卵母细胞成熟的调控机理

本文着重介绍卵母细胞成熟调控机理的研究进展,主要讨论成熟分裂阻滞、成熟分裂恢复和成熟促进因子与成熟分裂恢复的关系。     

牛卵母细胞的成熟与激活

本文综述了牛卵母细胞生长成熟过程和卵母细胞激活的形态学变化,并从分子调控机制上进行探讨。卵母细胞成熟经历了四个阶段：（１）减数分裂启动及在核网期的阻滞;（２）卵母细胞生长期;（３）减数分裂的恢复;（４）减数分裂在ＭⅡ停滞。一些细胞因子和小分子物质参与并调控这一过程的完成,卵母细胞激活的形态学标志是释放第二极体并形成原核,其实质是形成减数分裂向有丝分裂的转变。     

卵母细胞成熟及其成熟调控因子

卵母细胞成熟过程是个复杂的减数分裂过程,许多因子参与这个过程的调控.为了探讨哺乳动物卵母细胞体外成熟的机理,指导家畜体外胚胎生产,本文就卵母细胞体外成熟培养(in vitro maturation IVM)及影响因子等方面进行综述.认为在卵母细胞体外成熟过程中,仍需对胞质成熟、基因调控及影响因子等方面进行深入研究.     

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。     

胰岛素样生长因子-Ⅰ(ⅠGF-Ⅰ)对犬卵母细胞体外成熟的影响

促性腺激素浓度达到峰值时哺乳动物卵母细胞在体内开始成熟。体外成熟过程和体内相似,但是需要许多决定卵母细胞减数分裂和发育能力的因子相互作用。体外成熟技术需要理解这些因子间的作用机理。促性腺激素是卵母细胞核成熟的主要调控因素。但它只是影响卵母细胞功能及核成熟的众多因     

家畜卵泡卵母细胞体外成熟研究进展

卵母细胞的体外成熟是体外受精技术的重要环节之一。众多的研究证明，卵母细胞受精后三个细胞周期的细胞合成代谢均由卵母细胞中的母源基因及其产物如mRNA和rRNA完成．许多研究认为，卵母细胞在成熟过程中细胞质的退化及不完全成熟是造成体外受精胚胎生产效率低的主要原因，因此，卵母细胞成熟的质量是影响体外受精的关键因素。本文就家畜卵泡卵母细胞体外成熟的研究现状、影响成熟的主要因素及存在的问题等做以论述。     

在成熟过程中添加牛血清和猪卵泡液对猪卵母细胞核成熟及体外受精后早期胚胎发育的影响

研究目的在于探讨在成熟过程中添加牛血清和猪卵泡液对猪卵母细胞核成熟、卵丘细胞扩散及体外受精后早期胚胎发育的影响。卵母细胞·卵丘细胞复合体在含FSH和LH的以下处理组的成熟液中成熟培养 2 3～ 2 4h :(1)对照组-改良TCM - 199+0 .1%PVA ;(2 )试验组 1-改良TCM - 199+10 %新生牛血清 ;(3)试验组 2 -改良TCM - 199+10 %猪卵泡液 ,再移至无FSH和LH的不同处理组的成熟液中成熟培养 2 3～ 34h。试验 1中 ,卵母细胞在 4 6～ 4 8h成熟培养后 ,观察卵丘细胞扩散情况 ,并对卵母细胞进行固定和染色 ,鉴定卵母细胞减数分裂情况 :试验 2中 ,对在不同处理组的成熟液中成熟培养 4 6～ 4 8h的卵母细胞进行体外受精 ,再培养 8d。受精后第 2天检查分裂率、第 6天检查桑椹胚 /囊胚率、第 8天检查囊胚率。 4 6～ 4 8h成熟培养后试验组 1和试验组 2的大部分卵母细胞 -卵丘细胞复合体的卵丘细胞完全扩散 ,而对照组的卵丘细胞只有 5 0 %扩散。试验组 1和试验组 2的卵母细胞核成熟率分别为 39.9% (77/ 193)和 4 4 .3% (93/ 2 10 ) ,与对照组的卵母细胞核成熟率 4 8.1% (99/ 2 0 6 )相比没有显著差异 (P <0 .0 5 )。卵母细胞分裂率试验组 1(5 0 .0± 1.8) %和试验组 2 (49.9± 2 .6 ) %与对照组的卵母细胞分裂率 (49.0± 2     

牛卵母细胞体外成熟

卵母细胞体外成熟培养研究主要集中在提高体外成熟卵母细胞支持胚胎发育的能力上。本文主要从成熟机理、卵母细胞的来源、卵母细胞的成熟培养环境及培养时间等方面进行了综述,介绍了目前卵母细胞体外成熟培养的进展情况。     

雌激素及其受体调控卵母细胞成熟的研究进展

目前很多女性受到不孕不育、自然流产和出生缺陷等诸多生殖相关疾病的困扰,其主要原因为生殖细胞减数分裂异常和卵母细胞质量下降。雌激素不仅参与调控哺乳动物的发情和性行为,也参与调控卵母细胞的生长发育。雌激素受体作为类固醇激素受体的一种,在介导雌激素发挥基因组效应和非基因组效应中有着重要的作用。目前关于雌激素受体调控卵母细胞成熟的报道多集中于硬骨鱼类,特别是新型膜受体GPR30,而在哺乳动物方面的研究鲜有报道。因此作者介绍了雌激素核受体ERα、ERβ与新型膜受体GPR30的结构与分布,简述了膜受体GPR30的不同类型配体的研究进展,综述了雌激素受体在调控卵母细胞发育中的相互作用,发现雌激素的核受体与膜受体在卵母细胞成熟的过程中可能发挥不同的调控作用,并进一步阐述了雌激素及其受体在卵母细胞成熟过程中的潜在调控机制。     

lncRNA在卵母细胞成熟过程中作用的研究进展

长链非编码RNA(lncRNA)是一种长度大于200个核苷酸的RNA调控分子,它存在于细胞核和细胞质中,不具备蛋白编码能力,可以被剪切,以RNA的形式调控基因表达,并参与细胞内多种生物学调控过程。本文主要综述了lncRNA的作用机制及生物学功能、lncRNA在卵母细胞成熟过程中的研究,为进一步探讨lncRNA调控卵母细胞成熟提供理论依据。     

MPF与MAPK在生殖细胞减数分裂中相互作用研究进展

许多研究显示生殖细胞减数分裂是由MPF调控的,MAPK对细胞增殖、细胞分裂和细胞周期调控同样起到关键性作用.以前都是将MPF与MAPK作为两种行使不同功能的物质,分别进行研究,但经过近年的研究发现,MPF与MAPK在卵母细胞成熟中有一定的关系,不能将两者绝对割裂开.文章就生殖细胞减数分裂调节机制研究中有关MPF与MAPK的相互关系进行了综述.     

Effect of EGF on expression and localization of maturation‐promoting factor,mitogen‐activated protein kinase,p34cdc2 and cyclin B during different culture periods on in vitro maturation of canine oocytes

This study aimed to investigate the localization of MPF, MAPK, p34^cdc2 and cyclin B1 proteins, before and after treatment with EGF during different moments of oocyte maturation. The ovaries obtained from 350 domestic dogs were aseptically isolated, immersed in physiological solution and transported at 4°C. In the laboratory, the ovaries were sectioned for the release of cumulus–oocyte complexes. Cumulus–oocyte complexes were selected and divided into treatment groups with and without EGF and cultured for 24, 48 and 72 hr. Immunofluorescence was used for the detection and the localization of MAPK, MPF, p34^cdc2 and cyclin B1 proteins. We observed that the expression and localization of MPF, MAPK, p34^cdc2 and cyclin B1 proteins are associated with meiosis resumption and cell cycle progression, and that EGF influences cell signalling pathways by promoting alterations in the localization of these proteins, improving the acquisition of oocyte competence. This is the first report of the localization of crucial proteins for meiosis progression in domestic dogs and identification of the expression and localization of proteins for cell cycle progression performed in this study represented a step of great importance to elucidate the mechanisms involved in the meiosis block in domestic dogs, allowing the advance in this research area.     

哺乳动物卵母细胞非整倍体产生机制的研究进展

雌性生殖细胞进行减数分裂时易发生染色体分离错误而产生非整倍体卵母细胞,其受精后会产生非整倍体胚胎,导致出生缺陷或胚胎致死,是影响哺乳动物繁殖的重要因素。卵母细胞在第一次减数分裂前期发生同源染色体联会,此时DNA双链断裂引发重组。重组时缺乏交叉、重组事件数量的减少及交叉靠近端粒或着丝粒导致染色体发生同向分离或不分离,从而产生非整倍体卵母细胞。减数分裂期间,当染色体的端粒共向于同一极或没有完全附着在纺锤体微管上时,纺锤体组装检查点（spindle assembly checkpoint,SAC）被激活,E3泛素连接酶APC/Cyclome （APC/C）沉默,保护分离酶抑制蛋白（securin）和细胞周期蛋白B （cyclin B）不被降解,从而抑制分离酶和染色体的分离。直到所有染色体与纺锤体实现稳定的双极定向并正确排列到赤道板上,SAC关闭,染色体正确分离。卵母细胞中SAC蛋白缺失,导致SAC不能有效地监测端粒在纺锤体上的正确附着,发生染色体分离错误,从而产生非整倍体卵母细胞。因此,通过现代分子技术手段解析非整倍体卵母细胞所涉及的机制是保护哺乳动物生育的重要目标。作者主要介绍了卵母细胞减数分裂的特点,详细阐述了卵母细胞非整倍体发生的染色体分离错误的分子机制,以期为开发卵母细胞非整倍体的治疗手段提供参考。     

Inhibition of mitogen activated protein kinase activity induces parthenogenetic activation and increases cyclin B accumulation during porcine oocyte maturation

The inhibition of mitogen activated protein kinase (MAPK) activation during porcine oocyte maturation leads to decreased maturation promoting factor (MPF) activity and to the induction of parthenogenetic activation. In the present study, in order to analyze the mechanism underlying the suppression of MPF activity in MAPK-inhibited porcine oocytes, we injected mRNA of SASA-MEK, a dominant negative MAPK kinase, or antisense RNA of c-mos, a MAPK kinase kinase, into immature porcine oocyte cytoplasm. The injection of SASA-MEK mRNA or c-mos antisense RNA inhibited the MAPK activity partially or completely, respectively, decreased the MPF activity slightly or significantly, respectively, and induced parthenogenetic activation in 17.1% or 96.6% of mature oocytes, respectively, although no parthenogenetic activation was observed in the control oocytes. Immunoblotting experiments revealed that cyclin B accumulation in these MAPK-suppressed porcine oocytes was increased significantly after 50 h of culture and that a considerable amount of MPF was converted into inactive pre-MPF by hyperphosphorylation. These results indicate that the inhibition of MAPK activity in porcine oocytes did not promote cyclin B degradation but rather suppressed it; also the decrease in MPF activity in MAPK-suppressed porcine oocytes correlated with the conversion of active MPF into inactive pre-MPF.     

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.     

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。     

Activation with ethanol improves embryo development of ICSI-derived oocytes by regulation of kinetics of MPF activity

Developmental potential of bovine embryos that are not artificially activated after intracytoplasmic sperm injection (ICSI) is generally very low. In this study, we investigated effects of artificial activation with ethanol on kinetics of maturation promoting factor (MPF) activity (p34(cdc2) kinase activity) and development of bovine oocytes following ICSI. Treatment of oocytes with ethanol at 4 h after ICSI improved their first cleavage and further preimplantation development (51% vs. 13%, 14% vs. 4%: treatment with vs. without ethanol, respectively). MPF activity of oocytes was lowered until at least 2 h after ICSI. In oocytes without activation after ICSI, MPF activity temporarily elevated at 6 h after ICSI, whereas this phenomena was not observed in the oocytes treated with ethanol. Furthermore, MPF activity was elevated 20 h after ICSI in oocytes activated with ethanol, whereas this elevation of MPF activity was not shown in oocytes without activation. These results indicate that the stimulus of sperm was sufficient to lower MPF activity of oocytes following ICSI, and moreover the activation treatment of bovine oocytes with ethanol after ICSI served to maintain the low levels of MPF activity until the next cell cycle started.     

Bovine Oocyte Meiotic Inhibition Before In Vitro Maturation and Its Value to In Vitro Embryo Production: Does it Improve Developmental Competence?

The efficiency of bovine in vitro embryo production has remained low despite extensive effort to understand the effects of culture conditions, media composition and supplementation. As bovine oocytes resume meiosis spontaneously when cultured, it was hypothesized that preventing meiosis in vitro before in vitro maturation (IVM) and in vitro fertilization (IVF) would allow more oocytes to acquire developmental competence. This article reviews some of the factors involved in meiotic arrest as well as the effects of meiotic inhibition before IVM on bovine oocytes developmental competence following IVF. Follicular components and cAMP-elevating agents can delay or inhibit meiosis in various proportions of oocytes; however, few studies have examined their effects on development following IVM and IVF because they are not practical (follicular components) or have a transient effect on meiosis (cAMP-elevating agents). Protein synthesis or phosphorylation inhibition prevented meiosis in high percentages of oocytes; however, these non-specific inhibitions led to lower developmental competence compared with non-arrested oocytes. Maturation promoting factor (MPF) inhibition with specific inhibitors has been examined in several studies. Despite faster maturation following removal from inhibition and some structural damage to the oocytes, MPF inhibition generally led to blastocyst rates similar to control, non-arrested oocytes. Future work will involve evaluating the effects on arrested oocytes of molecules that can improve developmental competence in non-arrested oocytes. It is also anticipated that new IVM systems that take into consideration new knowledge of the mechanisms involved in the control of meiosis will be developed. Moreover, global gene expression analysis studies will also provide clues to the culture conditions required for optimal expression of developmental competence.     

Identification of candidate miRNAs and expression profile of yak oocytes before and after in vitro maturation by high‐throughput sequencing

Small RNA represents several unique non‐coding RNA classes that have important function in a wide range of biological processes including development of germ cells and early embryonic, cell differentiation, cell proliferation and apoptosis in diverse organisms. However, little is known about their expression profiles and effects in yak oocytes maturation and early development. To investigate the function of small RNAs in the maturation process of yak oocyte and early development, two small RNA libraries of oocytes were constructed from germinal vesicle stage (GV) and maturation in vitro to metaphase II‐arrested stage (M II) and then sequenced using small RNA high‐throughput sequencing technology. A total of 9,742,592 and 12,168,523 clean reads were obtained from GV and M II oocytes, respectively. In total, 801 and 1,018 known miRNAs were acquired from GV and M II oocytes, and 75 miRNAs were found to be significantly differentially expressed: 47 miRNAs were upregulated and 28 miRNAs were downregulated in the M II oocytes compared to the GV stage. Among the upregulated miRNAs, miR‐342 has the largest fold change (9.25‐fold). Six highly expressed miRNAs (let‐7i, miR‐10b, miR‐10c, miR‐143, miR‐146b and miR‐148) were validated by real‐time quantitative PCR (RT‐qPCR) and consistent with the sequencing results. Furthermore, the expression patterns of two miRNAs and their potential targets were analysed in different developmental stages of oocytes and early embryos. This study provides the first miRNA profile in the mature process of yak oocyte. Seventy‐five miRNAs are expressed differentially in GV and M II oocytes as well as among different development stages of early embryos, suggesting miRNAs involved in regulating oocyte maturation and early development of yak. These results showed specific miRNAs in yak oocytes had dynamic changes during meiosis. Further functional and mechanistic studies on the miRNAs during meiosis may beneficial to understanding the role of miRNAs on meiotic division.