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Intracytoplasmic Sperm Injection of Monkeys   

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摘要:胞浆内单精子注射(Intracytoplasmic Sperm Injection,ICSI),是指通过显微操作技术将单个精子注入卵母细胞胞浆内,从而使卵母细胞受精的一种辅助生殖技术。与传统的体外受精相比,ICSI对精子活力、数量及受精能力的要求较低,在临床上是第 2 代“试管婴儿”的关键技术。在科研领域,ICSI 可结合其他生物工程技术,制备基因编辑动物模型。此外 ICSI 也是精子冷冻复苏构建受精胚胎的重要辅助手段。本文详细描述了猴胞浆内单精子注射的操作方法及流程。

关键词: 精子, 卵母细胞, 受精, 辅助生殖

材料与试剂

  1. 3.5 cm 培养皿(CORNING,catalog number: 430165)
  2. 3.5 cm 操作皿(FALCON,catalog number: 353001)
  3. Borosilicate Glass(B100-75-10)
  4. 矿物油(Sigma,catalog number:M8410)
  5. 口吸管(北京正天,catalog number:BJ-40)
  6. 移液枪 (Eppendorf)
  7. 15 ml 离心管(Corning,catalog number:430791)
  8. 一次性使用无菌注射器带针(KDL 1ml)
  9. PVP(Sigma,catalog number:PVP360)
  10. 水银
  11. TH3液(见溶液配方)
  12. T3液(见溶液配方)
  13. HECM9aa培养液(见溶液配方)
  14. NaCl(Sigma-Aldrich,catalog number:S7653)
  15. KCl(Sigma-Aldrich,catalog number:P5405)
  16. CaCl2·2H2O(Sigma-Aldrich,catalog number:C7902)
  17. MgCl2·6H2O(Sigma-Aldrich,catalog number:M2670)
  18. NaH2PO4(Sigma-Aldrich,catalog number:S8282)
  19. NaHCO3(Sigma-Aldrich,catalog number:S5761)
  20. HEPES(Sigma-Aldrich,catalog number:H3375)
  21. D-Glucose(Sigma-Aldrich,catalog number:G7528)
  22. Sodium Pyruvate(Sigma-Aldrich,catalog number:P5280)
  23. Sodium lactate (60% syrup)(Sigma-Aldrich,catalog number:L4263)
  24. BSA(absin,catalog number:abs9156)
  25. penicillin-streptomycin (100X)(gibco,catalog number:15070-063)
  26. PVA(Sigma-Aldrich,catalog number:P8136)
  27. HCl(沪试,catalog number:7647-01-0)
  28. Taurine(Sigma-Aldrich,catalog number:T8691)
  29. Asparagine(Sigma-Aldrich,catalog number:A4159)
  30. Cysteine(Sigma-Aldrich,catalog number:C7352)
  31. Histidine(Sigma-Aldrich,catalog number:H6034)
  32. Lysine(Sigma-Aldrich,catalog number:L5501)
  33. Proline(Sigma-Aldrich,catalog number:L5607)
  34. Serine(Sigma-Aldrich,catalog number:S4311)
  35. Aspartic acid(Sigma-Aldrich,catalog number:A8949)
  36. Glycine(Sigma-Aldrich,catalog number:G8790)
  37. Glutamic acid(Sigma-Aldrich,catalog number:G8415)
  38. Glutamine(Sigma-Aldrich,catalog number:G8540)
  39. Pantothenate(Sigma-Aldrich,catalog number:P5155)
  40. Phenol Red(Sigma-Aldrich,catalog number:P0290)

仪器设备

  1. 培养箱(Thermo Scientific,model:Forma 3111、Forma 371)
  2. 拉针仪(Sutter Instrument,model:P97)
  3. 断针仪(Narishige,model:MF-900)
  4. 倒置显微镜(Olympus,model:IX-73)
  5. 体视显微镜(Olympus,model:SZX7)
  6. 一对显微操作臂(Narishige)
  7. 微量注射器(Narishige IM-9B)
  8. 离心机(Eppendorf centrifuge,model:5702)
  9. 压电冲击驱动系统(Prime Tech)

实验步骤

实验流程

  1. 超排母猴及获取卵母细胞;
  2. 猴子精子的采集与获能处理;
  3. 完成注射,将卵子移入培养碟中放置培养箱培养;
  4. 做好详细记录。

单精注射步骤
一、精子获能处理

  1. 将采集的精子液化15-20 min,取液化好的精子上清加入到15 ml离心管内;
  2. 在精子离心管内加入4 ml的TH3,轻轻吹打,1,800 rpm,离心5分钟;
  3. 离心完后去上清,再加入4 ml的TH3,轻轻吹打,1,800 rpm,离心5分钟;
  4. 离心完后去上清,再加入4 ml的T3,轻轻吹打,1,800 rpm,离心5分钟;
  5. 离心完后去上清,再加入2 ml的T3,轻轻吹打,1,800 rpm,离心5分钟;
  6. 离心完后等待精子上游,上游3分钟;
  7. 上游后取中层的精子,在培养皿内制备精子滴,盖上矿物油,放入培养箱待用。

二、单精子显微注射

  1. 将TH3显微注射操作皿做好预热:取200 μl TH3液,在显微操作碟内制备4-5滴TH3液滴和一个柱状TH3液滴,再做一个10% PVP的液滴,每滴体积15-20 μl,并覆盖矿物油;
  2. 将HECM9aa培养皿做好预热:取200 μl HECM9aa培养液,在培养皿内制备10-13个液滴,每滴体积约15-20 μl,并覆盖矿物油。
  3. 用拉针仪将1 mm外径的薄壁硼硅酸毛细玻璃管(B100-75-10)拉制成需要的内外径后,在断针仪上断成外径为100-110 μm和内径为15-20 μm,打弯成20-30度的持卵针;
  4. 用拉针仪将1 mm外径的薄壁硼硅酸毛细玻璃管(B100-75-10)拉制成需要的内外径后,在断针仪上断成外径为7-8 μm和内径为5-6 μm,打弯成20-30度的精子注射针;
  5. 用1 ml注射器从注射针的末端装入1-2 mm水银待用;
  6. 将制备的显微注射皿放置于载物台上,从低倍视野开始观察整个显微操作滴;
  7. 将持卵针安装于操作仪左侧的IM9B,缓慢深入注射滴中,并调整与操作皿底部水平;
  8. 将注射针安装于操作仪右侧的piezo压电冲击驱动系统的微注射器上,缓慢深入注射滴中,并调整与操作皿底部水平,保持与持卵针在同一水平线;
  9. 用胚胎转移管将一组MII期卵母细胞转移到操作皿TH3液滴中,同时将处理的3-5 μl获能精子加到另一个TH3液滴内;
  10. 镜下在10% PVP内清洗注射针并调整注射针到精子滴中,用注射针尖轻击精子尾部来制动精子后,将精子尾先头后吸入注射针内,移动注射针到放置卵母细胞的注射滴中(当注射针内的精子用完后,重复此步骤);
  11. 用持卵针抓取并固定MII期卵母细胞,并调整第一极体在六点或十二点的方向,注射针利用脉冲震荡刺穿破透明带,将精子推到注射针口后通过脉冲震荡辅助注射入卵母细胞内,以尽量少的液体吐出精子;(见图1、图2)


    图1. 极体在约 6 点方向,脉冲震荡刺穿破透明带


    图2. 精子推到注射针口,准备进行注射

  12. 完成注射后,持卵针放开卵母细胞;
  13. 重复上述操作直至完成所有的MII期卵母细胞的精子注射,将胚胎转移到HECM9aa培养液中在 37 °C,5% CO2培养箱中培养;
  14. 培养箱中培养六个小时以上,在显微镜下检查原核形成情况,出现两个原核的胚胎视为正常受精。(见图3)


图3. 受精卵,箭头所指处为两原核位置

注:本文以7只供体数,共获取103枚卵母细胞进行ICSI,其中有91枚正常受精,受精率达88%。(见表1)

表1. ICSI数据统计
供体数 注射卵母细胞数 受精数 受精率
1#65
2#2014
3#22
4#2019
5#55
6#2725
7#2321
总计 1039188%


溶液配方

  1. Tyrode's Albumin Lactate Pyruvate medium with HEPES (TH3)(表2)

    表2. Tyrode's Albumin Lactate Pyruvate medium with HEPES (TH3) 配制表
    成分/100ml/50ml备注
    NaCl3.329 ml1.665 ml200 mg/ml stock
    KCl239 μl119.5 μl100 mg/ml stock
    CaCl2·2H2O294 μl147 μl100 mg/ml stock
    MgCl2·6H2O102 μl51 μl100 mg/ml stock
    NaH2PO4 48 μl24 μl100 mg/ml stock
    NaHCO3 210.6 mg105.3 mg
    HEPES240 mg120 mg
    D-Glucose90 mg45 mg
    Sodium Pyruvate12 mg6 mg
    Sodium lactate (60% syrup)0.17 ml85 μl
    BSA300 mg150 mg
    penicillin-streptomycin (100X)1 ml500 μl
    Phenol Red (0.01%)10 μl5 μl
    pH7.4

  2. Tyrode's Albumin Lactate Pyruvate medium without HEPES (T3)(表3)

    表3. Tyrode's Albumin Lactate Pyruvate medium without HEPES (T3) 配制表
    成分/100ml/10ml备注
    NaCl3.329 ml333 μl200 mg/ml stock
    KCl239 μl23.9 μl100 mg/ml stock
    CaCl2·2H2O294 μl29.4 μl100 mg/ml stock
    MgCl2·6H2O102 μl10.2 μl100 mg/ml stock
    NaH2PO4 48 μl4.8 μl100 mg/ml stock
    NaHCO3 210.6 mg21.06 mg
    Glcose90 mg9 mg
    Sodium Pyruvate12 mg1.2 mg
    Sodium lactate (60% syrup)0.17 ml17 μl
    BSA300 mg30 mg
    penicillin-streptomycin (100X)1 ml100 μl
    Phenol Red (0.01%)10 μl1 μl
    pH7.4

  3.  HECM-9(表4)

    表4. HECM-9 配制表
    成分/100ml终浓度
    NaCl0.6639 g113.6 mmol/L
    KCl0.0224 g3 mmol/L
    CaCl2·2H2O0.0279 g1.9 mmol/L
    MgCl2·6H2O0.0102 g0.5 mmol/L
    NaHCO3 0.2100 g25 mmol/L
    Sodium lactate (60% syrup)0.0504 g4.5 mmol/L
    PVA0.01 g0.1 mg/ml
    1 mol/L HCl0.14 ml1.4 mmol/ml
    11 AA with Pantothenate1 ml
    100X AA stock
    成分MW100X 母液浓度终浓度
    Taurine12550 mmol/L0.5 mmol/L
    Asparagine1321 mmol/L0.01 mmol/L
    Cysteine1211 mmol/L0.01 mmol/L
    Histidine1551 mmol/L0.01 mmol/L
    Lysine1461 mmol/L0.01 mmol/L
    Proline1151 mmol/L0.01 mmol/L
    Serine1051 mmol/L0.01 mmol/L
    Aspartic acid1331 mmol/L0.01 mmol/L
    Glycine751 mmol/L0.01 mmol/L
    Glutamic acid1471 mmol/L0.01 mmol/L
    Glutamine14620 mmol/L0.2 mmol/L
    Pantothenate2380.3 mmol/L3 μmol/L
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Copyright: © 2023 The Authors; exclusive licensee Bio-protocol LLC.
引用格式:王燕, 许玉婷, 李春杨, 孙强. (2023). 猴胞浆内单精子注射. // 实验动物胚胎操作实验手册. Bio-101: e1010985. DOI: 10.21769/BioProtoc.1010985.
How to cite: Wang, Y., Xu, Y. T., Li, C. Y., and Sun, Q. (2023). Intracytoplasmic Sperm Injection of Monkeys. // Embryo Manipulation Manual of Laboratory Animals. Bio-101: e1010985. DOI: 10.21769/BioProtoc.1010985.
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