化学辅助去核法对绵羊卵母细胞去核率和重构胚发育率的影响

为提高绵羊体细胞核移植的效率,本研究采用一种新的去核方法—化学辅助去核法,对绵羊体外成熟的卵母细胞进行去核,研究了化学诱导剂秋水仙素的处理浓度、作用时间、卵母细胞的成熟时间对去核效果及重构胚发育的影响。结果表明:1)卵母细胞在0.4μg/mL的秋水仙素溶液中分别孵育0.5h和1h,胞质突起率和去核率没有显著的差异,突起率可高达85.4%,去核率达到100%;2)0.2μg/mL或0.4μg/mL秋水仙素溶液将卵母细胞处理0.5h,对去核效果没有显著影响;3)对于体外成熟18～23h的卵母细胞,随着成熟时间的延长,盲吸法的去核率降低,但没有影响秋水仙素诱导胞质突起的比率和去核率;4)两种去核方法对重构胚的发育没有产生显著影响,但成熟21～23h卵母细胞重构胚囊胚的发育率显著高于成熟18～20h卵母细胞重构胚囊胚的发育率。综上所述,本试验优化了绵羊卵母细胞化学辅助的去核程序,利用化学辅助去核法对高卵龄的绵羊卵母细胞进行去核,提高了去核率和重构胚的体外发育率。     

Demecolcine化学辅助去核技术在牛体细胞核移植中的初步研究

本实验目的是研究demecolcine辅助去核的卵母细胞能否支持牛的核移植胚胎的发育。通过化学药物demecolcine处理牛MII期卵母细胞来辅助去除牛卵母细胞核,并用去核的卵母细胞做受体,进行核移植的研究。实验结果显示,demecolcine辅助去核后的卵母细胞质膜有明显一个或二个突起,并且突起内都含有卵母细胞染色体组,显示去核效果较好（57.89%～73.3%）。药物处理一小时为最适时间,去核率可达73.3%。对demecolcine辅助去核的卵母细胞的核移植胚胎发育情况显示囊胚率较盲吸法核移植胚胎较好（12.5%VS10.2%）,但二者差异不显著(p>0.05)。Demecolcine药物处理后的卵母细胞能够支持核移植胚胎的发育。Demecolcine辅助去核可以在牛体细胞核移植中的到应用。     

卵母细胞的生发泡移植

生发泡(germinal vesicle,GV)移植到去核的GV期卵母细胞后,获得重构卵,重构卵在体外能成熟,受精和进行胚胎发育。GV移植到去核的第二次减数分裂中期(metaphase Ⅱ,MII)卵母细胞后,重构卵能发生GV破裂,但难以排出第一极体。GV移植后,通过连续核移植,重构合子具有发育到终期的能力。GV移植为研究卵母细胞的发育提供了一种重要工具。     

牛卵母细胞孤雌激活及马-牛异质克隆胚构建

首先用不同的激活剂孤雌激活体外成熟培养的牛卵母细胞,经试验获得:离子霉素、A23187和7%乙醇联合6-DMAP可有效地激活牛卵母细胞,并支持其发育到囊胚,但离子霉素激活效率显著优于其他两种(P<0.05);以10?S SOFaa 颗粒细胞为发育体系培养激活的成熟牛卵母细胞可得到较高的卵裂率和囊胚率(72.30%,14.91%)。其次,通过体外培养成年马皮肤成纤维细胞,将获得的成纤维细胞经血清饥饿培养后,作为核供体移入去核牛卵母细胞透明带下,电融合后,能得到融合的马牛重构胚,在交流电脉冲起始电压20V,持续时间10s,频率0.2MHz,结束电压15V,2次脉冲和融合间隔为0.125s的条件下,当融合电压为2.0kV/cm,脉冲时程为40μs时,重组胚的融合率和卵裂率最高(52.27%,71.74%)。     

脱羰秋水仙碱诱导昆明白小鼠卵母 细胞去核的研究

在已有的demecolcine(以下简称Deme)诱导去核技术路线的基础上,以昆明白小鼠卵母细胞为实验材料,对影响去核率的几个因素（包括Deme浓度、Deme处理起始时间和作用时间、卵母细胞的卵龄）逐次进行实验。结果表明：（1）激活卵母细胞在浓度为0.4μg/mL和0.5μg/mL Deme-KSOM液中处理60 min均能有效去核,但0.5μg/mL组获得更高的去核率（33.3%）。（2）卵母细胞在激活后0~5 min之内迅速放入0.5μg/mL Deme-KSOM液中,处理60~180 min得到了相对较高的去核率（31.9%~24.5%）。（3）昆明白小鼠hCG注射后17~18 h收集的卵母细胞更有利于Deme诱导去核,去核率为27.1%。经比较分析,建立了优化的Deme诱导去核程序。 Abstract: On the basis of exiting technique pathway of demecolcine-induced enucleation(IE),several factors(Demecolcine concentration、time of demecolcine inition and treatment、oocytes age) affecting the IE rate were tested using Kunming mouse oocytes.The experiments’ results demonstrated that: In experiment 1,activated oocytes could be enucleated efficiently by treating with KSOM medium containing 0.4μg/mL or 0.5μg/mL demecolcine for 60 min,but 0.5μg/mL group gained the higher IE rate(33.3%).In experiment 2,maximum IE rate (31.9%~24.5%) were obtained when oocytes were exposed to 0.5μg/mL demecolcine between 0 and 5 min postactivition and treated for 60~180 min.In experiment 3,oocytes collected from Kunming mouse at 17~18h after hCG administration were favoriate to demecolcine-IE(27.1%). By comparision and analysis of the data,we established the optimized IE procedure.     

克隆胚胎中核有丝分裂器蛋白(NuMA)的动态变化

核有丝分裂器蛋白(NuMA)是一种负责纺缍体极装配的蛋白质。供体细胞核移入去核卵母细胞后,在供体核中未发现NuMA,在重构胚的原核中出现NuMA。克隆胚胎卵裂后,NuMA存在于卵裂球的核中。在克隆胚胎中,NuMA的缺乏会导致有丝分裂纺缍体异常,染色体排列混乱,这些异常影响克隆胚胎的正常发育。     

用改进的细胞核移植方法构建重构胚

为了能够找出一种既容易操作,又不需要特殊设备的核移植方法,对以前的操作进行了改进。首先以预先吸有细胞核或细胞的注射针在固定于持卵针上的卵母细胞透明带上穿刺两个孔,然后一边缓慢地将注射针回拔至卵周隙中,一边逐渐增加持卵针中的负压,直至极体与目标核质被完整吸入持卵针中而完成去核,最后在不拔出注射针的情况下直接注射细胞核或完整细胞进而完成重构胚的构建。用此方法对200个卵母细胞进行注核和注细胞操作,平均完成一个重构胚的构建各自耗时约40s和30s,成功率分别为62.6%和86.0%。用核染料Hoechst 33342 对卵母细胞的去核效率进行验证,去核成功率达到73.3%。实验证明,用此方法可以在只有倒置显微镜和显微操作仪的条件下一次性快速完成去核和注核,大大提高了细胞核移植的效率和重构胚成活率;更重要的是该方法操作简单,新手可以很快掌握该技术,易于在实际工作中推广应用。     

小鼠体细胞核移植程序的研究

采用直接去核法、透明带切割法、PMM法3种核移植方法进行小鼠卵母细胞去核的研究。3种方法都未对卵母细胞核进行示踪,仅以第一极体作为参照进行去核,都属于盲吸法范畴,在去核率上没有显著差异。直接去核法对卵母细胞造成较大的伤害,去核操作过程中极易使卵母细胞膜破裂,发生崩解;透明带切割法分步操作使操作变得柔和,对卵母细胞的刺激减小,去核卵母细胞的存活率较高;PMM法靠脉冲电压在透明带上打孔进行辅助去核,脉冲参数稍大或压透明带太紧都极易在击破透明带的同时击破卵膜,使卵母细胞发生崩解。在构建重构胚的过程中,胞质内注射法较电融合法而言,程序简单,带入的供体胞质较少,构建重构胚的效率更高。     

用改进的细胞核移植方法构建重构胚

为了能够找出一种既容易操作,又不需要特殊设备的核移植方法,对以前的操作进行了改进。首先以预先吸有细胞核或细胞的注射针在固定于持卵针上的卵母细胞透明带上穿刺两个孔,然后一边缓慢地将注射针回拔至卵周隙中,一边逐渐增加持卵针中的负压,直至极体与目标核质被完整吸入持卵针中而完成去核,最后在不拔出注射针的情况下直接注射细胞核或完整细胞进而完成重构胚的构建。用此方法对200个卵母细胞进行注核和注细胞操作,平均完成一个重构胚的构建各自耗时约40s和30s,成功率分别为62·6%和86·0%。用核染料Hoechst33342对卵母细胞的去核效率进行验证,去核成功率达到73·3%。实验证明,用此方法可以在只有倒置显微镜和显微操作仪的条件下一次性快速完成去核和注核,大大提高了细胞核移植的效率和重构胚成活率;更重要的是该方法操作简单,新手可以很快掌握该技术,易于在实际工作中推广应用。     

牛体细胞核移植显微操作环节的优化

本研究从牛卵母细胞去核方法(纺锤体观测仪法&Hoechst33342染色法)、供体细胞核引入去核卵细胞质的方法(卵细胞质注射法和电融合法)和重构胚胎电融合(3组参数)等3个环节对牛体细胞核移植的显微操作过程及相关参数进行了筛选优化。以核移植胚胎的卵裂率、囊胚发育率作为检测指标,对不同的方法所获得的克隆胚胎的卵分裂率与囊胚发育率进行比较,最后筛选获得1个优化的牛体细胞核移植操作程序,即采用Spindle view系统对牛卵母细胞进行去核操作,将供核体细胞注射到卵周隙,然后通过电融合法将供体核引入去核卵细胞质(电融合参数为1.9kV/cm,脉冲时程10μs,方波2次间隔2s)。以此核移植程序进行牛体细胞核移植实验,自获得克隆胚胎中筛选80枚优质囊胚移植到33头受体牛子宫内,最后2头母牛产下2头克隆牛犊,结果表明利用该优化的显微操作环节进行牛体细胞核移植可以获得体细胞克隆牛犊。     

Hypertonic medium treatment for localization of nuclear material in bovine metaphase II oocytes

Oocytes enucleated at the second metaphase stage (MII) are often used as recipient cytoplasts for nuclear transfer. The oocyte's nuclear material has been traditionally removed blindly by aspirating the first polar body (Pb1) along with a portion of the cytoplasm. However, the Pb1-guided enucleation method is unreliable because the position of the Pb1 is variable. A previous study showed that pretreatment of mouse oocytes with 3% (0.09 M) sucrose allowed visualization of the metaphase spindle and chromosomes under standard light microscopy and led to a 100% enucleation rate. The same sucrose treatment, however, did not produce the same effect in bovine oocytes. In this study, we increased the concentration of sucrose to 0.3-0.9 M in PBS containing 20% fetal bovine serum (SPF) and found that the majority of the treated bovine oocytes (75%-86%) formed a small transparent bud into the perivitelline space, as compared with the 0.1 M sucrose (6%) or the no sucrose (0%) control groups. Staining of DNA with Hoechst 33342 revealed that these projections coincided with the position of the metaphase chromosomes in 100% of sucrose-treated oocytes, whereas only 31% of oocytes showed alignment of the position of Pb1 with their nuclear materials. Furthermore, 95% of oocytes treated in 0.3 M SPF were successfully enucleated by removing a small amount of cytoplasm adjacent to the projection. This is a significantly higher enucleation rate than that obtained by conventional Pb1-guided enucleation, even when a larger amount of cytoplasm was removed. For nuclear transfer, the enucleated oocytes treated with sucrose did not differ from the control oocytes in rates of fusion, cleavage, or development to blastocysts, or in the average cell numbers in blastocysts. This study demonstrated that 0.3 M sucrose treatment of bovine oocytes facilitates the localization of metaphase chromosomes under normal light microscopy and hence increases enucleation efficiency without compromising the in vitro development potential of cloned embryos by nuclear transfer.     

Preparation of young preactivated oocytes with high enucleation efficiency for bovine nuclear transfer

To improve the enucleation rate in newly matured bovine oocytes, we investigated the position of cytoplasmic chromatin in relation to the polar body and the consequent enucleation efficiency before and after sequential activation with calcium ionophore A23187 and cycloheximide. Oocytes aspirated from the follicles of slaughterhouse-collected ovaries were cultured for 18 to 20 h. With Hoechst staining, only 40.7% of the chromatin material was found adjacent to the first polar body in metaphase II oocytes, while 100% was located adjacent to the second polar body in oocytes after the activation. Enucleation trials after activation showed a higher enucleation rate (91.5%) than that before activation (59.9%). The following experiment determined the effect of using both kinds of cytoplast on the in vitro development of nuclear transfer embryos. Blastomeres of the 32-cell-stage in vitro-produced embryos were transferred, fused to the activated cytoplasts and cultured in vitro. No significant difference was detected in fusion, cleavage or development to blastocysts obtained 7 d (174 h) post fusion. In conclusion, this study showed that young in vitro-matured bovine oocytes sequentially activated with calcium ionophore and cycloheximide have cytoplasmic chromatin material adjacent to the second polar body, leading to a high enucleation rate.     

Vitrification of bovine oocytes and its application to intergeneric somatic cell nucleus transfer

We determined the efficacy of a microdrop vitrification procedure for cryopreservation of bovine oocytes, using vitrified oocytes as cytoplasts for intraspecies and intergeneric somatic cell nucleus transfer (NT). In vitro matured bovine MII oocytes were vitrified in microdrops with a vitrification solution containing 35% ethylene glycol, 5% polyvinyl pyrrolidone, and 0.4 M trehalose. After warming, approximately 80% of the vitrified oocytes were morphologically normal, and their enucleation rate was similar to that of fresh oocytes. The NT embryos constructed with bovine cumulus cells and the vitrified oocytes developed similar to blastocysts constructed with fresh oocytes, although the cell number of NT blastocysts originating from vitrified oocytes was lower than that of the fresh control. In a second experiment, we examined the development of NT embryos constructed with vitrified bovine oocytes and bovine fibroblasts (intraspecies NT embryos) or swamp buffalo fibroblasts (intergeneric NT embryos). There were no differences between the intraspecies and intergeneric NT embryos in fusion, cleavage and development to blastocysts, except for lower cell numbers in the intergeneric NT blastocysts. In conclusion, the efficacy of this microdrop vitrification procedure and the production of swamp buffalo NT blastocysts using vitrified bovine oocytes was demonstrated.     

The effects of chemical enucleation combined with whole cell intracytoplasmic injection on panda-rabbit interspecies nuclear transfer

Conventional methods of somatic cell nuclear transfer either by electrofusion or direct nucleus injection have very low efficiency in animal cloning, especially interspecies cloning. To increase the efficiency of interspecies somatic cell nuclear transfer, in the present study we introduced a method of whole cell intracytoplasmic injection (WCICI) combined with chemical enucleation into panda-rabbit nuclear transfer and assessed the effects of this method on the enucleation rate of rabbit oocytes and the in vitro development and spindle structures of giant panda-rabbit reconstructed embryos. Our results demonstrated that chemical enucleation can be used in rabbit oocytes and the optimal enucleation result can be obtained. When we compared the rates of cleavage and blastocyst formation of subzonal injection (SUZI) and WCICI using chemically enucleated rabbit oocytes as cytoplasm recipients, the rates in the WCICI group were higher than those in the SUZI group, but there was no statistically siginificant difference (p > 0.05) between the two methods. The microtubule structures of rabbit oocytes enucleated by chemicals and giant panda-rabbit embryos reconstructed by WCICI combined with chemical enucleation were normal. Therefore the present study suggests that WCICI combined with chemical enucleation can provide an efficient and less labor-intensive protocol of interspecies somatic cell nuclear transfer for producing giant panda cloned embryos.     

Comparison of bulk enucleation methods for porcine oocytes

Cloning of mammalian oocytes requires that the recipient oocyte is enucleated to remove all genetic material associated with the chromosomes. The procedure currently used in most species requires careful micromanipulation of oocytes treated with cytochalasin B to prevent structural damage. Although functional, this procedure requires time and limits the number of oocytes available for cloning, and our ability to understand the mechanisms of nuclear reprogramming. Therefore, this study aimed at evaluating different procedures to enucleate large pools of oocytes in a time-efficient manner. Two different approaches were tested. The first approach involved centrifugation of zona-free oocytes through a percoll gradient to separate the portion containing the chromatin from the cytoplasmic portion. The second used etoposide to prevent chromatin segregation at first metaphase and resulting in the expulsion of all chromosomes in the polar body. Using the chemical approach an average enucleation rate of 39.4 +/- 7.5% was obtained, while the centrifugation approach resulted in an average enucleation rate of 66.9 +/- 6. In terms of time efficiency, the control manipulation method takes 0.11 min and the centrifugation took an average of 0.52 min per oocyte. The MPF activity at the end of procedure was estimated through the measurement of H1 activity and as expected, the etoposide-cycloheximide treated oocytes had lower H1 activity which was restored by further incubation in the maturation medium for 5 hr while the centrifugation gave a nonsignificant intermediary result. In conclusion, the results presented suggest that both the chemical and the mechanical methods are usable alternatives to micromanipulation of oocytes to generate a large number of chromosome free cytoplasm for biochemical analysis. Mol. Reprod. Dev. 67: 70-76, 2004.     

A Comparative Study on the Efficiency of Two Enucleation Methods in Pig Somatic Cell Nuclear Transfer: Effects of the Squeezing and the Aspiration Methods

In this study, two enucleation methods, the squeezing and the aspiration methods, were compared. The efficiency of these two methods to enucleate pig oocytes and the in vitro and in vivo viability of somatic cell nuclear transfer (SCNT) pig embryos, were evaluated. In the squeezing method, the zona pellucida was partially dissected and a small amount of cytoplasm containing metaphase II (MII) chromosomes and the first polar body (PB) were pushed out. In the aspiration method, the PB and MII chromosomes were aspirated using a beveled micropipette. After injection of fetal fibroblasts into the perivitelline space, reconstructed oocytes were fused and activated electrically, and then cultured in vitro for 6 days or transferred to surrogates. The squeezing method resulted in a higher proportion of degenerated oocytes than the aspiration method (14% vs. 5%). The squeezing method took longer to enucleate 100 oocytes (306 minutes) than the aspirating method (113 minutes). Fusion rate (72–78%) and cleavage rate (67%) were not influenced by the enucleation method but blastocyst formation was improved (P < 0.05) in oocytes enucleated by the aspiration method (5 vs. 9%). When SCNT embryos were transferred to recipients, pregnancy rates to term were similar (27%, 3/11 and 27%, 3/11) in both methods with the birth of 10 piglets/3 litters and 16 piglets/3 litters in the squeezing and the aspiration methods, respectively. Our results indicate that the aspiration method for oocyte enucleation is more efficient than the squeezing method in producing a large number of pig SCNT embryos with normal in vivo viability.     

A comparative study on the efficiency of two enucleation methods in pig somatic cell nuclear transfer: effects of the squeezing and the aspiration methods

In this study, two enucleation methods, the squeezing and the aspiration methods, were compared. The efficiency of these two methods to enucleate pig oocytes and the in vitro and in vivo viability of somatic cell nuclear transfer (SCNT) pig embryos, were evaluated. In the squeezing method, the zona pellucida was partially dissected and a small amount of cytoplasm containing metaphase II (MII) chromosomes and the first polar body (PB) were pushed out. In the aspiration method, the PB and MII chromosomes were aspirated using a beveled micropipette. After injection of fetal fibroblasts into the perivitelline space, reconstructed oocytes were fused and activated electrically, and then cultured in vitro for 6 days or transferred to surrogates. The squeezing method resulted in a higher proportion of degenerated oocytes than the aspiration method (14% vs. 5%). The squeezing method took longer to enucleate 100 oocytes (306 minutes) than the aspirating method (113 minutes). Fusion rate (72-78%) and cleavage rate (67%) were not influenced by the enucleation method but blastocyst formation was improved (P < 0.05) in oocytes enucleated by the aspiration method (5 vs. 9%). When SCNT embryos were transferred to recipients, pregnancy rates to term were similar (27%, 3/11 and 27%, 3/11) in both methods with the birth of 10 piglets/3 litters and 16 piglets/3 litters in the squeezing and the aspiration methods, respectively. Our results indicate that the aspiration method for oocyte enucleation is more efficient than the squeezing method in producing a large number of pig SCNT embryos with normal in vivo viability.     

Development of enucleated mouse oocytes reconstituted with embryonic nuclei

The chromosomes of mouse oocytes at telophase of the first meiotic division were removed using micromanipulation and differential interference microscopy. The enucleated oocytes were used as recipients for nuclear transplantation, after culture for 4-6 h. The newly synthesized proteins of the enucleated oocytes showed the same pattern as those of secondary oocytes matured in vivo. When the enucleated oocytes received a nucleus from late 2- and 8-cell embryos, or a cell from the inner cell mass (ICM) of blastocysts, 23, 4 and 10%, respectively, of reconstituted embryos developed to blastocysts. After transfer to recipient females, live young were produced from the reconstituted eggs that received a nucleus from late 2-cell embryos.     

利用IVF废弃胚胎为核移植受体构建人体细胞克隆胚胎

目的：探讨利用IVF废弃胚胎构建人体细胞克隆胚胎的发育潜能及其在人治疗性克隆应用的可能性。方法：收集2008年7-12月在广州医学院第三附属医院进行体外受精-胚胎移植周期中的多精受精胚胎和MII期体外受精失败卵母细胞,运用显微操作技术构建人体细胞克隆胚胎,观察胚胎发育情况。结果：多精受精胚胎为核移植受体的克隆胚胎能够发育到8-细胞期,受精失败MII期卵母细胞为核移植受体的克隆胚胎能够激活,但不能够卵裂。两种IVF废弃的胚胎构建的人体细胞克隆胚胎在去核成功率,注核成功率上无显著差异（P＆gt;0.05）,但卵裂率和8细胞率上具有显著差异（P＆lt;0.05）。结论：多精受精胚胎比MII期体外受精失败卵母细胞更适合作为人核移植受体细胞。