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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.

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.

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.

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.

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.

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.

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.

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.

Human oocyte development was evaluated after a reduced time exposure to spermatozoa in vitro. A total of 119 patients were assigned to two study groups in a randomized prospective study in which each patient's oocytes were exposed to spermatozoa for either 1 h (group 1 - 58 patients) or the standard 16 h incubation period (group 2 - 61 patients). The fertilization rate obtained in group 1 was higher than in group 2 (285/393, 73%, and 272/410, 66% respectively), suggesting that the spermatozoa-oocyte interaction occurs within 1 h. This was confirmed in a study in vitro using fluorescently labelled spermatozoa and normal oocyte-cumulus complexes. Spermatozoa enter the cumulus complex within 15 min, traverse the cumulus layer within 3 h, and first appear in the oocyte cortex at 4 h post-insemination. The incidence of polyspermy was higher in oocytes exposed to spermatozoa for 16 h (3%) than for 1 h (1%). There was no difference in the cleavage rate or morphological characteristics of embryos from both study groups. However, when evaluating the timing of embryo development, group 1 generated a significantly higher percentage of four to five cell embryos when compared to group 2 (55 versus 39%; P < 0.001), documented at 40 h post-insemination. The implantation and pregnancy rates for group 1 were 11 and 28%, while the corresponding rates for group 2 were 8 and 15%. This suggests that a reduced exposure of oocyte to spermatozoa favours embryo viability, possibly due to a decrease in potential damage from sperm metabolic waste products.

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.

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.

Spermiogenesis is the last phase of the process that give rise to a mature and competent spermatozoon. It occurs from a dramatic morphological and structural change of the spermatid, in particular replacement of DNA-linked histones by protamines leading to a highly compact chromatin structure consisting of DNA and heterogeneous nucleoproteins. The target of a fertilizing spermatozoon is to deliver into the oocyte the paternal genome and regulatory factors that are required for proper embryonic development [1]. To do this the sperm must be capable of undergoing decondensation at a peculiar moment of the fertilization process. Increasing evidence on the strong paternal effect on preimplantation embryo development [2] focus on the importance to identify a reliable sperm quality parameter. Although many different causes may give rise to male infertility [3], traditionally in IVF centres, routine laboratory investigations evaluate seminal parameters such as concentration, motility and morphology in order to assess semen quality prior to undergo assisted reproduction. Recent acquisitions correlate poor chromatin condensation to a failure in fertilization, embryo development and repeated miscarriages indicating even that sperm DNA damage over 30% impedes natural pregnancy.

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.

During the early time of IVF, sperm competence was defined as the ability to fertilize an oocyte. However, with the advent of ICSI, despite the capacity to reach 2–4cell stage, it is impossible for numerous patients, to establish a pregnancy. Instead the consensus now is that male fertility has to be defined as the capacity to produce sperm cells able to establish a full term pregnancy [1–3] Basic parameters such as concentration, motility and morphology are of limited value in determining sperm capacity to allow full embryonic development to term. Determination of DNA changes like fragmentation and condensation are independent parameters [2] and obviously of major importance. DNA fragmentation, related or not to reactive oxygen species (ROS) insult is only one piece of the problem. Indeed, sperm chromatin tertiary structure seems to be critical for correct epigenetic regulation and maintenance, and further on for male fertility [4, 5]. During very early embryogenesis, an adequate chromatin structure is also necessary for the very first cleavages [6–9]. Methylation is one of the most important regulators of the tertiary structure and imprinting; it affects sperm DNA and histones packaging it; in this way a correct recycling of homocysteine is mandatory during spermatogenesis and embryogenesis. The sperm is not just a carrier of paternal genome: it has a relevant epigenetic contribution. A defective wrapping of DNA is often linked to immaturity. Hyaluronic acid (HA) binding ability has been proposed as a tool to select the more mature spermatozoa having reached their final nuclear and cytoplasmic maturation [10, 11], even if controversies exist [12], and it is used as well in veterinary medicine [13]. In this case report we have tested the relation between HA binding and methylation effector supplementation, by comparison to what we have observed with such a supplementation for patients with poor sperm condensation.

Events of reproduction occurring from meiotic resumption of the immature oocyte up to its exit from the second meiotic block following activation will be revealed. Morphological modifications of the oocyte during maturation will be related to signal transduction mechanisms involving primary or secondary messengers. A detailed view will be addressed to genetic and epigenetic control of oocyte maturation. At fertilization, reciprocal gamete activation and novel knowledge related to sperm factor and cascade mechanisms occurring in the oocyte will be focused. A detailed description of ion currents occurring during fertilization will depict another point of view of oocyte activation mechanisms, further supporting for their complexity. Finally, all basic information related to this short time lapse will be considered in relation to clinical application of assisted reproductive technologies. New frontiers, such as stem cells and cloning technologies, will be analyzed and future applications and improvements will be hypothesised.

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.

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.

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.

By using the whole cell voltage-clamp technique, we studied changes in plasma membrane permeability at different meiotic stages of bovine oocytes. Follicular oocytes were matured in vitro and activated by Ca2+ionophore. Oocytes at germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), and meiosis exit were used for electrophysiological recording. By clamping the oocytes at −30 mV, we found that the L-type voltage-dependent Ca2+channels were active at the GV stage and that their activity decreased after the GVBD stage. Furthermore, the resting potential decreased from the GV to the MI stage and increased again at MII. A significant decrease of the steady-state conductance occurred from the GV to the MI stage, followed by a sharp increase at the MII stage. With the addition of organic L-type Ca2+channel blockers (nifedipine and verapamil), we inhibited the Ca2+currents. However, only in the case of verapamil was there a decrease of in vitro maturation efficiency. Our results suggest that, in addition to the cumulus-oocyte junctions, the plasma membrane channels provide another mode of Ca2+entry into bovine oocytes during meiosis.

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.

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.