猪卵母细胞透明带弹性系数—杨氏模量测定

 【目的】为了探讨猪卵母细胞的生物物理特性，该文研究了猪卵母细胞透明带的力学性质。【方法】在39℃环境下，利用微管吸吮的方法在倒置显微镜下通过显微操作装置用显微玻璃管吸吮猪卵母细胞，通过自制的专用于卵母细胞透明带杨氏模量测量软件，测量了经过44～48 h体外成熟培养的核成熟和核未成熟猪卵母细胞（n=76，共7次重复）的透明带杨氏模量。【结果】计算得到猪卵母细胞透明带的弹性模量为（1.43±0.29）×104Pa。【结论】该研究结合了生殖生物学和力学的内容，为评判哺乳动物卵母细胞和胚胎质量开拓了新的思路和内容。     

Zona pellucida dissolution enzymes of the rabbit sperm head

Enzymes which dissolve the zona pellucida have been extracted from the rabbit sperm head and characterized as having the enzymic properties of hyaluronidase and trypsin. Their combined actions produce a rapid and complete dissolution of the zona pellucida, but the vitellus and its membrane are unaffected by prolonged exposure to this enzyme complex. Inhibition of the proteolytic component with lima bean or soybean inhibitors of trypsin prevents dissolution of the zona pellucida by the extract.     

Scintigraphy of normal mouse ovaries with monoclonal antibodies to ZP-2, the major zona pellucida protein

The zona pellucida is an extracellular glycocalyx, made of three sulfated glycoproteins, that surrounds mammalian oocytes. Parenterally administered monoclonal antibodies specific for ZP-2, the most abundant zona protein, localize in the zona pellucida. When labeled with iodine-125, these monoclonal antibodies demonstrate a remarkably high target-to-nontarget tissue ratio and provide clear external radioimaging of ovarian tissue.     

Improving the stages of bovine cloning technology with the use of oocytes without the zona pellucida

Bovine embryos are reconstructed with the use of fetal fibroblasts as donors of nuclei and enucleated oocytes without the zona pellucida as recipients. The stages of electrofusion and in vitro culturing of embryos without the zone pellucida up to the blastocyst stage is perfected.     

Inhibition of sperm-egg interaction by specific antibody

Treatment of hamster ova, in vitro, with ovary specific antibody interferes with sperm-egg interaction by inhibiting sperm attachment to the zona pellucida and subsequent zona penetration. This inhibition results from precipitation of the zona by the specific antibody.     

Vaccination with a synthetic zona pellucida peptide produces long-term contraception in female mice

The zona pellucida surrounding mouse oocytes is an extracellular matrix composed of three sulfated glycoproteins, ZP1, ZP2, and ZP3. It has been demonstrated that a monoclonal antibody to ZP3 injected into female mice inhibits fertilization by binding to the zona pellucida and blocking sperm penetration. A complementary DNA encoding ZP3 was randomly cleaved and 200- to 1000-base pair fragments were cloned into the expression vector lambda gt11. This epitope library was screened with the aforementioned contraceptive antibody, and the positive clones were used to map the seven-amino acid epitope recognized by the antibody. Female mice were immunized with a synthetic peptide containing this B cell epitope coupled to a carrier protein to provide helper T cell epitopes. The resultant circulating antibodies to ZP3 bound to the zona pellucida of immunized animals and produced long-lasting contraception. The lack of ovarian histopathology or cellular cytotoxicity among the immunized animals may be because of the absence of zona pellucida T cell epitopes in this vaccine.     

Penetration,adhesion, and fusion in mammalian sperm-egg interaction

Fertilization is the sum of the cellular mechanisms that pass the genome from one generation to the next and initiate development of a new organism. A typical, ovulated mammalian egg is enclosed by two layers: an outer layer of approximately 5000 cumulus cells and an inner, thick extracellular matrix, the zona pellucida. To reach the egg plasma membrane, sperm must penetrate both layers in steps requiring sperm motility, sperm surface enzymes, and probably sperm-secreted enzymes. Sperm also bind transiently to the egg zona pellucida and the egg plasma membrane and then fuse. Signaling in the sperm is induced by sperm adhesion to the zona pellucida, and signaling in the egg by gamete fusion. The gamete molecules and molecular interactions with essential roles in these events are gradually being discovered.     

ES细胞囊胚注射后脱透明带移植对小鼠产仔率和嵌合率的影响

[目的]提高ES细胞囊胚注射的移植出生率。[方法]对照组采用正常囊胚ES细胞注射,试验组注射后的囊胚经酸性台氏’液消化透明带后直接移植,研究Es细胞囊胚注射后脱透明带移植对小鼠产仔率和嵌合率的影响。[结果]对照组产仔率为15．96％,嵌合率为57．89％。试验组小鼠产仔率为17．95％,嵌合率为57．14％,出生率提高2％。[结论]嵌合率无显著差异,酸性台氏液消化透明带的方法可简化操作流程。     

Requirement of phospholipase Cdelta4 for the zona pellucida-induced acrosome reaction

Several phospholipase C (PLC) isoforms have been found in male and female mammalian gametes, and splicing isoforms of PLCdelta4 are predominantly expressed in testis. Here we report that male mice in which the PLCdelta4 gene had been disrupted either produced few small litters or were sterile. In vitro fertilization studies showed that insemination with PLCdelta4-/- sperm resulted in significantly fewer eggs becoming activated and that the calcium transients associated with fertilization were absent or delayed. PLCdelta4-/- sperm were unable to initiate the acrosome reaction, an exocytotic event required for fertilization and induced by interaction with the egg coat, the zona pellucida. These data demonstrate that PLCdelta4 functions in the acrosome reaction that is induced by the zona pellucida during mammalian fertilization.     

哺乳动物卵母细胞和胚胎冷冻损伤研究进展

哺乳动物卵母细胞和胚胎的冷冻保存在胚胎工程技术方面有重要意义。卵母细胞和胚胎在冷冻保存过程中很容易受到冷冻损伤的危害,甚至会影响细胞超微结构。冷冻损伤包括细胞膜、细胞质、细胞核以及细胞外透明带、卵丘细胞的损伤。阐述了目前在这些方面的研究进展,并探讨了冷冻损伤的机制,以期为今后改善冷冻保存技术提供理论依据。     

Blastocyst axis is specified independently of early cell lineage but aligns with the ZP shape

The mechanisms controlling the establishment of the embryonic-abembryonic (E-Ab) axis of the mammalian blastocyst are controversial. We used in vitro time-lapse imaging and in vivo lineage labeling to provide evidence that the E-Ab axis of the mouse blastocyst is generated independently of early cell lineage. Rather, both the boundary between two-cell blastomeres and the E-Ab axis of the blastocyst align relative to the ellipsoidal shape of the zona pellucida (ZP), an extraembryonic structure. Lack of correlation between cell lineage and the E-Ab axis can be explained by the rotation of the embryo within the ZP.     

小鼠胚胎切割取样后体外培养发育能力的研究

本文对小鼠胚胎切割取样细胞数量的多少，取样后胚胎在室温中停留时间以及透明带存留与否的体外发育情况进行了研究，目的在于为牛胚胎性别鉴定切割取样时供借鉴和探讨。实验中的胚胎依据切割取样的细胞数分１０个细胞以内和大于１０个细胞以上两个组。其体外培养发育率分别为９５．７％（６７／７０）和７６．２％（３２／４２），两者差异极显著（Ｐ＜０．０１）；其中晚期桑椹胚、早期羹胚、扩展囊胚的体外发育率分别为８７．７％（６４／７３）、８６．２％（２５／２９）、１００％（１０／１０），三者之间差异不显著（Ｐ＞０．０５）；培养前在室温下停留３～７ｈ和９ｈ的体外培养发育率分别为９４．８％（７３／７７）、５７．１％（２０／３５），两者差异极显著（Ｐ＜０．０１）；胚胎取样后透明带存留和脱失的体外培养发育率分别为８７．９％（５１／５８）和８３．３％（４５／５４），两者差异不显著（Ｐ＞０．０５）。     

延边黄牛体细胞克隆囊胚中总基因组RNA的提取

以延边黄牛体细胞克隆囊胚为试验材料,采用试剂盒GMS12106的使用方法提取试材的总基因组RNA,在琼脂糖凝胶电泳检测后,得到最适的囊胚数为5个。通过试验发现,生殖细胞的特殊生理结构限制了RNA提取的效果。为提取完整、纯度高的RNA,采用链蛋白酶对囊胚的透明带进行了消化处理,最终得出链蛋白酶处理的最适时间为10min,提取的RNA质量好,可为后续试验奠定基础。     

Human sperm binding is mediated by the sialyl-Lewis(x) oligosaccharide on the zona pellucida

Human fertilization begins when spermatozoa bind to the extracellular matrix coating of the oocyte, known as the zona pellucida (ZP). One spermatozoan then penetrates this matrix and fuses with the egg cell, generating a zygote. Although carbohydrate sequences on the ZP have been implicated in sperm binding, the nature of the ligand was unknown. Here, ultrasensitive mass spectrometric analyses revealed that the sialyl-Lewis(x) sequence [NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc], a well-known selectin ligand, is the most abundant terminal sequence on the N- and O-glycans of human ZP. Sperm-ZP binding was largely inhibited by glycoconjugates terminated with sialyl-Lewis(x) sequences or by antibodies directed against this sequence. Thus, the sialyl-Lewis(x) sequence represents the major carbohydrate ligand for human sperm-egg binding.     

小鼠冻胚分割试验

研究了分割所用溶液、冷冻前胚胎发育时期和分割后将半胚是否装入透明带对冻胚分割效果的影响。结果证明:①将解冻囊胚分割后,无论是否将半胚装入透明带对冻胚分割的效果均无显著影响(P>0.05);②在PBS中分割解冻桑椹胚和囊胚的效果无显著差异(P>0.05);③与分割解冻桑椹胚相比,在蔗糖液中分割解冻囊胚可显著提高冻胚分割效果(P<0.05)。将在蔗糖液中分割冻囊胚获得10对半胚移植于5只受体,结果有2只妊娠,共获得半胚仔鼠6只,其中2对为同卵双生。     

不同浓度的FBS和hLIF对小鼠原核期受精卵体外发育率的影响

通过在mM16培养液中添加不同浓度的FBS及hLIF,探讨小鼠原核期受精卵的体外培养条件,旨在建立稳定的小鼠原核期受精卵的体外培养系统。实验结果表明:在mM16中添加10%或15%的FBS都可降低胚胎在体外的发育率,其体外囊胚发育率与对照组mM16(未加血清组)差异显著(73.3%对32.6%;73.3%对35.8%);5001、0001、500 IU/ml 3种不同浓度的hLIF都可提高小鼠受精卵体外发育率,三者之间差异不显著,但以添加1 000 IU/ml hLIF组中,桑椹胚/囊胚率及囊胚脱出率为最高。结论:在mM16中添加血清不能有效地支持小鼠受精卵在体外的发育,添加1 000 IU/ml hLIF能够很好地支持小鼠受精卵的体外发育。     

玻璃化冷冻对猪卵母细胞超微结构的影响

猪体外成熟培养的卵母细胞用平皿-微滴法玻璃化冷冻,冷冻解冻后对其超微结构损伤和正常的体外成熟培养的卵母细胞进行了电镜观察比较。结果表明,玻璃化冷冻的卵母细胞解冻后有不同程度的结构损伤,包括:透明带破裂;微绒毛几乎消失;有的部位质膜模糊甚至破裂;质膜下皮质颗粒减少;线粒体膨胀,电子致密度下降,嵴消失;大量囊泡在质膜周围变形融合;细胞基质出现空白区。玻璃化冷冻引起卵母细胞结构损伤是导致卵母细胞解冻后存活力下降和受精率降低的主要原因。     

发情期蓝狐卵泡发育的显微结构观察(英文)

[目的]对发情期蓝狐卵巢的组织结构和卵泡发育过程的变化进行研究。[方法]采集10只发情期蓝狐的左侧卵巢共计10枚,利用光学显微镜对卵巢的组织结构和卵泡的发育过程进行观察,同时利用目镜测微尺和显微照相技术分别对30个原始卵泡、30个初级卵泡、15个生长卵泡、15个囊状卵泡、15个成熟卵泡及各级卵泡中的卵母细胞进行测量和拍照。[结果]蓝狐卵巢由生殖上皮、白膜、皮质和髓质构成。皮质内分布着大量的间质细胞及不同发育阶段的卵泡,位于卵巢的外周;髓质位于皮质下层,分布着较多的血管。卵泡发育过程中,各级卵泡和卵母细胞的直径差异较大(卵泡为45.45~974.55μm,卵母细胞为30.23~147.27μm),在初级卵泡阶段出现透明带物质。卵泡闭锁发生于卵泡生长的各个时期,主要表现为卵母细胞的皱缩,细胞核固缩;颗粒细胞松散、退化。[结论]为揭示雌性蓝狐的生殖机理提供了组织学依据。     

牛卵母细胞发育的超微结构研究

本实验根据卵细胞的数量及分布、透明带的变化,卵泡大小等把牛卵母细胞的整个发育过程分为六个阶段,在超微结构的水平上进行了研究。随着卵母细胞的发育,高尔基体、线粒体、滑面内质网、皮质颗粒等细胞器数量不断增加,且逐渐迁移到皮质区。卵母细胞成熟时。高尔基体及粗面内质网消失,皮质颗粒开始在质膜下呈线状排列,而线粒体又向胞质中央区扩散。直径2毫米以上卵泡卵母细胞中,少嵴圆形线粒体一律转变为带帽线粒体,核仁发生致密化。此外,牛卵母细胞中有带有界膜的、均匀分布的囊泡。皮质颗粒成团发生和分布也是牛卵母细胞的特点之一。实验事实证明,皮质颗粒的形成与滑面内质网关系更为密切。     

发情期蓝狐卵泡发育的显微结构观察

[目的]对发情期蓝狐卵巢的组织结构和卵泡发育过程的变化进行研究。[方法]采集发情期蓝狐的左侧卵巢共计10枚,利用光学显微镜对卵巢的组织结构和卵泡的发育过程进行观察,同时利用目镜测微尺和显微照相技术分别对30个原始卵泡3、0个初级卵泡、15个生长卵泡、15个囊状卵泡、15个成熟卵泡及各级卵泡中的卵母细胞进行测量和拍照。[结果]蓝狐卵巢由生殖上皮、白膜、皮质和髓质构成。皮质内分布着大量的间质细胞及不同发育阶段的卵泡,位于卵巢的外周;髓质位于皮质下层,分布着较多的血管。卵泡发育过程中,各级卵泡和卵母细胞的直径差异较大（卵泡为45.45～974.55μm,卵母细胞为30.23～147.27μm）,在初级卵泡阶段出现透明带物质。卵泡闭锁发生于卵泡生长的各个时期,主要表现为卵母细胞的皱缩,细胞核固缩;颗粒细胞松散、退化。[结论]为揭示雌性蓝狐的生殖机理提供了组织学依据。