Keys molecules implicated in apoptotic pathway of sea urchin embryos

Sea urchin embryos cadmium exposed as an experimental model system for studying the relationship between autophagy and apoptosis

Gadolinium toxicity on sea urchin embryos of geographically and phylogenetically distant species

Pharmaceuticals are a class of emerging environmental contaminants. Gadolinium (Gd) is a metal of the lanthanide series of the elements whose chelates are commonly employed as contrast agents for magnetic resonance imaging, and subsequently released into the aquatic environment. We investigated the consequences of sea urchin embryo exposure to sublethal Gd concentrations, comparing the effects on the development of four phylogenetically and geographically distant species: two Mediterranean species, Paracentrotus lividus and Arbacia lixula, and two species living in the East coast of Australia, Heliocidaris tuberculata and Centrostephanus rodgersii. Measures of the Gd and Ca content inside e…

Effects of cadmium exposition on sea urchin development by SSH and RT-qPCR techniques

Cadmium (Cd) is a toxic heavy metal contaminating coastal environments. It has been suggested that the mechanisms of acute Cd toxicity involve the depletion of glutathione and protein-bound sulfhydryl groups, resulting in enhanced production of ROS. Cd-increased ROS in turn produces lipid peroxidation, and results in DNA damage. However, little is known about direct evidence and mechanism for Cd-generated radicals until recently. In order to study the early defense strategies activated by P. lividus 30 hours embryos, in response to exposition to sub-lethal doses of Cd (100μM), we analyzed the induced transcriptome comparing it to that of control embryos by Suppression Subtractive Hybridizat…

HPV in semen: evaluation of the link among infection, sperm parameters and viral genome integration

Human papillomaviruses (HPVs) are agents of the most common sexually transmitted diseases. Both in men and in women, HPVs cause a variety of clinical symptoms ranging from warts to cancer. It has been shown that infection with oncogenic HPV is the major cause of development of cervical cancer in women and less frequently of penile cancers in men. HPV infects epithelial cells and can bind other cell types. Its presence in semen is widely known, but the effects on fertility and reproductive function are still controversial. Partners of women with high-grade squamous intraepithelial lesions were enrolled. From each subject seminal parameters were evaluated. HPV was detected and genotyped both …

Sea Urchin Mitochondrial Matrix Contains a 56-kDa Chaperonine-like Protein

Abstract Paracentrotus lividus mitochondrial matrix contains a constitutive hsp of 56-KDa which cross reacts with a serum anti-hsp-60 chaperonine from yeast mitochondria. The localization of hsps preexisting or newly synthesized in different subcellular fractions of gastrula embryos is also analyzed by two-dimensional electrophoresis.

Morte cellulare programmata in oociti umani dopo ICSI

Toxicity of manganese on embryos of the sea urchin, Paracentrotus lividus.

Manganese-exposed embryos as blueprints to study signaling pathways involved in development

Cadmium accumulation induces apoptosis in sea urchin embryos and larvae

Implication of specific isoforms of p38 in termothollerance and in the response to different stresses in sea urchin embryo

Toxicity of vanadium during development of sea urchin embryos: bioaccumulation, calcium depletion, ERK modulation and cell-selective apoptosis

Vanadium toxicology is a topic of considerable importance as this metal is widely used in industrial and biomedical fields. However, it represents a potential emerging environmental pollutant because wastewater treatment plants do not adequately remove metal compounds that are subsequently released into the environment. Vanadium applications are limited due to its toxicity, so it is urgent to define this aspect. This metal is associated with sea urchin embryo toxicity as it perturbs embryogenesis and skeletogenesis, triggering several stress responses. Here we investigated its bioaccumulation and the correlation with cellular and molecular developmental pathways. We used cytotoxic concentra…

Achievement of thermotolerance through hsps phosphorylation in sea urchin embryos.

TPA treatment of sea urchin embryos is able to induce thermotolerance. Evidence is provided that TPA treatment induces phosphorylation of a constitutive stress protein of 38 KDa.

Vanadium perturbs the fertilization outcome and the metalloproteinase activity in sea urchin embryos

Metal toxicology represents a current major topic due to the disper- sion of these elements in the environment. Metals are released from both natural sources and industrial activities. Some of them have also a clinical interest due to their application as metallodrugs (i.e., Pt, Cu, Au, Ru, and Y) or in medical diagnosis (Gd).1,2 Recently, V derivatives are considered as potential therapeutic factors in some diseases (e.g., obesity, diabetes, cancer, neurodegenerative and heart disorders). As a consequence, pharmaceutical residues could repre- sent emerging pollutants of aquatic environments, as wastewater treatment plants do not sufficiently remove these compounds3. Embryonic models repres…

Sea urchin as in vivo model for the assessment of Manganese effect in embryos development

Cadmium induces an apoptotic response in sea urchin embryos.

Cadmium is a heavy metal toxic for living organisms even at low concentrations. It does not have any biological role, and since it is a permanent metal ion, it is accumulated by many organisms. In the present paper we have studied the apoptotic effects of continuous exposure to subacute/sublethal cadmium concentrations on a model system: Paracentrotus lividus embryos. We demonstrated, by atomic absorption spectrometry, that the intracellular amount of metal increased during exposure time. We found, using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, that long treatments with cadmium triggered a severe DNA fragmentation. We demonstrated, by immunocytochemistry …

P38 MAPK plays a key role in skeletogenesis of Paracentrotus lividus embryo

Selection of the best three oocytes for intracytoplasmic sperm injection (ICSI) using apoptotic analysis of cumulus cells

Introduction: We studied the apoptosis rate of the cumulus cells of individual cumulus-oocyte complex (COC), to verify a relationship with clinical outcomes, in terms of pregnancy and implantation rates. Usually oocytes are selected using morphological criteria. We tried to verify if cumulus cell apoptotic rate could be used as a molecular criteria in selecting oocytes with higher implantation potentiality. Material and Methods: The study design consisted in two different trials: in the first we investigated apoptosis rate in cumulus cells of the three selected oocytes, according to Italian law, to be fertilized by intracytoplasmic sperm injection (ICSI). In a second trial, average apoptosi…

Sea urchin embryos as an in vivo model for the assessment of manganese toxicity: developmental and stress response effects.

Manganese (Mn), one of the most abundant metals in nature present in rocks, soil and water, is also found in soft bottom sediments of the oceans. It represents a trace element that is accumulated and utilized by all forms of life and plays multiple roles ranging from bone mineralization to cellular protection. Although Mn is an essential nutrient, exposure of cells/organisms to high levels of Mn cause toxicity. In the marine environment, increased concentrations of bio-available Mn often result from anthropogenic activities, and consequently, Mn represents a new important factor in environmental contamination. Emission of Mn into the marine environment occurs from metallurgic and chemical i…

Environmental factors as possible causes of DNA fragmentation in human sperms

Literature data demonstrated that some environmental factors could have a key role in the remarkable and continuous decline of sperm quality observed in the last fifty years. Specifically, in the Taranto area, data about the detrimental effects of environmental pollution are alarming because of the high level of poisons released in the atmosphere. Pollution coming from the plant causes health and fertility risks, mainly due to the exposure to the dioxin. Our study analyzed sperm samples from three patients groups: i) workers of local steel factories; ii) Taranto residents; iii) Controls. Results demonstrate that patients from the “factory workers” group, constantly exposed to environmental …

Cadmium effects on sea urchin embryos

Apoptotic pathway induced by cadmium in sea urchin embryos

The toxic effect of gadolinium ions on sea urchin embryos: comparison among phylogenetically distant species and focus on mechanisms regulating stress response and skeletogenesis.

Gadolinium (Gd) is a metal of the lanthanide series of the elements whose chelates are commonly employed as contrast agents for magnetic resonance imaging, and subsequently released into the aquatic environment. We investigated the consequences of sea urchin embryo exposure to sublethal Gd concentrations, comparing the effects on the development of four phylogenetically and geographically distant species: two Mediterranean species, Paracentrotus lividus and Arbacia lixula, and two species living in the East coast of Australia, Heliocidaris tuberculata and Centrostephanus rodgersii. Measures of the Gd content inside embryos by ICP-MS showed a time- and dose-dependent increase. In all these s…

Lower apoptosis rate in human cumulus cells, after r-LH administration in ovarian stimulation for IVF procedure

Gadolinium-induced stress response causes a time-dependent miss-expression of regulative and structural genes involved in the development of the sea urchin P. lividus

A relationship between human sperm morphology and DNA fragmentation in infertile patients

Vanadium Toxicity Monitored by Fertilization Outcomes and Metal Related Proteolytic Activities in Paracentrotus lividus Embryos

Metal pharmaceutical residues often represent emerging toxic pollutants of the aquatic environment, as wastewater treatment plants do not sufficiently remove these compounds. Recently, vanadium (V) derivatives have been considered as potential therapeutic factors in several diseases, however, only limited information is available about their impact on aquatic environments. This study used sea urchin embryos (Paracentrotus lividus) to test V toxicity, as it is known they are sensitive to V doses from environmentally relevant to very cytotoxic levels (50 nM; 100 nM; 500 nM; 1 µM; 50 µM; 100 µM; 500 µM; and 1 mM). We used two approaches: The fertilization test (FT) and …

Effects of exposure to gadolinium on the development of geographically and phylogenetically distant sea urchins species.

Gadolinium (Gd), a metal of the lanthanide series used as contrast agent for magnetic resonance imaging, is released into the aquatic environment. We investigated the effects of Gd on the development of four sea urchin species: two from Europe, Paracentrotus lividus and Arbacia lixula, and two from Australia, Heliocidaris tuberculata and Centrostephanus rodgersii. Exposure to Gd from fertilization resulted in inhibition or alteration of skeleton growth in the plutei. The similar morphological response to Gd in the four species indicates a similar mechanism underlying abnormal skeletogenesis. Sensitivity to Gd greatly varied, with the EC50 ranging from 56 nM to 132 μM across the four species…

Pollution exposure and high DNA fragmentation index in human sperms: a case-control study

Study question: Could pollution exposure play a key role on sperm quality, in terms of DNA fragmentation index (DFI)? Summary answer: Patients exposed to high level of pollution show an higher percentage of sperm DNA fragmentation index in contrast with patient from control group. What is known already: Taranto area is characterized by a number of steel factories and petrochemical industries. Data about the detrimental effects of environmental pollution are alarming. Pollution coming from the industrial plants causes health and fertility risks, mainly due to the exposure to several pollutant (PM10, heavy metals, etc). Toxic substances can affect DNA directly or indirectly, through oxidative…

Heavy Metals and Metalloids as Autophagy Inducing Agents: Focus on Cadmium and Arsenic

In recent years, research on the autophagic process has greatly increased, invading the fields of biology and medicine. Several markers of the autophagic process have been discovered and various strategies have been reported studying this molecular process in different biological systems in both physiological and stress conditions. Furthermore, mechanisms of metalloid- or heavy metal-induced toxicity continue to be of interest given the ubiquitous nature and distribution of these contaminants in the environment where they often play the role of pollutants of numerous organisms. The aim of this review is a critical analysis and correlation of knowledge of autophagic mechanisms studied under …

Sperm DNA fragmentation: An early and reliable marker of air pollution.

Environmental factors could have a key role in the continuous and remarkable decline of sperm quality observed in the last decades. This study compared the seminal parameters and sperm DFI in men living in areas with different levels of air pollution. Results demonstrate that both steel plants workers and patients living in a high polluted area show a mean percentage of sperm DNA fragmentation above 30%, highlighting a clear sperm damage. In this work, two different techniques were used to measure sperm DNA damage in patients’ groups, finding in both cases a high sperm DFI in patients living in polluted areas. We candidate sperm DNA fragmentation as a valuable early marker of the presence…

Autophagy is related to apoptosis in sea urchin embryos exposed to cadmium

The sea urchin embryo is a suitable model system that offers an excellent opportunity to investigate different defense strategies activated in stress conditions. We previously showed that cadmium treatment provokes the accumulation of metal in dose-time dependent manner in embryonic cells and the activation of defense systems, such as the synthesis of HSPs and/or the initiation of apoptosis. Analyzing autophagy, by neutral red, acridine orange and LC3-detection, we demonstrated that Cd-exposed embryos adopt this process as an additional stratagem to safeguard the developmental program. We observed that embryos treated with subletal Cd concentration activate massive autophagic response after…

Manganese: A New Emerging Contaminant in the Environment

The environment is composed of the atmosphere, earth and water. According to the World Health Organization, more than 100,000 chemicals are released into the global ambient every year as a consequence of their production, use and disposal. The fate of a chemical substance depends on its chemical application and physical-chemical properties, in combination with the characteristics of the environment where it is released. Chemical substances or contaminants discharged into the environment may be “natural” or “manmade”. One of the most misunderstood concepts regarding contamination is the missinterpretation of term “natural”. A “natural” contaminant is one substance that can occur without huma…

Apoptosis rate in human germinal cells: quality assay.

Apoptosis is a process that eliminates superfluous or genetically compromised cells. Cell death is one of the most important aspects of organization of the developing, as alteration in timing, level, or pattern of cell death can lead to developmental anomalies. In our studies, we investigated apoptosis in follicular somatic and germ cells of patients with infertility problems. We found that cell death occur in human germinal vesicle, in some mature oocytes and in the oocytes that failed fertilization after ICSI (Intracytoplasmic Sperm Injection), this failure was strictly correlated with apoptosis. Still, after the Italian law number 40 of 2004, it was not available oocytes to research, and…

Effects of work exposure to electromagnetic waves and organic compounds on sperm DNA fragmentation in patients undergoing ICSI

Apoptosi in oociti umani non fecondati dopo ICSI

Autophagy is related to apoptosis in Paracentrotus lividus embryos cadmium exposed

P. lividus embryo offers an excellent opportunity to investigate the adaptive response of cells exposed to different stress. We previously demonstrated that cadmium treatment triggers the accumulation of metal in embryonic cells and the activation of defense system depending on concentration and exposure time, through the synthesis of HSPs and/or the initiation of apoptosis. Analysing autophagy, by neutral red, acridine orange and LC3-detection, we demonstrated that Cd-exposed embryos adopt this process as an additional stratagem to safeguard the developmental program. We observed that embryos treated at subletal Cd concentration activate a massive autophagic response after 18h, which decre…

Sperm DNA integrity after conventional freezing vs vitrification with and without cryoprotectors

DNA fragmentation index, pAKT and pERK1/2 in cumulus cells are related to oocyte competence in patients undergoing in vitro fertilization programme

SummaryActivated pERK1/2 and pAKT are key players in supporting cell survival and proliferation pathways. Translocation of pERK1/2 into the nucleus, where it interacts with transcription factors and DNA itself, is instrumental in exerting an anti-apoptotic effect. In this study, pAKT levels, pERK1/2 nuclear localization and DNA fragmentation index (DFI) in cumulus cells of single cumulus–oocyte complexes of patients undergoing in vitro fertilization programmes were evaluated and correlated with the clinical outcome of the related embryos. For a positive clinical outcome of blastocyst development, pERK1/2 nuclear localization and DFI value had a significant inverse relationship, whereas the …

Relationship between apoptosis and survival molecules in human cumulus cells as markers of oocyte competence

SummaryTo select from a single patient the best oocytes able to reach the blastocyst stage, we searched for valuable markers for oocytes competence. We evaluated the DNA fragmentation index (DFI) and the level of some survival molecules, such as AKT, pAKT and pERK1/2, in individual cumulus cell–oocyte complexes (COC). The study included normo-responder women. The average age of the patients was 34.3. DFI in cumulus cells was evaluated using the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) assayin situ. AKT, pAKT and pERK1/2 were measured by immunological assay and densitometric analysis of fluorescent signals using NIS-Elements BR 3.10 image software. Statisti…

Toxic effects induced by vanadium on sea urchin embryos

Vanadium, a naturally occurring element widely distributed in soil, water and air, has received considerable interest because its compounds are often used in different applications, from industry to medicine. While the possible medical use of vanadium compounds is promising, its potential harmful effects on living organisms are still unclear. Here, for the first time, we provide a toxicological profile induced by vanadium on Paracentrotus lividus sea urchin embryos, reporting an integrated and comparative analysis of the detected effects reflecting vanadium-toxicity. At the morphological level we found a dose-dependent induction of altered phenotypes and of skeletal malformations. At the mo…

Asimmetric skeleton patterns induced by gadolinium ions in sea urchin embryos: focus on mechanisms regulating skeletogenesis and comparison among phylogenetically distant species

Autophagy is required for sea urchin oogenesis and early development.

SummaryAutophagy is a major intracellular pathway for the degradation and recycling of cytosolic components. Emerging evidence has demonstrated its crucial role during the embryo development of invertebrates and vertebrates. We recently demonstrated a massive activation of autophagy in Paracentrotus lividus embryos under cadmium stress conditions, and the existence of a temporal relationship between induced autophagy and apoptosis. Although there have been numerous studies on the role of autophagy in the development of different organisms, information on the autophagic process during oogenesis or at the start of development in marine invertebrates is very limited. Here we report our recent …

Cadmium insult and defence mechanisms in Paracentrotus lividus embryos and larvae.

Sea urchin embryos as a model system for studying autophagy induced by cadmium stress

It is well known that sea urchin embryos are able to activate different defense strategies against stress. We previously demonstrated that cadmium treatment triggers the accumulation of metal in embryonic cells and the activation of defense systems depending on concentration and exposure time, through the synthesis of heat shock proteins and/or the initiation of apoptosis. Here we show that Paracentrotus lividus embryos exposed to Cd adopt autophagy as an additional stratagem to safeguard the developmental program. At present, there are no data focusing on the role of this process in embryo development of marine organisms. In this paper we utilized different techniques to detect autophagy i…

Sea urchin embryos as an in vivo model for the assessment of manganese toxicity: developmental and stress response effects.

In the marine environment increasing concentrations of bio-available compounds often result from anthropogenic activities. Among metal ions, manganese represents a new emergent factor in environmental contamination. Here, we studied the effects of manganese on Paracentrotus lividus sea urchin embryos using biological and biochemical approaches for the analysis of impact on development, tissue accumulation and stress markers. Embryos were continuously exposed from fertilization to manganese at concentrations ranging from 1.0 to 61.6 mg l(-1), monitored for developmental abnormalities at 48 h after fertilization, and used for atomic spectrometric analysis at various times from 6 to 72 h. We f…

Studies on heat shock proteins in sea urchin development

Work on stress proteins in sea urchin embryos carried out over the last 20 years is reviewed and the following major results are described. Entire sea urchin embryos, if subjected to a rise in temperature at any postblastular stage undergo a wave of heat shock protein (hsp) synthesis and survive. If subjected to the same rise between fertilization and blastula formation, they are not yet able to synthesize hsp and die. Four clones coding for the major hsp, hsp70, have been isolated and sequenced; evidence for the existence of a heat shock factor has been provided, and a mechanism for the developmental regulation of hsp synthesis discussed. Intra- embryonic and intracellular hsp location has…

Gadolinium induces autophagy in sea urchin embryos

Lower sperm DNA fragmentation after r-FSH administration in functional hypogonadotropic hypogonadism

Abstract PURPOSE: An observational clinical and molecular study was designed to evaluate the effects of the administration of recombinant human FSH on sperm DNA fragmentation in men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. METHODS: In the study were included 53 men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In all patients, sperm DNA fragmentation index (DFI), assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end-labelling (TUNEL) assay, was evaluated before starting the treatment with 150 IU of recombinant hum…

Cadmium accumulation induces apoptosis in P. lividus larvae.

Apoptosis during early development of sea urchin.

Apoptosis is a genetic program of cell death that eliminates superfluous or compromised cells during development and adult life of many organisms. In sea urchin embryos, apoptosis is not only a physiological event during larval metamorphosis, but also a process induced by cadmium accumulation or other stressor like TPA (12-O-tetradecanoylphorbol-13-acetate) followed by an increase of temperature to 31°C. Apoptosis is a highly conserved process usually operated by a proteolytic cascade that involves caspase activation by two different pathways: extrinsic and intrinsic. The first one involves membrane death receptors, while the second involves mitochondria. In this work we analyzed the possib…

Apoptosis rate in cumulus cells can be considerate as an indicator for the selection of embryos to improve ongoing pregnancy and implantation rate

Interactive effects of increased temperature and gadolinium pollution in Paracentrotus lividus sea urchin embryos: a climate change perspective

Gradual ocean warming and marine heatwaves represent major threats for marine organisms already facing other anthropogenic-derived hazards, such as chemical contamination in coastal areas. In this study, the combined effects of thermal stress and exposure to gadolinium (Gd), a metal used as a contrasting agent in medical imaging which enters the aquatic environment, were investigated in the embryos and larvae of the sea urchin Paracentrotus lividus. Embryos were exposed to six treatments of three temperatures (18 °C, 21 °C, 24 °C) and two Gd concentrations (control: 0 μM; treated: 20 μM). With respect to developmental progression, increased temperature accelerated development and achievemen…

L'apoptosi nelle cellule del cumulo ooforo dopo somministrazione di r-LH.

AN INNOVATIVE STRATEGY IN SELECTING OOCYTES WITH HIGH IMPLANTATION POTENTIALITY FOR INTRACYTOPLASMIC SPERM INJECTION PROCEDURE

The aim of the research was to investigate the apoptosis rate of individual cumulus cell-oocyte complexes (COC), associated to the level of pAKT, to verify the difference between oocytes who produce embryos able to reach the blastocyst stage compared with embryos arrested during the in vitro culture. It was demonstrated that DNA fragmentation in cumulus cells was remarkably lower in patients who achieved a pregnancy after ICSI cycles, related to the quality of oocytes and embryos1,2. AKT pathway plays a critical role in the regulation of cell survival, and most growth factors activate this pathway3. The study focused on 53 patients, involved after informed consent. In this prospective and r…

APOPTOTIC ANALYSIS OF CUMULUS CELLS FOR THE SELECTION OF COMPETENT OOCYTES TO BE FERTILIZED BY INTRACYTOPLASMIC SPERM INJECTION (ICSI)

Oocyte quality is one of the main factors for the success of in vitro fertilization protocols. Apoptosis is known to affect oocyte quality and may impair subsequent embryonic development and implantation. The aim of this study was to investigate the apoptosis rate of single and pooled cumulus cells of cumulus cell–oocyte complexes (COCs), as markers of oocyte quality, prior to intracytoplasmatic sperm injection (ICSI).We investigated the apoptosis rate by TUNEL assay (DNA fragmentation) and caspase-3 immunoassay of single and pooled cumulus cells of COCs. The results showed that DNA fragmentation in cumulus cells was remarkably lower in patients who achieved a pregnancy than in those who di…

EGTA treatment causes the synthesis of heat shock proteins in sea urchin embryos.

Paracentrotus lividus embryos, at post-blastular stage, when subjected to a rise in temperature from physiologic (20 degrees C) to 31 degrees C, synthesize a large group of heat shock proteins (hsps), and show a severe inhibition of bulk protein synthesis. We show, by mono- and two-dimensional electrophoresis, that also EGTA (ethylene glycol-bis[beta-aminoethyl ether] tetraacetic acid) treatment induces in sea urchin embryos both marked inhibition of bulk protein synthesis and the synthesis of the entire set of hsps. Furthermore, EGTA-treated sea urchin embryos are able to survive at a temperature otherwise lethal (35 degrees C) becoming thermotolerant. Because incubation with a different c…

Induction of apoptosis by cadmium in Paracentrotus lividus embryos and larvae

Esiste una correlazione tra apoptosi e morfologia negli spermatozoi umani di pazienti infertili?

Induction of apoptosis by cadmium in sea urchin embryos: possible involvement of extrinsic pathway

L’autofagia durante l’oogenesi e i primi stadi di sviluppo embrionale in Paracentrotus lividus

Molecular approaches to elucidate the early response of P. lividus embryos to sublethal Cadmium exposition.

HUMAN SPERM CELL AS EARLY BIOINDICATOR OF MALE HEALTH AND REPRODUCTION

Environmental factors could have a key role in the remarkable and continuous decline of sperm quality observed in the last fifty years. This study addressed the gap of knowledge on the effect of air pollutants on sperm DNA fragmentation, comparing the seminal parameters from men living in locations with different levels of air pollution. The detrimental effects of environmental pollution in the Taranto area are alarming: the high level of pollutants released from the steel plants in the atmosphere can cause health and fertility issues. Our study analyzed sperm samples from three groups of patients: i) workers of Taranto steel plants; ii) Taranto residents; iii) Palermo residents, assumed as…

Apoptosis in Human Cumulus Cells

Implication of p38 MAPK in skeletogenesis of Paracentrotus lividus embryo

Mitochondria and Development

Autophagy during development of sea urchin embryos

Autophagy is a highly regulated mechanism that enhances cell eukaryotic survival under various environmental and cellular stresses, by breakdown and recycling of macromolecules and organelles. Here we report that in Paracentrotus lividus embryos autophagic process occur, at a lesser extend during physiological development and at greater levels after a cadmium treatment. By Acridine Orange staining, we found that embryonic cells exposed to cadmium display green fluorescence in cytoplasm and nucleus, and show considerable red fluorescent dots in cytoplasm. This evidence suggests formation of acidic auto-phagolysosomal vacuoles. By Neutral Red vital staining, specific for lysosomes and cellula…

Deciliation: A stressful event for Paracentrotus lividus embryos.

In this report, by using mono- and two-dimensional electrophoretic analysis, we demonstrate that deciliation on sea urchin embryos induces a stress response. Deciliation indeed causes not only the activation of ciliary subroutine, but also a transient decrease of bulk protein synthesis. This decrease is in agreement with our previous results on heat shock response in sea urchin, although deciliation does not induce the expression of the same main hsp set. We were able to characterize one main deciliation-stress protein of 40 kDa whose expression is transiently induced by deciliation and whose localisation is likely to be nuclear.

Identification of new members of metallothionein gene family in sea urchin

Heavy metals are common marine pollutants that emanate from such sources as industrial and sewage treatment discharges and anti-fouling paints. Cadmium (Cd2+) serves no essential function in biological organisms and it is a highly toxic and carcinogenic metal. In Paracentrotus lividus sea urchin embryos cadmium causes development arrest or severe malformations. At lower doses, cadmium activates different responses (HSPs synthesis, autophagic or apoptotic processes) that can allow embryo survival. This study was conducted to elucidate the gene transcription activation/upregulation or repression/downregulation inducted in sea urchin embryos grown under cadmium stress. Comparison between trans…

Sequence of a sea urchin hsp70 gene and its 5' flanking region.

We report the nucleotide sequence of a 4470-bp fragment derived from a sea urchin genomic clone containing part of a heat-shock protein 70 (Hsp70)-encoding gene. This fragment, named hsp70 gene II, contains 1271 bp of the flanking region and 3299 bp of structural gene sequence interrupted by five introns and encoding the N-terminal 371 amino acids (aa) of the protein. The 5' flanking region contains a putative TATA element, two CCAAT boxes, four heat-shock consensus sequence elements (hse) and one consensus sequence for binding of Sp1. Remarkable homologies were observed for deduced aa sequence and intron-exon organization between hsp70 gene II and rat hsc73 gene.

Maria Carmela Roccheri

Research focus Control of autophagy during both physiological development and in stress conditions in the sea urchin embryo. Model system Sea urchin embryo and larvae in development. Education and career 1971, biology degree, University of Palermo, Italy; advisor: G. Giudice. 1972–1973, fellowship from the Italian Ministry of Public Education, Institute of Comparative Anatomy, University of Palermo. 1974–1980, associate scientist at the Institute of Comparative Anatomy, University of Palermo. 1981–2005, associate professor of cell biology, Department of Cell and Development Biology, University of Palermo. 2000–2005, member of the scientific committee for PhD in “Biotechnology applied to pre…

The early response to sublethal Cadmium exposition of P. lividus embryos: a molecular approach

Lower apoptosis rate in human cumulus cells after administration of recombinant luteinizing hormone to women undergoing ovarian stimulation for in vitro fertilization procedures.

Objective: To investigate the effects of recombinant (r-) LH supplementation in “low responder” patients undergoing ovarian stimulation with r-FSH for an IVF program. The apoptosis rate in cumulus cells was used as an indicator of oocyte quality. Design: Comparison of the rate of DNA fragmentation and caspase-3 activity in cumulus cells in women stimulated with r-LH and r-FSH, versus patients treated with r-FSH alone (control). Setting: In vitro fertilization (IVF) laboratory. Patient(s): Forty patients undergoing assisted fertilization programs treated with a GnRH agonist, or r-FSH treatment begun on day 3 of the cycle (control). In the r-LH group, from day 8 of gonadotropin stimulation, 1…

Gadolinium perturbs expression of skeletogenic genes, calcium uptake and larval development in phylogenetically distant sea urchin species

Chelates of Gadolinium (Gd), a lanthanide metal, are employed as contrast agents for magnetic resonance imaging and are released into the aquatic environment where they are an emerging contaminant. We studied the effects of environmentally relevant Gd concentrations on the development of two phylogenetically and geographically distant sea urchin species: the Mediterranean Paracentrotus lividus and the Australian Heliocidaris tuberculata. We found a general delay of embryo development at 24 h post-fertilization, and a strong inhibition of skeleton growth at 48 h. Total Gd and Ca content in the larvae showed a time- and concentration-dependent increase in Gd, in parallel with a reduction in C…

Effects of cadmium exposure on sea urchin development assessed by SSH and RT-qPCR: metallothionein genes and their differential induction

In order to study the defense strategies activated by Paracentrotus lividus embryos in response to sub-lethal doses of CdCl2, we compared the induced transcripts to that of control embryos by suppression subtractive hybridization technique. We isolated five metallothionein (MT) cDNAs and other genes related to detoxification, to signaling pathway components, to oxidative, reductive and conjugative biotransformation, to RNA maturation and protein synthesis. RT-qPCR analysis revealed that two of the five P. lividus MT (PlMT7 and PlMT8) genes appeared to be constitutively expressed and upregulated following cadmium treatment, whereas the other three genes (PlMT4, PlMT5, PlMT6) are specifically…

The autophagic demand during oogenesis and early development of sea urchin

The autophagic pathway is an evolutionarily conserved homeostatic process, responsible for degradation and recycling of long-term proteins and cytoplasmic organelles in eukaryotic cells. This process constitutively occurs at basal levels and is involved in cell survival. Increased autophagy is induced by environmental cues, such as starvation and many stress agents, while excessive levels of autophagy can lead to autophagic Programmed Cell Death, with features that differ from those of the apoptotic process. We recently demonstrated massive activation of autophagy in P. lividus embryos, in cadmium stress conditions, and the existence of a temporal relationship between induced autophagy end …

Effect of environmental stress on sea urchin embryos and larvae: from developmental to molecular biology analyses

Manganese Toxicity: sea urchin embryos and Stress Response Effects.

Response to cadmium stress in sea urchin embryos

Apoptosis in human unfertilized oocytes after ICSI

Hsp56 expression in Paracentrotus lividus embryos

Apoptosi associata alla morfologia negli spermatozoi umani di pazienti infertili

Territorial localization of heat shock mRNA production in sea urchin gastrulae.

In situ hybridization experiments with a labeled DNA probe indicate that the ability to respond to heat shock with the production of the mRNA for the 70 kd heat shock protein is segregated into the ectodermal cells already at the gastrula stage or earlier during the embryonic development of Paracentrotus lividus.

Isolation and characterization of a Paracentrotus lividus cDNA encoding a stress-inducible chaperonin

Chaperonins are ubiquitous proteins that facilitate protein folding in an adenosine triphosphate–dependent manner. Here we report the isolation of a sea urchin cDNA (Plhsp60) coding for mitochondrial chaperonin (Cpn60), whose basal expression is further enhanced by heat shock. The described cDNA corresponds to a full-length mRNA encoding a protein of 582 amino acids, the first 32 of which constitute a putative mitochondrial targeting leader sequence. Comparative analysis has demonstrated that this protein is highly conserved in evolution.

Apoptosis: focus on sea urchin development

It has been proposed that the apoptosis is an essential requirement for the evolution of all animals, in fact the apoptotic program is highly conserved from nematodes to mammals. Throughout development, apoptosis is employed by multicellular organisms to eliminate damaged or unnecessary cells. Here, we will discuss both developmental programmed cell death (PCD) under normal conditions and stress induced apoptosis, in sea urchin embryos. Sea urchin represent an excellent model system for studying embryogenesis and cellular processes involved in metamorphosis. PCD plays an essential role in sculpting and remodelling the embryos and larvae undergoing metamorphosis. Moreover, this marine organi…

Sea urchin embryo as a model system for studing autophagy induced by cadmium stress

p38 MAPK activity during sea urchin embryo development (Paracentrotus lividus)

Apoptosis rate of human cumulus cells from single oocytes in infertile patients as criteria for the selection of oocytes to be treated by intracytoplasmic sperm injection (ICSI).

The Role of Autophagy and Apoptosis During Embryo Development

Programmed cell death (PCD) and cell survival are two sides of the same coin. Autopha‐ gy and apoptosis are crucial processes during embryo development of Invertebrates and Vertebrates organisms, as they are necessary for the formation of a new organism, start‐ ing from a fertilized egg. Fertilization triggers cell remodeling from each gamete to a toti‐ potent zygote. During embryogenesis, the cells undergo various processes, thus allowing the transformation of the embryo into an adult organism. In particular, cells require the appropriate tools to suddenly modify their morphology and protein content in order to respond to intrinsic and external stimuli. Autophagy and apoptosis are involved…

Localization of mitochondrial Hsp56 chaperonin during sea urchin development.

We have previously demonstrated that Paracentrotus lividus nuclear genome encodes for the heat shock inducible chaperonin homolog Hsp 56 (1) and that the mature protein is localized in the mitochondrial matrix (2). In this paper we report that constitutive Hsp56 is maternally inherited, in fact it is present in the in unfertilized eggs, and that it has a perinuclear specific localization during cleavage. In the later stages both the constitutive and the heat shock inducible chaperonin has a specific territorial distribution. Moreover following heat shock, the Hsp56 appears in the cytoplasm and in the postmitochondrial supernatant beside the mitochondrial fraction.

L'embrione di riccio di mare come modello di studio dell'autofagia indotta da stress

Gli embrioni di riccio di mare, Paracentrotus lividus, sono in grado di attivare differenti strategie di difesa come risposta a stress chimico/fisici. Recentemente abbiamo dimostrato che il cadmio, metallo pesante altamente embriotossico, induce la sintesi di specifiche hsps e/o l’innesco di processi apoptotici, via via che si accumula nelle cellule embrionali. Nel presente lavoro, mostriamo che gli embrioni di P. lividus sono in grado di attivare l’autofagia come un aggiuntivo meccanismo atto a salvaguardare il programma di sviluppo, in seguito a esposizione a dosi citotossiche di CdCl2. L’autofagia è un meccanismo molecolare che può stimolare la sopravvivenza, attraverso la degradazione e…

Apoptosis in sea urchin embryos.

Abstract It is demonstrated by DNA electrophoresis analysis, morphological observations and TdT in situ reaction, that Paracentrotus embryos if treated with TPA plus heat undergo an apoptotic reaction. Indication is also obtained that non treated embryos undergo spontaneous apoptosis at the early pluteus stage, expecially in the districts of arms and intestine. The possible meaning of this latter observation is discussed.

Manganese interferes with calcium, perturbs ERK signaling, and produces embryos with no skeleton.

Manganese (Mn) has been associated with embryo toxicity as it impairs differentiation of neural and skeletogenic cells in vertebrates. Nevertheless, information on the mechanisms operating at the cellular level remains scant. We took advantage of an amenable embryonic model to investigate the effects of Mn in biomineral formation. Sea urchin (Paracentrotus lividus) embryos were exposed to Mn from fertilization, harvested at different developmental stages, and analyzed for their content in calcium (Ca), expression of skeletogenic genes, localization of germ layer markers, and activation of the extracellular signal-regulated kinase (ERK). By optical and immunofluorescence microscopy, we found…

Cadmium induces autophagy during development of Paracentrotus lividus embryos.

FSH administration reduces significantly sperm apoptosis only in the case of high DFI value: a study in idiopathic dispermic patients

Introduction: In the last decades sperm DNA quality has been recognized as one of the most important markers of male reproductive potential (Lewis and Aitken, 2005; Ozmen, 2007; Tarozzi, 2007), in contrast to standard semen parameters as sperm density, motility and morphology, which do not act as powerful discriminators between fertile and infertile men. DNA damage in the male germ line is a major contributor to infertility, miscarriage and birth defects in the offspring. In animal models, it has been unequivocally demonstrated that the genetic integrity of the male germ line plays a major role in determining the normality of embryonic development. In humans, many studies showed that sperm …

Mitochondria during sea urchin oogenesis.

SummarySea urchin represents an ideal model for studies on fertilization and early development, but the achievement of egg competence and mitochondrial behaviour during oogenesis remain to be enlightened. Oocytes of echinoid, such as sea urchin, unlike other echinoderms and other systems, complete meiotic maturation before fertilization. Mitochondria, the powerhouse of eukaryotic cells, contain a multi-copy of the maternally inherited genome, and are involved directly at several levels in the reproductive processes, as their functional status influences the quality of oocytes and contributes to fertilization and embryogenesis. In the present paper, we report our latest data on mitochondrial…

Evaluation of DNA damage in human spermatozoa: a sperm quality assay.

Several studies have demonstrated the relationship between gonadotrophine serum levels and the apoptosis rate in germinal cells. In human, lower FSH serum levels are associated with reduced Sperm Standard Parameters in term of concentration, motility and morphology and higher sperm DNA fragmentation. Hypogonadotrope hypogonadism is a clinical condition associated with male infertility, characterized by low serum levels of gonadotrophins. In this pilot study we investigated the spermatozoa quality of 11 patients with development of hypogonadotrope hypogonadism urdergoing in vitro fertilization programs. Recombinant-FSH administered to improve the sperm parameters, apoptosis rate, aiming to i…

Skeleton growth inhibition in sea urchin embryos Paracentrotus lividus after manganese exposure.

In the sea urchin embryos, skeleton is specified by interactions between PMCs and patterning cues derived from the ectoderm. PMCs use spatial and temporal information to organize the proper animal-vegetal and oral-aboral position and orientation of the two tri-radiate skeletal spicules. Many experiments have demonstrated that exposure to metals, such as lithium, zinc and nickel, can disrupt skeleton patterning information. Here, we have investigated the effects of manganese (Mn) on Paracentrotus lividus embryo development. We found that Mn exposure prevents skeleton growth producing spicule-lacking embryos. Normal skeleton growth was partially rescued after Mn removal from the culture. To d…

Risposta da stress in embrioni di Paracentrotus lividus esposti a cadmio

Autophagy

Klionsky, Daniel J. et al.

Apoptosis in human unfertilized oocytes after intracytoplasmic sperm injection

Objective To investigate the presence of programmed cell death in unfertilized oocytes after intracytoplasmic sperm injection (ICSI), assuming that previous apoptotic events could be correlated with the fertilization failure. Design Comparison of the rate of DNA fragmentation in human oocytes at different stages of maturation soon after pick-up (control) and in unfertilized oocytes after ICSI treatment. Setting In vitro fertilization (IVF) laboratory with extensive ICSI experience. Patient(s) Sixty-three patients undergoing assisted fertilization by ICSI. Intervention(s) Terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay and anticaspase-3 cleaved…

Cadmium stress effects indicating marine pollution in different species of sea urchin employed as environmental bioindicators

In recent years, researches about the defense strategies induced by cadmium stress have greatly increased, invading several fields of scientific research. Mechanisms of cadmium-induced toxicity continue to be of interest for researchers given its ubiquitous nature and environmental distribution, where it often plays the role of pollutant for numerous organisms. The presence in the environment of this heavy metal has been constantly increasing because of its large employment in several industrial and agricultural activities. Cadmium does not have any biological role and, since it cannot be degraded by living organisms, it is irreversibly accumulated into cells, interacting with cellular comp…

Sea urchin deciliation induces thermoresistance and activates the p38 mitogen-activated protein kinase pathway.

In this study, we demonstrate by a variety of approaches (ie, morphological analysis, Western blots, immunolocalization, and the use of specific antibodies) that hyperosmotic deciliation stress of sea urchin embryos induces a thermotolerant response. Deciliation is also able to activate a phosphorylation signaling cascade the effector of which might be the p38 stress-activated protein kinase because we found that the administration of the p38 inhibitor SB203580 to sea urchin deciliated gastrula embryos makes the hyperosmotic deciliation stress lethal.

Synthesis of heat-shock proteins in developing sea urchins.

Heating sea urchin embryos at 31°C greatly reduces the synthesis of the bulk proteins, whereas it highly stimulates the synthesis of some new proteins, the main ones being two closely migrating proteins of about 70,000 daltons. The production of heat-shock proteins is obtained only if the embryos are heated after hatching. Stages which produce heat-shock proteins survive heating, whereas earlier stages, not producing heat-shock proteins, do not survive. Heat-shock proteins are not produced in the presence of actinomycin D.

Physiological and cadmium induced apoptosis in Paracentrotus lividus larvae

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Seuls les 100 premiers auteurs dont les auteurs INRA ont été entrés dans la notice. La liste complète des auteurs et de leurs affiliations est accessible sur la publication.; International audience; In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues…

Gene Expression and Apoptosis Levels in Cumulus Cells of Patients with Polymorphisms of FSHR and LHB Undergoing in Vitro Fertilization Program

Background/Aims: FSH receptor (FSHR) Ala307Thr and Asn680Ser and LHβ chain (LHB) Trp28Arg and Ile35Thr polymorphisms affect the response to pharmacological ovarian stimulation with r-FSH in women undergoing assisted reproductive treatment (ART). Here, we evaluated the expression level of selected genes involved in follicle maturation and the possible onset of apoptosis in cumulus cells of patients with single and double FSHR and LHB polymorphisms, as potential markers of oocyte competence. Methods: Cumulus cells from 36 stimulated patients were collected and SNP genotyping performed by PCR. Gene expression was evaluated through real-time PCR, and apoptosis estimated via TUNEL assay, and cle…

Effetti del cadmio sugli embrioni di riccio di mare

Apoptosis rate in human spermatozoa: a sperm quality assay.

Several studies have demonstrated the relationship between gonadotrophine serum levels and the apoptosis rate in germinal cells (Bosco et al 2005). In human, lower FSH serum levels are associated with reduced Sperm Standard Parameters in term of concentration, motility and morphology and higher sperm DNA fragmentation. Hypogonadotrope hypogonadism is a clinical condition associated with male infertility, characterized by low serum levels of gonadotrophins. In this pilot study we investigated the spermatozoa quality of 11 patients with development of hypogonadotrope hypogonadism urdergoing in vitro fertilization programs. Recombinant-FSH administered to improve the sperm parameters, apoptosi…

Effects of magnesium deprivation on development and biomineralization in the sea urchin Arbacia lixula

Echinoderms have an extensive endoskeleton composed of magnesian calcite and occluded matrix proteins. As biomineralization in sea urchin larvae is sensitive to the Magnesium:Calcium ratio of seawater, we investigated the effects of magnesium deprivation on development and skeletogenesis in the sea urchin Arbacia lixula. We focused on the localization of the skeletogenic cells (primary mesenchyme cells) and the spatial expression of associated genes. Embryos reared in Mg-free seawater exhibited developmental delay from 6-h post-fertilization and at 24 h embryos showed complete lack of biomineral formation. Larvae (48–72 h) exhibited severe skeleton malformations. Fluorescent labelling revea…

A new method to select the best three oocytes to be treated for intracytoplasmic sperm injection (ICSI)

Apoptosis rate of cumulus cells can be considered as an indicator for the selection of embryos to improve ongoing pregnancy and implantation rates.

Cumulus cells apoptosis rate an adjunct to morphology evaluation for the embryo selection on day 3 could be considered a new tool, compared with embryo selection by morphology alone, to select the embryos with higher implantation potential to increase the clinical outcomes after ICSI. Several studies have demonstrated a lower cumulus cell apoptotic rates in women who achieved pregnancy compared with women who did not become pregnant after ICSI. A prospective randomized observational study on 76 ICSI patients was performed before Ovum Pick-Up. Patients were randomized into either the control group (embryo selection by morphology only, A group: 48 patients) or the treatment group (morphology …

Vanadium induces calcium depletion and cell selective apoptosis during development of sea urchin embryos

Vanadium (V) is a metal widely distributed in soil, water and air. It has recently received growing interest because its compounds are often used in different applications, from industry to medicine.1 Here, using atomic absorption spectrometry, we demonstrate the predisposition of V to accumulate directly into embryonic cells, interfering with Ca uptake. At the morphological level, we observed dose- and time-dependent effects on phenotypes and on skeletal malformations. At the molecular level, V-exposed embryos showed the activation of the cellular stress response, inducing Hsp 60 and Hsp 70 synthesis and the activation of autophagy and apoptosis. The Hsps-mediated stress response to V appe…

Confocal microscopy study of the distribution, content and activity of mitochondria during Paracentrotus lividus development

Summary In the present paper we applied confocal microscopy andfluorescence technologies for studying the distribution andthe oxidative activity of sea urchin ( Paracentrotus lividus )mitochondria during development, by in vivo incubating eggsand embryos with cell-permeant MitoTracker probes. Wecalculated, by a mathematical model, the intensity values, the variations of intensity, and the variation index of incorporatedfluorochromes. Data demonstrate that mitochondrial massdoes not change during development, whereas mitochondrialrespirationincreases.Inaddition,startingfrom16blastomeresstage, some regions of the embryo contain organelles moreactive in oxygen consumption. Introduction The con…

Dosi citotossiche di manganese ed effetti sullo sviluppo embrionale. Modello sperimentale: Riccio di mare Paracentrotus lividus

DOSI CITOTOSSICHE DI MANGANESE ED EFFETTI SULLO SVILUPPO EMBRIONALE. MODELLO SPERIMENTALE: RICCIO DI MARE PARACENTROTUS LIVIDUS Annalisa Pinsino1, Francesca Trinchella2, Maria Carmela Roccheri1 1 Dipartimento di Biologia Cellulare e dello Sviluppo “A. Monroy”, Università degli Studi di Palermo, Italia 2 Dipartimento delle Scienze Biologiche, Università degli Studi di Napoli Federico II, Italia Il manganese (Mn) è uno degli elementi più abbondanti in natura, presente nelle rocce, nel suolo e nelle acque. E’ un elemento in traccia appartenente alla categoria dei nutrienti o metalli essenziali; utilizzato ed accumulato da tutte le forme di vita, è coinvolto nel normale funzionamento di meccani…

A possible role of autophagy for the execution of apoptosis in cadmium-exposed sea urchin embryos

The sea urchin embryo is a suitable model system that offers an excellent opportunity to investigate different defence strategies activated in stress conditions. We previously showed that cadmium treatment provokes the accumulation of metal in dose- and time-dependent manner in embryonic cells and the activation of defence systems, such as the synthesis of HSPs and/or the initiation of apoptosis. Analysing autophagy, by neutral red, acridine orange and LC3-detection, we demonstrated that Cd-exposed embryos adopt this process as an additional stratagem to safeguard the developmental program. We observed that embryos treated with subletal Cd concentration activate a massive autophagic respons…

Differences beetween physiological and induced apoptosis in sea urchin embryos.

P38 mapk activity during sea urchin embryo development

Autophagy induction in sea urchin embryos exposed to gadolinium ions

Sea urchin embryos are highly sensitive to several kinds of stressors, and able to activate different defense strategies. Gadolinium (Gd) is a metal of the lanthanide series of the elements: its chelates are employed as contrast agents for magnetic resonance imaging since the 1980s. Gd complexes are released in the aquatic environment, making Gd an emergent environmental pollutant. In this study we focused on the effects of Gd ions on sea urchin embryos development. The study was conducted looking at three different processes: general development, apoptosis and autophagy. At the whole morphological level, Paracentrotus lividus sea urchin embryos continuously exposed to Gd ions displayed mor…

P. lividus oogenesis and early development require autophagy

It is well known that autophagy is a major intracellular pathway for the degradation and recycling of cytosolic components, in both basal and stress conditions. We have recently demonstrated the activation of autophagy in P. lividus embryos: at high levels, after a stress induced by cadmium, and at basal levels, during the physiological development (1) (2). Here we report ours recent data about autophagy during oogenesis and segmentation. In order to detect autophagolysosomes (AVOs) and autolysosomes, we respectively performed incubation in vivo with acridine orange (AO), and in situ immunofluorescence (IF) with the anti-LC3 antibody (autophagy marker). All observations were carried out by …

r-lh administration in multiple follicular stimolation for ivf: effects of apoptosis in cumulus cells, oocytes quality and clinical outcome

The effects of manganese on embryos of the sea urchin, Paracentrotus lividus: stress or not?.

Il fallimento della fecondazione: un evento predeterminato?

Heat-Shock Proteins in Sea Urchin Embryos

The production of heat-shock proteins in sea urchin embryos is accompanied by the appearance at the polysomal level of their relative mRNAs, as shown by their translation in a cell-free system; thus suggesting that the regulation of their production occurs at a transcriptional level. The mechanism for the inhibition of the bulk protein synthesis and for its reversal on the other hand should be looked for at a posttranscriptional level, since both these phenomena occur also in the presence of actinomycin D. The heat-shock proteins produced as early as at the mesenchyme blastula stage persist within the embryo at least till the pluteus stage.

Manganese overload affects p38 MAPK phosphorylation and metalloproteinase activity during sea urchin embryonic development.

Abstract In the marine environment, manganese represents a potential emerging contaminant, resulting from an increased production of manganese-containing compounds. In earlier reports we found that the exposure of Paracentrotus lividus sea urchin embryos to manganese produced phenotypes with no skeleton. In addition, manganese interfered with calcium uptake, perturbed extracellular signal-regulated kinase (ERK) signaling, affected the expression of skeletogenic genes, and caused an increase of the hsc70 and hsc60 protein levels. Here, we extended our studies focusing on the temporal activation of the p38 mitogen-activated protein kinase (p38 MAPK) and the proteolytic activity of metalloprot…

Effect of ovarian stimulation with recombinant LH/ FSH, recombinant FSH and human menopausal gonadotrophin (hMG) in reducing apoptosis rate in cumulus cells of patients undergoing ICSI. a pilot study

Stress induced and physiological apoptosis during early development of sea urchin Paracentrotus lividus.

HPV infection in semen: results from a new molecular approach

AbstractHuman papillomavirus (HPV) is the agent of the most common sexually transmitted diseases causing a variety of clinical manifestations ranging from warts to cancer. Oncogenic HPV infection is the major cause of cervical cancer and less frequently of penile cancers. Its presence in semen is widely known, but the effects on fertility are still controversial. We developed a new approach to evaluate virus localisation in the different semen components. We analysed also the specific genotype localisation and viral DNA quantity by qPCR. Results show that HPV DNA can be identified in every fraction of semen: spermatozoa, somatic cells and seminal plasma. Different samples can contain the HP…

Toxicological Impact of Rare Earth Elements (REEs) on the Reproduction and Development of Aquatic Organisms Using Sea Urchins as Biological Models

The growing presence of lanthanides in the environment has drawn the attention of the scientific community on their safety and toxicity. The sources of lanthanides in the environment include diagnostic medicine, electronic devices, permanent magnets, etc. Their exponential use and the poor management of waste disposal raise serious concerns about the quality and safety of the ecosystems at a global level. This review focused on the impact of lanthanides in marine organisms on reproductive fitness, fertilization and embryonic development, using the sea urchin as a biological model system. Scientific evidence shows that exposure to lanthanides triggers a wide variety of toxic insults, includi…

Autophagy as defence strategy against cadmium stress in Paracentrotus lividus embryos.

Sea urchin embryo is a developmental model that offers an excellent opportunity to investigate the possible adaptive response of cells exposed to different stress during differentiation. These embryos are able to respond to many stress by synthesizing a set of highly conserved proteins, the hsps and/or by activation of apoptosis. The exposure to cadmium trigger the accumulation of metal in embryo cells and the activation of both defence mechanisms depending on concentration and exposure time [1-4]. Recent experimental evidences demonstrate that by autophagy, a highly regulated mechanism that enhances cell survival under various environmental and cellular stress, the breakdown and recycling …

Autophagy as a defense strategy against stress: focus on Paracentrotus lividus sea urchin embryos exposed to cadmium

Autophagy is used by organisms as a defense strategy to face environmental stress. This mechanism has been described as one of the most important intracellular pathways responsible for the degradation and recycling of proteins and organelles. It can act as a cell survival mechanism if the cellular damage is not too extensive or as a cell death mechanism if the damage/stress is irreversible; in the latter case, it can operate as an independent pathway or together with the apoptotic one. In this review, we discuss the autophagic process activated in several aquatic organisms exposed to different types of environmental stressors, focusing on the sea urchin embryo, a suitable system recently in…

Synthesis of RNA in isolated nuclei of sea urchin embryos.

It is demonstrated that isolated nuclei of sea urchin embryos are able to incorporate radioactive nucleotides into RNA. Some properties of the incorporation system are described.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) 1

Contains fulltext : 232759.pdf (Publisher’s version ) (Closed access) In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to…

Cellular, biochemical and molecular effects of cadmium on marine invertebrates: focus on Paracentrotus lividus sea urchin development

Cadmium is a heavy metal that is toxic for living organisms even at low concentrations. The presence in the environment of this metal has grown because of its large employment in some industrial and agricultural activities. Although heavy metals are terrestrially produced, they flow into the sea through effluents and sewage or are directly discharged from industries placed on the seawater front. In addition to its release into costal waters, cadmium fallout, following atmospheric events, contributes to the pollution of marine ecosystems. It should be considered that cadmium concentrations determined in the field vary widely according to different seawater latitudes and depths and can be str…

Relationship between autophagy and apoptosis in Paracentrotus lividus embryos cadmium exposed

Cadmium is a strong toxicant for living organism since it does not have biological roles and can cause several cellular damages comprising genotoxicity, oxidative stress, and other biochemical dysfunctions. Marine invertebrate embryos represent a suitable model system where to investigate the effects of many stressors on development and cell viability. Here we investigated the toxic effect of cadmium on sea urchin Paracentrotus lividus embryos focusing our attention on metal-inducead autophagy and the possible temporal and functional relationship with apoptosis. Using several techniques to detect autophagy (neutral red, acridine orange and LC3-detection) we demonstrated that Cd-exposed P. l…

Sperm DNA integrity after conventional freezing vs vitrification with and without cryoprotectors in oligoastenospermic patients

Manganese effects on sea urchin embryos.

A new strategy in selecting oocytes using cumulus cells analysis of specific molecules of the apoptotic pathway, according to the ability to reach blastocyst stage.

Study question: The aim of the research was to investigate the apoptosis rate of individual cumulus cell–oocyte complexes (COC), associated to the level of p-Akt, to verify the difference between oocytes who produce embryos able to reach the blastocyst stage compared with embryos arrested during the in vitro colture. Summary answer: It was demonstrated that DNA fragmentation in cumulus cells was remarkably lower in patients who achieved a pregnancy after ICSI cycles, related to the quality of oocytes and embryos. Akt pathway plays a critical role in the regulation of cell survival, and most growth factors activate this pathway. What is known already: Studies on oocyte maturation have shown …

Apoptosi indotta da cadmio in embrioni di riccio di mare

Autophagy

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide…

Induction of skeletal abnormalities and autophagy in Paracentrotus lividus sea urchin embryos exposed to gadolinium.

Abstract Gadolinium (Gd) concentration is constantly increasing in the aquatic environment, becoming an emergent environmental pollutant. We investigated the effects of Gd on Paracentrotus lividus sea urchin embryos, focusing on skeletogenesis and autophagy. We observed a delay of biomineral deposition at 24 hours post fertilization (hpf), and a strong impairment of skeleton growth at 48 hpf, frequently displayed by an asymmetrical pattern. Skeleton growth was found partially resumed in recovery experiments. The mesodermal cells designated to biomineralization were found correctly migrated at 24 hpf, but not at 48 hpf. Western blot analysis showed an increase of the LC3-II autophagic marker…

Cadmium induces apoptosis in P. lividus embryos

Ruolo dell’autofagia nell’oogenesi di riccio di mare Paracentrotus lividus

Isoforme specifiche di p38 implicate nella termotolleranza e nella risposta a differenti stress nell'embrione di riccio di mare

Erratum

Author(s): Klionsky, DJ; Abdelmohsen, K; Abe, A; Abedin, MJ; Abeliovich, H; Arozena, AA; Adachi, H; Adams, CM; Adams, PD; Adeli, K; Adhihetty, PJ; Adler, SG; Agam, G; Agarwal, R; Aghi, MK; Agnello, M; Agostinis, P; Aguilar, PV; Aguirre-Ghiso, J; Airoldi, EM; Ait-Si-Ali, S; Akematsu, T; Akporiaye, ET; Al-Rubeai, M; Albaiceta, GM; Albanese, C; Albani, D; Albert, ML; Aldudo, J; Algul, H; Alirezaei, M; Alloza, I; Almasan, A; Almonte-Beceril, M; Alnemri, ES; Alonso, C; Altan-Bonnet, N; Altieri, DC; Alvarez, S; Alvarez-Erviti, L; Alves, S; Amadoro, G; Amano, A; Amantini, C; Ambrosio, S; Amelio, I; Amer, AO; Amessou, M; Amon, A; An, Z; Anania, FA; Andersen, SU; Andley, UP; Andreadi, CK; Andrieu-Ab…

Acquisition of thermotolerance in sea urchin embryos correlates with the synthesis and age of the heat shock proteins.

Preheating at 31 degrees C induces thermotolerance in Paracentrotus lividus embryos, which therefore become able to withstand 1-h treatment at the otherwise lethal temperature of 35 degrees C, and to develop normally. The acquisition of thermotolerance is positively correlated with the amount of heat shock proteins produced during the 31 degrees C treatment. Evidence is provided that the heat shock proteins, although present in the embryo for long periods after synthesis, lose their effect on thermotolerance within 3 h of the cessation of synthesis.

Relazione tra autofagia e apoptosi in embrioni di riccio di mare esposti a stress

L’embrione di riccio di mare Paracentrotus lividus è stato utilizzato come organismo modello della biologia dello sviluppo per molti anni ed è considerato il deuterostoma più primitivo, con scheletro calcificato, correlato a protocordati e vertebrati. Tale sistema offre un'eccellente opportunità per studiare le numerose strategie di difesa che gli embrioni mettono in atto contro diverse condizioni di stress. In precedenza, abbiamo riportato che l’esposizione di embrioni a dosi citotossiche di cadmio, provoca l'accumulo intracellulare del metallo e l'attivazione del sistema difensivo, in modo dose-tempo dipendente, attraverso la sintesi di specifiche HSPs e/o l’innesco di apoptosi. Mediante …

Toxicity induced by Gadolinium ions on sea urchin embryos: comparison among phylogenetically distant species and focus on stress response and skeletogenesis.

Pharmaceuticals are a class of emerging environmental contaminants. Gadolinium (Gd) is a lanthanide metal whose chelates are employed as contrast agents for magnetic resonance imaging, and subsequently released into the aquatic environment. We investigated the effects of exposure to sublethal Gd concentrations on the development of four phylogenetically and geographically distant sea urchin species: two Mediterranean, Paracentrotus lividus and Arbacia lixula, and two from Australia, Heliocidaris tuberculata and Centrostephanus rodgersii. Sensitivity to Gd greatly varied, with EC50 ranging from 56 nM to 132 µM across the four species. Measures of the Gd and Ca content inside embryos showed a…

Apoptotic pathway in sea urchin embryos

In human cumulus cells, the apoptotic rate may be considered an indicator for the selection of embryos to improve ongoing pregnancy and implantation rates

In order to identify the embryos with higher implantation potential to increase the clinical outcomes after ICSI, the apoptotic rate in human cumulus cells for the embryo selection on day 3, as an adjunct to morphology evaluation, could be considered a new tool compared with embryo selection by morphology alone. Several studies have demonstrated a lower cumulus cell apoptotic rate in women who achieved pregnancy compared with women who did not become pregnant after ICSI. A prospective randomized observational study on 76 ICSI patients was performed before Ovum Pick-Up. Patients were randomized into either the control group (embryo selection by morphology only, A group: 48 patients) or the t…

Sea urchin embryos exposed to cadmium as an experimental model for studying the relationship between autophagy and apoptosis

The sea urchin embryo is a suitable model that offers an excellent opportunity to investigate different defence strategies activated in stress conditions. We previously showed that cadmium accumulates in a dose- and time-dependent manner into embryonic cells, activating different stress and defence mechanisms, including the synthesis of HSPs and the onset of apoptosis and/or autophagy. In this paper we investigated the functional relationship between autophagy and apoptosis, evaluating apoptosis signals in cadmium-exposed Paracentrotus lividus embryos with inhibited autophagy. We found that the inhibition of autophagy produced the concurrent reduction of apoptosis, suggesting that the two p…

Hsp40 Is Involved in Cilia Regeneration in Sea Urchin Embryos

In a previous paper we demonstrated that, in Paracentrotus lividus embryos, deciliation represents a specific kind of stress that induces an increase in the levels of an acidic protein of about 40 kD (p40). Here we report that deciliation also induces an increase in Hsp40 chaperone levels and enhancement of its ectodermal localization. We suggest that Hsp40 might play a chaperoning role in cilia regeneration.

Developmental abnormalities induced by Gadolinium causes a time-dependent miss-expression of regulative and structural genes in P. lividus sea urchin embryos

Gadolinium (Gd) is a metal of the lanthanide series of the elements whose chelates are commonly used as contrast agents for magnetic resonance imaging. Its release into the aquatic milieu has posed serious concerns regarding its noxious effects, and therefore Gd is now considered an emerging environmental pollutant. The sea urchin embryo is an excellent model used in both toxicological and developmental research. We analysed the consequences of embryo exposure to sublethal concentrations of Gd on embryo development, focusing on skeletogenesis and developmental symmetry. We observed a strong inhibition of skeleton growth, frequently displayed by an asymmetrical pattern. Continuous exposure t…

Proteolytic activity and MMP-14-like protein levels are affected by Vanadium in Paracentrotus lividus Embryo

The increasing industrial use of vanadium (V), as well as its recent medical use in various pathologies has intensified its environmental release, making it an emerging pollutant [1]. The sea urchin embryo has long been used to study the effects induced by metals, including V. In this study we used an integrated approach that correlates the biological effects on embryo development with proteolytic activities of gelatinases that could better reflect any metal induced imbalances. V-exposure caused morphological/morphometric aberrations, mainly concerning the correct dis-tribution of embryonic cells, the development of the skeleton and the embryo volume [2]. Moreover, V induced a concentration…

Apoptosis rate in human spermatozoa is not related to nuclear vacuoles

P–178 Mural granulosa cells of the human follicles indirectly show death molecular signals not depending on different ovarian stimulation protocols

Abstract Study question Could the expression of the anti-apoptotic molecules AKT, p-Akt and ERK1/2 in Mural Granulosa Cells (MGC) be considered as marker of oocyte quality? Summary answer MGCs activate cell death pathways in analyzed follicles and it is not influenced by different stimulation protocols and it is not correlated to oocyte competence. What is known already It has been previously demonstrated that apoptosis rate of mural granulosa or cumulus cells (CC) were correlated with follicular oocyte number, age, embryo numbers in IVF/ICSI and also clinical pregnancy. Moreover, our previous data demonstrated that in selected patients, who received recombinant LH associated with recombina…

Cadmium induces the expression of specific stress proteins in sea urchin embryos.

Abstract Marine organisms are highly sensitive to many environmental stresses, and consequently, the analysis of their bio-molecular responses to different stress agents is very important for the understanding of putative repair mechanisms. Sea urchin embryos represent a simple though significant model system to test how specific stress can simultaneously affect development and protein expression. Here, we used Paracentrotus lividus sea urchin embryos to study the effects of time-dependent continuous exposure to subacute/sublethal cadmium concentrations. We found that, between 15 and 24 h of exposure, the synthesis of a specific set of stress proteins (90, 72–70, 56, 28, and 25 kDa) was ind…

Apoptosi in larve di Paracentrotus lividus.

Exposure to cytotoxic concentrations of manganese and effects on development

Manganese is one of the most abundant metals present in nature in trace amounts, which is accumulated and utilized by all forms of life. It plays a multitude of biological roles, but exposure of cells/organisms to high levels causes toxicity. In the marine environment, increasing concentrations of bio-available ions often result from anthropogenic activities, and, consequently, manganese represents a new emergent factor in environmental contamination. To study cyto-toxicity of manganese on development, we used an excellent model system as sea urchin embryos Paracentrotus lividus. In this work, we show results from two-dimensional gel electrophoresis (2DE) analysis that highlighted qualitati…

Apoptosi in cellule del cumulo come possibile bio-marcatore molecolare di competenza ovocitaria: risultati di uno studio pilota condotto su pazienti sottoposte a diversi protocolli di stimolazione ovarica.

L'apoptosi come criterio di valutazione comparativa tra attività LH e hCG.