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

Early germline differentiation in bivalves: TDRD7 as a candidate investigational unit for Ruditapes philippinarum germ granule assembly

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

Microtubule organization and nucleation in the differentiating ovarian follicle of the lizard Podarcis sicula

-We analyzed the organization of the microtubular cytoskeleton and the distribution of centrosomes at the different stages of differentiation of the ovarian follicle of the lizard Podarcis sicula by examining immunolabeled a- and g-tubulins using confocal microscopy. We observed that in the follicular epithelium the differentiation of the nurse pyriform cells is accompanied by a reorganization of the microtubules in the oocyte cortex, changing from a reticular to a radial pattern. Furthermore, these cortical microtubules extend in the cytoplasm of the connected follicle cells through intercellular bridges. Radially oriented microtubules were still more marked in the oocyte cortex during the final stages of oogenesis, when the yolk proteins were incorporated by endocytosis. The nucleation centres of the microtubules (centrosomes) were clearly detectable as g-tubulin immunolabeled spots in the somatic cells of the germinal bed. A diffuse cytoplasmic immunolabeling together with multiple labeled foci, resembling the desegregation of the centrosomes in early oogenesis of vertebrates and invertebrates, was revealed in the prediplotenic germ cells. In the cytoplasm of growing oocytes, a diffuse labeling of the g-tubulin antibody was always detectable. In the growing ovarian follicles, immunolabeled spots were detected in the mono-layered follicle cells which surrounded the early oocytes. In follicles with a polymorphic follicular epithelium, only the small follicle cells showed labeled spots. A weak and diffuse labeling was observed in the pyriform cells while in the enlarging intermediate cells the centrosomes degenerated like in the early oocytes. Our observations confirm that in P. sicula most of the oocyte growth is supported by the structural and functional integration of the developing oocyte with the pyriform nurse cells and suggest that their fusion with the oocyte results in an acquirement by these somatic cells of characteristics typical of the germ cells

Response of Olfactory Sensory Neurons to Mercury Ions in Zebrafish: An Immunohistochemical Study

Olfactory sensory neurons (OSNs) of fish belong to three main types: ciliated olfactory sensory neurons (cOSNs), microvillous olfactory sensory neurons (mOSNs), and crypt cells. Mercury is a toxic metal harmful for olfaction. We exposed the olfactory epithelium of zebrafish to three sublethal Hg2+concentrations. Molecular markers specific for the different types of OSNs were immunohistochemically detected. Image analysis of treated sections enabled counting of marked cells and measurement of staining optical density indicative of the response of OSNs to Hg2+exposure. The three types of OSNs reacted to mercury in a different way. Image analysis revealed that mOSNs are more susceptible to Hg2+exposure than cOSNs and crypt cell density decreases. Moreover, while the ratio between sensory/nonsensory epithelium areas is unchanged, epithelium thickness drops, and dividing cells increase in the basal layer of the olfactory epithelium. Cell death but also reduction of apical processes and marker expression could account for changes in OSN immunostaining. Also, the differential results between dorsal and ventral halves of the olfactory rosette could derive from different water flows inside the olfactory chamber or different subpopulations in OSNs

LA DOPPIA EREDITA\u300 UNIPARENTALE NEI MOLLUSCHI BIVALVI (DUI): UN UTILE MODELLO PER STUDIARE IL RUOLO DEI MITOCONDRI NELLA FORMAZIONE DELLA LINEA GERMINALE

Durante le prime divisioni embrionali, le cellule che ereditano una precisa struttura citoplasmatica detta nuage daranno origine alla linea germinale. Il nuage e\u300 formato da proteine ed RNA prodotti da geni altamente conservati nei metazoi e la sua colocalizzazione coi mitocondri suggerisce un loro ruolo nella formazione della linea germinale. Abbiamo determinato il posizionamento dei mitocondri dello spermatozoo durante l\u2019embriogenesi di Venerupis philippinarum, tramite analisi al TEM e marcatura in vivo. Abbiamo anche studiato la localizzazione di Vasa, una proteina determinante della linea germinale, ed il pattern delle tubuline in relazione al posizionamento dei mitocondri dello spermatozoo nell\u2019embrione in divisione. Durante lo sviluppo degli embrioni maschi, i mitocondri derivati dallo spermatozoo si aggregano in corrispondenza del primo piano di divisione. L\u2019analisi effettuata suggerisce un ruolo delle tubuline nel posizionamento di questi organelli. L\u2019esame morfologico al TEM ha inoltre permesso il riconoscimento di strutture tipiche coinvolte nella formazione della linea germinale. I microtubuli sono coinvolti nel posizionamento dei mitocondri dello spermatozoo nella linea germinale, e la colocalizzazione di Vasa suggeri- sce un ruolo di entrambi nella sua formazione. Strutture tipiche indicano un meccanismo condiviso per la determinazione delle cellule germinali primordiali, rendendo la DUI un utile modello per capire meccanismi biologici generali

Crypt cell markers in the olfactory organ of Poecilia reticulata: analysis and comparison with the fish model Danio rerio

Olfactory crypt neurons have been observed in several bony fishes and chondrichtyans. Although their morphology is uniform in all fish, very few is known about their antigenic properties, usually studied in zebrafish, but quite overlooked in other species. We tested in Poecilia reticulata (guppy) the two antibodies recognized to mark zebrafish crypt cells: while anti-S100 showed an immunohistochemical pattern comparable to what reported in zebrafish, anti-TrkA gave no signal. Western blot analysis revealed that S100-antiserum bound an antigen of expected weight, probably belonging to the S100 family. On the contrary, anti-TrkA detected more bands, but the protein/s might be too much diffused and/or diluted in the tissue to be detected with immunohistochemistry. Because of the high level of conservation in the Trk family proteins of the kinase domain, on which anti-TrkA was produced, we also tested anti-TrkB to exclude cross reactivity. Immunohistochemistry and Western blot confirmed that anti-TrkB displayed high specificity to its target and a different staining pattern compared to anti-TrkA, but, as anti-TrkA, it did not label crypt neurons. Finally, we documented that calretinin, a known marker of zebrafish ciliated and microvillous olfactory cells, in the guppy is expressed also by a subpopulation of S100-positive crypt neurons. These results reveal differences in antigen expression between zebrafish and guppy crypt cells. Together with the already known species-specific projections to the olfactory bulb and a heterogeneous panel of odorants, our findings support the possibility that crypt cells are functionally less uniform as supposed

MITOCHONDRIAL INHERITANCE AND GERMLINE DETERMINATION IN A BIVALVE SPECIES WITH DOUBLY UNI PARENTAL INHERITANCE (DUI).

Metazoan mitochondria are inherited maternally, but a noteworthy exception is Doubly Uniparental Inheritance (DUI). In DUI males, during zygote segmentation, sperm mitochondria (M) aggregate and end up in the primordial germ cells, thereafter being transmitted through sperm, while they degrade in females, which transmit egg mitochondria only (F). The molecular dynamics of this segregation are unknown, but they are thought to be similar to those selecting mitochondria to enter the germline of all metazoans (i.e. the “mitochondrial bottleneck”). Moreover, a role of M mitochondria in germline determination has been proposed for DUI. Using in vivo staining, we confirmed the M displacement pattern in Venerupis philippinarum, a well-known DUI species. We also evidenced a role of microtubules in the movement of M mitochondria in male embryos: actually, a microtubule bunch is formed next to the cleavage furrow, where sperm mitochondria are localized. Moreover, we searched by TEM for a cytoplasm cloud-like material called nuage, which is transferred to germ cells only and includes specific proteins (i.e. Vasa-like proteins), miRNAs and other post-transcriptional regulatory elements. The nuage is normally located in the chromatoid body (Cb) in spermatocytes, and in the Balbiani body (Bb) or mitochondrial cloud in developing oocytes. There are evidences that some of the Bb-associated mitochondria are taken up by primordial germ cells, suggesting a selection for a specific sub-population of mitochondria to be transmitted to offspring. We identified both Cb and Bb by TEM analyses on V. philippinarum gonads, but, surprisingly, two Bb were found. Further analyses are planned to verify whether this unusual characteristic is related to DUI or not. All that considered, DUI is an useful model system to study mitochondrial inheritance, because of the unique chance to analyze separately two distinct mitochondrial lineages, understanding where they localize and which ones enter the germline