黑眶蟾蜍卵母细胞体外成熟过程中生发泡迁移的研究(英文)

生发泡迁移（ＧＶＭ）是大多数两栖类动物中卵母细胞成熟之前都可以观察到的、涉及细胞核行为的现象。本实验在光镜水平上对激素诱导下的黑眶蟾蜍卵母细胞的ＧＶＭ现象、以及细胞骨架解聚剂类药物———秋水仙素、细胞松弛Ｂ（ＣＢ）对这种激素诱导作用的影响进行了研究。同时,采用ＡＺＡＮ染色法观察了ＧＶＭ过程中生发泡周边纤维骨架的结构变化。将取自刚脱离冬眠期雌体的卵母细胞按不同的培养液、分三个实验组,体外培养不同的时间后,固定、染色、观察。对照组培养液成分为Ｒｉｎｇｅｒ液中加入人绒毛膜促性腺激素和脑垂体;实验组分别增加秋水仙素或ＣＢ。Ｔａｂ．１和ＰｌａｔｅＩ１,４,５,６,７,８,９表明：经过体外培养４ｈ,各组生发泡均向动物极表面发生了迁移。但是,秋水仙素的作用在培养的前２ｈ,对ＧＶＭ表现为促进效应（ＰｌａｔｅＩ５）;而培养的后２ｈ,却表现为抑制（ＰｌａｔｅＩ８）;ＣＢ的作用始终是抑制（ＰｌａｔｅＩ６＆９）。６ｈ后,各组生发泡均告破裂（ＰｌａｔｅＩ１０,１１,１２）。正常情况下,生发泡周围被一环形纤维包围,其外侧有两个纤维化小体（ＰｌａｔｅＩ２）。发育较快者,纤维化小体消失,植物极附近纤维逐渐加厚（ＰｌａｔｅＩ２     

Oocyte Maturation in Starfish: Sequential Changes of Oocyte Shape Induced by 1-Methyladenine Associated with Germinal Vesicle Breakdown

Observation of the early events of starfish oocyte maturation revealed a sequential change of the oocyte shape induced by 1-methyladenine (1-MeAde). The lengths of two diameters of the whole oocyte, one parallel to the egg axis (a–v diameter) and the other which is perpendicular to a–v diameter (e diameter) were measured by taking photographs successively. About 10 min after 1–MeAde administration, a sudden decrease of the a–v diameter (shortening of oocyte) occurred followed by a sudden increase (elongation of oocyte). Germinal vesicle breakdown (GVBD) occurred when the a–v diameter suddenly increased. The change of the oocyte shape occurred differentially in the animal and vegital halves i.e. when the egg-axis diameter shortened the surface of the vegital pole side shrank and the surface of the animal pole side expanded. GVBD was suppressed under hypertonic conditions and facilitated under hypotonic conditions. Cytological examination of the process of GVBD revealed (1) separation of the nuclear membrane from the granular cytoplasmic mass, (2) depression of several parts of the nuclear membrane and the increase of amorphous matrix on the outside of the nuclear membrane, and (3) disappearance (fragmentation) of nuclear membrane. The morphological changes were not the same along the egg-axis temporally and spacially. These observations suggest firstly that the cytoplasm, which has been in a certain quiescent state with a certain rigidity before 1–MeAde administration, not only undergoes a decrease in stiffness but also acquires a contractile property after 1–MeAde administration, and secondly that contractile or cytoskeletal components do not evenly function or are not evenly arranged in the animal and vegital halves.     

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。     

Energy Metabolism and Pyridine Nucleotide Levels during Xenopus Oocyte Maturation*, 1

The rate of oxygen consumption increased in maturing Xenopus oocytes within 2 hr after progesterone addition, well before GVBD. This suggested an early requirement for energy metabolism during maturation, similar to the situation in sea urchin eggs during fertilization. Yet, the absence of similar increases in glucose-6-phosphate levels, glucose-6-phosphate dehydrogenase activity, glucose conversion to CO₂, and the conversion of NAD(H) to NADP(H), indicated that carbohydrate metabolism was not being stimulated in Xenopus oocytes during maturation. The oxidation of other energy yielding substrates is discussed which might account for the finding that, within 5 min of progesterone addition, both reduced forms of the pyridine nucleotides increased 20% over control levels. This was later followed by a drop in NADH levels and a rise in NAD relative to controls. The significance of these changes in pyridine nucleotide levels and their relationship to a number of maturation events are discussed.     

Accumulation of Annulate Lamellae in the Subcortical Layer during Progesterone-induced Oocyte Maturation in Xenopus Laevis

Changes in the fine structure, the location and the number of stacks of annulate lamellae during progesterone-induced maturation of oocytes of Xenopus were determined by electron microscopy. longitudinal sections of full-grown oocytes, about 260 stacks of annulate lamellae were observed with marked concentration in the subcortical layer, particularly in the vegetal hemisphere. After exposure to. progesterone, annulate lamellae increased and accumulated further in the subcortical layer. A significant increase of annulate lamellae around the vegetal side of the germinal vesicle seen 3 h after progesterone application. In oocytes 6 h after progesterone application, an average of 320 stacks of annulate lamellae were seen in longitudinal sections and more than two-thirds of the pore complexes of annulate lamellae were localized in the subcortical layer less than 50 from the oocyte surface, the rest being distributed in the deeper ooplasm. At the time of ger- minal vesicle breakdown, all the annulate lamellae underwent complete decomposition. The results were discussed from the view point of comparative developmental biology.     

Proteomics-Based Systems Biology Modeling of Bovine Germinal Vesicle Stage Oocyte and Cumulus Cell Interaction

Background

Oocytes are the female gametes which establish the program of life after fertilization. Interactions between oocyte and the surrounding cumulus cells at germinal vesicle (GV) stage are considered essential for proper maturation or ‘programming’ of oocytes, which is crucial for normal fertilization and embryonic development. However, despite its importance, little is known about the molecular events and pathways involved in this bidirectional communication.

Methodology/Principal Findings

We used differential detergent fractionation multidimensional protein identification technology (DDF-Mud PIT) on bovine GV oocyte and cumulus cells and identified 811 and 1247 proteins in GV oocyte and cumulus cells, respectively; 371 proteins were significantly differentially expressed between each cell type. Systems biology modeling, which included Gene Ontology (GO) and canonical genetic pathway analysis, showed that cumulus cells have higher expression of proteins involved in cell communication, generation of precursor metabolites and energy, as well as transport than GV oocytes. Our data also suggests a hypothesis that oocytes may depend on the presence of cumulus cells to generate specific cellular signals to coordinate their growth and maturation.

Conclusions/Significance

Systems biology modeling of bovine oocytes and cumulus cells in the context of GO and protein interaction networks identified the signaling pathways associated with the proteins involved in cell-to-cell signaling biological process that may have implications in oocyte competence and maturation. This first comprehensive systems biology modeling of bovine oocytes and cumulus cell proteomes not only provides a foundation for signaling and cell physiology at the GV stage of oocyte development, but are also valuable for comparative studies of other stages of oocyte development at the molecular level.     

In vitro Induction of Germinal Vesicle Breakdown in Xenopus laevis Oocytes by Melittin

The bee venom, melittin, is an amphipathic polypeptide comprising 26 amino acids with known sequence. It consists of a hydrophobic and a basic hydrophilic segment, possesses lipolytic activity, and stimulates Na⁺-K⁺ pump activity. At 1.5 μM melittin induces 98% germinal vesicle breakdown (GVBD) in stage VI (Dumont) oocytes and 96% in stage IV oocytes. Progesterone (30 μM) induced 100% GVBD in stage VI oocytes and none in stage IV oocytes. GVBD occurs earlier with melittin than with progesterone, i.e., 3 h compared to 5 h. An unusual morphologic change observed with melittin is the occurrence of mottling of the animal pole. The inner boundary of the melanin layer appears irregular with projections extending into the cytoplasm.
When stage VI oocytes were microinjected with 60 nl of 3 mM melittin only 48% showed GVBD indicating that the effectiveness of melittin was dependent upon the route of administration. On the other hand, 60% of stage VI oocytes underwent GVBD when microinjected with the cytosol fraction obtained from melittin-treated oocytes. Dissolution of isolated germinal vesicles did not occur when they were incubated in modified Earth's medium containing 3 mM melittin. The present results suggest that melittin induces GVBD by promoting the production of maturation promoting factor.     

CPEB and miR-15/16 Co-Regulate Translation of Cyclin E1 mRNA during Xenopus Oocyte Maturation

Cell cycle transitions spanning meiotic maturation of the Xenopus oocyte and early embryogenesis are tightly regulated at the level of stored inactive maternal mRNA. We investigated here the translational control of cyclin E1, required for metaphase II arrest of the unfertilised egg and the initiation of S phase in the early embryo. We show that the cyclin E1 mRNA is regulated by both cytoplasmic polyadenylation elements (CPEs) and two miR-15/16 target sites within its 3’UTR. Moreover, we provide evidence that maternal miR-15/16 microRNAs co-immunoprecipitate with CPE-binding protein (CPEB), and that CPEB interacts with the RISC component Ago2. Experiments using competitor RNA and mutated cyclin E1 3’UTRs suggest cooperation of the regulatory elements to sustain repression of the cyclin E1 mRNA during early stages of maturation when CPEB becomes limiting and cytoplasmic polyadenylation of repressed mRNAs begins. Importantly, injection of anti-miR-15/16 LNA results in the early polyadenylation of endogenous cyclin E1 mRNA during meiotic maturation, and an acceleration of GVBD, altogether strongly suggesting that the proximal CPEB and miRNP complexes act to mutually stabilise each other. We conclude that miR-15/16 and CPEB co-regulate cyclin E1 mRNA. This is the first demonstration of the co-operation of these two pathways.     

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.     

The Cytoskeleton and Nuclear Disassembly during Germinal Vesicle Breakdown in Starfish Oocytes

In response to maturation-inducing hormone, prophase-arrested oocytes of the starfish Pisaster ochraceus resume meiosis and undergo nuclear disassembly during a process referred to as germinal vesicle breakdown (GVBD). Time-lapse video recordings of maturing oocytes reveal that the nucleus lengthens along the animal-vegetal axis of the oocyte directly prior to GVBD. Neither taxol (10 μM) nor microtubule-depolymerizing agents [colcemid (50 μM), colchicine (250 μM), or nocodazole (1 μM)] prevent the pre-GVBD changes in nuclear shape from occurring, although correlative microscopical studies demonstrate that microtubules are nucleated (taxol) or depolymerized (colcemid, colchicine, nocodazole) at the concentrations listed above. The microtubule-altering drugs also do not affect the time at which GVBD begins or ends. A 10 μM solution of the microfilament-disrupting drug cytochalasin B (CB), on the other hand, essentially eliminates the pre-GVBD elongation of the nucleus. CB also slightly delays the onset of GVBD and significantly lengthens the time required to complete GVBD. Such studies suggest that: (i) drug-sensitive microtubules are not required for GVBD to proceed in a normal fasion; (ii) the pre-GVBD changes in nuclear shape involve microfilament-mediated events; and (iii) cytochalasin-induced depolymerization of microfilaments retards the normal timing of GVBD.     

Assessment of Mouse Germinal Vesicle Stage Oocyte Quality by Evaluating the Cumulus Layer,Zona Pellucida,and Perivitelline Space

To improve the outcome of assisted reproductive technology (ART) for patients with ovulation problems, it is necessary to retrieve and select germinal vesicle (GV) stage oocytes with high developmental potential. Oocytes with high developmental potential are characterized by their ability to undergo proper maturation, fertilization, and embryo development. In this study, we analyzed morphological traits of GV stage mouse oocytes, including cumulus cell layer thickness, zona pellucida thickness, and perivitelline space width. Then, we assessed the corresponding developmental potential of each of these oocytes and found that it varies across the range measured for each morphological trait. Furthermore, by manipulating these morphological traits in vitro, we were able to determine the influence of morphological variation on oocyte developmental potential. Manually altering the thickness of the cumulus layer showed strong effects on the fertilization and embryo development potentials of oocytes, whereas manipulation of zona pellucida thickness effected the oocyte maturation potential. Our results provide a systematic detailed method for selecting GV stage oocytes based on a morphological assessment approach that would benefit for several downstream ART applications.     

Polysaccharide Complexes in Full-Grown Oocytes of the Newt, Pleurodeles, and Changes in their Distribution During Progesterone-Induced Maturation

Immature full-grown oocytes of Pleurodeles waltlii contain large amounts of small electron-dense polysaccharidic granules. These granules lack a limiting membrane, and have a dense but heterogeneous matrix and an apparent diameter of 24–36 nm. Their structure, organization and distribution strongly suggest that they are glycogen granules. On the other hand, mature oocytes both after oviposition or 22–24 hr after in vitro progesterone stimulation contain no polysaccharide granules or complexes. During the first 9–10 hr after hormonal stimulation, granules were abundant and present both individually and as large strands occupying most of the space between the organelles. Granules were frequently found packed together and arranged in regularly arrayed stacks within large subcortical ant cortical vacuoles. Between 4 and 6 hr after progesterone addition, oocytes released the contents of vacuoles to the outside. Between about 11 and 14 hr after progesterone addition, oocytes still contained large amounts of polysaccharide complexes, but the vacuoles were empty. From about 15 hr after progesterone treatment until the end of maturation, the complexes progressively disappeared from the cytoplasm, coincident with the detachment of the follicle cell layer from the oocytes and a reduction in the number and size of microvilli.     

哺乳动物卵母细胞成熟和受精中细胞骨架重组和作用

卵母细胞成熟和受精是动物生殖过程的核心环节。细胞骨架是遍布于卵母细胞胞质中的一种复杂的蛋白质纤维网络,研究表明,卵母细胞成熟和受精过程中伴随着广泛的胞质骨架重组。哺乳动物卵母细胞和早期胚胎中细胞骨架具有其独特的分布和功能,使卵母细胞和胚胎呈现出不同的变化特点。微丝、微管的分布变化与卵母细胞成熟和受精中遗传物质的重组密切相关。近年来,对哺乳动物不同物种间卵母细胞和胚胎中细胞骨架成分的研究取得了很大的进展,结合这些研究成果,对哺乳动物卵母细胞成熟和受精过程中细胞骨架的重组、分布和作用进行了介绍。同时,对多种信号转导途径参与卵母细胞成熟和受精中细胞骨架系统的调控也作了探讨。     

Multiple Requirements of PLK1 during Mouse Oocyte Maturation

Polo-like kinase 1 (PLK1) orchestrates multiple events of cell division. Although PLK1 function has been intensively studied in centriole-containing and rapidly cycling somatic cells, much less is known about its function in the meiotic divisions of mammalian oocytes, which arrest for a long period of time in prophase before meiotic resumption and lack centrioles for spindle assembly. Here, using specific small molecule inhibition combined with live mouse oocyte imaging, we comprehensively characterize meiotic PLK1’s functions. We show that PLK1 becomes activated at meiotic resumption on microtubule organizing centers (MTOCs) and later at kinetochores. PLK1 is required for efficient meiotic resumption by promoting nuclear envelope breakdown. PLK1 is also needed to recruit centrosomal proteins to acentriolar MTOCs to promote normal spindle formation, as well as for stable kinetochore-microtubule attachment. Consequently, PLK1 inhibition leads to metaphase I arrest with misaligned chromosomes activating the spindle assembly checkpoint (SAC). Unlike in mitosis, the metaphase I arrest is not bypassed by the inactivation of the SAC. We show that PLK1 is required for the full activation of the anaphase promoting complex/cyclosome (APC/C) by promoting the degradation of the APC/C inhibitor EMI1 and is therefore essential for entry into anaphase I. Moreover, our data suggest that PLK1 is required for proper chromosome segregation and the maintenance of chromosome condensation during the meiosis I-II transition, independently of the APC/C. Thus, our results define the meiotic roles of PLK1 in oocytes and reveal interesting differential requirements of PLK1 between mitosis and oocyte meiosis in mammals.     

Reorganization of the Endoplasmic Reticulum during Meiotic Maturation of the Mouse Oocyte

The endoplasmic reticulum (ER) of live metaphase II mouse eggs and prophase I-arrested oocytes was compared using the fluorescent, lipophilic dicarbocyanine dye, DiI. DiI, dissolved in soybean oil, was microinjected into oocytes and eggs; the dye diffused throughout the cytoplasm to label the ER, which was imaged by confocal microscopy. The mature egg had a fine reticular network of ER throughout the cell and numerous dense accumulations of membrane in the cortex. These ER accumulations, 1-2 μm in diameter, were generally absent deeper in the cytoplasm. A similar staining pattern was observed when the eggs were fixed within 1 min of injection, providing evidence that the cortical accumulations of membrane are part of a continuous ER membrane system, since membrane trafficking could not occur in a fixed egg. Cortical ER accumulations were localized to the same region of the egg as the cortical granules and were not observed in the cortical granule-free region adjacent to the meiotic spindle. In contrast, ER accumulations were rarely found in the cortex of the immature, prophase I-arrested oocyte, but larger and less well-defined membrane clusters were found throughout the deeper cytoplasm of the oocyte. The appearance of ER clusters in the egg cortex following oocyte maturation correlates with an increased ability of the mature egg to release calcium at fertilization. Since the ER is a calcium store, structural reorganization of the ER may be necessary to permit the large release of calcium and resulting cortical granule exocytosis at fertilization.     

Cdc42和球形肌动蛋白在卵母细胞胞质分裂中的定位分析

研究活性Cdc42与球形肌动蛋白(G-actin)在爪蟾卵母细胞胞质分裂中的定位关系。分别用GFP-wGBDmRNA与罗丹明-594-微管蛋白、Alexa-488-球形肌动蛋白与罗丹明-594-微管蛋白、GFP-wGBDmRNA与Alexa-594-球形肌动蛋白共同显微注射爪蟾卵母细胞。利用共聚焦显微镜,时间延迟摄影方法,分别观察活体卵母细胞中活性Cdc42、球形肌动蛋白在胞质分裂过程中的定位,以及活性Cdc42与球形肌动蛋白在胞质分裂中的定位关系。在卵母细胞胞质分裂中,活性Cdc42与球形肌动蛋白存在空间上共定位现象,并且在时相上具有一致性。结果提示活性Cdc42和球形肌动蛋白在卵母细胞胞质分裂过程中密切相关。