Three Phases of Cortical Maturation during Meiosis Reinitiation in Starfish Oocytes

Oocytes of the starfish, Asterina pectinifera , respond differently to calcium ionophore A23187 depending upon their stage of maturation. Oocytes not-treated with 1-methyladenine (1-MA) formed only a partial fertilization envelope (FE) in response to A23187. Those treated with 1-MA formed no FE if the ionophore was introduced to them before germinal vesicle breakdown (GVBD), in contrast with which they did fully elevate the FE if it was introduced after GVBD. Similar stage-dependent results were obtained if the intracellular concentration of calcium was increased by microinjection of calcium-EGTA buffers. In good accordance with the FE formation, a stage-dependent protease release from oocytes by the ionophore was observed.
It is concluded from these results that, in starfish oocytes, their ability to undergo the exocytosis of cortical granules in response to an increase in intracellular calcium greatly changes along the way of maturation.     

Urea-Induced Meiosis Reinitiation in Oocytes of the Starfish, Marthasterias glacialis

In the absence of hormone stimulation, prophase-blocked oocytes of Marthasterias glacialis have been induced to undergo meiosis reinitiation up to female pronucleus formation by pulse incubation in isoosmotic urea-sea water solutions. Even when this procedure was not effective all along the breeding season, it could trigger full maturation when applied to so-called "incompetent oocytes" that did not complete maturation following microinjection-induced mixing of their nucleoplasm and cytoplasm.
³²P phosphate incorporation into proteins and cell fusion experiments demonstrate that this treatment produces an increased protein phosphorylation which appears tightly associated with the production of M-phase promoting factor (MPF). Instead, when oocytes are maintained in the inducing medium, dephosphorylation soon occurs and MPF is no longer present to support meiosis. Under these conditions, the GV-disrupted oocytes present a permanent nucleolus and do not form a meiotic spindle. The same cytological aspect was also obtained when the oocytes were treated in the presence of 90 μM emetine or 150 μM of the intracellular chelator Quin 2-AM.
These data suggest that urea-induced maturation may involve an intracellular Ca²⁺ shift which would be required to activate both MPF precursor molecules and the resting female centers which stand in the animal cortex outside the nucleus and give rise to the poles of the first maturation spindle. They also show that nuclear disruption alone, without protein phosphorylation, cannot trigger meiosis reinitiation of incompetent oocytes.     

DNA Synthesis during Maturation of Starfish Oocytes

IN Xenopus, nuclear DNA is replicated at an early stage of meiosis and there is no measurable DNA synthesis during the long diplotene stage characterized by the presence of the lampbrush chromosomes^1–3; this is probably a general phenomenon in animals. But in the case of pollen formation in plants, autoradiographic data suggest that, in addition to normal replication during S phase, some chromosomal DNA is synthesized throughout meiosis⁴. Limited DNA synthesis also follows replication during the S phase in Lilium anthers⁵.     

Activation of Starfish Oocytes Modifies their Hormone Dependent Period for 1-Methyladenine in Meiosis Reinitiation

Starfish oocytes reinitiate the meiosis when they are exposed to the hormone, 1-methyladenine (1-MA): 1-MA must be present for a period of about 10–20 min (20°C) known as the hormone dependent period (HDP). We investigated several conditions which affect the duration of the HDP in the starfish Asterina pectinifera . Fertilization lengthened the HDP about 30–80%, when spermatozoa were added to the oocytes at the same time as 1-MA was applied. When oocytes were inseminated before 1-MA treatment, HDP was reduced progressively correlating with the time of the insemination coming earlier, and eventually became shorter than the control. Similarly, A23187 and caffeine lengthened the HDP if they were added to the oocytes at the same time as 1-MA was applied, and shortened it if they are applied sufficiently in advance to 1-MA treatment. These modifications of HDP are likely to be brought by changes in the intracellular calcium concentration. A23187 did not lengthen the HDP, if it was administered 10 min after 1-MA application. Thus the effect of A23187 on the HDP occurred specifically during the first 5 or 10 min after 1-MA application. These results indicate that the HDP comprises at least two distinct phases.     

Spindle Dynamics during Meiosis in Drosophila Oocytes

Mature oocytes of Drosophila are arrested in metaphase of meiosis I. Upon activation by ovulation or fertilization, oocytes undergo a series of rapid changes that have not been directly visualized previously. We report here the use of the Nonclaret disjunctional (Ncd) microtubule motor protein fused to the green fluorescent protein (GFP) to monitor changes in the meiotic spindle of live oocytes after activation in vitro. Meiotic spindles of metaphase-arrested oocytes are relatively stable, however, meiotic spindles of in vitro–activated oocytes are highly dynamic: the spindles elongate, rotate around their long axis, and undergo an acute pivoting movement to reorient perpendicular to the oocyte surface. Many oocytes spontaneously complete the meiotic divisions, permitting visualization of progression from meiosis I to II. The movements of the spindle after oocyte activation provide new information about the dynamic changes in the spindle that occur upon re-entry into meiosis and completion of the meiotic divisions. Spindles in live oocytes mutant for a lossof-function ncd allele fused to gfp were also imaged. The genesis of spindle defects in the live mutant oocytes provides new insights into the mechanism of Ncd function in the spindle during the meiotic divisions.     

pH in the Jelly Layer of Starfish Oocytes

Using liquid ion exchanger micro-electrodes we have studied the pH in the vicinity of starfish oocytes and eggs and also in their jelly layers. Although highly variable, the pH in these zones was consistently lower (6.3–7.6) than the pH of sea water (7.8–8.2). Aged oocytes/eggs presented more acid environments than fresh oocytes. The pH indicator Phenol Red shows that the ovarian fluid is more acid than seawater, ranging from 6.0 to 7.5. Our data suggests that starfish oocytes, from their time in the ovary until several hr after their release, are surrounded by an acid environment. This micro-environment may play a role in metabolic repression in the ovary and in the regulation of sperm-egg interaction following spawning.     

Geminin Stabilizes Cdt1 during Meiosis in Xenopus Oocytes

During the mitotic cell cycle, Geminin can act both as a promoter and inhibitor of initiation of DNA replication. As a promoter, Geminin stabilizes Cdt1 and facilitates its accumulation leading to the assembly of the pre-replication complex on DNA. As an inhibitor, Geminin prevents Cdt1 from loading the mini-chromosome maintenance complex onto pre-replication complexes in late S, G₂, and M phases. Here we show that during meiosis Geminin functions as a stabilizer of Cdt1 promoting its accumulation for the early division cycles of the embryo. Depletion of Geminin in Xenopus immature oocytes leads to a decrease of Cdt1 protein levels during maturation and after activation of these oocytes. Injection of exogenous recombinant Geminin into the depleted oocytes rescues Cdt1 levels demonstrating that Geminin stabilizes Cdt1 during meiosis and after fertilization. Furthermore, Geminin-depleted oocytes did not replicate their DNA after meiosis I indicating that Geminin does not act as an inhibitor of initiation of DNA replication between meiosis I and meiosis II.In eukaryotes, initiation of DNA replication involves the formation and activation of the pre-replication complex (pre-RC)³ at the origins of replication. Pre-RCs are formed by the sequential binding of the origin recognition complex components, Cdc6, Cdt1, and mini-chromosome maintenance complex (MCM 2–7) proteins, to DNA. After loading the MCM complex, the pre-RCs are activated by S phase kinases (Dbf4-dependent kinase and Cdks) to initiate DNA replication (1). Replication of DNA, limited to only once per cell cycle, is critical to maintain genomic stability. Redundant mechanisms exist to ensure that DNA replication is tightly regulated during the cell cycle (1, 2). A small protein named Geminin has been shown to play a significant role in such regulatory mechanisms during mitosis (2–6). Geminin, a multifunctional 25-kDa protein, was first identified in a screen for proteins degraded during mitosis in Xenopus laevis egg extracts (7). Geminin is present in higher eukaryotes, but its presence in yeast has not yet been reported (7–10). Geminin plays a major role in regulating the function of Cdt1, one of the pre-RC components (8, 11–13). Numerous studies suggest that in higher eukaryotes the interaction between Geminin and Cdt1 is pivotal to restrict DNA replication to only once per cell cycle (6, 14–22). Furthermore, in Xenopus egg extracts, the Geminin/Cdt1 ratio seems to control the assembly of pre-RCs at replication origins and to determine whether the origins are licensed or not (23). The positive and negative roles of Geminin in origin licensing and DNA replication are made possible by their temporal separation during the cell cycle. Pre-RC formation occurs during late M and early G₁ phase, whereas pre-RC inhibition occurs from late S to mid M phase.As a positive regulator of DNA replication, Geminin has been shown to stabilize Cdt1. In human osteosarcoma cells, silencing of GEMININ expression limits CDT1 accumulation during mitosis and therefore the formation of pre-RCs in the subsequent cell cycle. This stabilizing effect is the result of a direct interaction between CDT1 and GEMININ preventing CDT1 ubiquitination and degradation (13). Similar findings were also recently observed in normal human cells and various cancer cells (24). However, in both human normal and tumor cells, the low level of CDT1, generated by the absence of GEMININ, did not always prevent cellular proliferation or re-replication of the genome (5, 24, 25). Therefore, one might question the importance of the role of GEMININ in stabilizing CDT1 in human cells. Beyond its role as a stabilizer of Cdt1 levels, Geminin has also been shown to participate directly in the formation of pre-RCs in Xenopus egg extracts. A complex between Cdt1 and Geminin binds to chromatin and supports pre-RC assembly. However, the recruitment of additional Geminin molecules to this complex on the chromatin blocks further pre-RC formation. These results indicate that the stoichiometry of Cdt1 and Geminin in this complex regulates its activity as a promoter or inhibitor of pre-RC assembly and DNA replication (23, 26). Several mechanisms have been shown to modulate the Geminin/Cdt1 balance on the chromatin. In Xenopus the binding of Cdt1 to the MCM9 protein seems to block the recruitment of an excess of Geminin to the chromatin and therefore favors pre-RC assembly (27). Similarly, the inactivation of Geminin by either ubiquitination or degradation also has a positive effect on pre-RC assembly (8, 11, 28–30). On the other hand, the replication-dependent degradation of Cdt1 has the opposite effect and prevents refiring of replication origins during S and G₂ phases of the mitotic cell cycle (18, 20, 31).Although the role of Geminin during mitosis has been extensively studied, not much is known about its function during meiosis. The expression pattern of Geminin during oocyte maturation is unclear. The presence of Geminin in immature stage VI Xenopus oocytes is controversial, but the protein is fully expressed in mature oocytes arrested in metaphase of meiosis II (7, 32). To form haploid gametes, DNA replication has to be inhibited between meiosis I (MI) and meiosis II (MII). In Xenopus oocytes, cyclin B-dependent kinase 1 (Cdk1) also known as maturation-promoting factor (MPF) plays a role in preventing DNA replication between the two meiotic divisions (33–36). Inhibition of Cdk1 activity between MI and MII leads to the formation of interphase nucleus and DNA replication. However, the role of Geminin in preventing DNA replication between meiotic divisions has not been tested so far. Finally, the possibility that Geminin stabilizes Cdt1 during meiosis and ensures its accumulation for the early embryonic divisions has not been formally examined.Here we show that the levels of Geminin and Cdt1 proteins increase significantly during meiosis in Xenopus oocytes and that the primary role of geminin is to promote the accumulation of Cdt1 and not to repress DNA replication between meiosis I and meiosis II. Depletion of Geminin in Xenopus immature oocytes does not lead to DNA replication after the first meiotic division but to a decrease in Cdt1 stability during the maturation and activation of these oocytes. Rescue of Cdt1 levels in these Geminin-depleted oocytes is achieved by injection of exogenous recombinant Geminin protein confirming the role of Geminin as a stabilizer of Cdt1 during meiosis and the early embryonic division cycles. These results provide further support for the idea that Geminin functions universally in stabilizing Cdt1. Although the stabilizing role of Geminin might not be its most important function in somatic cells, we show here that stabilizing Cdt1 is a dominant function for Geminin in Xenopus oocytes undergoing meiosis. This stabilizing role of Geminin is essential for the stockpiling of Cdt1 before fertilization that is required to sustain the rapid divisions of the early embryo.     

The Cytoskeleton and Nuclear Disassembly during Germinal Vesicle Breakdown in Starfish Oocytes

In response to maturation-inducing hormone, prophase-arrested oocytes of the starfish Pisaster ochraceus resume meiosis and undergo nuclear disassembly during a process referred to as germinal vesicle breakdown (GVBD). Time-lapse video recordings of maturing oocytes reveal that the nucleus lengthens along the animal-vegetal axis of the oocyte directly prior to GVBD. Neither taxol (10 μM) nor microtubule-depolymerizing agents [colcemid (50 μM), colchicine (250 μM), or nocodazole (1 μM)] prevent the pre-GVBD changes in nuclear shape from occurring, although correlative microscopical studies demonstrate that microtubules are nucleated (taxol) or depolymerized (colcemid, colchicine, nocodazole) at the concentrations listed above. The microtubule-altering drugs also do not affect the time at which GVBD begins or ends. A 10 μM solution of the microfilament-disrupting drug cytochalasin B (CB), on the other hand, essentially eliminates the pre-GVBD elongation of the nucleus. CB also slightly delays the onset of GVBD and significantly lengthens the time required to complete GVBD. Such studies suggest that: (i) drug-sensitive microtubules are not required for GVBD to proceed in a normal fasion; (ii) the pre-GVBD changes in nuclear shape involve microfilament-mediated events; and (iii) cytochalasin-induced depolymerization of microfilaments retards the normal timing of GVBD.     

Regulation of Separase in Meiosis: Separase is Activated at the Metaphase I-II Transition in Xenopus Oocytes during Meiosis

Separase is a cysteine protease conserved in all eukaryotes and functions to remove the sisterchromatid cohesion in anaphase by cleaving the SCC1 subunit of the cohesin complex. Theregulation of separase activity as the degradation of its inhibitor, securin, and the downregulationof the inhibitory phosphorylation has never been directly investigated in themeiotic cell cycle of vertebrates. In this study, we cloned the full-length gene encodingXenopus separase from an oocyte cDNA library. Purified xSeparase can cleave the human ?-kleisin subunit of cohesin in vitro but cannot bind with hSecurin when these two proteins areco-expressed in 293T cells. Similar to its human counterpart, xSeparase cleaves itself uponactivation but at a single site. The cleavage site is conserved with one of the three selfcleavagesites in hSeparase. Using self-cleavage as a reporter for its activation, wedemonstrated that xSeparase was transiently activated between the two meioses and may beinvolved in the homologous chromosome separation, as observed in other organisms. Takingthe advantage of the inability of xSecurin to interact with hSeparase, we demonstrated that theCSF extract re-inhibit both full-length and auto-cleaved hSeparase, indicating thatphosphorylation inhibition of separase does occur under the physiological condition. Inaddition, we found that the endogenous xSecurin was accumulated in response toprogesterone-induced oocyte maturation, and was degraded at both the anaphase I and II in anAPC/C-dependent manner.     

Mass Isolation of Germinal Vesicles from Starfish Oocytes*

A rapid, simple and efficient isolation procedure for germinal vesicles was developed using fully grown oocytes from the starfish, Asterina pectinifera. It depends on removal of the vitelline coat by trypsin digestion, gentle cell lysis by hypotonic treatment and centrifugation on a discontinuous sucrose gradient. The germinal vesicles isolated by this method are not clumped, fairly uniform in size and morphology, and rimmed with a very thin layer of cytoplasmic embroidery. They appear morphologically very similar to those in the oocytes. Potential applications of this method and possible functions of the cytoplasmic embroidery are discussed.     

Acid Release at Activation and Fertilization of Starfish Oocytes

Fertilization or activation by ionophore A 23187 induces a transient acid release in prophase-blocked and in maturing oocytes of Asterias rubens and Marthasterias glacialis. 1-Methyladenine-induced maturation is not accompanied by acid release. There is no significant difference in the kinetic and amount of acid release related to the nature of activation or the stage of oocytes in each species. The amount of acid released per oocyte volume is smaller than total "fertilization acid" of sea urchin eggs but comparable to its Na-insensitive component. Cortical reaction can be initiated without significant acid release in ammonia treated oocytes. A burst of sodium influx occurs at activation or fertilization of oocytes. Kinetic and amount of Na influx are comparable to acid release. Vitelline membrane elevation is impaired upon activation of oocytes in the absence of extracellular sodium but a significant although smaller release of acid occurs. This suggests that starfish oocytes release acid by a mechanism differing from the Na⁺-H⁺ exchange of sea urchin eggs.     

Nature of the 1-Methyladenine-Requiring Phase in Maturation of Starfish Oocytes

Reinitiation of meiosis in starfish oocytes requires the continuous presence of 1-methyladenine (1-MeAde) in the surrounding medium for a definite period. The length of the 'hormone-dependent phase' (HDP) in Asterina pectinifera , which was defined as the time necessary for induction of 50% germinal vesicle breakdown (GVBD), was found to be about 11 min at 17°C, and 8 min at 20°C. Repeated treatments for shorter periods with 1-MeAde revealed that the action of this agent was cumulative, and that stable intermediate states between the unstimulated and fully stimulated levels existed during the HDP. Measurement of the stiffness of oocytes also demonstrated this stable intermediate state. Thus, there may be a factor(s) in the cytoplasm that accumulates continuously during the HDP and triggers GVBD when it reaches a critical level(s). When dithiothreitol (DTT) was used as an artificial maturation-inducing agent, the intermediate state was far less stable, suggesting a difference in the modes of action of 1-MeAde and DTT. Isotonic CaCl₂, the Ca²⁺ ionophore (A 23187) and methylxanthines, which are known to cause increase in intracellular Ca²⁺, had additive effects with 1-MeAde. These results suggest that part of the action of 1-MeAde is to release Ca²⁺ in the oocyte cytoplasm.     

Emi1 Class of Proteins Regulate Entry into Meiosis and the Meiosis I to Meiosis II Transition in Xenopus Oocytes

Xenopus oocytes are arrested at the G2/prophase boundary of meiosis I and enter meiosis in response to progesterone. A hallmark of meiosis is the absence of DNA replication between the successive cell division phases meiosis I (MI) and meiosis II (MII). After the MI-MII transition, Xenopus eggs are locked in metaphase II by the cytostatic factor (CSF) arrest to prevent parthenogenesis. Early Mitotic Inhibitor 1 (Emi1) maintains CSF arrest by inhibiting the ability of the Anaphase Promoting Complex (APC) to direct the destruction of cyclin B. To investigate whether Emi1 has an earlier role in meiosis, we injected Xenopus oocytes with neutralizing antibodies against Emi1 at G2/prophase and during the MI-MII transition. Progesterone-treated G2/prophase oocytes injected with anti-Emi1 antibody fail to activate Maturation Promoting Factor (MPF), a complex of cdc2/cyclin B, and the MAPK pathway, and do not undergo germinal vesicle breakdown (GVBD). Injection of purified ?90 cyclin B protein or blocking anti-Emi1 antibody with purified Emi1 protein rescues these meiotic processes in Emi1-neutralized oocytes. Acute inhibition of Emi1 in progesterone treated oocytes immediately after GVBD causes rapid loss of cdc2 activity with simultaneous loss of cyclin B levels and inactivation of the MAPK pathway. These oocytes decondense their chromosomes and enter a DNA replication phase instead of progressing to MII. Prior ablation of Cdc20, addition of methyl-ubiquitin, or addition of indestructible ?90 cyclin B rescues the MI-MII transition in Emi1 inhibited oocytes.     

罗氏海盘车(Asterias rollestoni)中几种化合物的提取分离和结构鉴定

罗氏海盘车的95％EtOH提取物用大孔吸附树脂柱、凝胶柱和硅胶柱进行反复分离得到4个化合物,用NMR、MS和IR分析技术分别鉴定为苯丙氨酸、2′-脱氧胸腺嘧啶核苷、2′脱氧次黄嘌呤核苷-5′-单磷酸盐和5-甲氧基-1-十三醇。     

Nucleation Promoting Factors Regulate the Expression and Localization of Arp2/3 Complex during Meiosis of Mouse Oocytes

The actin nucleation factor Arp2/3 complex is a main regulator of actin assembly and is involved in multiple processes like cell migration and adhesion, endocytosis, and the establishment of cell polarity in mitosis. Our previous work showed that the Arp2/3 complex was involved in the actin-mediated mammalian oocyte asymmetric division. However, the regulatory mechanisms and signaling pathway of Arp2/3 complex in meiosis is still unclear. In the present work, we identified that the nucleation promoting factors (NPFs) JMY and WAVE2 were necessary for the expression and localization of Arp2/3 complex in mouse oocytes. RNAi of both caused the degradation of actin cap intensity, indicating the roles of NPFs in the formation of actin cap. Moreover, JMY and WAVE2 RNAi decreased the expression of ARP2, a key component of Arp2/3 complex. However, knock down of Arp2/3 complex by Arpc2 and Arpc3 siRNA microinjection did not affect the expression and localization of JMY and WAVE2. Our results indicate that the NPFs, JMY and WAVE2, are upstream regulators of Arp2/3 complex in mammalian oocyte asymmetric division.     

Cortical Granule Translocation during Maturation of Starfish Oocytes Requires Cytoskeletal Rearrangement Triggered by InsP3-Mediated CaRelease

Cortical granules (secretory vesicles located under the cortex of mature oocytes) release their contents to the medium at fertilization. Their exocytosis modifies the extracellular environment, blocking the penetration of additional sperm. The granules translocate to the surface during the maturation process, and it has been suggested that they move to the cortex via cytoskeletal elements. In this paper we show that the increase in intracellular Ca²⁺, which the maturing hormone 1-methyladenine (1-MA) induces in starfish through the activation of inositol 1,4,5-trisphosphate (InsP₃) receptors, triggers changes in filamentous actin, which then direct the correct movement and reorientation of the cortical granules and the elevation of the fertilization envelope.     

Role of Contents of the Germinal Vesicle in Male Pronuclear Development and Cleavage of Starfish Oocytes

During 1-methyladenine induced germinal vesicle breakdown, contents of the germinal vesicle of starfish oocytes are mixed with the surrounding cytoplasm. Upon injection of contents of the germinal vesicle from immature (fully grown) oocytes into enucleated and inseminated oocytes, incorporated spermatozoa were not observed to change structurally. Alternatively, after treatment of the above oocytes with 1-methyladenine, sperm asters and male pronuclei were developed and subsequent cleavage was also detected. From these results, it is concluded that both action of 1-methyladenine and participation of contents of the germinal vesicle are indispensable for male pronuclear development and subsequent cleavage.