen, glycogen synthase kinase-3 (GSK3) and mitogen-activated protein kinases, as well as proteins involved in apoptosis in the cortex from 10-day-old rats. We show that undernutrition results in an enhanced glycogen content which is confined to astrocytes, according to our histochemical approaches. Cortical phospho-GSK3 is also increased. In addition to glycogen synthesis, GSK3 regulates crucial cellular processes. Therefore, its elevated degree of phosphorylation may have an impact on these processes and, consequently, on the cortical development. Phospho-p38 and both total JNK and phospho-JNK, which regulate apoptosis, are reduced following undernutrition. However, cleaved caspase 3 is not altered, which suggests that this condition does not induce extensive modifications to the cortical apoptosis. Thus, our results indicate that undernutrition gives rise to molecular alterations that may have repercussions on cerebral cortex development and functions.

Histochemical approach of cryobiopsy for glycogen distribution in living mouse livers under fasting and local circulation loss conditions

Soluble proteins and glycogen particles, which are easily lost upon conventional chemical fixation, have been reported to be better preserved in paraffin-embedded sections by 'cryobiopsy' combined with freeze-substitution fixation (FS). In this study, we examined the distribution of glycogen in living mouse livers under physiologic and pathologic conditions with periodic acid-Schiff (PAS) staining by cryobiopsy. The livers of the fully fed mice showed high PAS-staining intensity in the cytoplasm of all hepatocytes. The PAS-staining intensity gradually decreased away from hepatocytes around portal tracts, depending on treatments with different alpha-amylase concentrations. At 6 or 12 h after fasting, PAS-staining intensity markedly decreased in restricted areas of zone I near the portal tracts. The cryobiopsy was repeatedly performed not only on different mice, but also on individuals. Next, glycogen distributions were evaluated by temporarily clipping of liver tissues of anesthetized mice, followed by recovery of blood circulation. In the liver tissues in which blood was recirculated for 1 h after the 30 min anoxia, PAS staining was still observed in zone II and also in restricted areas of zone I far from the portal tracts. In PAS-unstained hepatocytes, the immunoglobulin-kappa light chain was not detected in the cytoplasm, indicating that cell membrane permeability was retained and that glycogen metabolism was related to the functional state of blood circulation. We propose that the level of consumption or production of glycogen particles could vary in zone I, depending on the distance from the portal tracts. Thus, cryobiopsy combined with FS enabled us to examine time-dependent changes in glycogen distribution in the liver tissues of living mice. This combination might be applicable to the clinical evaluation of human liver tissues.

In Situ Imaging of Metals in Cells and Tissues

In Situ Imaging of Metals in Cells and Tissues

A Histochemical Approach to the Diagnosis of Visceral Pleural Infiltration by Non-small Cell Lung Cancer

Although invasion of the visceral pleura (VPI) by non-small cell lung cancer (NSCLC) is a TNM-relevant diagnostic criterion and is known to affect the patients' prognoses, until recently there were no standardized or internationally accepted guidelines. This resulted in a diagnostic ambiguity leading to different tumor staging systems and to hardly comparable patient collectives in research studies world wide. The major problem in this issue is to exactly define what constitutes for the diagnosis of VPI with respect to anatomical landmarks. In order to address this problem we investigated the pleural infiltration depth of 173 NSCLC specimens without lymph node metastases and proven tumor-related death using elastic stains and a scoring system referring to prominent pleural elastic layers, the lamina elastica externa and interna, as anatomical landmarks. Performing comparative Kaplan-Meier survival analyses for each patient collective we could not find any significant difference in the patients' survival. This indicates that a differential evaluation of the tumor infiltration depth according to the elastic layers is not practicable. Our findings support the consequent application of the recently proposed, pragmatic approach of the international staging committee for lung cancer (IASLC) to define an internationally accepted and standardized staging system for VPI.

Rapid combined light and electron microscopy on large frozen biological samples

The use of large unfixed frozen tissue samples (10 x 10 x 5 mm(3)) for combined light microscopy (LM) and electron microscopy (EM) is described. First, cryostat sections are applied for various LM histochemical approaches including in situ hybridization, immunohistochemistry and metabolic mapping (enzyme histochemistry). When EM inspection is needed, the tissue blocks that were used for cryostat sectioning and are stored at -80 degrees C, are then fixed at 4 degrees C with glutaraldehyde/paraformaldehyde and prepared for EM according to standard procedures. Ultrastructurally, most morphological aspects of normal and pathological tissue are retained whereas cryostat sectioning at -25 degrees C does not have serious damaging effects on the ultrastructure. This approach allows simple and rapid combined LM and EM of relatively large tissue specimens with acceptable ultrastructure. Its use is demonstrated with the elucidation of transdifferentiated mouse stromal elements in human pancreatic adenocarcinoma explants grown subcutaneously in nude mice. Combined LM and EM analysis revealed that these elements resemble cartilage showing enchondral mineralization and aberrant muscle fibres with characteristics of skeletal muscle cells.

Larval organogenesis of flatfish brill Scophthalmus rhombus L: Histological and histochemical aspects

The present tabular overview of organogenesis in larvae of the flatfish brill Scophthalmus rhombus (L.) provides valuable information on its structural status during ontogeny, and it can be useful for establishing the functional systemic capabilities and physiological requirements of larvae for optimal welfare and growth. The organogenesis of the brill larvae was studied during the first month of larval life by means of zootechnical, histological, and histochemical approaches. Based upon its feeding mode, and analysing the main morphohistological characteristics of the organs and systems, larval development in brill was divided into four stages from hatching: Stage 1: 0–1 days after hatching (DAH); Stage 2: 2–9 DAH; Stage 3: 10–22 DAH and Stage 4 from 23 DAH onwards. As in most marine fish species, brill larvae at hatching had an undifferentiated digestive tract composed of monostratified epithelium cells, each containing a basal nucleus and evident apical microvilli. At the beginning of stage 2, both the mouth and the anus opened in conjunction with the differentiation of the digestive tract. During stages 2 and 3, the digestive tract differentiated and buccopharyngeal cavity, oesophagus, incipient stomach, and anterior, mid- and posterior intestine became distinguishable. At this time, prey capture began and the digestive processes continued to develop (e.g. gut mucosa folds, lipid infranuclear vesicles and protein supranuclear inclusions). The first taste buds appeared at the onset of the exotrophic stage, at 8 DAH, while the first pharyngeal and mandibular teeth were apparent at 4 DAH. Histological and histochemical observations suggest that digestive tract development of the brill larvae, involving the presence of functional liver (glycogen lipids), exocrine pancreas (proteins) and gall bladder, enabled early larvae (from 4 DAH) to ingest, digest, and assimilate the first exogenous food, even before endogenous reserves were completely resorbed. The appearance of the digestive mucous cells (from 3 DAH), containing neutral and/or acid mucosubstances, and the functional gastric gland differentiation (23 DAH) resembled such developments in other flatfish species. The most critical ontogenetic events occurred at stage 2. Primordial gills were detected at 2 DAH while gill filament, vascular structures, and lamellae differentiated from 6 DAH. At the beginning of stage 2, four defined cardiac cavities (sinus venosus, atrium, ventricle, and bulbus arteriosus) were discernible. The primordial swim bladder was differentiated from the dorsal wall of the digestive tube at 2 DAH and at this time renal tubules between interstitial kidney tissue were detected. Endocrine elements (Langerhans islets and thyroid follicles) were also evident at stage 2. From stage 3 onwards, most organs essentially exhibited an increase in tissue structure number and size.

Distribution of ectonucleotidases in the rodent brain revisited

Nucleotides comprise a major class of signaling molecules in the nervous system. They can be released from nerve cells, glial cells, and vascular cells where they exert their function via ionotropic (P2X) or metabotropic (P2Y) receptors. Signaling via extracellular nucleotides and also adenosine is controlled and modulated by cell-surface-located enzymes (ectonucleotidases) that hydrolyze the nucleotide to the respective nucleoside. Extracellular hydrolysis of nucleotide ligands involves a considerable number of enzymes with differing catalytic properties differentially affecting the nucleotide signaling pathway. It is therefore important to investigate which type of ectonucleotidase(s) contributes to the control of nucleotide signaling in distinct cellular and physiological settings. By using a classical enzyme histochemical approach and employing various substrates, inhibitors, and knockout animals, we provide, for the first time, a comparative analysis of the overall distribution of catalytic activities reflecting four ectonucleotidase families: ecto-5'-nucleotidase, alkaline phosphatases, ectonucleoside triphosphate diphosphohydrolases (E-NTPDases), and ectonucleotide pyrophyphatases/phosphodiesterases (E-NPPs). We place into perspective the earlier literature and provide novel evidence for a parenchymal localization of tissue non-specific alkaline phosphatase, E-NPPs, and E-NTPDases in the mouse brain. In addition, we specify the location of ectonucleotidases within the brain vasculature. Most notably, brain vessels do not express ecto-5'-nucleotidase. The preponderance of individual enzymes differs considerably between brain locations. The contribution of all types of ectonucleotidases thus needs to be considered in physiological and pharmacological studies of purinergic signaling in the brain.

Histochemical approaches to understanding central regulation of food intake by melanocortin signaling

Histochemical approaches to understanding central regulation of food intake by melanocortin signaling

Histochemical correlations between egg capsule laminae and the female gonoduct reveal the process of capsule formation in the Muricidae (Neogastropoda: Mollusca)

Summary Limited information is currently available on the process of encapsulation and degree of variation in the micromorphology of egg capsules deposited by muricid gastropods. In this study, a histochemical approach was employed to determine the origin of egg capsule laminae within the female gonoduct of Dicathais orbita. Comparisons of capsule micromorphology from this Australian Rapaninae, with species from the Muricinae and Ocenebrinae, suggest that whole capsule and lamina 1 (L1) thickness, L2 fiber orientation and intracapsular fluid biochemistry display interspecific variability. Furthermore, the method of capsule laminae deposition was found to be substantially different to previous descriptions of muricid capsule formation. Simultaneous examination of capsule and gonoduct biochemistry in D. orbita revealed that the intracapsular fluid nd inner capsule wall (L3) contain secretions from the posterior lobe of the capsule gland; the thick middle lamina (L2) is derived from the lateral capsule gland lobes; and the outer layers (L1 and L0) include dorsal capsule lobe, albumen gland and pedal gland components. The insight gained into the processes involved in capsule formation imply that interspecific differences in capsule micromorphology are dependent on the timing of egg entry during capsule formation, and the protein content of posterior lobe secretory products coupled with the presence or absence of albumen gland secretions. This investigation supplements existing evidence on the origin of capsule components and variability in capsule micromorphology in the Muricidae by providing a simple method for deciphering the complex process of encapsulation in neogastropods.

Paraxial organ of a scorpion: structural and ultrastructural studies of Euscorpius tergestinus paraxial organ (Scorpiones, Euscorpiidae)

Summary By combining ultrastructural and histochemical approaches, the paraxial organs of Euscorpius tergestinus (syn. E. carpathicus; Scorpiones, Euscorpiidae) have been studied for the first time. The paraxial organs are symmetrical structures in the male genital system. They are located on each side of the mesosoma and end at the genital aperture. The paraxial organs have a glandular function. They are directly involved in the secretion of the two hemispermatophores, which are deposited in the glandular lumen. When the hemispermatophores are extruded, they constitute the spermatophore, which is involved in sperm transfer. The epithelium of the paraxial organs is complex, being either unistratified or pseudostratified. Moreover, in the different parts of the gland, such as at the pars bulbosa, the epithelium can show folds. In addition, the appearance of the epithelial cells varies with secretory activity. The cells deposit the different components of the hemispermatophore in the lumen. We show that the cells have two successive methods of secretion: firstly merocrine and then apocrine. This has never been described before. The histochemical observations of the hemispermatophore show its complex structure with the presence of different constituents.