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.     

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.     

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.     

Role of dihydropyridine-sensitive calcium channels in meiosis and fertilization in the bivalve molluscs Ruditapes philippinarum and Crassostrea gigas

Prophase-arrested oocytes of Ruditapes philippinarum can not be fertilized or stimulated by a depolarizing agent such as an excess of KCl, in contrast to the situation found in Crassostrea gigas. We have performed a comparative study between the two situations found in these species. In vitro, both of these oocytes can be triggered to reinitiate meiosis following a treatment by serotonin which promotes an intracellular calcium surge. Ruditapes and Crassostrea oocytes further arrest in metaphase I, at which stage they can be either activated by sperm or by excess KCl. These treatments trigger an intracellular calcium increase. This suggests that functional voltage-operated Ca2+ channels are expressed in Ruditapes during the course of maturation between prophase and metaphase I. Results obtained using pharmacological tools and direct binding of specific dihydropyridines, strongly suggest that these channels are dihydropyridine-sensitive calcium channels. In Ruditapes they become functional after 5-HT stimulation, their number increasing before GVBD. In Crassostrea the dihydropyridine-sensitive Ca2+ channels are already present at prophase stage and their density is constant from prophase to metaphase I. Moreover, we have shown for Ruditapes and Crassostrea that: 1) the addition of 10 microM of S(-)BayK8644, an agonist of dihydropyridine-sensitive calcium channels to metaphase-arrested oocytes releases them from metaphase block; and 2) incubating these oocytes with nicardipine, a potent blocker of dihydropyridine-sensitive Ca2+ channels, inhibits both their activation by excess KCl or fertilization. Taken together these data suggest that the absence of dihydropyridine-sensitive Ca2+ channels in the membrane of prophase-arrested oocytes of Ruditapes may account for their inability to be fertilized at this stage, while the presence of dihydropyridine-sensitive Ca2+ channels in prophase-arrested oocytes of Crassostrea may explain their fertilizability at this stage.     

Meiosis, egg activation, and nuclear envelope breakdown are differentially reliant on Ca2+, whereas germinal vesicle breakdown is Ca2+ independent in the mouse oocyte

During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.     

Fertilization causes a single Ca2+ increase that fully depends on Ca2+ influx in oocytes of limpets (Phylum Mollusca, Class Gastropoda)

Mature limpet oocytes arrested at the first metaphase (MI) of meiosis are activated by the stimulation of fertilizing sperm. The aim of the present study was to clarify the spatiotemporal property and mechanism of intracellular Ca2+ increase in limpet oocytes, which is a prerequisite signal for initiation of development at fertilization. In all of the five limpet species tested, the initial Ca2+ rising phase just after fertilization took the form of a centripetal Ca2+ wave spreading from the whole cortex to the center (cortical flash), yielding a homogeneous Ca2+ elevation throughout the oocyte. The Ca2+ level remained high during the subsequent plateau phase lasting for several minutes and then returned nearly to the original value. No additional Ca2+ increase followed the plateau phase at least by the time of first cleavage. Both rising and plateau phases of Ca2+ increase at fertilization were inhibited by removal of external Ca2+, suggesting that continuous Ca2+ entry occurs throughout the Ca2+ increase. Injection of inositol 1,4,5-trisphosphate (IP3) was effective in generating a Ca2+ increase in mature limpet oocytes arrested at MI; however, their ability to show an IP3-induced Ca2+ increase was extremely low, as compared with other animals. Responsiveness to IP3 injection in immature oocytes arrested at the first prophase (PI) was similar to that in the mature oocytes, suggesting that the IP3-induced Ca2+ release system does not develop during the process of meiotic maturation in limpet oocytes. Caffeine, cyclic adenosine diphosphate ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), the agents known to stimulate internal Ca2+ release mechanisms distinct from an IP3-dependent pathway, had no effect on intracellular Ca2+ changes in mature limpet oocytes. Labeling of the endoplasmic reticulum (ER) with DiI revealed that cortical ER clusters are only present in the localized region around meiotic chromosomes in mature oocytes. These data strongly suggest that Ca2+ release and its propagating mechanisms are undeveloped in limpet oocytes and that Ca2+ influx is the only Ca2+-mobilizing system available and functioning at fertilization.     

Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase.

In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.     

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.     

Metaphase I arrest of starfish oocytes induced via the MAP kinase pathway is released by an increase of intracellular pH

Reinitiation of meiosis in oocytes usually occurs as a two-step process during which release from the prophase block is followed by an arrest in metaphase of the first or second meiotic division [metaphase I (MI) or metaphase II (MII)]. The mechanism of MI arrest in meiosis is poorly understood, although it is a widely observed phenomenon in invertebrates. The blockage of fully grown starfish oocytes in prophase of meiosis I is released by the hormone 1-methyladenine. It has been believed that meiosis of starfish oocytes proceeds completely without MI or MII arrest, even when fertilization does not occur. Here we show that MI arrest of starfish oocytes occurs in the ovary after germinal vesicle breakdown. This arrest is maintained both by the Mos/MEK/MAP kinase pathway and the blockage of an increase of intracellular pH in the ovary before spawning. Immediately after spawning into seawater, activation of Na+/H+ antiporters via a heterotrimeric G protein coupling to a 1-methyladenine receptor in the oocyte leads to an intracellular pH increase that can overcome the MI arrest even in the presence of active MAP kinase.     

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.     

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.     

Zinc maintains prophase I arrest in mouse oocytes through regulation of the MOS-MAPK pathway

Meiosis in mammalian females is marked by two arrest points, at prophase I and metaphase II, which must be tightly regulated in order to produce a haploid gamete at the time of fertilization. The transition metal zinc has emerged as a necessary and dynamic regulator of the establishment, maintenance, and exit from metaphase II arrest, but the roles of zinc during prophase I arrest are largely unknown. In this study, we investigate the mechanisms of zinc regulation during the first meiotic arrest. Disrupting zinc availability in the prophase I arrested oocyte by treatment with the heavy metal chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) causes meiotic resumption even in the presence of pharmacological inhibitors of meiosis. We further show that the MOS-MAPK pathway mediates zinc-dependent prophase I arrest, as the pathway prematurely activates during TPEN-induced meiotic resumption. Conversely, inhibition of the MOS-MAPK pathway maintains prophase I arrest. While prolonged zinc insufficiency ultimately results in telophase I arrest, early and transient exposure of oocytes to TPEN is sufficient to induce meiotic resumption and bypass the telophase I block, allowing the formation of developmentally competent eggs upon parthenogenetic activation. These results establish zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation, including the maintenance of and release from the first and second meiotic arrest points.     

Thapsigargin induces meiotic maturation in surf clam oocytes.

I report here that thapsigargin, an inhibitor of Ca(2+)-ATPase activities in internal Ca2+ stores, induces meiotic maturation in prophase I-arrested surf clam (Spisula solidissima) oocytes. The half-maximal dose for triggering germinal vesicle breakdown (GVBD) is 120 nM. Thapsigargin-induced GVBD is followed by all normal subsequent steps of meiotic maturation including extrusions of first and second polar bodies, with almost normal timing as compared with K(+)-induced activation. Thapsigargin-induced GVBD requires the presence of external Ca2+ at a half-maximal concentration of 0.6 mM. In normal sea water, thapsigargin-induced activation is accompanied by a slightly increased 45Ca2+ uptake by the oocytes and by an intracellular pH rise of 0.3 U. These results show that thapsigargin-sensitive Ca2+ pools regulating Ca2+ fluxes exist in surf clam oocytes, and they also further establish that Ca2+ ions are the major initial trigger for meiosis resumption in this species.     

Inositol 1,4,5-triphosphate microinjection triggers activation, but not meiotic maturation in amphibian and starfish oocytes

Inositol 3,4,5-triphosphate (InsP3) brought about cortical granule exocytosis and elevation of a fertilization membrane, due to a rapid increase of free calcium in cytoplasm, when injected into oocytes of the amphibian Xenopus laevis arrested at second meiotic metaphase. The same result was observed when injection was performed into oocytes of the starfish Marthasterias glacialis arrested either at the first meiotic prophase or after completion of meiosis. Although meiotic maturation was induced in both animals by specific hormones which have been previously shown to release Ca2+ within cytoplasm, InsP3 microinjection into prophase-arrested oocytes did not release them from prophase block.     

HORMONAL CONTROL OF OOCYTE MATURATION IN ARENICOLA MARINA L. (ANNELIDA, POLYCHAETA) II. MATURATION AND FERTILIZATION

In Arenicola marina (Annelida, Polychacta) the oocytes arc arrested in the first prophase stage of meiosis until spawning. Oocyte maturation is under hormonal control: when incubated in vitro in a brain extract oocytes proceed to the first metaphase at which they remain arrested until fertilization. The prophase arrested oocytes can neither be fertilized nor parthcnogenetically activated by ionophore A23187 or 1 M glycerol. On the contrary the metaphase-arrested oocytes can be fertilized and parthenogenetically activated. Fertilizability thus appears during maturation; it seems to be linked to microvilli retraction. A study of spermatozoa "capacitation" and oocyte fertilization or activation is reported. A scanning electron microscope study of early contact and penetration of spermatozoa is presented.     

MAP kinase, meiosis, and sperm centrosome suppression in Urechis caupo

Although MAP kinase is an important regulatory enzyme in many somatic cells, almost nothing is known about its functions during meiosis, except in frog and mouse oocytes. We investigated MAPK activation and function in oocytes of the marine worm Urechis caupo that are fertilized at meiotic prophase. Activity was first detected at 4-6 min after fertilization in immunoblots with anti-active MAPK, prior to germinal vesicle breakdown (GVBD). MAPK activation did not require new protein synthesis and was dependent on the increases in both intracellular pH and intracellular Ca(2+) that normally occur during activation. When MAPK activation was inhibited with PD98059 or U0126, GVBD still occurred, but meiosis was abnormal and there was a dramatic premature enlargement of sperm asters, which normally do not appear until second polar body formation. Failure of polar body formation and premature sperm aster enlargement also occurred when MAPK activation was inhibited by an entirely different treatment which involved lowering the pH of external seawater to interrupt the normal cytoplasmic pH increase. Thus, in Urechis, active MAPK appears to be required for (1) normal meiotic divisions and (2) suppressing the paternal centrosome until after the egg completes meiosis, a general phenomenon whose mechanism has been unknown.     

Comparative proteomic analysis of proteins involved in oocyte meiotic maturation in mice

After birth, oocytes stay at the diplotene stage in prophase of meiosis I. Meiosis resumes about 1 day before ovulation, and arrests in metaphase II (MII) after ovulation. The mature, MII oocytes are then ready for fertilization and to provide materials for early embryonic development. Proteomic characterization of oocytes can help identify proteins that are important for female meiotic maturation and early embryonic development. In this study, we compared the proteomic profiles between the germinal vesicle and MII mouse oocytes by two-dimensional electrophoresis; 95 differentially expressed protein spots corresponding to 63 proteins were identified. Many of these proteins are known to be essential for oocyte meiosis and early embryonic development, such as adenylosuccinate synthetase, nucleoplasmin-2, and protein-arginine deiminase type-6. Of the 12 proteins that were identified and are highly expressed in oocytes, a novel protein, E330034G19Rik, was found to be oocyte-specific. According to analysis by bioinformatics, it may regulate chromosome segregation during meiosis or cleavage. An in-depth study of these proteins will help us better understand the mechanisms of oocyte meiotic maturation, fertilization, and early embryogenesis. It will also help us understand the mechanisms of diseases that stem from abnormal oocyte maturation, such as polycystic ovary syndrome and premature ovary failure.     

Ca(2+) response to cADPr during maturation and fertilization of starfish oocytes.

During the reinitiation of the meiotic cycle (maturation) induced by the hormone 1-methyladenine (1-MA), starfish oocytes undergo structural and biochemical changes in preparation for successful fertilization. Previous work has shown that the sensitivity of internal Ca(2+) stores to InsP(3) increases during maturation of the oocytes. Since Astropecten auranciacus oocytes also respond to cADPr, we have studied whether the response to cADPr also changes during maturation. We have found that the photoactivation of injected cADPr in immature oocytes immediately induces multiple patches of Ca(2+) release in the cortical region. The Ca(2+) signal then spreads from these initial points of increase to the entire cell. In mature oocytes, the uncaging of cADPr induces instead a single (or at most a dual) initial point of Ca(2+) release, which is immediately followed by the formation of a cortical Ca(2+) flash and then by the globalization of the wave and by the elevation of the fertilization envelope. External Ca(2+) plays a role in the Ca(2+) responses. Inhibition of L-type Ca(2+) channels does not affect the initial Ca(2+) release, but abolishes the cortical flash and impairs the elevation of the fertilization envelope. External Ca(2+) has other effects, as shown by the irregular appearance of the surface of oocytes incubated in Ca(2+)-free sea water. The sequence of Ca(2+) responses induced by cADPr in mature oocytes mimics those seen at fertilization, i.e., a first localized Ca(2+) increase followed by a cortical flash and by the globalization of the Ca(2+) signal. As in the case of maturation, L-type Ca(2+) channel blockers abolish the sperm induced cortical flash.     

Synthesis and function of mos: The control switch of vertebrate oocyte meiosis

One distinguishing feature of vertebrate oocyte meiosis is its discontinuity; oocytes are released from their prophase I arrest, usually by hormonal stimulation, only to again halt at metaphase II, where they await fertilization. The product of the c-mos proto-oncogene, Mos, is a key regulator of this maturation process. Mos is a serine-threonine kinase that activates and/or stabilizes maturation-promoting factor (MPF), the master cell cycle switch, through a pathway that involves the mitogen-activated protein kinase (MAPK) cascade. Oocytes arrested at prophase I lack detectable levels of Mos, which must be synthesized from a pool of maternal mRNAs for proper maturation. While Mos is necessary throughout maturation in Xenopus, it seems to be required only for meiosis II in the mouse. The translational activation of c-mos mRNA at specific times during meiosis requires cytoplasmic polyadenylation. Cis- and trans-acting factors for polyadenylation are, therefore, essential elements of maturation.     

Ca(2+)-promoted cyclin B1 degradation in mouse oocytes requires the establishment of a metaphase arrest

CDK1-cyclin B1 is a universal cell cycle kinase required for mitotic/meiotic cell cycle entry and its activity needs to decline for mitotic/meiotic exit. During their maturation, mouse oocytes proceed through meiosis I and arrest at second meiotic metaphase with high CDK1-cyclin B1 activity. Meiotic arrest is achieved by the action of a cytostatic factor (CSF), which reduces cyclin B1 degradation. Meiotic arrest is broken by a Ca2+ signal from the sperm that accelerates it. Here we visualised degradation of cyclin B1::GFP in oocytes and found that its degradation rate was the same for both meiotic divisions. Ca2+ was the necessary and sufficient trigger for cyclin B1 destruction during meiosis II; but it played no role during meiosis I and furthermore could not accelerate cyclin B1 destruction during this time. The ability of Ca2+ to trigger cyclin B1 destruction developed in oocytes following a restabilisation of cyclin B1 levels at about 12 h of culture. This was independent of actual first polar body extrusion. Thus, in metaphase I arrested oocytes, Ca2+ would induce cyclin B1 destruction and the first polar body would be extruded. In contrast to some reports in lower species, we found no evidence that oocyte activation was associated with an increase in 26S proteasome activity. We therefore conclude that Ca2+ mediates cyclin B1 degradation by increasing the activity of an E3 ubiquitin ligase. However, this stimulation occurs only in the presence of the ubiquitin ligase inhibitor CSF. We propose a model in which Ca2+ directly stimulates destruction of CSF during mammalian fertilisation.