Search

SummaryThe integrity of chromatin in the spermatozoon is essential for reproductive outcome. The aim of this study was to evaluate the most effective and cost-effective method to reduce the percentage of spermatozoa with defects in chromatin decondensation for use in assisted reproductive technologies (ART) procedures. Sperm samples from 15 sub-fertile males were examined at CFA Naples to determine the sperm decondensation index (SDI), using the aniline blue test, before and after preparation, comparing density gradients with two different swim-up approaches. All three techniques led to a reduction in decondensed spermatozoa with no statistical difference (P > 0.05) between the control and the treated sperm. In contrast, we found a highly significant decrease in SDI (P < 0.01) after the two swim-up methods in all the samples, confirming the efficacy of these methods in lowering the percentage of chromatin compaction damage. There was no statistical difference between the two swim-up methods, however swim-up from the pellet led to improved count, motility and the percentage of normal condensed spermatozoa. We suggest that swim-up from the pellet be used in ART on sub-fertile males, both to reduce cell stress by multiple centrifugation and improve the recovery rate of mature spermatozoa.

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.

Voltage-gated ion channels are plasma membrane proteins that generate electrical signals following a change in the membrane voltage. Since they are involved in several physiological processes, their dysfunction may be responsible for a series of diseases and pain states particularly related to neuronal and muscular systems. It is well established for decades that bioactive peptides isolated from venoms of marine mollusks belonging to the Conus genus, collectively known as conotoxins, can target different types and isoforms of these channels exerting therapeutic effects and pain relief. For this reason, conotoxins are widely used for either therapeutic purposes or studies on ion channel mechanisms of action disclosure. In addition their positive property, however, conotoxins may generate pathological states through similar ion channel modulation. In this narrative review, we provide pieces of evidence on the pathophysiological impacts that different members of conotoxin families exert by targeting the three most important voltage-gated channels, such as sodium, calcium, and potassium, involved in cellular processes.

The aim of the present work is to demonstrate the practical importance of a multidisciplinary approach and weighted criteria to synthesize and integrate different typologies of data (or lines of evidence, LOEs), including chemical levels in marine sediments, their bioavailability to specific indicator species, ecotoxicological effects measured through subcellular biomarkers and batteries of bioassays, and potential impacts of pollution on local benthic communities. The area of Bagnoli (Gulf of Naples, Southern Italy) was selected as a model case-study, as it is a coastal area chronically impacted by massive industrial contamination (trace metals and hydrocarbons), and dismissed decades ago without any subsequent remediation or habitat restoration. The results of each LOE were elaborated to provide specific hazard indices before their overall integration in a weight of evidence (WOE) evaluation. Levels of some trace metals and PAHs revealed a severe contamination in the entire study area. Bioavailability of hydrocarbons was evident particularly for high molecular weight PAHs, which also caused significant variations of cellular biomarkers, such as cytochrome P450 metabolization in fish, lysosomal membrane destabilization in mussels, genotoxic effects both in fish and molluscs. The results of a battery of bioassays indicated less marked responses compared to those obtained from chemical and biomarkers analyses, with acute toxicity still present in sediments close to the source of contamination. The analysis of benthic assemblages showed limited evidence of impact in the whole area, indicating a good functioning of local ecosystems at chronic contamination. Overall, the results of this study confirm the need of combining chemical and biological data, the quantitative characterization of various typologies of hazard and the importance of assessing an integrated environmental WOE risk, to orientate specific and scientifically-supported management options in industrialized areas.

The ongoing increase of CO2 in the atmosphere is inducing a progressive lowering of marine water pH that is predicted to decrease to 7.8 by the end of this century. In marine environment, physical perturbation may affect reproduction, which is crucial for species' survival and strictly depends on gamete quality. The effects of seawater acidification (SWAc) on gamete quality of broadcast spawning marine invertebrates result largely from experiments of gamete exposure while the SWAc impact in response to adult exposure is poorly investigated. Performing microcosm and in field experiments at a naturally acidified site, we investigated the effects of adult SWAc exposure on sperm quality parameters underlying fertilization in Mytilus galloprovincialis. These animals were exposed to pH 7.8 over 21 days and collected at different times to analyze sperm parameters as concentration, motility, viability, morphology, oxidative status, intra- and extra-cellular pH and mitochondrial membrane potential. Results obtained in the two experimental approaches were slightly different. Under field conditions, we found an increase in total sperm motility and mitochondrial membrane potential on days 7 and 14 from the start of SWAc exposure whereas, in microcosm, SWAc group showed an increase of total motility on day 14. In addition, sperm morphology and intracellular pH were affected in both experimental approaches; whereas oxidative stress was detected only in spermatozoa collected from mussels under natural SWAc. The overall analysis suggests that, in mussels, SWAc toxic mechanism in spermatozoa does not involve oxidative stress. This study represents the first report on mussel sperm quality impairment after adult SWAc exposure, which may affect fertilization success with negative ecological and economic consequences; it also indicates that, although naturally acidified areas represent ideal natural laboratories for investigating the impact of ocean acidification, microcosm experiments are necessary for examining action mechanisms.

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.

Ion channels are proteins expressed in the plasma membrane of electrogenic cells. In the zygote and blastomeres of the developing embryo, electrical modifications result from ion currents that flow through these channels. This phenomenon implies that ion current activity exerts a specific developmental function, and plays a crucial role in signal transduction and the control of embryogenesis, from the early cleavage stages and during growth and development of the embryo. This review describes the involvement of ion currents in early embryo development, from marine invertebrates to human, focusing on the occurrence, modulation, and dynamic role of ion fluxes taking place on the zygote and blastomere plasma membrane, and at the intercellular communication between embryo cell stages.

In this retrospective study of 873 men enrolled for assisted reproduction techniques, relationships between sperm quality parameters, motile sperm organelle morphology examination (MSOME), DNA damage and live birth rate were evaluated. The presence of vacuoles in the sperm heads was detected by MSOME. Either chromatin decondensation or DNA fragmentation was used to study DNA damage. Results show that age significantly affected some of the examined parameters. In particular, sperm concentration was positively correlated (R = 0.088; P = 0.01) and chromatin decondensation was negatively correlated (R = -0.102; P = 0.003) with age. Furthermore, live birth rate was significantly lower in men aged 40 years or older (P < 0.02) compared with the younger age groups. The presence of sperm head vacuoles was not associated with head morphology, main sperm quality parameters, DNA fragmentation and live birth rate. Considering sperm heads in relation to the shape (normal/abnormal) and vacuoles (presence/absence), no significant variations in the occurrence of vacuoles in either normal or abnormal heads were found. These data suggest that vacuoles are physiological features that do not alter sperm functionality, and it seems that MSOME is not necessary for increasing the success of assisted reproduction techniques.

Global warming is an increasingly serious problem underlying ecological change in marine flora and fauna. Mytilus galloprovincialis is an intertidal species that colonizes coasts in moderate and warm climates, and can thus withstand extreme climatic conditions; however, it successfully reproduces only within a certain temperature range. The effects of prolonged exposure to 28 °C, a temperature unsuitable for breeding activity, on sperm quality were evaluated in this study. Such heat stress induced the following: a significant reduction in concentration; a biphasic pattern of motility and mitochondrial membrane potential that first increased, and then collapsed; a decrease in the intracellular calcium concentration; a rapid increase in lipid peroxidation that was normalized after the third week of heat stress; an increase in DNA fragmentation after the third week of heat stress; and atypical morphology (i.e., sperm with a globular head, asymmetrical tail, and acrosome loss). Currently, these elevated-temperature conditions are achieved along the Mediterranean coast during the late summer, when the reproductive activity of M. galloprovincialis is suspended after massive spawning in the spring. The increasing global temperature, however, may shift their breeding season, thus significantly impacting marine ecosystems and mussel production.

Rising atmospheric CO2 is causing a progressive decrease of seawater pH, termed ocean acidification. Predicting its impact on marine invertebrate reproduction is essential to anticipate the consequences of future climate change on species fitness and survival. Ocean acidification may affect reproductive success either in terms of gamete or progeny quality threating species survival. Despite an increasing number of studies focusing on the effects of ocean acidification on the early life history of marine organisms, very few have investigated the effects on invertebrate gamete quality. In this study, we set up two experimental approaches simulating the ocean conditions predicted for the end of this century, in situ transplant experiments at a naturally acidified volcanic vent area along the Ischia island coast and microcosm experiments, to evaluate the short-term effects of the predicted near-future levels of ocean acidification on sperm quality of the ascidian Ciona robusta after parental exposure. In the first days of exposure to acidified conditions, we detected alteration of sperm motility, morphology and physiology, followed by a rapid recovery of physiological conditions that provide a new evidence of resilience of ascidian spermatozoa in response to ocean acidification. Overall, the short-term tolerance to adverse conditions opens a new scenario on the marine species capacity to continue to reproduce and persist in changing oceans.

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.

In all vertebrates, mature oocytes arrest at the metaphase of the II meiotic division, while some invertebrates arrest at metaphase-I, others at prophase-I. Fertilization induces completion of meiosis and entry into the first mitotic division. Several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the regulation of the cytostatic factor (CSF) and the maturation promoting factor (MPF) in metaphase I or II. Recently, we proposed the oocytes of ascidian Ciona intestinalis as a new model to study the meiotic division. Here, taking advantage of the recent publication of the C. intestinalis genome, we presented a phylogenetic analysis of key molecular components of the CSF-related machinery. We showed that the Mos/MAP kinase pathway is perfectly conserved in ascidians. We demonstrated the presence of a CSF-like activity in metaphase-I arrested C. intestinalis oocytes able to block cell division in two-cell embryos. We further investigated the regulation of CSF by demonstrating that both CSF and MPF inactivation, at the exit of metaphase-I, are independent from protein synthesis, indicating the absence of short-lived factors that regulate metaphase stability, as in other invertebrate species. The results obtained suggest that meiotic regulation in C. intestinalis resembles that of vertebrates, such as Xenopus accordingly to the position of this organism in the evolutionary tree.

Growing oocytes are arrested at the first prophase of meiosis which is morphologically identified by the presence of a large and vesicular nucleus, called the germinal vesicle. The dissolution of the germinal vesicle marks the resumption of meiosis during which the oocyte undergoes massive modifications up to the second meiotic block, which is removed at fertilization. The interval between the first and the second meiotic block is defined as maturation and the events occurring during this period are crucial for ovulation, fertilization, and embryo development. Oocytes are excitable cells that react to stimuli by modifying their electrical properties as a consequence of ion currents flowing through ion channels on the plasma membrane. These electrical changes have been largely described at fertilization whereas little information is available during oocyte maturation. The aim of this review is to give an overview on the involvement of ion channels and ion currents during oocyte maturation in species from invertebrates to mammals. The results summarized here point to the possible functional role of ion channels underlying oocyte growth and maturation.

Ion channels are proteins expressed in the plasma membrane of electrogenic cells. In the zygote and blastomeres of the developing embryo, electrical modifications result from ion currents that flow through these channels. This phenomenon implies that ion current activity exerts a specific developmental function, and plays a crucial role in signal transduction and the control of embryogenesis, from the early cleavage stages and during growth and development of the embryo. This review describes the involvement of ion currents in early embryo development, from marine invertebrates to human, focusing on the occurrence, modulation, and dynamic role of ion fluxes taking place on the zygote and blastomere plasma membrane, and at the intercellular communication between embryo cell stages.

An intracellular calcium ([Ca(2+)]i) rise has been described in cumulus-oocyte complexes (COCs) following luteinizing hormone (LH) exposure. Together with cAMP, Ca(2+) is a candidate signal for resumption of meiosis. Here, we analyzed if the most common hormones involved in oocyte maturation can induce the same Ca(2+) signal. In addition, we characterized the source of this signal. Immature, in vitro-matured, and roscovitine-meiotically arrested COCs were loaded with Fluo-4 AM, stimulated with hormones/growth factors, and tested for [Ca(2+)](i) variations in cumulus cells. Reagents known to inhibit or stimulate [Ca(2+)](i) rises were used to characterize these [Ca(2+)](i) dynamics. Finally, expression of LH receptors (LHRs) in COCs was analyzed by immunofluorescence. In immature COCs, follicle-stimulating hormone (FSH) elicited a single [Ca(2+)](i) rise that was higher than those induced by LH and growth hormone (GH), whereas epithelial growth factor failed to induce any changes in [Ca(2+)](i). The [Ca(2+)](i) rise induced by FSH was higher in immature COCs; was reduced in roscovitine-arrested, immature COCs; and was negligible in gonadotropin-induced, in vitro-matured COCs. In the case of spontaneous- and GH-matured COCs, however, FSH stimulation caused a lower [Ca(2+)](i) rise. The hormone-induced [Ca(2+)](i) rise was due to: (i) external Ca(2+) entry; (ii) intercellular communication; and (iii) intracellular Ca(2+) stores. Immunofluorescence revealed that LHRs were expressed throughout the cumulus cells. The above results show that: (i) gonadotropins and GH cause a [Ca(2+)](i) rise in cumulus cells; (ii) this [Ca(2+)](i) rise results from extra-, inter-, and intra-cellular cumulative Ca(2+) fluxes; and (iii) LHRs are distributed on either outer or inner cumulus cells.

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.

Background The first clues to the process of gamete activation date back to nearly 60 years ago. The mutual activation of gametes is a crucial event during fertilization. In the testis and ovaries, spermatozoa and oocytes are in a state of meiotic and metabolic quiescence and require reciprocal signals in order to undergo functional changes that lead to competence for fertilization. First, the oocyte activates sperm by triggering motility, chemoattraction, binding and the acrosome reaction, culminating with the fusion of the two plasma membranes. At the end of this cascade of events, collectively known as sperm capacitation, sperm-induced oocyte activation occurs, generating electrical, morphological and metabolic modifications in the oocyte. Objective and rationale The aim of this review is to provide the current state of knowledge regarding the entire process of gamete activation in selected specific animal models that have contributed to our understanding of fertilization in mammals, including humans. Here we describe in detail the reciprocal induction of the two activation processes, the molecules involved and the mechanisms of cell interaction and signal transduction that ultimately result in successful embryo development and creation of a new individual. Search methods We carried out a literature survey with no restrictions on publication date (from the early 1950s to March 2016) using PubMed/Medline, Google Scholar and Web of Knowledge by utilizing common keywords applied in the field of fertilization and embryo development. We also screened the complete list of references published in the most recent research articles and relevant reviews published in English (both animal and human studies) on the topics investigated. Outcomes Literature on the principal animal models demonstrates that gamete activation is a pre-requisite for successful fertilization, and is a process common to all species studied to date. We provide a detailed description of the dramatic changes in gamete morphology and behavior, the regulatory molecules triggering gamete activation and the intracellular ions and second messengers involved in active metabolic pathways in different species. Recent scientific advances suggest that artificial gamete activation may represent a novel technique to improve human IVF outcomes, but this approach requires caution. Wider implications Although controversial, manipulation of gamete activation represents a promising tool for ameliorating the fertilization rate in assisted reproductive technologies. A better knowledge of mechanisms that transform the quiescent oocyte into a pluripotent cell may also provide new insights for the clinical use of stem cells.

Global warming is an increasingly serious problem underlying ecological change in marine flora and fauna. Mytilus galloprovincialis is an intertidal species that colonizes coasts in moderate and warm climates, and can thus withstand extreme climatic conditions; however, it successfully reproduces only within a certain temperature range. The effects of prolonged exposure to 28 °C, a temperature unsuitable for breeding activity, on sperm quality were evaluated in this study. Such heat stress induced the following: a significant reduction in concentration; a biphasic pattern of motility and mitochondrial membrane potential that first increased, and then collapsed; a decrease in the intracellular calcium concentration; a rapid increase in lipid peroxidation that was normalized after the third week of heat stress; an increase in DNA fragmentation after the third week of heat stress; and atypical morphology (i.e., sperm with a globular head, asymmetrical tail, and acrosome loss). Currently, these elevated-temperature conditions are achieved along the Mediterranean coast during the late summer, when the reproductive activity of M. galloprovincialis is suspended after massive spawning in the spring. The increasing global temperature, however, may shift their breeding season, thus significantly impacting marine ecosystems and mussel production.

Under the influence of gonadotropins or growth factors, a close cooperation develops between cumulus cells and the oocyte that is implicated in transmitting signals involved in maintaining or releasing the meiotic arrest in the oocyte. While cyclic adenosine 5'-monophosphate (cAMP) is a key molecule in maintaining the meiotic arrest, calcium (Ca(2+)) may play a role in controlling either spontaneous or gonadotropin-induced oocyte maturation, possibly by modulating intracytoplasmic cAMP concentrations via Ca(2+)-sensitive adenylate cyclases. This review focuses on the mechanisms related to the origin of the Ca(2+) wave that travels from the cumulus cells to the oocyte, and discusses the source of variations affecting the dynamics of this wave.

SUMMARY Electrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the postvitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous ‘‘rosette’’ embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development.

Infertile couples make up approximately 10% of the worldwide population, and around 1% of current live births are a result of assisted reproductive technology (ART). Since the time that this technology was first applied, many studies have been performed in order to determine the risk associated with infertility treatments. Short- and long-term risks have been identified, which confirm that the major complications are due to multiple pregnancies. In a previous study we described in detail the main reproductive processes, the techniques for in vitro fertilization (IVF) and the risks associated with each of them, with a focus on intracytoplasmic sperm injection (ICSI). In this review we provide an update from 2007 to the present. In particular, in addition to new information on post-pregnancy complications and infant morbidity and malformations, we report data on rare syndromes, including recent case reports. Although data are controversial, an association between IVF and a minor increase in the incidence of birth defects has been confirmed. Several lines of evidence also suggest that there may be a link between ART and psychological disorders in the parents and the child. Finally, recent findings draw attention to the need for accurate clinical and psychological counselling of couples before any treatment decisions are made.

Marine organisms represent an important source of novel bioactive compounds, often showing unique modes of action. Such drugs may be useful tools to study complex processes such as reproduction; which is characterized by many crucial steps that start at gamete maturation and activation and virtually end at the first developmental stages. During these processes cytoskeletal elements such as microfilaments and microtubules play a key-role. In this review we describe: (i) the involvement of such structures in both cellular and in vitro processes; (ii) the toxins that target the cytoskeletal elements and dynamics; (iii) the main steps of reproduction and the marine drugs that interfere with these cytoskeleton-mediated processes. We show that marine drugs, acting on microfilaments and microtubules, exert a wide range of impacts on reproductive events including sperm maturation and motility, oocyte maturation, fertilization, and early embryo development.

Electrophysiological techniques were used to study ion currents in the ascidian Ciona intestinalis oocyte plasma membranes during different stages of growth and meiosis. Three stages (A, B, C) of immature oocytes were discriminated in the ovary, with the germinal vesicle (GV) showing specific different features of growth and maturation. Stage A (pre-vitellogenic) oocytes exhibited the highest L-type Ca(2+)current activity, and were incompetent for meiosis resumption. Stage B (vitellogenic) oocytes showed Na(+) currents that remained high during the maturation, up to the post-vitellogenic stage C oocytes. The latter had acquired meiotic competence, undergoing spontaneous maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation plays a specific role in embryo development. Spontaneous maturation was inhibited at low pH whereas trypsin was able to trigger germinal vesicle breakdown (GVBD) regardless of pH; in addition spontaneous maturation was not affected by removal of follicle cells or by inhibiting junctional communication between oocyte and follicle cells. Taken together these results imply: (i) Ca(2+) and Na(+) currents are involved in meiotic progression, growth, and acquisition of meiotic competence; (ii) trypsin-like molecules may have a role as candidates for providing the physiological stimulus to resume meiosis. Finally, we provide evidence that follicle cells in Ciona are not involved in triggering GVBD as it occurs in other ascidians.

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.

It was shown that local application of agonists of the 3rd type receptors 5-HTQ and quipazine into the interblastomere cleft of Paracentrotus lividus embryos evoked specific membrane currents. At the same time, ligands of 5-HT 3 - receptors specifically affected the cleavage patterns of half-embryos, i.e., imitated or avoided the interblastomere signal. In the view of the data obtained, we discuss a more precise concept of pro- tosynapse, where the distribution of membrane serotonin receptors is restricted to the period of blastomere for- mation during cleavage and localized in the area of interblastomere contact.

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.

The increase of pollutants in the oceans as well as the global change around the world is having a negative impact on the biodiversity of living organisms. There is a crescent alarm in the scientific community for the different types of pollution (biological, chemical and physical) and for the changes occurring at the level of species and communities. Many parameters endangering marine wildlife contribute to create an unhealthy marine environment that induces reproductive disorders that, in turn, may influence even the survival of marine wildlife.

Reproduction is a stepwise process that starts at the formation of gametes and ends at the completion of meiosis and the first mitotic divisions of the zygote. The involvement of main cytoskeletal elements in mediating the reproductive processes is now well established. This chapter summarizes recent advances in the field related to cytoskeletal dynamics underpinning the basic events of reproduction such as gametogenesis, gametes reciprocal activation, sperm-oocyte interaction and zygote formation. We have described the distinct patterns of distribution of microfilaments and microtubules and their rearrangements along the entire reproductive process. Evidences are provided that these elements play a dynamic role in the establishment and regulation of basic structure and functions of gametes and zygote during the processes of maturation and fertilization.

An important component of quality of care is the use of evidence-base d practices. Three studies from three different continents (Africa, Asia and Latin America) have reported the suboptimal use of evidence - based interventions in obstetric care, proving that despite scientific evidence and dissemination efforts, there are harmful and/or unnecessary procedures still used, while others that are beneficial are ignored. This illustrates the existing bridges and barriers between practice guidelines based on research findings and practitioners. A theoretical framework could help explain these barriers and possibly help target interventions to specific barriers. A review of existing literature shows that an intervention aimed to increase the use of evidence-base d practices should have the following components: increase birth attendant concern about the effectiveness of routine clinical practices, stimulate their desire to review the effectiveness of their practice, provide them with skills to perform evidence based clinical guidelines appraisal and development and establish mechanisms through key hospital leaders to implement the guidelines and sustain them over time.

Using the whole-cell voltage clamp technique, the electrical changes in oocyte and embryo plasma membrane were followed during different meiotic and developmental stages in Ciona intestinalis. We show, for the first time, an electrophysiological characterization of the plasma membrane in oocytes at the germinal vesicle (GV) stage with high L-type calcium (Ca2+) current activity that decreased through meiosis. Moreover, the absence of Ca2+ reduced germinal vesicle breakdown (GVBD), which is consistent with a role of Ca2+ currents in the prophase/metaphase transition. In mature oocytes at the metaphase I (MI) stage, Ca2+ currents decreased and then disappeared and sodium (Na+) currents first appeared remaining high up to the zygote stage. Intracellular Ca2+ release was higher in MI than in GV, indicating that Ca2+ currents in GV may contribute to fill the stores which are essential for oocyte contraction at fertilization. The fertilization current generated in Na+ free sea water was significantly lower than the control; furthermore, oocytes fertilized in the absence of Na+ showed high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, suggesting that signaling pathways that mediate first cleavage do not rely on ion current activities. At the 8-cell stage embryo, a resumption of Na+ current activity and conductance occurred, without a correlation with specific blastomeres. Taken together, these results imply: (i) an involvement of L-type Ca2+ currents in meiotic progression from the GV to MI stage; (ii) a role of Na+ currents during electrical events at fertilization and subsequent development; (iii) a major role of plasma membrane permeability and a minor function of specific currents during initial cell line segregation events.

Using the whole-cell voltage clamp technique, we have studied the Ca2+ currents and the steady-state conductance during different oocyte growth stages and during the reproductive cycle of the female of Octopus vulgaris. Evidence is presented that L-type Ca2+ currents are high in small pre-vitellogenic oocytes (80-150 microm diameter) and significantly lower in early vitellogenic oocytes (180-300 microm diameter). Similarly, a significant decrease of the steady-state conductance occurred from the pre to early- vitellogenic oocytes. Octopus oocytes showed larger Ca2+ currents in the reproductive rather than non-reproductive periods. These data indicates that ion and L-type Ca2+ currents play a role in oocyte growth and cytoplasmic maturation, and possibly in preparing the plasma membrane to the interaction with the spermatozoon. By using fluorescent microscopy, we show that oocytes from 80 to 400 microm diameter have the large germinal vesicle characteristic of the immature oocytes. In subsequent stages of growth (up to 1000 microm diameter) the nucleus is no more visible and the metaphase spindle appears. These data demonstrate that Octopus vulgaris oocytes are arrested in the first meiotic prophase up to the early-vitellogenic stage and resume meiosis at this stage up to a second block presumably in metaphase I. We discuss a possible role for progesterone as the hormonal stimulus for the first prophase-metaphase meiotic transition.

The ubiquitous and pleiotropic dual specificity protein kinase CK2 has been studied and characterized in many organisms, from yeast to mammals. Generally, the enzyme is composed of two catalytic (α and/or α′) and two regulatory (β) subunits, forming a differently assembled tetramer. Although prone to controversial interpretation, the function of CK2 has been associated with fundamental biological processes such as signal transduction, cell cycle progression, cell growth, apoptosis, and transcription. Less known is the role of CK2 during meiosis and the early phase of embryogenesis. In this work, we studied CK2 activity during oocyte activation, a process occurring at the end of oocyte maturation and triggered by fertilization. In ascidian Ciona intestinalis, an organism whose complete genome has been published recently, CK2 was constitutively active in unfertilized and fertilized oocytes. The enzymatic activity oscillated through meiosis showing three major peaks: soon after fertilization (metaphase I exit), before metaphase II, and at the exit from metaphase II. Biochemical analysis of CK2 subunit composition in activated oocytes indicated that CK2-α was catalytically active as a monomer, independently from its regulatory subunit β; however, CK2-β was only detectable in unfertilized oocytes where it was associated with a bona fide identified ascidian mitogen-activated protein kinase. After fertilization, CK2-β was undetectable, suggesting its rapid degradation. Protein sequence analysis of CK2-α and -β cDNA indicated a high identity compared with vertebrate homologs. In addition, the absence of putative phosphorylation sites for Cdc2 kinase on both α and β subunits suggested an important role for CK2 in regulating meiotic cell cycle in C. intestinalis oocytes.

Gamete cells are electrogenic, i.e. capable of responding to electrical stimuli and modifying their electrical properties during the crucial periods of maturation and fertilization. Ion channels have been widely demonstrated on the plasma membrane of the oocyte and spermatozoon in all animals studied, and electrical modifications in gametes are due to ion currents that are modulated via these ion channels. The modification of intracellular calcium levels in gametes has been extensively studied, and these modifications are recognized to be a second messenger system for gamete maturation and fertilization. Other ions also move through the plasma membrane, either in association with or independent of calcium, and these generate typical features such as fertilization currents and oscillation of resting potential. These modifications were first studied in marine invertebrates, and the observations subsequently compared with mammalian systems, including human. The precise role played by these currents in the processes of maturation and fertilization is still poorly understood; however, recent research opens new frontiers for their clinical and technological application.

The effects of bioactive aldehydes from diatoms, unicellular algae at the base of the marine food web, were studied on fertilization and early development processes of the ascidian Ciona intestinalis. Using whole-cell voltage clamp techniques, we show that 2-trans-4-trans-decadienal (DD) and 2-trans-4-cis-7-cis-decatrienal (DT) inhibited the fertilization current which is generated in oocytes upon interaction with the spermatozoon. This inhibition was dose-dependent and was accompanied by inhibition of the voltage-gated calcium current activity of the plasma membrane. DD and DT did not inhibit the subsequent contraction of the cortex. Moreover, DD specifically acted as a fertilization channel inhibitor since it did not affect the steady state conductance of the plasma membrane or gap junctional (GJ) communication within blastomeres of the embryo. On the other hand, DD did affect actin reorganization even though the mechanism of action on actin filaments differed from that of other actin blockers. Possibly this effect on actin reorganization was responsible for the subsequent teratogenic action on larval development. The effect of DD was reversible if oocytes were washed soon after fertilization indicating that DD may specifically target certain fertilization mechanisms. Thus, diatom reactive aldehydes such as DD may have a dual effect on reproductive processes, influencing primary fertilization events such as gating of fertilization channels and secondary processes such as actin reorganization which is responsible for the segregation of cell lineages. These findings add to a growing body of evidence on the antiproliferative effects of diatom-derived aldehydes. Our results also report, for the first time, on the action of a fertilization channel blocker in marine invertebrates.

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