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。     

哺乳动物卵母细胞减数分裂恢复的诱导

促使哺乳动物卵母细胞减数分裂恢复的机制尚不十分清楚。有腔卵泡中发育充分的卵母细胞被减数分裂抑制因子阻滞在生发泡（GV）期,环一磷酸腺苷（cAMP)是研究得最为清楚的减数分裂抑制因子。然而,其它因子也参与了卵母细胞减数分裂的阻滞。虽然排卵前的促黄体素（LH）峰诱导卵母细胞减数分裂恢复已成定论,但是参与该事件的各种过程非常复杂,因而还没有完全确定。目前,有两种主要但并不互相排斥的假说。第一种假说认为,LH对颗粒细胞的刺激作用终止减数分裂抑制因子流向卵母细胞,从而使卵母细胞膈离这些抑制因子并进而促使减数分裂恢复,第二种假设认为LH刺激颗粒细胞产生一种减数分裂诱导信号,该信号进而克服或者破坏减数分裂抑制因子的作用。权衡这两种假说,目前的证据倾向于支持阳性信号假说,而且最近的研究暗示,该种阳性信号的产生发生在颗粒细胞中LH诱导的cAMP水平上升和MAPK激酶激活之后。     

ROS和丁内酯-Ⅰ抑制山羊卵母细胞的减数分裂

本文研究了ROS（Roscovitine）和丁内酯-Ⅰ（ButyrolactoneⅠ,BL-Ⅰ）两种细胞周期依赖性激酶抑制剂对山羊卵母细胞减数分裂恢复的抑制作用,并研究了抑制对卵母细胞成熟、激活和发育的影响。结果表明：ROS和BL-Ⅰ对山羊卵母细胞减数分裂恢复的抑制作用具有浓度依赖性;200μmol／LROS、100μmol／LBL-Ⅰ、100μmol／LROS＋6．25μmol／LBL-Ⅰ和50μmol／LROS＋25μmol／LBL-Ⅰ都能有效抑制山羊卵母细胞减数分裂的恢复,24h的抑制率分别为78．4％、80．9％、80．3％和77．8％。用ROS和BL-Ⅰ抑制24h后转为正常培养24h,各处理组卵母细胞的成熟率（分别为81．3％、81．9％、83．2％和85．2％）与对照组（83．0％）无显著差异;成熟卵母细胞的化学激活率分别为93．3％、96．2％、92．5％和90．5％,与对照组（97．8％）无显著差异。然而,抑制处理后卵母细胞的卵裂率和桑椹胚率降低,未能发育到囊胚。ROS和BL-Ⅰ抑制山羊卵丘扩展,并且转为正常培养后卵丘不能再扩展。ROS和BL-Ⅰ能够浓度依赖性地抑制山羊卵母细胞减数分裂,二者既可单独,又可降低浓度联合使用,但抑制山羊卵母细胞的浓度远高于牛和猪卵母细胞的;ROS和BL-Ⅰ抑制24h不影响山羊卵母细胞的成熟和激活能力,但影响卵母细胞的卵丘扩展和胚胎发育能力。因此,山羊卵母细胞减数分裂调控可能比它动物更精细。     

ROS和丁内酯-I抑制山羊卵母细胞的减数分裂

本文研究了ROS(Roscovitine)和丁内酯-I(ButyrolactoneI,BL-I)两种细胞周期依赖性激酶抑制剂对山羊卵母细胞减数分裂恢复的抑制作用,并研究了抑制对卵母细胞成熟、激活和发育的影响。结果表明:ROS和BL-I对山羊卵母细胞减数分裂恢复的抑制作用具有浓度依赖性;200μmol/LROS、100μmol/LBL-I、100μmol/LROS+6·25μmol/LBL-I和50μmol/LROS+25μmol/LBL-I都能有效抑制山羊卵母细胞减数分裂的恢复,24h的抑制率分别为78·4%、80·9%、80·3%和77·8%。用ROS和BL-I抑制24h后转为正常培养24h,各处理组卵母细胞的成熟率(分别为81·3%、81·9%、83·2%和85·2%)与对照组(83·0%)无显著差异;成熟卵母细胞的化学激活率分别为93·3%、96·2%、92·5%和90·5%,与对照组(97·8%)无显著差异。然而,抑制处理后卵母细胞的卵裂率和桑椹胚率降低,未能发育到囊胚。ROS和BL-I抑制山羊卵丘扩展,并且转为正常培养后卵丘不能再扩展。ROS和BL-I能够浓度依赖性地抑制山羊卵母细胞减数分裂,二者既可单独,又可降低浓度联合使用,但抑制山羊卵母细胞的浓度远高于牛和猪卵母细胞的;ROS和BL-I抑制24h不影响山羊卵母细胞的成熟和激活能力,但影响卵母细胞的卵丘扩展和胚胎发育能力。因此,山羊卵母细胞减数分裂调控可能比它动物更精细[动物学报52(2):342-348,2006]。     

哺乳动物卵母细胞老化的研究进展

卵母细胞的老化能够导致妊娠率降低、流产率增加和出生缺陷.研究排卵后卵母细胞在老化过程中发生的变化及卵母细胞老化的机制,探讨延缓卵母细胞老化的有效措施,对于改进卵母细胞体外成熟和受精系统,完善辅助生殖技术并减少出生缺陷具有重要的理论和现实意义.本文将从卵母细胞老化研究所用的方法,老化过程中纺锤体相关蛋白、线粒体、泛素、组蛋白修饰和DNA甲基化修饰的变化,卵母细胞老化与皮质反应、多精受精及凋亡的关系,老化的影响因素和分子机制等几个方面对哺乳动物卵母细胞老化的研究进展做一综述.     

哺乳动物卵母细胞及早期胚胎蛋白质组学研究进展

雌性生殖细胞发育是动物繁殖的基石,哺乳动物卵母细胞和早期胚胎在其生长发育过程中有许多独特的现象和规律,涉及一系列蛋白质合成/降解和磷酸化等状态的动态改变。对卵母细胞分裂、成熟调控机理以及植入前胚胎发育规律的研究是发育生物学领域的一项重要课题。蛋白质组学是以细胞或组织内全部的蛋白质为研究对象,系统鉴定、定量蛋白质并研究这些蛋白质功能的科学。随着蛋白质分离、鉴定技术的快速发展,蛋白质组学为卵母细胞发生、分化、成熟以及质量控制等相关研究提供了新的方法和内容,如在蛋白质定量、修饰、定位和相互作用等方面提供其他组学技术不可获得的重要信息。这些信息将有助于揭示哺乳动物卵母细胞成熟和早期胚胎发育的分子机制,对于进一步完善卵母细胞的体外成熟培养体系,提高胚胎体外生产、体细胞克隆和转基因动物生产效率具有重要意义。     

Ca^2＋与哺乳动物卵母细胞成熟分裂的关系

哺乳动物在胎儿期或出生后不久,初级卵母细胞进入并停滞于第一次成熟分裂前期的双线期,直到排卵。在促性腺激素的作用下,第一次成熟分裂重新启动,但完成第一次成熟分裂后又停滞在第二次成熟分裂的中期,必须在精子或化学信号的作用下,才完成第二次成熟分裂。当第一次成熟分裂重新启动后,卵母细胞发生明显的形态变化:生发泡破裂(GVBD),核仁消失,排出第一极体等。     

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。     

山羊卵母细胞的减数分裂进程

The meiotic progression of goat oocytes from follicles of different diameters was investigated in this study. The results were summarized as follows: (1) The in vitro meiotic maturation capacity was different among oocytes from follicles of different diameters. And thus oocytes from < or = 0.5 mm follicles were unable to resume meiosis; oocytes from 0.8-1.2 mm follicles were capable to resume meiosis, but could develop only to MI stage (60% at 24 h); oocytes from 1.5-5 mm follicles had acquired full-meiotic maturation capacity and 91% of them developed to M II stage at 24 h of culture. (2) The percentage of oocytes with intact-germinal vesicles from 1.5-5 mm follicles decreased significantly during 2-8 h of in vitro maturation and the decrease was even more rapid during 4-6 h of culture (from 60% to 19%, p < 0.0005). The percentage of oocytes at M I-stage increased from 24% to 61% during 6-12 h of in vitro maturation, and it then decreased. By 24 h of culture, only 2% oocytes remained at M I-stage. Twenty one percent of the oocytes in this group developed to M II-stage at 16 h of culture, and by 24 h of culture, 91% were at M II-stage. (3) Statistic analysis of the meiotic progression (the duration of each cell cycle stage) of oocytes from 1.5-5 mm follicles showed that GV stage lasted from 0 to 3 h of culture, prometaphase-I stage was from 3.0 to 7.0 h, metaphase-I stage was from 7.0 to 14.6 h, anaphase-I/telophase-I was from 14.6 to 18.4 h and metaphase-II stage lasted from 18.4 to 24 h. (4) Whether the oocytes capable of GVBD and entrance of M I developed to M II, the timing of meiotic progression prior to M I was similar. In summary, our results provided necessary data for studies on the mechanisms and control of meiosis in mammalian oocytes.     

哺乳动物卵母细胞的冷冻保存方法

卵母细胞的冷冻保存与精子和胚胎的冷冻保存一样,具有许多潜在的应用价值。八十年代以后,卵母细胞体外成熟、体外受精、核移植,基因转移等相继研究成功。把优良品种动物废弃卵巢中的大量卵母细胞冷冻保存,可为这些生物技术提供充足的材料。卵母细胞的冷冻保存也为稀有动物,濒危动物和其它有价值的雌性个体(如基因工程个体)的遗传资源的长距离国际间运输及长期保存提供了可能。在人,卵母细胞冷冻保存可以克服胚胎冷冻保存所引起的一系列伦理道德及法律问题。     

Protein phosphorylation in meiotically competent and incompetent mouse oocytes

Specific changes in the two-dimensional gel electrophoretic pattern of mouse oocyte phosphoproteins precede germinal vesicle breakdown (GVBD). We report that changes in the relative abundance of phosphoamino acids occurred prior to GVBD. We also report data that further strengthen the close association of the changes in phosphoprotein patterns with resumption of meiosis. The calmodulin antagonist W7, which transiently inhibits GVBD, inhibited partially at least two of the maturation-associated phosphoprotein changes, the dephosphorylation of a 60,000 Mr phosphoprotein and the phosphorylation of a 36,000 Mr protein. In oocytes from juvenile mice that were incompetent to resume meiosis, neither these changes nor the phosphorylation of proteins of Mr 24,000 and 28,000 occurred; all these changes occurred, however, in oocytes from juvenile mice that were competent to resume meiosis. The microinjection of the heat-stable inhibitor of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKI), which induces GVBD in fully grown oocytes, did not induce GVBD in meiotically incompetent oocytes. Microinjected PKI did not induce the increased protein phosphorylations associated with maturation, but it did induce the dephosphorylation of the 60,000 Mr phosphoprotein. These results provide molecular markers for commitment to resume meiosis in GV-intact oocytes and indicate a potential basis for meiotic incompetence.     

Nuclear maturation and development of IVM/IVF canine embryos in synthetic oviductal fluid or in co-culture with buffalo rat liver cells

In vitro embryo production in the domestic bitch can provide valuable insights for conservation of endangered canids. In the present study, canine oocytes underwent in vitro maturation (IVM) in simple or complex media, with production of in vitro matured and fertilized (IVM/IVF) canine embryos. Cumulus–oocyte complexes (COCs) were harvested from ovaries by slicing and subjected to IVM in four media (SOF, TCM 199, Ham-F10, and DMEM/F12). After culture for 48 h, oocytes were stained and examined for nuclear maturation. There were no significant differences in the mean (±S.D.) percentage of nuclear maturation (metaphase II) of oocytes cultured in SOF (18.6 ± 7.6%), TCM 199 (18.3 ± 4.5%), Ham-F10 (13.9 ± 8.2%), or DMEM/F12 (11.9 ± 4.2%). For assessment of embryo development, oocytes were matured for 48 h in synthetic oviductal fluid (SOF), fertilized with frozen-thawed sperm, and presumptive zygotes were cultured for 7 d, either in SOF or as co-cultures with BRL cells in TCM 199. Percentages of IVM/IVF oocytes that developed to the 2-cell, 3–4-cell, and 5–7-cell stages were higher (P < 0.05) following culture in SOF versus BRL cell co-cultures (33.6 ± 1.2% vs 13.7 ± 1.2%, 24.7 ± 0.5% vs 8.7 ± 1.1%, and 15.1 ± 2.2% vs 4.3 ± 1.3%, respectively). However, none of the embryos developed beyond the 8–16-cell stage. In conclusion, simple or complex media successfully induced resumption of meiosis and nuclear maturation of canine oocytes. Furthermore, SOF supported in vitro development of IVM/IVF canine embryos to the 8–16-cell stage.     

cAMP/PKA诱导卵母细胞减数分裂停滞的机制

大多数物种的卵母细胞在减数分裂前都要经历长时间停滞,其中cAMP对卵母细胞减数分裂停滞具有重要作用,本研究关注c AMP对卵母细胞减数分裂的影响及其机制。本研究通过将卵母细胞与cAMP预孵育,再用胰岛素刺激研究胰岛素诱导的卵母细胞成熟的影响,接着本研究通过显微注射和Zeiss 100TV显微镜分析cAMP对PKA在卵母细胞中定位的影响,并且本研究用Western blotting的方法研究cAMP/PKA对mos蛋白的表达和MAPK蛋白磷酸化的影响。结果显示,本研究通过亲和层析得到了高纯度的PKA蛋白,且cAMP/PKA能够抑制卵母细胞的成熟,而PKA的热稳定抑制剂PKI能够解除PKA对卵母细胞减数分裂的抑制,cAMP/PKA也能够影响mos的积累以及MAPK的磷酸化。cAMP能够影响PKA在卵母细胞中的定位,cAMP/PKA能够通过影响mos积累抑制卵母细胞的减数分裂,这可能与cAMP能够抑制MAPK磷酸化有关。     

前培养对猪卵母细胞生发泡染色质构型和体外成熟的影响

在体外成熟培养时使卵母细胞过早接触高浓度激素可能影响体外成熟卵母细胞的质量。本实验研究了推迟卵母细胞与激素接触时间对卵母细胞GV染色质构型、卵母细胞核成熟质量及极体形态的影响。结果表明：猪体外成熟卵母细胞的极体形态不同,分为完整、退化、碎裂和无极体四种。在不含激素培养液中前培养12h,A类卵母细胞的GV-1比例(48．2％)明显高于在含激素培养液中前培养12h卵母细胞(27．1％);而前者的GV-3比例(19．6％)却明显低于后者(50．8％);前者成熟卵母细胞的极体完整率(59．6％)也明显高于后者(27．5％)。这说明推迟卵母细胞与激素接触时间可能提高体外成熟卵母细胞的质量和发育同步水平。     

Effects of gonadotropins and granulosa cell secretions on the maturation and fertilization of rat oocytes in vitro

Fully grown germinal vesicle-stage oocytes are induced to resume meiosis and acquire the capacity to undergo fertilization in response to a surge of gonadotropins. The present study examined possible direct and indirect roles of gonadotropins in the maturation and fertilization of rat oocytes by determining 1) the effect of exogenous administration of gonadotropins (priming) to immature rats prior to oocyte collection on the capacity of oocytes to undergo maturation and fertilization in vitro, 2) the effect of follicle-stimulating hormone (FSH) in the maturation media on the resumption of meiosis and subsequent capacity of oocytes to undergo fertilization, and 3) the capacity of oocytes to undergo maturation and fertilization following culture in preovulatory follicular fluid or in conditioned media obtained from gonadotropin-stimulated granulosa cell (GC) cultures. In the first experiment, oocytes from unprimed rats underwent spontaneous meiotic maturation in vitro and 17% underwent subsequent fertilization. Priming increased the proportion of oocytes undergoing fertilization. Maturation of oocytes in media supplemented with various concentrations of FSH or for various lengths of time (6-16 h) in medium with 500 ng FSH/ml indicated that FSH slowed the rate of meiotic maturation, but had no effect on the capacity of the oocytes to be fertilized. Oocytes obtained from primed animals and cultured in the presence of preovulatory follicular fluid were fertilized in proportions similar to those cultured in serum-containing medium. In the third experiment, medium conditioned by FSH-stimulated GC for 40 h slowed the rate of meiotic maturation; the addition of luteinizing hormone (LH) to the FSH-stimulated cells produced a medium in which the rate of oocyte maturation was not different from that of control oocytes (in medium from unstimulated cells). Medium conditioned by FSH- or LH-stimulated GC, but not fibroblasts, increased the proportions of oocytes undergoing fertilization following maturation in those media. FSH + LH stimulation of GC increased the fertilization of oocytes to proportions significantly higher than with either gonadotropin alone. These data suggest that GC respond to gonadotropin stimulation by providing a factor(s) that regulates the rate of oocyte maturation and promotes the capacity of oocytes to undergo fertilization.     

嗜热四膜虫减数分裂研究进展

减数分裂是真核生物有性生殖过程的关键步骤,染色体的行为变化贯穿整个减数分裂的过程。近些年来,借助先进的分子生物学技术和细胞学实验手段,通过对突变细胞株的筛选和评价,单细胞真核模式生物原生动物嗜热四膜虫(Tetrahymena thermophila)减数分裂方面的研究取得了长足的进展。本文主要介绍嗜热四膜虫减数分裂的过程,以及在此过程中伴随染色体行为变化的相关基因的功能,从而为进一步探讨嗜热四膜虫减数分裂的分子机制提供有效信息。     

兔卵母细胞体外成熟和体外受精的研究

用贴壁和悬浮生长二种培养系统,分析发情兔血清、滤泡液和激素对体外培养的兔卵母细胞的成熟、原核形成和发育能力的影响,并分析了不同浓度的激素对兔卵母细胞的作用。在贴壁生长的培养系统,滤泡液和激素对卵母细胞有明显的促成熟作用。但用这种卵母细胞体外受精,其原核形成率和发育率都较低。但在体外培养8小时后转移到体内受精,其原核形成和发育率大大提高,三者差别不大。在悬浮培养系统,卵母细胞成熟率、及体外受精后原核形成和发育率都远比贴壁生长的高,尤以原核形成率更甚。兔卵母细胞对激素的耐受力很小,以含FSH(2μg)、LH(1μg)、E_2-17B(1μg)和PRL组合的培波和含低hCG(7IU)的较适宜,高中浓度的FSH、LH和hCG都有促使卵母细胞变性和老化的作用。文中还讨论了二种培养系统不同的机制。     

Cytochalasin D treatment induces meiotic resumption in follicular sheep oocytes

Ovine cumulus-enclosed oocytes collected from antral follicles (3-5 mm in diameter) were cultured in vitro with 2 x 10(6) granulosa cells/ml in the presence or absence of gonadotropins or in the presence of cytochalasin D (CD). The maturation rate was assessed after 24 h of culture. In the control group, in the presence of gonadotropins (follicle-stimulating hormone-luteinizing hormone (FSH-LH; -10 micrograms/ml) 100% of the oocytes reached metaphase II. Whereas intercellular junctions were no longer present after 6-7 h of culture, germinal vesicle breakdown (GVBD) occurred by the same time. In contrast, in the absence of gonadotropin, the majority of the oocytes (59%) remained blocked in GV stage. The inhibition exerted by the granulosa cells on meiotic resumption was overcome when the cumulus-oocyte complexes (COCs) were incubated in CD (5 micrograms/ml) for 6 h at the beginning of the culture. Under these conditions, 85% of the oocytes matured with extrusion of the first polar body. Cytological analysis by cytofluorescence (NBD phallacidin) and electron microscopy showed that, after 6 h of treatment, CD provoked a redistribution of the microfilaments, mainly in the cumulus cells and to a lesser extent in the oocyte cortex. Intercellular junctions disappeared concomitantly with a significant decrease of the intercellular transport of tritiated uridine. The initiation of GVBD occurred at the same time. These results indicate that the resumption of meiosis was correlated with a loss of both junctional complexes (intermediate and gap junctions) between the cumulus cells and the oocyte.