140 research outputs found
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, UniversitĂ 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 meccanismi cellulari come la replicazione, la mineralizzazione e la protezione cellulare. D’altro canto, se la sua presenza è fondamentale per la vita, l’esposizione di cellule/organismi ad elevate concentrazioni di Mn causa tossicità . Il manganese rappresenta oggi, un “nuovo” importante fattore di contaminazione ambientale, conseguenza dell’intensa attività antropica dell’ultimo secolo.
Il trattamento di embrioni di riccio di mare della specie Paracentrotus lividus con dosi citotossiche di Mn influisce sullo sviluppo embrionale producendo un alternativo morfotipo di sviluppo, attivando le principali MAPK (ERK e p38) coinvolte sia nella regolazione dello sviluppo che nello stress, provocando una modulazione nei livelli di sintesi delle principali proteine da stress HSC70 e HSC60. Nello specifico, troviamo che dosi crescenti di Mn (da 7,7 a 61,6 mg/L) provocano ritardi e malformazioni nello sviluppo embrionale secondo un andamento dose-effetto, chiaramente caratterizzato dall’aumento in frequenza del numero di embrioni con totale assenza o ridotto allungamento dello scheletro di CaCO3 (spicole). Il processo di spicologenesi sembra comunque ripristinarsi quando il Mn viene allontanato dal mezzo di coltura, per i diversi tempi di trattamento/ sviluppo, anche se dopo 40 ore di trattamento gli embrioni non riescono a recuperare il corretto disegno della struttura scheletrica. Mediante l’uso di marcatori territorio-specifici per i tre foglietti embrionali già differenziati in ecto-, meso- ed endoderma (anticorpi monoclonali Ecto V, UH2-95, 1D5) è stato osservato che tutti gli antigeni specifici per i vari territori sono presenti, sia negli embrioni controllo che negli embrioni trattati (61,6 mg Mn/L), in una posizione spaziale e per un tempo di sviluppo appropriato. Invece, le ibridazioni in situ per i trascritti codificanti per msp130, sm30 ed sm50 (geni espressi esclusivamente nelle cellule deputate alla secrezione delle spicole, PMC), hanno messo in evidenza, per i trattati, una forte inibizione nei livelli di espressione di sm30, l’unico dei tre geni che risponde a segnali mediati dalla matrice extracellulare.
Inoltre, ERK e p38 rimangono nella forma fosforilata per tutto lo sviluppo degli embrioni trattati: questo risultato potrebbe dipendere da una quantità di calcio fisiologico non sufficiente a regolare gli eventi di fosfo-/defosforilazione delle MAPK, dovuto all’accumulo di Mn negli embrioni (AAS analisi). Per concludere l’accumulo di Mn negli embrioni di P. lividus stimola una duplice soluzione adattativa: attivazione di una risposta da stress e regolazione di pathways alternativi per consentire il proseguo dello sviluppo.
http://www.unisi.it/eventi/gei/programma.pd
Cadmium induces the expression of specific stress proteins in sea urchin embryos
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 induced, with an increase in the rate of synthesis of 72-70 kDa (hsps), 56 kDa (hsp), and 25 kDa, which was dependent on the lengths of treatment. Recovery experiments in which cadmium was removed showed that while stress proteins continued to be synthesized, embryo development was resumed only after short lengths of exposure
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 possible involvement of extrinsic and intrinsic apoptotic pathways in physiological and stressful conditions in Paracentrotus lividus embryos. By fluorescent TUNEL assays we demonstrate that apoptosis is part of cadmium and TPA+31°C stress response. We find that Cd and TPA+31°C treatments induce apoptosis through caspase-3 activation, while caspase-7 is the main effector of physiological apoptosis. Caspase-10 is active only in physiological apoptosis, while caspase-8 is mainly involved in stress-induced apoptosis. In addition, we did not find any involvement of mitochondria.
Moreover we observed, in Cd-treated embryos, a Reactive Oxygen Species (ROS) increase, that could be related to the induction of apoptosis
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 uM; 50 uM;
100 uM; 500 uM; and 1 mM).We used two approaches: The fertilization test (FT) and a protease detection
assay after 36 h of exposure. V affected the fertilization percentage and increased morphological
abnormalities of both egg and fertilization envelope, in a dose-dependent manner. Moreover, a total
of nine gelatinases (with apparent molecular masses ranging from 309 to 22 kDa) were detected,
and their proteolytic activity depended on the V concentration. Biochemical characterization shows
that some of them could be aspartate proteases, whereas substrate specificity and the Ca2+/Zn2+
requirement suggest that others are similar to mammalian matrix metalloproteinases (MMPs)
SELECTION OF THE BEST OOCYTES FOR INTRACYTOPLASMICSPERM 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 molecular criteria in selecting oocytes with higher
implantation potentiality (1;2).
Materials 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, to be fertilized by
intracytoplasmic sperm injection (ICSI); in a second trial, average apoptosis rate of the cumulus
cells coming from the three selected oocytes to be fertilized by ICSI and the pooled remaining
oocytes were compared, when more than 5 COCs were aspirated. In a first trial we included 22
consecutive couples undergoing ICSI cycles, 20 in a second one, for a total of 42 patients. We
selected the three oocytes for (ICSI) on the basis of the morphological appearance of the
cumulus, according to Veek’s criteria. The cumulus cells of each COC were submitted to
apoptotic assays (3). The patients were classified, on the basis of pregnancy success, in A Group
(pregnant patients) and B Group (patients with negative βhCG).
Results: Both trials showed that apoptosis in the cumulus cells was remarkably lower in the A
Group if compared with B Group. The apoptosis rate in the selected COCs was similar to pooled
COCs for each patient, confirming that apoptosis rate in cumulus cells is characteristic for
patient. Out of 22 patients involved in the first trial, 8 were pregnant (36.3% A Group) and 14
were not pregnant (B Group). In the second trial 4 of a total of 20 patients were pregnant (20%).
In the first trial a total of 58 metaphase II oocytes and 56 in the second trial were studied. In the
second trial 38 oocytes where pooled to compare apoptosis rate with the three selected oocytes
pools. In the first trial the incidence of DNA fragmentation, evaluated by TUNEL assay (fig. 1),
of the cumulus cells from individual treated oocytes, was lower in A Group than in B Group
(6.7% ranging between 2.2–13.3 vs 13.19% ranging between 6.2–34.9 respectively, p<0.05). To
confirm if DNA fragmentation was related to apoptosis process, we performed caspase-3
immunoassay in the same cells (fig. 2). Data showed a lower capase-3 activity in cumulus cells
of pregnant than in those of non-pregnant patients (5.2% ranging between 1.2–8.6 vs 11.8%
ranging between 5.6–14.8, p<0.05). It is noteworthy to underline that pregnant patients usually
exhibited, at least, one COC with a DNA fragmentation rate (TUNEL) less than 10% and
caspase-3 activity rate less than 7%. Four (A Group) of 20 patients involved in the second trial
were pregnant but two aborted at 8–9 weeks. The low number of pregnant patients did not allow
us to have a powerful statistical analysis of apoptotic rate in cumulus cells, but it seems evident
that a higher apoptotic rate in cumulus cells is associated to the pregnancy failure (B Group) and
in aborted patients of A Group, ranging from 10 to 60.3%.
Conclusion: The data seem to demonstrate that apoptosis may be a marker for the selection of
the best oocytes to be submitted to ICSI treatment. All pregnant patients showed a lower
apoptosis rate in cumulus cells if compared with patients with pregnancy failure.
86° CONGRESSO NAZIONALE SIBS - PALERMO 24-25 OTTOBRE 2013
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References
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FIGURE 1. Apoptosis evaluation using TUNEL assay in human cumulus cells. (A1, A2, A3) A
group; (B1, B2, B3) B group; (C1, C2, C3) positive control for TUNEL assay. (A1, B1, C1)
fragmented DNA; (A2, B2, C3) propidium iodide staining; (A3, B3, C3) merge. Scale bar = 15
ÎĽm.
FIGURE 2. Apoptosis evaluation using Cleaved caspase 3 immunofluorescence in situ assay in
human cumulus cells. (A1, A2, A3) A group; (B1, B2, B3) B group; (A1, B1) Cleaved caspase 3;
(A2, B2) propidium iodide staining; (A3, B3) merge. Scale bar = 15 ÎĽm
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