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\u2013oocyte 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 did not. Cumulus cell apoptosis rate could be a marker for the selection of the best oocytes to be fertilized by intracytoplasmatic sperm injection

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. Autophagy and apoptosis are crucial processes during embryo development of Invertebrates and Vertebrates organisms, as they are necessary for the formation of a new organism, starting from a fertilized egg. Fertilization triggers cell remodeling from each gamete to a totipotent 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 physiological 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 machinery 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 entire organelles, that was mainly detected in those tissues where abundant cell death is required. 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 between 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 tissues of vertebrates undergo PCD preferentially through an apoptotic mechanisms. In this chapter, we will briefly describe some specific features of apoptotic and autophagic processes. We will focus our attention in some useful model systems of invertebrates and vertebrates organisms, where autophagy and apoptosis occur both in physiological and stress conditions; specifically, we will analyze embryos of: the nematode Caenorhabditis elegans, the insect Drosophila melanogaster, the sea urchin Paracentrotus lividus, the fish Danio Rerio, the mouse mammalian model, and finally we will consider the differentiation of the male and female embryonic germlines in humans

APOPTOSIS RATE IN CUMULUS CELLS AS POSSIBLE MOLECULAR BIOMARKER FOR OOCYTE COMPETENCE.

Several lines of evidence showed that apoptosis rate of cumulus cells in oocytes derived by assisted reproductive technologies could be used as an indicator of fertilizing gamete quality. Aim of the study was to investigate the effects of three different ovarian stimulation protocols on the biological and clinical outcome in hyporesponder patients. Collected data showed a higher significant rate of DNA fragmentation index (DFI) in U group (patients treated with Highly Purified human Menopausal Gonadotrophin) than in P group (treated with recombinant human Follicle Stimulating Hormone (r-hFSH) combined with recombinant human Luteinizing Hormone (r-hLH)). Both groups R (treated with r-hFSH alone) and P showed a significant increase in collected and fertilized oocytes number, embryo quality number. This study showed that combined r-hFSH/r-hLH therapy could represent the best pharmacological strategy for controlled ovarian stimulation and suggests to use DFI as a biomarker of ovarian function in hyporesponder patients

MITOCHONDRIAL MASS, DISTRIBUTION AND ACTIVITY DURING SEA URCHIN OOGENESIS

The sea urchin egg is a favourite model for studies of the molecular biology and physiology of fertilization and early development, yet we know sparingly little of its oocytes and of mitochondria behaviour during oogenesis. The process of oogenesis in most echinoderms is asynchronous so each ovary lobe has hundreds of oocytes at all stages of development. At the beginning of oogenesis, the oocyte is about 10 \ub5m in diameter. During the vitellogenic phase of oogenesis, the oocyte accumulate yolk proteins and grow to ten times their original size to 80 to 100 \ub5m in sea urchins. The oocyte, arrested at the prophase of the first meiotic division, is apparent with its large nucleus, the germinal vesicle (GV), containing a prominent nucleolus. Echinoid (such as sea urchin) and Holothurian oocytes complete meiotic maturation prior to fertilization, distinct from other echinoderms and almost all others animals. As maturation progresses, it occurs the GV breaks down (GVBD). These eggs may then be stored for weeks to months within the female before they are spawned, and the proportion of eggs in the ovary increases from early to late season, as the numbers of oocytes decline [1]. Mitochondria, generally known as the powerhouses of eukaryotic cells, play a primary role in cellular energetic metabolism, homeostasis and death. These organelles, with their multicopy genome maternally inherited, are directly involved at several levels in the reproductive process since their functional status influences the quality of oocytes and contributes to the process of fertilization and embryonic development. It has been demonstrated that the number of maternal mitochondria is sufficient to support development until late stages without new synthesis of mitochondrial DNA or production of new organelles [2]. During embryogenesis mitochondrial mass does not change, whereas mitochondrial respiration increases [3]. The behaviour of these organelles during oogenesis remains at moment unclear. In the present paper we studied, by Confocal Laser Scanning Microscopy tecnologies (CLSM), the mass and distribution, the activity and the DNA content of sea urchin Paracentrotus lividus mitochondria during oogenesis, by in vivo incubating oocytes of different size with cell-permeant probes specific for mitochondria and for DNA and by immunodetection of hsp60 chaperonine, a well known mitochondrial marker. In particular the oocytes were grouped in six classes: < 10, 20/30, 40/50, 60/70, 80/90 \ub5m, and 90 \ub5m ovulated egg, on the base of diameters. Microscopic observations were performed capturing 2 \ub5m thick layers of oocytes. Of the several thousands oocytes we observed, 20 for each different oogenesis stage were analyzed and processed. In order to interpret results and to draw unequivocal conclusions, we measured by IMAGE J software analysis the intensity values of fluorescent signals, as suggested in Agnello et al 2008 [4]. The mitochondria of oocytes with a diameter between 20 and 70 \ub5m, appeared to give rise to clusters that disappear in that of 80 \ub5m. In the oocytes between 60 and 90 \ub5m the red fluorescence seems to be more evident around the germinal vesicle (the merge tends to red), suggesting an increasing oxidative phosphorylation activity. In the ovulated eggs, red and green fluorescence are uniformly distributed suggesting that mitochondria are dispersed in the cytoplasm. In addition the merge of green and red colours shows that the whole mitochondrial population is consuming oxygen at the same level (the resulting colours tends to yellow), figure 1. In order to calculate the total mitochondrial mass and activity we integrated the values of pixel intensities for all captured sections and used the arithmetic means to draw a statistical analysis. Results suggest a parallel rise of mitochondrial mass and activity, suggesting that the amount and activity of organelles change remarkably during oogenesis. Figure 1. shows the distribution of hsp60 protein, detected by immunofluorescence analysis (A), the mitochondrial and genomic DNA, after in vivo incubation with PicoGreen probe (B) and the merge of green and red fluorescence signal, respectively due to mitochondrial mass and activity, after in vivo incubation with Mitotraker Green and Orange (C). The size of the oocytes reported is 80 \ub5m. Results suggest that mitochondria are actively duplicating and that mitochondrial DNA is replicating during the different oogenesis phases. It is noteworthy that around the germinal vesicle, especially in the larger oocytes, next to the germinal vesicle breakdown, the organelles are more active in oxygen consumption, probably due to the major energetic needing in this key moment of gametogenesis. [1] Wessel G.M., Voronina E., and Brooks J.M. (2004) Obtaining and handling echinoderm oocytes. In \u201cMethods in Cell Biology\u201d, Elsevier. Vol.74, Chapter 5, pp. 87-114. [2] Matsumoto L., Kasamatsu H., Pik\ub4o L. and Vinograd J. (1974) Mitochondrial DNA replication in sea urchin oocytes. J. Cell Biol. 63: 146\u2013159. [3] Morici G., Agnello M., Spagnolo F., Roccheri M.C., Di Liegro C.M. and Rinaldi A.M. (2007) Confocal microscopy study of the distribution, content and activity of mitochondria during Paracentrotus lividus development. Journal of Microscopy. 228: 165-173. [4] Agnello M., Morici G., Rinaldi A.M. (2008) A method for measuring mitochondrial mass and activity . Cytotechnology. 56: 145-149. Maria Carmela Roccheri: Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche. Universit\ue0 degli Studi di Palermo, Viale delle Scienze Ed.16, Palermo, Italy; tel: 09123897414; e-mail: [email protected]

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. These different sensitivities highlight the importance of testing toxicity in several species for risk assessment. The strong negative effects of Gd on calcification in plutei, together with the plethora of marine species that have calcifying larvae, indicates that Gd pollution is urgent issue that needs to be addressed

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

It was previously demonstrated that Paracentrotus lividus Hsp56 mitochondrial chaperonin is con- stitutively expressed during development, that it increases after heat-shock and cadmium treatment, and that it has a speci\ufb01c 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 en- dodermal cells. Moreover, by \u201cin situ\u201d 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 identi\ufb01ed a cytoplasmic factor that binds to the region of Hsp56 messenger containing the 5\u2019UT

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, including reproductive performance, fertilization, redox metabolism, embryogenesis, and regulation of embryonic gene expression. This was thoroughly demonstrated for gadolinium, the most widely used lanthanide in diagnostic medicine, whose uptake in sea urchin embryos occurs in a time-and concentration-dependent manner, correlates with decreased calcium absorption and primarily affects skeletal growth, with incorrect regulation of the skeletal gene regulatory network. The results collected on sea urchin embryos demonstrate a variable sensitivity of the early life stages of different species, highlighting the importance of testing the effects of pollution in different species. The accumulation of lanthanides and their emerging negative effects make risk assessment and consequent legislative intervention on their disposal mandatory