Parthenogenetic activation of porcine oocytes after nuclear transfer

Mature porcine oocytes are arrested at metaphase II of meiosis. At fertilization, like all mammalian oocytes they exhibit a low frequency Ca(2+) oscillation lasting several hours. This oscillation is thought to be the signal that triggers resumption of meiosis and activates the developmental program of the oocyte. The signal transduction mechanism of the sperm-induced Ca(2+) signal is not known in detail, and attempts to generate the oscillation artificially have met with little success. Nevertheless, artificial activation of the oocyte is a crucial step during nuclear transfer. Methods are available to induce a transient elevation in the intracellular free Ca(2+) concentration to surpass the meiotic arrest and induce development of the constructed embryo. Further studies concentrating on the mechanism of Ca(2+) signaling during fertilization will help to improve the efficiency of the procedures used for parthenogenetic activation of the oocyte.     

Intracellular Ca2+ response of rabbit oocytes to electrical stimulation.

Electrical stimulation is known to cause activation in mammalian oocytes, possibly by eliciting an elevation in intracellular calcium (Ca2+). This study reports intracellular Ca2+ concentrations in mature rabbit oocytes using the Ca2+ indicator fura-2. Calcium levels were determined prior to, during, and after the administration of an electrical pulse (3.6 kV/cm for 60 microseconds). Baseline Ca2+ levels ranged from 30 to 90 nM. The intracellular Ca2+ transient evoked by a pulse, peaked at 11 sec, was highly variable in amplitude (40-300 nM) and returned to prepulse levels within 300 sec. Electrically stimulated oocytes did not exhibit repetitive Ca2+ transients. The size of the cytoplasmic Ca2+ rise was influenced by the duration of the pulse, the field strength and the concentrations of external Ca2+ rise was influenced by the duration of the pulse, the field strength and the concentrations of external Ca2+ (P less than 0.05). Oocytes electrically stimulated in the presence of 100 microM CaCl2, which evoked Ca2+ transients with a mean magnitude of 120 nM, activated at a higher rate (P less than 0.05) than oocytes stimulated in the presence of either higher or lower levels of external Ca2+. Although oocytes electrically shocked at 16-18 hr after administration of human chorionic gonadotropin (hphCG) activated at a lower rate than oocytes stimulated at 22-24 hphCG (P less than 0.05), their intracellular Ca2+ response to the pulse was similar (P less than 0.05). These results indicate that electrical pulse parameters and extracellular Ca2+ concentrations can be used to modulate intracellular Ca2+ levels and optimize oocyte activation rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Egg activation events are regulated by the duration of a sustained [Ca2+]cyt signal in the mouse

Although the dynamics of oscillations of cytosolic Ca2+ concentration ([Ca2+]cyt) play important roles in early mammalian development, the impact of the duration when [Ca2+]cyt is elevated is not known. To determine the sensitivity of fertilization-associated responses [i.e., cortical granule exocytosis, resumption of the cell cycle, Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, recruitment of maternal mRNAs] and developmental competence of the parthenotes to the duration of a [Ca2+]cyt transient, unfertilized mouse eggs were subjected to a prolonged [Ca2+]cyt change for 15, 25, or 50 min by means of repetitive Ca2+ electropermeabilization at 2-min intervals. The initiation and completion of fertilization-associated responses are correlated with the duration of time in which the [Ca2+]cyt is elevated, with the exception that autonomous CaMKII activity is down-regulated with prolonged elevated [Ca2+]cyt. Activated eggs from 25- or 50-min treatments readily develop to the blastocyst stage with no sign of apoptosis or necrosis and some implant. Ca2+ influx into unfertilized eggs causes neither Ca2+ release from intracellular stores nor rapid removal of cytosolic Ca2+. Thus, the total Ca2+ signal input appears to be an important regulatory parameter that ensures completion of fertilization-associated events and oocytes have a surprising degree of tolerance for a prolonged change in [Ca2+]cyt.     

Improved postimplantation development of rabbit nuclear transfer embryos by activation with inositol 1,4,5-trisphosphate

Cloned rabbit embryos are characterized by their extremely poor postimplantation development, despite their high survivability until the blastocyst stage in vitro. This study examined whether the developmental failure of cloned rabbit embryos in vivo can be overcome by technical improvements to the activation protocol. Freshly collected cumulus cells were transferred into enucleated oocytes by intracytoplasmic injection. One to two hours later, the oocytes were activated by electroporation with Ca(2+) or inositol 1,4,5-trisphosphate (IP3), which is known to induce repeated rises in intracellular Ca(2+), as in normal fertilization. After transfer of embryos at the two- to four-cell stages, well-defined implantation sites with remnant fetal tissue were observed at term (day 28) only in the IP3-stimulation groups (0.9% and 5.8% per transferred embryo for single and triple stimulation groups, respectively). When some recipients in the same group were examined at days 16-20, a viable cloned fetus (day 19) with normal organogenesis was obtained. These findings clearly demonstrate that the oocyte activation protocol using IP3 enhances the postimplantation development of nuclear-transferred rabbit embryos.     

L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.     

Factors shaping the confocal image of the calcium spark in cardiac muscle cells.

The interpretation of confocal line-scan images of local [Ca2+]i transients (such as Ca2+ sparks in cardiac muscle) is complicated by uncertainties in the position of the origin of the Ca2+ spark (relative to the scan line) and by the dynamics of Ca(2+)-dye interactions. An investigation of the effects of these complications modeled the release, diffusion, binding, and uptake of Ca2+ in cardiac cells (producing a theoretical Ca2+ spark) and image formation in a confocal microscope (after measurement of its point-spread function) and simulated line-scan images of a theoretical Ca2+ spark (when it was viewed from all possible positions relative to the scan line). In line-scan images, Ca2+ sparks that arose in a different optical section or with the site of origin displaced laterally from the scan line appeared attenuated, whereas their rise times slowed down only slightly. These results indicate that even if all Ca2+ sparks are perfectly identical events, except for their site of origin, there will be an apparent variation in the amplitude and other characteristics of Ca2+ sparks as measured from confocal line-scan images. The frequency distributions of the kinetic parameters (i.e., peak amplitude, rise time, fall time) of Ca2+ sparks were calculated for repetitive registration of stereotyped Ca2+ sparks in two experimental situations: 1) random position of the scan line relative to possible SR Ca(2+)-release sites and 2) fixed position of the scan line going through a set of possible SR Ca(2+)-release sites. The effects of noise were incorporated into the model, and a visibility function was proposed to account for the subjective factors that may be involved in the evaluation of Ca(2+)-spark image parameters from noisy experimental recordings. The mean value of the resulting amplitude distributions underestimates the brightness of in-focus Ca2+ sparks because large numbers of out-of-focus Ca2+ sparks are detected (as small Ca2+ sparks). The distribution of peak amplitudes may split into more than one subpopulation even when one is viewing stereotyped Ca2+ sparks because of the discrete locations of possible SR Ca(2+)-release sites in mammalian ventricular heart cells.     

In intact mammalian photoreceptors, Ca2+-dependent modulation of cGMP-gated ion channels is detectable in cones but not in rods

In the mammalian retina, cone photoreceptors efficiently adapt to changing background light intensity and, therefore, are able to signal small differences in luminance between objects and backgrounds, even when the absolute intensity of the background changes over five to six orders of magnitude. Mammalian rod photoreceptors, in contrast, adapt very little and only at intensities that nearly saturate the amplitude of their photoresponse. In search of a molecular explanation for this observation we assessed Ca2+-dependent modulation of ligand sensitivity in cyclic GMP-gated (CNG) ion channels of intact mammalian rods and cones. Solitary photoreceptors were isolated by gentle proteolysis of ground squirrel retina. Rods and cones were distinguished by whether or not their outer segments bind PNA lectin. We measured membrane currents under voltage-clamp in photoreceptors loaded with Diazo-2, a caged Ca2+ chelator, and fixed concentrations of 8Br-cGMP. At 600 nM free cytoplasmic Ca2+ the midpoint of the cone CNG channels sensitivity to 8BrcGMP, 8BrcGMPK1/2, is approximately 2.3 microM. The ligand sensitivity is less in rod than in cone channels. Instantly decreasing cytoplasmic Ca2+ to <30 nM activates a large inward membrane current in cones, but not in rods. Current activation arises from a Ca2+ -dependent modulation of cone CNG channels, presumably because of an increase in their affinity to the cyclic nucleotide. The time course of current activation is temperature dependent; it is well described by a single exponential process of approximately 480 ms time constant at 20-21 degrees C and 138 ms at 32 degrees C. The absence of detectable Ca2+-dependent CNG current modulation in intact rods, in view of the known channel modulation by calmodulin in-vitro, affirms the modulation in intact rods may only occur at low Ca2+ concentrations, those expected at intensities that nearly saturate the rod photoresponse. The correspondence between Ca2+ dependence of CNG modulation and the ability to light adapt suggest these events are correlated in photoreceptors.     

Identification of a Ras GTPase-activating protein regulated by receptor-mediated Ca2+ oscillations

Receptor-mediated increases in the concentration of intracellular free calcium ([Ca2+]i) are responsible for controlling a plethora of physiological processes including gene expression, secretion, contraction, proliferation, neural signalling, and learning. Increases in [Ca2+]i often occur as repetitive Ca2+ spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca2+ spikes increase their frequency with the amplitude of the receptor stimuli, a phenomenon that appears critical for the induction of selective cellular functions. Here we report the characterisation of RASAL, a Ras GTPase-activating protein that senses the frequency of repetitive Ca2+ spikes by undergoing synchronous oscillatory associations with the plasma membrane. Importantly, we show that only during periods of plasma membrane association does RASAL inactivate Ras signalling. Thus, RASAL senses the frequency of complex Ca2+ signals, decoding them through a regulation of the activation state of Ras. Our data provide a hitherto unrecognised link between complex Ca2+ signals and the regulation of Ras.     

A class of parametrically excited calcium oscillation detectors.

Intracellular Ca2+ oscillations are often a response to external signals such as hormones. Changes in the external signal can alter the frequency, amplitude, or form of the oscillations suggesting that information is encoded in the pattern of Ca2+ oscillations. How might a cell decode this signal? We show that an excitable system whose kinetic parameters are modulated by the Ca2+ concentration can function as a Ca2+ oscillation detector. Such systems have the following properties: (1) They are more sensitive to an oscillatory than to a steady Ca2+ signal. (2) Their response is largely independent of the signal amplitude. (3) They can extract information from a noisy signal. (4) Unlike other frequency sensitive detectors, they have a flat frequency response. These properties make a Ca(2+)-sensitive excitable system nearly ideal for detecting and decoding Ca2+ oscillations. We suggest that Ca2+ oscillations, in concert with these detectors, can act as cellular timekeepers to coordinate related biochemical reactions and enhance their overall efficiency.     

小鼠卵受精和人工激活过程中胞质游离Ca^2＋变化及其在卵激活中的作用

休止于第二次成熟分裂中期（ＭＩ）的小鼠卵母细胞分别乙醇,钙离子载体Ａ２３１８７、电刺激或精子激活并用Ｃａ＾２＋特异荧光探针－Ｆｕｒａ２／ＡＭ测定细胞内游离Ｃａ＾２＋的变化。结果表明,受精诱导ＭⅡ卵内游离Ｃａ＾２＋浓度多次跃升（ｏｓｃｉｌｌａｔｉｏｎ）乙醇,钙离子载体及１次电刺激仅诱导胞内Ｃａ＾２＋１次升高,人工诱导激活的卵可象正常受精卵一样卵裂并发育至囊胚,用ＥＧＴＡ阻止受精和人工激活过程中卵内游     

Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilization in mouse eggs

In mammalian eggs, sperm-induced Ca2+ oscillations at fertilization are the primary trigger for egg activation and initiation of embryonic development. Identifying the downstream effectors that decode this unique Ca2+ signal is essential to understand how the transition from egg to embryo is coordinated. Here, we investigated whether conventional PKCs (cPKCs) can decode Ca2+ oscillations at fertilization. By monitoring the dynamics of GFP-labeled PKCalpha and PKCgamma in living mouse eggs, we demonstrate that cPKCs translocate to the egg membrane at fertilization following a pattern that is shaped by the amplitude, duration, and frequency of the Ca2+ transients. In addition, we show that cPKC translocation is driven by the C2 domain when Ca2+ concentration reaches 1-3 microM. Finally, we present evidence that one physiological function of activated cPKCs in fertilized eggs is to sustain long-lasting Ca2+ oscillations, presumably via the regulation of store-operated Ca2+ entry.     

Interaction between injected Ca2+ and intracellular Ca2+ stores in Xenopus oocytes.

Upon two repetitive deep injections of Ca2+ into Xenopus oocyte (200-300 microns under the membrane), the amplitude of the transient Cl- current induced by the second injection is several-fold higher than that of the first one. This 'potentiation' persists even at 60-90 min intervals between injections. However, in oocytes permeabilized to Ca2+ by the ionophore A23187 in a Ca2(+)-free solution, the potentiation completely disappears after 30 min. It is proposed that the injected Ca2+ is largely taken up by the stores, whereas following the second injection, a higher proportion of Ca2+ reaches the membrane, since the stores are already loaded. In ionophore-treated oocytes, the stores lose the accumulated Ca2+ over several minutes and are then ready to take up Ca2+ again, hindering its arrival at the membrane.     

The dynamic complexity of the TRPC1 channelosome

A rise in cytoplasmic [Ca2+] due to store-operated Ca2+ entry (SOCE) triggers a plethora of responses, both acute and long term. This leads to the important question of how this initial signal is decoded to regulate specific cellular functions. It is now clearly established that local [Ca2+] at the site of SOCE can vary significantly from the global [Ca2+] in the cytosol. Such Ca2+ microdomains are generated by the assembly of key Ca2+ signaling proteins within the domains. For example, GPCR, IP 3 receptors, TRPC3 channels, the plasma membrane Ca2+ pump and the endoplasmic reticulum (ER) Ca2+ pump have all been found to be assembled in a complex and all of them contribute to the Ca2+ signal. Recent studies have revealed that two other critical components of SOCE, STIM1 and Orai1, are also recruited to these regions. Thus, the entire machinery for activation and regulation of SOCE is compartmentalized in specific cellular domains which facilitates the specificity and rate of protein-protein interactions that are required for activation of the channels. In the case of TRPC1-SOC channels, it appears that specific lipid domains, lipid raft domains (LRDs), in the plasma membrane, as well as cholesterol-binding scaffolding proteins such as caveolin-1 (Cav-1), are involved in assembly of the TRPC channel complexes. Thus, plasma membrane proteins and lipid domains as well as ER proteins contribute to the SOCE-Ca2+ signaling microdomain and modulation of the Ca2+ signals per se. Of further interest is that modulation of Ca2+ signals, i.e. amplitude and/or frequency, can result in regulation of specific cellular functions. The emerging data reveal a dynamic Ca2+ signaling complex composed of TRPC1/Orai1/STIM1 that is physiologically consistent with the dynamic nature of the Ca2+ signal that is generated. This review will focus on the recent studies which demonstrate critical aspects of the TRPC1 channelosome that are involved in the regulation of TRPC1 function and TRPC1-SOC-generated Ca2+ signals.     

The role of Ca2+ in the contractility of rabbit small intestine in vitro.

This study evaluated the role of Ca2+ in spontaneous and ACh- and KCl-induced contractions in longitudinal and circular smooth muscle from rabbit small intestine in vitro. In the first experiment, the amplitude, frequency and tone of spontaneous contractions in longitudinal and circular smooth muscle of small intestine were determined and, in the second experiment, the ACh- and KCl-induced responses of longitudinal and circular smooth muscle were measured. Atropine and guanethidine reduced the amplitude and tone of contractions in longitudinal and circular muscle, but reduced the frequency of contractions in circular muscle, only. TTX attenuated the amplitude of contractions and decreased the tone of contractions in longitudinal muscle, but increased the tone in circular muscle. Ca2+-free solutions, verapamil, nifedipine and caffeine diminished the three parameters of spontaneous contractions. Thapsigargin and cyclopiazonic acid increased the amplitude and tone of contractions in ileum longitudinal muscle, only, and cyclopiazonic acid increased the amplitude of contractions in circular muscle. Ca2+-free solutions, verapamil, nifedipine, thapsigargin, cyclopiazonic acid, and caffeine diminished ACh- and KCl-induced contractions. Those results suggest that extracellular Ca2+ plays a role in spontaneous contractions, and extracellular and intracellular Ca2+ participate in the ACh- and KCl-induced contractions of rabbit small intestine.     

Ion conductances related to development of repetitive firing in mouse retinal ganglion neurons in situ

ABSTRACT: In the retina, the ability to encode graded depolarizations into spike trains of variable frequency appears to be a specific property of retinal ganglion neurons (RGNs). To deduce the developmental changes in ion conductances underlying the transition from single to repetitive firing, patch-clamp recordings were performed in the isolated mouse retina between embryonic day 15 (E15) and postnatal day 5 (P5). Immature neurons of the E15 retina were selected according to their capacity to generate voltage-activated Na+ currents (I(Na)(v)). Identification of P5 RGNs was based on retrograde labeling, visualization of the axon, or the amplitude of I(Na)(v). At E15, half of the cells were excitable but none of them generated more than one spike. At P5, all cells were excitable and a majority discharged in tonic fashion. Ion conductances subserving maintenance of repetitive discharge were identified at P5 by exposure to low extracellular Ca2+, Cd2+, and charybdotoxin, all of which suppressed repetitive discharge. omega-Conotoxin GVIA and nifedipine had no effect. We compared passive membrane properties and a variety of voltage-activated ion channels at E15 and P5. It was found that the density of high voltage-activated (HVA) Ca2+ currents increased in parallel with the development of repetitive firing, while the density of Ni2+-sensitive low voltage-activated (LVA) Ca2+ currents decreased. Changes in density and activation kinetics of tetrodotoxin-sensitive Na+ currents paralleled changes in firing thresholds and size of action potentials, but seemed to be unrelated to maintenance of repetitive firing. Densities of A-type K+ currents and delayed rectifier currents did not change. The results suggest that HVA Ca2+ channels, and among them a toxin-resistant subtype, are specifically engaged in activation of Ca2+-sensitive K+ conductance and thereby account for frequency coding in postnatal RGNs.     

Changes in miniature endplate potential frequency during repetitive nerve stimulation in the presence of Ca2+, Ba2+, and Sr2+ at the frog neuromuscular junction

Miniature endplate potentials (MEPPs) were recorded from frog sartorious neuromuscular junctions under conditions of reduced quantal contents to study the effect of repetitive nerve stimulation on asynchronous (tonic) quantal transmitter release. MEPP frequency increased during repetitive stimulation and then decayed back to the control level after the conditioning trains. The decay of the increased MEPP frequency after 100-to 200-impulse conditioning trains can be described by four components that decayed exponentially with time constants of about 50 ms, 500 ms, 7 s, and 80 s. These time constants are similar to those for the decay of stimulation-induced changes in synchronous (phasic) transmitter release, as measured by endplate potential (EPP) amplitudes, corresponding, respectively, to the first and second components of facilitation, augmentation, and potentiation. The addition of small amounts of Ca2+ or Ba2+ to the Ca2+-containing bathing solution, or the replacement of Ca2+ with Sr2+, led to a greater increase in the stimulation-induced increases in MEPP frequency. The Sr-induced increase in MEPP frequency was associated with an increase in the second component of facilitation of MEPP frequency; the Ba-induced increase with an increase in augmentation. These effects of Sr2+ and Ba2+ on stimulation-induced changes in MEPP frequency are similar to the effects of these ions on stimulation- induced changes in EPP amplitude. These ionic similarities and the similar kinetics of decay suggest that stimulation induced changes in MEPP frequency and EPP amplitude have some similar underlying mechanisms. Calculations are presented which show that a fourth power residual calcium model for stimulation-induced changes in transmitter release cannot readily account for the observation that stimulation- induced changes in MEPP frequency and EPP amplitude have similar time- courses.     

The MEK inhibitor, U0126, alters fertilization-induced [Ca2+]i oscillation parameters and secretion: differential effects associated with in vivo and in vitro meiotic maturation

Although mitogen-activated protein kinase (MAPK) is a well-known cell cycle regulator, emerging studies have also implicated its activity in the regulation of intracellular calcium concentration ([Ca2+](i)) and secretion. Those studies raise the hypothesis that MAPK activity during oocyte maturation and early fertilization is required for normal egg Ca2+ oscillations and cortical granule (CG) secretion. We extend the findings of [Lee, B., Vermassen, E., Yoon, S.-Y., Vanderheyden, V., Ito, J., Alfandari, D., De Smedt, H., Parys, J.B., Fissore, R.A., 2006. Phosphorylation of IP(3)R1 and the regulation of [Ca2+](i) responses at fertilization: a role for the MAP kinase pathway. Development 133, 4355-4365] by demonstrating acute effects on Ca2+ oscillation frequency, amplitude, and duration in fertilized mouse eggs matured in vitro with the MAPK inhibitor, U0126. Frequency was increased, whereas amplitude and duration were greatly decreased. These effects were significantly reduced in eggs matured in vivo and fertilized in the presence of the inhibitor. Ionomycin studies indicated that intracellular Ca2+ stores were differentially affected in eggs matured in vitro with U0126. Consistent with these effects on [Ca2+](i) elevation, fertilization-induced CG exocytosis and metaphase II exit were also reduced in in vitro-matured eggs with U0126, but not in those similarly treated after in vivo maturation. These results indicate that MAPK targets Ca2+ regulatory proteins during both maturation and fertilization, as well as provide a new hypothesis for MAPK function, which is to indirectly regulate events of early development by controlling Ca2+ oscillation parameters.     

Ca2+ and Na+ current patterns during oocyte maturation, fertilization, and early developmental stages of Ciona intestinalis

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.