c-Mos and cyclin B/cdc2 connections during Xenopus oocyte maturation.

Fully-grown G2 arrested Xenopus oocytes can be induced to enter and progress into meiotic cell cycle by progesterone stimulation. This process is termed oocyte maturation. An early response to progesterone is the synthesis of the onco-protein c-Mos, defined as the candidate initiator of Xenopus oocyte maturation, which triggers the MAPK cascade, MPF activation and promotes CSF activity. Here we review our current knowledge on the synthesis, activation and functions of c-Mos in connection with MPF activation during maturation. We also discuss our recent results concerning the dispensability of cyclin B degradation in meiosis I-meiosis II transition and the stabilization of c-Mos through its direct phosphorylation by cyclin B/cdc2.     

Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation

Degradation of proteins mediated by ubiquitin-proteasome pathway (UPP) plays important roles in the regulation of eukaryotic cell cycle. In this study, the functional roles and regulatory mechanisms of UPP in mouse oocyte meiotic maturation, fertilization, and early embryonic cleavage were studied by drug-treatment, Western blot, antibody microinjection, and confocal microscopy. The meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated by two potent, reversible, and cell-permeable proteasome inhibitors, ALLN and MG-132. The metaphase I spindle assembly was prevented, and the distribution of ubiquitin, cyclin B1, and polo-like kinase 1 (Plk1) was also distorted. When UPP was inhibited, mitogen-activated protein kinase (MAPK)/p90rsk phosphorylation was not affected, but the cyclin B1 degradation that occurs during normal metaphase-anaphase transition was not observed. During oocyte activation, the emission of second polar body (PB2) and the pronuclear formation were inhibited by ALLN or MG-132. In oocytes microinjected with ubiquitin antibodies, PB2 emission and pronuclear formation were also inhibited after in vitro fertilization. The expression of cyclin B1 and the phosphorylation of MAPK/p90rsk could still be detected in ALLN or MG-132-treated oocytes even at 8 h after parthenogenetic activation or insemination, which may account for the inhibition of PB2 emission and pronuclear formation. We also for the first time investigated the subcellular localization of ubiquitin protein at different stages of oocyte and early embryo development. Ubiquitin protein was accumulated in the germinal vesicle (GV), the region between the separating homologous chromosomes, the midbody, the pronuclei, and the region between the separating sister chromatids. In conclusion, our results suggest that the UPP plays important roles in oocyte meiosis resumption, spindle assembly, polar body emission, and pronuclear formation, probably by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.     

The subcellular localization of cyclin B2 is required for bipolar spindle formation during Xenopus oocyte maturation

Cyclins B1 and B2 are subtypes of cyclin B, a regulatory subunit of a maturation/M-phase promoting factor, and they are also highly conserved in many vertebrate species. Cyclin B1 is essential for mitosis, whereas cyclin B2 is regarded as dispensable. However, the overexpression of the cyclin B2 N-terminus containing the cytoplasmic retention signal, but not cyclin B1, inhibits bipolar spindle formation in Xenopus oocytes and embryos. Here we show that endogenous cyclin B2 was localized in and around the germinal vesicle. The perinuclear localization of cyclin B2 was perturbed by the overexpression of its N-terminus containing the cytoplasmic retention signal, which resulted in a spindle defect. This spindle defect was rescued by the overexpression of bipolar kinesin Eg5, which is located at the perinuclear region in the proximity of endogenous cyclin B2. These results demonstrate that the proper localization of cyclin B2 is essential for bipolar spindle formation in Xenopus oocytes.     

A link between MAP kinase and p34(cdc2)/cyclin B during oocyte maturation: p90(rsk) phosphorylates and inactivates the p34(cdc2) inhibitory kinase Myt1.

M-phase entry in eukaryotic cells is driven by activation of MPF, a regulatory factor composed of cyclin B and the protein kinase p34(cdc2). In G2-arrested Xenopus oocytes, there is a stock of p34(cdc2)/cyclin B complexes (pre-MPF) which is maintained in an inactive state by p34(cdc2) phosphorylation on Thr14 and Tyr15. This suggests an important role for the p34(cdc2) inhibitory kinase(s) such as Wee1 and Myt1 in regulating the G2-->M transition during oocyte maturation. MAP kinase (MAPK) activation is required for M-phase entry in Xenopus oocytes, but its precise contribution to the activation of pre-MPF is unknown. Here we show that the C-terminal regulatory domain of Myt1 specifically binds to p90(rsk), a protein kinase that can be phosphorylated and activated by MAPK. p90(rsk) in turn phosphorylates the C-terminus of Myt1 and down-regulates its inhibitory activity on p34(cdc2)/cyclin B in vitro. Consistent with these results, Myt1 becomes phosphorylated during oocyte maturation, and activation of the MAPK-p90(rsk) cascade can trigger some Myt1 phosphorylation prior to pre-MPF activation. We found that Myt1 preferentially associates with hyperphosphorylated p90(rsk), and complexes can be detected in immunoprecipitates from mature oocytes. Our results suggest that during oocyte maturation MAPK activates p90(rsk) and that p90(rsk) in turn down-regulates Myt1, leading to the activation of p34(cdc2)/cyclin B.     

Maturation-promoting factor and p34cdc2 kinase during oocyte maturation of the Japanese quail

Maturation-promoting factor and a homolog of fission yeast cdc2+ gene product (p34cdc2) were investigated during the final 24 hr of maturation of quail oocytes. Kinase activity of p34cdc2 in the oocyte germinal disk (GD) increased 15 times at maturation. Two bands, at 32 and 34 kDa, were detected in immature oocytes by immunoblotting of SDS-PAGE with anti-p34cdc2 monoclonal antibody. A new band, which is close to the 32-kDa band but with a slightly faster mobility, appeared during maturation. No p34cdc2 could be detected outside the GD. Microinjection of GD extract from mature oocytes caused maturation of Xenopus oocytes.     

Acquisition of meiotic competence in mouse oocytes: absolute amounts of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent and competent oocytes

M-Phase promoting factor (MPF) is a complex of p34(cdc2) and cyclin B. Results of previous studies in which relative mass amounts of these cell cycle regulators were determined suggested that the accumulation of p34(cdc2), rather than cyclin B, could be a limiting factor in the acquisition of meiotic competence in mouse oocytes. Nevertheless, in the absence of measurements of the absolute amount of these components of MPF, it is possible that the molar amount of p34(cdc2) is in excess to that of cyclin B, i.e., the accumulation of p34(cdc2) is not a limiting factor. We report measurements of the absolute mass of p34(cdc2) and cyclin B1, as well as the two proximal regulators of MPF, namely cdc25C and wee1, in meiotically incompetent and competent mouse oocytes. We find that the numbers of molecules of p34(cdc2), cyclin B1, cdc25C, and wee1 in meiotically incompetent oocytes are 1.4 x 10(6), 11.3 x 10(6), 24.6 x 10(6), 15. 6 x 10(6), respectively, and in meiotically competent oocytes the numbers are 14.3 x 10(6), 95.5 x 10(6), 80.0 x 10(6), 40.1 x 10(6), respectively. Thus, the concentration of cyclin B1 is always in excess to that of p34(cdc2), and this is consistent with the hypothesis that the accumulation of p34(cdc2) plays a role in the acquisition of meiotic competence. Last, the concentration of cdc25C is greater than that of wee1 and the concentration of each is greater than that of p34(cdc2) in both meiotically incompetent and competent oocytes.     

Synthesis and accumulation of p34cdc2 and cyclin B in mouse oocytes during acquisition of competence to resume meiosis

This study tests the hypothesis 033 that growing murine oocytes, which are incompetent to resume meiosis, are deficient in their content of p34^cdc2 and/or cyclin B, the two subunits of maturation promoting factor (MPF). Accumulation of the two MPF components occurred in an asynchronous manner in growing oocytes. Cyclin B content reached maximal levels in oocytes that were not yet competent to undergo germinal vesicle breakdown (GVB), the first obvious morphological manifestation of the resumption of meiosis. Thus, the amount of cyclin B is not the limiting factor rendering these growing oocytes incompetent to undergo GVB. In contrast, synthesis and accumulation of p34^cdc2 increased during the period of oocyte growth in vivo when they became competent to undergo GVB. A similar increase in the amount of p34^cdc2 also occurred in cultured granulosa cell-free oocytes despite the lack of oocyte growth, but these cultured oocytes did not become GVB competent. Thus, the accumulation of p34^cdc2 is probably necessary, but not sufficient, for mouse oocytes to become competent to undergo GVB. This accumulation occurs autonomously in oocytes independently of growth or of the participation of follicular somatic cells. © 1995 Wiley-Liss, Inc.     

In vitro maturation and fertilization of porcine oocytes after a 48 h culture in roscovitine, an inhibitor of p34cdc2/cyclin B kinase

Maintaining oocytes at the germinal vesicle (GV) stage in vitro may permit enhanced acquisition of the developmental competence. The objective of the current study was to evaluate the nuclear and cytoplasmic maturation in vitro of porcine oocytes after pretreatment with S-roscovitine (ROS). Cumulus oocyte complexes (COC) were treated with 50 microM ROS for 48 h and then matured for various lengths of time in a conventional step-wise in vitro maturation (IVM) system by using dibutyryl cyclic AMP. The COC that were matured in the same system for 44 h without pretreatment with ROS were used as the control group. At various periods after the start of IVM, oocytes were assessed for the meiotic stages and subjected to in vitro fertilization (IVF) with fresh spermatozoa. The ROS treatment inhibited GV breakdown of 94.4% oocytes, with the majority arrested at the GV-I stage (67.4%). Maximum maturation rate to the metaphase-II stage after ROS treatment was achieved by 44 h of IVM (92.1%) and no differences were observed with control oocytes (95.0%). Penetration rate was correlated to the maturation rate. The duration of IVM had no effects on polyspermy and male pronuclear (MPN) formation rates at 8 h post insemination (hpi), whereas both rates increased at 22 hpi. Direct comparison with controls assessed at 22 hpi confirmed a lesser MPN formation in ROS-treated oocytes (73.7% compared with 53.6%). Glutathione (GSH) concentrations were less in oocytes treated with ROS than in control oocytes (5 compared with 7.7 pmol/oocyte) as well as blastocyst rate (22.0% compared with 38.1%, respectively). These results demonstrate that cytoplasmic maturation in porcine oocytes pretreated with ROS for 48 h did not equal that of control oocytes in the current IVM system.     

Characterization of polo-like kinase 1 during meiotic maturation of the mouse oocyte

We have characterized plk1 in mouse oocytes during meiotic maturation and after parthenogenetic activation until entry into the first mitotic division. Plk1 protein expression remains unchanged during maturation. However, two different isoforms can be identified by SDS-PAGE. A fast migrating form, present in the germinal vesicle, seems characteristic of interphase. A slower form appears as early as 30 min before germinal vesicle breakdown (GVBD), is maximal at GVBD, and is maintained throughout meiotic maturation. This form gradually disappears after exit from meiosis. The slow form corresponds to a phosphorylation since it disappears after alkaline phosphatase treatment. Plk1 activation, therefore, takes place before GVBD and MAPK activation since plk1 kinase activity correlates with its slow migrating phosphorylated form. However, plk1 phosphorylation is inhibited after treatment with two specific p34(cdc2) inhibitors, roscovitine and butyrolactone, suggesting plk1 involvement in the MPF autoamplification loop. During meiosis plk1 undergoes a cellular redistribution consistent with its putative targets. At the germinal vesicle stage, plk1 is found diffusely distributed in the cytoplasm and enriched in the nucleus and during prometaphase is localized to the spindle poles. At anaphase it relocates to the equatorial plate and is restricted to the postmitotic bridge at telophase. After parthenogenetic activation, plk1 becomes dephosphorylated and its activity drops progressively. Upon entry into the first mitotic M-phase at nuclear envelope breakdown plk1 is phosphorylated and there is an increase in its kinase activity. At the two-cell stage, the fast migrating form with weak kinase activity is present. In this work we show that plk1 is present in mouse oocytes during meiotic maturation and the first mitotic division. The variation of plk1 activity and subcellular localization during this period suggest its implication in the organization and progression of M-phase.     

Localization and function of mSpindly during mouse oocyte meiotic maturation

Spindly was first identified in Drosophila; its homologues are termed SPDL-1 in Caenorhabditis elegans and Hs Spindly/hSpindly in humans. In all species, Spindly and its homologues function by recruiting dynein to kinetochores and silencing SAC in mitosis of somatic cells. Depletion of Spindly causes an extensive metaphase arrest during somatic mitoses in Drosophila, C. elegans and humans. In Drosophila, Spindly is required for shedding of Rod and Mad2 from the kinetochores in metaphase; in C. elegans, SPDL-1 presides over the recruitment of dynein and MDF-1 to the kinetochores; in humans, Hs Spindly is required for recruiting both dynein and dynactin to kinetochores but it is dispensable for removal of checkpoint proteins from kinetochores. The present study was designed to investigate the localization and function of the Spindly homologue (mSpindly) during mouse oocyte meiotic maturation by immunofluorescent analysis, and by overexpression and knockdown of mSpindly. We found that mSpindly was typically localized to kinetochores when chromatin condensed into chromosomes after GVBD. In metaphase of both first meiosis and second meiosis, mSpindly was localized not only to kinetochores but also to the spindle poles. Overexpression of mSpindly did not affect meiotic progression, but its depletion resulted in an arrest of the pro-MI/MI stage, failure of anaphase entry and subsequent polar body emission, and in abnormal spindle morphology and misaligned chromosomes. Our data suggest that mSpindly participates in SAC silencing and in spindle formation as a recruiter and/or a transporter of kinetochore proteins in mouse oocytes, but that it needs to cooperate with other factors to fulfill its function.     

Polo-like kinase-1 is a pivotal regulator of microtubule assembly during mouse oocyte meiotic maturation,fertilization, and early embryonic mitosis

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that have been activated through phosphorylation. The activity of these kinases has been shown to be required for regulating multiple stages of mitotic progression in somatic cells. In this experiment, the changes in Plk1 expression were detected in mouse oocytes through Western blotting. The subcellular localization of Plk1 during oocyte meiotic maturation, fertilization, and early cleavage as well as after antibody microinjection or microtubule assembly disturbance was studied by confocal microscopy. The quantity of Plk1 protein remained stable during meiotic maturation and decreased gradually after fertilization. Plk1 was localized to the spindle poles of both meiotic and mitotic spindles at the early M phase and then translocated to the middle region. At anaphase and telophase, Plk1 was concentrated at the midbody of cytoplasmic cleavages. Plk1 was concentrated between the male and female pronuclei after fertilization. Plk1 disappeared at the spindle region when microtubule formation was inhibited by colchicine or staurosporine, while it was concentrated as several dots in the cytoplasm after taxol treatment. Plk1 antibody injection decreased the germinal vesicle breakdown rate and distorted MI spindle organization. Our results indicate that Plk1 is a pivotal regulator of microtubule organization during mouse oocyte meiosis, fertilization, and cleavage and that its functions may be regulated by other kinases, such as staurosporine-sensitive kinases.     

Relocation and distinct subcellular localization of p34cdc2-cyclin B complex at meiosis reinitiation in starfish oocytes.

M phase promoting factor (MPF) is a major element controlling entry into the M phase of the eukaryotic cell cycle. MPF is composed of two subunits, p34cdc2 and cyclin B. Using indirect immunofluorescence staining with specific antibody against starfish cyclin B, we monitored the dynamics of the subcellular distribution of MPF during meiosis reinitiation in starfish oocytes. We found that all of the cyclin B is already associated with p34cdc2 in immature oocytes arrested at the G2/M border and that this inactive complex is present exclusively in the cytoplasm. After its activation, part of the p34cdc2-cyclin B complex moves into the germinal vesicle before nuclear envelope breakdown, independently of either microtubules or actin filaments. Thereafter, some part of the complex accumulates in the nucleolus and condensed chromosomes. Another portion of the complex accumulates on meiotic asters and spindles, while the rest is still present throughout the cytoplasm. As these patterns of localization are detected in the detergent-extracted oocytes, we propose at least four distinct subcellular states of the p34cdc2-cyclin B complex: freely soluble, microtubule-associated, detergent-resistant cytoskeleton-associated and chromosome-associated. Thus, in addition to the intramolecular modification of p34cdc2-cyclin B complex, its intracellular relocation plays a key role in promoting the M phase.     

雷公藤多甙对小鼠卵母细胞成熟和体外受精的影响

采用超排卵技术研究雷公藤多甙（GTW）对小鼠卵母细胞的成熟和体外受精以及脏器等的影响,GTW对小鼠卵母细胞生发泡破裂没有影响,但可以抑制卵母细胞第一极体的释放,影响卵母细胞的存活率并可降低体外受精率和超排卵的卵母细胞数量。GTW可以破坏卵母细胞成熟,降低卵母细胞的体外受精能力,影响小鼠的正常生殖功能。     

Nuf2 is required for chromosome segregation during mouse oocyte meiotic maturation

Nuf2 plays an important role in kinetochore-microtubule attachment and thus is involved in regulation of the spindle assembly checkpoint in mitosis. In this study, we examined the localization and function of Nuf2 during mouse oocyte meiotic maturation. Myc₆-Nuf2 mRNA injection and immunofluorescent staining showed that Nuf2 localized to kinetochores from germinal vesicle breakdown to metaphase I stages, while it disappeared from the kinetochores at the anaphase I stage, but relocated to kinetochores at the MII stage. Overexpression of Nuf2 caused defective spindles, misaligned chromosomes, and activated spindle assembly checkpoint, and thus inhibited chromosome segregation and metaphase-anaphase transition in oocyte meiosis. Conversely, precocious polar body extrusion was observed in the presence of misaligned chromosomes and abnormal spindle formation in Nuf2 knock-down oocytes, causing aneuploidy. Our data suggest that Nuf2 is a critical regulator of meiotic cell cycle progression in mammalian oocytes.     

Activation of p34cdc2 kinase by cyclin A

Functional clam cyclin A and B proteins have been produced using a baculovirus expression system. Both cyclin A and B can induce meiosis I and meiosis II in Xenopus in the absence of protein synthesis. Half-maximal induction occurs at 50 nM for cyclin A and 250 nM for cyclin B. Addition of 25 nM cyclin A to activated Xenopus egg extracts arrested in the cell cycle by treatment with RNase or emetine activates cdc2 kinase to the normal metaphase level and stimulates one oscillatory cell cycle. High levels of cyclin A cause marked hyperactivation of cdc2 kinase and a stable arrest at the metaphase point in the cell cycle. Kinetic studies demonstrate the concentration of cyclin A added does not affect the 10 min lag period required for kinase activation or the timing of maximal activity, but does control the rate of deactivation of cdc2 kinase during exit from mitosis. In addition, exogenous clam cyclin A inhibits the degradation of both A- and B-type endogenous Xenopus cyclins. These results define a system for investigating the biochemistry and regulation of cdc2 kinase activation by cyclin A.     

Subcellular localization of nardilysin during mouse oocyte maturation

We have previously shown that the peptidase, nardilysin, contains a bipartite nuclear localization signal that permits the enzyme to cycle between the nucleus and cytoplasm. In the present study, we report that nardilysin accumulates in the nucleus of an oocyte as a function of its maturation. Nardilysin is predominantly localized in the cytoplasm of an oocyte when initially placed into culture. The enzyme starts to accumulate in the nucleus within 30 min of in vitro culture. After 3 h, nardilysin is found as a spherical structure surrounded by condensed chromosomal DNA. After 18 h of in vitro culture, it co-localizes with beta-tubulin at the spindle apparatus. Cilostamide, a phosphodiesterase 3A inhibitor that inhibits meiosis, blocks accumulation of nuclear nardilysin. This finding demonstrates that the nuclear entry of nardilysin is tightly controlled in the oocyte. Taken together, these experiments strongly suggest a role for nardilysin in meiosis through its dynamic translocation from cytosol to nucleus, and then to the spindle apparatus.     

A specific inhibitor of p34(cdc2)/cyclin B suppresses fertilization-induced calcium oscillations in mouse eggs

Fertilization-induced Ca(2+) oscillations in mouse eggs cease at the time of pronuclear formation when maturation-promoting factor (MPF) is inactivated, but the Ca(2+) oscillations are ceaseless if eggs are arrested at metaphase by colcemid, which maintains the activity of MPF. To determine the possible role of MPF in regulation of cytoplasmic Ca(2+) excitability, roscovitine, a specific inhibitor of p34(cdc2)/cyclin B kinase, was used to inactivate MPF, and its effect on fertilization-induced Ca(2+) oscillations was investigated. Our results showed that roscovitine at >/= 50 microM suppressed fertilization-induced Ca(2+) oscillations in normal and colcemid-treated metaphase II (MII) eggs after the first 1-2 Ca(2+) spikes. Roscovitine inhibition of fertilization-induced Ca(2+) oscillations could be reversed by extensive washing of the eggs. Histone H1 kinase activity in colcemid-treated MII eggs was similarly inhibited by roscovitine, which suggested that the cessation of fertilization-induced Ca(2+) oscillations is due to the inactivation of MPF. Thimerosal-induced Ca(2+) oscillations in Ca(2+)-, Mg(2+)-free medium was also suppressed by roscovitine, suggesting a general inhibitory effect of roscovitine on Ca(2+) oscillations. The inhibition may be achieved by disruption of Ca(2+) release and refilling of the calcium store. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca-ATPase, induced significantly less Ca(2+) release in roscovitine-treated eggs than in the non-drug-treated eggs. Taken together, our results suggest that MPF plays an important role in regulation of the cytoplasmic Ca(2+) excitability in mouse eggs.     

Polo-like kinase-1 in porcine oocyte meiotic maturation, fertilization and early embryonic mitosis.

Polo-like kinases (Plks) are a family of serine/threonine protein kinases that regulate multiple stages of mitosis. Expression and distribution of polo-like kinase 1 (Plk1) were characterized during porcine oocyte maturation, fertilization and early embryo development in vitro, as well as after microtubule polymerization modulation. The quantity of Plk1 protein remained stable during meiotic maturation. Plk1 accumulated in the germinal vesicles (GV) in GV stage oocytes. After germinal vesicle breakdown (GVBD), Plk1 was localized to the spindle poles at metaphase I (MI) stage, and then translocated to the middle region of the spindle at anaphase-telophase I. Plk1 was also localized in MII spindle poles and on the spindle fibers and on the middle region of anaphase-telophase II spindles. Plk1 was not found in the spindle region when colchicine was used to inhibit microtubule organization, while it accumulated as several dots in the cytoplasm after taxol treatment. After fertilization, Plk1 concentrated around the female and male pronuclei. During early embryo development, Plk1 was found to be in association with the mitotic spindle at metaphase, but distributed diffusely in the cytoplasm at interphase. Our results suggest that Plk1 is a pivotal regulator of microtubule organization and cytokinesis during porcine oocyte meiotic maturation, fertilization, and early embryo cleavage in pig oocytes.