Aldo Donizetti

Transcriptional profiling of endometriosis tissues identifies genes related to organogenesis defects

Endometriosis is a common benign pathology, characterised by the presence of endometrial tissue outside the endometrial cavity with a prevalence of 10–15% in reproductive‐aged women. The pathogenesis is not completely understood, and several theories have been proposed to explain the aetiology. Our group has recently described the presence of ectopic endometrium in a consistent number of human female foetuses analysed by autopsy, reinforcing the hypothesis that endometriosis may be generated by defects during the organogenesis of the female reproductive trait. Herein, in order to identify, at molecular level, changes involved in the disease, we compared the transcriptional profiling of ectopic endometrium with the corresponding eutopic one. Statistical analyses lead us to identify some genes specifically deregulated in the ectopic endometrium, that are involved in gonad developmental process or in wound healing process. Among them, we identified BMP4 and GREM1. BMP4 was never associated before to endometriosis and is involved in the mesoderm‐Müllerian duct differentiation. GREM1 is needed for the initial step of the ureter growth and perhaps could possibly be involved in Müller ducts differentiation. These molecules might be related to the endometriosis aetiology since we showed that their expression is not related to the menstrual cycle phase both at RNA and at protein levels. These data support the theory that embryological defects could be responsible of the endometriosis generation. J. Cell. Physiol. 228: 1927–1934, 2013.

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.

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 48u2003h 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 72u2003hpf. 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.

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.

Cortical spreading depression differentially affects lysine methylation of H3 histone at neuroprotective genes and retrotransposon sequences

Recently cortical spreading depression (CSD) has been hypothesized to involve epigenetic control of gene expression, by inducing an overall decrease of H3K4 and increase of H3K9 di-methylation. Here we evaluated the H3K4 and H3K9 di-methylation level at specific loci in rat brains 24 h after CSD induction. Analysis of two selected neuroprotective genes, iNOS and HIF-1α, showed marked increase in lysine 4 di-methylation and decrease in lysine 9 di-methylation of H3 histone. In addition, di-methylation of H3K4 increased moving toward 5 end of the genes in CSD-induced rat hemispheres. Such behavior may reflect an epigenetic molecular memory of actively transcribed genes. We extended our analysis on the H3K4 and H3K9 di-methylation levels of two long interspersed sequences (LINEs). We showed that CSD induction led to di-methylation decrease in lysine 4 and increase in lysine 9 of H3 histone, a trend which reflected the overall chromatin changes previously demonstrated. In conclusion, our data corroborate the hypothesis that epigenetic regulation of gene expression can be affected by CSD and that might be a pivotal molecular mechanism of CSD-induced preconditioning phenomenon which induces tolerance to a subsequent episode of ischemia. In such control, we evidenced two effects: i) a molecular memory of transcribed neuroprotective genes, ii) an epigenetic silencing of retrotransposable sequences.

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.

Expression of prothymosin alpha in meiotic and post-meiotic germ cells during the first wave of rat spermatogenesis.

Prothymosin alpha (PTMA) is a highly acidic small polypeptide, that is, widely distributed and conserved among mammals. Its possible involvement in male gametogenesis has been mentioned but not clarified yet; in particular, it has been suggested that, in non‐mammalian vertebrates, it could play a role during GC meiosis and differentiation. In the present work we investigated the possible association between PTMA and meiotic and post‐meiotic phases of mammalian spermatogenesis. Three different time points during postnatal development of rat testis were analyzed, that is, 27u2009dpp (completed meiosis), 35u2009dpp (occurring spermiogenesis), and 60u2009dpp (first wave of spermatogenesis definitely ended). RT‐PCR and Western blot analyses showed that the expression levels of both Ptma mRNA and corresponding protein decrease in total extracts from 27 to 60u2009dpp. The in situ hybridization localized the transcript in interstitial Leydig cells, peritubular myoid cells and, inside the tubules, in germ cells from pachytene spermatocytes to newly formed haploid spermatids. The immunohistochemistry analysis localized the protein in the same cell types at 27u2009dpp, while at 35 and 60u2009dpp the haploid cells remain the only germ cells that still express it. In particular, PTMA specific localization in the heads of spermatids and epididymal spermatozoa, associated with the acrosome system, supports for the first time the hypothesis of a direct function in male germ cells. J. Cell. Physiol. 224: 362–368, 2010.

Characterization and developmental expression pattern of the relaxin receptor rxfp1 gene in zebrafish

We report the gene characterization, the cDNA cloning and the temporal and spatial expression pattern of the relaxin receptor rxfp1 gene in the zebrafish Danio rerio. The zebrafish rxfp1 gene has the same syntenic genomic organization, and a similar exon‐intron structure to the homologue human gene. Furthermore, the deduced Rxfp1 protein sequence shows a high degree of amino acid similarity when compared with the human protein and the conservation of all amino acid identity necessary for the binding with relaxin. Our results show that rxfp1 gene is active either during embryogenesis or in the adult organism, showing a wide expression pattern. Moreover, we provide the first description of rxfp1 spatial expression pattern during embryo development, showing that the transcript is already present at the early developmental stage and is distributed in all of the embryonic cells until somitogenesis. Starting at the pharyngula stage the gene expression becomes mainly restricted in the brain territories. In fact, at the larval stage, the transcript is detectable in the epiphysis, postoptic region, posterior tuberculum, hypothalamus, optic tectum, tegmentum/pons, medulla and also in the structure of a peripheral nervous system, the terminal nerve. The rxfp1 expression pattern in Danio rerio embryos is very similar to that reported in the adult mammalian brain, suggesting a pivotal role of this receptor in the neurophysiology processes already at very early developmental stages.

First Evidence of DAAM1 Localization During the Post-Natal Development of Rat Testis and in Mammalian Sperm

Dishevelled‐associated activator of morphogenesis 1 (DAAM1) is a formin‐family protein involved in nucleation of unbranched actin filaments and in cytoskeletal organization through Wnt‐Dishevelled PCP pathway, which participates in essential biological processes, such as cell polarity, movement, and adhesion during morphogenesis and organogenesis. While its role has been investigated during development and in somatic cells, its potential association with the germinal compartment and reproduction is still unexplored. In this work, we assessed the possible association of DAAM1 with the morphogenesis of rat testis. We studied its expression and profiled its localization versus actin and tubulin, during the first wave of spermatogenesis and in the adult gonad (from 7 to 60u2009dpp). We show that, in mitotic phases, DAAM1 shares its localization with actin in Sertoli cells, gonocytes, and spermatogonia. Later, during meiosis, both proteins are found in spermatocytes, while only actin is detectable at the forming blood‐testis barrier. DAAM1, then, follows the development of the acrosome system throughout spermiogenesis, and it is finally retained inside the cytoplasmic droplet in mature gametes, as corroborated by additional immunolocalization data on both rat and human sperm. Unlike the DAAM1, actin keeps its localization in Sertoli cells, and tubulin is associated with their protruding cytoplasm during the process. Our data support, for the first time, the hypothesis of a role for DAAM1 in cytoskeletal organization during Mammalian testis morphogenesis and gamete progression, while also hinting at its possible investigation as a morphological marker of germ cell and sperm physiology. J. Cell. Physiol. 231: 2172–2184, 2016.

Long Non-coding RNA in Neurons: New Players in Early Response to BDNF Stimulation.

Brain-derived neurotrophic factor (BDNF) is a neurotrophin family member that is highly expressed and widely distributed in the brain. BDNF is critical for neural survival and plasticity both during development and in adulthood, and dysfunction in its signaling may contribute to a number of neurodegenerative disorders. Deep understanding of the BDNF-activated molecular cascade may thus help to find new biomarkers and therapeutic targets. One interesting direction is related to the early phase of BDNF-dependent gene expression regulation, which is responsible for the activation of selective gene programs that lead to stable functional and structural remodeling of neurons. Immediate-early coding genes activated by BDNF are under investigation, but the involvement of the non-coding RNAs is largely unexplored, especially the long non-coding RNAs (lncRNAs). lncRNAs are emerging as key regulators that can orchestrate different aspects of nervous system development, homeostasis, and plasticity, making them attractive candidate markers and therapeutic targets for brain diseases. We used microarray technology to identify differentially expressed lncRNAs in the immediate response phase of BDNF stimulation in a neuronal cell model. Our observations on the putative functional role of lncRNAs provide clues to their involvement as master regulators of gene expression cascade triggered by BDNF.