猪第一极体核物质参与卵母细胞受精及其胚胎发育的研究

旨在探索猪卵母细胞第一极体核物质能否参与体外受精及其进一步的胚胎正常发育.第一极体(FPB)获自猪卵巢卵泡中卵丘卵母细胞复合体(COCs)体外成熟培养的MⅡ期卵母细胞.存活极体从成熟卵母细胞内分离后采用显微操作术注入去核卵母细胞胞质中.第一极体重组卵母细胞分别采用常规体外受精和单精子胞浆注射法进行受精,来自极体核重组的受精卵进行体外培养,观察和分析其进一步体外卵裂发育的能力.结果显示:极体显微操作重组的成功率约为80%,20%左右的重组卵母细胞于重组后的受精和发育培养过程中退化、崩解;常规体外受精和单精子注射后2-细胞卵裂率分别达到12.9%和29.8%.结论:猪第一极体核物质具有参与卵母细胞受精和进一步胚胎发育的能力.据此推测利用第一极体将有可能繁殖正常仔猪后代.     

兔卵母细胞去核方法的研究

本研究观察了兔卵母细胞的纺锤体在不同条件下的变化，并对不同去核方法的去核效率进行了比较。结果表明，在卵母细胞去核时应用piezo-driven操作系统，能有效减少去核对卵母细胞的机械挤压损伤，使卵母细胞去核后存活率达98％。卵母细胞中极体与核的相对距离随着卵龄的增加而加大，卵龄16h的卵母细胞只有70.4％的核位于极体附近，当卵龄增加到19h时，这一比例下降到60.5％。用spindle view系统能有效提高去核效率，使去核率从盲吸法的83.8％提高到98.4％，且发现在注射hCG后19h左右去核较为合适。纺锤体对环境变化敏感，适宜的温度为36～37℃，温度不适容易使纺锤体解聚。     

不同去核方法对猪手工克隆重构胚发育效果的影响

为探讨不同去核方法对猪手工克隆(HMC)重构胚发育效果的影响,本研究比较了盲切法,第一极体(Pb1)定位法及脱羰秋水仙碱(DM)辅助去核法3种不同的去核方法的去核效率及HMC胚胎发育的影响。结果表明:第一极体定位法和DM辅助去核法的整体去核效率显著高于盲切法去核法(P0.05);应用0.4μg/m L DM处理卵母细胞60 min的突起率、整体去核效率显著高于其他浓度和时间处理组(P0.05);DM辅助去核与pb1定位去核法2种不同去核方法对HMC重构胚的发育效果的影响差异不显著。本研究表明,添加适量的DM可以提高徒手克隆去核效率,且对后期重构胚发育没有显著影响。     

小鼠卵母细胞化学去核及手工构建核移植胚胎

为优化脱羰秋水仙碱（DC）诱导去核程序,研究以DC去核卵母细胞为核受体的、无透明带体细胞核移植方法在小鼠体细胞核移植中的应用,试验比较了乙醇、SrCl2两种激活方法及脱羰秋水仙碱处理开始时间对小鼠MⅡ期卵母细胞去核效率的影响;将DC诱导去核成功的卵母细胞去除透明带,与胎儿成纤维细胞粘合、电融合和SrCl2激活后,体外培养重构胚。结果显示,7%乙醇激活后0 min起始DC处理可得到最高的诱导去核率（66.4%）;而在8 mmol/L SrCl2中激活15 min后用DC处理可得到最高的诱导去核率（64.3%）;目前重构胚可以体外发育到8-细胞。试验结果首次证明了SrCl2在小鼠卵母细胞DC诱导去核中的作用效果与乙醇相当,初步证明了将DC诱导去核技术与无透明带技术相结合手工克隆生产小鼠重构胚的可能性,它的成功将大大简化核移植程序。     

脱羰秋水仙碱(DM)辅助去牛卵母细胞核的研究

受体细胞去核是核移植关键步骤之一。利用脱羰秋水酰碱（Demecolcine,DM）显示牛卵母细胞染色体的位置进行去核,主要从以下几个方面进行了试验：卵母细胞在DM中孵育浓度、卵母细胞在DM中孵育时间及卵母细胞成熟时间对显示效果的影响。结果表明：（1）成熟牛卵母细胞用离子霉素激活5 min,DM孵育2 h完全诱导去核,结果没有见到完全去核的卵母细胞;（2）挑选有第一极体的成熟牛卵母细胞,在浓度为0.5μg/mL的DM中孵育2 h显示率可达76.54%;（3）牛卵母细胞在成熟18 h可以获得73.86%的显示效果。（4）在没有去卵丘细胞的卵母细胞中添加DM取得了更好的显示效果（80.82%）,囊胚的发育率也较其它组高,可达18.60%,说明未去卵丘细胞的卵母细胞添加DM更有利于显示染色体的位置,而且更有利于囊胚的发育。利用DM显示染色体的位置不仅可以高效快速的去核,同时也避免了传统去核时荧光对卵母细胞的照射。     

微丝调控卵母细胞减数分裂成熟的分子机制研究进展

卵母细胞在与精子融合形成合子前会经历2轮减数分裂。与有丝分裂不同的是,2次减数分裂都是不对称的,最终会产生1个大体积的具有全能性单倍体卵母细胞和2个小体积的注定退化的极体。肌动蛋白丝作为卵母细胞中的细胞骨架,与分裂过程中的囊泡转运、细胞核定位、纺锤体迁移与锚定、极体排出和染色体分离等生物学事件存在重要联系。本文以哺乳动物为模型,总结了肌动蛋白在卵母细胞减数分裂成熟过程中的重要调节机制与信号通路,以期为进一步研究卵母细胞成熟过程的调控机制提供参考。     

6-DMAP对延边黄牛末期卵母细胞核移植的影响

为探讨6-二甲基氨基嘌呤（6-DMAP）在延边黄牛末期去核的体细胞克隆中的作用,本试验主要研究了单独使用离子霉素处理与离子霉素联合添加6-DMAP处理对体外成熟的老化延边黄牛卵母细胞的激活率的不同影响;以及不同时期添加6-DMAP对重构胚后期发育能力的影响。试验结果显示,体外成熟的老化卵母细胞,使用离子霉素单独处理后91%被激活,而在离子霉素联合添加6-DMAP处理后却没有细胞被激活,也就是没有第二极体的排出。另外,融合后使用6-DMAP处理,所得重构胚的囊胚发育率最低,而激活后的卵母细胞立即用6-DMAP处理,所得重构胚的发育能力与无6-DMAP处理的相近。综上所述,离子霉素联合添加6-DMAP可抑制老化的延边黄牛卵母细胞的激活,阻止第二极体的排出,但对重构胚的后期发育没有影响。而融合使用添加6-DMAP的培养液,抑制了重构胚的后期发育。     

绵羊去透明带卵母细胞体细胞核移植研究

为探索简化的核移植程序,本研究分析不同成熟培养时间、去核过程中紫外光照时间、融合电压强度、激活剂种类、不同培养方法对绵羊去透明带卵母细胞核移植的影响。结果表明:卵母细胞成熟培养18～19 h后去除透明带,其极体排出和附着效果最佳。采用1.9 kV/cm直流电压融合去核卵母细胞与颗粒细胞,融合率为88.2%,效果最好。离子霉素对重构胚具有较好的激活效果,卵裂率为82.1%,囊胚率为10.4%;压制WOW(s孔中孔)发育组卵裂率和囊胚发育率与四孔板培养组相比无显著差异,但卵裂率和囊胚发育率并不高;去除透明带的绵羊卵母细胞采用衰减1/4的UV照射10 s辅助去核后,卵裂率为35.6%,但重构胚未能发育至囊胚。结果显示,通过去透明带辅助显微操作去核的方法进行的绵羊体细胞克隆程序较易掌握。     

猪卵母细胞去核方法的改进

对猪体细胞克隆技术中关键步骤之一的去核方法作了较深入的研究。结果表明，由本实验室改进的Willadsen去核法——二步挤压去核法，无论在去核操作成功率（73．9％）上还是在去核效率（81．2％）上都明显好于McGrath-Solter去核法（42．5％、67．4％，P〈0．05）；同时，本试验还发现，卵母细胞成熟培养36h后去核组去核效率（89．1％）显著地高于成熟培养44h后去核组（55．8％，P〈0．05），而核移植重构胚的发育率2个组间无显著差异。     

昆明鼠卵母细胞的去核及其去核率的观察

本文研究昆明鼠卵母细胞的去核方法，并通过对去核卵母细胞的固定染色，显示其中期Ⅱ染色体，由此观察统计其确定的去核率。     

体细胞核移植技术在转基因动物研究中的应用

利用基因工程技术将目的基因整合入动物体细胞染色体中，并将其作为供体核移植入受体－－去核卵母细胞构成重建胚，然后将其移植入假孕母体，待其妊娠、分娩，便可得到经定向遗传修饰的转基因克隆动物。本文就这一领域的发展历史、研究现状、应用前景及存在的问题等方面作一概述。     

Somatic Cell Nuclear Transfer in Horses

The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids and the present status of this technique. The first step is competent oocyte maturation, this is followed by oocyte enucleation and reconstruction, using either zona-enclosed or zona-free oocytes, by efficient activation to allow high cleavage rates and finally by a suitable in vitro embryo culture technique. Cloning of the first equid, a mule, was achieved using an in vivo -matured oocytes and immediate transfer of the reconstructed embryo, i.e. at the one cell stage, to the recipient oviduct. In contrast, the first horse offspring was obtained using a complete in vitro procedure from oocyte maturation to embryo culture to the blastocyst stage, followed by non-surgical transfer. Later studies on equine cloning report high efficiency relative to that for other species. Cloned equid offspring reported to date appear to be normal and those that have reached puberty have been confirmed to be fertile. In summary, horse cloning is now a reproducible technique that offers the opportunity to preserve valuable genetics and notably to generate copies of castrated champions and therefore, offspring from those champions that would be impossible to obtain otherwise.     

牛胚胎原代和继代细胞核移植结果比较

比较了原代和继代核移植的操作各环节以及核移植胚胎在体外发育能力上的差异。通过显微操作将体外受精发育而来的８～３２细胞期胚胎的单个卵裂球注入激活的去核卵母细胞的卵周隙内,并用８０Ｖ/mm、４０us２次电脉冲诱导卵裂球与去核卵母细胞融合,借此进行牛胚胎的原代核移 体外发育来的８～３２细胞期的原代核移植胚胎作为供体,用原代核移植相同的方法进行牛胚胎的继代移植。原代核移植的存活率和融合率（８７．３％和６８．５     

Experimental approach to prezygotic chromosome screening using only a single pair of gametes in mice

During in vitro embryo production, chromosome screening is essential to prevent pregnancy losses caused by embryonic chromosome aberrations. When the chromosome screening is completed before fertilization, gametes are effectively utilized as genetic resources. The aim of this study was to investigate whether chromosome screening of gametes accompanied by fertilization would be feasible using a single mouse spermatozoon and oocyte. Metaphase II oocytes were divided into a cytoplast and a karyoplast. For genome cloning of the gametes, androgenic and gynogenic embryos were produced by microinjection of sperm into cytoplasts and parthenogenetic activation of karyoplasts, respectively. Pairs of blastomeres from androgenic and gynogenic embryos were fused electrically to produce diploid embryos, which were transferred into pseudopregnant surrogate mothers to examine fetal development. Blastomeres from androgenic and gynogenic embryos were individually treated with calyculin A—a specific inhibitor of type 1 and 2A protein phosphatases—for 2 h to induce premature chromosome condensation. Thereafter, chromosome analysis of blastomeres, reflecting the genetic constitution of individual spermatozoa and oocytes, was performed, and we confirmed that most of the androgenic and gynogenic 2-cell embryos had a haploid set of chromosomes in their sister blastomeres. The reconstructed embryos from blastomeres of androgenic and gynogenic 2-cell embryos could be implanted and develop into live fetuses, albeit at low efficiency. This study indicates that prezygotic chromosome screening and embryo production using a single pair of gametes may be practicable.     

Supplement of autologous ooplasm into porcine somatic cell nuclear transfer embryos does not alter embryo development

Somatic cell nuclear transfer (SCNT) is considered as the technique in which a somatic cell is introduced into an enucleated oocyte to make a cloned animal. However, it is unavoidable to lose a small amount of the ooplasm during enucleation step during SCNT procedure. The present study was aimed to uncover whether the supplement of autologous ooplasm could ameliorate the oocyte competence so as to improve low efficiency of embryo development in porcine SCNT. Autologous ooplasm‐transferred (AOT) embryos were generated by the supplementation with autologous ooplasm into SCNT embryos. They were comparatively evaluated with respect to embryo developmental potential, the number of apoptotic body formation and gene expression including embryonic lineage differentiation, apoptosis, epigenetics and mitochondrial activity in comparison with parthenogenetic, in vitro‐fertilized (IVF) and SCNT embryos. Although AOT embryos showed perfect fusion of autologous donor ooplasm with recipient SCNT embryos, the supplement of autologous ooplasm could not ameliorate embryo developmental potential in regard to the rate of blastocyst formation, total cell number and the number of apoptotic body. Furthermore, overall gene expression of AOT embryos was presented with no significant alterations in comparison with that of SCNT embryos. Taken together, the results of AOT demonstrated inability to make relevant values improved from the level of SCNT embryos to their IVF counterparts.     

Cytoskeletal Changes in Oocytes and Early Embryos During in vitro Fertilization Process in Mice

The cytoskeleton plays crucial roles in the development and fertilization of germ cells and in the early embryo development. The growth, maturation and fertilization of oocytes require an active movement and a correct localization of cellular organelles. This is performed by the re-organization of microtubules and actin filaments. Therefore, the aim of the present study was to determine the changes in cytoskeleton during in vitro fertilization process using appropriate immunofluorescence techniques. While the chromatin content was found to be scattered throughout the nucleus during the oocyte maturation period, it was seen only around nucleolus following the completion of the maturation. Microtubules, during oocyte maturation, were regularly distributed throughout the ooplasm which was then localized in the subcortical region of oocytes. Similarly microfilaments were scattered throughout the ooplasm during the oocyte maturation period whereas they were seen in the subcortical region around the polar body and above the meiotic spindle throughout the late developmental stages. In conclusion, those changes occurred in microtubules and microfilaments might be closely related to the re-organization of the genetic material during the oocyte maturation and early embryo development.     

An intracytoplasmic injection of deionized bovine serum albumin immediately after somatic cell nuclear transfer enhances full-term development of cloned mouse embryos

In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in vitro development of cloned embryos to the blastocyst stage compared with that of a conventional nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine 12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear reprogramming.     

Effect of activation methods for bovine oocytes after intracytoplasmic injection

A principal nuclear transfer procedure is to inject a donor cell into the perivitelline space in an enucleated oocyte and then electric fusion is performed (cell fusion method). The effects of activation methods in reconstructed oocytes for the serum-starved somatic cell cloning procedure were investigated in this study by means of intracytoplasmic injection (i.c.i.). Bovine oocytes were enucleated at 18-22 h for in vitro maturation, and subsequently the nucleus of cumulus cell collected from Japanese Black Bulls (JBCC) after 5-7 days of starved culture was injected into the recipient cytoplast with a piezo-micromanipulator. At 1 h after i.c.i., reconstructed oocytes were stimulated with ethanol (ET) or calcium ionophore (CaI) as the first activation treatment, followed by cycloheximide (CHX) or 6-dimethylaminopurin (DMAP) treatment as the second activation. In the experiment on the first activation method, the proportion of reconstructed oocytes developing to the blastocyst stage was significantly (p<0.01) higher in the ET activation method than that with CaI (10.5% and 4.7%, respectively). And the experiment on the second activation method after ET treatment showed similar proportions of blastocyst development in both CHX and DMAP treatments (5.9% and 2.8%, respectively). The present results indicated that combined activation treatment with ET and CHX was efficient for reconstructed bovine oocytes by i.c.i.     

Nucleus reprogramming/remodeling through selective enucleation (SE) of immature oocytes and zygotes: a nucleolus point of view

It is now approximately 25 years since the sheep Dolly, the first cloned mammal where the somatic cell nucleus from an adult donor was used for transfer, was born. So far, somatic cell nucleus transfer, where G1-phase nuclei are transferred into cytoplasts obtained by enucleation of mature metaphase II (MII) oocytes followed by the activation of the reconstructed cells, is the most efficient approach to reprogram/remodel the differentiated nucleus. In general, in an enucleated oocyte (cytoplast), the nuclear envelope (NE, membrane) of an injected somatic cell nucleus breaks down and chromosomes condense. This condensation phase is followed, after subsequent activation, by chromatin decondensation and formation of a pseudo-pronucleus (i) whose morphology should resemble the natural postfertilization pronuclei (PNs). Thus, the volume of the transferred nuclei increases considerably by incorporating the content released from the germinal vesicles (GVs). In parallel, the transferred nucleus genes must be reset and function similarly as the relevant genes in normal embryo reprogramming. This, among others, covers the relevant epigenetic modifications and the appropriate organization of chromatin in pseudo-pronuclei. While reprogramming in SCNT is often discussed, the remodeling of transferred nuclei is much less studied, particularly in the context of the developmental potential of SCNT embryos. It is now evident that correct reprogramming mirrors appropriate remodeling. At the same time, it is widely accepted that the process of rebuilding the nucleus following SCNT is instrumental to the overall success of this procedure. Thus, in our contribution, we will mostly focus on the remodeling of transferred nuclei. In particular, we discuss the oocyte organelles that are essential for the development of SCNT embryos.     

The developmental ability of vitrified oocytes from different mouse strains assessed by parthenogenetic activation and intracytoplasmic sperm injection

Assessment of the developmental ability of oocytes following freezing and thawing is an important step for optimizing oocyte cryopreservation techniques. However, the in vitro fertilization of frozen-thawed mouse oocytes is often inefficient because of incomplete capacitation of spermatozoa in the absence of surrounding cumulus cells. This study was undertaken to determine whether the oocyte cryopreservation efficiency of different strains of mice could be assessed from the development of oocytes following parthenogenetic activation and intracytoplasmic sperm injection (ICSI). Oocytes were collected from hybrid (C57BL/6 x DBA/2) F1 or inbred (C57BL/6J, C3H/HeN, DBA/2J and BALB/cA) strains and were vitrified in a solution containing ethylene glycol, DMSO, Ficoll and sucrose. In the first series of experiments, oocytes were activated parthenogenetically by Sr(2+) treatment after warming. The oocytes from the inbred strains, but not those of the F1 hybrid, were diploidized by cytochalasin treatment to obtain a sufficient number of blastocysts. In all strains tested, parthenogenetic embryos derived from vitrified oocytes developed into blastocysts at rates between 23 and 68%. In the second series of experiments, vitrified oocytes from each strain were injected with homologous spermatozoa after warming. Normal offspring were obtained from all strains at rates between 5 and 26% per embryo transferred. Thus, the feasibility of oocyte cryopreservation protocols can be assessed easily by in vitro development of parthenogenetic embryos or by in vivo development of ICSI embryos. Moreover, the oocytes of these four major inbred strains of mice can be cryopreserved safely for production of offspring.