Localization of cholecystokinin in the zebrafish retina from larval to adult stage.

The peptide hormone cholecistokinin (CCK) plays a key role in the central and peripheral nervous system. It is known to be involved in the digestive physiology and in the regulation of food intake. Moreover, the CCK expression has also been detected in the retina of different vertebrates, including fish, although its biological activity in this tissue remains to be elucidated. In literature no data are yet available about the CCK-immunoreactivity in the zebrafish retina during development. Therefore, the aim of the study was to investigate the distribution of sulfated cholecystokinin octapeptide (CCK8-S) as a well preserved form during evolution in the zebrafish retina from 3days post hatching (dph) until adult stage, using immunohistochemistry in order to elucidate the potential role of this protein in the development and maintenance of normal retinal homeostasis. The cellular distribution of CCK in the retina was similar from 3 dph to 40days post fertilization (dpf) when immunoreactivity was found in the photoreceptors layer, in the outer plexiform layer, in the inner plexiform layer and, to a lesser extent, in the ganglion cell layer (GCL). Immunohistochemical localization at 50 dpf as well as in the adult stage was observed in a subpopulation of amacrine cells in the proximal inner nuclear layer, in the inner plexiform layer, in displaced amacrine cells and in retinal ganglion cells in the GCL. Our results demonstrate for the first time the occurrence of CCK in the zebrafish retina from larval to adult stage with a different pattern of distribution, suggesting different roles of CCK during retinal cells maturation.

Induction of mild enterocolitis in zebrafish Danio rerio via ingestion of Vibrio anguillarum serovar O1.

Vibrio anguillarum is the etiological agent of a fatal hemorrhagic disease known as vibriosis that affects a wide range of fish species, causing severe economic losses. Several investigations have been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease-prevention strategies. The aim of our study was to evaluate the most effective way to induce mild enteritis in a fish model, in order to allow further applications. The experiments were carried out using 2 methods of administration of V. anguillarum serotype O1 to adult zebrafish Danio rerio: via intraperitoneal injection and via ingestion of infected Artemia nauplii. The results showed that the intraperitoneal administration often caused massive fish death due to severe systemic involvement. In our experiments, the effect of intraperitoneal infection was evident 48 h post infection, with cumulative mortality within 7 d post infection with severe histopathological changes in kidney hematopoietic tissue and in the intestine. In contrast, oral infection via Artemia did not show systemic involvement and only a moderate degree of inflammatory influx of the mucosa, a partial recovery at 12 d post infection, and no mortality. For these reasons, oral infection with live food appears to be the most effective method to induce mild enteritis with a local inflammatory response.

Fine structure of spermatozoa in the blackspot sea bream Pagellus bogaraveo (Brünnich, 1768) with some considerations about the centriolar complex.

Scanning and transmission electron microscopy were used to investigate the fine structure of the sperm of the sparid fish Pagellus bogaraveo. The spermatozoon of P. bogaraveo belongs, like that of the other sparid fish, to the teleostean "type I" spermatozoon with the flagellar axis insert perpendicular to the nuclear fossa. It has an ovoidal head, a short, cylindrically shaped midpiece and a long tail region. The nucleus reveals a deep invagination (nuclear fossa), in which the centriolar complex is located, and a satellite nuclear notch shaped like a golf club. The two centrioles are perpendicular to each other and show a conventional "9+0" pattern. The distal centriole is attached to the nuclear envelope by means of basal feet and radial fibers made of electron-dense material. Below the basal plate, plasma membrane pinches in, and the necklace, a specialized connection joining axonemal doublets to the plasma membrane, is visible. The short midpiece houses one mitochondrion. The flagellum is perpendicularly and eccentrically with respect to the nucleus and contains the conventional "9+2" axoneme.

Ontogeny of the digestive tract in sharpsnout sea bream Diplodus puntazzo (Cetti, 1777).

The ontogeny of the digestive tract was studied histologically and histochemically in sharpsnout sea bream Diplodus puntazzo from hatching (0 DAH, Days After Hatching) until day 57 (57 DAH). At hatching, the digestive tract appeared as a histologically undifferentiated straight tube lying dorsally to the yolk sac. When the mouth opened at 3 DAH, the digestive tract was differentiated into buccopharynx, oesophagus, incipient stomach and intestine. The pancreas, liver and gall bladder were also differentiated at this stage and both the bile and pancreatic duct had opened into the anterior intestine. Active feeding began in 50% of larvae at 4 DAH, although permanence of yolk reserves until 7 DAH suggests a period of both endogenous and exogenous feeding. Nutrient absorption was first visible from 5 DAH, as colourless supra- and infranuclear vacuoles in the anterior intestinal mucosa, suggesting a lipid content, as well as supranuclear, eosinophilic vacuoles, containing protein, in the posterior intestinal mucosa. Early caecal development could be detected from 10 DAH, whereas gastric glands appeared at 30 DAH, indicating the transition from larval to juvenile stage and the acquisition of an adult mode of digestion. Goblet cells appeared in the digestive tract of sharpsnout sea bream larvae shortly after first feeding. The mucus content of goblet cells varied with the digestive region and, in the buccal cavity and oesophagus, also with the developmental phase. This study provides knowledge for better husbandry practices in the aquaculture industry, as well as for the implementation of future nutritional studies.

Fine structure of spermatozoa in the gilthead sea bream (Sparus aurata Linnaeus, 1758) (Perciformes, Sparidae).

Scanning and transmission electron microscopy were used to investigate the fine structure of the sperm of the sparid fish Sparus aurata L. The mature spermatozoon of gilthead sea bream belongs, like that of the other sparid fish, to a "type I" as defined by Mattei (1970). It has a spherical head which lacks an acrosome, a short, irregularly-shaped midpiece and a long cylindrical tail. The nucleus reveals a deep invagination (nuclear fossa) in which the centriolar complex is located. The two centrioles are approximately perpendicular to each other and show a conventional "9+0" pattern. The proximal centriole is associated with a cross-striated cylindrical body lying inside a peculiar satellite nuclear notch which appears as a narrow invagination of the nuclear fossa. The distal centriole is attached to the nuclear envelope by means of a lateral plate and radial fibres made of an electron-dense material. The short midpiece houses one mitochondrion. The flagellum is inserted perpendicularly into the base of the nucleus and contains the conventional 9+2 axoneme.

Pored domes in adrenocortical capillaries. An ultrastructural study in mammals.

The adrenocortical gland is one of the most vascularized organs of the mammalian body. It undergoes continuous morphological changes dynamically dependent upon special permeability conditions related to various physiological and physiopathological events. The adrenal cortex of different adult mammals (3 pigs, 2 mice, 3 sheep) was studied by means of scanning (SEM) and transmission (TEM) electron microscopy. As seen by SEM it is formed of polyhedral cells which delimit a characteristic continuous labyrinth system of intercellular lacunae occupied by sinusoid-like capillaries. The capillary wall is fenestrated and is lined by flattened endothelial cells with their nuclear part bulging into the capillary lumen. The fenestrae are round or oval pores measuring 50-100 nm in diameter. They are usually clustered to form sieve plates and characteristically present a thin membranous diaphragm. Irregular microelevations and dome-like projections are often seen. These structures are made of thin cytoplasmic plates interrupted by numerous small pores which show a sieve plate profile. The pored-domes on the nuclear portion seem to be identical in structure to those found in the thinner endothelial part; some of them appear to detach from the endothelial cell. The pored-domes are structurally comparable to those reported both in the renal glomerular endothelium and in liver sinusoids. These endothelial structures may be an expression of the high rate of filtration of these tissues and may be also related to the final step of the replacement of the sieve plates during endothelial regeneration.

Fine structure of spermatozoa in the common pandora (Pagellus erythrinus Linnaeus, 1758) (Perciformes, Sparidae).

Scanning and transmission electron microscopy were used to investigate the fine structure of the sperm of the Sparid fish Pagellus erythrinus L. The spermatozoon of pandora has a spherical head lacking an acrosome, a cone-shaped midpiece and a long tail. The midpiece houses a single mitochondrion. The centriolar complex lies inside the nuclear fossa and is composed of a proximal and a distal centriole which are arranged at right angles to each other. The flagellum is inserted medio-laterally into the head, contains the conventional 9+2 axoneme and possesses one pair of lateral fins. On the basis of its ultrastructural organization, the pandora sperm can be regarded as an evolved form of the primitive spermatozoon found in Teleosts. According to the morphological classification proposed by Mattei (1970), the sperm of pandora belongs to a "type I" designation, like that of the other Sparid fish.

A new morpho-functional classification of the Fallopian tube based on its three-dimensional myoarchitecture.

The recent direct observations, under scanning electron microscopy (SEM), of the three-dimensional architecture of myosalpinx in different mammals allows us classify salpinxes according to the myoarchitecture of their tubo-uterine junction (TUJ) and isthmus segments. Based upon the myoarchitecture of the outer wall of the TUJ we could find barrier-like species (rat and sow), sphincter-like species type a (rabbit and ewe) and sphincter-like species type b (cow and woman). The different architecture of TUJ can be explained by the different nature of the mating process. Based upon the myoarchitecture of the isthmus we could distinguish type 1 (rat) and type 2 (rabbit, ewe, sow, cow and woman) salpinxes. In the latter the close fusion of musculature deriving from the meso (extrinsic musculature) with the musculature of salpinx (intrinsic musculature) suggests the existence of a unique mesosalpinx contractile system. The myosalpinx is mostly made up of a single network of muscular fibers. Such a plexiform structure, owing to the uneven distribution of fibers, rather than producing a series of regular contraction waves, is more likely to generate random contraction waves. The random propagation of muscular network contraction may deform the plexiform wall of the myosalpinx causing the stirring of tubal contents. By such a stirring movement the contact between hormones and nutrients and the eggs or embryos is intensified, thus favoring a correct fertilization and early embryo development. Taken all together, these systematic results probably suggest an additional and rather new function for the musculature of the tube, namely to increase fertility in a large number of species.

Dp116, talin, vinculin and vimentin immunoreactivities following nerve transection.

The time course of the expression of Dp116, talin, vinculin and vimentin in rat sciatic nerve was investigated after experimental transection. Dp116 was still found at 5 days after experiment in some degenerating myelinated fibers of both proximal and distal stumps. The findings are consistent with the known preservation of electrical excitability of the distal nerve in the first days after injury. Some regenerating nerve fibers into the neuroma also expressed Dp116 at 25 and 40 days after nerve transection. Talin and vinculin markedly and diffusely immunostained the neuroma. Talin in the distal stump and vimentin in both proximal and distal stumps were found decreased during the time course of the experiment. Vinculin binding increased in the distal stump, due to a real overexpression or simply to a cross-reaction to degeneration products.

Microarchitecture of the cat testis with special reference to Leydig cells: a three-dimensional study by alkali maceration method and scanning electron microscopy.

Testes from adult cats were studied by means of parallel transmission and scanning electron microscopy (SEM) after NaOH digestion technique, which selectively removed connective tissues or cells. The testis is covered by a dense fibroconnective tunica albuginea that partially divides the organ in lobules by sending septa into the parenchyma. The lamina propria of the seminiferous tubules consisted of one or two rows of cells. The interstitium was made up of randomly arranged collagen bundles. The most significant feature was the numerous Leydig cells rich in lipid droplets and displaying epithelioid features. Following alkali digestion and SEM these cells showed a cord-like arrangement. The cords were formed by one or two closely apposed cells, in between which some labyrinthine or canalicular-like spaces were left that in some areas opened in wide perivascular spaces. This particular arrangement of Leydig cells and the labyrinthine intercellular spaces is very likely designed to improve cell secretion of hormones, facilitating their transport into the blood, as well as the traffic of fluids and metabolites. The present techniques allowed the visualization of a real three-dimensional testicular microarchitecture and microtopography, not detectable with other methods. Such a study may help to better highlight the testicular morphophysiology.

The architecture of the myosalpinx in the sow as revealed by scanning electron microscopy.

OBJECTIVE: To provide a definitive settlement of data on the architecture of myosalpinx in the sow in consideration of controversial data existing in literature. STUDY-DESIGN: To allow direct visualization of muscular architecture, segments of tube from fifteen sows were investigated by means of scanning electron microscopy after the removal of interstitial connective tissue with NAOH digestion. RESULTS: In the extramural portion of the tubo-uterine junction, in the isthmus and ampulla, the myosalpinx is mainly constituted by oblique bundles of variable length, which run around the tube and merge into the surrounding musculature, giving origin to a plexiform arrangement. In the ampulla the fibers join in short bundles variously oriented. CONCLUSION: The three-dimensional architecture of the sow myosalpinx consists of muscular bundles independent of one another which follow multiple spatial arrangements and form a complex network. Such a muscular structure is likely more suitable for stirring rather than pushing the embryo and gametes.

Morphometric comparison between contralateral sciatic nerves in the male and female rabbit.

Some morphometrical parameters of the axons making up the contralateral sciatic nerves, both in the male and female rabbits were calculated and compared by means of a Zeiss Vidas image analyser (Ober Kochen, Germany). The results show that the fibres constituting the left nerve have a greater mean diameter but a lower mean density that those constitution the right nerve. This suggests that the diameter of the myelinated fibres and the density of both the myelinated and unmyelinated fibres do not vary from male to female. On the other hand, the G ratio and the diameter of the unmyelinated axons do, since the nerves on the right side (in both sexes) have higher morphometric values, on average, than the contralateral ones.

The three-dimensional architecture of the myosalpinx in the cow as revealed by scanning electron microscopy.

The three-dimensional architecture of the myosalpinx in the cow has been investigated by means of scanning electron microscopy after removal of interstitial connective tissue with NaOH digestion. In the extramural portion of the tubo-uterine junction, in the isthmus and ampulla, the myosalpinx is made up of oblique bundles of variable orientation and length, which are loosely distributed in the tubo-uterine junction and densely packed in both isthmus and ampulla. These bundles intersect and merge into the surrounding musculature. Our observations demonstrate how myosalpinx consists mainly of bundles of muscular fibers independent one of another, which show a multiple spatial arrangement and form a complex network. Such a muscular architecture is likely more suitable for stirring rather than pushing the embryos and gametes through the tube.

The three-dimensional architecture of the myosalpinx in the sheep as revealed by scanning electron microscopy.

The three-dimensional (3-D) architecture of the myosalpinx in the sheep was studied by scanning electron microscopy after the removal of interstitial connective tissue with NaOH digestion. In the extramural portion of the tubo-uterine junction, the myosalpinx is made up of densely packed, circularly arranged superficial fibers and of innermost loose fiber bundles following an almost longitudinal course. In the isthmus and ampulla, the myosalpinx is constituted by oblique bundles of variable length, which run around the tube and merge into the surrounding musculature, giving rise to a plexiform arrangement. The present 3-D observations demonstrate that the sheep myosalpinx consists mainly of single muscular fibers and more complex bundles which show a multiple spatial arrangement and a plexiform distribution. Such a muscular architecture is more likely suited for stirring rather than pushing the embryos and gametes through the tube.

The three-dimensional architecture of the myosalpinx in the rat (Rattus norvegicus) as revealed by scanning electron microscopy.

The three-dimensional (3-D) architecture of myosalpinx in the rat has been investigated by means of scanning electron microscopy after microdissection and removing interstitial connective tissue with 6N NaOH digestion. In the extramural portion of tube-uterine junction the myosalpinx shows circularly arranged fibers originating from the uterus, together with oblique fibers typical for the salpinx, which occur more frequently in the deeper layers. As fibers approach the mucous folds they assume a plexiform arrangement, which is maintained through all tubal segments. In the isthmus surface fibers form wide muscle rings around the elbow of loops, peculiar to the rat tubal morphology. Surface fibers in the ampulla and pre-ampulla have an even circular course. Our 3-D results reveal that the muscular architecture of rat tube is mainly organized in concentric, monolayered shells with a plexiform arrangement tightly fastened together. Functionally, this muscular arrangement seems to be capable of stirring rather than pushing the embryo and gametes. Finally, such a plexiform network might work as a mechanism of "tube locking" in proximity of isthmic loops as well as at the level of the ampullary-isthmic junction.

The collagen skeleton of the cat testis. A scanning electron microscopy study after 2N-NaOH maceration.

Testes from adult cats were studied by means of scanning electron microscopy (SEM) after 2N-NaOH maceration method which selectively digests cells and tissue compounds. As seen by this technique, the testis appears covered by a dense fibroconnective tunica albuginea that divides partially the organ by sending septa into the parenchyma. The interstitium is made up by a rich connective tissue composed by randomly-arranged fine collagen bundles that clearly outline the spaces occupied by the seminiferous tubules. The basement membrane of the seminiferous tubules consists in two rough layers of collagen fibers that in some places leave small fenestrations. The three-dimensional arrangement of collagen fibers in the interstitium outlines a system of cavities likely to be lacunar spaces for Leydig cells and vascular imprints. Especially, the above chemical technique followed by deep observations under the SEM allowed a clear and complete view of the real three-dimensional microarchitecture of the connective tissue of the testis. Therefore, the collagen component was revealed to actually form a unique and complex skeleton for the whole organ. This three-dimensional figure closely follows indeed the classical histological compartmentalization of the testis with a better insight of its spatial microtopographical features. Moreover, this structural pattern is also likely to give rise to very fine morphofunctional subcompartmentalization, especially regarding the basement membrane of the seminiferous tubules and the lacunar spaces for Leydig cells including satellite vessels.

Localization of vinculin and talin at perineurial cells of human sural nerve.

Immunolocalization of spectrin, vinculin, talin, desmin and titin was investigated in human sural nerve. No binding for spectrin and titin was seen in any structure of the nerve. Antibody against desmin immunostained sporadic epineurial vessels only. Endoneurial and epineurial vessels were intensely positive for vinculin and talin. We found expression of vinculin and talin at the perineurial cells, using immunocytochemistry and gold immunoelectron microscopy. Since vinculin and talin are known to be involved in cell-cell and cell-extracellular matrix transmembrane connections, we propose that they, possibly together with other cytoskeletal proteins, may be implicated in the permeability barrier property of the perineurium. In pathological conditions, perineurium plays an as yet unknown role. Future studies are needed to investigate expression of vinculin and talin in neuropathies.

The three-dimensional organization of the smooth musculature in the ampulla of the human fallopian tube: a new morpho-functional model.

The three-dimensional organization of the smooth musculature around the human ampulla is revealed by means of scanning electron microscopy after NaOH maceration and ultrasonic microdissection of the interstitial connective tissue. The muscular wall of the ampulla appears as a continuous network of randomly anastomosed smooth muscle cell bundles that showed a multidirectional arrangement. The smooth muscle cell bundles modify their orientation along their course, intertwine repeatedly with each other and dichotomize, generating new bundles with a different orientation from that at the origin. These results demonstrate that the myosalpinx of the human ampulla is not organized into clear cut longitudinally, circularly or spirally arranged layers, as suggested in previous light microscopy studies. In contrast, the presence of a network of multidirectional smooth muscle cell bundles revealed in this study suggests that there is no morphological evidence for unidirectional peristalsis, and that the musculature is probably structurally designed to stir rather than push the tubal contents. These morphological findings better explain the random pattern of propagation of the contraction waves and the electrical impulses through the smooth musculature of the human ampulla, as postulated in early experimental physiological studies. Further, they suggest a specific function for the ampullar musculature which may not be only strictly related to tubal content transport.

The collagen fibrils arrangement in the Wharton's jelly of full-term human umbilical cord.

In the present study, a cell maceration/scanning electron microscopy method is employed in order to reveal the three-dimensional organization of the collagen fibrils in the Wharton's jelly of the human umbilical cord at term. By this method the jelly appears as a three-dimensional network of collagen fibrils that runs continuously from the amniotic membrane to the umbilical vessels. A diverse architecture of the collagen fibrillar network in the inner and outer region of the cord is observed suggesting an anatomical as well as a functional compartmentalization. In fact, the network is soft in the inner part and it is characterized by canalicular structures whereas in the outer region the collagen is dense and the network is characteristically composed by a sponge-like system of cavernous spaces. It is suggested that these cavities might store the ground substance and allow its diffusion during twisting or compression of the cord. Furthermore they may facilitate the flow of metabolites throughout the jelly from and to the umbilical vessels and the amniotic cavity.

An ultrastructural study of the fibromuscular capsule of the corpus luteum in mice.

The presence and distribution of contractile cells in mature corpora lutea (CL) of pregnant and normal cycling mice were studied by transmission (TEM) and scanning (SEM) electron microscopy. In the connective tissue surrounding the CL, SEM observations made after maceration in alkali, revealed long, flat, spindle-shaped elements arranged parallel to each other, forming a continuous capsule. TEM of these elements revealed ultrastructural features of mature smooth muscle cells (SMC), as well as structures intermediate between fibroblasts, fibrocytes and SMC. Discrete bundles of SMC were seen to exit the external capsule of the CL and to penetrate the luteal tissue. The extensive branches of these bundles of SMC appeared to make contact with numerous vessels. Therefore, three-dimensional reconstruction, based on TEM and SEM analysis, revealed that the SMC of the CL were organized such that they form a wide fibromuscular skeleton around and within the luteal mass. It is possible that pulsatile or tonic contraction of this structure may help to regulate the secretory activity of the luteal tissue by modifying the pressure inside the CL.