Increased sensitivity and clustering of elementary Ca2+ release events during oocyte maturation

The universal signal for egg activation at fertilization is a rise in cytoplasmic Ca(2+) with defined spatial and temporal kinetics. Mammalian and amphibian eggs acquire the ability to produce such Ca(2+) signals during a maturation period that precedes fertilization and encompasses resumption of meiosis and progression to metaphase II. In Xenopus, immature oocytes produce fast, saltatory Ca(2+) waves that can be oscillatory in nature in response to IP(3). In contrast, mature eggs produce a single continuous, sweeping Ca(2+) wave in response to IP(3) or sperm fusion. The mechanisms mediating the differentiation of Ca(2+) signaling during oocyte maturation are not well understood. Here, I characterized elementary Ca(2+) release events (Ca(2+) puffs) in oocytes and eggs and show that the sensitivity of IP(3)-dependent Ca(2+) release is greatly enhanced during oocyte maturation. Furthermore, Ca(2+) puffs in eggs have a larger spatial fingerprint, yet are short lived compared to oocyte puffs. Most interestingly, Ca(2+) puffs cluster during oocyte maturation resulting in a continuum of Ca(2+) release sites over space in eggs. These changes in the spatial distribution of elementary Ca(2+) release events during oocyte maturation explain the continuous nature and slower speed of the fertilization Ca(2+) wave.     

STIM1 is required for Ca2+ signaling during mammalian fertilization

During fertilization in mammals, a series of oscillations in the oocyte's intracellular free Ca(2+) concentration is responsible for oocyte activation and stimulation of embryonic development. The oscillations are associated with influx of Ca(2+) across the plasma membrane that is probably triggered by the depletion of the intracellular stores, a mechanism known as store-operated Ca(2+) entry. Recently, STIM1 has been identified in oocytes as a key component of the machinery that generates the Ca(2+) influx after store depletion. In this study, the involvement of STIM1 in the sperm-induced Ca(2+) oscillations and its significance in supporting subsequent embryo development were investigated. Downregulation of STIM1 levels in pig oocytes by siRNA completely inhibited the repetitive Ca(2+) signal triggered by the fertilizing sperm. In addition, a significantly lower percentage of oocytes cleaved or formed blastocysts when STIM1 was downregulated prior to fertilization compared to the control groups. Restoring STIM1 levels after fertilization in such oocytes by means of mRNA injection could not rescue embryonic development that in most cases was arrested at the 2-cell stage. On the other hand, STIM1 overexpression prior to fertilization did not alter the pattern of sperm-induced Ca(2+) oscillations and development of these fertilized oocytes up to the blastocyst stage was also similar to that registered in the control group. Finally, downregulation of STIM1 had no effect on oocyte activation when activation was stimulated artificially by inducing a single large elevation in the oocyte's intracellular free Ca(2+) concentration. These findings suggest that STIM1 is essential for normal fertilization as it is involved in the maintenance of the long-lasting repetitive Ca(2+) signal.     

Maturation and fertilization in Lottia gigantea oocytes: intracellular pH, Ca(2+), and electrophysiology

Intracellular pH and Ca(2+) were measured with BCECF- and Calcium Green-dextran during maturation and fertilization of oocytes of the limpet Lottia gigantea. Maturation of oocytes from prophase to metaphase I of meiosis was induced in seawater adjusted to pH 9 with NH(4)OH. Intracellular pH rose during maturation induction, and maturation was also induced by microinjecting pH 8, but not pH 7, HEPES buffer. Intracellular Ca(2+) rose during NH(4)OH-induced maturation, but maturation was not inhibited when the increase was blocked by microinjection of BAPTA. When the metaphase I oocytes were fertilized(), there was an abrupt increase in intracellular Ca(2+), and activation (polar body formation) failed to occur in BAPTA-injected oocytes. Intracellular pH did not rise during fertilization. These observations show that maturation from prophase to metaphase I of meiosis is pH-dependent and activation of the metaphase I oocytes is Ca(2+)-dependent. A Ca(2+) action potential was present in both immature and mature oocytes but was more prominent in mature oocytes whose input resistance was higher. Fertilization produced a long-lasting (17-20 min) Na(+)-dependent fertilization potential with superimposed oscillations resembling Ca(2+) action potentials.     

Ca(2+)(cyt) negatively regulates the initiation of oocyte maturation

Ca(2+) is a ubiquitous intracellular messenger that is important for cell cycle progression. Genetic and biochemical evidence support a role for Ca(2+) in mitosis. In contrast, there has been a long-standing debate as to whether Ca(2+) signals are required for oocyte meiosis. Here, we show that cytoplasmic Ca(2+) (Ca(2+)(cyt)) plays a dual role during Xenopus oocyte maturation. Ca(2+) signals are dispensable for meiosis entry (germinal vesicle breakdown and chromosome condensation), but are required for the completion of meiosis I. Interestingly, in the absence of Ca(2+)(cyt) signals oocytes enter meiosis more rapidly due to faster activation of the MAPK-maturation promoting factor (MPF) kinase cascade. This Ca(2+)-dependent negative regulation of the cell cycle machinery (MAPK-MPF cascade) is due to Ca(2+)(cyt) acting downstream of protein kinase A but upstream of Mos (a MAPK kinase kinase). Therefore, high Ca(2+)(cyt) delays meiosis entry by negatively regulating the initiation of the MAPK-MPF cascade. These results show that Ca(2+) modulates both the cell cycle machinery and nuclear maturation during meiosis.     

Calcium signaling differentiation during Xenopus oocyte maturation

Ca(2+) is the universal signal for egg activation at fertilization in all sexually reproducing species. The Ca(2+) signal at fertilization is necessary for egg activation and exhibits specialized spatial and temporal dynamics. Eggs acquire the ability to produce the fertilization-specific Ca(2+) signal during oocyte maturation. However, the mechanisms regulating Ca(2+) signaling differentiation during oocyte maturation remain largely unknown. At fertilization, Xenopus eggs produce a cytoplasmic Ca(2+) (Ca(2+)(cyt)) rise that lasts for several minutes, and is required for egg activation. Here, we show that during oocyte maturation Ca(2+) transport effectors are tightly modulated. The plasma membrane Ca(2+) ATPase (PMCA) is completely internalized during maturation, and is therefore unable to extrude Ca(2+) out of the cell. Furthermore, IP(3)-dependent Ca(2+) release is required for the sustained Ca(2+)(cyt) rise in eggs, showing that Ca(2+) that is pumped into the ER leaks back out through IP(3) receptors. This apparent futile cycle allows eggs to maintain elevated cytoplasmic Ca(2+) despite the limited available Ca(2+) in intracellular stores. Therefore, Ca(2+) signaling differentiates in a highly orchestrated fashion during Xenopus oocyte maturation endowing the egg with the capacity to produce a sustained Ca(2+)(cyt) transient at fertilization, which defines the egg's competence to activate and initiate embryonic development.     

Defective calcium release during in vitro fertilization of maturing oocytes of LT/Sv mice

Oocytes of LT/Sv mice have anomalous cytoplasmic and nuclear maturation. Here, we show that in contrast to the oocytes of wild-type mice, a significant fraction of LT/Sv oocytes remains arrested at the metaphase of the first meiotic division and is unable to undergo sperm-induced activation when fertilized 15 hours after the resumption of meiosis. We also show that LT/Sv oocytes experimentally induced to resume meiosis and to reach metaphase II are unable to undergo activation in response to sperm penetration. However, the ability for sperm-induced activation developed during prolonged in vitro culture. Both types of LT/Sv oocytes, i.e. metaphase I and those that were experimentally induced to reach metaphase II, underwent activation when they were fertilized 21 hours after germinal vesicle breakdown (GVBD). Thus, the ability of LT/Sv oocytes to become activated by sperm depends on cytoplasmic maturation rather than on nuclear maturation i.e. on the progression of meiotic division. We also show that sperm penetration induces fewer Ca(2+) transients in LT/Sv oocytes than in control wild-type oocytes. In addition, we found that the levels of mRNA encoding different isoforms of protein kinase C (alpha, delta and zeta), that are involved in meiotic maturation and signal transduction during fertilization, differed between metaphase I LT/Sv oocytes which cannot be activated by sperm, and those which are able to undergo activation after fertilization. However, no significant differences between these oocytes were found at the level of mRNA encoding IP(3) receptors which participate in calcium release during oocyte fertilization.     

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     

Orai1 mediates store-operated Ca2+ entry during fertilization in mammalian oocytes

The presence of the store-operated Ca(2+) entry channel Orai1 and its function in signal transduction during fertilization have been investigated in mammalian oocytes using the pig as a model. RT-PCR cloning and sequence analysis revealed that Orai1 is expressed in the oocytes with a coding sequence of 921bp. After indirect immunocytochemistry or the overexpression of EGFP-tagged Orai1, the fluorescent signal was present primarily in the cell cortex consistent with plasma membrane localization of the protein. Western blot and real-time PCR results showed that Orai1 expression decreases during oocyte maturation; this is associated with the oocytes gaining the ability to generate a large Ca(2+) influx after store depletion. Downregulation of Orai1 expression by siRNA microinjection blocked Ca(2+) influx after store depletion and subsequent Ca(2+) add-back; the Ca(2+) oscillations induced by the fertilizing sperm were also inhibited in oocytes with downregulated Orai1 levels. At the same time, overexpression of Orai1 in the oocytes also modified store-operated Ca(2+) entry and had an inhibitory effect on the fertilization Ca(2+) signal. The abnormal Ca(2+) signaling due to Orai1 downregulation had a strong negative impact on subsequent embryo development. Co-overexpression of Orai1 and STIM1 on the other hand, led to a dramatic increase in Ca(2+) entry after store depletion. The findings indicate that Orai1 is a plasma membrane-resident Ca(2+) channel that is responsible for mediating Ca(2+) entry after the mobilization of intracellular Ca(2+) in oocytes. Orai1 and a functional store-operated Ca(2+) entry pathway are required to maintain the Ca(2+) oscillations at fertilization and to support proper embryo development.     

Inactivation of Ca(2+) action potential channels by the MEK inhibitor PD98059

In a previous study involving the inhibition of mitogen-activated protein kinase (MAPK) activation during fertilization of the marine worm Urechis caupo, we found that PD98059, but not U0126, caused multiple sperm penetrations in oocytes (Gould and Stephano, 1999, Dev. Biol. 216, 348-358). Since these oocytes are protected against polyspermy by a positive shift in membrane potential at fertilization (Gould-Somero et al., 1979, J. Cell Biol. 82, 426-440), we investigated the effects of PD98059 on the electrical properties of the oocyte membrane. PD98059, but not U0126, selectively blocked the voltage-dependent Ca(2+) channels that participate in the electrical polyspermy block. We also noted previously that PD98059 had more serious effects than U0126 on chromosome behavior during meiosis. This, too, could be explained by the effect on Ca(2+) channels, since when U0126-treated eggs were fertilized in low Ca(2+) seawater to reduce Ca(2+) uptake, similar effects were produced. These results show that PD98059 has side effects unrelated to the inhibition of MAPK activation and underscores the need for caution in interpreting the results of experiments with this widely used MEK inhibitor.     

Two phases of calcium requirement during starfish meiotic maturation

During meiosis in oocytes of the starfish, Asterina pectinifera, a Ca(2+) transient has been observed. To clarify the role of Ca(2+) during oocyte maturation in starfish, an intracellular Ca(2+) blocker, TMB-8, was applied. The oocyte maturation induced by 1-methyladenine (1-MA) was blocked by 100 microM TMB-8. Reinitiation of meiosis with germinal vesicle breakdown (GVBD) and the following chromosome condensation did not take place. Maturation-promoting factor (MPF) activity did not increase and GVBD and chromosome condensation did not occur. Ca(2+) transient observed immediately after 1-MA application in control oocytes was also blocked by TMB-8. When calyculin A, which activate the MPF directly, was applied to the oocytes instead of 1-MA in seawater containing 100 microM TMB-8, GVBD and chromosome condensation were blocked. Cytoplasmic transplantation studies confirmed that MPF was activated, although TMB-8 blocked GVBD. These results show that TMB-8 blocked the increase of MPF activity induced by 1-MA and the process of active MPF inducing GVBD and subsequent chromosome condensation. Together with the above phenomena, it is conceivable that there are two phases of Ca(2+) requirement during starfish oocyte maturation. These are the activation of MPF, moreover, GVBD, and the subsequent chromosome condensation.     

Activation of amphibian oocytes by sperm extracts

In the fertilization of most animals, egg activation is accompanied by an increase in cytoplasmatic Ca2+; however, the mechanism through which the fertilizing sperm induce this phenomenon is still controversial. An increase in intracellular free Ca2+ is required to trigger egg activation events, a process that includes cortical granule exocytosis, resumption and completion of meiosis and DNA replication, and culminates in the first mitotic cleavage. In this work, we investigated the effect of microinjection and incubation of different fractions of homologous sperm extract on the activation of Bufo arenarum oocytes matured in vitro. Two heat treatment-sensitive fractions obtained by chromatography were able to induce oocyte activation. The sperm fraction, which contained a 24 kDa protein, induced 90% activation when it was microinjected into the oocytes. Whilst the sperm fraction, which contained a 36 kDa protein, was able to induce about 70% activation only when it was applied on the oocyte surface.     

Mammalian oocyte activation by the synergistic action of discrete sperm head components: induction of calcium transients and involvement of proteolysis

Sperm-borne oocyte-activating factor (SOAF) elicits activation sufficient for full development and originates from sperm head submembrane matrices. SOAF comprises discrete, heat-sensitive and -stable components (referred to here respectively as SOAF-I and -II) which are each necessary but not sufficient to activate oocytes. The heat-sensitive SOAF component, SOAF-I(m), becomes solubilized from the perinuclear matrix under reducing conditions (the SOAF transition) to generate SOAF-I(s). Although calcium transients likely play an important role in oocyte activation at fertilization, the question is open as to whether demembranated heads or SOAF-I(s) and/or SOAF-II can induce calcium transients. We now report that injection of demembranated sperm heads into mouse oocytes efficiently induced Ca(2+) oscillations. When injected independently, SOAF-I(s) and demembranated heads heated to 48 degrees C failed to generate Ca(2+) oscillations. However, co-injection of SOAF-I(s) and 48 degrees C-heated heads induced oscillations, mirroring their synergistic ability to activate oocytes. This suggests that SOAF-mediated activation proceeds via pathways resembling those at fertilization and provides the first direct evidence that multiple sperm components are required to induce Ca(2+) oscillations. We probed the SOAF-I(s) liberation at the center of this activation and show that in vitro it was sensitive to a profile of serine protease inhibitors. These findings support a model in which mammalian oocyte activation, including the induction of calcium transients, involves proteolytic processing of SOAF from sperm head submembrane compartments.     

Induction of maturation-promoting factor during Xenopus oocyte maturation uncouples Ca(2+) store depletion from store-operated Ca(2+) entry.

During oocyte maturation, eggs acquire the ability to generate specialized Ca(2+) signals in response to sperm entry. Such Ca(2+) signals are crucial for egg activation and the initiation of embryonic development. We examined the regulation during Xenopus oocyte maturation of store-operated Ca(2+) entry (SOCE), an important Ca(2+) influx pathway in oocytes and other nonexcitable cells. We have previously shown that SOCE inactivates during Xenopus oocyte meiosis. SOCE inactivation may be important in preventing premature egg activation. In this study, we investigated the correlation between SOCE inactivation and the Mos-mitogen-activated protein kinase (MAPK)-maturation-promoting factor (MPF) kinase cascade, which drives Xenopus oocyte maturation. SOCE inactivation at germinal vesicle breakdown coincides with an increase in the levels of MAPK and MPF. By differentially inducing Mos, MAPK, and MPF, we demonstrate that the activation of MPF is necessary for SOCE inactivation during oocyte maturation. In contrast, sustained high levels of Mos kinase and the MAPK cascade have no effect on SOCE activation. We further show that preactivated SOCE is not inactivated by MPF, suggesting that MPF does not block Ca(2+) influx through SOCE channels, but rather inhibits coupling between store depletion and SOCE activation.     

Human round spermatids from azoospermic men exhibit oocyte-activation and Ca2+ oscillation-inducing activities

During mammalian fertilization, intracellular Ca2+ oscillations are important for both oocyte activation and embryonic development. As the ability of round spermatids (ROS) to induce Ca2+ oscillations and oocyte activation is different between species, we examined Ca2+ oscillation- and oocyte activation-inducing abilities of human ROS originating from patients with non-obstructive azoospermia. Human ROS from 11 non-obstructive azoospermic patients were collected during their TESE-ICSI cycles. Following injection into mature unfertilized mouse oocytes, we examined the oocyte-activating and Ca2+ oscillation-inducing activities of ROS by using Ca2+ imaging and confocal laser scanning microscopy (mouse test). In these 11 cases, clinical TESE-ICSI using mature testicular spermatozoa was successful, with the exception of one case in which only one sperm-injected oocyte was not fertilized. The mean fertilization rate was 70.1% (40-100%); the mean cleavage rate was 97.9% (46/47). Two pregnancies were established from 10 transfer cycles (PR; 20%). When the ROS from these patients were injected into mouse oocytes, the ROS from all patients induced at least some intracellular Ca2+ oscillations (25-100%). In all patients, 40 out of 82 oocytes injected with ROS exhibited normal oscillation patterns of [Ca2+]i.Human spermatogenetic cells acquired oocyte-activating and Ca2+ oscillation-inducing abilities at the round spermatid stage, an earlier stage than found for rodent cells. These data indicate that human ROS might be useful for clinical treatments of non-obstructive azoospermic patients exhibiting mature spermatozoa in biopsied specimens.     

Intercellular communication between follicular angiotensin receptors and Xenopus laevis oocytes: medication by an inositol 1,4,5- trisphosphate-dependent mechanism

In Xenopus laevis oocytes, activation of angiotensin II (AII) receptors on the surrounding follicular cells sends a signal through gap junctions to elevate cytoplasmic calcium concentration ([Ca2+]i) within the oocyte. The two major candidates for signal transfer through gap junctions into the oocyte during AII receptor stimulation are Ins(1,4,5)P3 and Ca2+. In [3H]inositol-injected follicular oocytes, AII stimulated two- to fourfold increases in phosphoinositide hydrolysis and production of inositol phosphates. Injection of the glycosaminoglycan, heparin, which selectively blocks Ins(1,4,5)P3 receptors, prevented both AII-stimulated and Ins(1,4,5)P3-induced Ca2+ mobilization in Xenopus follicular oocytes but did not affect mobilization of Ca2+ by ionomycin or GTP. These results indicate that the AII-regulated process of gap junction communication between follicular cells and the oocyte operates through an Ins(1,4,5)P3-dependent mechanism rather than through transfer of Ca2+ into the ooplasm and subsequent Ca(2+)-induced Ca2+ release.     

Starting a new life: sperm PLC-zeta mobilizes the Ca2+ signal that induces egg activation and embryo development: an essential phospholipase C with implications for male infertility

We have discovered that a single sperm protein, phospholipase C-zeta (PLCζ), can stimulate intracellular Ca(2+) signalling in the unfertilized oocyte ('egg') culminating in the initiation of embryonic development. Upon fertilization by a spermatozoon, the earliest observed signalling event in the dormant egg is a large, transient increase in free Ca(2+) concentration. The fertilized egg responds to the intracellular Ca(2+) rise by completing meiosis. In mammalian eggs, the Ca(2+) signal is delivered as a train of long-lasting cytoplasmic Ca(2+) oscillations that begin soon after gamete fusion and persist beyond the completion of meiosis. Sperm PLCζ effects Ca(2+) release from egg intracellular stores by hydrolyzing the membrane lipid PIP(2) and consequent stimulation of the inositol 1,4,5-trisphosphate (InsP(3) ) receptor Ca(2+) -signalling pathway, leading to egg activation and early embryogenesis. Recent advances have refined our understanding of how PLCζ induces Ca(2+) oscillations in the egg and also suggest its potential dysfunction as a cause of male infertility.     

Spontaneous and LH-induced maturation in Bufo arenarum oocytes: importance of gap junctions

It has been demonstrated in Bufo arenarum that fully grown oocytes are capable of meiotic resumption in the absence of a hormonal stimulus if they are deprived of their follicular envelopes. This event, called spontaneous maturation, only takes place in oocytes collected during the reproductive period, which have a metabolically mature cytoplasm. In Bufo arenarum, progesterone acts on the oocyte surface and causes modifications in the activities of important enzymes, such as a decrease in the activity of adenylate cyclase (AC) and the activation of phospholipase C (PLC). PLC activation leads to the formation of diacylglycerol (DAG) and inositol triphosphate (IP(3)), second messengers that activate protein kinase C (PKC) and cause an increase in intracellular Ca(2+). Recent data obtained from Bufo arenarum show that progesterone-induced maturation causes significant modifications in the level and composition of neutral lipids and phospholipids of whole fully grown ovarian oocytes and of enriched fractions in the plasma membrane. In amphibians, the luteinizing hormone (LH) is responsible for meiosis resumption through the induction of progesterone production by follicular cells. The aim of this work was to study the importance of gap junctions in the spontaneous and LH-induced maturation in Bufo arenarum oocytes. During the reproductive period, Bufo arenarum oocytes are capable of undergoing spontaneous maturation in a similar way to mammalian oocytes while, during the non-reproductive period, they exhibit the behaviour that is characteristic of amphibian oocytes, requiring progesterone stimulation for meiotic resumption (incapable oocytes). This different ability to mature spontaneously is coincident with differences in the amount and composition of the phospholipids in the oocyte membranes. Capable oocytes exhibit in their membranes higher quantities of phospholipids than incapable oocytes, especially of PC and PI, which are precursors of second messengers such as DAG and IP(3). The uncoupling of the gap junctions with 1-octanol or halothane fails to induce maturation in follicles from the non-reproductive period, whose oocytes are incapable of maturing spontaneously. However, if the treatment is performed during the reproductive period, with oocytes capable of undergoing spontaneous maturation, meiosis resumption occurs in high percentages, similar to those obtained by manual defolliculation. Interestingly, results show that LH is capable of inducing GVBD in both incapable oocytes and in oocytes capable of maturing spontaneously as long as follicle cells are present, which would imply the need for a communication pathway between the oocyte and the follicle cells. This possibility was analysed by combining LH treatment with uncoupling agents such as 1-octanol or halothane. Results show that maturation induction with LH requires a cell-cell coupling, as the uncoupling of the gap junctions decreases GVBD percentages. Experiments with LH in the presence of heparin, BAPTA/AM and theophylline suggest that the hormone could induce GVBD by means of the passage of IP(3) or Ca(2+) through the gap junctions, which would increase the Ca(2+) level in the oocyte cytoplasm and activate phosphodiesterase (PDE), thus contributing to the decrease in cAMP levels and allowing meiosis resumption.     

Regulation of inositol 1,4,5-trisphosphate receptor function during mouse oocyte maturation

At the time of fertilization, an increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) underlies egg activation and initiation of development in all species studied to date. The inositol 1,4,5-trisphosphate receptor (IP(3)R1), which is mostly located in the endoplasmic reticulum (ER) mediates the majority of this Ca(2+) release. The sensitivity of IP(3)R1, that is, its Ca(2+) releasing capability, is increased during oocyte maturation so that the optimum [Ca(2+)](i) response concurs with fertilization, which in mammals occurs at metaphase of second meiosis. Multiple IP(3)R1 modifications affect its sensitivity, including phosphorylation, sub-cellular localization, and ER Ca(2+) concentration ([Ca(2+)](ER)). Here, we evaluated using mouse oocytes how each of these factors affected IP(3)R1 sensitivity. The capacity for IP(3)-induced Ca(2+) release markedly increased at the germinal vesicle breakdown stage, although oocytes only acquire the ability to initiate fertilization-like oscillations at later stages of maturation. The increase in IP(3)R1 sensitivity was underpinned by an increase in [Ca(2+)](ER) and receptor phosphorylation(s) but not by changes in IP(3)R1 cellular distribution, as inhibition of the former factors reduced Ca(2+) release, whereas inhibition of the latter had no impact. Therefore, the results suggest that the regulation of [Ca(2+)](ER) and IP(3)R1 phosphorylation during maturation enhance IP(3)R1 sensitivity rendering oocytes competent to initiate oscillations at the expected time of fertilization. The temporal discrepancy between the initiation of changes in IP(3)R1 sensitivity and acquisition of mature oscillatory capacity suggest that other mechanisms that regulate Ca(2+) homeostasis also shape the pattern of oscillations in mammalian eggs.     

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.