卵细胞体外成熟(IVM)在小鼠生物净化中的应用

目的 在小鼠生物净化中,从超排后未排卵的卵巢,取卵泡内未成熟卵采用卵母细胞体外成熟(in vitro maturation,IVM)使之在体外成熟并具备受精能力,以提高卵子利用率和作为常规促超排失败的一种补救措施。方法 在小鼠生物净化中,取注射PMSG和HCG后未排卵卵巢,在实体显微镜下划破卵泡挑选卵丘卵母细胞复合物(COCs),置于成熟液滴中在体外发育成熟。同时以注射PMSG和HCG后正常超排卵、只注射PMSG后取未成熟卵体外成熟和注射PMSG和HCG后取疑似成熟卵和裸卵体外成熟作为对照。经体外受精、体外胚胎发育后,将2-细胞胚移植到受体输卵管,使其在受体内发育成为成熟的个体。结果 注射PMSG和HCG后未排卵组(A组),卵细胞体外成熟率为87.0%±3.2%,二胞率为55.1%±12.3%,囊胚率为23.1%,移植41枚二细胞胚至2只受体鼠,出生5只幼崽,产仔率12.2%。只注射PMSG,48 h后取未成熟卵组(B组),体外成熟率为83.9%±3.9%,二胞率为51.8%±9.3%,囊胚率为38.5%±13.9%。注射PMSG和HCG正常超排组(C组),其二胞率为78.9%±0.6%,囊胚率为78.0%±3.8%。注射PMSG和HCG未排卵卵巢,取裸卵和疑似成熟卵(D组),体外成熟培养0 h,6 h和16～18 h,其成熟率、二胞率和囊胚率与其它三组相比均较低且有极显著性差异。A组和B组与正常对照C组相比,二胞率均有显著性差异,囊胚率均有极显著性差异。结论 卵母细胞体外成熟(IVM)可以作为小鼠生物净化中促超排失败的一种补救措施,并且可以提高珍稀品系小鼠的卵子利用率。     

促减数分裂甾醇（MAS）对猪卵母细胞体外成熟及其胚胎发育能力的影响

利用促减数分裂甾醇(MAS)合成代谢过程中的抑制剂AY9944累积FF-MAS的原理,在猪卵母细胞体外成熟过程中添加AY9944,间接地研究了内源性MAS对猪卵母细胞体外成熟质量的影响.猪卵丘卵母细胞复合体(cumulus oocyte complexes,COCs)培养在NCSU23成熟培养液中,并添加不同浓度(0,10,20,40μmol/L)的AY9944培养44h.培养结束后,成熟的卵母细胞进行孤雌激活和以胎儿成纤维细胞为核供体重构胚胎,分别于48和144h观察胚胎发育情况,统计卵裂率和囊胚率及囊胚/2-细胞胚比率.结果如下:(1)随着AY9944添加浓度的增加,退化的卵母细胞增多,40μmol/L AY9944处理的退化卵显著,卵母细胞成熟率显著下降.(2)成熟培养液添加20和40μmol/L AY9944处理的孤雌激活胚胎的囊胚形成率和2-细胞胚发育到囊胚的比率显著增加(P<0.05).(3)以对照组(0μmol/L AY9944)和20μmol/L AY9944处理的卵母细胞为胞质受体,发现20μmol/L AY9944处理的克隆胚的发育能力囊胚率和2-细胞胚发育到囊胚的比率有所提高,但无显著差异(P>0.05).以上结果表明,猪卵母细胞体外成熟过程中添加AY9944提高了猪卵母细胞体外成熟的胞质质量,胚胎的发育能力提高.     

水牛卵母细胞孤雌激活及孤雌胚与体外受精胚发育的比较

目的对MII期水牛卵母细胞进行人工诱导激活,可以间接判断体外成熟卵母细胞质量的优劣,并且,卵母细胞的充分激活也是提高核移植效率的关键因素之一。比较了水牛卵母细胞体外成熟时间对孤雌激活胚发育能力的影响以及不同化学激活方法对水牛卵母细胞孤雌激活效果的影响,并在相同条件下,对孤雌激活胚与体外受精胚的发育能力进行了比较。结果表明,水牛卵母细胞体外成熟27 h或30 h的囊胚发育率(19.0%,17.7%)明显高于体外成熟21 h或24 h的囊胚发育率(12.3%,13.8%);Ion联合6_DMAP激活水牛卵母细胞的效果优于其他几组激活方法;在相同培养条件下,孤雌激活胚与体外受精胚的发育能力存在着差异,其中卵裂率差异不显著,但孤雌激活胚的囊胚发育率显著高于体外受精胚(21.7%,13.0%)。     

猪卵核移植的研究

以湖北白猪为受体，杜洛克为供体进行了猪卵核移植的研究。结果表明，半胱氨酸对卵具有保护作用。卵龄对核移植效率有显影响，以４２－４８ｈ为宜，激活后卵母细胞做受体可提高移核胚的发育率；供体核发育期对移核胚的发育有显影响。核移植后的重组胚移入受体后，经体内发育得到了５只核移植仔猪。     

克隆绵羊遇到的问题及其分析

本研究以体外成熟不同时间的绵羊卵母细胞为胞质受体,对去核、融合、不同激活方法、卵裂率、桑/囊胚率和克隆胚受胎率等作了研究与分析.观察妊娠受体母羊的状态、胎儿体内发育情况,分析影响胎儿发育的原因.结果显示,成熟培养18h、22h、24h卵母细胞的盲吸去核率无显著性差异(84.9%,86.6%,84.3%,p>0.05),但显著高于26h组的去核率(71.7%).离子酶素联合cycloheximide(CHX)对重构胚进行激活,卵裂率显著高于A23187联合6-DMAP的激活方法(84.6%vs 63.2%,p<0.05),但二者处理对桑/囊胚率发育无显著差异(25.5%vs18.7%,p>0.05).作为胞质受体,成熟20h的卵母细胞与成熟24h和26h的卵母细胞相比,不利于重构胚的体外发育,桑/囊胚率差异显著(12.3%vs 25.3%,23.4%,p<0.05).分别以美丽奴、杜泊肉羊的体细胞为供体细胞进行克隆生产,90天以后的妊娠率为10%-17%,发育到期率为10%以上.出生羔羊的体重普遍较大.     

不同状态的牛供核细胞对核移植胚胎发育的影响

比较了不同状态的牛供核细胞对核移植胚胎发育的影响．结果表明：体外培养2-5,6—10,11—15,16v20代的供核细胞的融合率和卵裂率没有差异,但随着体外培养代数的增加,重构卵发育到囊胚的比例下降．16—20代供核细胞组的囊胚率显著低于2—5和6—10代供核细胞组的囊胚率（分别为19．61％,26．67％和28．57％,P〈0．05）;来源于原代牛（G0）,克隆一代牛（G1）和克隆二代牛（G2）的供核细胞的融合率,卵裂率和囊胚率没有明显差异;对于新鲜供核细胞和冷冻供核细胞,胞质内注射法的重构卵率要明显高于透明带下注射法的重构卵率（分别为81．31％,67．96％和82．08％,52．94％,P〈0．05）,但各组重构卵的卵裂率和囊胚率没有差异,冷冻供核细胞结合胞质内注射法可以获得较好的核移植胚胎发育．     

精子和酒精对小鼠卵母细胞激活的比较

对不同卵龄的小鼠卵母细胞被精子和酒精激活后的激活率进行了比较。结果显示,卵母细胞对常规的体外受精和酒精的人工激活的激活率存在卵龄的差异。注射hCG后15～24h的卵母细胞容易被酒精的人工刺激所激活,20h卵龄的卵母细胞激活率最高,平均为81．6％,且速即卵裂率也最高,平均为48％,卵龄更大的卵母细胞激活率降低,而13h的卵母细胞难以被酒精激活。另一方面,13～15h的卵母细胞容易被精子激活而受精,卵龄较大的卵母细胞在体外难以被精子激活受精。这表明,精子和酒精对卵母细胞的激活机制有所不同。     

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     

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)。     

C57BL/6J超排小鼠取卵时间对体外受精效果的影响

目的 探讨雌性小鼠注射绒毛膜促性腺激素(HCG)后，取卵时间对体外受精率的影响。方法 选用3～4周龄的野生型C57BL/6J雌鼠，体质量10～13 g,通过腹腔注射血清促性腺激素(PMSG)和HCG联合使用^[1]做超排处理。我们将超排后雌鼠取卵时间分为6个时间段，分别为13、14、15、16、17、18 h后取卵母细胞与新鲜精子做体外受精。取3月龄雄性小鼠附睾里精子，在HTF液里获能1 h后，用于体外受精。实验共分3组，每组12只雌鼠用于超排处理，共得到2-细胞胚胎数分别为258、199和243枚；分别移植到当天见栓的假孕鼠输卵管内，得到出生仔鼠分别为98只、87只、95只；出生率分别为37.98%、43.72%和39.09%。结果 总取卵数和2-细胞发育率，超排后15 h取卵发育受精率最高，这说明小鼠卵母细胞超排后14 h少数成熟，15～16 h完全成熟，排卵17 h后则开始出现退化。结论 3组实验最高受精率比较：第1组15 h后取的卵母细胞团受精率最高为79.17%、第2组15 h后取的卵母细胞团受精率最高为75.68%、第3组16 h后取的卵母细胞团受精率最...     

Effects of different nuclear recipients on developmental potential of mouse somatic nuclear transfer embryos

Somatic cell nuclear transfer has been succeeded in procedures of nuclear transfer. One is single nucleartransfer, the other is serial nuclear transfer. Viable animals have been cloned in different species using both me-thods[1—6]. Different nuclear recipients and donors wereused in serial nuclear transfer, namely, transferring thenuclear of reconstructed embryo into enucleated MⅡoocytes[7], transferring the nuclear of reconstructed em-bryos at one cell stage into enucleated zygote[4] and t…     

成纤维细胞和ES细胞在小鼠和兔去核卵母细胞内发育

以昆明白小鼠成纤维细胞和胚胎干（ES）细胞作为供核细胞,以昆明白小鼠和日本大耳白兔的MⅡ期去核卵母细胞作为受体,采用核移植方法,构楚了克隆胚胎．在同种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率明显低于以成纤维细胞为供核细胞的克隆胚胎卵裂率（24．4％相对于56．9％,P〈0．05）,1．8％的ES细胞克隆胚胎发育到囊胚阶段,而成纤维细胞克隆胚胎没能发育到囊胚阶段;在异种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率（89．6％）和囊胚发育率（18．8％）明显高于以成纤维细胞为供核细胞的克隆胚胎卵裂率（54．2％）和囊胚发育率（4．2％）．     

Effects of different nuclear transfer and activation methods on the development of mouse somatic cell cloned embryos

A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect of different nuclear transfer (NT) and activation methods on the development of mouse cloned embryos, embryos were reconstructed using two traditional NT methods (electrofusion and direct injection) and four activation treatments (electric pulse, ethanol, SrCl2 and electric pulse combined with SrCl2). The data showed that the efficiency of reconstruction using the direct injection method is significantly higher (90.7%) than that of the electrofusion method (49.7%). Parthenogenetic embryos can develop to blastocyst stage with three activation conditions, including ethanol, electric pulse and SrCl2; however, the rates of development to blastocyst after ethanol and electric pulse acti-vation (52.4%, 54.2%) are significantly lower than after SrCl2 activation (76.9%). Treatment of embryos for 6 h with 10 mmol/L SrCl2 was found to be the best condition for activation of parthenogenetic as well as reconstructed embryos. By contrast, reconstructed embryos failed to develop to blastocyst stage after being activated by ethanol. The use of either injection or electrofusion for embryo reconstruction affected the pre-implantation development. However, after transfer in pseudopregnant mice, cloned mice were obtained from both methods.     

Effect of leptin on oocyte maturation and subsequent pregnancy rate of cloned embryos reconstructed by somatic cell nuclear transfer in pigs

Cloning pigs by somatic cell nuclear transfer （SCNT） has wide applications in basic research, human medicine and agricultural production. To improve cloning efficiency, the effect of two basic maturation media, NCSU-23 and TCM199, was compared, and TCM199 was selected for the following experiments with leptin. We systematically studied the effects of leptin supplementation on oocytes in vitro maturation （IVM）, in vitro development of parthenogenetically activated （PA） and SCNT embryos and in vivo development of SCNT embryos after embryo transfer （ET）. The results showed that supplementation of 100 or 200 ng/ml leptin into the maturation medium did not greatly affect nuclear maturation of oocytes, or cleavage rates of PA and SCNT （P 〉 0.05）. Blastocyst rates of PA and SCNT embryos were significantly improved when 100 or 200 ng/ml leptin was added to maturation medium, and the number of cells in PA blastocysts was also improved （P 〈 0.05）. The number of cells in blastocyst of SCNT was improved, when 100 ng/ml leptin was added （P 〈 0.05）. Furthermore, supplementation of 100 or 200 ng/ml leptin to the IVM medium may improve pregnancy rate and the delivery rate inpig cloning.     

小鼠四倍体半克隆胚胎发育研究

半克隆(Semi-Cloned)胚胎是通过注射体细胞核到未去核的卵母细胞中产生的。在半克隆胚胎中,体细胞被用来作为精子的替代物。然而,由于异常的染色体分离,构建的半克隆胚胎在激活后形成了非整倍体而导致胚胎发育受到严重影响,不能发育到期。本研究通过抑制小鼠半克隆胚胎在激活过程中染色体数目减半,避免非整倍体胚胎形成,研究四倍体半克隆(TetraploidSemi-cloned,TSC)胚胎的发育和体细胞核的掺入对胚胎发育的影响。结果显示,TSC胚胎的体外发育率显著高于二倍体半克隆胚胎,与正常受精卵及孤雌激活对照无显著性差异,但TSC胚胎的细胞数在桑椹胚和囊胚期比正常二倍体受精胚胎和孤雌激活胚胎少。通过Oct-4染色发现,TSC胚胎囊胚期内细胞团(InnerCellMass,ICM)细胞很少或者没有。移植63个四倍体半克隆胚胎到3只假孕母鼠体内,得到20个胎盘,但没有得到胎儿。组蛋白乙酰化和DNA甲基化检测显示,部分TSC胚胎在囊胚期没有形成正常受精胚胎在ICM和滋养外胚层(Trophectoderm,TE)之间的差异分布。TSC胚胎的基因表达不依赖于细胞分裂次数而依赖于发育时间。虽然TSC胚胎避免了二倍体半克隆胚胎形成非整倍体现象,但由于TSC胚胎没有ICM细胞或ICM细胞很少,所以只能形成胎盘而不能形成胎儿。本实验第一次较为全面地研究了TSC胚胎的发育,同时也为研究体细胞核再程序化、基因打靶技术提供了一种新的途径。     

Cloned pigs derived from somatic cell nuclear transfer embryos cultured in vitro at low oxygen tension

Great progress have been made in animal cloning in China, as evidenced by the live births of cloned cat- tle[1,2], goats[3,4], and sheep[5]. In contrast, pig cloning is still in its infancy though limited fundamental studieshave been conducted[6]. It is g…     

Production of transgenic calves by somatic cellnuclear transfer

Bovine fetal oviduct epithelial cells were transfected with constructed double marker selective vector(pCE-EGFP-IRES-Neo-dNdB) containing the enhanced green fluorescent protein (EGFP) and neomycin-resistant(Neo^r) genes by electroporation, and a transgenic cell line was obtained. Somatic cell nuclear transfer (SCNT) was cartied out using the transgenic cells as nuclei donor. A total of 424 SCNT embryos were reconstructed and 208 (49.1%) of them developed to blastocyst stage. 17 blastocysts on D 7 after reconstruction were transferred to 17 surrogate calves,and 5 (29.4%) recipients were found to be pregnant. Three of them maintained to term and delivered three cloned calves.PCR and Southern blot analysis confirmed the integration of transgene in all of the three cloned calves. In addition, expression of EGFP was detected in biopsy isolated from the transgenic cloned calves and fibroblasts derived from the biopsy. Our results suggest that transgenic calves could be efficiently produced by SCNT using transgenic cells as nuclei donor. Furthermore, all cloned animals could be ensured to be transgenic by efficiently pre-screening transgenic cells and SCNT embryos using the constructed double marker selective vector.     

Green fluorescent protein （GFP） transsenic pig produced by somatic cell nuclear transfer

Transgenic somatic cell nuclear transfer is a very promising route for producing transgenic farm animals. Research on GFP transgenic pigs can provide useful information for breeding transgenic pigs, human disease models and human organ xenotransplantation. In this study, a liposomal transfecUon system was screened and transgenic embryos were reconstructed by nuclear transfer of GFP positive cells into enucleated in vitro matured oocytes. The development of reconstructed embryos both in vitro and in vivo was observed, and GFP expression was determined. The results showed that porcine fe- tal-derived fibroblast cells cultured with 4.0 μL/mL liposome and 1.6 μg/mL plasmid DNA for 6 h resulted in the highest transfecUon rate （3.6%）. The percentage of GFP reconstructed embryos that de- veloped in vitro to the blastocyst stage was 10%. Of those the GFP positive percentage was 48%. Reconstructed transgenic embryos were transferred to 10 recipients. 5 of them were pregnant, and 3 delivered 6 cloned piglets in which 4 piglets were transgenic for the GFP as verified by both GFP protein expression and GFP DNA sequence analysis. The percentage of reconstructed embryos that resulted in cloned piglets was 1.0%; while the percentage of piglets that were transgenic was 0.7%. This is the first group of transgenic cloned pigs born in China, marking a great progress in Chinese transgenic cloned pig research.     

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。