Search

Mitochondrial heteroplasmy is the presence of more than one type of mitochondrial genome (mtDNA) within an individual, and in most of the reported cases it seems to be an unfavourable condition. For example, heteroplasmy in humans increases the risk of common age-related disorders as neurodegenerative diseases. The only evolutionarily stable and natural heteroplasmic system in Metazoa is the Doubly Uniparental inheritance (DUI), typical of some bivalves, in which two mitochondrial lineages are present: one transmitted through eggs (F-type) and the other through sperm (M-type). While females are homoplasmic for the F-type, males have M-type-homoplasmic gametes, but heteroplasmic soma. So far, no study has investigated mitochondrial heteroplasmy at the protein level, and no analysis has been performed to clarify if it is present at tissue, cell, or organelle level. We characterized the expression of three mitochondrially-encoded proteins (ND5, CYTB, COX3) in the DUI species Ruditapes philippinarum. Specific antibodies were produced to discriminate, with immunolocalization, between the F and M form of the same protein (variants highly divergent in DUI species) in germ line and somatic tissues of females and males in different developmental stages. Unexpectedly, M-type antibodies labelled mitochondria in female primordial stem cells (PriSCs), undifferentiated germ cells, and early oocytes, while mature eggs and female somatic cells expressed only the F-type. M- and F-type expression in male somatic tissues showed mitochondrial heteroplasmy at the cell level. Interestingly, F-labelled mitochondria were detected in male PriSCs and early germ cells. We hypothesize that PriSCs and undifferentiated germ cells carry both types of mtDNA, but during gamete maturation one of the two genomes disappears, and only the sexspecific mtDNA remains, maintaining the homoplasmy of the germ line. This would imply a selective degradation of mitochondria in adulthood.

The life cycle of many organism features an early moment in which cells split between two lineages, the somatic line and the germ line. These represent, respectively, the beginning of body architecture in the present generation, and the beginning of body architecture in the following generation. The separation of germ cells is a key point for the development of an organism and occurs by specification. This process has two main modes of action, the ancestral one, called epigenesis, and the derived one, called preformation. In this work we investigated bivalves (Mollusca Bivalvia), a clade in which preformation has evolved. Despite the relevance of these animals in marine ecosystems, their mechanism of germ line speci cation and seasonal gonad reconstitution lacks a detailed study. We thus analyzed bivalve germ line development by tracking the expression of VASA, a protein often used as germ line marker. We used specific antisera produced against the VASA homolog of Ruditapes philippinarum (Subclass Heterodonta, Family Veneridae). We compared the known developmental pattern of R. philippinarum to that of two species of the Subclass Pteriomorphia, Anadara kagoshimensis (Family Arcidae) and Crassostrea gigas (Family Ostreidae), and another species of the Subclass Heterodonta, Mya arenaria (Family Myidae). The immunohistological data obtained support for the two Subclasses a similar mechanism of primordial germ cells proliferation among the columnar epithelium of the gut. Indeed, given the taxonomic separation of the analyzed species, pertaining to two highly divergent clades, we suggest that the observed seasonal migration to the reconstituting gonad, associated with the timing of gonad maturation, could be a shared feature of bivalve molluscs. The comparative study of bivalve reproductive biology using the germ line determinant VASA can add details on the preformation mechanism and help to depict a more comprehensive picture of the role that this protein plays during cell specification.

Acoustic organ of European anchovy from Adriatic Sea was described through histological and immunohistochemical analyses, in order to define a new approach for future studies of quali-quantitative variations in specimens according to sex, age and distribution.

Quali-quantitative ultrastructural changes during ontogenesis of the sagitta otoliths of the inner ear European anchovy from the Adriatic Sea were analyzed by SEM. Crystalline arrangement area presented a complex modification of the crystal growth dimension (TL around 40 mm) and crystalline arrangement complexity. These changes in the ear could be related to ecological changes in mobility and feeding.

Chronic renal disease causes olfactory impairment through an unknown mechanism. Chondrichthyes, a class of vertebrates characterized by high plasma urea concentration, lack olfactory cilia and the G protein alpha subunit olfactory-type (Gαolf - marker of the olfactory receptor neurons in vertebrates) in their olfactory neuroepithelium (ONE). Despite of that, there is no data in the literature about a direct action of urea on the ONE. In this study, Danio rerio embryos were exposed to 75 mM urea concentrations from 24 to 96 hours post fertilization. The endocytosis of Neutral Red dye in the olfactory mucosa were detected in control and urea-exposed larvae as a marker of binding and internalization of the Olfactory Receptors. The presence and distribution of Gαolf were investigated in the olfactory epithelium of control and urea-exposed larvae, using a commercial antibody. As Nitric oxide (NO) has important roles in the ONE and urea is known to affect its production, we evaluated, in control and urea-treated larvae, the presence of NO synthase (NOS) in the ONE using immunohistochemistry and a histoenzymatic reaction. We also exposed D. rerio larvae to a NOS-inhibitory molecule, NG-L-Nitro-Arginine (L-NArg), comparing them to the control and urea-treated fishes. Both Gαolf and NOS were increased in urea- and L-NArg-treated fishes than in the control, while the Neutral Red internalization was unaffected. Different hypotheses can be made about the effect of urea on NO production in the olfactory epithelium and, more speculatively, also about the role of NO on the Gαolf synthesis/degradation. Further investigations are needed in order to understand the real effects of urea on the olfactory system of pathologically and physiologically uremic vertebrates.

Crypt cells are the most recently described morphotype of fish olfactory neurons, not found in tetrapods. These cells are less abundant than ciliated and microvillar ones in the sensory epithelium. They are suspected to be involved in reproductive behaviours, even if electrophysiological investigations demonstrated some sensitivity to amino acids but failed to identify response to pheromones. In adult Poecilia reticulata the number of crypt cells is different in the two sexes. Using immunohistochemical markers, Gαolf, calretinin and S100 to identify ciliated, microvillar and crypt cells, we compared the size of their populations in juvenile guppies at various steps from birth to 90 days, when their gonads reach maturity. We observed that the number of crypt cells is sex specific, with independent developmental dynamics between males and females, while ciliated and microvillar cell populations have similar pathways. In the guppy calretinin appeared to be expressed also in a subfraction of S100-positive crypt cells (calretinin(+)-S100(-) crypt cells were not observed). We examined the changes in the amount of calretinin(+)-crypt neurons during growth: cell density reflects the variations observed in the whole population of S100(+) crypt cells in both sexes in the first 45 days. However it decreases reaching 3 months of age, in contrast with the increment registered for calretinin(-)-S100(+) cells. We estimated the total number of calretinin(+) crypt neurons and we observed that it remains very close to the value calculated for all crypt cells until 21 days, but it only slightly rises in the next 2 months, while calretinin(-) crypt neurons sensibly increment their number. We hypothesize that this new identified phenotype, percentually more represented in the first weeks of life than later, could be involved in early non-reproductive function. The known sensitivity for amino acids might indicate a role in the perception of food-related stimuli. On the contrary, calretinin(-) crypt cells become predominant reaching sexual maturity, in line with their supposed involvement in reproductive activity.

Chronic renal disease is known to alter olfactory function. Of the uraemic toxins, high urea blood levels have been suspected to induce a hyposmic condition. However, with the exception of olfactometric analysis on human subjects, no other examinations have been conducted, particularly histomorphological observations of the olfactory organ. Even if hematic administration could better mimic pathologic conditions, as first approach we opted for direct nasal exposure, to minimize potential systemic interference. In this study, we describe the effects of environmental exposure to elevated concentrations of urea (7 g/L, 13.5 g/L and 20 g/L) on the sensory mucosa of zebrafish (considered a good predictive model of mammalian toxicity) in acute (48h and 96h) and chronic (30 days) toxicity tests. We observed that lamellae maintained structural integrity and epithelial thickness was slightly reduced only after 30 days at highest urea concentration. However the ratio between the volumes of sensory and non-sensory epithelial regions sensibly decreased during exposure. Pan-neuronal labelling with anti-Hu was negatively correlated with high doses of urea (13.5 g/L and 20 g/L), thus, we investigated whether distinct neuron subtypes were equally sensitive to the toxicant. Using densitometric analysis we evaluated and compared the immunolabelling of Golf -, TRPC2- and S100-expressing cells, as representatives of ciliated, microvillous and crypt neurons, respectively. The three subpopulations responded differently to urea. In particular, crypt cells were more severely affected than the other cell types, and Golf -immunoreactivity increased when fish were exposed to low doses of urea. The moderate sensory toxicity of urea seems to be in accordance with the olfactometric measurements reported in the literature. We counted also the dividing PCNA+-cells, whose density remained constant: it is possible that other non-neuronal cells substituted olfactory neurons preserving epithelial integrity.

Introduction Urea treatment in Danio rerio affects the expression or degradation of the G protein alpha subunit olfactory type (Gαolf), involved in olfactory transduction, causing an increased presence of this protein in the olfactory cilia (Ferrando et al, 2014; Bettini et al 2015). The effects of uremia on human olfactory functions have been evaluated in some clinical studies (Landis et al 2011). We here attempt to determine a specific action of urea, among all the uremic toxins, on the olfactory capability of a vertebrate.The OAT on CTRL showed a sudden burst of swimming. The U5m fishes showed a reaction to the stimuli similar to the CTRL. The U20m did not show an appreciable response to the stimuli. The unresponsiveness lasted during the 5 days of treatment. A recovery of 15 min. in clean water did not restore the responsiveness, while 1h was enough to show an appreciable response to the stimuli, still not equal to the CTRL. Although HIC is not quantitative, it was possible to observe that the Gαolf immunoreactivity (ir) was similar in the CTRL and in the U20m fishes, while it was more intense in the U5d and the U5dR1h adults.Urea altered the response to the OAT of D. rerio with a quite rapid and reversible effect. This effect is probably independent from a mere interference of urea on the receptor-ligand binding (Ferrando et al 2014). The Gαolf ir level after 5 days was higher than control and 5-20 min. This suggests that two different mechanisms would be involved in effect of urea on olfaction capability in the long and short term. Since in the rat striatum the activation of Gαolf cause its degradation (Hervé et al 2001) we speculate that in our experimental setting the Gαolf ir increase could depend by the lack of Gαolf activation after the receptor binding with odorant molecules or by an alteration of further post signal transduction cascade.

Objectives. Mesenchymal stem cells (MSC) are currently being investigated in numerous pre-clinical and clinical settings of regenerative medicine. We had previously reported (Revoltella et al., Cell Transplant. 2008; 17, 665-678) that human umbilical cord blood CD133+ stem cells transplanted IV into pre-irradiated nod-scid mice made permanently deaf by ototoxic treatment with kanamycin or intense sound, were able to engraft the cochlea and contribute to inner ear restoration, in vivo. We further investigated here whether human adult MSC derived from either bone marrow or fat (lipid suction), if injected IV to deafened nod-scid mice pre-treated with kanamycin , were able to engraft the damaged cochlea regaining hearing. Materials, Methods & Results. We tested HLA-DQa1 DNA and three human microsatellites (CODIS) as indicators of engrafted cells, finding polymerase chain reaction evidence of chimaerism in various tissues of the host, including the Organ of Corti in the damaged cochlea, at 7, 31 and 60 days following MSC transplant. After 31 days, histology, immunohistochemistry, and lectin staining confirmed the repair process and stimulation ex novo of morphological recovery in the inner ear, contrasting with the lack of morphological repair in control similarly injured but non-transplanted mice. FISH analysis, to detect human genomic sequences from different chromosomes, confirmed persistent engraftment of the regenerating inner ear with a very limited number of chimaeric cells. Dual color FISH analysis provided evidence of a limited positive engraftment in the inner ear and in other mouse tissues, also revealing small numbers of possible heterokaryons, probably resulting from unstable clones derived from fusion of donor with endogenous cells, up to 2 months following transplantation. Stem cells and differentiation pathways focused PCR arrays favoured to select MSC inducing the best response. Conclusions. These findings support the concept that transplanted MSC migrating to the damaged inner ear area may provide conditions for the resumption of a damaged cochlea , emerging as a potential strategy for hearing rehabilitation.