哺乳动物卵母细胞纺锤体的形成及纺锤体检验点的作用机制

染色体精确分离是在纺锤体的正确组装和纺锤体检查点(spindle assembly checkpoint,SAC)的监控下完成的,对于哺乳动物卵母细胞来说,纺锤体的形成和SAC都是保证染色体精确分离的重要因素,如果染色体分离错误将直接导致自发性流产或其他出生缺陷。卵母细胞中心体缺失后,细胞依然能够依靠独立于中心体而围绕染色体成核的微管反向平行排列能形成双极纺锤体,即自我组装纺锤体。由微观组织中心(microtubue organizing center,MTOC)召集微管聚集,成熟促进因子(maturation promoting factor,MPF)维持两次减数分裂过程中纺锤体的形成过程,细胞静止因子(cytostatic factor,CSF)维持分裂中期结构,使纺锤体在染色体没有全部集合到赤道板时保持稳定。大体积的卵母细胞容易产生非整倍体,且卵母细胞中不含有中心体这一特殊性导致卵母细胞中是否存在SAC在很长一段时间内存在争议,但现在SAC是确保卵母细胞染色体精确分离的机制之一已被初步证明。在减数分裂中期染色体之间存在一种黏连,细胞会产生"等待-后期"信号抑制SAC活性,从而保持这种黏连稳定,直至所有染色体完成与纺锤体的连接,"等待-后期"信号失活,SAC启动,使染色体间的黏连失活,进而在纺锤体的作用下染色体分离。作者综述了减数分裂过程中纺锤体的特异性组装过程和纺锤体检查点的组成及作用机制,丰富了减数分裂的相关知识,并为减数分裂过程中非整倍体的形成机制提供依据。     

哺乳动物卵母细胞纺锤体的形成及纺锤体检验点的作用机制

染色体精确分离是在纺锤体的正确组装和纺锤体检查点(spindle assembly checkpoint,SAC)的监控下完成的,对于哺乳动物卵母细胞来说,纺锤体的形成和SAC都是保证染色体精确分离的重要因素,如果染色体分离错误将直接导致自发性流产或其他出生缺陷。卵母细胞中心体缺失后,细胞依然能够依靠独立于中心体而围绕染色体成核的微管反向平行排列能形成双极纺锤体,即自我组装纺锤体。由微观组织中心(microtubue organizing center,MTOC)召集微管聚集,成熟促进因子(maturation promoting factor,MPF)维持两次减数分裂过程中纺锤体的形成过程,细胞静止因子(cytostatic factor,CSF)维持分裂中期结构,使纺锤体在染色体没有全部集合到赤道板时保持稳定。大体积的卵母细胞容易产生非整倍体,且卵母细胞中不含有中心体这一特殊性导致卵母细胞中是否存在SAC在很长一段时间内存在争议,但现在SAC是确保卵母细胞染色体精确分离的机制之一已被初步证明。在减数分裂中期染色体之间存在一种黏连,细胞会产生"等待-后期"信号抑制SAC活性,从而保持这种黏连稳定,直至所有染色体完成与纺锤体的连接,"等待-后期"信号失活,SAC启动,使染色体间的黏连失活,进而在纺锤体的作用下染色体分离。作者综述了减数分裂过程中纺锤体的特异性组装过程和纺锤体检查点的组成及作用机制,丰富了减数分裂的相关知识,并为减数分裂过程中非整倍体的形成机制提供依据。     

微丝调控卵母细胞减数分裂成熟的分子机制研究进展

卵母细胞在与精子融合形成合子前会经历2轮减数分裂。与有丝分裂不同的是,2次减数分裂都是不对称的,最终会产生1个大体积的具有全能性单倍体卵母细胞和2个小体积的注定退化的极体。肌动蛋白丝作为卵母细胞中的细胞骨架,与分裂过程中的囊泡转运、细胞核定位、纺锤体迁移与锚定、极体排出和染色体分离等生物学事件存在重要联系。本文以哺乳动物为模型,总结了肌动蛋白在卵母细胞减数分裂成熟过程中的重要调节机制与信号通路,以期为进一步研究卵母细胞成熟过程的调控机制提供参考。     

低温和冷冻保护剂(DMSO)对卵细胞的影响

实验以小鼠为动物模型 ,研究延长小鼠卵母细胞在含有冷冻保护剂的液体中的平衡时间、并且降低平衡时的温度是否能影响卵母细胞完成减数分裂时染色体的倍性。结果表明 :①在含有DMSO的液体中平衡卵母细胞的非整倍体率同对照组没有明显差异 ,但是卵母细胞在没有DMSO的液体中平衡 ,非整倍体率明显增加 ;②有DMSO时 0℃平衡15和 6 0min后卵母细胞非整倍体率没有发生明显变化 ;③卵母细胞在 1.5mol/L的DMSO液体中 2 4℃平衡 ,卵母细胞的非整倍体率明显增加。这些结果表明 :1.5mol/L的DMSO 0℃平衡对卵母细胞的染色体倍性有保护作用     

蛋白激酶C在哺乳动物卵母细胞第1次减数分裂成熟过程中的调节作用

蛋白激酶C（PKC）是一个广泛分布在真核细胞中的丝氨酸/苏氨酸蛋白激酶家族。它在卵母细胞的生发泡破裂（GVBD）、染色体凝集、MⅠ期纺锤体组装和第一极体排放等过程中起着重要的调节作用。PKC的活性变化调节着GVBD的发生，GVBD标志着第1次减数分裂的启动。PKC活性在卵母细胞成熟过程中逐渐升高，在第1次减数分裂中/后期转变时活性下降，使卵母细胞得以释放出第一极体，至此卵母细胞完成第1次减数分裂进入第2次减数分裂。作者就PKC在卵母细胞第1次减数分裂成熟过程中的作用综述如下。     

哺乳动物卵母细胞低温存活的影响因素

哺乳动物卵母细胞的有效冷冻保存,会大大提高众多繁殖技术的实用性。那些最初用于哺乳动物卵裂期胚胎的平衡和非平衡冷冻保存方法,已经用于卵母细胞,但受精率和发育率显著低于没有冷冻的卵母细胞。在家畜上还未曾有过从冷冻卵母细胞发育到幼畜的研究报导。细胞骨架元素、减数分裂纺锤体和其它卵皮质组分,对遇冷和防冻剂的敏感性,不利于卵母细胞冷冻保存。降温过程中其它物理因素也会影响卵母细胞的存活。那些影响卵母细胞渗透反应和冷冻生物学反应的生物物理参数的估计将日益重要,有助于创造新的冷冻方法。     

哺乳动物卵母细胞老化机制的研究进展

 哺乳动物卵母细胞的老化,导致胚胎非整倍体的比率和流产率增加。研究卵母细胞老化的机制,探讨延缓卵母细胞老化的措施,对提高卵母细胞体外成熟和受精效率等具有重要的理论意义。论文从卵母细胞老化的生物学特性、影响老化的因素以及延缓老化的措施等几个方面进行综述。     

AuroraA激酶在猪卵母细胞成熟分裂过程中的动态分布及其与微管蛋白相关性分析

为探讨Aurora A激酶在猪卵母细胞成熟过程中的动态分布及其与细胞微管蛋白相关性,采用间接免疫荧光结合激光共聚焦显微成像技术,检测Aurora A蛋白的动态分布与亚细胞定位,然后分别使用Aurora A特异性抑制剂(MLN-8054)、微管蛋白抑制剂(秋水仙素)处理猪卵母细胞,研究Aurora A与细胞骨架分布的相关性。结果显示:在生发泡(GV)期,Aurora A主要分布在细胞质中,在第一次和第二次减数分裂中期,即MⅠ和MⅡ期,Aurora A则主要伴随着纺锤体分布,与纺锤体有着相似的亚细胞定位;使用Aurora A特异性抑制剂MLN-8054处理细胞后,与对照组相比,纺锤体形态异常的比例显著增加;使用秋水仙素处理细胞后,α-微管蛋白结构紊乱地分布在染色体周围或消失,而Aurora A蛋白或以异常形态伴随纺锤体分布,或消失。结果表明:猪卵母细胞成熟分裂过程中Aurora A与纺缍体微管的分布密切相关,并具有明显的阶段性特点,Aurora A可能通过参与调节纺锤体微管的组装进而参与猪卵母细胞减数分裂的调控。     

哺乳动物早期胚胎端粒酶活性的研究进展

端粒位于真核染色体末端，是稳定染色体末端的重要元件。端粒酶（TER）是一种特殊的细胞核蛋白（RNP）反转录酶（RT），其核心酶包括蛋白亚基和RNA元件。在DNA复制过程中的端粒丢失可以被有活性的端粒酶补偿回来。哺乳动物端粒酶在发育中受调控，端粒的重编程可能是由于早期胚胎不同时期的端粒酶活性而造成的，因此，研究胚胎发育早期端粒和端粒酶重编程是非常重要的。本文对端粒和端粒酶的结构和功能，及其与哺乳动物早期胚胎发育的关系进行了综述．并在此基础上展望了端粒和端粒酶在克隆动物胚胎发育上的基础作用。     

细胞周期检查点激酶1的表达对猪卵母细胞体外成熟的影响

细胞周期检查点激酶1(Chk1)作为DNA复制检查点和DNA损伤反应的重要成员,在有丝分裂和减数分裂中都具有重要作用。为探究Chk1对猪卵母细胞减数分裂的影响,本研究首先采用免疫荧光染色和实时荧光定量PCR技术检测了Chk1在猪卵母细胞不同时期的表达和定位,随后利用chir-124抑制Chk1的表达,研究其对猪卵母细胞减数分裂的影响,以及相关基因的表达和纺锤体的变化。结果表明,Chk1在4个时期(GV、GVBD、MI、MII)均有表达,GV期主要定位在生发泡内,GVBD期主要定位在核周围,MI期和MII期主要定位在纺锤体上;用不同浓度的chir-124处理卵母细胞来抑制Chk1,发现0.5μmol/L的chir-124对卵母细胞生发泡破裂(GVBD)的影响最大,可显著提高猪卵母细胞处于GV-IV期的比例(P0.05),并且可显著提高卵母细胞达到GVBD期的比率(P0.05),但对后续的成熟没有显著影响;随后对MII期卵母细胞的基因表达进行检测发现,Chk1的抑制可调节cdk1、cdc25C、cyclin b1基因的表达,并使gwl基因的表达显著下降(P0.05),从而调节卵母细胞恢复减数分裂的进程;Chk1的表达会激活纺锤体组装检查点(SAC),阻止同源染色体分离,将猪卵母细胞定位在MI期,而减少Chk1的表达后,猪卵母细胞可渡过MI期,进入MII期。综上,在GV期使用chir-124抑制Chk1的表达可促进猪卵母细胞恢复减数分裂,并促进后续的成熟。     

Roles of microtubules and microfilaments in spindle movements during rat oocyte meiosis

Spindle movements, including spindle migration from the center to the cortex of oocytes during first meiosis and spindle rotation during second meiosis, are required for asymmetric meiotic divisions in many species. However, little is currently known in relation to the rat oocyte. To explore how spindles move and the mechanism controlling spindle movements in rat oocytes, we observed the spindle dynamics during the two meiotic divisions in the rat oocyte by confocal microscopy. Drugs that depolymerize microtubules or microfilaments were employed to further determine the roles of these two cytoskeletons in spindle movements. The results showed that peripheral spindle migration took place during first meiosis and spindle rotation took place during second meiosis in the rat oocytes. Microfilament inhibitor inhibited both spindle migration and spindle rotation, and depolymerization of microtubules inhibited spindle rotation. Severe depolymerization of microtubules inhibited spindle migration, while migration was achieved by partial but not complete depolymerization of microtubules. We thus conclude that microfilaments are important for both spindle migration and spindle rotation and that spindle microtubules are essential for spindle movements in rat oocytes.     

Spindle formation and microtubule organization during first division in reconstructed rat embryos produced by somatic cell nuclear transfer

The present study was conducted to demonstrate the spindle formation and behavior of chromosomes and microtubules during first division in reconstructed rat embryos produced by somatic cell nuclear transfer (SCNT) with cumulus cell nuclei. To demonstrate the effect of oocyte aging after ovulation on the cleavage of SCNT embryos, micromanipulation was carried out 11, 15 and 18 h after injection of hCG. SCNT oocytes were activated by incubation in culture medium supplemented with 5 microM ionomycin for 5 min followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) in mR1ECM for 2-3 h. For immunocytochemical observation, the SCNT embryos were incubated with monoclonal anti-alpha-tubulin antibody and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Cleavage rates were significantly higher for oocytes collected after 15 and 18 h rather than for those collected 11 h after injection of hCG (56 and 53%, respectively vs. 28%; P<0.05). Premature chromosome condensation occurred before activation of the SCNT oocytes, but adequate spindle formation was only rarely observed. The distribution of microtubules in SCNT embryos after activation was different from those of fertilized and parthenogenic oocytes, i.e., a dense microtubule organization shaped like a ring was observed. Eighteen to 20 h post-activation, most SCNT embryos were in the 2-cell stage, but no nucleoli were clearly visible, which was quite different from the fertilized oocytes. In addition, first division with and without small cellular bodies containing DNA was observed in the rat SCNT embryos in some cases. The present study suggests that reorganization of transferred nuclei in rat SCNT embryos may be inadequate in terms of formation of the mitotic assembly and nucleolar reorganization.     

Localization of gamma-tubulin in mouse eggs during meiotic maturation, fertilization, and early embryonic development

Gamma-tubulin, a member of the tubulin superfamily, is a peri-centriolar component which is considered to be essential for microtubule nucleation. The dynamics of gamma-tubulin during mouse oocyte meiotic maturation, fertilization, and early cleavage as well as the co-localization of gamma-tubulin and alpha-tubulin during the formation of the meiotic I spindle were studied by confocal microscopy. We found that gamma-tubulin was evenly distributed in the germinal vesicle (GV) stage oocyte. After germinal vesicle breakdown (GVBD) gamma-tubulin dots were localized in both the cytoplasm and the vicinity of the condensed chromosomes, and aligned at both poles of the meiotic spindle at prometaphase I and metaphase I. At anaphase I and telophase I, gamma-tubulin was detected between the separating chromosomes, while it was absent in the midbody. At the MII stage, gamma-tubulin was again accumulated at the spindle poles. Alpha-tubulin had a similar distribution pattern as gamma-tubulin in the cytoplasm and radiated from gamma-tubulin foci close to the chromosomes during the meiotic spindle formation. After fertilization, gamma-tubulin was translocated from spindle poles to the area between separating chromatids and distributed around the pronuclei. It aggregated into some dots during the interphase, but was distributed on the mitotic spindle poles in early embryos. Our results suggest that gamma-tubulin is essential for microtubule nucleation and spindle formation during mouse oocyte meiosis, fertilization, and early embryo cleavage.     

SRT1720 enhances maturity and quality of oocytes in aged mice

This study aims to investigate the morphology and distribution of mitochondria, spindles, and chromosomes in oocytes of aged mice and examine the effects of SRT1720 on oocyte maturation. C57BL/6J mice were divided into young (4–8 weeks) and aged groups (48–52 weeks). In vitro maturation media contained (0.05, 0.1, and 1.0 μM) SRT1720 and 0.1-μM dimethyl sulfoxide (DMSO control). The rate of chromosome misalignment and spindle misorientation in oocytes of aged mice were significantly higher than that of young mice (P < 0.01). Fluorescence intensity of mitochondria from oocytes of aged mice was significantly lower than that of young mice (P < 0.01). SRT1720 at 0.1 μM significantly improved oocyte maturation, fertilization, and blastocyst formation in aged mice compared with young mice (P < 0.01). Additionally, immunofluorescence intensity of mitochondria, normal spindle morphology, and chromosome alignment were notably enhanced with SRT1720 when compared with the DSMO control group for metaphase II (MII)-stage oocytes matured in vitro (P < 0.01); 0.1-μM SRT1720 enhanced the expression level of SRIT1 in oocytes from aged mice. In summary, the aged mice oocytes showed increased nuclear and cytoplasmic defects, whereas SRT1720 enhanced oocyte maturation and quality. We concluded that 0.1-μM SRT1720 was an appropriate concentration for in vitro maturation media.     

Effects of maturation conditions on spindle morphology in porcine MII oocytes

Incomplete cytoplasmic maturation of in vitro matured (IVM) oocytes has been known to cause microtubule and microfilament alterations, which may result in abnormal pronuclear formation and failed embryonic development. We examined the influences of maturation conditions on meiotic spindle morphology at metaphase of meiosis II (MII) in porcine oocytes. Porcine oocytes were matured under various conditions, i.e., in vitro or in vivo, with different amounts of cumulus cells, with or without hormonal supplements, and with various exposure durations to the hormones, to examine the effects on spindle morphology in MII oocytes by immunofluorescence under confocal laser microscopy. Interpolar spindle length (microm) and spindle area (microm2) were compared among these maturation conditions. The spindle length was significantly shorter in IVM oocytes compared to those matured in vivo. Oocytes collected from cumulus oocyte complexes (COCs), which were poor in cumulus cells, showed smaller spindle areas than those from cumulus-rich COCs. The spindle length and area were both significantly reduced in oocytes grown without hormonal supplements. When oocytes were grown with hormonal supplements for either 6 or 22 hours for the first half of culture, there was no difference in the spindle morphology between these oocytes. These results suggested that maturation conditions significantly influence morphogenesis of MII spindles in porcine oocytes. Oocytes matured in poor conditions were more likely to have a shorter spindle length (long axis) and smaller spindle areas.     

Molecular mechanisms underlying pig oocyte maturation and fertilization

Since the pig is not only an important farm animal, but also a model animal for biomedical applications, the development of reproductive technologies in this species has been very important. In vitro oocyte maturation and fertilization (IVM-IVF) are basic techniques for a number of oocyte- or embryo-related technologies. The practical aspects for pig oocyte IVM-IVF have been reviewed, while the molecular mechanisms underlying oocyte meiotic maturation and fertilization have not been well summarized, although accumulating data have been obtained in recent one decade. This review will focus on what is known about the molecular mechanisms of porcine oocyte maturation and fertilization such as first meiosis resumption, meiotic spindle assembly, second meiosis metaphase (MII) arrest during oocyte maturation, sperm-egg recognition and fusion, sperm acrosome reaction, second meiosis resumption, sperm chromatin decondensation, and pronucleus formation during fertilization, as well as the establishment of polyspermy block.     

Sirt1 protects pig oocyte against in vitro aging

Sirtuins have been widely reported to be involved in multiple biological processes. However, their function during pig oocyte aging has not been reported yet. Here, we first identify that sirt1 expression is dramatically reduced in pig in vitro‐aged oocytes. Furthermore, by confocal scanning and quantitative analysis, we find the increased frequency of spindle defects and chromosome misalignment, disturbed redistribution of cortical granules and mitochondria during oocyte in vitro‐aging. Importantly, these aging‐associated defective phenotypes can be ameliorated through resveratrol (sirt1 activator) treatment during pig oocyte maturation, providing the evidence for the hypothesis that decreased sirt1 is one of a number of factors contributing to oocyte in vitro‐aging. In summary, our data indicate a role for sirt1 in pig oocytes and uncover a striking beneficial effect of sirt1 expression on aged oocytes.     

Equine Aging and the Oocyte: A Potential Model for Reproductive Aging in Women

Numerous similarities in reproductive aging have been documented between the mare and woman. Aging is associated with a decline in fertility. In mares and women, oocyte transfer procedures were initially used to establish that oocyte donor age is associated with oocyte quality. Age-associated differences in oocytes include altered morphology, gene expression, and developmental potential. Reactive oxygen species and mitochondrial dysfunction are thought to be important contributors to loss of oocyte quality. In the woman, aneuploidy is a primary consideration with maternal aging. Although misalignment of chromosomes during meiosis has been observed in the mare, less is known in this area. Reproductive aging will be reviewed in the mare and compared with the woman with emphasis on factors that affect oocyte quality and developmental potential. Areas in which the mare could be used as a research model to study reproductive aging in women will be highlighted.