γ-微管蛋白在猪卵母细胞成熟和活化中的分布(英)

微管蛋白（tubulin）是一蛋白质超家族,其中α-,β-微管蛋白是主要的微管蛋白,而γ-微管蛋白主要在微管组装中起作用. 我们利用蛋白质印迹和激光共聚焦技术研究了γ-微管蛋白在猪卵母细胞成熟、受精和活化中的分布. γ-微管蛋白存在于猪卵母细胞中,并且在减数分裂成熟各个时期的量保持不变. 它聚集在微管上,特别是中期纺锤体的两极和后末期的中板. 体外受精和孤雌活化后,γ-微管蛋白聚集在雌雄原核的周围.另外它也存在于精子的顶体帽和颈部.在早期卵裂中,γ-微管蛋白聚集在胚胎的细胞核周围.实验结果表明,γ-微管蛋白在猪卵母细胞、精子和胚胎的微管组装中起重要的调节作用,在猪受精过程中,精子和卵子都向受精卵贡献中心体物质.     

γ-微管蛋白在猪卵母细胞成熟和活化中的分布

微管蛋白(tubulin)是一蛋白质超家族,其中α-,β-微管蛋白是主要的微管蛋白,而γ-微管蛋白主要在微管组装中起作用. 我们利用蛋白质印迹和激光共聚焦技术研究了γ-微管蛋白在猪卵母细胞成熟、受精和活化中的分布. γ-微管蛋白存在于猪卵母细胞中,并且在减数分裂成熟各个时期的量保持不变. 它聚集在微管上,特别是中期纺锤体的两极和后末期的中板. 体外受精和孤雌活化后,γ-微管蛋白聚集在雌雄原核的周围.另外它也存在于精子的顶体帽和颈部.在早期卵裂中,γ-微管蛋白聚集在胚胎的细胞核周围.实验结果表明,γ-微管蛋白在猪卵母细胞、精子和胚胎的微管组装中起重要的调节作用,在猪受精过程中,精子和卵子都向受精卵贡献中心体物质.     

小鼠孤雌胚早期发育过程中γ-微管蛋白的动态变化

微管蛋白是构成微管的主要蛋白,其中α、β亚单位形成异二聚体,而γ-微管蛋白在微管组装中起作用。为了研究小鼠早期孤雌胚中廿微管蛋白的动态变化,本实验采用了免疫荧光化学染色与激光共聚焦显微镜观察相结合的方法,在SrCl2激活的卵母细胞减数分裂以及早期孤雌胚有丝分裂过程中对γ-微管蛋白进行了定位观察。结果显示,SrCl2和细胞松弛素B（cytochalasin B,CB）诱导的第二次减数分裂中期（metaphase Ⅱ ofmeiosis,MII）小鼠卵母细胞恢复减数分裂,并且纺锤体始终与质膜平行,表明纺锤体旋转被抑制,但核分裂不受影响。减数分裂过程中γ-微管蛋白主要定位于中期纺锤体两极和后期分开的染色单体之间;孤雌活化两雌原核形成以后,γ-微管蛋白聚集在两雌原核周围。在早期孤雌胚有丝分裂间期无定形的γ-微管蛋白均匀分布于核;前中期γ-微管蛋白向两极移动,遍布于整个纺锤体区。有丝分裂中期、后期和末期廿微管蛋白的分布变化与减数分裂相似。结果表明,SrCl2和CB激活的MII卯母细胞产生杂合二倍体;γ-微管蛋白具有促微管负极帽形成和稳定微管的功能,从而促进纺锤体的形成;分裂后期和末期廿微管蛋白的重新分布可能是由纺锤体牵引同源染色体分离所诱导的：γ-微管蛋白负责两雌原核的迁移靠近。     

电刺激家兔卵母细胞孤雌活化的研究

采用电刺激使家兔卵母细胞孤雌活化,并对电刺激参数和电刺激介质进行选择,发现在电刺激电压为1.5kV/cm,脉冲持续时间为80us,电刺激3次,电刺激介质为M16的条件下,兔卵母细胞孤雌活化效果最好,可使95％的兔卵母细胞激活,体外培养有89.5％的激活卵发育到正常的8—16细胞期,12％的激活卵发育到囊胚,在体内培养的条件下,26％的激活卵发育到囊胚。采用不同的电刺激介质,卵母细胞最佳激活条件和发育情况不同,表明电刺激介质中Ca~(++)和Mg~(++)浓度似乎与卵母细胞的孤雌活化和维持早期发育有关。家兔在注射LH后17—19小时取卵,电刺激效果最好,激活卵在含细胞松弛素B的培养基中培养3小时,电镜观察已有原核形成,细胞膜附近的细胞器向中央移动。光镜检查表明:约有85％的激活卵具有二个原核,约15％的激活卵有四个原核。     

Oct4基因在猪孤雌激活和体外受精早期胚胎中的表达特征

目的研究植入前胚胎发育重要基因Oct4在猪孤雌和体外受精胚胎中的表达特征。方法收集成熟卵母细胞、孤雌和体外受精2细胞、4细胞、8细胞胚胎和囊胚,做荧光即时定量PCR检测,以体外成熟的猪卵母细胞做对照分析相对表达量。结果孤雌组和体外受精组胚胎在8细胞期Oct4表达量均最高(P<0.05),在孤雌和体外受精组囊胚相对于其他时期Oct4表达量最低(P<0.05)。在同一时期孤雌和体外受精胚胎上Oct4表达并没有差异。结论多能性基因Oct4在卵裂发育时期表达量动态变化,孤雌胚胎在一定程度上可作为体外胚胎基因表达的模型,且不同的胚胎培养条件可能导致基因表达的差异。     

卵母细胞激活

卵母细胞可被精子、化学物质和电脉冲等激活,激活后,胞质内发生Ca2＋振荡、皮质颗粒释放、pH值增长以及成熟促进因子和有丝分裂原活化蛋白激酶失活等一系列重要事件。卵母细胞激活是人工构建孤雌胚胎和克隆胚胎的必需步骤。     

母源物质对重编程作用研究进展

卵母细胞发生过程中会积累大量的物质,即所谓的母源物质(maternal materials),自然状态下,这些母源物质对受精以及之后的发育具有重要的生物学功能。雌雄配子融合后,精子核与卵母细胞中单倍体染色体组均会发生剧烈的表观遗传修饰变化,这个过程也同样发生在体细胞核移植到卵母细胞质之后,这种变化被称之为重编程。重编程奠定了新个体发生发育全部程序的基础,因此是一个备受重视的生物学过程。重编程包括DNA去甲基化、染色质重塑和组蛋白修饰等。受精后,卵母细胞与精子的基因组均会在一定时间和空间范围内经历相应的重编程过程,清除各自基因组在配子形成中保留的表观遗传学修饰,调控基因表达并形成正常发育的全能性胚胎。受精后,卵母细胞成熟中积累的多种母源物质聚集在雄原核周围,调控其基因组的重编程。体细胞核移植胚胎中供体细胞核注到去核卵母细胞后也将在卵母细胞中蛋白质、mRNA、酶类等母源物质的作用下进行重编程。现总结了母源物质对雄原核及供体细胞核重编程作用的研究进展,并探讨了母源物质作用的可能机制。     

两种激活处理促进小鼠孤雌胚胎原核形成

不同人工处理方法激活哺乳动物卵母细胞的机理相似,但其激活效率存在差异。本研究以昆明(KM)、129/Sv×KM F1和C3H×KM F1雌鼠来源的卵母细胞为对象,利用氯化锶(SrCl2,Sr2+)联合细胞松弛素B(cytochalasin B,CB)(Sr2++CB)和离子霉素(ionomycin,Ion)联合6-二甲胺基嘌呤(6-dimethylaminopurine,6-DMAP)(Ion+6-DMAP)两种激活方法处理下对比分析不同品系小鼠卵母细胞的激活效率,并以卵母细胞原核形成率、原核数量和孤雌胚胎体外发育来评价两种激活剂的激活效率。研究结果表明,Ion+6-DMAP激活卵的1原核比率显著高于2原核(p0.05),Sr2++CB激活卵的2原核比率显著高于1原核(p0.05);KM、129/Sv×KM F1和C3H×KM F1各组孤雌胚胎卵裂率和激活率没有显著差异(P0.05),但129/Sv×KM F1和C3H×KM F1囊胚发育率显著高于KM组(p0.05)。3种小鼠品系的卵母细胞用Sr2++CB处理的孤雌胚胎发育率显著高于Ion+6-DMAP。结果证明,Sr2++CB处理小鼠卵母细胞的激活效率明显优于Ion+6-DMAP;129/Sv×KM F1和C3H×KM F1的孤雌胚胎体外发育率显著高于KM小鼠,为研究小鼠遗传背景影响孤雌胚胎发育的机理提供参考。     

中国对虾受精过程中精卵核的细胞学变化

中国对虾精子以其棘部顶端随机附着在卵上,精子在凝胶膜形成后,第一极体排出前入卵,精子入卵后,絮状的精核经过重建形成雄原核,中国对虾卵子排放时处于第一次成熟分裂的中期,卵子入海水时,纺锤体的长轴与质膜平行,卵子激活后,纺锤体的长轴开始旋转,旋转至纺鲑体长轴与质膜垂直时,由纺锤丝牵引着染色体向两极移动,外侧的染色体由质膜包裹形成第一极体,受膜举起后,由次级卵母细胞排放出第二极体,此后,单倍雌核重建形成雌原核,雄原核形成早于雌原核,雌雄原核于卵子中央联会形成联合核,受精后的50分钟纺锤丝牵关染色体称向两极,质膜内缢断裂形成两个细胞的胚胎。     

卵母细胞激活

卵母细胞可被精子、化学物质和电脉冲等激活,激活后,胞质内发生Ca z 振荡、皮质颗粒释放、pH值增长以及成熟促进因子和有丝分裂原活化蛋白激酶失活等一系列重要事件.卵母细胞激活是人工构建孤雌胚胎和克隆胚胎的必需步骤.     

Microtubule distribution during fertilization in the rabbit.

The distribution of microtubules was studied during fertilization of the rabbit oocyte by immunofluorescence microscopy after staining with an anti-alpha-tubulin antibody. In ovulated oocytes, microtubules were found exclusively in the meiotic spindle. At fertilization, the paternal centrosome generated sperm astral microtubules. During pronuclear development, the sperm aster increased in size, and microtubules extended from the male pronucleus to the egg center and towards the female pronucleus. These observations indicate that microtubules emanating from the sperm centrosome were involved in the movements leading to the union of the male and female pronuclei. At late pronuclear stage, microtubules surrounded the adjacent pronuclei. The mitotic spindle that emerged from the perinuclear microtubules contained broad anastral poles.     

Cell-cycle-dependent subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse

M phase or maturation promoting factor (MPF), a kinase complex composed of the regulatory cyclin B and the catalytic p34cdc2 kinase, plays important roles in meiosis and mitosis. This study was designed to detect and compare the subcellular localization of cyclin B1, phosphorylated cyclin B1 and p34cdc2 during oocyte meiotic maturation and fertilization in mouse. We found that all these proteins were concentrated in the germinal vesicle of oocytes. Shortly after germinal vesicle breakdown, all these proteins were accumulated around the condensed chromosomes. With spindle formation at metaphase I, cyclin B1 and phosphorylated cyclin B1 were localized around the condensed chromosomes and concentrated at the spindle poles, while p34cdc2 was localized in the spindle region. At the anaphase/telophase transition, phosphorylated cyclin B1 was accumulated in the midbody between the separating chromosomes/chromatids, while p34cdc2 was accumulated in the entire spindle except for the midbody region. At metaphase II, both cyclin B1 and p34cdc2 were horizontally localized in the region with the aligned chromosomes and the two poles of the spindle, while phosphorylated cyclin B1 was localized in the two poles of spindle and the chromosomes. We could not detect a particular distribution of cyclin B1 in fertilized eggs when the pronuclei were initially formed, but in late pronuclei cyclin B1 was accumulated in the pronuclei. p34cdc2 and phosphorylated cyclin B1 were always concentrated in one pronucleus after parthenogenetic activation or in two pronuclei after fertilization. At metaphase of 1-cell embryos, cyclin B1 was accumulated around the condensed chromosomes. Cyclin B1 was accumulated in the nucleus of late 2-cell embryos but not in early 2-cell embryos. Furthermore, we also detected the accumulation of p34cdc2 in the nucleus of 2- and 4-cell embryos. All these results show that cyclin B1, phosphorylated cyclin B1 and p34cdc2 have similar distributions at some stages but different localizations at other stages during oocyte meiotic maturation and fertilization, suggesting that they may play a common role in some events but different roles in other events during oocyte maturation and fertilization.     

Kinetochore appearance during meiosis,fertilization and mitosis in mouse oocytes and zygotes

The events of mammalian fertilization overlap with the completion of meiosis and first mitosis; the pronuclei never fuse, instead the parental genomes first intermix at the mitotic spindle equator at metaphase. Since kinetochores are essential for the attachment of chromosomes to spindle microtubules, this study explores their appearance and behavior in mouse oocytes, zygotes and embryos undergoing the completion of meiosis, fertilization and mitoses. Kinetochores are traced with immunofluorescence microscopy using autoimmune sera from patients with CREST (CREST = calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) scleroderma. These sera cross-react with the 17 kDa centromere protein (CENP-A) and the 80 kDa centromere protein (CENP-B) found at the kinetochores in human cell cultures. The unfertilized oocyte is ovulated arrested at second meiotic metaphase and kinetochores are detectable as paired structures aligned at the spindle equator. At meiotic anaphase, the kinetochores separate and remain aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis is lost. The female pronucleus and the second polar body nucleus each receive a detectable complement of kinetochores. Mature sperm have neither detectable centrosomes nor detectable kinetochores, and shortly after sperm incorporation kinetochores become detectable in the decondensing male pronucleus. In pronuclei, the kinetochores are initially distributed randomly and later found in apposition with nucleoli. At mitosis, the kinetochores behave in a pattern similar to that observed at meiosis or mitosis in somatic cells: irregular distribution at prophase, alignment at metaphase, separation at anaphase and redistribution at telophase. They are also detectable in later stage embryos. Colcemid treatment disrupts the meiotic spindle and results in the dispersion of the meiotic chromosomes along the oocyte cortex; the chromosomes remain condensed with detectable kinetochores. Fertilization of Colcemid-treated oocytes results in the incorporation of a sperm which is unable to decondense into a male pronucleus. Remarkably kinetochores become detectable at 5 h post-insemination, suggesting that the emergence of the paternal kinetochores is not strictly dependent on male pronuclear decondensation.     

Microtubule and centrosome distribution during sheep fertilization

The distribution of microtubules and centrosomes was studied during sheep fertilization by electron and immunofluorescence microscopy. Tubulin and centrosomal material was identified with monoclonal anti-alpha-tubulin and MPM-2 antibodies, respectively. In ovulated eggs, microtubules were exclusively found in the meiotic spindle and centrosomal material at each of its poles. At fertilization, sperm centrosomes were incorporated into the egg and organized the sperm astral microtubules. During pronuclear development and migration, the sperm aster increased in size; microtubules of the sperm aster extended from the male pronucleus to the egg center and towards the female pronucleus. The position of the sperm aster during pronuclear migration suggests that it plays a role in this process. When the pronuclei were in apposition in the egg center, a dense array of microtubules and the centrosomal material were present between the two pronuclei. The proximal centriole of the sperm was identified by electron microscopy, between the apposed pronuclei. The centrosomal material extending around the centriole and the sperm neck and proximal mid-piece, apparently contained several foci from which microtubules radiated. These data suggest that in sheep unlike in mice, centrosomal material originating from the sperm is involved in the fertilization events.     

Behavior of sperm nuclei in intact and bisected metaphase II mouse oocytes fertilized in the presence of colcemid

Our objective was to examine the developmental fate of sperm nuclei in oocytes fertilized under conditions of meiotic arrest. Therefore zona-free metaphase II oocytes and oocyte fragments (nucleate and anucleate) were fertilized in the presence of colcemid. In anucleate oocyte fragments, normal male pronuclei develop. In contrast, in intact oocytes and nucleate fragments sperm nuclei after initial decondensation undergo secondary condensation. This state is maintained as long as the oocytes are treated with colcemid. When the drug is removed 3 h after insemination, the meiotic spindle(s) is reconstructed, the second polar body(ies) is extruded, and a female pronucleus (or micronuclei) forms. At the same time the sperm nucleus decondenses again and transforms into a male pronucleus. In addition oocytes fertilized in the presence of colcemid could not be refertilized. These observations suggest that oocytes and oocyte fragments fertilized in the presence of colcemid undergo activation despite the failure of pronucleus formation. The inhibitory effect of colcemid on the formation of pronuclei is expressed only in the presence of oocyte chromosomes. We suggest that colcemid stabilizes factors responsible for chromosome condensation that are associated with oocyte chromosomes but not factors (whether the same or different) present in the cytoplasm.     

Cytoskeleton and chromatin reorganization in horse oocytes following intracytoplasmic sperm injection: patterns associated with normal and defective fertilization

Intracytoplasmic sperm injection (ICSI) is the method of choice for fertilizing horse oocytes in vitro. Nevertheless, for reasons that are not yet clear, embryo development rates are low. The aims of this study were to examine cytoskeletal and chromatin reorganization in horse oocytes fertilized by ICSI or activated parthenogenetically. Additional oocytes were injected with a sperm labeled with a mitochondrion-specific vital dye to help identify the contribution of the sperm to zygotic structures, in particular the centrosome. Oocytes were fixed at set intervals after sperm injection and examined by confocal laser scanning microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle and the first polar body. After sperm injection, an aster of microtubules formed adjacent to the sperm head and subsequently enlarged such that at the time of pronucleus migration and apposition it filled the entire cytoplasm. During syngamy, the microtubule matrix reorganized to form a mitotic spindle on which the chromatin of both parents aligned. Finally, after nuclear and cellular cleavage were complete, the microtubule asters dispersed into the interphase daughter cells. Sham injection induced parthenogenetic activation of 76% of oocytes, marked by the formation of multiple cytoplasmic microtubular foci that later developed into a dense microtubule network surrounding the female pronucleus. The finding that a parthenote alone can produce a microtubule aster, whereas the aster invariably forms at the base of the sperm head during normal fertilization, indicates that both gametes contribute to the formation of the zygotic centrosome in the horse. Finally, 25% of sperm-injected oocytes failed to complete fertilization, mostly due to absence of oocyte activation (65%), which was often accompanied by failure of sperm decondensation. In conclusion, this study demonstrated that union of the parental genomes in horse zygotes is accompanied by a series of integrated cytoskeleton-mediated events, failure of which results in developmental arrest.     

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.     

Maternal gamma (gamma)-tubulin is involved in microtubule reorganization during bovine fertilization and parthenogenesis

In this study, gamma-tubulin distribution was determined chronologically in conjunction with microtubule dynamics during bovine fertilization and parthenogenesis. In unfertilized bovine oocytes, gamma-tubulin was identified in the cytoplasm, mainly in the cortex and concentrated in the meiotic spindle. Following sperm penetration, gamma-tubulin in the cytoplasm was recruited by a sperm component. During pronuclear apposition, gamma-tubulin was localized as spots at the spindle poles. gamma-tubulin spots were observed in blastomeres of embryos cleaved in vitro. Following electrical stimulation, gamma-tubulin and microtubule matrix were noted in oocyte cortex. In the late pronuclear stage, considerably less gamma-tubulin and microtubules were detected in the cytoplasm. At the mitotic metaphase of parthenotes, gamma-tubulin was recruited to the condensed chromatin and concentrated in the spindle. The gamma-tubulin spots were not detected until the 8-cell stage of parthenotes. This suggests that maternal gamma-tubulin is recruited by a sperm component to reconstitute the zygotic centrosome. In the absence of sperm components, the cell cycle-related assembly of gamma-tubulin organizes microtubule nucleation for positioning the pronucleus and spindle protein of mitotic metaphase during the first cell cycle of bovine parthenotes.