Molecular characterization and metal induced gene expression of the novel glutathione peroxidase 7 from the chordate invertebrate Ciona robusta.

In the present study, we describe the identified and characterized the gene and the transcript of a novel glutathione peroxidase-7 (GPx7) from the solitary ascidian Ciona robusta, an invertebrate chordate widely distributed in temperate shallow seawater. The putative nucleotide and amino acid sequences were compared with those of GPx7 from other metazoans and phylogenetic analysis suggests the presence of a high evolutionary pressure in the contest of neutral evolution. The mRNA of CrGPx7 is located in hemocytes and ovarian follicular cells, as revealed by in situ hybridization. The time course of CrGPx7 mRNA levels in the presence of Cd, Cu and Zn, showed upregulation in the final stages of the experiments, suggesting a role of GPx7 in late protection from oxidative stress. Our in silico analyses of the crgpx7 promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and xenobiotic-responsive elements (XRE), suggesting that the transcription of these genes directly depends on metals. Cell-free extract from C. robusta tissues show the presence of selenium-independent GPx activity that is inhibited by the presence of metals.

Transcriptome of early embryonic invasion at implantation sites in a murine model.

Successful implantation relies on the interaction between a competent embryo and a receptive endometrium. The aim of the present study was to investigate genes differentially expressed in early invasive embryonic tissue versus decidual tissue in mice. Samples were obtained from the ectoplacental cone, the immediately surrounding deciduas and from deciduas from interimplantation sites. Microarray analysis showed that 817 genes were differentially expressed between extra-embryonic tissue and the surrounding decidua and that 360 genes were differentially expressed between the different deciduas, with a high representation of developmental processes. Genes differentially expressed in the maternal compartment included chemokines, lipoproteins, growth factors and transcription factors, whereas the embryonic invasive tissue expressed genes commonly observed in invasive tumour-like processes. These results provide information about genes involved in early embryonic invasion and the control exerted by the surrounding decidua. This information may be useful to find targets involved in pathologies associated with implantation failure and early pregnancy loss.

Transcription of genes involved in glutathione biosynthesis in the solitary tunicate Ciona intestinalis exposed to metals.

Exposure to metals is known to generate oxidative stress risk in living organisms, which are able to respond with the induction of antioxidant defenses, both enzymatic and non-enzymatic. Glutathione (GSH) is considered to be an important cellular component involved in protecting cells, both as metal chelating agent and oxygen radical scavenger. In this work we used molecular techniques to analyze the nucleotide and predicted amino acid sequences of genes involved in GSH biosynthesis, γ-glutamyl-cysteine ligase catalytic subunit (ci-gclc), γ-glutamyl-cysteine ligase modifier subunit (ci-gclm) and GSH synthase (ci-gs) in the solitary tunicate Ciona intestinalis. We also studied the transcription of the above genes after in vivo exposure to Cd, Cu and Zn by semiquantitativ RT-PCR to improve our knowledge about the relationship between metal-induced oxidative stress and GSH production and locate mRNA expression by in situ hybridization (ISH). These genes exhibit a good level of sequence conservation with metazoan homologs generally, especially for residues important for the activity of the enzymes. Phylogenetic analyses indicate that the three enzymes evolved in different ways, Ci-GCLC and Ci-GS being mostly correlated with invertebrate proteins, Ci-GCLM being as sister group of vertebrate GCLMs. Our in silico analyses of the ci-gs and ci-gclc promoter regions revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and antioxidant response elements (ARE), indicating that the transcription of these genes may directly depend on metals and/or reactive oxygen species. Results highlight a statistically significant increase in gene transcription, demonstrating that metal treatments have inducible effects on these genes. They can modulate gene transcription not only through MREs but also through AREs, as a consequence of metal-dependent ROS formation. The ISH location of Ci-GS and Ci-GCLC mRNAs shows that the cells most involved in glutathione biosynthesis are circulating hemocytes. The data presented here emphasize the importance of complex metal regulation of ci-gclc, ci-gclm and ci-gs transcription, which can create an efficient detoxification pathway allowing C. intestinalis to survive in continued elevated presence of metals in the environment.

beta-Microseminoprotein in human spermatozoa and its potential role in male fertility.

beta-Microseminoprotein (MSMB) is one of the most abundant proteins in human seminal plasma. The objectives of this study were: (1) to purify MSMB from seminal plasma (SP) and generate antibodies against the pure protein; (2) to investigate the interaction of MSMB with ejaculated spermatozoa and its possible effect on the spontaneous acrosome reaction (AR); and (3) to quantify MSMB content in SP and examine its relationship with the clinical sperm parameters. MSMB was purified from SP and its presence on the sperm surface was examined by indirect immunofluorescence using a specific polyclonal antibody. The effect of MSMB on the AR was evaluated using guinea pig epididymal spermatozoa as a model. MSMB quantification assay was performed with a two-site binding ELISA using two polyclonal antibodies against MSMB. MSMB was assessed in semen samples from fertile donors (controls) and subfertile patients according to World Health Organization criteria. MSMB was detected on the sperm surface and mainly localized to the acrosomal region of the head and neck. A significant spontaneous AR inhibition was observed when guinea pig epididymal spermatozoa were preincubated with MSMB. Finally, MSMB was significantly increased in subfertile patients when compared with fertile controls (P<0.02). The association of MSMB to the sperm surface, the inhibitor effect on the spontaneous AR and the increased MSMB levels found in SP in subfertile men suggests a relationship between this protein and semen quality and a possible role in the process of fertilization.

Multi response biomarker approach in the crab Carcinus aestuarii experimentally exposed to benzo a pyrene, polychlorobiphenyls and methyl mercury.

The aim of this study was to test a multi response biomarker approach for evaluating toxicological risk due to some of the main contaminants in the Mediterranean benzo a pyrene, polychlorobiphenyls and methyl mercury, using the Mediterranean crab Carcinus aestuarii as bioindicator organism. Forty crabs were injected with different doses of these contaminants. Several molecular, biochemical and genotoxic biomarkers were tested in different tissues and biological materials. The main conclusions were: 1 hepatopancreas, gills, haemolymph and excreta seem to be useful for biomarker studies in this species; 2 several biochemical, molecular and genotoxic biomarkers were found suitable for testing in these tissues; 3 several biomarkers were found suitable for evaluating chemical stress due to different Mediterranean contaminants.