Manganese: A New Emerging Contaminant in the Environment

Physiolog

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 72 References 1. Ruvolo G, Bosco L, Pane A, Morici G, Cittadini E, Roccheri MC. Lower apoptosis rate in human cumulus cells after administration of recombinant luteinizing hormone to women undergoing ovarian stimulation for in vitro fertilization procedures. Fertil Steril. 2007 Mar; 87(3):542-6. Epub 2006 Nov 27. 2. Host E, Gabrielsen A, Lindenberg S, Smidt-Jensen S 2002 Apoptosis in human cumulus cells in relation to zona pellucida thickness variation, maturation stage, and cleavage of the corresponding oocyte after intracytoplasmic sperm injection. Fertility and Sterility 77, 511-515. 3. Bosco L, Ruvolo G, Morici G, Manno M, Cittadini E, Roccheri MC. Apoptosis in human unfertilized oocytes after intracytoplasmic sperm injection. Fertil Steril. 2005 Nov; 84(5):1417- 23. 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 μm. 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 μm