钙离子信号对非细胞体系中小鼠卵母细胞游离生发泡减数分裂启动的影响

本文构建了包括HeLa裂解液和游离小鼠卵母细胞生发泡的实验体系,用于研究Ca2+及其下游信号对小鼠卵母细胞减数分裂启动的影响.游离的卵母细胞生发泡可以在M期细胞裂解液中发生减数分裂启动,表现为染色质的凝集.进一步的研究表明,Ca2+信号的存在对G2期细胞裂解液促进减数分裂启动是至关重要的,G2期中期的细胞裂解液只有经Ca2+诱导后才具有启动生发泡减数分裂的作用,而G2期晚期无论Ca2+存在与否均诱发减数分裂的启动,但是G2期早期的裂解液元启动减数分裂的作用.卵母细胞的体外培养实验分析也表明,抑制CaM和CaMKII的活性可以阻止GVBD和抑制第一极体的释放.免疫沉淀及Western Blotting结果显示,HeLa细胞裂解液中的MPF从G2期中期到M期均存在,且Cdc2亚基的Tyr由磷酸化向去磷酸化转变.结果进一步证明,卵母细胞减数分裂的启动可能是通过一种Ca2+/CaM依赖的途径来推动的.     

核糖体S6蛋白激酶p90rsk与卵母细胞减数分裂

丝裂原活化蛋白激酶(MAPK)信号途径对减数分裂有重要调节作用,p90rsk是迄今研究最清楚的MAPK下游靶分子,介导MAPK途径在卵母细胞减数分裂中的多种功能,包括卵母细胞减数分裂的启动、MⅠ/MⅡ期转化和MⅡ期阻滞的维持等.p90rsk的磷酸化是MAPK激活的结果,而细胞退出减数分裂时,p90rsk的去磷酸化也发生在MAPK失活以后.介绍了在卵母细胞中p90rsk的研究进展.     

核糖体S6蛋白激酶p90rsk与卵母细胞减数分裂

丝裂原活化蛋白激酶（MAPK）信号途径对减数分裂有重要调节作用,p90^rsk是迄今研究最清楚的MAPK下游靶分子,介导MAPK途径在卵母细胞减数分裂中的多种功能,包括卵母细胞减数分裂的启动、MⅠ/MⅡ期转化和MⅡ期阻滞的维持等.p90^rsk的磷酸化是MAPK激活的结果,而细胞退出减数分裂时,p90^rsk的去磷酸化也发生在MAPK失活以后.介绍了在卵母细胞中p90^rsk的研究进展.     

山羊卵母细胞的减数分裂进程

本实验以随机屠宰山羊的卵巢为实验材料,研究了不同直径卵泡卵母细胞的减数分裂进程。结果显示,不同直径卵泡卵母细胞在体外成熟培养条件下的减数分裂能力不同:≤0.5mm直径卵泡的卵母细胞不能恢复减数分裂;0.8-1.2mm卵泡的卵母细胞可恢复减数分裂,但只能发育到MⅠ期,培养24h发育到MⅠ期比率60%;1.5-5.0mm卵泡卵母细胞已经完全获得减数分裂能力,培养24h发育到MⅡ的比例91%。完全获得减数分裂能力的1.5-5.0mm卵泡卵母细胞处于生发泡(GV)期的比率在成熟培养2-8h期间明显下降;其中,4-6h期间GⅤ比率下降最为迅速(由61%降低到19%,p<0.0005);体外培养6-12h期间MⅠ比率由25%上升到60%,随后下降,到24h仅有2%卵母细胞处于MⅠ期;培养16h有21%卵母细胞进入MⅡ期,24h 91%卵母细胞到达MⅡ期。对卵母细胞体外核成熟进程的数据做折线图计算结果表明,1.5-5.0mm卵泡卵母细胞减数分裂进程(各细胞周期事件出现和维持的时间)为:0-3.0h为GⅤ期,3.0-7.0h为前中期Ⅰ,7.0-14.6h为MⅠ期,14.6-18.4h处于后期-Ⅰ和末期-Ⅰ,18.4-24h为MⅡ期。本实验还证明,部分获得减数分裂能力(0.8-1.2mm卵泡)与完全获得减数分裂能力(1.5-5mm卵泡)的卵母细胞,其各细胞周期事件一旦发生,所需的时间是相同的。这些结果为进一步研究山羊卵母细胞减数分裂机制及其调控提供了重要的基础数据。     

小鼠Cdc25B核输出序列

为探讨小鼠卵母细胞中Cdc25B(cell division cycle 25 homolog B)核输出序列在卵母细胞G2/M转换过程中的调控机制,应用显微注射方法将Cdc25B的野生型、N末端缺失1～51位氨基酸片段(Cdc25B-Δ51)、1～65位氨基酸片段(Cdc25B-Δ65)突变体的mRNA和pEGFP-Cdc25B-WT、pEGFP-Cdc25B-Δ51、pEGFP-Cdc25B-Δ65的融合质粒显微注射到含有完整生发泡的小鼠卵母细胞中,观察不同注射组小鼠卵母细胞发生生发泡破裂的情况及蛋白质亚细胞定位。结果显示Cdc25B-Δ51及Cdc25B-Δ65都丧失了诱导小鼠卵母细胞减数分裂的能力;同时亚细胞定位研究表明在G2期野生型Cdc25B主要分布在细胞浆中,Cdc25B-Δ51在核浆均有分布,Cdc25B-Δ65则主要分布于细胞核中。研究结果表明Cdc25B在52～65位氨基酸之间存在核输出序列(nuclear export sequence,NES),NES参与的核转运机制作为一种重要的调控机制控制着细胞的生理进程;N末端的氨基酸对减数分裂的重启动起促进作用。     

Cdc25B在小鼠受精卵的表达和定位

为阐明细胞分裂周期(Cdc)25B调控小鼠受精卵发育的机制,利用Western印迹检测小鼠受精卵各时期Cdc25B的表达及Cdc2-Tyr15的磷酸化状态。利用间接免疫荧光技术观察Cdc25B在小鼠受精卵的定位。构建pEGFP-Cdc25B融合表达载体并显微注射到受精卵中,观察Cdc25B在受精卵M期的定位变化。结果表明Cdc25B在G1和S期被磷酸化,在G2和M期去磷酸化。Cdc2-Tyr15在G1和S期处于磷酸化状态,G2期只检测到Cdc2-Tyr15轻微的磷酸化信号,M期未检测到任何Cdc2-Tyr15的磷酸化信号。Cdc25B在G1期定位于细胞质和细胞核中,S和G2期定位于细胞质的皮质部分,M期由细胞质转向核区。证明Cdc25B核输出后激活有丝分裂促进因子,从而启动小鼠受精卵的有丝分裂。     

腺嘌呤对体外小鼠卵母细胞减数分裂的抑制

本文研究了嘌呤类物质对小鼠卵母细胞减数分裂的影响,于卵母细胞的生发泡内显微注射腺嘌呤和腺嘌呤的类似物苄基腺嘌呤可显著抑制卵母细胞的分裂的重新启动。同时发现在腺嘌呤的作用过程中,腺苷酸环化酶的激活前氟化钠,可增强其对卵母细胞的抑制作用,表明ｃＡＭＰ途径在小鼠卵母细胞的抑制作用,腺嘌呤在不同培养液中的抑制效果不一,次黄嘌呤在ＤＭＥＭ和ＥＭＥＭ中对小鼠的卵丘细胞－卵母细胞复合体和无卵丘细胞的卵丘细胞－卵     

THE MECHANISM OF SPERM HEAD TRANSFORMATION DURING MOUSE OOCYTE FERTILIZATION IN VITRO

用Ｈｏｅｃｈｓｔ３３３４２标记及氨银反应的方法在光镜和电镜水平上研究不同发育阶段的小鼠卵母细胞转化精核的能力。生发泡期卵母细胞质不能诱发精子组蛋白替代鱼精蛋白及精核解聚。生发泡破裂后,卵母细胞获得使精核解聚的能力,但直到卵母细胞完成成熟前,雄原核均不能形成。进一步研究表明,卵母细胞成熟过程中持续的蛋白质合成是雄原核形成所必需的,鱼精蛋白质磷酸化是精核解聚中的关键步骤。     

Skp2参与HeLa细胞G_2/M周期检查点的调控

具癌基因特性的Skp2在大多数肿瘤组织和肿瘤细胞中异常高表达,它作为SCFSkp2复合物的底物识别亚基调控p27KIP蛋白的稳定性而促进细胞G1/S期转换.为进一步明确Skp2与G2/M周期检查点的关系,在HeLa细胞中过表达Skp2以及通过反义寡核苷酸抑制Skp2表达.结果发现:Skp2能促进细胞周期运转,表现为S期细胞增多和G2/M期细胞减少,其中F-box结构域具有重要的功能意义;反义寡核苷酸抑制Skp2表达后,HeLa细胞发生显著的G2/M期阻滞;MTT检测结果表明,400nmol/L的Skp2的反义寡核苷酸能明显抑制HeLa细胞的增殖活性;Western印迹结果表明,HeLa细胞中Skp2可能通过负调控p21WAF的稳定性来参与G2/M检查点调控,这在用放线菌素D处理HeLa细胞的实验中得到验证.这些结果初步揭示了Skp2参与HeLa细胞G2/M周期检查点调控的分子机制.     

不同年龄昆明白小鼠卵母细胞的生发泡互换

为探讨卵母细胞减数分裂异常及其与年龄相关变化之间的关系,对不同年龄段昆明白小鼠卵母细胞进行了生发泡(GV)移植研究。应用显微操作和电融合技术,将6~8周龄小鼠GV期卵母细胞分别与6月龄9、月龄和12月龄小鼠GV期卵母细胞进行GV互换,所形成的6种GV-胞质体复合体的融合率(89.7%~95.6%)和6种重组卵母细胞的成熟率(83.5%~88.2%)并不因小鼠年龄的改变而有所变化。成熟的6种重组卵母细胞经体外受精后,形成原核期胚和2-细胞期胚的比率(分别为80.0%~87.3%和42.7%~50.9%)并不因不同年龄小鼠卵母细胞GV互换所带来的细胞质或细胞核的改变而受到影响。     

Involvement of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes

Calcium signal is important for the regulation of meiotic cell cycle in oocytes, but its downstream mechanism is not well known. The functional roles of calcium/calmodulin-dependent protein kinase II (CaMKII) in meiotic maturation and activation of pig oocytes were studied by drug treatment, Western blot analysis, kinase activity assay, indirect immunostaining, and confocal microscopy. The results indicated that meiotic resumption of both cumulus-enclosed and denuded oocytes was prevented by CaMKII inhibitor KN-93, Ant-AIP-II, or CaM antagonist W7 in a dose-dependent manner, but only germinal vesicle breakdown (GVBD) of denuded oocytes was inhibited by membrane permeable Ca2+ chelator BAPTA-AM. When the oocytes were treated with KN-93, W7, or BAPTA-AM after GVBD, the first polar body emission was inhibited. A quick elevation of CaMKII activity was detected after electrical activation of mature pig oocytes, which could be prevented by the pretreatment of CaMKII inhibitors. Treatment of oocytes with KN-93 or W7 resulted in the inhibition of pronuclear formation. The possible regulation of CaMKII on maturation promoting factor (MPF), mitogen-activated protein kinase (MAPK), and ribosome S6 protein kinase (p90rsk) during meiotic cell cycles of pig oocytes was also studied. KN-93 and W7 prevented the accumulation of cyclin B and the full phosphorylation of MAPK and p90rsk during meiotic maturation. When CaMKII activity was inhibited during parthenogenetic activation, cyclin B, the regulatory subunit of MPF, failed to be degraded, but MAPK and p90rsk were quickly dephosphorylated and degraded. Confocal microscopy revealed that CaM and CaMKII were localized to the nucleus and the periphery of the GV stage oocytes. Both proteins were concentrated to the condensed chromosomes after GVBD. In oocytes at the meiotic metaphase MI or MII stage, CaM distributed on the whole spindle, but CaMKII was localized only on the spindle poles. After transition into anaphase, both proteins were translocated to the area between separating chromosomes. All these results suggest that CaMKII is a multifunctional regulator of meiotic cell cycle and spindle assembly and that it may exert its effect via regulation of MPF and MAPK/p90rsk activity during the meiotic maturation and activation of pig oocytes.     

Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro

We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.     

Evidence that multifunctional calcium/calmodulin-dependent protein kinase II (CaM KII) participates in the meiotic maturation of mouse oocytes

Calcium-dependent signaling pathways are thought to be involved in the regulation of mammalian oocyte meiotic maturation. However, the molecular linkages between the calcium signal and the processes driving meiotic maturation are not clearly defined. The present study was conducted to test the hypothesis that the multi-functional calcium/calmodulin-dependent protein kinase II (CaM KII) functions as one of these key linkers. Mouse oocytes were treated with a pharmacological CaM KII inhibitor, KN-93, or a peptide CaM KII inhibitor, myristoylated AIP, and assessed for the progression of meiosis. Two systems for in vitro oocyte maturation were used: (1) spontaneous gonadotropin-independent maturation and (2) follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest. FSH-induced, but not spontaneous germinal vesicle breakdown (GVB) was dose-dependently inhibited by both myristoylated AIP and KN-93, but not its inactive analog, KN-92. However, emission of the first polar body (PB1) was inhibited by myristoylated AIP and KN-93 in both oocyte maturation systems. Oocytes that failed to produce PB1 exhibited normal-appearing metaphase I chromosome congression and spindles indicating that CaM KII inhibitors blocked the metaphase I to anaphase I transition. Similar results were obtained when the oocytes were treated with a calmodulin antagonist, W-7, and matured spontaneously. These results suggest that CaM KII, and hence the calcium signaling pathway, is potentially involved in regulating the meiotic maturation of mouse oocytes. This kinase both participates in gonadotropin-induced resumption of meiosis, as well as promoting the metaphase I to anaphase I transition. Further evidence is therefore, provided of the critical role of calcium-dependent pathways in mammalian oocyte maturation.     

Calcium and meiotic maturation of the mammalian oocyte

The role of calcium in the regulation of both the meiotic and mitotic cell cycles has been the subject of considerable investigation in the nonmammalian field. In contrast, the mechanisms for signalling meiotic maturation in the mammalian oocyte are not as well documented nor as clearly defined. In the mammalian oocyte, calcium is associated with both spontaneous and hormone-induced meiotic maturation. A transient release of endogenously stored calcium precedes germinal vesicle breakdown and can override cyclic AMP maintained meiotic arrest; it thus may signal the resumption of meiosis. Additionally, extracellular calcium is apparently required for meiotic progression past metaphase I. The time sequence for meiotic resumption and progression is very varied between species. The timing of cell cycle protein synthesis during meiosis suggests that cyclins may be expressed in oocytes of some species much earlier in their development than in others. A generic model is proposed for the mechanism for triggering meiotic resumption in the mammalian oocyte. In this model, the critical components of meiotic resumption involve the temporal relationship of cyclin synthesis and the subsequent activation of the MPF complex by the calcium signal generated, which accounts for differences among species. © 1995 Wiley-Liss, Inc.     

Several signaling pathways are involved in the control of cattle oocyte maturation

The main limit of in vitro production of domestic mammal embryos comes from the low capacity of in vitro matured oocytes to develop after fertilization. As soon as they are separated from follicular environment, oocytes spontaneously resume meiosis without completion of their terminal differentiation. Roscovitine (ROS), an inhibitor of M-phase promoting factor (MPF) kinase activity reversibly blocks the meiotic resumption in vitro. However, in cattle maturing oocytes several cellular events such as protein synthesis and phosphorylation, chromatin condensation and nuclear envelope folding escape ROS inhibition suggesting the alternative pathways in oocyte maturation. We compared the level of synthesis and phosphorylation of several protein kinases during bovine cumulus oocyte complex (COC) maturation in vitro in the presence or not of epidermal growth factor (EGF) and ROS. We showed that during the EGF-stimulated maturation, ROS neither affected the decrease of EGF receptor (EGFR) nor did inhibit totally its phosphorylation in cumulus cells and also did not totally eliminate tyrosine phosphorylation in oocytes. However, ROS did inhibit the Phosphoinositide 3-kinase (PI3) activity when oocytes mature without EGF. Accumulation of Akt/PKB (protein kinase B), JNK1/2 (jun N-terminal kinases) and Aurora-A in oocytes during maturation was not affected by ROS. However, the phosphorylation of Akt but not JNKs was diminished in ROS-treated oocytes. Thus, PI3 kinase/Akt, JNK1/2 and Aurora-A are likely to be involved in the regulation of bovine oocyte maturation and some of these pathways seem to be independent to MPF activity and meiotic resumption. This complex regulation may explain the partial meiotic arrest of ROS-treated oocytes and the accelerated maturation observed after such treatment.     

Regulation of amphibian oocyte maturation

Xenopus oocyte maturation is a model system for studying the control of cell proliferation and the regulation of the cell cycle. Addition of progesterone or insulin to oocytes releases a G2 block and stimulates progression through meiosis to an unfertilized egg. The release of the G2 block is a consequence of a decrease in cAMP mediated entirely or in part by an inhibition of adenylate cyclase. The mechanism of cyclase inhibition involves a membrane steroid receptor controlling the rate of guanine nucleotide exchange. Subsequent events include an increase in intracellular pH and the phosphorylation of ribosomal protein S6. The latter event may play a role in translational control of maturation. Late events in maturation involve the appearance of the maturation-promoting factor (MPF), a cytoplasmic protein responsible for causing nuclear envelope breakdown, chromosome condensation, and spindle formation. MPF oscillates in meiotic and mitotic cell cycles. The events caused by MPF can now be obtained in crude extracts with retention of cell cycle control by calcium, providing a framework for rapid progress in characterizing MPF and its regulation.     

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.     

Nuclear responses to MPF activation and inactivation in Xenopus oocytes and early embryos

The cell cycle of most organisms is highlighted by characteristic changes in the appearance and activity of the nucleus. Structural changes in the nucleus are particularly evident when a cell begins to divide. At this time, the nuclear envelope is disassembled, the chromatin condenses into metaphase chromosomes, and the chromosomes associate with a newly formed spindle. Upon completion of cell division the nuclear envelope reassembles around the chromosomes as they form telophase nuclei, and subsequently interphase nuclei, in the daughter cells. The cytoplasmic control of nuclear behavior has been the theme of Yoshio Masui's research for much of his career. His pioneering demonstration that the cytoplasm of maturing amphibian oocytes causes the resumption of the meiotic cell cycle when it is injected into an immature oocyte provided unequivocal evidence that a cytoplasmic factor could initiate the transition from interphase to metaphase (M-phase) in intact cells. As described in several reviews in this and the previous issue of Biology of the Cell (see Beckhelling and Ford; Duesbery and Vande Woude; Maller), Masui initially called this activity maturation promoting factor (MPF), but when it was realized that it was a ubiquitous regulator of both mitotic and meiotic cell cycles, MPF came to stand for M-phase promoting factor. Biochemical evidence indicates that MPF activity is composed of a mitotic B-type cyclins and cyclin-dependent kinase 1. The increase in the protein kinase activity of cdk1 initiates the changes in the nucleus associated with oocyte maturation and with the entry into mitosis. This article will attempt to provide a brief summary of the responses of the nucleus to the activation of MPF. In addition, the effect of MPF inactivation on nuclear envelope assembly at the end of mitosis will be discussed. This article is written as a tribute to Yoshio Masui on his retirement from the University of Toronto, and as an expression of gratitude for his guidance while I was a student in his laboratory. I have felt very privileged to have known him as a mentor and a friend.