绵羊卵泡成分对卵母细胞体外减数分裂调控的研究

哺乳动物卵巢中的卵母细胞一直处于减数分裂的停滞状态,卵泡内各成分被认为是产生抑制因子的主要来源。本研究以绵羊卵泡各成分为研究对象,用共培养的方法对卵丘细胞、颗粒细胞、膜细胞在卵母细胞体外减数分裂过程中的作用加以探讨。结果表明：1．卵泡整体及卵泡分泌物在体外可以有效地维持减数分裂停滞,经过24h培养,这两个处理组中,处于GV期的卵母细胞分别为69．6％和49．1％。经抑制处理后的卵母细胞脱离抑制环境后可以继发成熟,MⅡ比率可达88．9％。去掉卵丘细胞的裸卵其减数分裂过程不能被卵泡分泌物有效抑制,24h培养后其GV期比例为17．8％。以上结果说明卵泡中的抑制因子主要是通过卵丘细胞束发挥其调控作用的。2．用颗粒细胞与卵母细胞共培养,结果发现具有颗粒细胞卵丘细胞缝隙连接的卵母细胞(COCGs)在培养24小时后47．4％达到MⅡ,与在不具有细胞连接的总浮颗粒细胞中共培养的卵母细胞之间存在无显差异,无论是紧密连接的颗粒细胞层还是悬浮在培养液中的颗粒细胞都不能有效抑制生发泡破裂(GVBD)的发生,只能将卵母细胞抑制在MⅡ以前的各个时期。以上结果说明颗粒细胞在体外分泌抑制图子的活力大大下降。3．卵泡膜细胞具有分泌抑制成熟分裂因子的能力,与膜细胞层共培养的卵母细胞在8h和24h时,其GV期的比例为34．4％和32．7％,显高于没有膜细胞层的对照组(4．5％和1.1％)。综上所述,绵羊卵泡中的抑制因子不仅来自于颗粒细胞,而且膜细胞也参与了成熟分裂的抑制,这些细胞在体外仍具有分泌抑制因子的能力,只是与体内分泌能力有所不同。     

环磷酸腺苷参与哺乳动物卵泡发育的研究进展

环磷酸腺苷(cyclic adenosine monophosphate,cAMP)是哺乳动物体内重要且保守的第二信使之一,可通过转导细胞外信号参与调节多种器官和组织的发育及生理功能.已有研究显示,雌性哺乳动物卵母细胞减数分裂进程与cAMP的水平变化密切相关且受到严格调控.卵母细胞中cAMP主要由腺苷酸环化酶3(ade...     

大熊猫卵泡及卵母细胞发育的研究

本文用５只大熊猫卵巢作了连续切片观察,测量分析了卵泡、卵母细胞和透明带的生长发育与成熟过程,以及它们之间的相互关系：（１）大熊猫卵泡及卵母细胞具哺乳类动物双相生长的共同特点,在生长过程中,仅 极少数优势卵泡被确定下来,其余卵泡均在不同时期成为闭锁卵泡,特别在发情期闭锁卵泡黄体化及囊肿现象居多。（２）透明带出现在单层卵泡细胞呈低柱状至柱状期,随着卵泡及卵母细胞的增长,透明带缓慢增厚。     

原始卵泡库的形成、维持和激活分子调控机制研究进展及其临床应用

卵泡是哺乳动物卵巢结构和功能的基本单位,由位于中央的卵子和周围的体细胞组成。原始卵泡是卵泡发育的最早阶段,在哺乳动物出生前后形成。由于数量有限,原始卵泡构成了卵巢的储备库,为女性的生殖周期提供成熟卵子。原始卵泡在大部分时间内处于休眠状态,直到被激活后开始生长和发育,最终成为可排卵的成熟卵泡。原始卵泡休眠与激活的动态平衡对于卵巢生殖功能的维持非常重要,而各种因素导致的原始卵泡库提前耗竭则可能引起女性的早发性卵巢功能不全。该文系统总结了过去二十年关于原始卵泡形成和激活分子机制的研究进展,全面概述了原始卵泡体外激活技术的发展和在治疗早发性卵巢功能不全或卵巢功能障碍导致的不孕症患者中的临床应用。此外,该文就如何调节原始卵泡库来保护卵巢储备、延缓卵巢衰老方面的研究也进行了讨论,为诊断和治疗与卵泡发育相关的疾病、卵巢性不孕不育的干预以及女性生育力保存提供了新的思路。     

小鼠卵巢冷冻移植后卵泡发育和卵母细胞成熟的研究

本研究探讨了冷冻保存的1日龄小鼠卵巢异体异位移植后,其原始卵泡重新启动生长发育的能力。一日龄B6C2F．小鼠卵巢分离冷冻后置液氮中保存,保存1周。6个月后解冻,并将卵巢移植到8-12周龄B6C2F．受体鼠。肾脏包膜下,移植至少14d。每侧肾囊移植2枚卵巢的40只受体鼠中卵巢的回收率为45．00％（72／160）,而每侧。肾囊移植l枚卵巢的20只受体鼠的回收率为82．50％（33／40）。移植卵巢上卵泡的发育基本与体外自然生长鼠的卵巢卵泡发育情况一致。对卵巢移植19d的受体鼠用孕马血清促性腺激素（pregnant mare serum gonadotrophin,PMSG）处理后,从移植卵巢上发育成熟卵泡中获得的卵母细胞在MEM0c培养基中培养16-17h,有40．90％的卵母细胞发生生发泡破裂（germinal vesicle breakdown,GVBD）,其中89．02％的卵母细胞发育到第二次减数分裂中期（metaphaseⅡ,MⅡ）。将剩余的卵母细胞继续培养到20～21h,又有50．83％的卵母细胞发生生发泡破裂,但其中只有21．40％的卵母细胞能够发育到MII期。以上结果说明,小鼠早期卵巢经过冷冻．解冻并异体异位移植后,其原始卵泡能够重新启动生长发育,发育后的卵泡卵母细胞能够在体外培养成熟。这些结果意味着原始卵泡或卵巢冷冻一移植技术有可能充分利用雌性生殖细胞用于濒危动物保种、建立动物基因库和人类辅助生殖等。     

缝隙连接在小鼠卵泡发育过程中的作用

缝隙连接广泛分布于各组织细胞中,由其构成的胞间通道允许小分子在胞间直接传递,在胞间通讯方面起着重要的作用。缝隙连接由连接蛋白(Cx)组成,已发现Cx家族有20多个成员。在哺乳动物卵泡发育过程中,卵母细胞与周围的颗粒细胞之间形成的缝隙连接,介导胞间通讯,对原始生殖细胞迁移、卵母细胞减数分裂能力恢复、颗粒细胞分层、卵泡成腔、黄体形成、促性腺激素信号传递均有非常重要的调节作用。本文根据近年来相关的研究报道,总结了不同的缝隙连接在小鼠卵泡发育过程中的调节作用。     

人重组卵泡刺激素和表皮生长因子对性未成熟小鼠卵母细胞和颗粒细胞复合体体外发育的作用

卵泡刺激素（FSH）对有腔卵泡和排卵前卵泡的促生命作用已被普遍接受,但关于其对腔前卵泡发育的作用报道结果不尽相同,关于表皮生长因子（EGF）对腔前卵泡的作用尚不确切,本研究目的在于探讨人重组卵泡刺激素（rechFSH)和EGF对早期卵泡发育的作用,利用胶原酶消化法从12日龄的小鼠卵巢中分离得到卵母细胞－颗粒细胞复合体（OGCs)(Fig.1)。体外每孔30－40个培养物并分别添加胎牛血清（FBS）,rechFSH和EGF。培养物每4天测量卵母细胞和OGCs直径,并每天照相,结果显示,rechFSH显著促进小鼠OGCs 及其卵母细胞的体外发育,这一作用可被EGF进一步增强（P＜0．05）（Fig.2),但到第八天培养结束时,培养后的OGCs卵母细胞要显著小于体内期生长对照组（P＜0．05）（Fig.3),说明FSH和EGF在卵泡早期发育中起重要作用。     

牦牛卵泡细胞及其卵母细胞不同发育时期的结构变化

采集成年母牦牛卵巢,通过光镜和电镜对牦牛卵泡及其卵母细胞不同发育时期的结构变化进行了观察。结果发现当卵母细胞被单层立方卵泡细胞包围时,微绒毛开始出现,而皮质颗粒、透明带则在包被2-4层卵泡细胞时开始出现。随着卵母细胞的继续发育,透明带增厚,微绒毛由粗短变为细长,密度增加;皮质颗粒、线粒体、滑面内质网等细胞器的数目不断增加,并逐渐移行到质膜下;在移行的过程中,皮质颗粒成团存在。在囊状卵泡中,卵母细胞皮质颗粒呈线形分布于质膜下,线粒体、滑面内质网又移向胞质中央。卵母细胞借助微绒毛穿过透明带与卵泡细胞胞质突起相联系。结果证明牦牛卵泡和卵母细胞不同发育时期的结构变化与其它哺乳动物的基本相似。     

哺乳动物卵泡发育模式及其调控机理

了解卵泡发育动脉及其调控机理对于提高动物繁殖率和控制人类生育,都具有重要的意义。近年来随着超声波扫描技术的发展,人们对家畜卵泡发育模式及其调控机理有了更深入的认识。本文简要综述了当前在家畜卵泡发育基本模式,参与卵泡发育调节的各种因子及卵泡发育的种属特异性等方面的研究进展。     

处理方式不同的颗粒细胞和卵泡液对牛卵母细胞体外受精后卵裂率和囊胚率的影响

将牛的卵母细胞置于添加有不同预处理颗粒细胞及含有卵泡液的培养液或不舍有卵泡液的培养液中进行体外成熟、受精及胚胎发育培养,研究了颗粒细胞、卵泡液及颗粒细胞与卵泡液交互作用对牛卵母细胞成熟、受精后卵裂率、囊胚率的影响。2178枚卵母细胞体外成熟受精后胚胎发育的对比观察结果表明：颗粒细胞、卵泡液及颗粒细胞与卵泡液交互作用对卵母细胞成熟受精后胚胎的卵裂具有显著影响（P〈0．05）;颗粒细胞对囊胚的发育有显著影响（P〈0．05）;卵泡液及颗粒细胞与卵泡液交互作用对囊胚的发育无显著影响（P〉0．05）。不同因素对卵裂率、囊胚率的影响表现为：颗粒细胞因素〉培养液因素〉培养液×颗粒细胞交互作用。结论：TCM199培养液中添加卵泡液和单层颗粒细胞组成的培养系统用于牛卵母细胞体外成熟及胚胎发育的效果较好。共培养体系中的单层颗粒细胞用经酶消化分散处理后在培养箱中孵育10min的颗粒细胞替代时,胚胎的发育效果并不受影响。     

Oogenesis in four species of Piscicola (Hirudinea, Rhynchobdellida)

Piscicola has a pair of elongated sac-shaped ovaries. Inside the ovaries are numerous small somatic cells and regularly spherical egg follicles. Each follicle is composed of three types of cells: many (average 30) germ cells (cystocytes) interconnected by intercellular bridges in clones (cysts), one intermediate cell, and three to five outer follicle cells (envelope cells). Each germ cell in a clone has one intercellular bridge connecting it to the central anucleate cytoplasmic mass, the cytophore. Each cluster of germ cells is completely embedded inside a single huge somatic follicle cell, the intermediate (interstitial) cell. The most spectacular feature of the intermediate cell is its development of a system of intracytoplasmic canals apparently formed of invaginations of its cell membrane. Initially the complex of germ cell cluster + intermediate cell is enclosed within an envelope composed of squamous cells. As oogenesis progresses the envelope cells gradually degenerate. All the germ cells that have terminated their mitotic divisions are of similar size and enter meiotic prophase, but one of the cystocytes promptly starts to grow faster and differentiates into the oocyte, whereas the remaining cystocytes withdraw from meiosis and become nurse cells (trophocytes). Numerous mitochondria, ER, and a vast amount of ribosomes are transferred from the trophocytes via the cytophore toward the oocyte. Eventually the oocyte ingests all the content of the cytophore, and the trophocytes degenerate. Little vitellogenesis takes place; the oocyte gathers nutrients in the form of small lipid droplets. At the end of oogenesis, an electron-dense fibrous vitelline envelope appears around the oocyte, among short microvilli. At the same time, electron-dense cortical granules occur in the oocyte cortical cytoplasm; at the end of oogenesis they are numerous, but after fertilization they disappear from the ooplasm. In the present article we point out many differences in the course of oogenesis in two related families of rhynchobdellids: piscicolids and glossiphoniids.     

Environmental factors for establishment of the dormant state in oocytes

Guaranteeing the sustainability of gametogenesis is a fundamental issue for perpetuating the species. In the mammalian ovary, sustainability is accomplished by keeping a number of oocytes “stocked” in the dormant state. Despite the evident importance of this state, the mechanisms underlying the oocyte dormancy are not fully understood, although it is presumed that both intrinsic and extrinsic factors are involved. Here, we review environmental factors involved in the regulation of oocyte dormancy. Consideration of the environmental factors illustrates the nature of the ovarian compartment, in which primordial follicles reside. This should greatly improve our understanding of the mechanisms and also assist in reconstitution of the dormant state in culture. Accumulating knowledge on the dormant state of oocytes will contribute to a wide range of research in fields such as developmental biology, reproductive biology and regenerative medicine.     

From primordial germ cell to primordial follicle: mammalian female germ cell development

In mammals, the final number of oocytes available for reproduction of the next generation is defined at birth. Establishment of this oocyte pool is essential for fertility. Mammalian primordial germ cells form and migrate to the gonad during embryonic development. After arriving at the gonad, the germ cells are called oogonia and develop in clusters of cells called germ line cysts or oocyte nests. Subsequently, the oogonia enter meiosis and become oocytes. The oocyte nests break apart into individual cells and become packaged into primordial follicles. During this time, only a subset of oocytes ultimately survive and the remaining immature eggs die by programmed cell death. This phase of oocyte differentiation is poorly understood but molecules and mechanisms that regulate oocyte development are beginning to be identified. This review focuses on these early stages of female germ cell development.     

果蝇原生殖细胞特化的分子机制

原生殖细胞在许多有性生殖动物的胚胎发育早期就已特化出来,并进一步分化为生殖细胞以产生新的子代。动物原生殖细胞的特化主要有生殖质决定和诱导两种模式,果蝇原生殖细胞的特化模式属于前者。研究表明,果蝇原生殖细胞特化过程中生殖质组装的关键基因是osk,其调控下游基因转录产物的定位和翻译,如vas和tud。此外,基因转录沉默是原生殖细胞特化过程的一个重要特征,其与生殖质中的成分如基因nos、gcl、pgc的表达产物密切相关。现对果蝇原生殖细胞特化分子机制进行综述。     

Mammalian oocyte growth and development in vitro

This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes from preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk. © 1996 Wiley-Liss, Inc.     

Developmental and ultrastructual characteristics of mouse oocytes grown <Emphasis Type="Italic">in Vitro</Emphasis> from primordial germ cells

The aim of this study was to clarify the developmental and ultrastructual characteristics of oocytes grown in vitro from primordial germ cells. The female genital ridges at 12.5 days post coitus were cultured for 18 days on an insert membrane in Waymouth's MB752/1 medium, supplemented with 15% fetal bovine serum and 1 mM sodium pyruvate; subsequently, the follicles isolated from the tissue were cultured for eight days in Waymouth's medium supplemented with 5 microg/ml insulin, 5 microg/ml transferrin, 5 ng/ml selenium, 10 mIU/ml follicle stimulating hormone, and 100 ng/ml stem cell factor. The primordial germ cells developed in vitro into oocytes of more than 60 microm in diameter. The transmission electron microscopic analysis indicated that the oocytes, which developed in vitro, showed no obvious abnormality in their ultrastructure and had organelles appropriate for the oocyte size. However, a delay in the progressive changes of morphology in some of the organelles during oocyte growth was often found when comparing them to oocytes grown in vivo.     

Oogenesis in the leech Glossiphonia heteroclita (Annelida, Hirudinea, Glossiphonidae). I. Ovary structure and previtellogenic growth of oocytes

Glossiphonia heteroclita has paired ovaries whose shape and dimensions change as oogenesis proceeds: during early previtellogenesis they are small and club-shaped, whereas during vitellogenesis they broaden and elongate considerably. During early oogenesis (previtellogenesis), each ovary is composed of an outer envelope (ovisac) that surrounds the ovary cavity and is filled with hemocoelomic fluid, in which a single and very convoluted ovary cord is bathed. The ovary cord consists of germline cells, including nurse cells and young oocytes surrounded by a layer of elongated follicle cells. Additionally, follicle cells with long cytoplasmic projections occur inside the ovary cord, where they separate germ cells from each other. The ovary cord contains thousands of nurse cells. Each nurse cell has one intercellular bridge, connecting it to a central anucleate cytoplasmic mass, the cytophore (rachis); it in turn is connected by one intercellular bridge with each growing oocyte. Numerous mitochondria, RER cisternae, ribosomes, and Golgi complexes are transported from the nurse cells, via the intercellular bridge and cytophore, to the growing oocytes. Oogenesis in G. heteroclita is synchronous with all oocytes in the ovary in the same stage of oogenesis. The youngest observed oocytes are slightly larger than nurse cells, and usually occupy the periphery of the ovary cord. As previtellogenesis proceeds, the oocytes gather a vast amount of cell organelles and become more voluminous. As a result, in late previtellogenesis the oocytes gradually protrude into the ovary cavity. Simultaneously with oocyte growth, the follicle cells differentiate into two subpopulations. The morphology of the follicle cells surrounding the nurse cells and penetrating the ovary cord does not change, whereas those enveloping the growing oocytes become more voluminous. Their plasma membrane invaginates deeply, forming numerous broad vesicles that eventually seem to form channels or conducts through which the hemocoelomic fluid can easily access the growing oocytes.     

哺乳动物原始生殖细胞体内特化和体外培养

原始生殖细胞(primordial germ cells, PGCs)是胚胎中最先出现的生殖细胞。PGCs来源于上胚层,最早出现在后肠,随后向生殖嵴迁移。这一过程伴随一系列复杂的分子调控机制,以及DNA甲基化重编程和组蛋白修饰等表观遗传过程。PGCs经过不断的分裂、发育及分化,最终形成配子。为了更好地研究PGCs发育与分化的调控和表观遗传过程,体外培养的研究变得越来越重要。本文以小鼠和人为例,介绍了哺乳动物PGCs的特化过程、PGCs特化过程中的表观遗传过程和PGCs的体外培养研究进展。     

原始卵泡形成与早期卵泡发育的遗传调控

原始卵泡形成与早期发育调控对卵母细胞发育来说非常重要,通过对原始卵泡的体外培养,可以了解原始卵泡发育的调节机制.原始卵泡向初级卵泡的转变不依赖于促性腺激素,而主要受卵巢旁分泌因子的调控,包括Kit/KL、GDF9等促进因子和AMH等抑制因子的调控.目前,原始卵泡发育的分子调控机理研究多以小鼠为模型,而对大型哺乳动物和人的研究甚少.概述了哺乳动物原始卵泡形成与发育启动调控机制的既有研究成果,详细阐述了原始卵泡发育过程中关键分子的调控作用,并介绍了小鼠及其他哺乳动物卵泡体外培养的研究进展.