No abstract

Mitochondria, responsible for the energygenerating process essential for the cell metabolism, differ for the number, localization and activity in animal cells and tissues in relation to the energetic needs. Using fluorescent probes specific for mitochondria, Mitotracker Green (MTG) and Orange (MTO), and Confocal Laser-Scanning Microscope (CLSM), we elaborated a method to measure in vivo the mitochondrial mass and activity, in sea urchin Paracentrotus lividus eggs and embryos. The analysis of captured images, revealed a variation of mitochondrial distribution and an increase of activity after fertilization.

SummaryIn 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.

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. 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. (Fertil Steril 2005;84:1417-23.

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.

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.

The dynamics of chondriome changes in oogenesis of the sea urchin Paracentrotus lividus were studied by electron microscopy. An oocyte-enriched fraction obtained by gonad mechanical dissociation without protease treatment was used. The shape, size and arrangement of mitochondria (Mt) in cells were quantitatively analysed on the basis of data from reconstruction experiments, with serial sections performed using a specific computer program. At all stages of oogenesis, the chondriome was shown to consist of rod-shaped Mt of various lengths and also of small amounts of globular Mt about 0.3 m in diameter. Chondriome transformation during oogenesis is shown to involve the following processes: (1) a 64-fold increase in number of Mt, with the ratio of cytoplasm to Mt volume quite constant in the course of oogenesis; (2) an increase in length of Mt to a maximum of 1.54 m in medium oocytes and successive considerable mitochondrial division; (3) changes in Mt ultrastructure; and (4) a clustering of Mt. In a mature egg, the modal value of Mt length was reduced and, unlike the oocytes, was more homogeneous, and the Mt were completely clustered.

No abstract