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The germline is a key feature of sexual animals and the ways in which it separates from the soma differ widely across Metazoa. However, at least at some point during germline differentiation, some cytoplasmic supramolecular structures (collectively called germ plasm-related structures) are present and involved in its specification and/or differentiation. The factors involved in the assembly of these granular structures are various and non-ubiquitous among animals, even if some functional patterns and the presence of certain domains appear to be shared among some. For instance, the LOTUS domain is shared by Oskar, the Holometabola germ plasm master regulator, and some Tudor-family proteins assessed as being involved in the proper assembly of germ granules of different animals. Here, we looked for the presence of LOTUS-containing proteins in the transcriptome of Ruditapes philippinarum (Bivalvia). Such species is of particular interest because it displays annual renewal of gonads, sided by the renewal of germline differentiation pathways. Moreover, previous works have identified in its early germ cells cytoplasmic granules containing germline determinants. We selected the orthologue of TDRD7 as a candidate involved in the early steps of germline differentiation through bioinformatic predictions and immunohistological patterning (immunohistochemistry and immunofluorescence). We observed the expression of the protein in putative precursors of germline cells, upstream to the germline marker Vasa. This, added to the fact that orthologues of this protein are involved in the assembly of germ granules in mouse, zebrafish, and fly, makes it a worthy study unit for investigations on the formation of such structures in bivalves. Supplementary Information The online version contains supplementary material available at 10.1007/s00418-021-01983-0.

Heteroplasmy is the presence of more than one type of mitochondrial genome within an individual, a condition commonly reported as unfavorable and affecting mitonuclear interactions. So far, no study has investigated heteroplasmy at protein level, and whether it occurs within tissues, cells, or even organelles. The only known evolutionarily stable and natural heteroplasmic system in Metazoa is the Doubly Uniparental Inheritance (DUI)—reported so far in ∼100 bivalve species—in which two mitochondrial lineages are present: one transmitted through eggs (F-type) and the other through sperm (M-type). Because of such segregation, mitochondrial oxidative phosphorylation proteins reach a high amino acid sequence divergence (up to 52%) between the two lineages in the same species. Natural heteroplasmy coupled with high sequence divergence between F- and M-type proteins provides a unique opportunity to study their expression and assess the level and extent of heteroplasmy. Here, for the first time, we immunolocalized F- and M-type variants of three mitochondrially-encoded proteins in the DUI species Ruditapes philippinarum, in germline and somatic tissues at different developmental stages. We found heteroplasmy at organelle level in undifferentiated germ cells of both sexes, and in male soma, whereas gametes were homoplasmic: eggs for the F-type and sperm for the M-type. Thus, during gametogenesis, only the sex-specific mitochondrial variant is maintained, likely due to a process of meiotic drive. We examine the implications of our results for DUI proposing a revised model, and we discuss interactions of mitochondria with germ plasm and their role in germline development. Molecular and phylogenetic evidence suggests that DUI evolved from the common Strictly Maternal Inheritance, so the two systems likely share the same underlying molecular mechanism, making DUI a useful system for studying mitochondrial biology.

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.

Which mitochondria are transmitted to progeny? A subset of inactivated mitochondria, or the most active ones? The need for a functional mitochondrial genome, protected from oxidative damage induced by mitochondrial activity, is at the base of the 'inactive theory' [1,2]. By contrast, for the 'active theory' mitochondrial activity is an indication of correct functioning, and it may be the phenotype under selection for the inheritance mechanism [3,4]. Inherited mitochondria appear to be a specific organelle subset. Mitochondria of the Balbiani body (Bb), a distinctive structure found in oocytes of many organisms, are taken up by embryo primordial germ cells (PGCs) together with germ line determinants [5]. The selective accumulation in the Bb of mitochondria with high inner membrane potential (Δψm) is consistent with the active theory [6]. Mitochondrial trafficking is tightly associated with the ability of mitochondria to produce ATP [7], so mitochondria with high Δψm, indicating mtDNA integrity, would have a better chance to attach to microtubules, be recruited into the Bb, and be preferentially carried into PGCs [4]. Moreover, it appears that the timing of germ line specification can influence this process. In animals with an early mechanism of germ line specification, the segregation of the most active mitochondria in gonadic presumptive blastomeres has to take place contextually, in some cases allowing sperm mitochondria segregation into PCGs [4]. In animals in which germ line specification is driven by inductive signals at a later developmental stage, if sperm mitochondria are not degraded they are segregated early into specific blastomeres, preventing their spread in the embryo [4]. In summary, Δψm may allow the most active mitochondria to reach specific locations, but the different timing of germ line specification can influence the outcome of the segregation mechanism.

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.

We used immunodeficient mice, whose dorsomedial olfactory region was permanently damaged by dichlobenil inoculation, to test the neuroregenerative properties of transplanted human adipose tissue-derived stem cells after 30 and 60 days. Analysis of polymerase chain reaction bands revealed that stem cells preferentially engrafted in the lesioned olfactory epithelium compared with undamaged mucosa of untreated transplanted mice. Although basal cell proliferation in untransplanted lesioned mice did not give rise to neuronal cells in the olfactory mucosa, we observed clusters of differentiating olfactory cells in transplanted mice. After 30 days, and even more at 60 days, epithelial thickness was partially recovered to normal values, as also the immunohistochemical properties. Functional reactivity to odorant stimulation was also confirmed through electro-olfactogram recording in the dorsomedial epithelium. Furthermore, we demonstrated that engrafted stem cells fused with mouse cells in the olfactory organ, even if heterokaryons detected were too rare to hypothesize they directly repopulated the lesioned epithelium. The data reported prove that the migrating transplanted stem cells were able to induce a neuroregenerative process in a specific lesioned sensory area, enforcing the perspective that they could become an available tool for stem cell therapy.

Down syndrome (DS) is a genetic condition caused by triplication of chromosome 21. Widespread neurogenesis reduction during brain development underlies the numerous neurological defects of DS. These defects start to manifest themselves at birth and worsen with age. However, unlike other brain functions, smell is impaired only at advanced life stages, suggesting preservation of olfactory bulb neurogenesis up to adulthood. To clarify this issue, in the current study we examined olfactory bulb (OB) neurogenesis and olfactory function by exploiting the Ts65Dn mouse, a widely used model of DS. We found that in young (15-day-old) Ts65Dn mice, in spite of a reduced proliferation rate in the subventricular zone (SVZ) in comparison with euploid mice, the number of neuroblasts traveling in the rostral migratory stream (RMS), en route to the OB, and the number of new granule neurons added to the OB were similar to those of euploid mice. In mid-age (13-month-old) Ts65Dn mice, however, the proliferation rate in the SVZ was more severely reduced in comparison with euploid mice and the number of neuroblasts in the RMS and new granule neurons added to the OB underwent a reduction. While in young Ts65Dn mice the olfactory function, assessed with the buried food pellet test, was similar to that of euploid mice, in mid-age mice it was significantly impaired. Taken together, results suggest that an age-related reduction in the renewal of OB granule cells may underlie the age-related smell impairment in DS.

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 intravenously (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. 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 cochlea, at 7 and 31 days following MSC transplantation. 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 and hearing loss 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 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 . These findings support the concept that transplanted MSC migrating to the damaged inner ear area provide conditions for the resumption of a damaged cochlea , emerging as a potential strategy for hearing rehabilitation.

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.

The herbicide dichlobenil selectively causes necrosis of the dorsomedial part of olfactory neuroepithelium (NE) with permanent damage to the underlying mucosa, whereas the lateral part of the olfactory region and the nasal respiratory mucosa remain undamaged. We investigated here whether human umbilical cord blood CD133+ stem cells (HSC) injected intravenously to nod-scid mice pretreated with dichlobenil may engraft the olfactory mucosa and contribute to the regeneration of the damaged NE. We tested HLA-DQα1 DNA and three human microsatellites (Combined DNA Index System) as indicators of engrafted cells, finding polymerase chain reaction evidence of chimaerism in various tissues of the host, including the olfactory mucosa and bulb, at 7 and 31 days following HSC transplantation. Histology, immunohistochemistry, and lectin staining revealed the morphological recovery of the dorsomedial region of the NE in dichlobenil-treated mice that received HSC, contrasting with the lack of regeneration in similarly injured areas as these remained damaged in control nontransplanted mice. FISH analysis, to detect human genomic sequences from different chromosomes, confirmed persistent engraftment of the regenerating olfactory area with chimeric cells. Electro-olfactograms in response to odorants, to test the functionality of the olfactory NE, confirmed the functional damage of the dorsomedial area in dichlobenil-treated mice and the functional recovery of the same area in transplanted mice. These findings support the concept that transplanted HSC migrating to the damaged olfactory area provide conditions facilitating the recovery from olfactory receptor cell loss. Disclosure of potential conflicts of interest is found at the end of this article.