Histochemical and morphological features of biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga, Brycon nattereri (Characiformes)

The aim of the present study was to characterize biopsied and stripped oocytes from the Brazilian endangered teleost pirapitinga (Brycon nattereri) using histochemical and morphological analyses. Biopsied oocytes had a mean diameter of 2.225 mm (modal diameter: 2.312 mm), complete vitellogenesis and a central or slightly eccentric nucleus. Neutral polysaccharides were detected in the follicular cells, zona radiata and yolk globules, while acidic polysaccharides were detected in the follicular cells and cortical alveoli. Ten out of the 19 females treated with two doses of carp pituitary extract (cPE) released oocytes, which were also analysed. Stripping occurred 292 +/- 39 degree-hours after the second dose of cPE and led to a mean spawning weight of 36.2 g, 10% spawning index, 241 oocytes/g of ova, 8222 oocytes/female and 23 oocytes/g of body weight. Stripped oocytes had a mean diameter of 2.33 mm and a mode at 2.375 mm, were weakly adhesive and coloration ranged from wine to brown. Under scanning electron microscopy, stripped oocytes exhibited a single funnel-shaped micropyle located at the animal pole and a zona radiata that measured 7.7 m in thickness with eight pore canals/m(2). Oocyte morphology in Brycon nattereri is similar to that found in other species of the genus, except for the larger size and weaker adhesiveness. These findings provide essential information for a better understanding of the reproductive biology of B. nattereri and the establishment of conservation measures for this threatened species.

Somatic embryogenesis of a wild passion fruit species Passiflora cincinnata Masters: histocytological and histochemical evidences

The characterization of cellular changes that occur during somatic embryogenesis is essential for understanding the factors involved in the transition of somatic cells into embryogenically competent cells and determination of cells and/or tissues involved. The present study describes the anatomical and ultrastructural events that lead to the formation of somatic embryos in the model system of the wild passion fruit (Passiflora cincinnata). Mature zygotic embryos were inoculated in Murashige and Skoog induction media supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine. Zygotic embryo explants at different development stages were collected and processed by conventional methods for studies using light, scanning, and transmission electron microscopy (TEM). Histochemical tests were used to examine the mobilization of reserves. The differentiation of the somatic embryos began in the abaxial side of the cotyledon region. Protuberances were formed from the meristematic proliferation of the epidermal and mesophyll cells. These cells had large nuclei, dense cytoplasm with a predominance of mitochondria, and a few reserve compounds. The protuberances extended throughout the abaxial surface of the cotyledons. The ongoing differentiation of peripheral cells of these structures led to the formation of proembryogenic zones, which, in turn, dedifferentiated into somatic embryos of multicellular origin. In the initial stages of embryogenesis, the epidermal and mesophyll cells showed starch grains and less lipids and protein reserves than the starting explant. These results provide detailed information on anatomical and ultrastructural changes involved in the acquisition of embryogenic competence and embryo differentiation that has been lacking so far in Passiflora.

Morphological, histological, histochemical and behavioral aspects during early development of red porgy Pagrus pagrus L. reared in mesocosm.

[EN]The present study provides a description of the morphological, histological and histochemical development, and the ecological implications of red porgy larvae reared in mesocosm, from hatching to 30 days after hatching (DAH).

Characteristics of aleveolar bone associated with physiological movement of molar in mice: a histological and histochemical study

Mouse molars undergo distal movement, during which new bone is formed at the mesial side of the tooth root whereas the preexisting bone is resorbed at the distal side of the root. However, there is little detailed information available regarding which of the bones that surround the tooth root are involved in physiological tooth movement. In the present study, we therefore aimed to investigate the precise morphological differences of the alveolar bone between the bone formation side of the tooth root, using routine histological procedures including silver impregnation, as well as by immunohistochemical analysis of alkaline phosphatase and tartrate-resistant acid phosphatase activity, and immunohistochemical analysis of the expression of the osteocyte markers dentin matrix protein 1, sclerostin, and fibroblast growth factor 23. Histochemical analysis indicated that bone formation by osteoblasts and bone resorption by osteoclasts occurred at the bone formation side and the bone resorption side, respectively. Osteocyte marker immunoreactivity of osteocytes at the surface of the bone close to the periodontal ligament differed at the bone formation and bone resorption sides. We also showed different specific features of osteocytic lacunar canalicular systems at the bone formation and bone resorption sides by using silver staining. This study suggests that the alveolar bone is different in the osteocyte nature between the bone formation side and the bone resorption side due to physiological distal movement of the mouse molar.

Histochemical and cytological techniques.

Mimeographed copy.

Histochemical and ultrastructural studies on the islets of langerhans of lean and obese hyperglycaemic mice with age

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Histochemical studies of chondrogenesis

The formation of the cartilage tissue depends on the coordination of cell to cell or cell to ECM interaction that cause to the cell polarity, migration and differentiation of precursor mesenchymal cells during chondrogenesis Many of these events are mediated by ECM components such as glycocojugates which with their suger residues such as galactose or aminosuger have a ligand role for regulatory molecules. The aim of this study was to identify the presence and distribution of some different glycoconjugates and their suger residues in the chondrogenesis by histochemistry and lectin-histochemistry techniques. For this purpose, embryos from pregnant wistar rats from E12-E20 were collected and fixed. Some of them were stained with alizarin red Salcin blue staining to demonstrate cartilage and bone formation in whole mount embryos. Other embryos with serial sections (5-7micm thikness) were stained by: 1-alcian blue (pH: l) for S-GAG,2-alcin blue (pH:2.5)for C-GAG, S-PAS alcian blue fora neutral and acidic sugers,4- tuloidin blue for metachromatic substances. Stained sections were graded according to the staining intensity (0-5 grading s method). Statistical analysis showed significant difference for those substances among experimental groups. Lectin histochemistry with MPA, VVA, SBA, OFA demonstrated differences between organs for suger residues during chondrogensis. It seems that synthesis and secretion of glycocojugates and change of their suger residues follows a spatiotemporal pattern and developmentaly regulated.

Spermathecae of the mangrove crab Ucides cordatus: a histological and histochemical view

Ucides cordatus is the most commercially, important mangrove crab in Brazil. In spite of its economic importance, there are few studies of its reproduction, in particular the female reproductive system. The present study describes the histology and histochemistry of the spermathecae of U. cordatus. Adult females were can lit monthly from July 2004 through June 2005, at Iguape, State of São Paulo. The crabs were anaesthetized, and their spermathecae removed and fixed in Davidson's fluid, following the histological routine for paraffin. The slides were stained with HE, xylidine Ponceau, PAS, alcian blue (pH 1.0 and 2.5), Sudan black B and picrosirius-haematoxylin. Histologically, the spermathecae possesses a capsule of conjunctive tissue, rich in collalgen fibres, which surrounds the secretory columnar epithelium. In the lumen, individual sperm packets are not observed; the spermatophores are intermixed with the seminal fluid and secretions of the spermathecae itself. A large proportion of the free spermatozoids and spermatophores are arranged in homogeneous masses in the proximal part of the spermathecae. The secretion produced by the columnar epithelium appears to promote the movement of the gametes to the fertilization chamber, in a ventral position, allowing fertilization of the oocytes. Histochemically, the secretion produced by the columnar epithelium was strongly positive for neutral polysaccharides, positive for acid polysaccharides, and weakly positive for proteins and lipids. Tills secretion forms a glycoprotein matrix which is associated with maintenance of the spermatophores, which can remain stored for long periods.

Anatomical, histochemical and immunohistochemical characterization of the outflow tract of ray hearts (Rajiformes; Chondrichthyes)

Recent work has shown that the cardiac outflow tract of sharks and chimaeras does not consist of a single myocardial component, the conus arteriosus, as classically accepted, but two, namely, the myocardial conus arteriosus and the non-myocardial bulbus arteriosus. However, the anatomical composition of the outflow tract of the batoid hearts remains unknown. The present study was designed to fill this gap. The material examined consisted of hearts of two species of rays, namely, the Mediterranean starry ray (Raja asterias) and sandy ray (Leucoraja circularis). They were studied using scanning electron microscopy, and histochemical and inmunohistochemical techniques. In both species, the outflow tract consists of two components, proximal and distal with regard to the ventricle. The proximal component is the conus arteriosus; it is characterized by the presence of compact myocardium in its wall and several transverse rows of pocket-shaped valves at its luminal side. Each valve consists of a leaflet and its supporting sinus. Histologically, the leaflet has two fibrosas, inner and outer, and a middle coat, the spongiosa. The distal component lacks myocardium. Its wall consists of smooth muscle cells, elastic fibers and collagen. Thus, it shows an arterial-like structure. However, it differs from the aorta because it is covered by the epicardium and crossed by coronary arteries. These findings indicate that the distal component is morphologically equivalent to the bulbus arteriosus of sharks and chimaeras. In contrast to foregoing descriptions, the valves of the first transverse row are distally anchored to the bulbus arteriosus and not to the ventral aorta. Our findings give added support to the notion that presence of a bulbus arteriosus at the arterial pole of the heart is common to all chondrichtyans, and not an apomorphy of actinopterygians as classically thought.

Establishment of antibody panels and histochemical techniques in routine tumor diagnosis in Veterinary Pathology

In Portugal, Veterinary Pathology is developing rapidly, and in recent years we assist to the emergence of private laboratories and the restructuring of universities,polytechnics and public laboratories.The Portuguese Society of Animal Pathology,through its actions and its associates has been keeping the discussion among its peers in order to standardizethe criteria of description,classification and evaluation of cases which are the subject of our daily work.One of the last challenges is associated with the use of routine histochemical techniques and immunohistochemistry, in an effort to establish standardized panels for tumour diagnosis, which could eventually reduce each analysis cost.For this purpose a simple survey was built, in which all collaborators answered questions about the markers used for carcinoma, sarcoma and round cell tumour diagnosis, as well as general questions related with the subject. We obtained twenty-one answered to the questions, from public and private laboratories.In general, in most cases immunohistochemical and histochemical methods are used for diagnosis.The wide spectrum cytokeratins are universally used to confirm carcinoma, and vimentin for sarcoma. The CD3 marker is used by all laboratories to identify T lymphocytes. For the diagnosis of B-cell lymphoma, the marker used is not consensual. In each laboratory there are different markers for more specific situations and only two labs perform PCR techniques for diagnosis. These data will be presented to promote extended discussion,namely to reach a consensus when different markers are used.

Histochemical characterization of 3D bioprinted meniscus

La cartilagine ad oggi rappresenta ancora una sfida per la medicina rigenerativa a causa della sua scarsa capacità di rigenerarsi quando sottoposta a gravi lesioni tissutali. Inoltre, la sua struttura complessa e l'ambiente biologico che offre sono ancora difficili da riprodurre con precisione mediante l’utilizzo di strutture bioartificiali e biomimetiche. Sebbene gli interventi per riparare i danni al tessuto cartilagineo siano ancora prevalentemente di stampo chirurgico, si stanno aprendo nuove strade nel campo dell'ingegneria tissutale volte a ricreare una struttura simile a quella originaria, che possa indurre la rigenerazione o sostituire direttamente il tessuto danneggiato. Lo studio condotto e discusso in questo elaborato mira a soddisfare quest'ultimo intento, ponendo le basi per un'eventuale evoluzione sperimentale della stampa del menisco. La ricerca si è basata sulla biostampa 3D di campioni di menisco, di dimensioni pari a circa 1 cm2, realizzati mediante un bioinchiostro a base di alginato e nanocellulosa, al cui interno sono stati immersi sferoidi/microtessuti di condrociti con diversa concentrazione. La stampa dei campioni è avvenuta in tre diverse condizioni: i) solo bioinchiostro, ii) bioinchiostro contenente sferoidi da 5000 cellule, iii) bioinchiostro contenente sferoidi da 10000 cellule. Al termine della stampa sono stati analizzati i campioni per verificare la sopravvivenza delle cellule e investigare l'attitudine di queste di ricreare matrice extracellulare cartilaginea nelle condizioni ambientali in cui sono state introdotte. A questo proposito, sono state eseguite delle analisi istochimiche, quali la RT-PCR e l’analisi istologica. Inoltre, a partire dalle immagini al microscopio degli sferoidi a seguito della loro formazione e dalle sezioni istologiche, sono state analizzate le dimensioni dei campioni per confrontare l'eventuale variazione volumetrica di questi prima della stampa e a seguito del processo di estrusione.

Fluorescence, Aggregation Properties And Ft-ir Microspectroscopy Of Elastin And Collagen Fibers.

Histological and histochemical observations support the hypothesis that collagen fibers can link to elastic fibers. However, the resulting organization of elastin and collagen type complexes and differences between these materials in terms of macromolecular orientation and frequencies of their chemical vibrational groups have not yet been solved. This study aimed to investigate the macromolecular organization of pure elastin, collagen type I and elastin-collagen complexes using polarized light DIC-microscopy. Additionally, differences and similarities between pure elastin and collagen bundles (CB) were investigated by Fourier transform-infrared (FT-IR) microspectroscopy. Although elastin exhibited a faint birefringence, the elastin-collagen complex aggregates formed in solution exhibited a deep birefringence and formation of an ordered-supramolecular complex typical of collagen chiral structure. The FT-IR study revealed elastin and CB peptide NH groups involved in different types of H-bonding. More energy is absorbed in the vibrational transitions corresponding to CH, CH2 and CH3 groups (probably associated with the hydrophobicity demonstrated by 8-anilino-1-naphtalene sulfonic acid sodium salt [ANS] fluorescence), and to νCN, δNH and ωCH2 groups of elastin compared to CB. It is assumed that the α-helix contribution to the pure elastin amide I profile is 46.8%, whereas that of the B-sheet is 20% and that unordered structures contribute to the remaining percentage. An FT-IR profile library reveals that the elastin signature within the 1360-1189cm(-1) spectral range resembles that of Conex-Toray aramid fibers.

Trichomes Related To An Unusual Method Of Water Retention And Protection Of The Stem Apex In An Arid Zone Perennial Species.

It is well known that trichomes protect plant organs, and several studies have investigated their role in the adaptation of plants to harsh environments. Recent studies have shown that the production of hydrophilic substances by glandular trichomes and the deposition of this secretion on young organs may facilitate water retention, thus preventing desiccation and favouring organ growth until the plant develops other protective mechanisms. Lychnophora diamantinana is a species endemic to the Brazilian 'campos rupestres' (rocky fields), a region characterized by intense solar radiation and water deficits. This study sought to investigate trichomes and the origin of the substances observed on the stem apices of L. diamantinana. Samples of stem apices, young and expanded leaves were studied using standard techniques, including light microscopy and scanning and transmission electron microscopy. Histochemical tests were used to identify the major groups of metabolites present in the trichomes and the hyaline material deposited on the apices. Non-glandular trichomes and glandular trichomes were observed. The material deposited on the stem apices was hyaline, highly hydrophilic and viscous. This hyaline material primarily consists of carbohydrates that result from the partial degradation of the cell wall of uniseriate trichomes. This degradation occurs at the same time that glandular trichomes secrete terpenoids, phenolic compounds and proteins. These results suggest that the non-glandular trichomes on the leaves of L. diamantinana help protect the young organ, particularly against desiccation, by deposition of highly hydrated substances on the apices. Furthermore, the secretion of glandular trichomes probably repels herbivore and pathogen attacks.

Characterisation Of The Membrane Transport Of Pilocarpine In Cell Suspension Cultures Of Pilocarpus Microphyllus.

Pilocarpine is an alkaloid obtained from the leaves of Pilocarpus genus, with important pharmaceutical applications. Previous reports have investigated the production of pilocarpine by Pilocarpus microphyllus cell cultures and tried to establish the alkaloid biosynthetic route. However, the site of pilocarpine accumulation inside of the cell and its exchange to the medium culture is still unknown. Therefore, the aim of this study was to determine the intracellular accumulation of pilocarpine and characterise its transport across membranes in cell suspension cultures of P. microphyllus. Histochemical analysis and toxicity assays indicated that pilocarpine is most likely stored in the vacuoles probably to avoid cell toxicity. Assays with exogenous pilocarpine supplementation to the culture medium showed that the alkaloid is promptly uptaken but it is rapidly metabolised. Treatment with specific ABC protein transporter inhibitors and substances that disturb the activity of secondary active transporters suppressed pilocarpine uptake and release suggesting that both proteins may participate in the traffic of pilocarpine to inside and outside of the cells. As bafilomicin A1, a specific V-type ATPase inhibitor, had little effect and NH4Cl (induces membrane proton gradient dissipation) had moderate effect, while cyclosporin A and nifedipine (ABC proteins inhibitors) strongly inhibited the transport of pilocarpine, it is believed that ABC proteins play a major role in the alkaloid transport across membranes but it is not the exclusive one. Kinetic studies supported these results.