Maria Agnello

Cadmium induces an apoptotic response in sea urchin embryos.

Abstract 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 on whole-mount embryos, that treatment with cadmium causes activation of caspase-3 and cleavage of death substrates α-fodrin and lamin A. Incubating the embryos since fertilization with Z-DEVD FMK, a caspase-3 inhibitor, we found, by immunocytochemistry, that cleavage by caspase-3 and cleavage of death substrates were inactivated.

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.

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 organism directly interacts with its environment, and is susceptible to effects of several aquatic contaminants. Apoptosis can be adopted as a defence mechanism against any environmental chemical, physical and mechanical stress, for removing irreversibly damaged cells. This review, while not comprehensive in its reporting, aims to provide an overview of current knowledge on mechanisms to regulate physiological and the induced apoptotic program in sea urchin embryos.

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 phenomena are functionally related. Considering the catabolic role of autophagy, an energetic hypothesis to explain the relationship was evaluated. Using a substrate for ATP production, we found that apoptosis, assessed by TUNEL and cleaved caspase-3 immunocytochemistry, was substantially restored in cadmium-treated embryos where autophagy was inhibited by 3-Methyladenine. On the basis of these results, we propose that, autophagy could play a crucial role in stress response of this organism because autophagy could energetically contribute to apoptotic execution through its catabolic role.

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.

Autophagy is required for sea urchin oogenesis and early development

Autophagy 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 data on the occurrence of autophagy at these key phases of development. In order to investigate autophagy trends we performed in vivo assays to detect autophagolysomes, as well as in situ analysis with anti-LC3 antibody to detect autophagosomes before the fusion with lysosomes. From data generated through confocal laser scanning microscopy and quantification of autophagic signals we have drawn several unequivocal conclusions. The results showed a copious and rising number of autophagic organelles that had specific localization. Interestingly the increase in autophagy that occurred just after fertilization has been proved to be crucial for correct initiation of the developmental programme: irreversible developmental delays and morphologic anomalies were induced by short autophagic inhibition. This work focused on the sea urchin model system and corroborates evidence on the need for self-digestion during development, enriching the knowledge on autophagy, a biological mechanism belonging to evolutionarily different organisms.

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 included into the guidelines for the use and interpretation of assays to monitor autophagy. After cadmium (Cd) exposure, a heavy metal recognized as an environmental toxicant, the sea urchin embryo is able to adopt different defense mechanisms, in a hierarchical way. Among these, autophagy is one of the main responses activated to preserve the developmental program. Finally, we discuss the interplay between autophagy and apoptosis in the sea urchin embryo, a temporal and functional choice that depends on the intensity of stress conditions.

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 in cell proliferation, differentiation and morphogenesis. Programmed cell death is a key physio‐ logical mechanism that ensures the correct development and the maintenance of tissues and organs homeostasis in multicellular organisms. PCD has been classified into three types, according to the morphology that the dying cells acquire and the molecular machi‐ nery involved: PCD type I or apoptosis; PCD type II or autophagy and PCD type III or necrosis (not involved in physiological development). These different types of cell death have specific features that can be used to be identified and characterized. Apoptosis is a highly conserved, genetically-controlled process through which certain cells destroy themselves. Autophagy is an evolutionarily conserved pathway used by eukaryotes for degrading and recycling various cellular constituents, such as long-lived proteins and en‐ tire organelles, that was mainly detected in those tissues where abundant cell death is re‐ quired. Both autophagy and apoptosis are induced under stress conditions as an adaptive response against stress. Usually, environmental stress cause severe effects on embryonic development. Embryos of different species, exposed to different types of physical or chemical stress, temporarily suspend their development and activate several protective strategies, including PCD II and PCD III. Research has yet to elucidate the interplay be‐ tween these key processes. Not all types of PCD are always detected in association with a developmental process. Unlike the degeneration of tissues of some invertebrates, the tis‐ sues of vertebrates undergo PCD preferentially through an apoptotic mechanisms. In this chapter, we will briefly describe some specific features of apoptotic and autophagic proc‐ esses. We will focus our attention in some useful model systems of invertebrates and ver‐ tebrates organisms, where autophagy and apoptosis occur both in physiological and stress conditions; specifically, we will analyze embryos of: the nematode Caenorhabditis el‐

MITOCHONDRIA DURING SEA URCHIN OVOGENESIS

Sea 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 distribution, content and activity during Paracentrotus lividus oogenesis. The analyses were carried out using confocal microscopy, in vivo incubating oocytes at different maturation stages with specific probes for mitochondria and mtDNA, and by immunodetection of Hsp56, a well known mitochondrial marker. Results show a parallel rise of mitochondrial mass and activity, and, especially in the larger oocytes, close to germinal vesicle (GV) breakdown, a considerable increase in organelle activity around the GV, undoubtedly for an energetic aim. In the mature eggs, mitochondrial activity decreases, in agreement with their basal metabolism. Further and significant information was achieved by studying the mitochondrial chaperonin Hsp56 and mtDNA. Results show a high increase of both Hsp56 and mtDNA. Taken together these results demonstrate that during oogenesis a parallel rise of different mitochondrial parameters, such as mass, activity, Hsp56 and mtDNA occurs, highlighting important tools in the establishment of developmental competence.

Hsp56 protein and mRNA distribution in normal and stressed P.lividus embryos

Abstract It was previously demonstrated that Paracentrotus lividus Hsp56 mitochondrial chaperonin is constitutively expressed during development, that it increases after heat-shock and cadmium treatment, and that it has a specific territorial distribution, both in normal and heat-shocked embryos, as shown by immunolocalization experiments. In this work, we analyzed by Western blot the territorial distribution of the protein in plutei exposed to heat-shock or sublethal cadmium concentrations, and we found that Hsp56 increases in both ectodermal and endodermal cells. Moreover, by “in situ” hybridization, we looked at Hsp56 mRNA during normal development and under stress conditions. We found that the territorial distribution of the messenger changes during development and that its amount is steadily increased in stressed embryos. Finally, by T1 RNase assay, we identified a cytoplasmic factor that binds to the region of Hsp56 messenger containing the 5’UTR.