不同品系小鼠的体外受精、胚胎冷冻及移植的比较研究

目的　探讨不同品系小鼠的体外受精、胚胎和精子的低温保存效果。方法　本实验分别在中国科学院上海实验动物中心 (SLAC)和日本熊本大学动物资源开发中心 (CARD)对 13个品系小鼠 (C57BL 6J、BALB c、C3H HeJ、ICR、KM、FVB、MRL、NOD、CBA、DBA 2、CD 1、BDF1、B6C3F1)的体外受精 (IVF)率、胚胎培养及移植成绩进行了比较研究。结果　各品系小鼠新鲜精子的IVF率 15 1%～ 87 9% ,冻融精子的IVF率 8%～ 80 % ;冷冻胚胎的复苏率4 2 6 %～ 83 9% ;冻融胚胎移植后的产仔率在 17 8%～ 5 1 8%。结论　遗传背景不同的小鼠体外受精率、冷冻胚胎复苏率和胚胎移植的产仔率差异有显著性。但同一品系两个实验室间的新鲜精子的IVF率、冷冻胚胎的复苏率及移植产仔率差异无显著性 (P >0 0 5 ) ;冻融精子的体外受精率CARD明显高于SLAC(P <0 0 1)。     

昆明小鼠精子冷冻的研究（简报）

胚胎工程技术是动物品种、品系培育,种质资源保存及转基因动物制备、保种的重要手段。配子的冷冻保存技术目前广泛应用于胚胎工程。和胚胎冷冻相比小鼠精子冷冻技术方便、高效尤其适用于转基因及突变系小鼠的保种。成功的精子冷冻要求复苏后通过体外受精(IVF)获得胚胎,再移植入受     

昆明小鼠精子冷冻的研究(简报)

胚胎工程技术是动物品种、品系培育,种质资源保存及转基因动物制备、保种的重要手段。配子的冷冻保存技术目前广泛应用于胚胎工程。和胚胎冷冻相比小鼠精子冷冻技术方便、高效尤其适用于转基因及突变系小鼠的保种。成功的精子冷冻要求复苏后通过体外受精(IVF)获得胚胎,再移植入受体,生长发育并产出幼仔。胚胎体外培养获得足够的晚桑葚胚和早期囊胚是胚胎干细胞建立与制备嵌合体小鼠的成功关键,暂时不用的胚胎应仍能耐受冷冻保存,待复苏移植,建立新的繁殖群。但小鼠精子冷冻研究在我国开展的十分有限,缺乏相关资料数据。本实验对不同周龄SPF级昆明(KM)小鼠进行精子冷冻及冻融精子IVF,并将IVF获得的2-细胞胚胎分别进行胚胎移植、体外培养、胚胎冷冻及复苏后移植,应用冻融小鼠精子进行胚胎工程实践。     

两种玻璃化胚胎冷冻方法对不同品系小鼠胚胎冷冻效果比较

目的探讨EFS和DAP两种玻璃化冷冻方法对不同品系小鼠胚胎冷冻的效果。方法6个品系小鼠（KM、ICR、BALB／c、C57BL／6J、OB／OB、LAP／~TAOF59）的2-cell胚胎分别用EFS和DAP两种玻璃化冷冻方法进行冷冻和复苏,比较两种冷冻方法的胚胎复苏率和着床率。结果6个品系小鼠冷冻胚胎EFS方法的平均复苏率为69．97％（47．9％～83．6％）,DAP方法的平均复苏率47．23％（26．3％-76．7％）,EFS方法明显优于DAP方法。其中KM、ICR和BALB／c小鼠EFS方法的冷冻复苏率显著高于DAP方法（P〈0．01）;冻融胚胎移植后EFS方法的平均着床率27．23％（1．75％一45．0％）,DAP方法的平均着床率31．43％（7．0％一46．3％）。除KM、ICR小鼠外,其他4个品系小鼠的着床率DAP方法高于EFS方法。结论KM和ICR远交群小鼠胚胎适合用EFS方法冷冻保存;C57BL／6J、OB／OB、LAP／aTAOF59三个品系小鼠DAP方法优于EFS方法,但差异不大;BALB／c小鼠两种玻璃化冷冻方法的冻融胚胎着床率均较低,需进一步研究。     

激光打孔技术在小鼠卵子冷冻保存上的运用

目的比较卵子冷冻复苏后、以及复苏卵子经过激光打孔后,与新鲜精子、冷冻复苏精子体外受精,受精率的变化。方法 （1）通过免疫荧光染色技术,判断卵子冷冻前后透明带糖蛋白-2、微丝以及细胞核的变化;（2）冷冻C57BL/6J小鼠卵子,复苏后一部分卵子打孔,一部分不打孔,然后与9个品系的新鲜精子和冷冻精子体外受精,比较各组受精率的变化。结果 （1）新鲜卵子组透明带糖蛋白-2、微丝及细胞核结构清晰,而冷冻组以及冷冻剂处理组,微丝结构略有变化,透明带糖蛋白-2受到不同程度的损伤,而细胞核在冷冻前后无明显变化;（2）9个品系的新鲜精子与C57BL/6J复苏卵子受精率为17.6%～42.9%,平均为29.6%;新鲜精子与复苏打孔卵子受精率为29.1%～72.3%,平均为49.7%,差异显著（P〈0.05）;9个品系复苏精子与复苏卵子体外受精率为5.4%～23%,平均为15.5%;复苏精子与复苏打孔卵子受精率为16.7%～48.6%,平均为28.8%,差异显著（P〈0.05）。结论 （1）冷冻对卵子的透明带糖蛋白-2有较大的损伤,对微丝有一定的影响,但是对染色体没有影响;（2）冷冻卵子复苏后,体外受精率下降明显,但是复苏卵子经过激光打孔后,可以显著提高体外受精率。     

两种抗冷冻剂对不同品系小鼠精子冷冻的影响

目的建立一套比较理想的小鼠精子冷冻复苏的方法。方法采用R18S3和FERTIUPTM-CPA两种保护剂,对DBA/2、C57BL/6J、KM和B6.129S7-Ldlrtm1Her/J四个品系的小鼠精子进行冷冻,冷冻精子用三种方法复苏,以体外受精率来评价精子冷冻的效果。结果以R18S3作为冷冻保护剂,DBA/2(73.3%,88.4%,55.6%)和KM(64.9%,60.2%,39.6%)品系小鼠冷冻精子复苏后体外受精率差异显著(P<0.05),C57BL/6J(3.0%,10.3%,3.7%)和B6.129S7-Ldlrtm1Her/J(0%,5.0%,0%)结果差异不显著(P>0.05);以FERTIUPTM-CPA作为冷冻保护剂,DBA/2(33.6%,14.1%,91.6%)、C57BL/6J(8.4%,21.0%,4.9%)和B6.129S7-Ldlrtm1Her/J(8.2%,10.0%,28.9%)品系小鼠冷冻精子复苏后体外受精率差异显著(P<0.05),而KM(48.1%,48.0%,48.1%)小鼠冷冻精子复苏后体外受精率差异不显著(P>0.05)。结论对于DBA/2和KM品系小鼠来说,用R18S3或FERTIUPTM-CPA冷冻精子,选择一种恰当的复苏方法,均可以得到较理想的体外受精率,而C57BL/6J和B6.129S7-Ldlrtm1Her/J品系小鼠无论采用哪种冷冻保护剂,选择何种方法复苏精子,得到的体外受精率都较低。     

Ⅰ型干扰素受体缺失小鼠体外受精实验研究

目的探索两种I型干扰素受体缺失对于小鼠体外受精的影响并对体外受精条件进行优化。方法对两种I型干扰素受体缺失小鼠(IFN-αR-/-、IFN-α/βR-/-)及背景野生型小鼠(C57BL/6)分别进行体外受精及胚胎移植,每组超排5只小鼠,3次重复,记录相关数据,分析干扰素受体缺失是否对小鼠体外受精存在影响。分别将两种I型干扰素受体缺失小鼠配子与C57BL/6小鼠配子进行体外受精杂交试验,每组5只小鼠,3次重复,探讨I型干扰素受体缺失对雌雄配子体外受精的影响。同时,优化体外受精条件,探索提高受精率技术方法,每组5只小鼠,三次重复。结果两种I型干扰素受体缺失小鼠平均体外受精率低于背景品系C57BL/6小鼠,组间差异具有显著性(P0. 05)。干扰素α受体缺失小鼠体外受精率高于干扰素α、β受体双缺失小鼠体外受精率,组间差异具有显著性(P0. 05)。两种I型干扰素受体缺失小鼠的精子与C57BL/6小鼠卵细胞体外受精率均高于其卵细胞与C57BL/6小鼠精子的体外受精率,组间差异具有显著性(P0. 05)。通过延长精子获能时间至1 h或在获能液及受精液中加入1 mmol/L还原型谷胱甘肽(GSH)能够提高体外体外受精率,相应组间差异具有显著性(P0. 05)。结论 I型干扰素受体缺失可能导致相应品系小鼠体外受精率降低,而且对卵细胞的影响较精子的影响更为显著,通过适当延长精子获能时间或改变获能及受精液成分,能够提高体外受精率。     

不同品系小鼠胚胎玻璃化冷冻保存的比较研究

目的　研究甘油作为冷冻保护剂、不同基因型小鼠对胚胎玻璃化冷冻的影响。方法　采用 6 5mol L的甘油作为冷冻保护剂 ,采用二步法对CBA、NOD、C57BL 6J、ICR及CD1小鼠 3 5d的胚胎进行玻璃化冷冻 ,并比较了不同品系小鼠胚胎的复苏率及移植受孕率。结果和结论　CBA、NOD、C57BL 6J,ICR及CD1的复苏率分别为 5 7 6 %、4 8%、31 3%、86 5 %及 88% ,移植受孕率为 2 1%、2 3 5 %、11%、38%和 35 5 % ,封闭群小鼠的胚胎复苏率、移植受孕率均显著高于近交系小鼠。这提示胚胎的复苏率及移植受孕率可能与小鼠的不同基因型有关。五个品系中 ,桑椹胚及早期囊胚的体外复苏率均显著高于扩张囊胚。这说明不同基因型及胚胎的不同发育阶段对胚胎玻璃化冷冻效果有影响     

小鼠冷冻胚胎和精子SNP遗传鉴定方法的建立

目的建立小鼠冷冻胚胎和精子SNP(single nucleotide polymorphism)分型方法,用于冷冻胚胎和精子快速遗传鉴定方案。方法以中科院上海实验动物中心(国家啮齿类实验动物种子中心上海分中心)提供的小鼠冷冻胚胎和精子为样本,采用全基因组扩增技术和PCR-LDR分型技术建立小鼠冷冻物SNP遗传鉴定方法。结果全基因组扩增技术能大幅度增加冷冻胚胎样本的DNA总量;PCR-LDR分型方法适用于小鼠全基因组45个SNPs的分型;分型确定C57BL/6,BALB/c,FVB/NJ等胚胎和精子各10种近交系,SNP位点信息与测序结果一致;小鼠冷冻胚胎个数与SNPs检出个数成正比,当胚胎数达到12以上时SNP检出率100%。结论实现近交系小鼠冷冻胚胎和精子快速SNP基因分型及遗传质量鉴定。     

小鼠8细胞期胚胎超低温（一1960G)冷冻保存

1972年W hittingham 和W ilmut在超低 温条件下冷冻保存小鼠早期胚胎分别取得成 功。由于这项工作的理论意义和实用价值,国 际上一些实验室相继开展了研究工作。迄今, 家兔、羊和牛早期胚胎冷冻保存都已成功,其中 牛的胚胎冷冻已发展到商业规模。用胚胎冷冻 保存技术保存小鼠近交系、突变系等遗传资源 有其特殊意义,因而受到普遍重视。用这一方 法维持重要品系可以避免因意外事件造成遗传 资源丢失和遗传污染,可减轻由于日益增加的 近交系、突变系数量给繁育管理工作带来的沉 重负担。国外有些实验室已建立小鼠冷冻胚胎 库,证明这是稳定、经济和安全地保存遗传资源 的行之有效的方法。     

三种不同冷冻保护剂对小鼠附睾冷冻效果的比较

目的探讨三种不同冷冻保护剂对C57BL/6J小鼠附睾冷冻的效果。方法性成熟并交配过的6~8周龄C57BL/6J雄鼠附睾,分别用3种不同冷冻保护剂二甲基亚砜(DMSO)、丙二醇(PROH)、R18S3进行玻璃化冷冻、复苏和精子采集,观察比较三个实验组的复苏精子形态、存活率以及生殖能力。结果三组冷冻复苏分离后的精子形态完整,具有镰刀头状结构;荧光染色后PROH组精子存活率为(88.17±3.43)%,明显高于DMSO组:(61.17±10.65)%和R18S3组(16.83±6.49)%(P0.05);经辅助体外受精后均具有受精能力。获得的胚胎移植后可得到正常子代小鼠(DMSO∶PROH∶R18S3=13∶8∶17)。结论 3种冷冻保护剂均适用于C57BL/6J小鼠附睾冷冻,但PROH冷冻效果较好。     

滇南小耳猪精液的直接冷冻保存

目的建立滇南小耳猪精液冷冻保存方法,加速云南省特有小型猪种的小型化选育。方法利用脉冲电刺激模式诱导公猪输精管自助收缩排精。利用直接冷冻新技术（DFM）研究不同冷冻方案对精子的运动度、精子顶体完整性和体内授精胚胎发育能力的影响。结果在直接冷冻方法中,3%甘油防冻剂的作用下,60 s植冰时间和1.5 mm/s的冷冻降温参数对精子运动度保护良好,精子运动复苏率达到61.7%。但是,3%乙二醇虽然与甘油一样对精子的顶体完整性都有很好的保护作用,对精子运动度保护能力较差。此外,3%甘油、60 s植冰、1.5mm/s冷冻速度的直接冷冻的冻精解冻,移植到超数排卵的母猪子宫颈口实施人工授精,获得卵的受精和胚胎发育潜能尚可。结论玻璃管直接冷冻可以完成滇南小耳猪精子的冷冻保存。     

Performance of ten inbred mouse strains following assisted reproductive technologies (ARTs)

Superovulation, in vitro fertilization, embryo cryopreservation, and embryo transfer are assisted reproductive technologies (ARTs) widely used in laboratory mice. Inbred strains of mice have inherent genetic differences that cause them to respond differently to these technologies. Knowing how common inbred strains will perform when used for ARTs will ensure the most efficient use of mice, time, and resources. In this study, we characterized the ability of 10 inbred strains: 129S1/SvImJ, A/J, BALB/cJ, BALB/cByJ, C3H/HeJ, C57BL/6J, DBA/2J, FVB/NJ, NOD/LtJ, and SJL/J to superovulate, fertilize in vitro, and produce live pups subsequent to embryo transfer. Three-week-old female mice were superovulated using eCG (5.0 IU) and hCG (5.0 IU). The resulting oocytes were fertilized in vitro in human tubal fluid medium with spermatozoa of the same strain. The following day, two-cell embryos were either transferred into pseudopregnant recipient females or cryopreserved. The cryopreserved embryos were later thawed and transferred into pseudopregnant recipient females. Differences in response to superovulation, fertilization, and number of live born produced after embryo transfer were observed between strains, substantiating the influence of genetic variability on ARTs. The response to the superovulation treatment varied among strains and ranged from 5+/-1(A/J) to 40+/-3 (129S1/SvImJ) normal oocytes per female. The average proportion of oocytes that fertilized ranged among strains from 24% (129S1/SvImJ) to 93% (DBA/2J and A/J). The average proportion of two-cell embryos that were transferred into recipient females and subsequently developed into live pups varied from 5% (A/J) to 53% (C57BL/6J) for fresh embryos and from 18% (BALB/cByJ) to 45% (129S1/SvImJ) for thawed embryos.     

Improved in vitro fertilization and development by use of modified human tubal fluid and applicability of pronucleate embryos for cryopreservation by rapid freezing in inbred mice

We examined in vitro fertilizability and development of 10 inbred mouse strains (C57BL/6J, C57BL/10, C57BL/10.D2/newSn, C57BL/10-Thy1.1, C57BL/10.Br/Sn, C3H/He, RFM/Ms, STS/A, BALB/c-nu and C.B-17/Icr), and the viability of frozen-thawed in vitro fertilized (IVF) embryos after embryo transfer (ET). In seven strains, fertilizability was significantly greater in modified human tubal fluid (mHTF) compared with modified Krebs-Ringer's bicarbonate solution (TYH medium). The TYH medium supported almost no fertilization in four strains. More than 80% of IVF embryos developed to the blastocyst stage by 120 h in potassium-enhanced simplex optimization medium (KSOM). Reciprocal fertilization between C57BL/6J and BALB/c-nu gametes in TYH medium yielded poor fertilization o f BALB/c-nu due to spermatozoal deficiencies. Increased concentrations of bovine serum albumin and spermatozoa during capacitation and Percoll washing did not drastically affect fertilization. The mHTF, but not TYH medium, supported BALB/c-nu spermatozoa penetration into the zona pellucida irrespective of capacitation media. In vitro fertilized embryos frozen-thawed rapidly were transferred to surrogate mothers at the two-cell stage. Compared with that of unfrozen controls, rapid freezing had no significant effect on fetus development except in C57BL/10.D2/newSn mice. These results suggest that mHTF medium is superior with respect to IVF of inbred mice, and that KSOM adequately supports in vitro fertilized embryo development in inbred mice. The data also indicate that rapid freezing of pronucleate embryos following IVF is suitable for cryopreservation and embryo banking of inbred mice and for the production of genetically modified mice.     

Effects of cryopreservation of mouse embryos and in vitro fertilization on genotypic frequencies in colonies

To evaluate the effects of cryopreservation and in vitro fertilization (IVF) on genotypic frequencies in mouse colonies, genotypic frequencies at 15 biochemical, 4 immunological and 20 microsatellite loci were examined in three colonies of MCH (ICR) mice derived from noncryopreserved embryos obtained by natural mating without the induction of superovulation, cryopreserved embryos obtained by natural mating with the induction of superovulation, and cryopreserved embryos obtained by the induction of superovulation and IVF. Three (Pgm-1, Ldr-1 and Hbb) out of the 15 biochemical loci, two (Thy-1 and H2K) out of four immunological loci and five (D5Mit18, D6Mit15, D12Mit5, D13Mit26, and D14Mit7) out of 20 microsatellite loci that showed polymorphisms in every colony were used for detection of genotypic frequencies. The genotypic frequencies of the loci in the three colonies did not differ from the predicted genotypic frequencies (P > 0.05). The results suggested that genetic drift does not occur among colonies established from treated and untreated embryos, and it was clear that the embryo banking by cryopreservation is suitable for preservation of outbred stock without genetic drift.     

Functionality of cryopreserved juvenile ovaries from mutant mice in different genetic background strains after allotransplantation

The rapid expansion of mutant mouse colonies for biomedical research has resulted in lack of space at laboratory animal facilities and increasing risks of losing precious lines. These challenges require cheap and effective methods in addition to freezing embryos and sperm to archive the expanding mutant mouse lines. Cryopreservation of mouse ovarian tissue has been reported, but the application in the diverse mutant lines and genetic backgrounds has not yet been studied. In this study, juvenile ovaries (10-day-old) collected from genetically modified mouse lines were cryopreserved using high concentrations of cryoprotectants (dimethyl sulfoxide (Me₂SO) and ethylene glycol (EG)) and instrumented ultra-rapid freezing. The validation of the frozen ovary batches was assessed by orthotopically transplanting a thawed ovary into a nearly completely ovariectomized mature female (congenic with the ovary donor). After 2 weeks of recovery, the ovary recipient was continuously paired with a male (congenic with the ovary donor) to evaluate the fertility of the recipient and delivered offspring were genotyped to evaluate the continued functionality of the grafted ovary. The recipient females delivered genetically modified offspring starting 6 weeks after ovary transplantation and lasting up to 6 months. The presented cryopreservation and transplantation protocols enabled retrieval of the genetic modification in 20 (from 22) genetically modified mutant mouse models on a C57BL/6 (17), FVB (2), or BALB/c (1) background. The thawed ovaries functioned after successful orthotopic allotransplantation to congenic wild-type recipients and produced mutant offspring, which allowed recreation of the desired genotype as a heterozygote on the proper genetic background. The results indicate that cryopreservation of mouse ovaries is a promising method to preserve genetic modification of the increasing number of mutant mouse models and can be used as a model for ovary cryopreservation using a variety of mouse mutants.     

Simple and efficient in vitro fertilization with cryopreserved C57BL/6J mouse sperm

Archiving of mouse stocks by cryopreservation of sperm has great potential, because it is simple, rapid, and cheap. However, for some of the most commonly used inbred strains, including C57BL/6J, the postthaw fertility of the sperm (0%-12%) is too low to be useful without recourse to zona nicking or intracytoplasmic sperm injection to aid penetration of the zona pellucida. In the present study, nonmotile sperm and cell debris were removed from thawed suspensions of C57BL/6J mouse sperm, and the remaining, largely progressively motile sperm were used for in vitro fertilization. These sperm fertilized 38%-88% of denuded, zona-intact eggs, and when 2-cell embryos were transferred to pseudopregnant recipient mice, 40%-63% produced live-born young. The production of 2-cell embryos and the birth of live pups at these rates indicate that cryopreservation of sperm is a practical way to archive the haploid genome of genetically altered C57BL/6J mice.