Differential nickel-induced responses of olfactory sensory neuron populations in zebrafish

The olfactory epithelium of fish includes three main types of olfactory sensory neurons (OSNs). Whereas ciliated (cOSNs) and microvillous olfactory sensory neurons (mOSNs) are common to all vertebrates, a third, smaller group, the crypt cells, is exclusive for fish. Dissolved pollutants reach OSNs, thus resulting in impairment of the olfactory function with possible neurobehavioral damages, and nickel represents a diffuse olfactory toxicant. We studied the effects of three sublethal Ni2+ concentrations on the different OSN populations of zebrafish that is a widely used biological model. We applied image analysis with cell count and quantification of histochemically-detected markers of the different types of OSNs. The present study shows clear evidence of a differential responses of OSN populations to treatments. Densitometric values for G\u3b1 olf, a marker of cOSNs, decreased compared to control and showed a concentration-dependent effect in the ventral half of the olfactory rosette. The densitometric analysis of TRPC2, a marker of mOSNs, revealed a statistically significant reduction compared to control, smaller than the decrease for G\u3b1 olf and without concentration-dependent effects. After exposure, olfactory epithelium stained with anti-calretinin, a marker of c- and mOSNs, revealed a decrease in thickness while the sensory area appeared unchanged. The thickness reduction together with increased densitometric values for HuC/D, a marker of mature and immature neurons, suggests that the decrements in G\u3b1 olf and TRPC2 immunostaining may depend on cell death. However, reductions in the number of apical processes and of antigen expression could be a further explanation. We hypothesize that cOSNs are more sensitive than mOSNs to Ni2+ exposure. Difference between subpopulations of OSNs or differences in water flux throughout the olfactory cavity could account for the greater susceptibility of the OSNs located in the ventral half of the olfactory rosette. Cell count of anti-TrkA immunopositive cells reveals that Ni2+ exposure does not affect crypt cells. The results of this immunohistochemical study are not in line with those obtained by electro-olfactogram

Analysis of clasp2 Transcription Pattern in Male Germ Cells during Spermatogenesis: A Comparative Study in Zebrafish (Danio rerio) and Guppy (Poecilia reticulata)

Cytoplasmic linker-associated protein-2 (CLASP2) is a member of the CLIP-associating proteins (CLASPs) family involved in the structure and function of microtubules and Golgi apparatus. Several studies performed using different mammalian and non-mammalian model organisms reported that CLASP2 controls microtubule dynamics and the organization of microtubule networks. In Drosophila and mice, an important role of CLASP2 during the development of germ cell lines has been uncovered. However, no study has clearly defined its role during fish germ cell differentiation. In the present study, we used two excellent aquatic animal models among teleost fish: zebrafish (Danio rerio) and guppy (Poecilia reticulata). Using qPCR, we found that the clasp2 transcript level is significantly high in the testis of both fish. Then, by in situ hybridization, we localized the clasp2 transcript in the spermatozoa of zebrafish and the spermatozeugmata of guppy. Our data suggest a potential role for this gene in the last stage of spermiogenesis in fis

Doubly Uniparental Inheritance of Mitochondria As a Model System for Studying Germ Line Formation

BACKGROUND: Doubly Uniparental Inheritance (DUI) of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI). DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos. METHODOLOGY/PRINCIPAL FINDINGS: We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow. CONCLUSIONS/SIGNIFICANCE: In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown, they could be a variation of the mechanism regulating the mitochondrial bottleneck in all metazoans

Differentiating male germ cells during spermatogenesis of a reptile

Male germ cells differentiation of Podarcis sicula was analyzed at confocal microscope with anti-alpha tubulin and TO-PRO3 nuclear dy

EXPRESSION OF PL10 PROTEIN IN MALE GERM CELLS OF PODARCIS SICULA (REPTILIA, LACERTIDAE) DURING THE REPRODUCTIVE-CYCLE PHASES

PL10 is a DEAD-box protein that functions as ATP-dependent RNA helicase. DEAD-box proteins are involved in many processes related to RNA metabolism. In particular, some of them regulate the translation of multiple mRNAs allowing specific molecules to direct the process of differentiation in male and female germ cells. VASA, PL10, and P68 DEAD-box proteins are members of three closely related subfamilies: Vasa, in several animals, is exclusively expressed to the germ cell lineage, while PL10 and P68 expression are documented also in somatic tissues. In mouse, three PL10 related genes are identified: DDX3 and DDX3Y located on chromosomes X and Y and expressed in germ and somatic cells and the autosomal retrogene PL10 specifically expressed in testicular tissues at the pachytene stage of male meiosis. In non-mammal animals, PL10 is the sole member of the subfamily and in Xenopus and in Danio rerio PL10 expression is well documented in male and female germ cells but also in most embryonic and adult tissues. At the moment, there is no available data regarding PL10 in reptiles. We isolated Podarcis sicula PL10 homologue gene (Ps-PL10), developed a specific antibody (anti-Ps-PL10) and analyzed, at confocal microscopy, the expression pattern of PL10 during spermatogenesis in all phases of adult reproductive cycle of P. sicula (1- full gonadal activity in the spring, 2- complete regression in the summer, and 3- slow autumnal recrudescence without spermiation) with the aim to identify when PL10 is expressed during the differentiation process of male germ cells. Moreover, to verify if this protein is also expressed in the somatic tissue of the testis, this analysis was extend to young testes when the walls of the seminiferous tubules were forming. The obtained results show that PL10 expression is present from spermatocytes I to spermatids. During full gonadal activity (spring), PL10 expression increases in spermatids in the final steps of spermiogenesis, the strong immunostaining accumulates in the cytoplasm of residual bodies. No stained is observed in spermatogonia and in spermatozoa as well as in the somatic cells of seminiferous epithelium in all phases of the reproductive cycle analyzed. The specific expression of PL10 in meiotic cells suggests that this protein is involved in the differentiation of germ cells, in particular, during the differentation process from spermatid to spermatozoa, given the massive expression observed in the cytoplasm of residual bodies

PL10 DEAD-Box Protein is Expressed during Germ Cell Differentiation in the Reptile Podarcis sicula (Family Lacertidae)

Among genes involved in the regulation of germ cell differentiation, those of DDX4/Vasa and the Ded1/DDX3 subfamilies encode for DEAD-box ATP-dependent RNA helicases, proteins involved in many mechanisms related to RNA processing. For the first time in reptiles, using specific antibodies at confocal microscopy, we analysed the localization pattern of a Ded1/DDX3 subfamily member in testis and ovary of Podarcis sicula (Ps-PL10) during the reproductive cycle. In testis, Ps-PL10 is expressed in the cytoplasm of spermatocytes and it is not detected in spermatogonia. Differently from Ps-VASA, in round spermatids, Ps-PL10 is not segregated in the chromatoid body but it accumulates in the cytoplasm of residual bodies, and mature spermatozoa are unstained. These observations suggest that in males, Ps-PL10 (1) is involved in spermatogenesis and (2) is then eliminated with residual bodies. In the ovary, Ps-PL10 is present with granules in the cytoplasm of early meiotic cells of the germinal bed (GB), while it is not present in oogonia and somatic cells of the GB stroma. In follicular cells of ovarian follicles, Ps-PL10 expression starts after their fusion with the oocyte. Numerous Ps-PL10 spots are visible in pyriform (nurse-like) cells concomitantly with the protein accumulation in the cytoplasm of differentiating oocyte. In pyriform cells, Ps-PL10 spots are present in the cytoplasm and nuclei, as observed for Ps-VASA, and in the nucleoli, suggesting for Ps-PL10 a role in rRNA processing and in the transport of molecules from the nucleus to cytoplasm and from nurse cells to the oocyte

EXPRESSION OF MITOCHONDRIAL AND NUCLEAR ELEMENTS DURING PRIMORDIAL GERM CELL PROLIFERATION AND DIFFERENTIATION IN THE BIVALVE SPECIES RUDITAPES PHILIPPINARUM (BIVALVIA VENERIDAE)

The first representatives of germ line to appear in the embryo are primordial germ cells (PGCs), and some conserved proteins play a role in their determination and specificity (e.g.: VASA). Also mitochondria have an active role in germ line development and to further understand this role, it is fundamental to deal with their inheritance dynamics. Metazoa generally experience strictly maternal inheritance (SMI) of mitochondria. The only known exception is represented by some bivalve molluscs that show doubly uniparental inheritance (DUI). Recently, a novel protein (RPHM21) was shown to be encoded by the male-transmitted mtDNA of the DUI species Ruditapes philippinarum. In silico analyses suggested its viral origin, and we hypothesized that the endogenization of a viral element provided sperm mitochondria with the ability to invade male germ line, thus being transmitted to the progeny. We investigated the dynamics of germ line development in relation to RPHM21 and expression patterns. We used specifically produced antibodies to detect germ cell proliferation and to compare the localization of VASPH (R. philippinarum VASA homolog) and RPHM21. Bivalves show a mechanism of seasonal gonad production, in which the gonad is re-adsorbed after spawning, and rebuilt de novo at the beginning of the subsequent reproductive season. Based on VASPH staining, we determined that in R. philippinarum the proliferation of PGCs begins among the simple columnar epithelium (batiprismatic cells) of the gut. Then PGCs appear to migrate in the connective tissue. Germ cells at initial stage of gametogenesis were also VASPH-stained. Since adult specimens showed an intense proliferation of PGCs in the gut, we deduced that this reservoir of PGCs migrate from the intestinal epithelium and reach the presumptive gonad localization where they rebuild the gonad at every reproductive season. Both VASPH and RPHM21 were localized in male PGCs, at one side of the cell cytoplasm, but while VASPH was detected in all PGCs, RPHM21 appeared to be expressed only in a subpopulation of them. Since RPHM21 was detected in all spermatozoa, we propose that the germ cells expressing it could gain advantage over the others during spermatogenesis. Specifically, RPHM21 might have a role in activation and proliferation of male PGCs, or might be involved in a process of active elimination of germ cells not expressing it, something that would resemble a meiotic drive

Localization and dynamics of primordial germ cells in the bivalve species Ruditapes philippinarum

no abstract provide

Endogenized mitochondrial gene expressed in the germ line

Germ line is the cell lineage that transmits genes from generation to generation in sexually reproducing animals. Mitochondria, among many other factors, are responsible of the specification of primordial germ cells (PGCs), and their inheritance dynamics are relevant to understand this process. We studied these issues in the Manila clam Ruditapes philippinarum, a species with Doubly Uniparental Inheritance (DUI) of mitochondria, in which a novel protein (RPHM21) is encoded by the male-transmitted mtDNA and expressed in spermatozoa. RPHM21 was predicted in silico to be of viral origin \u2013 namely an endogenized residual of an ancestral infection. We analyzed mitochondrial transcription and expression patterns using qPCR and antibodies targeting the germ line marker VASPH (VASA homolog) and RPHM21. qPCR allowed to discriminate the phases in which vasph begins to be transcribed, and VASPH immunolabelling allowed to identify PGCs. Both targets were localized in the PGCs of males, but while VASPH was detected in all PGCs, RPHM21 appeared to be expressed only in a subpopulation. In R. philippinarum we identified a source of germ cell precursors that migrate from the gut and reach the presumptive gonad localization, where they rebuild the gonad at every reproductive season. Since RPHM21 might have a role in activation, proliferation, and migration of male PGCs, we propose that the germ cells expressing RPHM21 could gain advantage over others during spermatogenesis. The investigation of how co-option of foreign elements modifies existing biological pathways is fundamental to assess the impact of such events on the evolution of new developmental features

Differential response of olfactory sensory neuron populations to copper ion exposure in zebrafish

The peripheral olfactory system of fish is in direct contact with the external aqueous environment, so dissolved contaminants can easily impair sensory functions and cause neurobehavioral injuries. The olfactory epithelium of fish is arranged in lamellae forming a rosette in the olfactory cavity and contains three main types of olfactory sensory neurons (OSNs): ciliated (cOSNs) and microvillous olfactory sensory neurons (mOSNs), common to all vertebrates, and a third minor group of olfactory neurons, crypt cells, absent in tetrapods. Since copper is a ubiquitously diffusing olfactory toxicant and a spreading contaminant in urban runoff, we investigated the effect of low copper concentration on the three different OSNs in the olfactory epithelium of zebrafish, a model system widely used in biological research. Image analysis was applied for morphometry and quantification of immunohistochemically detected OSNs. Copper exposure resulted in an evident decrease in olfactory epithelium thickness. Moreover, after exposure, the lamellae of the dorsal and ventral halves of the olfactory rosettes showed a different increase in their sensory areas, suggesting a lateral migration of new cells into non-sensory regions. The results of the present study provide clear evidence of a differential response of the three neural cell populations of zebrafish olfactory mucosa after 96 h of exposure to copper ions at the sublethal concentration of 30 \ub5g/L-1. Densitometric values of cONS, immunostained with anti-G \u3b1olf, decreased of about 60% compared to the control. When the fish were transferred to water without copper addition and examined after 3, 10 and 30 days, we observed a partial restoration of anti-G\u3b1olf staining intensity to normal condition. The recovery of cOSNs appeared sustained by neuronal proliferation, quantified with anti-PCNA immunostaining, in particular in the early days after exposure. The densitometric analysis applied to mOSNs, immunostained with anti-TRPC2, revealed a statistically significant decrease of about 30% compared to the control. For cOSNs and mOSNs, the decrement in staining intensity may be indicative of cell death, but reduction in antigen expression may not be excluded. In the post-exposure period of 1 month we did not find recovery of mOSNs. We hypothesize that cOSNs are more sensitive than mOSNs to copper treatment, but also more prompted to tissue repair. Anti-TrkA-immunopositive crypt cells appeared not to be affected by copper exposure since statistical analysis excluded any significant difference between the control and treated fish. Comparative studies on OSNs would greatly enhance our understanding of the mechanisms of olfaction