绵羊体外成熟,体外受精卵的体外发育及移植后的产羔

体外成熟、体外受精后的绵羊卵在体外长期培养或在2～4细胞期和桑堪～囊胚期移植给受体母羊,观察了培养卵的发育和移植后的受胎率。方法是将采自屠宰场的绵羊卵巢在1～12小时内带回实验室,抽取卵母细胞。从中选取卵丘细胞层完整的卵子,在二氧化碳培养箱内,用含有10％NSS(或FCS)、hCG和E_2,并以Hepes缓冲的M199培养24～26小时。再用以IonophoreA23187诱导获能处理过的新鲜公羊精于进行体外受精.7～10小时后移入发育培养基,即含有10％FCS(或NSS)和丙酮酸钠的Hepes缓冲的M199内继续培养,受精后将部分发育为2～4细胞胚和桑堪～囊胚期胚手术移植给受体母羊。另一部分卵子则在受精后48～72小时的不同时间内统计其卵裂卵的出现率,并继续培养7～12天详细观察卵裂卵的发育情况。在受精后48～72小时卵裂卵的出现率,FCS和NSS组分别为39.6％145/366)和52.4％(182/347),前者显著低于后者;将61枚2～4细胞期胚和桑椹～囊胚期胚分别移植给20只受体母羊,有10只受胎。共产羔11只,受胎率为50％;在发育培养液内继续培养的482枚卵裂卵中有312枚(64.7％)发育为桑椹～囊胚期胚(其中包括部分孵化囊胚)。     

小鼠附睾精子的显微受精

目的：建立显微受精的方法,并探讨小鼠附睾精子在显微注射进入卵母细胞后的受精能力。方法：用显微注射法把小鼠附睾头和附睾尾精子注入卵母细胞的胞质内或卵周隙进行显微受精。结果：把单个附睾尾精子注入卵细胞质中,培养后１４个存活的卵细胞中,有５个卵裂为２－细胞期胚胎;将单个附睾头精子注入卵母细胞中,有２５个存活,其中９个受精发育为２－细胞期胚胎;将附睾尾精子注入卵周隙进行带下受精,３０个存活的卵细胞中有４个     

松江鲈鱼(Trachidermus fasciatus)胚胎发育的初步观察

通过对上海松江鲈驯养繁育基地松江鲈鱼自然产卵受精及人工授精所得受精卵的群体试验观察,描述了受精卵的发育过程．松江鲈鱼刚产出的卵为淡黄、橘黄、橘红和淡红色等多种颜色．卵内有很多油球,显微镜下观察油球总是旋转到卵的上方．卵为粘性卵,遇水很快粘结成块．松江鲈鱼卵受精后8～10h胚盘隆起,之后1d内细胞多次分裂进入桑椹期．在卵受精后第1天和第4天分别到达囊胚期和原肠期．第7天胚体的脑开始分化．第11天体节出现．第16天消化道已经形成,但不连通．第17天胚体已可在卵内活动．受精卵在水温13．7～16．2℃下23d即可破膜孵出．同时对孵出6d内的仔鱼进行了观察．研究结果可为松江鲈鱼的大规模育苗生产提供必要的理论指导．     

牛胚胎体外生产技术的应用研究

利用屠宰牛卵巢抽出的卵母细胞，以卵裂率和囊胚发育为标准，对牛胚胎体外生产过程进行了简化试验，结果显示：不加卵丘细胞的高密度卵母细胞体外成熟（１００－２００枚／平皿）可代替加卵丘细胞标准密度培养，其卵裂率和囊胚发育率没有显著变化，体外授精时间可从成熟培养后２２小时延长至２７小时，卵裂率和囊胚发育率均没有显著变化。卵母细胞体外成熟后可以不经洗涤直接移入受精滴，原成熟培养皿内残留孵丘细胞再培养４８小时形     

唇(♀)×花(♂)杂交子一代胚胎发育

以唇为母本,花为父本进行属内远缘杂交,观察和记录了杂交F1受精卵胚胎发育情况,具体描述了各个时期的形态特征。结果表明,受精卵为粘性卵,卵径为2.50~2.80 mm,在水温在(20±1)℃条件下,受精后55 min,胚盘形成;1 h 35 min,细胞开始分裂,进入卵裂期;5 h 05 min,囊胚层形成,进入囊胚期;10 h 30 min,胚层下包,形成胚环,进入原肠期;17 h 25 min,神经板形成,胚体侧卧,进入神经胚期;19 h 15 min,胚孔关闭,进入胚孔封闭期;22 h 25 min,进入器官形成期;94 h 10 min,仔鱼开始出膜。研究认为唇(♀)×花(♂)杂交F1受精卵与母本胚胎发育时序和形态特征基本相似。     

唇(鱼骨)(♀)×花(鱼骨)(♂)杂交子一代胚胎发育

以唇(鱼骨)为母本,花为父本进行属内远缘杂交,观察和记录了杂交F1受精卵胚胎发育情况,具体描述了各个时期的形态特征.结果表明,受精卵为粘性卵,卵径为2.50～2.80 mm,在水温在(20±1)℃条件下,受精后55 rain,胚盘形成;1 h 35 min,细胞开始分裂,进入卵裂期;5 h 05 min,囊胚层形成,进入囊胚期;10 h 30 min,胚层下包,形成胚环,进入原肠期;17 h 25 min,神经板形成,胚体侧卧,进入神经胚期;19 h 15 min,胚孔关闭,进入胚孔封闭期;22 h 25 min,进入器官形成期;94 h 10 min,仔鱼开始出膜.研究认为唇(鱼骨)(♀)×花(鱼骨)(♂)杂交F1受精卵与母本胚胎发育时序和形态特征基本相似.     

鲈鲤胚胎发育特征观察

观察了水温（18±0．5）℃条件下,鲈鲤[Percocypris pingi pingi（ T chang ）]的胚胎发育特征。结果显示：成熟鲈鲤卵呈球形、桔黄色,为粘性卵,卵径2．2mm,卵膜遇水膨胀后,最大外膜径达3．2-3．8mm。受精卵在水温（18±0．5）℃条件下,受精2h28min后开始第1次卵裂,受精后45h23min开始形成器官,受精后126h28min孵出仔鱼。初孵仔鱼全长为10．4mm,卵黄囊大而侧扁。根据胚胎发育过程的形态特征,可将鲈鲤胚胎发育过程分为受精卵、卵裂、囊胚、原肠胚、神经胚、器官发生和出膜7个连续阶段。32个时期。     

杂交鳜胚胎发育观察

通过对斑鳜(♀)×翘嘴鳜(♂)人工杂交F1胚胎发育的观察,详细描述了其胚胎发育各个阶段的发育时序和形态特征.结果表明:杂交F1的卵裂方式属于盘状卵裂,在水温(24±1)℃条件下,受精后2 h 30 min进入2细胞期,受精后4h 30 min进入囊胚期,受精后13 h进入原肠期,受精后19 h 40 min进入神经胚期,受精后22 h 30 min进入器官形成期,受精后117 h 30 min仔鱼出膜.杂交F1的胚胎发育特征与母本基本一致.     

隆线溞孤雌卵胚胎发育的形态学研究

采用组织学方法较为系统地研究了隆线溞(Daphnia carinata)孤雌卵(夏卵)胚胎发育的全过程.隆线溞夏卵为中黄卵,室温24℃下,整个胚胎发育过程需45h左右.根据隆线溞胚胎内部结构特征及外部形态学变化,将其胚胎发育分为卵裂期、囊胚期、原肠期、前无节幼体期、后无节幼体期、复眼色素期和准备孵化期7个时期.卵排至孵育囊约40min后开始表面卵裂.卵裂至256细胞时,胚胎发育进入囊胚阶段,在卵表形成一薄层细胞层,囊胚腔则全被卵黄颗粒所充塞.囊胚后期,囊胚层细胞分裂加快,相互挤压向囊胚腔中移入形成原肠胚.随后,胚胎外部形态开始出现变化.首先在胚胎前端出现头部的三对附肢原基(两对触角原基及一对大颚原基),胚胎发育进入前无节幼体期;随后胸节分化,胚胎发育进入后无节幼体期,并形成胸肢、壳瓣和肠道等结构.复眼在复眼色素期的基础上,逐渐发育形成完整的复眼结构,同时其他各组织器官也不断发育完善.至准备孵化期的胚胎结构与幼体已基本相同.以上研究结果可为深入研究枝角类胚胎发育的机理积累基础生物学资料.     

梭鱼(Mugil so-iuy Basilewsky)胚胎发育的初步观察

从梭鱼人工繁殖所获之受精卵中,取得胚胎发育的观察材料。卵的直径约0.95毫米,具单个油球,油球的直径0.45—0.5毫米、受精卵在盐度为8—10‰的流水中孵化。水温在16.8—22℃的范围内,受精后1小时15分,开始第一次分裂;6小时,形成囊胚;10小时左右,出现胚环;24小时左右,原口闭合,胚体具有2—3对体节。受精后48小时,胚体脱膜而出,从而结束了胚胎发育阶段。孵出当天的鱼苗,长2.5毫米。     

冷藏雄鼠尸体应用于小鼠运输及意外死亡后品系恢复的可行性

目的 基于小鼠尸体4 ℃冷藏不同时长后精子的体外受精(IVF)效率,探讨低温冷藏小鼠尸体应用于小鼠运输及意外死亡后品系恢复的可行性。方法 实验组为5月龄C57BL/6 J雄鼠,脱颈处死后4 ℃冷藏24、48、72 h和96 h,取附睾尾精子IVF,对照组为正常5月龄C57BL/6 J雄鼠IVF,统计各组IVF受精率。结果 冷藏24 h后附睾尾精子具备正常受精能力,受精率为73.0%、60.0%和77.0%;48 h 组内差异大,受精率为0%、55.4%和3.2%;72 h受精率极低,受精率为3.2%、0%和1.7%;96 h 精子有活力,但无受精能力。所有受精卵均可发育至2细胞,冷藏24 h组和对照组分别移植受体妊娠率分别为60%和66%,证明4 ℃冷藏对胚胎发育无影响。结论 C57BL/6J鼠经4 ℃冷藏的附睾尾,在24 h内具备稳定的受精率,可用于小鼠运输及特殊情况品系恢复。     

Transient activation of calcineurin is essential to initiate embryonic development in Xenopus laevis

At fertilization, an increase of cytosolic calcium ions (Ca2+) triggers various activation responses in animal eggs. In vertebrates, these responses include exit from metaphase arrest in meiosis II (MII exit) and cortical remodelling initiated by cortical granule exocytosis. Although the essential requirement of Ca2+/calmodulin-dependent protein kinase II for inducing MII exit has been documented, a role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in egg activation has not been investigated. Here we show, using cell-free extracts from unfertilized eggs of Xenopus laevis, that calcineurin is transiently activated immediately after Ca2+ addition to a concentration that induces MII exit. When calcineurin activation is inhibited, cyclin-dependent kinase 1 (Cdk1) inactivation by means of cyclin B degradation is prevented and sperm chromatin incubated in the extracts remains condensed. Similarly, if calcineurin is inhibited in intact eggs, MII exit on egg activation is prevented. In addition, the activation contraction in the cortex is suppressed whereas cortical granule exocytosis occurs. We further demonstrate that, when a high level of calcineurin activity is maintained after activation, growth of sperm asters is prevented in egg extracts and, consistently, migration of male and female pronuclei towards each other is hindered in fertilized eggs. Thus, both activation and the subsequent inactivation of calcineurin in fertilized eggs are crucial for the commencement of vertebrate embryonic development.     

人工诱导四倍体泥鳅的研究

目前泥鳅的养殖热潮正在兴起,对其新品种的需求也日益强烈.采用热休克抑制受精卵第一次卵裂、Ag-NOR计数鉴定四倍体胚胎的方法,探索了诱发四倍体泥鳅的最佳条件是:在受精后20 min左右采用41℃热水持续处理泥鳅受精卵4 min.人工诱导泥鳅四倍体将会为新品种的培育提供原始素材.本研究首次提出了依据间期细胞核中的Ag-NOR的最高数目作为确定胚胎倍性的标准,该方法简便快速.     

Fertilization potential in golden hamster eggs consists of recurring hyperpolarizations

The fertilization potential, or activation potential, has been demonstrated in the eggs of various species, and it has been shown to block polyspermy in echinoderm, echiuran and frog eggs, but no studies have been reported of electrical phenomena occurring when mammalian eggs are fertilized. We report here the fertilization potential of golden hamster eggs in vitro. To correlate the change of potential with the interaction between sperm and egg, only one sperm was attached to each egg. We found that a sperm induces recurring hyperpolarizations, constituting a fertilization potential which differs from that in the eggs of other species.     

四面山棘腹蛙在渝西地区饲养繁殖初步研究

研究棘腹蛙在渝西地区的人工饲养及繁殖情况,通过对成蛙两年的饲养繁殖观察,发现其生长良好,能安全度夏和越冬;采用自然受精繁殖,获得棘腹蛙卵2 870粒（受精卵1 925粒,平均受精率为67.1%）,孵化出蝌蚪1 700尾（孵化时间为12天左右,平均孵化率为88.3%）,培育出变态幼蛙230只,越冬前未变态蝌蚪530尾（蝌蚪期为90天左右,平均变态率为39.6%,平均成活率为39.2%）.结果表明,在海拔低、气温高的渝西地区进行棘腹蛙人工饲养和繁殖是切实可行的.该研究对棘腹蛙的规模化养殖具有一定的实践指导意义.     

Calcium-releasing activity induced by nuclei of mouse fertilized early embryos

At fertilization,repectitive transient rises of intracellular calcium concentration occur in all mammals studied so far .It has been shown that calcium rises could be induced when mouse fertilized 1-,2-cell nuclei were trans-planted into unfertilized eggs and that the reconstituted embryo could be activated .Howerver,whecther the capability of inducing calcium rises occurs in all stages of mammalian embryos remains unknown ,In this study ,by using the nuclear transplantation technique and measurement of intracellular calcium rises in living cells,we showed that only the nuclei from mouse fertilized 1-cell and 2-cell embryos ,neither the nuclei from 4-,8-cell and ethanol activated parthe-nogenetic embryos nor 2 or 3 nuclei of electrofused 4-cell stage syncytium ,have calcium -releasing activity when they were transferred into unfertilized mature oocytes,Our results indicate that the calcium-releasing activity in nuclei of 1-,2-cell embryos is produced during fertilization and exists at the special stage of fertilized early embryos.These sug-gested that the capacity of inducting calcium release activity in fertilized early embryos is important for normal embryonic development.     

热休克诱导三倍体黄鳝的研究

采用热休克诱导方法研究了促使第二极体保留诱发三倍体黄鳝的最佳条件．在卵受精后５－６ｍｉｎ，采用４０．５℃热水处理３．５ｍｉｎ，不但能导致较高的三倍化率，而且胚胎存活率相当高，表明热休克是诱导多倍化的有效方法，需用设备简单，易于推广     

小鼠精子微注射受精卵的体外发育与移植的研究

用显微注射方法,将精子注入小鼠卵母细胞卵周隙中,使精卵体外受精获得受精卵。并对精子注射后卵母细胞的成活率、受精率和受精卵的发育率以及移植后受胎率等方面进行了系统的研究,结果表明:卵子存活率为75.36％(1309/1737),其中264枚卵裂,受精率为20·17％(264/1309),卵裂卵经体外培养后有192枚发育到桑椹胚及胚泡期,发育率为72.73％(192/264)。将其中73枚(桑椹胚10枚,肛泡63枚)胚胎移植到10只假孕受体子宫中,一只受体妊娠产仔9只,仔鼠发育正常。     

Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis

It has been suggested that the failure of parthenogenetic mouse embryos to develop to term is primarily due to their aberrant cytoplasm and homozygosity leading to the expression of recessive lethal genes. The reported birth of homozygous gynogenetic (male pronucleus removed from egg after fertilization) mice and of animals following transplantation of nuclei from parthenogenetic embryos to enucleated fertilized eggs, is indicative of abnormal cytoplasm and not an abnormal genotype of the activated eggs. However, we and others have been unable to obtain such homozygous mice. We investigated this problem further by using reconstituted heterozygous eggs, with haploid parthenogenetic eggs as recipients for a male or female pronucleus. We report here that the eggs which receive a male pronucleus develop to term but those with two female pronuclei develop only poorly after implantation. Therefore, the cytoplasm of activated eggs is fully competent to support development to term but not if the genome is entirely of maternal origin. We propose that specific imprinting of the genome occurs during gametogenesis so that the presence of both a male and a female pronucleus is essential in an egg for full-term development. The paternal imprinting of the genome appears necessary for the normal development of the extraembryonic membranes and the trophoblast.