Francesco Aniello

Occurrence and neuroendocrine role of D-aspartic acid and N-methyl-D-aspartic acid in Ciona intestinalis

Probes for the occurrence of endogenous D‐aspartic acid (D‐Asp) and N‐methyl‐D‐aspartic acid (NMDA) in the neural complex and gonads of a protochordate, the ascidian Ciona intestinalis, have confirmed the presence of these two excitatory amino acids and their involvement in hormonal activity. A hormonal pathway similar to that which occurs in vertebrates has been discovered. In the cerebral ganglion D‐Asp is synthesized from L‐Asp by an aspartate racemase. Then, D‐Asp is transferred through the blood stream into the neural gland where it gives rise to NMDA by means of an NMDA synthase. NMDA, in turn, passes from the neuronal gland into the gonads where it induces the synthesis and release of a gonadotropin‐releasing hormone (GnRH). The GnRH in turn modulates the release and synthesis of testosterone and progesterone in the gonads, which are implicated in reproduction.

Identification and developmental expression of Ci-msxb: a novel homologue of Drosophila msh gene in Ciona intestinalis.

We report the cloning and expression pattern of Ci-msxb the second Ciona intestinalis homeobox gene homologue to the Drosophila muscle segment homeobox (msh) gene. Northern blot analysis showed that transcripts appeared at gastrula stage, peaked in the early tailbud and decreased during the tailed stages. Whole mount in situ hybridization showed that the Ci-msxb expression first is detected at 110 cell-stage in the blastomeres that are precursors of different tissue (muscle, spinal cord, endodermal strand, brain, mesenchyme, pigmented cells and primordial pharynx). Transcript level declined in mesoderm cells after the completion of gastrulation, but mRNAs were still present in the folding neural plate during neurulation and in the pigmented cells. Later, at larval stage, transcripts were present around the otolith and ocellus, in a restricted part of the nervous system and in the primordial pharynx; the gene expression was conserved after metamorphosis in the juvenile.

Two Neuron Clusters in the Stem of Postembryonic Zebrafish Brain Specifically Express relaxin-3 Gene: First Evidence of Nucleus Incertus in Fish

We examined the spatial expression of the relaxin‐3 gene in the developing zebrafish brain, one of the vertebrate model systems in which this gene has been identified. Until the pharyngula stage, the gene is expressed diffusely in the brain, where, starting at about 40 hpf, the transcripts appear restricted in a midbrain cell cluster of the periaqueductal gray. Later, at 72 hpf, the transcripts are still evident in that cluster and distributed in a larger cell number; at this stage, the gene is also expressed posteriorly, in a smaller cell group that, as we report for the first time, could be homologous to mammalian nucleus incertus. The gene expression persists in both cell clusters at 96 hpf. This pattern indicates both conserved and divergent expression features of the relaxin‐3 gene among developing zebrafish and rat brains, where only scattered cells express the gene in the periaqueductal gray. Developmental Dynamics 237:3864–3869, 2008.

First evidence of prothymosin α in a non-mammalian vertebrate and its involvement in the spermatogenesis of the frog Rana esculenta ☆

A cDNA clone encoding for a Prothymosin alpha (Prot-alpha) has been isolated and characterized from the testis of the frog Rana esculenta. Frog Prothymosin alpha (fProt-alpha) predicted a 109 amino acid protein with a high homology to the mammalian Prot-alpha. fProt-alpha contains 28 aspartic and 25 glutamic acid residues and presents the typical basic KKQK amino acid sequence in the close carboxyl terminal region. Northern blot analysis revealed that fProt-alpha is highly expressed in the testis. A different expression of fProt-alpha transcript was found during the frog reproductive cycle with a peak in September/October in concomitance with germ cell maturation, strongly suggesting a role for this protein in the testicular activity. In situ hybridization evidenced that the only germ cells expressing fProt-alpha are the primary and secondary spermatocytes; in addition, the hybridization signal was stronger in the October testis. Taken together, our findings indicate that fProt-alpha might contribute to the efficiency of frog spermatogenesis with a role during the meiosis. This study is the first report on the isolation and characterization of a Prot-alpha in a non-mammalian vertebrate. In addition, our results indicate that the testis of the frog R. esculenta may be a useful model to increase the knowledge concerning the physiological role of Prot-alpha in vertebrates.

Duplicated zebrafish relaxin‐3 gene shows a different expression pattern from that of the co‐orthologue gene

Relaxin‐3 (Rln3) is thought to function as a neurotransmitter mainly produced in the mammalian nucleus incertus and is involved in different neural processes; among them, the stress response and food intake. Here, we report the expression pattern of the duplicated zebrafish rln3b gene and compare it to the previously analyszd spatial expression pattern of the rln3a gene. Both genes, during the embryogenesis and in the adult fish, are active and show relevant differences in their expression patterns. rln3b is diffusely expressed in the brain until the pharyngula period, when, at 48 h postfertilization (hpf), the expression becomes restricted to the periaqueductal gray, where it persists also at later developmental stages. No expression was observed in the nucleus incertus cells that express the rln3a gene from 72 hpf. In the adult, both genes are expressed in brain, but only rln3b transcript is revealed in testis at the similar expression level, whereas in the other analyzed tissues the transcript levels are lower or absent. Both the putative mature protein sequences are highly conserved, this feature and their differential expression patterns might indicate a sub‐functionalization during evolution with the consequent retention of the two paralogues genes.

Experimentally nonylphenol-polluted diet induces the expression of silent genes VTG and ERα in the liver of male lizard Podarcis sicula

Endocrine Disruptor Chemicals (EDCs) with estrogen-like properties i.e nonylphenol (NP) induce vitellogenin (VTG) synthesis in males of aquatic and semi-aquatic species. In the oviparous species VTG is a female-specific oestrogen dependent protein. Males are unable to synthesize VTG except after E2 treatment. This study aimed to verify if NP, administered via food and water, is able to induce the expression of VTG even in males of vertebrates with a terrestrial habitat such as the lizard Podarcis. By means of ICC, ISH, W/B and ELISA we demonstrated that NP induces the presence of VTG in the plasma and its expression in the liver. VTG, undetectable in untreated males, reaches the value of 4.34 μg/μl in the experimental ones. Expression analysis and ISH in the liver showed that an NP-polluted diet also elicits the expression of ERα in the liver which is known to be related to VTG synthesis in Podarcis.

Differential Expression of Duplicated Genes for Prothymosin Alpha During Zebrafish Development

We show that ptma, a single copy gene found in all organisms investigated so far, is duplicated in zebrafish. The two genes, ptmaa and ptmab, are individually controlled as indicated by their different expression patterns during embryonic development. Only the ptmab transcript is observed at 4 and 8 hpf of development in all embryonic cells, whereas both genes are expressed at later stages as revealed by in situ hybridization studies. In most cases, the two genes are expressed in the same territories, but only the ptmaa transcript was found in the trigeminal ganglion and in endodermal pouches. In the eye, at 72 hpf, the ptmaa and ptmab transcripts were found in amacrine cells, whereas only the ptmab transcript appeared in horizontal cells. The existence of two prothymosin genes indicates that their function in cell proliferation and differentiation is more complex in fishes than in mammals. Developmental Dynamics 237:1112–1118, 2008.

Evidence for the involvement of prothymosin α in the spermatogenesis of the frog Rana esculenta

Prothymosin alpha (PTMA) is a small acidic protein abundantly and ubiquitously expressed in mammals and involved in different biological activities. Until now, its specific function in spermatogenesis has never been properly investigated. Recently, the isolation of a cDNA encoding for PTMA from the testis of the frog Rana esculenta has been reported: ptma transcript is highly expressed throughout the frog reproductive cycle, peaking in September/October, in concomitance with the germ cell maturation; it is specifically localized in the cytoplasm of primary and secondary spermatocytes and, at a lower level, in the interstitial compartment, in Leydig cells.In this article we support the involvement of PTMA in the meiotic phases of frog spermatogenesis. The expression of ptma mRNA increases in the testis of frogs treated with the antiandrogen cyproterone acetate, which blocks the II meiotic division and induces an increase in SPC cysts; on the contrary, it highly decreases in the testis of animals kept at 4 degrees C and treated with human corionic gonadotropin, in concomitance with the induced block of spermatogenesis and the disappearance of meiotic cells in the tubules. Furthermore, for the first time we have also evidenced by immunohistochemistry the expression of PTMA in the nuclei of secondary spermatocytes, spermatids, and spermatozoa, as well as in the cytoplasm of interstitial Leydig cells. Taken together our data suggest for an important role of PTMA in germ cell maturation and/or differentiation during R. esculenta spermatogenesis.

Expression of Prothymosin alpha during the spermatogenesis of the spotted ray Torpedo marmorata

In this study, we show that Prothymosin alpha (Ptma), a small, unfolded, negatively charged protein, is present in the cartilaginous fish Torpedo marmorata. The ptma gene is functional and peculiarly controlled during the male spermatogenesis of T. marmorata, as revealed by in situ hybridization and by immunocytochemistry studies. The data show that the ptma transcript is present in stage-specific germ cells, i.e. spermatocytes II and round spermatids. The Ptma protein is detectable in spermatocytes II, in round and elongated spermatids as well as in spermatozoa before their release from cysts, while it is not evident in spermatozoa located in male genital tracts. The ptma transcript and protein are also evident in some Leydig cells, located among maturing cysts containing meiotic and differentiating male cells. No expression for ptma is observed within Sertoli cells. Furthermore, immunolocalization procedures demonstrate that the protein is preferentially localized in the cytoplasm, whereas a nuclear localization is observed in round and elongated spermatids. The possibility that Ptma is involved in testis activity is discussed.

Connexin 43 expression in the testis of the frog Rana esculenta.

Testicular cell-to-cell interactions play a key role in the regulation of spermatogenesis. In the testis, cell contacts are mediated through several mechanisms, including paracrine and direct contacts depending on gap junctional pathways. Gap junctions require connexin (Cx) channels and connexin-43 (Cx43) represent the most abundant Cx found in mammalian testis. Little is known about Cx expression in non-mammalian testis. Here we report the partial cloning of a Cx43 transcript of 381 bp from Rana esculenta testis. We also demonstrate that, in the frog testis, Cx43 transcript and protein show a parallel temporal and spatial pattern of expression throughout the reproductive annual cycle, with higher levels from September to January (when spermatogenesis is at a maximum level). In situ hybridization, carried out on testis collected in October, indicated that Leydig cells (LC) and Sertoli cells express Cx43 transcript, while the hybridization signal was less intense in germ cells. To obtain more information on Cx43 expression in the frog testis, we have used ethane-dimethane sulphonate (EDS), a toxin that specifically destroys LC. RT-PCR analysis shows a progressive decrease in Cx43 expression in EDS-treated testis from day 1 to day 4 after the injection, associated with LC destruction. Moreover, Cx43 expression returns to normal on day 28, when a new population of LC reappear in the interstitium, indicating that Cx43 is mainly expressed by LC. Taken together our data provide evidence that Cx43 is present in the frog testis with an important role in spermatogenesis.