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。     

延边黄牛卵母细胞去核方法的研究

用改进的Willaden去核法与McGrath-Solter去核方法对延边黄牛卵母细胞进行去核操作,比较其去核操作效率。结果表明,本实验改进的willaden去核法——挤压去核法,无论在去核操作时间(1．0 min／枚,P<0．05)上,还是在去核效率(94%,P<0．01)上都显著地高于McGrath-Solter去核法(1．4 min／枚、72%)。表明改进的Willaden去核方法是一种适用于延边黄牛卵母细胞操作的快速、简单、实用的去核方法。     

卵母细胞去核方法及其对卵母细胞发育的影响

自1938年Spermann为研究不同发育时期细胞核的发育能力而提出的细胞核移植构想以来,核移植技术已经取得了极大的成就,特别是1997年由英国科学家Wilmut等经体细胞核移植克隆出绵羊"多莉"而带来的克隆风暴更是以迅猛之势席卷全球。     

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。     

卵母细胞去核及卵丘细胞移核方法对猪体细胞核移植的影响

以猪体外成熟卵母细胞周围分离得到的卵丘细胞作为猪体细胞核移植的供核细胞,研究了卵母细胞不同去核方法(盲吸法、活性荧光染色去核法、盲吸法和活性荧光染色法联合去核法)的去核率差异,并比较了卵丘细胞透明带下注核和胞质内注核两种重建胚构建方法的胚胎裂解率、4 8细胞发育率和桑椹胚率的差异。结果表明:(1)以荧光染料染色法的去核率(87.18%)最高,与其他试验组相比差异显著(P<0.05);盲吸法去核所用时间最少(1.2 min/个),与其他试验组相比差异显著(P<0.05)。(2)透明带下注核比卵母细胞质内直接注核的卵裂率(40.11%/29.75%)和4 8细胞率(11.86%/6.81%)高,差异显著(P<0.05);桑椹胚率(1.75%/1.79%)无明显差异(P>0.05)。     

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。     

水牛卵母细胞玻璃化冷冻保存

以水牛MII期的卵母细胞为材料,利用玻璃化冷冻液EDS33(16.5%EG+16.5%DMSO+sucrose)对水牛MII期的卵母细胞进行两步法(玻璃毛细管(GMP)和拉细的开口塑料细管(OPS))玻璃化冷冻保存,即卵母细胞首先放入预平衡液(7.5%EG+7.5%DMSO+sucrose)中平衡3 min,再移入玻璃化冷冻液中30 s后装管直接投入液氮.解冻是在蔗糖浓度逐渐降低的解冻液中进行的.解冻后存活的卵母细胞孤雌激活,通过囊胚发育率作为评定卵母细胞冷冻效果的指标.结果发现,GMP和OPS法冷冻保存的水牛卵母细胞解冻后的存活率(分别为96.80%和97.41%)与对照组卵母细胞的存活率(100%)3者之间差异均不显著(P>0.05).GMP法和OPS法冷冻的水牛卵母细胞激活后的胚胎分裂率和囊胚发育率2者均明显低于对照组(分别为30.58%和28.32% vs50.94%,10.81%和9.38% vs 29.63%,P<0.05),而这2种方法冷冻的水牛卵母细胞激活后的分裂率和囊胚发育率差异均不显著(P>0.05).这表明GMP和OPS玻璃化冷冻方法可以用于水牛卵母细胞的冷冻,并且玻璃化冷冻的卵母细胞能继续分裂并发育到囊胚.     

水牛卵母细胞冷冻保存初步研究

①用EFS30、EFS40、EDFS30、EDFS40四种玻璃化冷冻液对MⅡ期水牛卵母细胞进行毒性试验,结果表明:试验组卵母细胞形态正常率与对照组均无显著性差异(P>0.05);对卵母细胞孤雌激活后EDFS30、EDFS40组的卵裂率与对照组(75.28%)及EFS30、EFS40组差异显著(P<0.05);利用4种冷冻保护剂采用OPS法冷冻保存MⅡ期水牛卵母细胞,其中以EDFS40作为冷冻液时,卵母细胞冷冻解冻后孤雌激活卵裂率最高,达31.60%;以EDFS40作为冷冻液,比较了GMP法和OPS法的冷冻效果,结果表明GMP法冷冻效果好于OPS法。②采用不同预处理时间和平衡时间使用细管法常规冷冻G V期卵母细胞,结果表明预处理5 min、平衡15min组的形态正常率和极体排出率相对较高,分别为72.73%、27.27%。     

改进的表面张力辅助去核法对小鼠卵母细胞去核的研究

为了探讨改进的小鼠表面张力辅助去核法（STA）的可靠性，本试验用改进的表面张力辅助去核法与表面张力辅助去核法对小鼠卵母细胞进行去核操作，比较其去核操作效率。结果发现，改进的表面张力辅助去核法能显著减少去核操作时间，去核率与表面张力去核法无显著差异。改进的小鼠表面张力辅助去核法是一种适用于小鼠卵母细胞操作的快速、简单、实用的去核方法。     

水牛卵母细胞在不同培养液中成熟与凋亡的比较研究

为了解卵母细胞体外成熟与凋亡过程,进而提高卵母细胞体外成熟率,本试验研究了在培养液中添加不同浓度的表皮生长因子(EGF)、胰岛素样生长因子-1(IGF-1)对水牛卵母细胞体外成熟和凋亡的影响。结果表明:(1)添加各种浓度的EGF(10,20,30,50,100 ng/mL)均可以提高水牛卵母细胞的成熟率,降低卵母细胞的凋亡率,其中50 ng/mL EGF有显著影响(P<0.05);(2)添加各种浓度的IGF-1(10,30,50,100 ng/mL)均能提高水牛卵母细胞体外成熟率,降低卵母细胞的凋亡率,以30 ng/mL效果明显(P<0.05);(3)添加20 ng/mL EGF+30ng/mL IGF-1组卵母细胞体外成熟率和凋亡率分别高于和低于单独添加IGF-1和EGF组。     

猪卵母细胞与猪、水牛及食蟹猴精子的胞质内显微受精

为探讨猪精子、水牛精子及食蟹猴精子分别注入到猪卵母细胞后原核形成及早期胚胎发育情况,利用屠宰场收集的猪卵母细胞,经体外成熟44～48h后,进行胞质内显微受精(ICSI)操作。试验1:体外培养18～19h,用Ho-echst33342荧光染色,检查原核形成情况。试验2:进行体外培养,ICSI后2d检查分裂率,7d记录囊胚率。试验1结果显示:在原核形成率上,猪同种显微受精,原核形成率(50.10%)显著高于注入水牛精子(37.06%)及食蟹猴精子(37.48%),且差异显著(P0.05),注入水牛精子和食蟹猴精子间差异不显著。试验2结果显示:猪同种显微受精所得的分裂率(86.58%)和囊胚率(29.93%)与水牛精子注入(70.98%,18.48%)、食蟹猴精子注入(78.69%,16.92%)差异显著(P0.05)。结果表明,水牛精子、食蟹猴精子分别注入到猪卵母细胞后,观察到雌雄原核和精子解聚;水牛精子注入猪卵母细胞与食蟹猴精子注入猪卵母细胞,经体外培养发育到囊胚。     

Early metaphase II oocytes treated with dibutyryl cyclic adenosine monophosphate provide suitable recipient cytoplasm for the production of miniature pig somatic cell nuclear transfer embryos

We investigated the effects of in vitro maturation duration and treatment with dibutyryl cyclic adenosine monophosphate (dbcAMP) on the blind enucleation efficiency and developmental competence of miniature pig somatic cell nuclear transfer (SCNT) embryos. Oocytes were cultured for 22 h in NCSU-23 medium with or without 1 mM dbcAMP and then additionally cultured in dbcAMP-free NCSU-23 for 14, 18, or 22 h. Regardless of dbcAMP treatment, the rate of nuclear maturation reached a plateau at 36 and 40 h. However, mitochondrial distribution, a marker for cytoplasmic maturation, differed between the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h. The metaphase II chromosomes were adjacent to the first polar body in 68.8% and 63.5% of the dbcAMP-untreated oocytes at 36 h and dbcAMP-treated oocytes at 40 h, respectively. Furthermore, the blind enucleation efficiency by removing a small volume of cytoplasm was significantly higher in the dbcAMP-untreated oocytes at 36 h (82.9%) and dbcAMP-treated oocytes at 40 h (89.9%) than other groups. The rate of blastocyst formation was highest in the dbcAMP-treated oocytes at 40 h. Hence, this study demonstrated that dbcAMP-treated early metaphase II oocytes are suitable for the production of miniature pig SCNT embryos.     

Cryopreservation of immature buffalo oocytes: Effects of cytochalasin B pretreatment on the efficiency of cryotop and solid surface vitrification methods

The aim of the present study was to compare the efficiency of the solid surface (SSV), cryotop (CT) vitrification methods and cytochalasin B (CB) pretreatment for cryopreservation of immature buffalo oocytes. Cumulus‐oocyte complexes (COCs) were placed for 1 min in TCM199 containing 10% dimethylsulfoxide (DMSO), 10% ethylene glycol (EG), and 20% fetal bovine serum, and then transferred for 30 s to base medium containing 20% DMSO, 20% EG and 0.5 mol/L sucrose. CB pretreated ((+)CB) or non‐pretreated ((?)CB) COCs were vitrified either by SSV or CT. Surviving vitrified COCs were selected for in vitro maturation (IVM) and in vitro fertilization (IVF). The rate of viable oocytes after vitrification in CT groups (82%) was significantly lower (P < 0.05) than that in a fresh control group (100%), but significantly higher (P < 0.05) than those in SSV groups (71–72%). Among vitrified groups, the highest maturation rate was obtained in the CT (?)CB group (32%). After IVF, the cleavage and blastocyst formation rates were similar among vitrified groups but significantly lower than those of the control group. In conclusion, a higher survival rate of oocytes after vitrification and IVM was obtained in the CT group compared with that in the SSV group, indicating the superiority of the CT method. Pretreatment with CB did not increase the viability, maturation or embryo development of vitrified oocytes.     

磷酸二酯酶抑制剂对水牛卵母细胞减数分裂成熟的影响

本研究探讨了磷酸二酯酶抑制剂米力农(milrinone)对水牛卵母细胞体外自发成熟和促性腺激素诱导成熟的影响,以便提高卵母细胞体外成熟质量。试验对水牛卵丘卵母细胞复合体(COCs)培养不同时间或采取不同处理后,取出卵母细胞剥光后固定,然后用间苯二酚蓝染色,观察卵母细胞核成熟情况。结果表明:(1)Milrinone对水牛COCs的自发成熟具有抑制作用,且具有剂量依赖关系;(2)Milrinone对水牛卵母细胞体外自发成熟的抑制作用随培养时间的延长没有减弱,可作为水牛卵母细胞成熟过程中的核成熟抑制剂;(3)Milrinone能显著抑制促卵泡激素(FSH)诱导的水牛COCs体外成熟,但是随着时间的延长,这种抑制作用可以部分被FSH克服。     

水牛体细胞连续核移植的效果

为探讨水牛体细胞连续核移植的效果,以水牛胎儿成纤维细胞为核供体,进行了水牛体细胞连续核移植。结果显示,连续核移植的融合率显著高于原代核移植(87.9%vs76.2%,P<0.05),但两者之间的分裂率和囊胚率没有显著差异(P>0.05);这说明水牛体细胞核移植胚胎可被再次克隆而不降低其发育能力,水牛体细胞连续核移植是可行的。     

水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响.结果显示,添加颗粒细胞传代接种第2天收集的20％颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P＜0.05);然...     

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。     

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.     

Effect of vitrification procedures on the subsequent development of in vitro matured swamp buffalo oocytes following in vitro fertilization

Nowadays, the efficiency of buffalo oocytes cryopreservation is still low. The purpose of this study was to evaluate effects of two combinations of cryoprotectant agents (CPAs) and two vitrification devices for vitrification of swamp buffalo oocytes on their survival after vitrification warming, and subsequent developmental ability after in vitro fertilization. In vitro matured (IVM) oocytes were vitrified by either Cryotop (CT) or solid surface vitrification (SSV) interacting with vitrification solution A (VA) or B (VB). In the VA or VB solution exposed test, the oocytes showed similar survival rates, but decreased blastocyst rates after in vitro fertilization compared with that of untreated oocytes. After vitrification, the CT method combined with VA solution yielded a higher survival rate (91.3 ± 5.84%) of vitrified oocytes than that combined with VB solution (69.8 ± 4.19%–75.8 ± 4.55%); however, all the vitrification treatments showed lower blastocyst rates (1.1 ± 0.07%–5.2 ± 0.24%) compared with that of untreated oocytes (18.0 ± 1.09%). Our results indicated that combined vitrification treatments in this study did not improve the decreased ability of vitrified oocytes developing to the blastocyst stage.