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Conotoxins (CnTX) are bioactive peptides produced by marine molluscs belonging to Conus genus. The biochemical structure of these venomous peptides is characterized by a low number of amino acids linked with disulfide bonds formed by a high degree of post-translational modifications and glycosylation steps which increase the diversity and rate of evolution of these molecules. CnTX different isoforms are known to target ion channels and, in particular, voltage-gated sodium (Na+) channels (Nav channels). These are transmembrane proteins fundamental in excitable cells for generating the depolarization of plasma membrane potential known as action potential which propagates electrical signals in muscles and nerves for physiological functions. Disorders in Nav channel activity have been shown to induce neurological pathologies and pain states. Here, we describe the current knowledge of CnTX isoform modulation of the Nav channel activity, the mechanism of action and the potential therapeutic use of these toxins in counteracting neurological dysfunctions.

Sperm quality assessment is the first step for evaluating male fertility and includes the estimation of sperm concentration, motility, and morphology. Nevertheless, other parameters can be assessed providing additional information on the male reproductive potential. This study aimed to evaluate and correlate the oxidative status, mitochondrial functionality, and motility in spermatozoa of two marine invertebrate (Ciona robusta and Mytilus galloprovincialis) and one mammalian (Bos taurus) species. By combining fluorescent staining and spectrofluorometer, sperm oxidative status was evaluated through intracellular reactive oxygen species (ROS) and plasma membrane lipid peroxidation (LPO) analysis. Mitochondrial functionality was assessed through the mitochondrial membrane potential (MMP). In the three examined species, a negative correlation emerged between sperm motility vs ROS levels and LPO. Sperm motility positively correlated with MMP in bovine, whereas these parameters were not related in ascidian or even negatively related in mussel spermatozoa. MMP was negatively related to ROS and LPO levels in ascidians, only to LPO in bovine, and positively related in mussel spermatozoa. These results suggest that energy sources for sperm motility vary between species and that ROS causes a decline in sperm motility via oxidative damage of membrane lipids. Overall, this study validates the use of fluorescent probes in combination with spectrofluorometer as a simple and powerful methodology for supplementary evaluation of sperm quality shedding light on new potential quality markers and provided relevant information on sperm energetic metabolism.

Microalgae are considered good bioindicators of marine environmental quality. Frequently, they are used to investigate the toxicity of sediment elutriates, but their sensitivity is disputed. This paper compared the sensitivity of Phaeodactylum tricornutum (diatom), Skeletonema costatum (diatom), and Dunaliella tertiolecta (green alga), analyzing 257 samples of elutriates (1:4 sediment: water ratio), considering growth inhibition (72 h) as the reference endpoint and sediment chemical (metals, metalloids and polyaromatic hydrocarbons) and grain size. Results of the toxicity tests showed that the microalgae sensitivity was not correlated. The integration of chemical data did not allow to discriminate toxicity effects but contributed to highlight that D. tertiolecta was the most sensitive microalgae (no cell wall) followed by P. tricornutum and S. costatum. Further analysis, including lines of evidence and weight of evidence approaches to calculate risk quotients of elutriate samples, confirmed these results.

Copper oxide nanoparticles (CuO NPs) are extensively used in various industrial and commercial applications. Despite their wide application may lead to the contamination of marine ecosystem, their potential environmental effects remain to be determined. Toxicity assessment studies have primarily focused on investigating the effects of CuO NPs on fertilization success and embryo development of different sea urchin species while the impact on sperm quality have never been assessed. In this line, this study aims to assess the effects of CuO NPs on the spermatozoa of the sea urchin Paracentrotus lividus.After sperm exposure to CuO NPs, biomarkers of sperm viability, cytotoxicity, oxidative stress, and genotoxicity as well as morphology were evaluated. Results showed that CuO NPs exposure decreased sperm viability, impaired mitochondrial activity and increased the production of reactive oxygen species (ROS) and lipid peroxidation. Furthermore, CuO NPs exposure caused DNA damage and morphological alterations. Together with the antioxidant rescue experiments, these results suggest that oxidative stress is the main driver of CuO NP spermiotoxic effects. The mechanism of toxicity is here proposed: the spontaneous generation of ROS induced by CuO NPs and the disruption of the mitochondrial respiratory chain lead to production of ROS that, in turn, induce lipid peroxidation and DNA damage, and result in defective spermatozoa up to induce sperm cytotoxicity. Investigating the effects of CuO NPs on sea urchin spermatozoa, this study provides valuable insights into the mechanism of reproductive toxicity induced by CuO NPs. Keywords: Copper oxide nanoparticles, Cytotoxicity, Genotoxicity, Oxidative stress, Sea urchin, Sperm quality

The sperm plasma membrane is a sensitive target to oxidative stress. The most representative reactive oxygen species (ROS) scavengers in the genital tract, hypotaurine and glutathione, require, for their synthesis, cysteine whose availability is associated with the 1-carbon cycle (1-CC). Human, bovine and ascidian spermatozoa were incubated with compounds supporting the 1-CC (Vitamin B6, Methylcobalamin, 5 Methyl Tetrahydrofolate, Zinc Bisglycinate and N-acetyl-cysteine) (TRT) and compared to the effects induced solely by N-acetyl-cysteine (NAC). In control groups (CNTRL), spermatozoa were incubated with medium alone. After 90 and 180 minutes of incubation, the mitochondrial membrane potential (ΔΨM) in TRT and NAC was significantly (P 2DCFDA evaluation, ROS production differed between species whereas, at 2-OH Ethidium, it significantly decreased in bovine TRT group. Intracellular pH (pHi) did not significantly vary in relation to treatment. In ascidian spermatozoa, the NAC supplementation decreased external pH, which in turn brought to a pHi lowering. Buffering seawater with NaHCO3 reversed the beneficial effects of N-acetyl-cysteine supplementation. In conclusion, both fully supporting the 1-CC and treatment with N-acetyl-cysteine alone improved kinetics, ΔΨM and ROS production in mammalian sperm demonstrating for the first time the direct in vitro effects of these compounds on sperm functionality.

Nickel nanoparticles (Ni NPs) are increasingly used in modern industries as catalysts, sensors, and in electronic applications. Due to this large use, their inputs into marine environment have significantly increased; however, the potential ecotoxicological effects in marine environment have so far received little attention. In particular, little is known on the impact of NPs on gamete quality of marine organisms and on the consequences on fertility potential. The present study examines, for the first time, the impact of Ni NPs exposure on sperm quality of the marine invertebrate Ciona intestinalis (ascidian). Several parameters related with sperm status such as plasma membrane lipid peroxidation, mitochondrial membrane potential (MMP), intracellular pH, DNA integrity, and fertilizing ability were assessed as toxicity end points after exposure to different Ni NPs concentrations. Ni NPs generate oxidative stress that in turn induces lipid peroxidation and DNA fragmentation, and alters MMP and sperm morphology. Furthermore, sperm exposure to Ni NPs affects their fertilizing ability and causes developmental anomalies in the offspring. All together, these results reveal a spermiotoxicity of Ni NPs in ascidians suggesting that the application of these NPs should be carefully assessed as to their potential toxic effects on the health of marine organisms that, in turn, may influence the ecological system. This study shows that ascidian sperm represent a suitable and sensitive tool for the investigation of the toxicity of NPs entered into marine environment, for defining the mechanisms of toxic action and for the environmental monitoring purpose.

Metal oxide nanoparticles, among them copper oxide nanoparticles (CuO NPs), are widely used in different applications (e.g. batteries, gas sensors, superconductors, plastics and metallic coatings), increasing their potential release in the environment. In aquatic matrix, the behavior of CuO NPs may strongly change, depending on their surface charge and some physical-chemical characteristics of the medium (e.g. ionic strength, salinity, pH and natural organic matter content). Ecotoxicity of CuO NPs to aquatic organisms was mainly studied on freshwater species, few tests being performed on marine biota. The aim of this study was to assess the toxicity of CuO NPs on suitable indicator species, belonging to the ecologically relevant level of consumers. The selected bioassays use reference protocols to identify Effect/Lethal Concentrations (E(L)C), by assessing lethal and sub-lethal endpoints. Mortality tests were performed on rotifer (Brachionus plicatilis), shrimp (Artemia franciscana) and copepod (Tigriopus fulvus). While moult release failure and fertilization rate were studied, as sub-lethal endpoints, on T. fulvus and sea urchin (Paracentrotus lividus), respectively. The size distribution and sedimentation rates of CuO NPs, together with the copper dissolution, were also analyzed in the exposure media. The CuO NP ecotoxicity assessment showed a concentration-dependent response for all species, indicating similar mortality for B. plicatilis (48hLC50 = 16.94 ± 2.68mg/l) and T. fulvus (96hLC50 = 12.35 ± 0.48mg/l), followed by A. franciscana (48hLC50 = 64.55 ± 3.54mg/l). Comparable EC50 values were also obtained for the sub-lethal endpoints in P. lividus (EC50 = 2.28 ± 0.06mg/l) and T. fulvus (EC50 = 2.38 ± 0.20mg/l). Copper salts showed higher toxicity than CuO NPs for all species, with common sensitivity trend as follows: P. lividus ≥ T. fulvus (sublethal endpoint) ≥ B. plicatilis >T. fulvus (lethal endpoint) >A. franciscana. CuO NP micrometric aggregates and high sedimentation rates were observed in the exposure media, with different particle size distributions depending on the medium. The copper dissolution was about 0.16% of the initial concentration, comparable to literature values. The integrated ecotoxicological-physicochemical approach was used to better describe CuO NP toxicity and behavior. In particular, the successful application of ecotoxicological reference protocols allowed to produce reliable L(E)C data useful to identify thresholds and assess potential environmental hazard due to NPs.

The Conus genus includes around 500 species of marine mollusks with a peculiar production of venomous peptides known as conotoxins (CTX). Each species is able to produce up to 200 different biological active peptides. Common structure of CTX is the low number of aminoacids stabilized by disulfide bridges and post-translational modifications that give rise to different isoforms. µ and µ-O CTX are two isoforms that specifically target voltage-gated sodium channels. These, by inducing the entrance of sodium ions in the cell, modulate the neuronal excitability by depolarizing plasma membrane and propagating the action potential. Hyperxcitability and mutations of sodium channels are responsible for perception and transmission of inflammatory and neuropathic pain states. In this review, we describe the current knowledge of µ-CTX interacting with the different sodium channels subtypes, the mechanism of action and their potential therapeutic use as analgesic compounds in the clinical management of pain conditions.

The viability of spermatozoa is a crucial parameter to evaluate their quality that is an important issue in ecotoxicological studies. Here, a new method has been developed to rapidly determine the viability of spermatozoa in three marine invertebrates: the ascidian Ciona intestinalis, the sea urchin Paracentrotus lividus and the mollusc Mytilus galloprovincialis. This method employed the dual DNA fluorescent staining coupled with spectrofluorimetric analysis. The dual fluorescent staining used the SYBR-14 stained live spermatozoa and propidium iodide stained degenerated cells that had lost membrane integrity. Stain uptake was assessed by confocal microscopy and then the percentage of live and dead spermatozoa was quantified by spectrofluorimetric analysis. The microscopic examination revealed three populations of spermatozoa: living-SYBR-14 stained, dead-PI stained, and dying-doubly stained spermatozoa. The fluorescence emission peak values recorded in a spectrofluorimeter provide the portion of live and dead spermatozoa showing a significant negative correlation. The stain combination was further validated using known ratios of live and dead spermatozoa. The present study demonstrated that the dual DNA staining with SYBR-14 and propidium iodide was effective in assessing viability of spermatozoa in marine invertebrates and that spectrofluorimetric analysis can be successfully employed to evaluate the percentage of live and dead spermatozoa. The method develop herein is simple, accurate, rapid, sensitive, and cost-effective, so it could be a useful tool by which marine pollutants may be screened for spermiotoxicity.

Fertilization and embryo development that occur in sea water are sensitive to xenobiotics from anthropogenic sources. In this work, we evaluated the influence of two antifouling biocides, tributyltin (TBT) and diuron, on the reproductive mechanisms of the marine invertebrate Ciona intestinalis. By using electrophysiological techniques, we examined the impact of these compounds on the electrical properties of the mature oocytes and of events occurring at fertilization. With different toxicity assays, we studied the effect of the two biocides on the gametes by evaluating fertilization rate and embryo development. Results show that sodium (Na+) currents were significantly reduced by either of the two biocides, whereas conductance was significantly increased. The fertilization current frequency and amplitude, fertilization rate and larval development were affected only by TBT. This study suggests that: (i) the two biocides affect either the electrical properties of the oocyte plasma membrane and the reproductive success representing a risk factor for the survival of the species exposed to environmental pollution; (ii) the ascidian Ciona intestinalis may represent a good model organism to test toxicity of marine pollutants. Possible mechanisms of action of the two biocides are discussed.

Gametes are electrogenic cells that modify their electrical properties in response to different stimuli. This behavior is due to the occurrence of ion currents flowing through ion channels located on the plasma membranes. The modulation of ion channels has been described during the processes of gamete maturation, activation and fertilization in most of the animal models studied. In particular, predominant ions involved in physiological events in oocyte and sperm have been recognized to be sodium, potassium and calcium. In this review, we give an overview on the occurrence, modulation and function of ion fluxes, from gametogenesis to early fertilization events, from marine animals to human. The implications for a dynamic role of ion currents in gamete physiology and their possible clinical and technological applications are discussed.

The analysis of differences between juvenile and adult oocytes may provide useful information on the acquisition of meiotic and developmental competence of the female gamete. In oocytes collected from either ewes or 40-day-old lambs, we evaluated membrane electrical properties, such as resting potential, conductance, activation ion currents, L-type Ca(2+) currents as well as calcium stores and IP3 sensitivity; in addition, the incidence of apoptosis in cumulus cells in these two age categories was compared. The analysis was carried out in oocytes both prior to and after in vitro maturation. Significant differences were found in all the examined parameters in relation to maturational stages whereas minor differences were recorded in relation to age of the donor. IP3 sensitivity strongly increased after in vitro maturation following a dose-dependent pattern from 1 to 500 micromol/L with a significant interaction (P < 0.01) between dose and maturational stage. The incidence of apoptosis in cumulus cells strongly increased after in vitro maturation and was greater in adult than in juvenile cumulus cells (39.2 +/- 5.8% vs. 21.9 +/- 3.5%; P < 0.01). In conclusion, all the examined parameters were greatly affected by the maturational stage, whereas minor differences were due to age-related oocyte quality, that is, at plasma membrane levels to conductance, activation current peaks and calcium currents, at cytosol level to calcium stores and IP3 sensitivity, and to incidence of apoptosis in cumulus cells. These parameters were compared with previous data in bovine to analyze oocyte quality in juvenile and adult individuals or between species.

To determine the level of expression for mRNAs that regulate DNA repair activity in oocytes at the germinal vesicle (GV) stage. Reactive oxygen species (ROS) have been shown to play a major role in the appearance of deleterious DNA decays, and this study focuses on the repair of damage linked to decay caused by the action of ROS. The oocyte needs a mechanism for repairing DNA decays in the early preimplantation embryo before the onset of genomic activation, since in the absence of repair, residual DNA damage would lead to either apoptosis or tolerance. Tolerance of DNA damage is a source of potential mutations.GV oocytes were selected for this study, both for the ethical reason that they are unsuitable for patient treatment, and because no transcription takes place during the period from GV to MII and then prior to genomic activation. The GV oocyte is therefore a good model for looking at DNA during the first cleavages of early preimplantation development. Six cohorts of GV oocytes were pooled for extraction of mRNA; the DNA was analysed using Affimetrix HG-UG133 Plus 2, containing 54,675 probe sets; spike and housekeeping genes were also added as internal controls.In GV oocytes, DNA repair pathways for oxidized bases are redundant. One step repair procedure (OSR), BER (base excision repair), MMR (mismatch repair) and NER (Nucleotide excision repair) are present. All the recognition proteins are also present. The chromatin assembly factors necessary for the maintenance of genomic stability are highly expressed.Gene expression analysis shows that the oocyte does not allow a high level of tolerance for DNA decays. This regulatory mechanism should avoid transmitting mutations into the next generation.

Metamorphosis in the ascidian Ciona intestinalis is a very complex process which converts a swimming tadpole to an adult. The process involves reorganisation of the body plan and a remarkable regression of the tail, which is controlled by caspase-dependent apoptosis. However, the endogenous signals triggering apoptosis and metamorphosis are little explored. Herein, we report evidence that nitric oxide (NO) regulates tail regression in a dose-dependent manner, acting on caspase-dependent apoptosis. An increase or decrease of NO levels resulted in a delay or acceleration of tail resorption, without affecting subsequent juvenile development. A similar hastening effect was induced by suppression of cGMP-dependent NO signalling. Inhibition of NO production resulted in an increase in caspase-3-like activity with respect to untreated larvae. Detection of endogenously activated caspase-3 and NO revealed the existence of a spatial correlation between the diminution of the NO signal and caspase-3 activation during the last phases of tail regression. Real-time PCR during development, from early larva to early juveniles, showed that during all stages examined, NO synthase (NOS) is always more expressed than arginase and it reaches the maximum value at late larva, the stage immediately preceding tail resorption. The spatial expression pattern of NOS is very dynamic, moving rapidly along the body in very few hours, from the anterior part of the trunk to central nervous system (CNS), tail and new forming juvenile digestive organs. NO detection revealed free diffusion from the production sites to other cellular districts. Overall, the results of this study provide a new important link between NO signalling and apoptosis during metamorphosis in C. intestinalis and hint at novel roles for the NO signalling system in other developmental and metamorphosis-related events preceding and following tail resorption.(c) 2007 Elsevier Inc. All rights reserved.

Reproduction and embryo development of marine animals occur in the sea water and are processes very sensitive to substances present in the environment that come from industrial and agricultural activities. These compounds are collectively known as xenobiotics due to their extraenvironmental origin. Among xenobiotics present in the sea water, a relevant role is played by the anti-fouling compounds. These are used to prevent settlement and growth of organisms on submerged structures of vessels and ships that otherwise should create serious problems of speed slow down.

The purpose of the present study was to determine whether Octopus vulgaris spermatozoa are activated by progesterone stimulation. Spermatozoa were collected from the spermatophores in the Needham's sac of the male lMSr and from the spermathecae of oviducal glands of the female lFSr. We used transmission lTEMr and scanning lSEMr electron microscopy to study the morphology of untreated, Ca2p ionophore A23187 and progesteronehtreated MS spermatozoa, and untreated FS spermatozoa. We showed that ionophore and progesterone stimulation of MS spermatozoa induce breakdown of the membranes overlapping the acrosomal region, exposing the spiralized acrosome. These modifications resemble the acrosome reaction observed in other species. FS stored in the spermathecae did not show the membranes covering the acrosomal region present in the MS spermatozoa. When ionophore and progesterone treatments were performed in Ca2phfree artificial sea water, no changes were observed, suggesting the role of external calcium in modifying membrane morphology. Lectin studies showed a different fluorescence distribution and membrane arrangement of FShuntreated spermatozoa with respect to the MS, suggesting that spermatozoa transferred in the female genital tract after mating, are stored in a prehactivated state. The plasma membrane of the untreated MS and FS spermatozoa was labelled with ProgesteronehBSAhFITC, indicating the presence of plasma membrane progesterone receptor. Taken together these data suggest that progesterone induces an acrosomeh like reaction in MS spermatozoa similar to that induced by calcium elevation. In addition progesterone may play a role in the prehactivation of spermatozoa stored in the female tract, further supporting the hypothesized parallelism between cephalopods and vertebrates. Mol. Reprod. Dev. 59:97–105, 2001. © 2001 WileyhLiss, Inc.

Using the whole-cell voltage-clamp technique we have studied electrical coupling and dye coupling between pairs of blastomeres in 16- to 128-cell-stage sea urchin embryos. Electrical coupling was established between macromeres and micromeres at the 16-cell stage with a junctional conductance ( G j ) of 26 nS that decreased to 12 nS before the next cleavage division. G j between descendants of macromeres and micromeres was 12 nS falling to 8 nS in the latter half of the cell cycle. Intercellular current intensity was independent of transjunctional voltage, nondirectional, and sensitive to 1-octanol and therefore appears to be gated through gap junction channels. There was no significant coupling between other pairs of blastomeres. Lucifer yellow did not spread between these electrically coupled cell pairs and in fact significant dye coupling between nonsister cells was observed only at the 128-cell stage. Since 1-octanol inhibited electrical communication between blastomeres at the 16- to 64-cell stage and also induced defects in formation of the archenteron, it is possible that gap junctions play a role in embryonic induction.

In this paper we use the nitric oxide (NO) donor sodium nitroprusside to examine the response of the unfertilised oocyte of the ascidian Ciona intestinalis to nitric oxide. We show that the release of NO triggers an inward current that displays similar properties to the ascidian fertilisation current. Furthermore, the production of NO causes the release of intracellular calcium through a ruthenium-red sensitive mechanism. Our data suggest that these effects are due to the stimulation of nicotinamide nucleotide metabolism, but the active second messenger is not cyclic adenosine diphosphate ribose (cADPr). Finally, we show that NO production increases at fertilisation. The results suggest that ascidian sperm trigger the release of NO and this second messenger causes the breakdown of nicotinamide nucleotides leading to the production of a second messenger which induces the fertilisation current and may assist in the production of the increase in calcium.

Using the whole cell clamp technique, we have measured calcium-dependent currents and steady-state conductance in early sea urchin blastomeres. The calcium currents in M phase decreased from 8.5 microA/cm2 at the four-cell stage to 5.4 microA/cm2 at the eight-cell stage. In 16-cell stage embryos, calcium currents were 7.4 microA/cm2 in the mesomeres, 2.3 microA/cm2 in the macromeres, and were not detected in the micromeres. In contrast, the micromeres had a two- to threefold higher steady-state conductance than the mesomeres or macromeres, which may be due to potassium ion conductivity. Nifedipine, an L-type channel antagonist, delays cleavage division at a concentration of 0.05-0.1 mM and causes developmental defects, such as poor skeletal differentiation in later sea urchin embryos.

SummaryThe process of oocyte maturation is underlined by a redistribution of cellular organelles, among which mitochondria play a functional role for the acquisition of fertilization and developmental competence. In this paper, we applied electron and confocal microscopy by using DIOC6and JC-1 stain to evaluate mitochondria distribution pattern and activity during different stages of oocyte growth in the ascidianStyela plicata. Three categories of oocytes at the germinal vesicle stage underlying the vitellogenic process were characterized on the basis of size, pigmentation and accessory cells. Mitochondria were spread throughout the cytoplasm at the smallest oocyte stage and gradually migrated to the periphery of the subcortical cytoplasm at the intermediate stage. At the fully grown oocyte stage, mitochondria were aggregated in the subcortical cytoplasm. This pattern of polarized mitochondria distribution correlates significantly with an increase in mitochondria potential and activity. In this paper we discuss the relationship of mitochondria to the acquisition of oocyte developmental competence.

Purpose: A retrospective study of 89 hypo-fertile male patients attending for in vitro fertilization was undertaken in order to evaluate possible correlations among sperm DNA damage, sperm analysis parameters and pregnancies. Methods: Sperm parameters (concentration, normal morphology and multiple anomalies) were evaluated according to the World Health Organization guidelines. DNA damages were simultaneously evaluated on each sperm sample by i) sperm chromatin decondensation test; ii) sperm DNA fragmentation evaluated by Sperm Chromatin Dispersion and Halo sperm Kit; iii) sperm DNA fragmentation evaluated by the Terminal Uridine Nick-End Labelling procedure. Results: Statistical analysis was performed by using analysis of variance and least squares regression. The sperm chromatin dispersion and fragmentation assays showed a statistically significant positive correlation (P=0.0017) in all the samples confirming the good efficacy of either of the two tests in detecting sperm DNA damage. Both the two test negatively correlated with normal sperm morphology (P=0.008), however only by using Halo sperm test we obtained a significant correlation with multiple sperm pathological morphologies (P=0.029) and an inverse correlation with pregnancies outcome (P=0.013). No correlation was detected among DNA damages, sperm concentration and chromatin decondensation. Conclusions: These data suggest a similar efficacy of sperm chromatin dispersion and Terminal Uridine Nick-End Labelling in detecting sperm DNA integrity. Due to the higher sensitivity of Halo test, its prognostic role in the diagnosis of fertilizing ability of a semen sample and possible pregnancy rate is discussed

Voltage-dependent calcium currents play a fundamental role during oocyte maturation, mostly L-type calcium currents, whereas T-type calcium currents are involved in sperm physiology and cell growth. In this paper, using an electrophysiological and pharmacological approach, we demonstrated, for the first time in oocytes, that T-type calcium currents are present with functional consequences on the plasma membrane of growing immature oocytes of the ascidian Styela plicata. We classified three subtypes of immature oocytes at the germinal vesicle stage on the basis of their size, morphology and accessory cellular structures. These stages were clearly associated with an increased activity of T-type calcium currents and hyperpolarization of the plasma membrane. We also observed that T-type calcium currents oscillate in the post-fertilization embryonic stages, with minimal amplitude of the currents in the zygote and maximal at 8-cell stage. In addition, chemical inhibition of T-type calcium currents, obtained by applying specific antagonists, induced a significant reduction in the rate of cleavage and absence of larval formation. We suggest that calcium entry via T-type calcium channels may act as a potential pacemaker in regulating cytosolic calcium involved in fertilization and early developmental events.

Using the fluorescent dye Calcium Green-dextran, we measured intracellular Ca2+ in oocytes of the ascidian Ciona intestinalis at fertilization and during progression through meiosis. The relative fluorescence intensity increased shortly after insemination in a single transient, the activation peak, and this was followed by several smaller oscillations that lasted for approximately 5 minutes (phase 1). The first polar body was extruded after the completion of the phase 1 transients, about 9 minutes after insemination, and then the intracellular calcium level remained at baseline for a period of 5 minutes (phase 2). At 14 minutes postinsemination a second series of oscillations was initiated that lasted 11 minutes (phase 3) and terminated at the time of second polar body extrusion. Phases 1 and 3 were inhibited by preloading oocytes with 5 mM heparin. Simultaneous measurements of membrane currents, in the whole-cell clamp configuration, showed that the 1–2 nA inward fertilization current correlated temporally with the activation peak, while a series of smaller oscillations of 0.1–0.3 nA amplitude were generated at the time of the phase 3 oscillations. Biochemical characterization of Maturation Promoting Factor (MPF) in ascidian oocytes led to the identification of a Cdc2-like kinase activity. Using p13suc1-sepharose as a reagent to precipitate the MPF complex, a 67 kDa (67×103Mr) protein was identified as cyclin B. Histone H1 kinase activity was high at metaphase I and decreased within 5 minutes of insemination reaching a minimum level during phase 2, corresponding to telophase I. During phase 3, H1 kinase activity increased and then decayed again during telophase II. Oocytes preloaded with BAPTA and subsequently inseminated did not generate any calcium transients, nonetheless H1 kinase activity decreased 5 minutes after insemination, as in the controls, and remained low for at least 30 minutes. Injection of BAPTA during phase 2 suppressed the phase 3 calcium transients, and inhibited both the increase in H1 kinase activity normally encountered at metaphase II and second polar body extrusion.

Neomycin, injected into ascidian oocytes to a final concentration of 10–50 mM, inhibits both the fertilization current and the surface contraction, showing that phosphoinositide hydrolysis is required for these early activation events. Sperm-activated fertilization currents are not inhibited in the presence of 100 μg/ml intracellular heparin, suggesting that these currents are not directly gated by InsP3. The sulfhydryl reagent thimerosal at 100 μM, in contrast, significantly increases the fertilization current presumably by sensitizing the channel receptor. Since heparin inhibits the surface contraction, InsP3 receptors are shown to play a role in the propagation of the activation response in ascidian oocyte. Depleting intracellular calcium stores by microinjecting 50 mM EGTA into oocytes does not activate fertilization channels; however, subsequent fertilization of these EGTA loaded oocytes leads to a significantly larger and faster fertilization current. Thus in contrast to somatic cells studied to date, second messenger operated plasma membrane channels in ascidian oocytes are not gated by calcium released from intracellular stores. © 1994 Wiley-Liss, Inc.

By using conventional intracellular microelectrodes, we show that the starfish germinal vesicle in situ has a resting potential of +4 mV to +30 mV with respect to the cytoplasm. Patch-clamp electrodes reveal that the surface of the isolated nucleus contains ion channels of 120 pS single-channel conductance. Under whole-cell voltage-clamp conditions, the starfish germinal vesicle generates asymmetric time-variant currents that depend on the ionic environment. Fluorescent dyes in the whole-cell clamp electrode fill the nucleus, showing that the time-variant and the steady-state conductances are properties of the outer and inner nuclear membranes or something that spans them. Because scanning electron microscopy shows a density of nuclear pores in the range 60—110 per square micrometre, and the steady-state resistance in the range is 30—100 MΩ, it seems probable that the pores are not freely permeable to ions. Furthermore, fluorescent dyes of molecular weight up to 500 Da micro-injected either into the cytoplasm or the nucleus of intact oocytes accumulate in the nucleus. Our results show that the nucleus acts as an electrically isolated compartment that separates charged low molecular mass molecules including inorganic ions.

Using a specific protein binding assay we have shown that a spermatozoon of the ascidian Ciona intestinalis contains 1.58 +/- 0.74 x 10(-19) moles of inositol 1,4,5-tri-phosphate (InsP3), while a human spermatozoon contains 6.4 +/- 0.14 x 10(-19) moles. Induction of the acrosome reaction (AR) in both species, by exposure to the calcium ionophore A23187, does not significantly alter levels of InsP3, suggesting that phosphatidylinositol (PI) turnover is not necessary for the calcium ionophore induced AR. Furthermore, PI turnover in ascidian spermatozoa appears to be insensitive to lithium and phorbol ester. The high intracellular concentration of InsP3 in spermatozoa, corresponding to 50-200 microM, suggests it may play a role in egg activation.

SummaryImmature oocytes are arrested at prophase I of the meiotic process and maturation onset is indicated by oocyte nuclear disassembly (germinal vesicle breakdown or GVBD). Signaling pathways that elevate intracellular cyclic AMP (cAMP) may either prevent or induce oocyte maturation depending on the species. In some marine invertebrates and, in particular, in ascidian oocytes, cAMP triggers GVBD rather than blocking it. In this paper, we tested different cAMP elevators in fully grown oocytes at the germinal vesicle stage (GV) of the ascidianCiona intestinalis. We demonstrated that through the activation of adenylate cyclase or the inhibition and phosphodiesterases the oocyte remained at the GV stage. This effect was reversible as the GV-arrested oocytes, rinsed and incubated in sea water, are able to undergo spontaneous maturation and extrusion of follicle cells. In addition, oocytes acquire the ability to be fertilized and start early development. However, morphology of follicle cells, embryos and larvae fromin vitromatured oocytes showed different morphology from those derived fromin vivomature oocytes. The role and the transduction mechanism of cAMP in the regulation of oocyte maturation were discussed. Finally, we indicated a variation of biological mechanisms present in the ascidian species; moreover, we sustain evidence proving that tunicates share some biological mechanisms with vertebrates. This information provided new hints on the importance of ascidians in the evolution of chordates.

Ion currents and cytosolic free calcium ([Ca(2+)](i)) elevations are crucial events in triggering the complex machinery involved in both gamete maturation and fertilization. Oocyte maturation is triggered by hormone signaling which causes ion currents and [Ca(2+)](i) increase. Extracellular calcium seems to be required for meiosis progression since: (i) calcium depletion in the maturation medium severely affects oocyte developmental competence; (ii) the activity of plasma membrane L-type Ca(2+) currents decreases during maturation; (iii) the exposure to verapamil, a specific Ca(2+) channel blocker, decreases in vitro maturation efficiency. In spermatozoa, maturation initiates inside the epididymis and ends in the female genital tract. During their journey through the female reproductive tract, sperm undergo a dramatic selection and capacitation achieving fertilization competence. Adhesion to the tubal epithelium extends sperm life through depression of [Ca(2+)](i) until capacitation signals trigger an [Ca(2+)](i) elevation followed by sperm release. At fertilization, egg-sperm interaction evokes well-described transient and almost simultaneous events: i.e., fertilization current, a change in resting potential, and an increase in free [Ca(2+)](i) concentration. These events, termed oocyte activation, are the direct consequence of sperm interaction via either activation of a receptor or entry of a sperm factor. The latter hypothesis has been recently supported by the discovery of PCLzeta, a sperm-specific isozyme triggering a dramatic [Ca(2+)](i) increase via inositol 1,4,5-trisphosphate (IP(3)) production. The course of ion currents and [Ca(2+)](i) transients during maturation and fertilization plays a pivotal role in correct embryo development.

In recent years, the impact of sperm DNA damage on fertility has become an important issue. The different technologies developed to check sperm DNA fragmentation lead to the same conclusion: DNA damage negatively impacts upon reproductive processes. Oocyte DNA repair capacity is one of the cues to understanding embryo developmental arrest. APEX/Ref-1 (apurinic/apyrimidic endonuclease) is an enzyme involved in the DNA base excision repair pathway removing the abasic sites, the most common DNA decays. In humans, APEX has a multifunctional role, including the control of the redox status of transcription factors. RT-PCR allowed us to detect human APEX transcripts in oocytes, spermatozoa and preimplantation blocked embryos. In parallel, a comparative study on sea squirt Ciona intestinalis (ascidian) indicated that APEX transcripts are clearly detectable in oocytes and embryos until the larva stage, but not in spermatozoa, suggesting the appearance of the paternal contribution to DNA repair during development having arisen only late in Vertebrate evolution. Of additional phylogenetic significance is the observation that sea squirt APEX appears to lack redox transcriptional activity.

Chlorothalonil is a widely used biocide in antifouling paint formulation that replaces tin-based compounds after their definitive ban. Although chlorothalonil inputs into the marine environment have significantly increased in recent years, little is known about its effect on marine animals and in particular on their reproductive processes. In this line, the aim of the present study was to investigate the effects of chlorothalonil exposure on the gamete physiology, fertilization rate and transmissible damage to offspring in the marine invertebrate Ciona intestinalis (ascidians). To identify a possible mechanism of action of chlorothalonil, electrophysiological techniques were used to study the impact on oocyte membrane excitability and on the electrical events occurring at fertilization. The pre-exposure of spermatozoa and oocytes to chlorothalonil did not affect the fertilization rate but caused damage to the offspring by inducing larval malformation. The highest toxicity was observed when fertilization was performed in chlorothalonil solutions with the lowest EC50 value. In particular, it was observed that low chlorothalonil concentrations interfered with embryo development and led to abnormal larvae, whereas high concentrations arrested embryo formation. In mature oocytes, a decrease in the amplitudes of the sodium and fertilization currents was observed, suggesting an involvement of plasma membrane ion currents in the teratogenic mechanism of chlorothalonil action. The risk estimation confirmed that the predicted no-effect concentration (PNEC) exceeded the predicted effect concentration (PEC), thus indicating that chlorothalonil may pose a risk to aquatic species.

During maturation, the last phase of oogenesis, the oocyte undergoes several changes which prepare it to be ovulated and fertilized. Immature oocytes are arrested in the first meiotic process prophase, that is morphologically identified by a germinal vesicle. The removal of the first meiotic block marks the initiation of maturation. Although a large number of molecules are involved in complex sequences of events, there is evidence that a calcium increase plays a pivotal role in meiosis re-initiation. It is well established that, during this process, calcium is released from the intracellular stores, whereas less is known on the role of external calcium entering the cell through the plasma membrane ion channels. This review is focused on the functional role of calcium currents during oocyte maturation in all the species, from invertebrates to mammals. The emerging role of specific L-type calcium channels will be discussed.

In mammals, sperm adhesion to the epithelial cells lining the oviductal isthmus plays a key role in the maintenance of motility and in the selection of superior quality subpopulations. In the bovine species, heparin and other sulfated glycoconjugates powerfully induce the synchronous release of sperm adhering to tubal epitheliumin vitroand may represent the signal which triggers release at ovulationin vivo. Sperm detachment may be due either to surface remodeling or to hyperactivation brought about by capacitation. In this paper, the dynamics of intracellular free Ca2+concentration ([Ca2+]i) and protein tyrosine phosphorylation in sperm during and after heparin-induced release fromin vitrocultured oviductal monolayers were assessed to determine whether this event is due to capacitation. Moreover, Ca2+-ionophore A23187, thapsigargin, thimerosal and caffeine were used to determine whether [Ca2+]iincrease and/or hyperactivation can induce sperm release. Results showed that: 1. heparin-released sperm have significantly higher [Ca2+]ithan adhering sperm; 2. heparin induces a [Ca2+]ielevation in the sperm head followed by detachment from the monolayers; 3. external Ca2+is not required for heparin-induced release; 4. [Ca2+]iincrease and/or hyperactivation are unable to release sperm; and 5. heparin-released sperm have an increased level of tyrosine phosphorylated proteins compared with adhering sperm. In conclusion, although heparin is considered a long-lasting capacitation agent, it quickly modulates the capacitation of bovine sperm adhering to the fallopian epithelium, probably leading to surface remodeling and therefore to the release of sperm selected and stored within the oviduct through adhesion.

One of the first events that occurs at fertilization is a transient modification of the electrical properties of the oocyte plasma membrane. The whole-cell voltage clamp technique was used to demonstrate an outward ion current and a hyperpolarization of the plasma membrane after fertilization in bovine oocytes. These electrical events, together with measurement of internal calcium concentrations, were also recorded after injection with sperm factor and exposure to parthenogenetic activators, such as Ca(2+) ionophore, ethanol and thapsigargin. Experiments were carried out simultaneously in immature and in vitro matured oocytes. Significant differences were recorded in the activation current and hyperpolarization among oocyte activators and between immature and matured oocytes. However, outward ion current and Ca(2+) release showed similar dynamics. The injection of the calcium chelator EGTA completely abolished both ion current and hyperpolarization, indicating that these electrical events are calcium dependent. Addition of specific calcium releasers, such as 1,4,5-inositol trisphosphate (IP(3)) and caffeine, triggered ion activation current and hyperpolarization indicating that IP(3) and ryanodine receptors are active in both immature and matured oocytes. Different ion channel inhibitors were used to characterize the channels underlying outward currents. Only addition of rIberiotoxin caused a complete inhibition of the current, indicating the involvement of high conductance Ca(2+)-activated K(+) channels in generating activation current. In conclusion, these findings provide evidence that bovine oocyte activation is associated with Ca(2+)-dependent electrical events. Oocytes have the potential to react to different activators even when immature; however, oocyte maturation seems to increase sensitivity to physiological activators, such as spermatozoa and sperm factor, and chemicals, such as ethanol.

In vivo bovine embryos were obtained by nonsurgical flushing of uterine horns of cows submitted to superovulatory treatment, while in vitro embryos were generated from oocytes collected from slaughtered donors. Lucifer Yellow injected into single blastomeres did not diffuse into neighboring cells until the morula stage in in vivo embryos and the blastocyst stage in in vitro embryos. In both cases diffusion was limited to a few cells. In contrast, diffusion was extensive in microsurgically isolated inner cell mass (ICM) but absent in the trophectoderm (TE). At the blastocyst stage, diffusion was always more extensive in in vivo than in in vitro embryos. Ultrastructural analyses confirmed these functional observations, and gap junction-like structures were observed at the blastocyst stage. These structures were diffuse in the ICM of in vivo embryos, scarce in the ICM of in vitro embryos and in the TE of in vivo embryos, and not observed in the TE of in vitro embryos. Blastomeres at all stages of development from the 2-cell stage to the blastocyst stage in in vitro embryos and at the morula and blastocyst stage in in vivo embryos were electrically coupled, and the junctional conductance (Gj) decreased in in vitro embryos from 4.18 +/- 1.70 nS (2-cell stage) to 0.37 +/- 0.12 nS (blastocyst stage). At each developmental stage, in vivo embryos showed a significantly (P < 0. 05) higher Gj than in vitro-produced embryos. Moreover, a significantly (P < 0.01) higher Gj was found in isolated ICM than in the respective blastocyst in both in vivo- and in vitro-produced embryos (3.5 +/- 1.4 vs. 0.7 +/- 0.3 and 2.6 +/- 1.6 vs. 0.37 +/- 0. 12 nS, respectively). The electrical coupling in absence of dye coupling in the early bovine embryo agrees with observations for embryos from other phyla. The late and reduced expression of intercellular communicative devices in in vitro-produced embryos may be one of the factors explaining their developmental low efficiency.

Fertilisation is characterised by an ion current across the oocyte plasma membrane (DeFelice et al., 1986), a transient increase in intracellular calcium (see Jaffe, 1985), and the release of the cell cycle block controlled at the level of MPF (Newport and Kirschner, 1984). The fertilisation calcium signal appears to trigger the resumption of the cell cycle at fertilisation in oocytes arrested at meiosis II or after the completion of meiosis (reviewed in Whitaker and Patel, 1990). However, the role of intracellular second messengers, including calcium, in the control of progression through the early meiotic cell cycle, is less well defined (see Whitaker and Patel, 1990; Whitaker, 1996).

Purpose: Our goal was to study the activation current in physiologically competent metaphase II human oocytes, i.e., not previously exposed to spermatozoa or aged in vitro, and, in particular, to determine whether a soluble sperm factor triggers a fertilization current comparable to that observed with intact spermatozoa and to characterize the current involved. Methods: The whole-cell voltage-clamp technique was used on spare metaphase II oocytes, obtained with patient consent from IVF programs. In this configuration a soluble fraction from human spermatozoa was microinjected, and the current recorded. Results: Metaphase II human oocytes generate bell-shaped outward currents of 400–1000 pA (X=706±322;n=10), following injection of a cytosolic extract from human spermatozoa. The amount of sperm extract injected was less than 10% of the total oocyte volume and was equivalent to 1–10 spermatozoa. A similar current was generated following exposure to 20 µM of the calcium ionophore A23187 (n=10). The steady-state conductance of the oocyte increased from 10 to 19.8 nS (n=10) following injection of the sperm factor and from 5.3 to 27.7 nS following ionophore exposure. Both sperm factor- and ionophore-induced currents were reduced in amplitude when the unfertilized oocyte was preexposed to 25–75 µM iberiotoxin (n=8) and eliminated at a concentration of 100 µM iberiotoxin. Conclusions: The data support the hypothesis of a soluble sperm factor involved in the activation of human oocytes and shows that the initial activation response in the human oocyte is the gating of Ca2+-activated K+ channels.

SummaryThe purpose of the present study was to determine whether the plasma membrance of the human oocyte is reorganised following fertlisation and during early cleavage. In order to characterise and localise the major sugar moieties on surface glycoporteins, oocytes and embroys were labelled with a range of flourescent lectins. Regional organisation of plasma membrane microvilli in oocytes and embryos was also studied using scanning electron microscopy (SEM). The plasma membrance of human oocytes, zygotes and early blastomeres stained strongly and homogeneously with concanavalin A andTriticum vulgarislectin (WGA), indicationg the presence of plasma membrance glycoconjugates with α-D-mannosyl residues, sialic acid and β-NAc-glucosaminyl groups. We did not observe regional domains in oocytes and zygotes, suggesting that the plasma membrane is not topographically reorganised following fertilisation. SEM shows the surface of the human zygote to be organised into short microvilli 0.2–3.0 μm in length and at a density of 5–20/μm2. In early cleavage stages the microvilli are shorter and less frequent (0.2–1.0 μm; 1–5/μm2); however, there is no evidence of polarisation at this level of organisation, at either stage of development. The surface of cell fragments, common in the human embryoin vitro, differs in having few microvilli and numerous cytoplasmic blebs. In conclusion, there are no obvious morphological signs of regionalisation in the plasma membrane of the human embryo before the 8-cell stage.

In this report we show that the first event of activation in the human oocyte, the Fertilization current (FC), is a slow transient outward current of 300 pA, which induces a gradual hyperpolarization of the plasma membrane from -20mV to -60mV, 60-120 min after insemination, followed by a repolarization to -20mV. Activation currents (AC) of 600-2,500 pA, induced by exposure to the calcium ionophore A23187 or by microinjection of InsP3 into the cytosol, are also outward. The AC are inhibited by preloading oocytes with EGTA suggesting they are calcium dependent. Since AC are 2-10-fold the amplitude of the FC the fertilizing spermatozoon in the human only activates a portion of the primary elements stored in the oocyte for triggering metabolic depression. Oocyte activation in the human resembles that in the hamster rather than other mammals or invertebrates studied to date.

Objective To study the molecular origin and functionality of the plasma membrane of round-head spermatozoa in the human. Design Clinical and laboratory study. Setting Patients in a clinical and academic environment. Patients Men with round-head spermatozoa. Results Pisum sativum lectin homogeneously stains the surface of round sperm; however, the staining pattern and transmission electron microscopy show that the plasma membrane does not alter after exposure to the calcium ionophore A23187. In a clinical program, round-head spermatozoa injected subzonally into metaphase II oocytes with or without pretreatment with the fusogen polyethylene glycol did not bind or fuse to the oocyte surface. Conclusion The data suggests that plasma membrane fusion in human gametes is regulated by specific surface molecules and that exposure of these molecules on the sperm surface cannot be triggered by elevating intracellular calcium alone.

SummarySpermatozoa from the mollusc Ostrea edulis are capable of fusing to and entering de-chorionated ascidian eggs. During interaction they generate activation currents, comparable to the fertilisation currents induced by homologous spermatozoa. Activation currents are inward at − 80 mV, with a mean initial slope of 111 ± 124 pA/s for Ciona intestinalis eggs and 47 ± 25 pA/s for Phallusia mammillata eggs, while the mean peak currents are 2782 ± 1132 pA and 1523 ± 1668 pA, respectively. The fertilisation and activation currents reverse at a holding potential of 0 mV to + 20 mV, suggesting that oyster sperm and ascidian sperm gate the same channel precursor, a non-specific, large conductance channel described previously (Dale & DeFelice, 1984). In contrast to homologous fertilisation, the activation current is not followed by a polarised contraction of the egg surface, nor other signs of egg activation. Staining eggs with Hoechst 33342 after insemination shows the female nucleus and a single oyster sperm nucleus at the antipode. This suggests a specialised predetermined site at the vegetal pole for sperm entry. Homologous and heterologous spermatozoa delivered, in a large pipette, to localised areas of the egg surface generate fast inward currents of 200–2000 pA, but do not induce contraction of the egg surface. This shows that although channel precursors are located globally over the egg surface, channel activation does not necessarily trigger the contraction wave. Subsequent induction of both a fertilisation current and a contraction by homologous sperm added to the bath, implies a regionalised activation site with an accumulation of channel precursors and a ‘pacemaker’ for the initiation of the contraction wave.

Using the whole-cell voltage clamp technique, we have studied junctional conductance (Gj), and Lucifer Yellow (LY) coupling in 2-cell and 32-cell ascidian embryos. Gj ranges from 17.5 to 35.3 nS in the 2-cell embryo where there is no passage of LY, and from 3.5 to 12.2 nS in the later embryo where LY dye spread is extensive. In both cases, Gj is independent of the transjunctional potential (Vj). Manually apposed 2-cell or 32-cell embryos established a junctional conductance of up to 10 nS within 30 min of contact. Furthermore, since we did not observe any significant number of cytoplasmic bridges at the EM and Gj is sensitive to octanol, it is probable that blastomeres in the 2-cell and 32-cell embryos are in communication by gap junctions. In order to compare Gj in the two stages and to circumvent problems of cell size, movement and spatial location, we used cytochalasin B to arrest cleavage. Gj in cleavage-arrested 2-cell embryos ranged from 25.0 to 38.0 nS and remained constant over a period of 2.5 h. LY injected into a blastomere of these arrested embryos did not spread to the neighbour cell until they attained the developmental age of a 32- to 64-cell control embryo. Our experiments indicate a change in selectivity of gap junctions at the 32cell stage that is not reflected by a macroscopic change in ionic permeability.

A preliminary study on intercecullar communicative devices in the early human embryo has been made using dye-coupling techniques and electron microscopy (EM). Lucifer yellow injected into single blastomeres of embryos at the 4-cell stage up to the late morula stage did not spread to neighbouring cells, indicating that gap junctions and cytoplasmic bridges are not significant pathways for information transfer. Dye spread was first observed in the blastocyst stage, where trophectoderm cells and inner mass cells were shown to be in communication through gap junctions. Studies at the EM level confirmed this finding. Tight junctions and desmosome-like structures, apparent from the 6-cell stage onward, were located both peripherally and centrally and were initially nonzonular. The role of intercellular devices in the primary differentiation of the human embryo is discussed.

In this paper we report on the mechanism of action of bonellin on sea urchin eggs. Bonellin acts in the same way as porphyrins, ie, by causing the crosslinking of membrane proteins and the peroxocompounds formation. A possible role of membrane organization related to cleavage formation is discussed.

1. 1. The hemolytic activity of suberitine, a neurotoxic protein isolated from Suberites domuncula , has been examined. 2. 2. The toxin has no phospholipase A activity. 3. 3. The hemolytic activity of suberitine does not exhibit the induction period and is completely inhibited by sucrose. 4. 4. A study of the effect of temperature on the rate of hemolysis shows that suberitine has two values of the critical thermal increment. The optimum pH for the hemolytic activity was 6.0. 5. 5. There was a correlation between the hemolytic and toxic activity.

A morphological classification of the immature cumulus-oocyte complex (COC), which grossly resembled the atresia grade of its follicle source, was used in bovine oocytes to determine 1) the developmental potential by either in vitro fertilization or parthenogenetic activation, 2) the calcium current activity by whole-cell voltage clamp technique, and 3) the intracytoplasmic calcium stores by microfluorimetric evaluation. The COC classification took into account some cumulus and ooplasm features, designated as follows: A) presence of a clear and compact cumulus and translucent ooplasm, B) dark and compact cumulus and dark ooplasm, and C) dark and expanded cumulus and dark ooplasm. We found no difference between in vitro fertilization and parthenogenetically activated oocytes in terms of cleavage rate and blastocyst production. Both protocols indicated a significant variability between the three compared COC categories. The B-COCs showed the highest embryo production efficiency as well as the greatest Ca(2+) current activity, whereas A-COCs showed an opposite pattern. The C-COCs, mostly attributed to atretic and heavily atretic follicles, showed morphological characteristics between those of A- and B-COCs. Stores of Ca(2+) were significantly greater in A-COCs than in B- and C-COCs in the case of immature oocytes, and greater in B-COCs than in C-and A-COCs in the case of in vitro-matured oocytes. These results demonstrate that in the bovine 1) the considered morphological criteria for oocyte classification are related to developmental competence, 2) plasma membrane Ca(2+) current in the immature oocyte is related to developmental potential, and 3) calcium stores are related to morphological quality in immature oocytes and to developmental competence in mature oocytes.