线粒体与卵母细胞发育

卵子发育、成熟是一个复杂的过程, 细胞核成熟和细胞质成熟过程必须同步化, 才能保证卵子的正常受精和进一步的发育。作为细胞质内最重要的细胞器, 线粒体在卵子成熟过程中的分布的变化、氧化磷酸化产生ATP的能力以及线粒体ＤＮＡ的含量和拷贝数或转录水平对卵母细胞发育成熟有着重要的影响。因此, 对卵子成熟过程中线粒体的分布和功能状况及线粒体DNA的研究, 有利于进一步了解生殖生理, 并为解决辅助生殖技术中及克隆胚胎技术所面临的困难提供新的思路。     

牛卵母细胞体外成熟的研究

牛卵母细胞的成熟过程中,包括细胞膜、细胞质、细胞核的成熟。其中细胞质的成熟最为复杂。线粒体、皮质颗粒数量的变化和位移,脂滴类型的变化和形态改变,空泡形态学的变化等是鉴别卵母细胞幼稚、成熟和老化的重要特征。透明带随着培养而外侧疏松,内侧致密,母卵细胞膜上伸出的微绒毛为膨大泡状和细长毛状两种。在培养１４小时后颗粒细胞与透明带脱离联系。根据综合指标判定,１８小时这前为成熟生长期,１８￣２６小时为成熟期,     

隆线溞精子发生及成熟精子的超微结构

用透射电镜和扫描电镜观察隆线精子的发生过程及成熟精子的超微结构。隆线精子发生经历精原细胞、精母细胞、精子细胞和成熟精子四个时期。精原细胞核染色质凝集成团,细胞质内有线粒体、粗面内质网分布。精母细胞核染色质分散,不均匀地分布于核中,细胞质内粗面内质网聚集。在精子细胞分化形成精子的早期,细胞纵向拉伸,核物质开始浓缩;中期精子细胞呈明显的长条形,精子细胞逐渐移入精巢管腔中央,外围包裹一厚层精子鞘;后期精子细胞已进入管腔中,核物质呈高度浓缩状,细胞质层较少,精子细胞间通过外围精子鞘相互粘连成片。成熟精子分散在精巢管腔中央,外形呈棒状,一端稍钝,一端稍尖,无鞭毛、棘突等附属物;核内染色质解聚,均匀分布在核中,具双层核膜,细胞质层很少,精子鞘为单层,无法确认顶体端。隆线雄性生殖细胞的结构及其发生过程均较高等甲壳动物简单和原始,但在功能上表现出相对的适应性,使以隆线为代表的枝角类能适应复杂多变的生活环境     

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。     

Hira基因产物在银鲫和彩鲫卵子发生过程中的动态变化

为进一步研究Hira基因在卵子发生和雌核发育过程中的作用,通过原位杂交和免疫荧光定位的方法检测了Hira mRNA和蛋白质在雌核发育银鲫和两性生殖彩鲫卵子发生过程中的动态变化。结果表明,银鲫和彩鲫卵子发生过程中Hira基因转录产物的变化基本一致,在Ⅰ期卵母细胞的细胞核中大量表达,至Ⅱ期卵母细胞时转至细胞质中均匀分布,在Ⅲ期卵母细胞中,杂交信号逐渐移向细胞的周边,到Ⅳ期时随着卵黄物质大量积累,杂交信号几乎不见。HIRA蛋白在银鲫和彩鲫卵子发生过程中的变化略有差别。HIRA蛋白在银鲫Ⅰ期卵母细胞中没有表达,在Ⅱ期卵母细胞的细胞质中有弱表达,在Ⅲ期早期卵母细胞的周边有强烈表达;而在彩鲫Ⅰ期卵母细胞中就有HIRA蛋白的弱表达,至Ⅱ期时HIRA蛋白在细胞质中大量表达,Ⅲ期早期卵母细胞的细胞质中有弱表达。在银鲫和彩鲫Ⅲ期末和Ⅳ期卵母细胞中都很难观察到荧光信号。Hira mRNA和蛋白质在银鲫和彩鲫早期卵母细胞中有较强表达,且在银鲫和彩鲫卵子发生过程中没有显著差异,说明其可能对于脊椎动物卵子发生和减数分裂没有显著影响,而是在受精和/或胚胎发育过程中起作用。       

扁豆成熟胚囊的超微结构

本文对扁豆（Dolichos lablab)成熟胚囊的超微结构进行了研究,在成熟胚囊中,卵细胞和助细胞仅在珠孔端1／3有细胞壁,靠近合点端,卵细胞一助细胞,卵细胞－中央细胞,助细胞－中央细胞之间没有细胞壁存在,相邻细胞的质膜靠在一起,在卵细胞和中央细胞的质膜间,有些地方存在中等电子密度的物质,卵细胞的细胞质中含有很多的线粒体和质体,内质网和高尔基体较少,助细胞的珠孔端有一复杂的丝状器,靠近珠孔端的细胞质中有很多管状的内质网,表明助细胞可能具有分泌功能,在助细胞的合点端,含有丰富的粗糙内质网,助细胞和卵细胞的质膜之间有很多囊泡状的结构,中央细胞内含有丰富的线粒体,高尔基体和内质网,中央细胞的壁向内形成突起,在周缘细胞质中含有丰富的脂滴。     

棉花小孢子发生过程中细胞质的超微结构变化：着重“细胞质改组”问题

棉花(Gossypium hirsutum L.)小孢子发生过程中,细胞质超微结构发生显著而有规律的变化。这些变化主要涉及细胞质中核糖体、质体和线粒体。减数分裂前期Ⅰ,细胞质中核糖体密度逐渐降低,质体和线粒体结构变得不明显。粗线期至双线期,细胞质中核糖体密度降至极低水平,同时质体和线粒体呈衰退结构状态。中期Ⅰ,细胞质中核糖体恢复致密,质体和线粒体也恢复了正常的形态和结构。来自细胞核的类核仁进入细胞质并扩散。这是恢复中期Ⅰ细胞质中核糖体密度的主要原因。内质网在核糖体数量变化中显示出有重要作用。这些细胞质超微结构的变化可认为与世代转变有关。     

小鼠卵母细胞成熟和受精过程中Ca^2＋分布变化的研究

用焦锑酸钾原位定位法、膜结合Ｃａ＾２＋荧光探针金霉素标记法,分别在电镜和光镜水平对小鼠卵成熟和卵受精过程中结合态Ｃａ＾２＋的分布及其变化进行了研究,发现：１）Ｃａ＾２＋分布于线粒体、胞质、内质网囊泡、微绒毛和透明带等部位,其中以线粒体基质中分布密度为最大;２）减数分裂Ｉ中、后期于纺锤体极区结合有较多的Ｃａ＾２＋;３）生发泡、纺锤体和原核内膜结合态Ｃａ＾２＋含量很少,但纺锤体和原核周围分布较多;４）     

灵长类卵母细胞体外发育潜能的调控

灵长类卵母细胞细胞质成熟调控研究较为滞后,使得体外成熟的灵长类卵母细胞的胚胎发育潜能十发低下。提高其潜能对治疗人类不育症有重要的应用价值,并可推动灵长类胚胎发育的研究。基于猕猴卵母细胞质成熟调控研究,讨论了无血清成熟培养基中雌激素和孕激素、能量物质、氨基酸对卵母细胞质成熟的影响,以及动物年龄和生殖周期与发育潜能的关系。从分子水平进一步解释这些因素的作用,阐明卵母细胞质成熟的机理将是今后工作的方向。     

中华蚱蜢精子发生过程中花生凝集素受体的分布与变化

采用生物素标记的花生凝集素(PNA)对中华蚱蜢(Acrida cinerea)精子发生过程中细胞质膜的糖蛋白(含有糖基Gal--β(1,3)-GalNAC)进行了标记,旨在认识精子发生过程中细胞质膜糖复合物的形成与变化规律,探讨其所具有的功能。结果表明,在中华蚱蜢的精子发生中,精母细胞能够自身合成PNA受体;精子细胞到成熟精子时期生精细胞质膜糖复合物发生了明显的修饰变化,这些变化与精子获得及卵子进行识别、粘附、结合等受精能力密切相关。     

Redistribution of mitochondria leads to bursts of ATP production during spontaneous mouse oocyte maturation

During mammalian oocyte maturation there are marked changes in the distribution of mitochondria that supply the majority of the cellular ATP. Such redistribution of mitochondria is critical for oocyte quality, as oocytes with a poor developmental potential display aberrant mitochondrial distribution and lower ATP levels. Here we have investigated the dynamics of mitochondrial ATP production throughout spontaneous mouse oocyte maturation, using live measurements of cytosolic and mitochondrial ATP levels. We have observed three distinct increases in cytosolic ATP levels temporally associated with discrete events of oocyte maturation. These changes in cytosolic ATP levels are mirrored by changes in mitochondrial ATP levels, suggesting that mitochondrial ATP production is stimulated during oocyte maturation. Strikingly, these changes in ATP levels correlate with the distribution of mitochondria undergoing translocation to the peri‐nuclear region and aggregation into clusters. Mitochondrial clustering during oocyte maturation was concomitant with the formation of long cortical microfilaments and could be disrupted by cytochalasin B treatment. Furthermore, the ATP production bursts observed during oocyte maturation were also inhibited by cytochalasin B suggesting that mitochondrial ATP production is stimulated during oocyte maturation by microfilament‐driven, sub‐cellular targeting of mitochondria. J. Cell. Physiol. 224: 672–680, 2010. © 2010 Wiley‐Liss, Inc.     

The consequences of metabolic changes in high-yielding dairy cows on oocyte and embryo quality

Unsatisfactory reproductive performance in dairy cows, such as reduced conception rates, in addition to an increased incidence of early embryonic mortality, is reported worldwide and has been associated with a period of negative energy balance (NEB) early post partum. Typically, NEB is associated with biochemical changes such as high non-esterified fatty acid (NEFA), high β-hydroxybutyrate (β-OHB) and low glucose concentrations. The concentrations of these and other metabolites in the follicular fluid (FF) of high-yielding dairy cows during NEB were determined and extensively analyzed, and then were replicated in in vitro maturation models to investigate their effect on oocyte quality. The results showed that typical metabolic changes during NEB are well reflected in the FF of the dominant follicle. However, the oocyte seems to be relatively isolated from extremely elevated NEFA or very low glucose concentrations in the blood. Nevertheless, the in vitro maturation models revealed that NEB-associated high NEFA and low glucose levels in the FF are indeed toxic to the oocyte, resulting in deficient oocyte maturation and developmental competence. Induced apoptosis and necrosis in the cumulus cells was particularly obvious. Furthermore, maturation in saturated free fatty acid-rich media had a carry-over effect on embryo quality, leading to reduced cryotolerance of day 7 embryos. Only β-OHB showed an additive toxic effect in moderately hypoglycemic maturation conditions. These in vitro maturation models, based on in vivo observations, suggest that a period of NEB may hamper the fertility of high-yielding dairy cows through increased NEFA and decreased glucose concentrations in the FF directly affecting oocyte quality. In addition to oocyte quality, these results also demonstrate that embryo quality is reduced following an NEB episode. This important observation may be linked to the typical diet provided to stimulate milk yield, or to physiological adaptations sustaining the high milk production. Research into this phenomenon is ongoing.     

Cytoplasmic remodelling and the acquisition of developmental competence in pig oocytes

The progression of oocyte meiosis is accompanied by major changes in the ooplasm that play a key role in the completion of a coordinate nuclear and cytoplasmic maturation. We review evidence from the literature and present data obtained in our laboratory on different aspects of pig oocyte cytoplasm compartmentalization during maturation and early embryo development. In particular, we will discuss the changes in adenosine triphosphate (ATP) concentration and distribution taking place during the maturation process and their possible significance for oocyte developmental competence. We describe two important aspects of cytoplasmic streaming: mitochondrial distribution patterns in oocytes and early embryos and the complex rearrangements of cytoplasmic microtubule networks, while discussing their possible correlations with ooplasm compartmentalization. Recent evidence indicates that the cytoskeleton is used to shuttle not only organelles but also mRNAs to specific sites within the oocyte cytoplasm. Localization is driven by specific molecular motors belonging to the kinesin superfamily and requires the involvement of the RNA targeting molecule Staufen. We present recent experimental evidence, obtained in our laboratory, on the pig orthologues for kinesin KIF5B and Staufen, describe their expression patterns and discuss their possible role in oocyte maturation.     

Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp.MPF activity was measured by using histone H1 as phosphorylation substrate.There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes,but there existed some subtle difference between them.The subtle difference was that the first peak of MPF kinase activity was kept to a longerlasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp.It was suggested that the difference may be related to the spindle behavior changes,such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.     

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。     

Increased sensitivity and clustering of elementary Ca2+ release events during oocyte maturation

The universal signal for egg activation at fertilization is a rise in cytoplasmic Ca(2+) with defined spatial and temporal kinetics. Mammalian and amphibian eggs acquire the ability to produce such Ca(2+) signals during a maturation period that precedes fertilization and encompasses resumption of meiosis and progression to metaphase II. In Xenopus, immature oocytes produce fast, saltatory Ca(2+) waves that can be oscillatory in nature in response to IP(3). In contrast, mature eggs produce a single continuous, sweeping Ca(2+) wave in response to IP(3) or sperm fusion. The mechanisms mediating the differentiation of Ca(2+) signaling during oocyte maturation are not well understood. Here, I characterized elementary Ca(2+) release events (Ca(2+) puffs) in oocytes and eggs and show that the sensitivity of IP(3)-dependent Ca(2+) release is greatly enhanced during oocyte maturation. Furthermore, Ca(2+) puffs in eggs have a larger spatial fingerprint, yet are short lived compared to oocyte puffs. Most interestingly, Ca(2+) puffs cluster during oocyte maturation resulting in a continuum of Ca(2+) release sites over space in eggs. These changes in the spatial distribution of elementary Ca(2+) release events during oocyte maturation explain the continuous nature and slower speed of the fertilization Ca(2+) wave.     

Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro

Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh-like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue-shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 μm during maturation and increased to 106 μm after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP-predominant pattern to a MV-predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination. © 1994 Wiley-Liss, Inc.     

Responses of oocytes and embryos to the culture environment

Embryo development is strongly influenced by events occurring during oocyte maturation. Although many immature oocytes are capable of completing meiosis in vitro, only a small percentage of the original pool of immature oocytes is competent to continue development to the blastocyst stage and subsequently result in a pregnancy. This indicates that maturation of oocytes in vitro may not be occurring in an entirely normal manner. Cytoplasmic changes occurring during maturation, collectively termed cytoplasmic maturation, are essential for embryonic development. The cytoplasm of the oocyte may play a crucial role in assembling the correct metabolic machinery for production of sufficient energy for cellular functions during maturation, cleavage and blastocyst formation. A better understanding of the structural, functional and metabolic characteristics of the oocyte during maturation, and the consequence of changes in these parameters on developmental competence is needed. Understanding the role of cytoplasmic changes during oocyte maturation will help increase the efficiency of in vitro embryo production. Better embryo production strategies will facilitate basic research into the control of early development, improve implementation in endangered species, provide a source of high quality oocytes for nuclear transfer and transgenic technologies and benefit the commercial embryo transfer industry.     

The mare model for follicular maturation and reproductive aging in the woman

Reproductive aging and assisted reproduction are becoming progressively more relevant in human medicine. Research with human subjects is limited in many aspects, and consequently animal models may have considerable utility. Such models have provided insight into follicular function, oocyte maturation, and reproductive aging. However, models are often selected based on factors other than physiological or functional similarities. Although the mare has received limited attention as a model for reproduction in women, comparisons between these species indicate that the mare has many attributes of a good model. As the mare ages, cyclic and hormonal changes parallel those of older women. The initial sign of reproductive aging in both species is a shortening of the reproductive cycle with elevated concentrations of FSH. Subsequently, cycles become longer with intermittent ovulations and elevated concentrations of FSH and LH. Reproduction ceases with failure of follicular growth and elevated gonadotropins, apparently because of ovarian failure. In the older woman and mare, oocytes have been maintained in meiotic arrest for decades -- approximately four to five for the woman and two to three for the mare; in both species, reduced oocyte quality is the end factor identified in age-associated infertility. After induction of oocyte maturation in vivo, the timeline to ovulation is the same for the mare and woman, suggesting a comparable sequence of events. The mare's anatomy, long follicular phase and single dominant follicle provide a foundation for studies in oocyte and follicular development. The aim of this review is to evaluate the mare as an animal model to study age-associated changes in reproduction and to improve our understanding of oocyte and follicular maturation in vivo.     

Proteins implicated in sperm capacitation

Spermatozoa after being deposited in the female reproductive tract spend a considerable time in this foreign environment prior to fertilization of the oocyte. Chang and Austin independently observed1,2 that this time spent by the spermatozoa in the female tract is not consequential but a necessary event in the life cycle of the male gamete, and Austin2 first called this maturation period of spermatozoa as 'Capacitation'. Ever since, attempts have been made to understand and unravel the molecular mechanism of capacitation. Based on the results obtained so far, it is clear that capacitation is guided by novel signal transduction pathways influencing varied aspects of spermatozoa. Capacitation could be, thus, defined as the cumulative molecular, cellular and physiological changes that occur in spermatozoa in the female reproductive tract to achieve the final competence to fertilize the oocyte. This review is structured so as to first understand the key features of capacitation and then to survey the players which bring about these changes during capacitation.