Identification and characterization of PlAlix, the Alix homologue from the Mediterranean sea urchin Paracentrotus lividus.

The sea urchin provides a relatively simple and tractable system for analyzing the early stages of embryo development. Here, we use the sea urchin species, Paracentrotus lividus, to investigate the role of Alix in key stages of embryogenesis, namely the egg fertilization and the first cleavage division. Alix is a multifunctional protein involved in different cellular processes including endocytic membrane trafficking, filamentous (F)-actin remodeling, and cytokinesis. Alix homologues have been identified in different metazoans; in these organisms, Alix is involved in oogenesis and in determination/differentiation events during embryo development. Herein, we describe the identification of the sea urchin homologue of Alix, PlAlix. The deduced amino acid sequence shows that Alix is highly conserved in sea urchins. Accordingly, we detect the PlAlix protein cross-reacting with monoclonal Alix antibodies in extracts from P. lividus, at different developmental stages. Focusing on the role of PlAlix during early embryogenesis we found that PlAlix is a maternal protein that is expressed at increasingly higher levels from fertilization to the 2-cell stage embryo. In sea urchin eggs, PlAlix localizes throughout the cytoplasm with a punctuated pattern and, soon after fertilization, accumulates in larger puncta in the cytosol, and in microvilli-like protrusions. Together our data show that PlAlix is structurally conserved from sea urchin to mammals and may open new lines of inquiry into the role of Alix during the early stages of embryo development

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, 150 IU per day of r-LH were administered. Intervention(s) Terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine-triphosphate (dUTP) nick-end labeling (TUNEL) assay, and anti-caspase-3 cleaved immunoassay, to detect apoptosis in human cumulus cells. Main Outcome Measure(s) Difference in DNA fragmentation rate between cumulus cells derived from r-LH treatment and cumulus cells derived from control patients. Result(s) No differences were observed between the two groups in the total amount of r-FSH administered and in the number of retrieved oocytes per patient. A statistically significant increase in the number of immature oocytes and in the E2 serum peak was observed in the control group. The number of transferred embryos was significantly higher in the r-LH group. Pregnancy and implantation rates were higher in the r-LH group, but without statistical significance. The apoptosis rate in cumulus cells was higher in the control group than in the r-LH group. Conclusion(s) This study suggests that supplementation with r-LH improves the chromatin quality of cumulus cells involved in the control of oocyte maturation

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 immunoassay to detect apoptosis in control and ICSI-treated oocytes. Main Outcome Measure(s) Differences in the percentage of oocytes demonstrating DNA fragmentation between control oocytes and unfertilized ICSI treated oocytes at different stages of maturation. Result(s) The DNA fragmentation, by TUNEL assay, appeared in all the immature control oocytes, but only 37% of mature oocytes showed DNA fragmentation. This DNA fragmentation was observed in 88.8% of the oocytes unfertilized after ICSI; furthermore, DNA fragmentation appeared as well in the sperm injected into the cytoplasm. Conclusion(s) The study has shown DNA fragmentation in human oocytes unfertilized after ICSI. The evidence is confirmed as well in control oocytes, free from in vitro culture or manipulation stress. Caspase-3 immunoassay suggests the presence of apoptosis. The high percentage of oocytes demonstrating DNA fragmentation in the unfertilized oocytes could be correlated with fertilization failure

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

In the present paper we applied confocal microscopy and fluorescence technologies for studying the distribution and the oxidative activity of sea urchin (Paracentrotus lividus) mitochondria during development, by in vivo incubating eggs and embryos with cell-permeant MitoTracker probes. We calculated, by a mathematical model, the intensity values, the variations of intensity, and the variation index of incorporated fluorochromes. Data demonstrate that mitochondrial mass does not change during development, whereas mitochondrial respiration increases. In addition, starting from 16 blastomeres stage, some regions of the embryo contain organelles more active in oxygen consumption

OUTCOMES OF ELDERLY PATIENTS WITH ST-ELEVATION OR NON-ST-ELEVATION ACUTE CORONARY SYNDROME UNDERGOING PERCUTANEOUS CORONARY INTERVENTION

Acute coronary syndromes have been classified according to the finding of ST-segment elevation on the presenting ECG, with different treatment strategies and practice guidelines. However, a comparative description of the clinical characteristics and outcomes of acute coronary syndrome elderly patients undergoing percutaneous coronary intervention during index admission has not been published so far

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 il riciclo di macromolecole e organelli o, alternativamente, la morte cellulare. Inoltre, è stato riportato che l’autofagia svolge un ruolo cruciale durante l’embriogenesi di alcuni organismi. Pertanto, le cellule multipotenti degli embrioni di echinodermi possono rappresentare un idoneo modello sperimentale per lo studio di questo processo. Diverse metodologie, qui di seguito riportate, sono state utilizzate per verificare l’induzione di processi autofagici in embrioni esposti a cadmio. Mediante saggi vitali con Neutral Red, sono stati evidenziati elevati livelli di organelli vescicolari acidi (AVOs), probabili autolisosomi. Analoghi risultati sono stati ottenuti mediante saggi vitali con Acridina Orange e microscopia confocale a scansione laser (CLSM); quest’ultima ha consentito di rilevare specifiche disposizioni spaziali nella localizzazione degli AVOs. L’induzione dell’autofagia è stata confermata mediante l’impiego di bafilomicina A1, uno specifico inibitore di questo processo. Questi dati sono stati rafforzati analizzando i livelli della proteina LC3, sicuro marker dell’autofagia, attraverso Western blotting e immunofluorescenza in situ. I risultati di queste indagini hanno evidenziato che gli embrioni di P. lividus sono in grado di attivare significativi processi autofagici in condizioni di stress da cadmio, in una fase temporale che precede la risposta apoptotica massiva. Inoltre, livelli autofagici basali sono stati evidenziati durante lo sviluppo fisiologico. In conclusione si può ipotizzare che l’autofagia svolga un ruolo fondamentale nella sopravvivenza cellulare, allo scopo di salvaguardare il programma di sviluppo o, in alternativa, possa servire a fornire l’ATP necessario a sostenere il processo apoptotico e/o operi la clearance dei corpi apoptotici