Immunolocalization of Vasa, PIWI, and TDRKH proteins in male germ cells during spermatogenesis of the teleost fish Poecilia reticulata.

Vasa, PIWI and TDRKH are conserved components of germ granules that in metazoans are involved in germline specification and differentiation, as documented by mutational experiments in some model animals. So far, investigations on PIWI during spermatogenesis of fish has been limited to a few species, and no information is available for TDRKH, another protein involved in the piRNA pathway. In this study, the immunolocalization of these three germline determinants was analyzed in male gonads of the teleost fish Poecilia reticulata to document their localization pattern in the different stages of germ cell differentiation. To analyze their distribution pattern during the different stages of spermatogenesis we performed immunohistochemistry (IHC) and immunofluorescence (IF) assays using primary polyclonal antibodies after testing their specificity with Western Blot. Moreover, sections of testis stained with haematoxylin and eosin clarified the structural organization of P. reticulata testis, while the use of the confocal microscope and the nuclear staining clarified the different stages of germ cell differentiation during spermatogenesis. The results showed that Vasa, PIWI and TDRKH were specifically immunolocalized in the germ cells of P. reticulata, with no specific signal detected in Sertoli cells and in other somatic cells of the gonad. These markers were detected in all stages of differentiation from early spermatogonia to advanced spermatids. Vasa staining was the strongest in spermatogonia, and then decreases throughout differentiation. Instead, both PIWI and TDRKH staining increases during differentiation, and their distribution pattern, similar to what observed in the mouse, suggests their concerted participation in the piRNA pathway also in this fish.

Primate embryonic stem cells create their own niche while differentiating in three-dimensional culture systems.

Rhesus monkey embryonic stem cells (ESCs) (R366.4), cultured on a three-dimensional (3D) collagen matrix with or without human neonatal foreskin fibroblasts (HPI.1) as feeder cells, or embedded in the collagen matrix, formed complex tubular or spherical gland-like structures and differentiated into phenotypes characteristic of neural, epithelial and endothelial lineages. Here, we analysed the production of endogenous extracellular matrix (ECM) proteins, cell-cell adhesion molecules, cell-surface receptors, lectins and their glycoligands, by differentiating ESCs, forming a micro-environment, a niche, able to positively influence cell behaviour. The expression of some of these molecules was modulated by HPI.1 cells while others were unaffected. We hypothesized that both soluble factors and the niche itself were critical in directing growth and/or differentiation of ESCs in this 3D environment. Creating such an appropriate experimental 3D micro-environment, further modified by ESCs and modulated by exogenous soluble factors, may constitute a template for adequate culture systems in developmental biology studies concerning differentiation of stem cells.

Cell proliferation and growth-associated protein 43 expression in the olfactory epithelium in Poecilia reticulata after copper solution exposure.

This study investigated the regeneration in the olfactory mucosa of the teleostean fish Poecilia reticulata when returned to dechlorinated tap water after 4-day exposure to 30 microg/L of Cu(2+). The regeneration process in the olfactory tissue was examined in fishes at 0, 3, 6 and 10 days of recovery in well water. Jade B staining permitted to evaluate the rate of the damage which was especially extended to olfactory neurons. Immediately after the end of exposure, a massive mitotic activity in the basal region of the mucosa was detected by immunostaining with PCNA. After 3 days of recovery the nuclei of the newly formed cells had already finished their migration to the upper portion of the epithelium, and cellular division was much less intense. Simultaneously, immunoreactivity for the neural growth-associated phosphoprotein GAP-43 increased respect to control levels, revealing that the new differentiating PCNA-positive elements belonged to immature neurons. After 6 days in well water no mitotic activity was detected, while the GAP-43 labelling appeared particularly concentrated in the apical surface of the olfactory epithelium. After 10 days the aspect of the olfactory epithelium was almost identical to the control. The present results suggest that after 10 days regeneration seems to be complete and integrity of the tissue restored. Furthermore, the epithelium reconstitution does not show apparent divergence from other fishes or mammals.

The difficult primary total knee arthroplasty: a review.

Primary total knee arthroplasty (TKA) is a reliable procedure with reproducible long-term results. Nevertheless, there are conditions related to the type of patient or local conditions of the knee that can make it a difficult procedure. The most common scenarios that make it difficult are discussed in this review. These include patients with many previous operations and incisions, and those with severe coronal deformities, genu recurvatum, a stiff knee, extra-articular deformities and those who have previously undergone osteotomy around the knee and those with chronic dislocation of the patella. Each condition is analysed according to the characteristics of the patient, the pre-operative planning and the reported outcomes. When approaching the difficult primary TKA surgeons should use a systematic approach, which begins with the review of the existing literature for each specific clinical situation.

Identification of surface glycoconjugates in the olfactory system of turtle.

Lectin binding histochemistry was performed on the olfactory system of Pseudemys scripta to investigate the distribution and density of defined carbohydrate terminals on the cell surface glycoproteins of the olfactory receptors and their terminals in the olfactory bulbs. The lectin staining patterns indicate that the receptor cells of the olfactory mucosa are characterized by glycoconjugates containing alpha-D-galactose and N-acetyl-D-glucosamine terminal residues. The vomeronasal receptor cells contain instead alpha-N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and alpha-D-galactose residues. The results demonstrate that the vomeronasal receptor cells contain high density of alpha-N-acetyl-D-galactosamine sugar residues that are not expressed by receptor cells of the olfactory mucosa. The presence of specific glycoproteins, whose terminal sugars are detected by lectin binding, might be related to the chemoreception and transduction of the odorous message into a nervous signal or in the histogenesis of the olfactory system. In fact, the olfactory receptors are the only known neurons in the vertebrate nervous system that undergo a continual cycle of proliferation not only in developing animals but also in mature ones. Moreover the results show that BSA-I-B4, an alpha-D-galactosyl-specific isolectin, targets the terminal sugar residues in the ramified microglial cells.

Lectin characterization of the olfactory system in brachiopterygian fish.

Lectin binding was performed on the olfactory system of Polypterus and Erpetoichthys, the living genera of the subclass of Brachiopterygii. The lectin histochemical patterns and the Western-blot analysis indicate that the receptor cells of the olfactory mucosa are characterized by high density of specific glycoconjugate residues. The presence of glycoproteins, whose terminal sugars are detected by lectin binding, might be related to the reception of an odor stimulus and its transduction into a nervous signal or to the histogenesis of the olfactory system.

Histochemical study by lectin binding of surface glycoconjugates in the developing olfactory system of rat.

Lectin-binding histochemistry was used to investigate the distribution and density of defined carbohydrate sequences on the cell surface glycoproteins of the olfactory receptors of rat during development. The olfactory and vomeronasal receptors showed a positive labelling after biotinylated Lycopersicum esculentum lectin binding on embryonic day 16 (E16), while horseradish peroxidase-labelled Glycine max, Bandeiraea simplicifolia (BSA-I) and its B4 isomer BSA-I-B4 agglutinins started to label from day 18 (E18). From this stage onward there was a progressive increase in the intensity and number of lectin-binding olfactory receptors. The first lectin-labelled bundles of axons penetrating the olfactory bulb were observed on E20; from E21 it was possible to identify the first labelled glomeruli that, on the first day (P1) of postnatal life, showed a feature very similar to that of the adult. The lectin staining patterns indicate that during development there are differences in the kind and distribution of saccharidic moieties on the surface of rat olfactory neurons. The possible role of carbohydrate-containing glycoproteins in the reception and transduction of the odours and in the modulation of the cell-cell interactions in the olfactory system is discussed.

Lectin-binding patterns in the olfactory system of the lizard, Physignathus lesueurii.

Lectin binding histochemistry was performed on the olfactory system of Physignathus lesueurii to investigate the distribution and density of defined carbohydrate terminals on the cell-surface glycoproteins of the olfactory and vomeronasal receptor cells and their terminals in the olfactory bulbs. The lectin staining patterns indicate that the vomeronasal and olfactory receptor cells are characterized by glycoconjugates containing alpha-D-galactose and N-acetyl-D-glucosamine terminal residues. The presence of specific glycoproteins, whose terminal sugars are detected by lectin binding, might be related to the chemoreception and transduction of the odorous message into a nervous signal or to the histogenesis and development of the olfactory system. The olfactory and vomeronasal receptor cells are vertebrate neurons that undergo a continual cycle of proliferation not only during development but also in mature animals.

Lectin cytochemical localisation of glycoconjugates in the olfactory system of the lizards Lacerta viridis and Podarcis sicula.

To investigate the presence of defined carbohydrate moieties on the cell surface of the olfactory and vomeronasal receptor cells and the projections of the latter into the olfactory bulbs, a lectin binding study was performed on the olfactory system of the lizards: Lacerta viridis and Podarcis sicula. Both lizards showed a high lectin binding for N-acetyl-glucosamine in the sensory neurons. The lectin binding patterns in Lacerta indicated that the main olfactory system possessed a moderate density of N-acetyl-galactosamine residues and detectable levels of galactose ones. The vomeronasal system on the other hand contained a high density of N-acetyl-galactosamine moieties and a moderate density of glucosamine ones. In Podarcis the main olfactory system and vomeronasal organ contained respectively detectable and moderate levels of galactose residues. The expression of specific glycoconjugates may be associated with outgrowth, guidance and fasciculation of olfactory and vomeronasal axons.

Lectin binding to olfactory system in a shark, Scyliorhinus canicula.

Lectin histochemical studies were performed on the olfactory system of Scyliorhinus canicula to identify specific glycoconjugates on the cell surface of primary olfactory neurons. The olfactory receptor cells, the olfactory nerve fibers and their terminals in the bulbs were labelled with SBA, BSA-I and BSA-I-B4. The lectin staining patterns indicate that the membranes of small-spotted catshark olfactory neurons had glycoproteins with alpha-galactose residues. This carbohydrate moiety could be related to modulation of the cell-cell interactions in the olfactory system.

A histochemical study of the microglial cells in the brain of Salamandra salamandra by lectin binding.

Seven biotinylated lectins were utilized as histochemical markers for the study of microglial cells in the brain of Salamandra salamandra. It has been demonstrated that SBA, BSA-I, BSA-I-B4 and RCA120 label the microglial cells and, on the basis of the binding selectivity of the single lectins for specific carbohydrates, it was found that alpha-galactosyl residues are present in high density on the microglial membrane of S. salamandra. The reaction was localized not only to the ramified microglial cells, but also to other round cells without extensions, interpreted as ameboid microglial cells. The results show that lectin binding is a reliable molecular probe for identifying microglial cells in urodels.

[Morphological research on the dental pulp of the calf]. / Ricerche morfologiche nella polpa dentaria di vitello.

Morphological researches have been carried out through histological studies with fine and semifine sections at the electronic scan microscope on the dental pulp of calves. The general aim was to identify the antioxidant properties of pulp and study cellular density and microfibrillar architecture. The dental pulps of calves of 5-6 months taken immediately after slaughtering were used with immersions into the fixing liquid. The scan microscope showed fibroblasts in the deepest regions of the pulp in a stroma of collagenic fibres, not organised but scattered among the cells of the connective tissue. The odontoblasts presented well ordered one beside the others with well visible details such as the swollen basal portion, the nucleus and a very tight villosity. No clear interodontoblastic connections were evident. The electron transmission microscope revealed typical cells with histiocyte appearance with microfilaments evident in the cytoplasma (myofibroblasts) presenting a wide variety of cytoplasmatic interconnections with interdigitations.

[Survey of childhood handicaps in the Unità Sanitaria Locale No. 22 "Valtiberina"]. / Rilevamento degli handicaps infantili nella U.S.L. N. 22 "Valtiberina".

An epidemiological research about the infantile handicaps has been carried out to make a quantitative and etiological valuation of the existing cases in the social, economic and cultural sphere of U.S.L. number 22 "Valtiberina". Considering an infantile population of 5,063 subjects, 55 cases, corresponding to 1.08% have been examined; 32 of them are male children and 23 are female children. The research which has been carried out shows that there are more cases of psychological handicaps (35 cases out of 55) rather than of organic handicaps. It seems that it is possible to find a connection between this high percentage and the social, economic and cultural situation of the territory. At the same time an efficacious work has been carried out by the Social and Medical Services of U.S.L. and by the school to recognise, to point out, to diagnose and to study these pathologies. Such a study can result useful if we want to plan a campaign to prevent and to treat as soon as possible the infantile handicaps.

Ultrastructural study and cholinesterase activity of paired capillaries in the newt brain.

We have investigated the ultrastructural and histochemical (AChE and BuChE) features of intracerebral vessels in newt. The blood vessels of the newt brain are paired and end in a closed loop. The two limbs, each of them has delineate the lumen by one endothelial cell, are enclosed within a single basement membrane and are separated from each other by a thin intercapillary wall. The brain capillaries are un-fenestrated and the overlapping endothelial cells were connected by clefts. Ependymal astrocytes extensively ensheath the surface of brain capillaries, but the sheats are incomplete. Pericytes and mast cells are frequently sandwiched in the endothelial basal lamina. Microglial cells are also present adjacent to cerebral vessels. The newt cerebral capillaries are characterized by high levels of AChE. This enzyme is localized in the basal membrane and in extracellular spaces between the overlapping endothelial cells. The vascular walls are instead deprived of BuChE activity. The non-nervous role of cholinesterases is discussed.

Acetylcholinesterase activity of the mesencephalic trigeminal nucleus of Triturus cristatus carnifex.

This study deals with acetylcholinesterase activity in mesencephalic trigeminal nucleus neurons of the newt. These cells appear clearly positive to histochemical reaction. Electron microscope investigations revealed the fine localization of the enzyme in cell bodies. On the basis of the ultrastructural pattern of AChE distribution some considerations on the possible sites of utilization are discussed.

The pattern of acetylcholinesterase distribution in the normal and retino-deprived optic tectum of newt.

A light and electron microscopic histochemical study has been carried out on the localization of acetylcholinesterase in the optic tectum of newt. The ultrastructural analysis reveals that the pyriform neurons in the periventricular area are provided with the metabolic pathways for AChE synthesis. The enzyme, synthetized in the rough reticulum and conveyed in dendritic branches is prevailingly released in two reaction bands in the neuropil of the superficial fibrous zone. The ablation of one eye gives rise to both the degeneration of retinal tracts and terminals and to the disappearance of the most external reaction band. Some modifications in AChE distribution are also evident in the only reaction band therein. The results obtained are interpreted on the basis of some hypotheses about possible cholinergic mechanisms being active in the optic tectum of Urodela.

Lectin histochemical study of olfactory neurons in the eel.

Lectin histochemical studies were performed on paraffin embedded sections of the olfactory system of the eel to identify specific glycoconjugates on the surface of primary olfactory neurons. The olfactory receptors, the olfactory nerve fibres and their terminals in the bulbs were labelled with the lectins (SBA, BSA-I, BSA-I-B4 and DBA) HRP-conjugated or biotinylated. The lectin staining patterns indicate that the membrane of olfactory neurons of the eel had oligosaccharides with alpha-galactose and alpha-N-acetyl-D-galactosamine residues. These findings represent the demonstration of a molecular probe that recognizes specific sets of neurons. The identical histochemical features previously described in the olfactory neurons in amphibians suggest that these carbohydrate moieties might to related to modulation of the cell-cell interactions in the olfactory system of vertebrates.

Structural and spatial organisation of brain parenchymal vessels in the lizard, Podarcis sicula: a light, transmission and scanning electron microscopy study.

The structure and 3-dimensional pattern of the intraparenchymal microvessels in the brain of the lizard, Podarcis sicula, were studied by a combination of light and transmission electron microscopy as well as scanning electron microscopy of vascular corrosion casts. The angioarchitecture pattern consists of narrow hairpin-shaped microvascular loops of different length originating from the meningeal surface. In each loop, descending and ascending vessels are closely apposed to one another throughout their length and are connected by a narrow U-shaped terminal loop at their tips. The 2 limbs of the vessel pairs show a slightly different diameter but lack other structural differences. While some paired vessels give rise to a secondary hairpin-shaped loop with 2 possible branching patterns, there are no anastomotic intraparenchymal connections with analogous neighbouring structures. The cerebral vascular pattern of Podarcis sicula resembles that found in a few representatives of other vertebrate classes. All cerebral vessels structurally appear to be capillaries. Also the observations carried out on semithin and thin sections strongly support the capillary loop model in the Podarcis brain vasculature and, in accordance with studies carried out on various vertebrates, the general submicroscopic features of the brain capillary wall suggest the presence of an endothelial type blood-brain barrier.