细胞周期和卵母细胞成熟调控机制研究的新进展

随着分子生物学和细胞生物学的飞速发 展,人们从单纯追求生物大分子的化学和物理 结构的研究,逐步转移到在分子水平上洞察和 认识生命活动的基本规律,自然也就包括了细 胞周期的调控问题。 自从细胞的分裂图象第一次在显微镜下被 观察到之后,关于细胞周期以及它与分化和发 育等的关系,就一直引起细胞生物学家和发育 生物学家的注意。按形态,细胞周期被划分为     

卵母细胞脂肪酸及其对卵母细胞成熟和发育的影响

脂肪酸是一类具有重要生物学功能的营养物质,它们影响机体脂类的代谢、基因表达及细胞膜功能。而且,许多研究揭示,日粮脂肪酸浓度、种类及比例影响动物卵母细胞的质量。目前,利用代谢组学预测卵母细胞质量成为繁殖生物学研究的热点,但主要集中在葡萄糖、氨基酸代谢研究。动物卵母细胞含有大量的内源脂肪酸,但目前关于卵母细胞脂肪酸的代谢作用研究较少,而且脂肪酸还作为信号分子调控基因表达,同时也是生物膜的重要组成成分。基于此,该文综述了卵母细胞脂肪酸主要种类及来源、脂肪酸的生理作用及对卵母细胞成熟和发育的影响,为未来科学合理地调控卵母细胞脂肪酸组成和利用、促进卵母细胞成熟和发育,进而提高动物繁殖能力提供理论基础。     

卵母细胞成熟调控的分子机制及进展

卵母细胞的成熟是人类配子发育成熟,进而形成胚胎的必然阶段。目前已知有多种因素调控卵母细胞的成熟。成熟促进因子(MPF)是卵母细胞成熟调控的最重要分子,它通过CDK1亚基的磷酸化及cyclin B累积合成调节卵母细胞的成熟。MAPK/Mos及cAMP均可通过影响MPF的活性从而间接调控卵母细胞成熟。这三者之间又存在相互影响相互作用,形成一个复杂的调控网络。阐明卵母细胞成熟的分子机制有利于为治疗女性不孕症及卵母细胞体外培养成熟提供可靠的理论依据。     

卵母细胞成熟过程中信号转导及调控研究进展

哺服动物卵母细胞成熟机制一直是生殖生物学研究的热点,目前仍有许多环节尚不十分清楚。本文对卵母细胞成熟过程中复杂的信号转导通路及蛋白质合成过程中的最新进展进行了综述。     

卵母细胞成熟发生机制的研究进展

卵母细胞体外成熟培养已成为现代胚胎生物技术的重要内容之一,是体外受精、核移植等生物技术的重要环节。卵母细胞体外成熟受到众多因素的调控,其调控机制十分复杂。本文主要针对卵母细胞成熟过程中卵母细胞胞质成熟、核成熟及其主要调控因子等方面的发生发展机制进行总结。     

原癌基因c-erbB2和c-myb经丝裂原活化蛋白激酶和成熟促进因子途径调节卵母细胞的成熟

本研究探讨了原癌基因c-erbB:和c-myb对小鼠卵母细胞成熟的影响及其在调控卵母细胞成熟中与丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和成熟促进因子(mamration promoting factor,MPF)的上下游关系.c-erbB2反义寡脱氧核苷酸(antisense oligodeoxynucleotide,ASODN)和c.myb ASODN均呈剂量依赖方式抑制卵母细胞的生发泡破裂(germinalvesicle breakdown,GVBD)率和第一极体(first polar body,PBl)排放率,并显著延迟其成熟时间.小鼠卵母细胞显微注射重组人c-erbB2蛋白和c-myb蛋白后,培养6 h其GVBD率分别比对照组上升了23.1%(P<0.05)和32.2%(P<0.05),.培养12 h其PBl排放率分别比对照组上升了17.3%(P<0.05)和23.5%(P<0.05).RT-PCR结果显示,小鼠卵母细胞中存在c-erbB2mRNA和c-myb mRNA表达;c-erbB2ASODN能明显抑制卵母细胞中c-erbB2mRNA和c-myb mRNA的表达,c-myb ASODN能明显抑制卵母细胞中c-myb mRNA的表达,对c-erbB2 mRNA无明显影响;MAPK抑制剂PD98059以及MPF抑制剂roscovitine在抑制卵母细胞成熟的同时,均能阻断显微注射重组人c-erbB:蛋白和重组人c-myb蛋白对卵母细胞成熟的促进作用,但对卵母细胞中c-erbB2mRNA和c-myb mRNA表达无明显影响.Western blot结果显示,c-erbB2ASODN、c-mybASODN、PD98059、roscovitine均使卵母细胞中MAPK磷酸化水平和cyclinB 1含量下降.结果提示,原癌基因c-erbB2、c-myb在卵母细胞成熟中起重要作用,可能是调控卵母细胞成熟中关键蛋白激酶如MAPK、MPF的上游激活物.     

灵长类卵母细胞体外发育潜能的调控

灵长类卵母细胞细胞质成熟调控研究较为滞后,使得体外成熟的灵长类卵母细胞的胚胎发育潜能十发低下。提高其潜能对治疗人类不育症有重要的应用价值,并可推动灵长类胚胎发育的研究。基于猕猴卵母细胞质成熟调控研究,讨论了无血清成熟培养基中雌激素和孕激素、能量物质、氨基酸对卵母细胞质成熟的影响,以及动物年龄和生殖周期与发育潜能的关系。从分子水平进一步解释这些因素的作用,阐明卵母细胞质成熟的机理将是今后工作的方向。     

成熟促进因子对克隆重构胚核重编程的调控

成熟促进因子(maturation promoting factor,MPF)由催化亚单位P34cdc2和调节亚单位cyclin组成,对细胞周期的调控起着重要作用。目前,在核移植研究中发现：供体核在MPF的作用下发生核膜破裂(nuclear envelop breakdown．NEBD)和早熟染色体凝集(premature chromosome condensation,PCC),促进了核、质蛋白质因子的交换,有利于核重编程的进行。PCC还会对供体核的倍性及形态产生影响。     

绵羊体外成熟卵母细胞的电激活及其在体外孤雌发育的研究

首先对绵羊卵母细胞收集及其体外成熟（ＩＶＭ）条件进行了摸索,然后对影响电刺激激活ＩＶＭ卵母细胞的因素进行了研究,观察激活后卵母细胞在体外的发育能力。结果表明,剥离法比注射器吸卵法所获得的卵母细胞成熟率高,激活更加正常。剥离法收集的卵母细胞体外成熟培养２７小时后,以含０．１ｍｍｏｌ／Ｌ ＣａＣｌ２、０．１ｍｍｏｌ／Ｌ ＭｇＳＯ４和１０ｍｍｏｌ／Ｌ组氨酸的０．２８ｍｏｌ／Ｌ肌醇（ｉｎｏｓｉｔｏｌ）作基     

SIRT1功能及其对卵泡发育和卵母细胞成熟的调控作用 *

沉默信息调节因子1(silent information regulator1, SIRT1)是NAD⁺ 依赖的去乙酰化酶,通过使底物发生去乙酰化而参与细胞众多生理功能的调节,在糖脂代谢、衰老、细胞凋亡、氧化应激等过程中发挥了重要作用。另外,众多研究表明,SIRT1是调控动物卵巢老化、卵泡发育和卵母细胞成熟的重要因子,SIRT1 表达下降或活性改变将导致卵母细胞老化,降低动物的繁殖力。为了充分理解SIRT1功能,并通过调控SIRT1活性而延缓卵巢和卵母细胞老化,从而提高动物繁殖力,简述了SIRT1的激活及其参与细胞内调控的生物过程,并从能量代谢、抗氧化胁迫、染色质重塑的角度讨论了SIRT1的主要功能,重点阐述了SIRT1对动物卵泡发育和卵母细胞成熟的调控作用。     

Oocyte nucleus controls progression through meiotic maturation

We analyzed progression through the meiotic maturation in oocytes manipulated to replace the prophase oocyte nucleus with the nucleus from a cumulus cell, a pachytene spermatocyte or the pronucleus from a fertilized egg. Removal of the oocyte nucleus led to a significant reduction in histone H1 kinase activity. Replacement of the oocyte nucleus by a pronucleus followed by culture resulted in premature pseudomeiotic division and occasional abnormal cytokinesis; however, histone H1 kinase activity was rescued, microtubules formed a bipolar spindle, and chromosomes were condensed. In addition to the anomalies observed after pronuclear transfer, those after transfer of the nucleus from a cumulus cell or spermatocyte included a dramatically impaired ability to form the bipolar spindle or to condense chromosomes, and histone H1 kinase activity was not rescued. Expression of a cyclin B-YFP in enucleated oocytes receiving the cumulus cell nucleus rescued histone H1 kinase activity, but spindle formation and chromosome condensation remained impaired, indicating a pleiotropic effect of oocyte nucleus removal. However, when the cumulus cell nucleus was first transformed into pronuclei (transfer into a metaphase II oocyte followed by activation), such pronuclei supported maturation after transfer into the oocyte in a manner similar to that of normal pronuclei. These results show that the oocyte nucleus contains specific components required for the control of progression through the meiotic maturation and that some of these components are also present in pronuclei.     

哺乳动物卵母细胞凋亡的研究进展

细胞凋亡是发育过程中的基本生命现象,除各种体细胞凋亡外,生殖细胞的发生过程中也发生细胞凋亡。就雌性生殖系而言,细胞凋亡是其发育过程中的一个重要组成部分。在哺乳动物中,超过99．9％的雌性生殖细胞都会在卵子发生的不同阶段发生凋亡。有三种学说解释这一现象：1)被忽视死亡;2)因缺陷死亡;3)自我牺牲死亡。本文主要综述了哺乳动物卵母细胞凋亡的现象、卵母细胞凋亡学说、线粒体遗传与卵母细胞凋亡的关系以及凋亡的分子机理,同时还探讨了卵母细胞凋亡的生物学意义。     

卵丘在卵母细胞成熟中的作用

卵丘是指在卵母细胞外周并与之进行代谢联系的颗粒细胞群;卵丘对于卵母细胞成熟有极其重要的作用。主要表现在卵丘参与维持卵母细胞减数分裂阻滞,诱导卵母细胞减数分裂恢复、支持卵母细胞细胞质的成熟。卵丘形态和卵丘扩展影响卵母细胞成熟。了解卵丘在卵母细胞成熟中的作用有助于帮助人们进一步揭示哺乳动物卵母细胞成熟的机制。     

哺乳动物卵母细胞减数分裂研究进展

介绍了国内外哺乳动物卵母细胞减数分裂研究的现状、内容、技术、方法及意义。特别指出了哺乳动物卵母细胞体外成熟及其相关技术在畜牧业生产、医疗卫生事业中的广泛应用前景和重要的实践意义。     

Maturation of the rat cumulus-oocyte complex: structure and function

The cumulus cells that surround the mammalian oocyte become dispersed following the preovulatory surge of the pituitary gonadotropin, luteinizing hormone (LH). We have examined cumulus-oocyte complexes of PMSG-primed immature rats before and at 1, 2, 3, 4, 6, and 8 hr after injection of human chorionic gonadotropin (hCG), which acts on the rat ovary like the pituitary gonadotropin. Associations between projections of the cumulus cells and the oocyte were analyzed in thin sections. We observed that some cumulus projections were greatly enlarged where they associate with the oocyte. These enlarged regions were filled with numerous small vesicles. Gap junctions between cumulus cell projections and the oocytes were small. We quantitated the number and size of gap junctions between cumulus cells. The number of small gap junctions (less than 1 microM) between cumulus cells did not change significantly over the 8-hr period after hCG administration. Larger gap junctions, however, showed a general downward trend beginning after the third hour post hCG. Light microscopic observations of plastic sections revealed that dispersion of the cumulus oophorus is not observed until after 4 hr post-hCG, but between 4 and 8 hr after gonadotropin administration the cumulus becomes markedly dispersed. In the majority of the oocytes in these complexes the germinal vesicle (GV) displayed some irregularity in shape at 2 hr post-hCG, although absence of the GV was not observed until later. Our observations suggest a new means of communication in the cumulus-oocyte complex by the vesicle-filled enlargements of the cumulus cell projections at the oocyte surface. They further indicate that the decrease in metabolic coupling observed in rat cumulus-oocyte complexes soon after exposure to LH is not associated with a change in number and size of the gap junctions between the cumulus cells. We suggest that it is either the disruption of the gap junctions at the region of contact of the cumulus cell projections with the oocyte surface or the operation of a gating mechanism that blocks the junctional channels without affecting their morphological appearance that is responsible for uncoupling of the oocyte from the cumulus cells.     

Synthesis and modification of D7 protein during Xenopus oocyte maturation.

The Xenopus maternal mRNA D7 is translationally repressed during oogenesis, only becoming recruited into polysomes during oocyte maturation, with D7 protein being detectable for the first time prior to germinal vesicle breakdown (GVBD). The synthesis of D7 protein was found to be induced by a variety of maturation-promoting agents including cyclin, c-mos and crude preparations of MPF. D7 protein induced by all these agents is post-translationally modified and exists as a number of variants of differing molecular weight. In contrast to endogenous D7 mRNA, D7 RNA injected into the stage VI oocyte is efficiently translated, resulting in the accumulation of predominantly unmodified D7 polypeptides, which become increasingly modified during oocyte maturation to produce a pattern of polypeptides similar to those derived from endogenous D7 mRNA. Thus, the system that results in the post-translational modification of the D7 protein is itself activated during oocyte maturation. The nature of the protein modification is not known but does not appear to be phosphorylation. The translation of exogenous D7 RNA in the stage VI oocyte does not lead to translational derepression of endogenous D7 mRNA.     

Changes in Electrophoretic Patterns of Oocyte Proteins during Oocyte Maturation in Oryzias latipes

Changes in the two-dimensional SDS-electrophoretic patterns of extracts of maturing denuded oocytes of the medaka ( Oryzias latipes ) were surveyed. In oocytes without follicular constituents several proteins became detectable in the area between the acidic and slightly basic proteins on the two-dimensional electrophoretograms, while a few of the protein spots disappeared during the process of oocyte maturation. The former proteins were detected also in oocytes that were induced to mature in vivo without breakdown of the germinal vesicle. Several proteins newly observed in extracts of post-vitellogenic oocytes during maturation after breakdown of the germinal vesicle were also identified by two-dimensional electrophoresis. Of several proteins that exhibited noticeable changes in maturing oocytes, only one spot incorporated ¹⁴C-labeled amino acid during maturation, suggesting that post-translational modification of many proteins occurred during oocyte maturation.     

In vitro Oocyte Maturation in the Zebrafish Brachydanio rerio and Fertilization and Development of the Mature Egg

Maturation process of zebrafish oocyte was investigated using in vitro incubation.In medium EM-199 containing 0.5 μg/ml of 17α-hydroxyprogesterone incubated under 80％ O_2 and at 25°C,germinal vesicles(GV)of oocytes in stage Ⅳ migrated from midway between the center and theperiphery ofoocytes to the periphery in 40 minutes and the oocytes went into stage V.Half an hourlater,the oocytes underwent germinel vesicle breakdown(GVBD)with a breakdown rate of 59％.Two more hours were needed for such oocytes to complete their final maturation.The mature eggscould not come off from the follicle layer surrounding them by themselves(ovulation).By removingthe follicle and adding active sperms for insemination,we could make the mature eggs fertilized.Thechorion was elevated and blastoderm formed on the animal pole.The cleavage and development ofthese fertilized eggs followed the same course as the naturally matured and fertilized eggs.Usingblastula formation as a marker of successful fertilization of the in vitro matured egg,the fertilizationrate was 78％.This is the first report on the successful in vitro incubation of mature oocytes inzebrafish.The establishment of this in vitro oocyte maturation technology has laid the foundationfor further investigation of the transfer of foreign genes in the germinal vesicles of oocytes.     

山羊卵母细胞体外成熟过程中线粒体的动态分布

目的研究山羊卵母细胞减数分裂过程中线粒体的动态分布。方法收集山羊卵母细胞,在M199中分别培养4、8、12、16、20和24 h,用特异性线粒体标记探针进行标记,用激光扫描共聚焦显微镜观察线粒体的分布情况。结果生发泡期线粒体多分散在卵母细胞的胞质内,并且距生发泡有一定的距离;生发泡破裂期线粒体逐渐移向染色质;第一次减数分裂中期与第二次减数分裂中期线粒体成簇密布在染色体周围。排出的第一极体中也含有大量的线粒体。结论同其他哺乳动物卵母细胞体外成熟过程中线粒体分布情况相比,线粒体在山羊卵母细胞中的分布具有明显的相似性。线粒体密布在成熟卵母细胞染色体周围可能与极体的排出和受精后染色体的迁移有关。