黏性卵鱼类受精卵一种快速高效的显微注射方法

黏性卵鱼类受精卵遇水后产生的黏性和卵壳变硬的现象严重影响着大批量显微注射操作的速度和随后的取材。研究建立了一种高效的黏性受精卵快速脱黏显微注射方法, 并利用荧光标记葡聚糖Alex-Fluor488-dextran评估了消化脱黏、直接注射和脱壳注射三种方法的技术特点和适用范围。结果表明: 在23℃, 用0.25%胰蛋白酶(pH=7.1-7.4)消化4min可获得脱黏受精卵。与直接注射和脱壳注射方法相比, 研究建立的消化脱黏方法兼具二者的优点: 在受精后5min可以开始显微操作, 无黏性, 容易进针, 胚盘清晰便于观察、注射后容易培养和取材。实验方法适用于研究与黏性卵鱼类卵子发生、卵-胚转换和早期胚胎发育密切相关基因的功能, 亦可满足追踪受精过程中核质细微变化研究的需要。     

蛋白激酶A对小鼠受精卵早期发育过程中M期促进因子活性的影响

为研究小鼠体内 1 细胞期受精卵M期蛋白激酶A(PKA)对M期促进因子 (MPF)活性的影响 ,应用PKA激动剂cAMP及热稳定性抑制剂PKI显微注射入 1 细胞期受精卵内 ,观察MPF及PKA活性变化 .未经注射的对照组MPF活性在分裂期增高 ,分裂间期下降 ;而PKA活性在进入分裂期下降 ,分裂间期升高 .cAMP组PKA活性维持高峰值 ,直至注射HCG后 2 8h ,MPF活性高峰延迟 30min出现 ;PKI显微注射组PKA活性低 ,而MPF活性在注射HCG后 2 7 5h即达高峰 ,且维持高峰时间达1 5h .结果表明 ,PKA活性在细胞周期中也呈波动性 ,间期活性高 ,分裂期活性低 ;PKA高活性抑制MPF活性 ,而抑制PKA活性则MPF活性高峰提前出现 .     

通过双原核显微注射提高转基因小鼠研制效率的实验研究

目的建立高效的转基因小鼠制备技术,为开展遗传工程动物模型研究奠定技术基础。方法通过向小鼠受精卵原核中注入不同浓度的DNA分子,筛选最适注射用DNA浓度;将K14/hCTLA4-Ig基因表达载体分子通过显微注射分别导入小鼠受精卵雌、雄原核,并设立单原核注射对照组;利用输卵管腹壶部穿刺移植法将注射后的小鼠受精卵移植于同期发情的受体母鼠;利用PCR对出生的转基因首建小鼠进行筛选。结果最适DNA分子浓度为10ng/μl;在单、双原核注射组胚胎2细胞卵裂率分别为52.3%(132/253)和45.0%(108/240),差异有显著性(P<0.05);注射胚胎移植后体内存活率分别为18.1%(24/132)和16.7%(18/108),差异无显著性;转基因首建小鼠阳性率分别为3/24和5/18,转基因阳性小鼠占总注射胚胎的比例为1.2%(3/253)和2.08%(5/240),差异有极显著性(P<0.01)。结论尽管双原核注射对胚胎的2细胞卵裂率有一定影响,但通过双原核注射可有效提高转基因小鼠的制备效率。     

人CD55基因的克隆及其转基因构件的组装

本实验采用人肝组织作为RNA的来源 ,经RT -PCR扩增得到CD55基因的cDNA片段。与人α -珠蛋白启动子及其polyA序列重组 ,插入质粒载体pGEM - 5zf,获得了可用于受精卵原核显微注射的基因构件 ,为建立人CD55转基因动物模型奠定了基础。     

激肽释放酶转基因大鼠模型的建立

目的建立KLK1转基因大鼠模型。方法腹腔注射PMSG-HCG（150-150IU/kg）超排不同周龄（4-8周）SD大鼠比较超排效果的差异,以可用于注射胚胎数作为评判标准确定最佳超排周龄。构建pBC-klk1转基因构件,经酶切、纯化后通过显微注射方法导入SD大鼠受精卵原核并移植到同期受孕的SD受体母鼠输卵管内。出生后仔鼠用PCR和Southern方法检测鼠尾DNA鉴定基因型,通过RT-PCR和免疫组化方法检测klk1基因表达。结果4-8周SD大鼠超排后分别获得受精卵14±14.8、30±15.2、13.3±13.7、13±14.7、8±5.7,5周龄大鼠超排效果最好,与其他周龄大鼠相比有显差异,P〈0.05;显微注射1538枚卵,移植685（44.53%）枚卵于31只受体输卵管中,12（12/31,38.7%）只怀孕,共产仔62只,经PCR检测获得6只阳性鼠,Southern检测3只阳性。对Southern检测阳性转基因大鼠子代进行RT-PCR检测和免疫组化分析证明klk1基因在肾脏、胰腺和乳腺内表达。结论成功建立klk1基因表达的转基因大鼠模型,该模型是高血压病研究的理想动物模型。     

绿色荧光蛋白基因在转基因小鼠体内的表达

建立绿色荧光蛋白（GFP）转基因小鼠,继而传代建系。采用显微注射法,将GFP基因注入FVB／NJ小鼠受精卵原核内,获得子代鼠。分娩后3周剪取仔鼠尾,提取基因组DNA,应用PCR、Southern印迹技术进行整合检测。结共用雌性小鼠200只,注射受精卵1586枚,移植卵数386枚,受体鼠32只,怀孕鼠4只,子代鼠18只,有4只为阳性：取2只首建鼠的胚胎,在荧光显微镜下观察GFP表达明显,表明初步获得了转绿色荧光蛋白基因小鼠,     

受精卵反义寡核苷酸显微注射影响因素的研究

以金鱼受精卵为材料,通过胚胎培养探讨反义寡核苷酸显微注射剂量、受精卵质量、胰酶消化浓度等对受精卵反义寡核苷酸显微注射的影响．研究表明,反义寡核苷酸注射剂量在4．6nL、胰酶消化浓度在0.4％的情况下,选用质量较好的受精卵注射,显微注射效果较好,可为之后的基因功能研究提供材料保障．利用显微注射法导入反义寡核苷酸来研究基因的功能．是研究基因功能的有效途径．     

实验小鼠受精卵染色体的观察及制作法

哺乳动物精子穿人卵细胞后,精子头部膨 胀,进一步发育,出现雄性原核和雌性原核。受 精卵第一次成熟分裂时制作的染色体可以单独 分析雄性和雌性染色体．小鼠受精卵染色体制 作国外已有报道[2,3.87,国内尚未见报道。本文 根据Tarkowski的方法〔BI略加修改,能获得清晰 的小鼠受精卵染色体,可为胚胎发育、发育遗传 学和遗传毒理学等方面研究提供实验手段。     

绿色荧光蛋白转基因小鼠模型的建立及胚胎冷冻保种

目的建立绿色荧光蛋白转基因小鼠模型,并采取胚胎冷冻的方法进行保种。方法通过原核显微注射法,把线性化、纯化后的外源基因pEGFP注射入BDF1小鼠受精卵中,胚胎移植给同期发情的假孕受体母鼠,获得子代小鼠。经鉴定对有表达的转基因鼠进行胚胎冷冻保种。结果移植注射胚胎385枚给30只假孕小鼠共出生了306只后代鼠,经PCR和southern blot检测得到5只阳性小鼠。F2代转基因鼠胚胎冷冻240枚胚胎。结论通过显微注射法使外源基因pEGFP在小鼠基因组中得到整合,建立了转pEGFP的转基因小鼠模型。     

转MDV特异性磷蛋白基因兔的获得及遗传

显微注射ＭＤＶ特异性磷蛋白基因进入２１枚受精卵原核,移入受体,共获８只仔兔,２只很快死亡,６只发育到性成熟,用非同位素Ｄｉｇｏｘｉｇｅｕｉｎ标记核酸探针,经斑点杂交证实３只性成熟兔整合注射基因。２只当代转基因兔分别与正常免回交,所产后代,经同样的方法证实都获得第一代转基因兔,这表明ＭＤｖ特异性磷蛋白基因已经整合到它们的生殖细胞系。     

Immunocytochemical localization of cyclin/proliferating cell nuclear antigen (PCNA) in fertilized eggs of mice.

Immunolocalization of cyclin/PCNA (proliferating cell nuclear antigen) was performed with monoclonal antibody using immunogold methods on ultrathin cryosections of fertilized mouse eggs. Immunolabeling in pronuclei was checked 20, 22, 24 and 26 h after HCG injection. A relation between onset of pronuclei migration (early S-phase) and appearance of colloidal particle clusters was found. Afterwards, (mid S-phase) the increase of labelling and the localization of cyclin/PCNA were found throughout the pronuclei, except in the nucleolar bodies. Lower labelling appeared at the time of close reciprocal pronuclei contact (late S-phase). It is concluded that bulk and distribution of cyclin/PCNA in pronuclei is closely related to the progression of first interphase after fertilization.     

Pronuclear synthesis of DNA in fertilized and parthenogenetically activated mouse eggs : A cytophotometric study

Mouse one-cell embryos were taken 1, 1.5, 2, 3, 4, 6, 8, 10, 13 and 18 h after insemination. One-cell parthenogenones were induced by treatment of mouse eggs obtained 20 h after HCG injection with hyaluronidase and cultured for 0.5, 1, 3, 4.5, 6, 8, 10, 12 and 24 h. Some parthenogenones were pulse-labelled with tritiated thymidine, cut and autoradiographed. Both the embryos and parthenogenones were Feulgen-stained, and integrated relative optical absorption of either pronuclei or nuclei of polar bodies was measured with a cytophotometer. In some fertilized eggs and parthenogenones the DNA synthesis sets in 4–6 h after either insemination or parthenogenetic stimulus. Between the 8th and 13th hour after insemination the fraction of DNA synthesizing embryonic pronuclei remained at the level 30–40%. Most parthenogenones duplicated their DNA content between the 8th and 12th hour after hyaluronidase treatment. The DNA synthesis time in pronuclei of embryos was determined to be 3.5–4.0 h and that of pronuclei of parthenogenones approx. 4 h. The minimal time of the G2 phase was estimated to be 3–5 h. The first labelled pronuclei of parthenogenones were detected 6 h after stimulus. Male pronuclei started and ended DNA synthesis earlier than female pronuclei. Differences in the DNA content between pronuclei of the parthenogenones (when there are two in one parthenogenone) were observed beginning with the 10th h after hyaluronidase treatment.The DNA content in the nuclei of the second polar bodies (PB) of embryos increased slowly between the 8th and 22nd hour after insemination, up to an overall value of 1.4 C. That of the nuclei of the polar bodies of parthenogenones accompanied the synthesis of DNA in pronuclei to the 10th hour after hyaluronidase treatment, up to an overall value of 1.4 C.     

Fertility, ovulation and maturation of eggs in mares injected with HCG

Pony mares were observed from January to August for incidence of oestrus, duration of oestrus, length of the oestrous cycle and for ovulation and fertility after injection of HCG. From January to 15 May most mares showed oestrus but the duration of oestrus was quite variable and few mares ovulated in response to HCG. From 15 May to 17 August oestrous cycles were more regular and ovulation was induced within 40-50 h by an intramuscular injection of 1500-5000 i.u. HCG. Pregnancy was established by one mating at a fixed time after HCG in 20 of 69 mares. Degenerate eggs were recovered from the oviducts of anoestrous recently ovulated, mated, unmated and pregnant mares. The first polar body was formed before ovulation in 2 eggs and had not formed in 2 recently ovulated eggs flushed from the oviduct. The second polar body formed after sperm penetration 10-12 h after ovulation. After formation of pronuclei, the first cleavage division occurred at 20 h and the second at 32 h after ovulation. Oestrus was inhibited by progesterone administered by vaginal devices but occurred within 1-3 days in 12 of the 20 mares after withdrawal of the devices.     

雌核发育和两性融合发育鱼卵调控精核受精发育的生化特性研究

两性融合生殖的鱼卵受精后,精核能疏松、解凝,形成雄性原核：雌核发育银鲫卵子受精后,精核发育受到抑制,无法形成原核。采用显微注射去膜精核以及细胞学和电镜观察的方法,本文对两类鱼卵受精后精核早期发育的生化性质进行了初步探讨,并着重研究了雌核发育银鲫卵子控制精核发育的生化特征。实验结果显示,两性融合生殖鱼类卵质中,一定量的Ｃａ２＋的存在,二硫键的还原作用对于精核的发育显然是必要的;而在雌核发育银鲫卵中,Ｃａ２＋的功能和二硫键的还原作用与精核发育受到抑制之间并无直接联系。银鲫卵质中似乎显示出异常的磷酸酶脂解活性,导致磷酸化过程无法进行,使精核解凝受到阻碍。另外,两性融合生殖的鱼卵重质层中具有大量诱导精核原核化的有关因子,而银鲫卵质中则缺少该因子（或活性极低）。银鲫卵质中还可能缺乏某些与雄性原核的核膜重组装有关的大分子物质。     

Effect of egg composition on the developmental capacity of androgenetic mouse embryos.

Analysis of the developmental capacities of androgenetic and gynogenetic mouse embryos (bearing two paternal or two maternal pronuclei, respectively) revealed a defect in blastocyst formation of androgenetic, but not gynogenetic, embryos that was a function of the maternal genotype. Androgenetic embryos constructed using fertilized eggs from C57BL/6 or (B6D2)F1 mice developed to the blastocyst stage at frequencies similar to those previously reported, whereas androgenetic embryos constructed with fertilized eggs from DBA/2 mice developed poorly, the majority failing to progress beyond the 16-cell stage and unable to form a blastocoel-like cavity, regardless of whether the male pronuclei were of C57BL6 or DBA/2 origin. This impaired development was observed even in androgenetic embryos constructed by transplanting two male pronuclei from fertilized DBA/2 eggs to enucleated C57BL/6 eggs, indicating that the defect cannot be explained as the lack of some essential component in the DBA/2 cytoplasm that might otherwise compensate for androgeny. Rather, the DBA/2 egg cytoplasm apparently modifies the incoming male pronuclei differently than does C57BL/6 egg cytoplasm. Several specific alterations in the protein synthesis pattern of DBA/2 androgenones were observed that reflect a defect in the regulatory mechanisms that normally modulate the synthesis of these proteins between the 8-cell and blastocyst stages. These results are consistent with a model in which cytoplasmic factors present in the egg direct a strain-dependent modification of paternal genome function in response to epigenetic modifications (genomic imprinting) established during gametogenesis and indicate that preimplantation development can be affected by these modifications at both the morphological and biochemical levels.     

Cytoplasmic and sperm nuclear transformations in fertilized ammonia-activated sea urchin (Arbacia punctulata) eggs

Studies examining cytoplasmic and sperm nuclear transformations in sea urchin (Arbacia punctulata) eggs inseminated at different periods after ammonia activation have been caried out at the light- and electron-microscopic levels of observation. Arbaca eggs treated with ammonia-seawater demonstrated chromosome condensation after DNA synthesis and underwent a chromosome cycle similar to that described for Lytechinus [Mazia, 1947]. Cortical granule reaction, fertilization cone formation, and sperm aster development in eggs fertilized at 20 (interphase), 50 (prometaphase), and 180 (interphase) min after ammonia activation were structurally simialr to processes in untreated zygotes. Cyclical changes in the formation of fertilization cones and sperm asters, as reported for eggs fertilized after activation by agents that induce a cortical granule reaction, were not observed. Although sperm nuclear transformations were prolonged (14 vs 18 min), male pronuclei that developed in eggs fertilized 20 min after ammonia activation were morphologically similar to those observed in fertilized, untreated ova and incorporated ³H-thymidine. Sperm incorporated into eggs at 50 min after ammonia activation underwent nuclear envelope breakdown and chromatin despersion; however, ³H-thymidine incorporation was not observed, and male pronuclei rarely developed (less than 5% of all specimens examined). Subsequent to dispersion, the paternal chromatin condensed into chromosomes which were associated with an aster. These results demonstrate that although ammonia-activated eggs inseminated at interphase or prometaphase undergo similar cytoplasmic alterations, sperm nuclear transformations vary with the chromosome cycle of the egg.     

胞浆内精子注射技术生产小鼠

以piezo操作系统为技术支撑 ,在掌握小鼠卵母细胞胞浆内精子注射技术 (ICSI)的基础上 ,进行了ICSI技术生产试管小鼠的尝试。来自成年昆明 (KM)小鼠附睾尾的新鲜精子 ,剪切去尾后 ,直接将精子头注射到B6D2F1小鼠卵母细胞质中 ,注射后 1h ,83.3%的卵母细胞存活。6h时 ,84.0 %的成活卵子成功受精 ,形成原核 ,排出PB2 体外培养的ICSI胚胎 ,卵裂率 (98%vs 94.7% )和 4-细胞期胚胎比率 (89.5%vs 92.1% )均与培养的体内受精卵没有差异 (P >0.05 ) ;但是 ,桑椹胚(63.8%vs84.2% )和囊胚发育率 (25.7%vs68.4% )极显著地 (P <0.01)低于对照组。120枚原核期胚胎移植给 7只假孕受体后 ,4只受孕小鼠共产出 28只ICSI小鼠 (23.3% )。健康成年的 25只ICSI小鼠都没有明显的生理和行为异常。随机选择其中的 20只小鼠 ,分别进行ICSI小鼠间、ICSI与KM小鼠间共 12组的交配 ,结果所有雌鼠妊娠产仔。在成功建立小鼠ICSI技术的基础上,成功获得了我国的首例ICSI小鼠,并且证明这些ICSI小鼠都具有正常的繁殖后代的能力。     

Full-term development of mouse eggs transplanted with male pronuclei derived from round spermatids: the effect of synchronization between male and female pronucleus on embryonic development

Pronucleus transplanted mice have been produced, but their donor male pronuclei were derived from mature sperm and were completely synchronous with female pronuclei because both male and female pronuclei came from the same fertilized oocyte. The present study firstly produced male pronuclei by introducing round spermatids into enucleated mouse oocytes, then transferred the male pronuclei into mouse oocytes at three activation stages and finally compared the effect of three kinds of oocytes on the development of reconstructed embryos. Our results indicate that, in enucleated oocytes, mouse round spermatid nuclei can transform to male pronuclei in a higher proportion, and the synchronization between male and female pronucleus does not significantly influence the early cleavage but the later and full-term development of reconstructed embryos.     

Nascent protein requirement for completion of meiotic maturation and pronuclear development: examination of fertilized and A-23187-activated surf clam (Spisula solidissima) eggs

The involvement of newly synthesized proteins and calcium in meiotic processes, sperm nuclear transformations, and pronuclear development was examined in emetine-treated, fertilized, and A-23187-activated Spisula eggs by observing changes in the morphogenesis of the maternal and paternal chromatin. Emetine treatment (50 micrograms/ml) initiated 30 min before fertilization or A-23187 activation inhibited incorporation of [3H]leucine into TCA-precipitable material and blocked second polar body formation. Sperm incorporation and the initial enlargement of the sperm nucleus were unaffected; however, the dramatic enlargement and transformation of the sperm nucleus into a male pronucleus, which normally follow polar body formation, were delayed 10 to 20 min. Unlike the situation in untreated, control eggs, male pronuclear development took place while the maternally derived chromosomes remained condensed. It was not until approximately 20 min after the normal period of pronuclear development that the maternal chromosomes dispersed and formed a female pronucleus in emetine-treated, fertilized eggs. Formation of pronuclei, however, was unaffected in both emetine-treated, A-23187-activated eggs and fertilized eggs incubated with A-23187. These observations indicate that germinal vesicle breakdown, first polar body formation, and initial transformations of the sperm nucleus are independent of newly synthesized proteins. Inhibition of second polar body formation and the delay in pronuclear development brought about by emetine, as well as the appearance of silver grains over pronuclei in autoradiographs of control eggs incubated with [3H]leucine demonstrate that nascent proteins are involved with the completion of meiotic maturation and the development of male and female pronuclei. The ability of A-23187 to override the inhibitory effects of emetine on pronuclear development suggests that both nascent protein and calcium signals are involved in regulating the status of the maternal and paternal chromatin during pronuclear development.     

Distribution of proteins labelled during meiotic maturation in rabbit and pig eggs at fertilization

The fate of proteins formed during meiotic maturation was examined after fertilization. Rabbit ovarian oocytes were labelled in vitro with [3H]lysine and fertilized after transfer to recipients. A significant accumulatin of the label was detected autoradiographically only in fully grown male and female pronuclei. Pig oocytes at the germinal vesicle and metaphase I stages were labelled with [3H]lysine, [3H]methionine or [3H]tryptophan and fertilized. Pronuclei were labelled by all 3 precursors. During cleavage, eggs labelled with [3H]lysine lost the nuclear label by the 4-cell stage. However the [3H]methionine label was present in the cytoplasm and marked in the nuclei at the 4-cell stage, while the [3H]tryptophan label was still clear in 8-cell embryos.