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Feb 2021

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Efficient Superovulation and Egg Collection from Mice
高效的超排卵和小鼠卵子收集   

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Abstract

Superovulation is a method used to reduce the number of mice used per experiment by increasing the egg number. Conventionally, superovulation for obtaining mouse eggs involves the use of equine chorionic gonadotropin (eCG) for stimulation and human CG for induction. Female mice of the C57BL/6 inbred strain spontaneously ovulate approximately 10 eggs. The average number of eggs ovulated using the conventional superovulation method is approximately twice as high as that obtained by spontaneous ovulation. Here, we describe the conventional and non-conventional methods of intraperitoneal injection of superovulation reagents in mice and subsequent egg collection. The non-conventional superovulation method combining anti-inhibin serum (AIS) plus eCG for stimulation is more efficient than conventional superovulation. Appropriate intervals from each injection to sampling induce large numbers of high-quality eggs. Immediately after ovulation, eggs are surrounded by cumulus cells, forming an egg-cumulus complex. These cumulus cells are then removed from the egg-cumulus complex by treatment with hyaluronidase to obtain the exact number of eggs. This protocol is suitable for further manipulations such as intracytoplasmic sperm injection and cryopreservation of eggs, as well as for the analyses of responsivity to superovulation reagents in genetically modified mice obtained by genome editing.

Keywords: Mouse (), Egg (卵细胞), Oocyte (卵母细胞), Cumulus cells (积云细胞), Superovulation (排卵过多), Anti-inhibin serum (抗抑制素血清), Chorionic Gonadotropin (绒毛膜促性腺激素), Hyaluronidase (透明质酸酶)

Background

Superovulation, involving the stimulation of follicle development and induction of ovulation, is used to increase the number of eggs in animals, including mice. Conventionally, superovulation of mice involves the use of equine chorionic gonadotropin (eCG) that has a follicle-stimulating hormone (FSH)-like activity and human CG (hCG) for stimulation and induction, respectively. Anti-inhibin serum (AIS) neutralizes the function of inhibin, regulates FSH secretion, and increases the number of ovulated eggs (Wang et al., 2001). Thus, an efficient superovulation method involving the combined use of AIS with eCG for stimulation, increases the number and improves the quality of ovulated eggs, and reduces the number of mice used in animal experiments compared with the conventional superovulation method (Takeo and Nakagata, 2015). Furthermore, we and other researchers have compared the responsivity of multiple mouse strains to these superovulation reagents (Takeo and Nakagata, 2015; Shindo et al., 2021). Eggs obtained from superovulation reagent-treated female mice are used for subsequent applications such as intracytoplasmic sperm injection (ICSI) and cryopreservation of eggs. Considering that the demand for mouse egg manipulation will continue increasing to produce mouse modelsthat reproduce human diseases and to perform in vivo functional studies using genome editing techniques in the future, as well as the application for in vitro fertilization, we describe an efficient protocol for the superovulation of mice and subsequent egg collection.

Materials and Reagents

  1. Glass pipettes (Drummond Scientific Company, MICROCAPS®, catalog number: 1-000-0500)

  2. 1.5 mL microcentrifuge tubes (WATSON, catalog number: 131-815C)

  3. 50 mL tubes (Greiner, catalog number: 227261)

  4. 10 mL plastic pipettes (FALCON, catalog number: 357551)

  5. 1,000 µL pipette tips (WATSON, catalog number: 110-7-6C)

  6. 200 µL pipette tips (WATSON, catalog number: 110-705C)

  7. 10 µL pipette tips (WATSON, catalog number: 110-207C)

  8. Kimwipes (NIPPON PAPER CRECIA Co., Ltd., catalog number: 62020)

  9. Paper towels (ASKUL, catalog number: 1944368)

  10. 35 mm dishes (IWAKI, catalog number: 1000-035)

  11. 60 mm dishes (CORNING, catalog number: 351007)

  12. Syringes with needles (Terumo Corporation, 1 mL syringe with 26-gauge 1/2-inch needle, catalog number: SS-01T2613S)

  13. Disposable gloves (AXEL, catalog number: 61-7347-30)

  14. C57BL/6N and C57BL/6J female mice (Japan SLC, Inc.): 4–6 and ≥10 weeks of age (excluding 7–9 weeks of age because they are unsuitable for superovulation)

  15. eCG (ASKA Animal Health Co., Ltd., SEROTROPIN®, 1,000 IU × 10 vials, catalog number: No application), store at 4°C

  16. AIS plus eCG (Kyudo Co., Ltd., CARD HyperOva®, 1 mL, catalog number: F021), store at -20°C

  17. hCG (ASKA Animal Health Co., Ltd., Gonadotropin for animal, 3,000 IU × 5 vials, catalog number: No application), store at 4°C

  18. Saline (Otsuka Pharmaceutical Co., Ltd., 20 mL × 50 vials, catalog number: Not applicable): Store at room temperature

  19. CARD mHTF medium (Kyudo Co., Ltd., 2 mL, catalog number: GA017), store at 4°C

  20. M2 medium (Merck, EmbryoMax® M2 Medium, catalog number: MR-015-D), store at -20°C

  21. Hyaluronidase (Sigma-Aldrich, catalog number: H3506-100MG), store at -20°C

  22. Liquid paraffin (Nacalai Tesque, Inc., Specially prepared reagent, catalog number: 26137-85): Store at room temperature and in the dark, away from sunlight

  23. 70% ethanol (Yoshida Pharmaceutical Company, Ecosyoueta disinfectant solution, catalog number: 14987288980046)

  24. Aluminum foil (AXEL, catalog number: 6-713-01)

Equipment

  1. Protective equipment (e.g., masks, goggles, and lab coats)

  2. CO2 incubator (ASTEC Co., Ltd., model: SCA-165DS)

  3. Stereo microscope (Nikon, model: SMZ645)

  4. Dry-heat sterilizer (Panasonic, model: MOV-212S-PJ)

  5. Precision balance (AXEL, model: 1-1726-01)

  6. Ampoule cutter (AXEL, model: 5-124-22)

  7. Alcohol lamp (AXEL, model: 6-487-01)

  8. Pipette controller (FALCON, model: 357469)

  9. P-1000 pipette (GILSON, model: F120602)

  10. P-200 pipette (GILSON, model: F123601)

  11. Micropipette (Eppendorf, model: 4920000024)

  12. Mouth pipette (Drummond, model: 2-040-000)

  13. Filter (Millipore, model: SLGPR33RS)

  14. Ampoule glass cutter (AXEL, model: 5-124-22)

  15. Large straight scissors (Natsume Seisakusho Co., Ltd., model: B-3)

  16. Small straight scissors (Natsume Seisakusho Co., Ltd., model: B-12)

  17. Curved tip tweezers (AXEL, model: 6-531-19)

  18. Tweezers (AXEL, model: 2-529-12)

  19. Precision tweezers (DUMONT, model: NO.5-INOX)

  20. Needle (AXEL, model: 2-013-01)

  21. Dispenser for paraffin liquid (Nichiryo Co., Ltd., model: 00-DP-2B)

  22. Mechanical tally counter (AXEL, model: 63-1584-34)

  23. Plastic cages (Clea Japan, Inc., model: CL-0103-2 Mouse TPX)

Software

  1. Microsoft Excel (Microsoft Corporation)

Procedure

  1. Animals

    Breed the C57BL/6 strain (2–4 mice per cage) under the following specific pathogen-free conditions: at 23 ± 1°C controlled temperature, 12 h light-dark cycles (light on at 8:00 and off at 20:00), and ad libitum access to food and water.


  2. Preparation of eCG (7.5 IU/100 µL)

    1. Open three vials containing eCG powder and two bottles of saline.

    2. Add 2 mL of saline into each eCG vial using a P-1000 pipette with a 1,000 µL tip, mix by pipetting, and transfer the solution (a total volume of 6 mL) to a 50 mL tube.

    3. Repeat Step B2 twice to collect all the eCG, i.e., dissolve eCG powder (3,000 IU) in 18 mL of saline.

    4. Add 22 mL of saline into the 50 mL tube using a pipette controller with a 10 mL plastic pipette to obtain an eCG working solution, i.e., dissolve eCG powder (3,000 IU) in 40 mL of saline.

    5. Prepare 1.2 mL aliquots (7.5 IU/100 µL per mouse) in 1.5 mL microcentrifuge tubes and store at -20°C until use, for up to six months.

    6. When needed, thaw the eCG solution completely at room temperature and mix well before use.


  3. Preparation of hCG (7.5 IU/100 µL)

    1. Open a vial containing hCG powder and two bottles of saline.

    2. Add 2 mL of saline into the hCG vial using the P-1000 pipette with a new 1,000 µL tip, mix by pipetting, and transfer the solution to a new 50 mL tube.

    3. Repeat Step C2 twice to collect all the hCG, i.e., dissolve hCG powder (3,000 IU) in 6 mL of saline.

    4. Add 34 mL of saline into the 50 mL tube using the pipette controller with a new 10 mL plastic pipette to obtain the hCG working solution, i.e., dissolve hCG powder (3,000 IU) in 40 mL of saline.

    5. Prepare 1.2 mL aliquots (7.5 IU/100 μL per mouse) in 1.5 mL microcentrifuge tubes and store at -20°C until use, for up to six months.

    6. When needed, thaw the hCG solution completely at room temperature and mix well before use.


  4. Preparation of M2 medium containing hyaluronidase

    1. Measure 0.5 mg of hyaluronidase and add to a new 50 mL tube.

    2. Thaw M2 medium and add 50 mL to the tube containing hyaluronidase (1% solution).

    3. Mix thoroughly by gentle inversion.

    4. Prepare 1 mL aliquots in 1.5 mL microcentrifuge tubes and store at -20°C until use.

    5. When needed, thaw the solution completely at room temperature and mix well before use.
      Note: Hyaluronidase is sterile but non-filtered because this reagent is adjusted on a clean bench.


  5. Preparation of glass pipettes for egg handling (see Video 1)

    1. Cut the glass pipettes in the middle using an ampoule glass cutter (Figure 1A).

    2. Heat the cut edge of glass pipettes with the flame of an alcohol lamp to smooth.

    3. Hold both sides of the glass pipette with one hand and a pair of curved tip tweezers.

    4. Heat the middle of the pipette until it is softened slightly (Figure 1B), remove from the flame, and immediately pull both ends horizontally.

    5. Cut the excess part of the pipette, heat, and smooth the tip slightly. The smooth tip is less likely to damage the eggs and dishes.

    6. Check the tip under a stereomicroscope (Figure 1C) and choose glass pipettes with an inner diameter of approximately 150–200 µm.

    7. Wrap the glass pipettes with aluminum foil (Figure 1D), dry-heat sterilize (180°C, 30 min), and store in a new 15 mL tube at room temperature.

      Note: Pipettes with proper inner diameter enable to handle eggs easily. Multiple glass pipettes should be prepared for one experiment to replace the pipette with a new one when operation is not easy (e.g., cumulus cells adhere to the tip).


    Video 1. Making glass pipettes.


    Figure 1. Making glass pipettes.

    (A) Cut glass pipettes. (B) Heat and soften a glass pipette. Insert, pulled glass pipettes. (C) Tips of glass pipettes. Insert, an enlarged view of the pipette tips. (D) Wrap glass pipettes.


  6. Superovulation

    1. Thaw eCG or CARD HyperOva® solutions at room temperature and mix well.

    2. Aspirate the required amount of eCG or HyperOva® (100 μL per mouse) with a syringe; for example, aspirate 1 mL of eCG or HyperOva® for 10 female mice (Figure 2A).

    3. Hold a mouse and inject 100 µL of eCG or HyperOva® into the peritoneal cavity (Figure 2B and 2C).

    4. Return mice to their cages.

    5. At 48 h after injecting eCG or HyperOva®, inject 100 µL of hCG in the same manner (Figure 2A, 2B, and 2C).

    6. Return mice to their cages.

      Note: Either wrong dosage or inadequate timing of hormone injection reduces the number of normal eggs ovulated.



      Figure 2. Superovulation procedure and egg collection.

      (A) Filling a syringe. (B) Intraperitoneal injection of female mice. (C) Time schedule of superovulation reagent injections and egg collection. AIS, anti-inhibin serum; eCG, equine chorionic gonadotropin; hCG, human chorionic gonadotropin.


  7. Preparation of dishes

    1. Add paraffin liquid to an empty 35 mm dish (Figure 3A and 3B).

    2. Place four and six CARD mHTF medium drops (100 µL per drop) in two 60 mm dishes and cover these drops with paraffin liquid (Figure 3A, 3C, 3D, and 3E).

    3. Incubate these dishes in a CO2 incubator at 37°C with 5% CO2 for at least 30 min before collecting the egg-cumulus complex (Figure 3F).

    4. Thaw the M2 medium containing hyaluronidase and place four drops (200 µL per drop) in a new 60 mm dish (Figure 3G and 3H).

    5. Note: Prepare this just before washing the eggs. There is no need to cover M2 medium drops with paraffin liquid and to place them in the CO2 incubator. As M2 medium is a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-based medium, it can be used for a long time at room temperature outside the CO2 incubator (Carter et al., 1993).



      Figure 3. Dishes utilized in this experiment.

      (A) Dispensing paraffin liquid. (B) Keeping dish for isolated oviducts. (C, E) Dish for collecting the egg-cumulus complex. (D) Dish for washing eggs. (F) CO2 incubator. (G, H) Dish for removing cumulus cells from the egg.


  8. Collection of the egg-cumulus complex

    1. Place scissors and tweezers on the bench (Figure 4A) and wear gloves (and other protective equipment as necessary).

    2. At 14–16 h after injecting hCG, sacrifice female mice (Figure 2C).

    3. Disinfect the abdomen with 70% ethanol and cut the abdominal skin of mice using large scissors.

    4. Pull two sides of the cut skin and access the mouse peritoneum.

    5. Cut the peritoneum using small scissors.

    6. Locate the V-shaped uterus, oviducts, and ovaries by shifting the position of the gut and internal organs.

    7. Hold the utero-tubal junction with a pair of precision tweezers, cut the utero-tubal and isthmic-ampullary junctions, and separate the oviducts from the uterus (Figure 5A and 5B).

    8. Clean the oviducts by removing adipose tissue, blood, and tissue fluid, and transfer oviducts to the keeping dish moved from the CO2 incubator.

      Note: Reduce the exposure time of the collecting dish to room temperature using the keeping dish.

    9. Move the collecting dish from the CO2 incubator to the bench and transfer oviducts to liquid paraffin on the collecting dish using the pair of precision tweezers and place the oviducts on the bottom of this dish.

    10. Take out the egg-cumulus complex from the ampulla using a needle under the stereomicroscope (Video 2 and Figure 4B) and add the egg-cumulus complex to one drop of CARD mHTF medium on the collecting dish (Figure 6A and 6B). Superovulation treatment induces an average of 20–50 eggs per female mouse (Shindo et al., 2021), and one drop includes <100 eggs.

    11. Move the collecting dish to the CO2 incubator.

    12. Repeat Steps H2–H11 for all female mice.

      Note: Prepare the CARD mHTF medium drops corresponding to the number of superovulated mice.



      Figure 4. Tools utilized in this experiment.

      (A) Anatomical set for abdominal exploration. (B) Stereo microscope. Insert, handling set for the egg-cumulus complex. (C) Mouth pipetting set for egg transfer. (D) Glass pipette with aspirated medium. Arrow, liquid surface of the medium.



      Figure 5. Pictures of ovary, oviduct, and uterus.

      (A) Serial view of ovary, oviduct, and uterus. Each junction is represented by dashed lines. (B) Separated view of the oviduct. Arrow indicates the ampulla.


      Video 2. Removing the egg-cumulus complex from the ampulla.


  9. Removing cumulus cells and counting eggs

    1. Move the collecting and washing dishes from the CO2 incubator to the bench.

    2. Aspirate the M2 medium containing hyaluronidase using a mouth pipetting set (Figure 4C), and transfer the egg-cumulus complex from the CARD mHTF medium drop on the collecting dish to the drop of M2 medium with hyaluronidase on the removing dish.

      Note: Aspirate the M2 medium containing hyaluronidase until the wide part of the glass pipette for easy handling of the eggs (Figure 4D).

    3. Remove cumulus cells from the egg-cumulus complex by mouth pipetting and transfer the eggs to the drops of CARD mHTF medium on the washing dish (Figure 6C).

    4. Wash the eggs by moving them between three drops of CARD mHTF medium on the washing dish.

    5. Repeat steps I2–I4 for all egg-cumulus complexes.

    6. Count the number of normal eggs per drop using a mechanical tally counter and record the results in Microsoft Excel. Normal and abnormal eggs are easily distinguished by morphology (Figure 6D and 6E).

      Note: If the number of normal eggs is reduced, check the expiration date for solutions used, the timing of superovulation, and whether the age of mice is suitable for superovulation.



    Figure 6. Eggs manipulation.

    (A) Removing the egg-cumulus complex from the ampulla. This drop contains 47 eggs. (B) The egg-cumulus complex before hyaluronidase treatment. (C) Eggs after hyaluronidase treatment. (D) Normal eggs. (E) Abnormal eggs.

Data analysis

Perform Student’s t-test or Mann-Whitney’s U-test using Microsoft Excel (Shindo et al., 2021).

Notes

  1. Inject twice the amount (200 µL per mouse) of superovulation reagents into mice with a body weight of 35 g or more.

  2. Apply one drop for each mouse if you need to count the number of eggs per mouse.

  3. Reduce the exposure time of eggs to room temperature to avoid a reduction in developmental efficiency when performing ICSI.

  4. Take pictures with a microscope camera if necessary.

Acknowledgments

This work was supported in part by JSPS KAKENHI (Grant Numbers: JP19K09682 and JP19H01067) and the National Center for Child Health and Development (2021C-27). This protocol was adapted from two original articles ( Shindo et al., 2019 and 2021). We would like to thank Editage (www.editage.com) for English language editing.

Competing interests

The authors declare no competing interests.

Ethics

All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the National Research Institute for Child Health and Development (experimental numbers, A2005-007 and A2020-005).

References

  1. Carter, D. A. (1993). Preparation of culture media for fertilized one-cell mouse eggs. Methods Mol Biol 18: 141-143.
  2. Shindo, M., Inui, M., Kang, W., Tamano, M., Tingwei, C., Takada, S., Hibino, T., Yoshida, M., Yoshida, K., Okada, H., et al. (2019). Deletion of a seminal gene cluster reinforces a crucial role of SVS2 in male fertility. Int J Mol Sci 20(18): 4557.
  3. Shindo, M., Tsumura, H., Miyado, K., Kang, W., Kawano, N., Yoshida, T., Fukami, M. and Miyado, M. (2021). Similar responsiveness between C57BL/6N and C57BL/6J mouse substrains to superovulation. MicroPubl Biol 2021.
  4. Takeo, T. and Nakagata, N. (2015). Superovulation using the combined administration of inhibin antiserum and equine chorionic gonadotropin increases the number of ovulated oocytes in C57BL/6 female mice. PLoS One 10: e0128330.
  5. Wang, H., Herath, C. B., Xia, G., Watanabe, G. and Taya, K. (2001). Superovulation, fertilization and in vitro embryo development in mice after administration of an inhibin-neutralizing antiserum. Reproduction 122(5): 809-816.

简介


[摘要] 超排卵是一种通过增加卵子数量来减少每次实验使用的小鼠数量的方法。传统上,获得小鼠卵子的超排卵涉及使用马绒毛膜促性腺激素 ( eCG ) 进行刺激和使用人 CG 进行诱导。 C57BL/6 近交系雌性小鼠自发排卵约 10 个卵子。使用传统的超排卵方法排卵的平均卵子数量大约是自然排卵获得的卵子数量的两倍。 在这里,我们描述了在小鼠腹腔注射超排卵试剂和随后的卵子收集的常规和非常规方法。抗抑制素血清(AIS)联合心电图的非常规超排卵方法 用于刺激 比传统的超排卵更有效。从每次注射到取样的适当间隔可诱导大量优质鸡蛋。排卵后,卵子立即被卵丘细胞包围,形成卵丘复合体。然后通过用透明质酸酶处理将这些卵丘细胞从卵丘复合体中去除以获得准确的卵子数量。该协议适用于进一步的操作,如胞浆内精子注射和卵子的冷冻保存,以及分析通过基因组编辑获得的转基因小鼠对超排卵试剂的反应性。


[背景] 超排卵,包括刺激卵泡发育和诱导排卵,用于增加卵子的数量 在动物中,包括老鼠。传统上,小鼠的超排卵涉及使用马绒毛膜促性腺激素 ( eCG ) 和人类 CG ( hCG ) 分别用于刺激和诱导。抗抑制素血清 (AIS) 中和抑制素的功能,调节 FSH 分泌,并增加排卵数 (Wang et al ., 2001)。因此,一种有效的超排卵方法涉及结合使用 AIS 和eCG 用于刺激,增加排卵数量并提高排卵质量,减少小鼠数量 与传统的超排卵方法相比,用于动物实验(Takeo 和Nakagata ,2015 年)。此外,我们和其他研究人员已经比较了多个小鼠品系对这些超排卵试剂的响应性(Takeo 和Nakagata ,2015; Shindo 等人,2021)。从超排卵试剂处理的雌性小鼠获得的卵子用于后续应用,例如胞浆内精子注射 (ICSI) 和卵子的冷冻保存。考虑到对小鼠卵子操作的需求将继续增加,以生产复制人类疾病的小鼠模型并使用基因组编辑技术进行体内功能研究 将来,以及体外受精的应用,我们描述了一种用于小鼠超排卵和随后的卵子收集的有效方案。

关键字:鼠, 卵细胞, 卵母细胞, 积云细胞, 排卵过多, 抗抑制素血清, 绒毛膜促性腺激素, 透明质酸酶



材料和试剂


1. 玻璃移液管(Drummond Scientific Company, MICROCAPS ® ,目录号:1-000-0500)
2. 1.5 mL微量离心管(WATSON,目录号:131-815C)
3. 50 mL管(Greiner,目录号:227261)
4. 10 mL塑料移液管(FALCON,目录号:357551)
5. 1,000 µL 移液器吸头(WATSON,目录号:110-7-6C)
6. 200 µL 移液器吸头(WATSON,目录号:110-705C)
7. 10 µL 移液器吸头(WATSON,目录号:110-207C)
8. Kimwipes(NIPPON PAPER CRECIA Co., Ltd.,目录号:62020)
9. 纸巾(ASKUL,目录号:1944368)
10. 35毫米盘子(IWAKI,目录号:1000-035)
11. 60 mm 培养皿(CORNING,目录号:351007)
12. 带针头的注射器(Terumo Corporation,带 26 号 1/2 英寸针头的 1 mL 注射器,目录号:SS-01T2613S)
13. 一次性手套(AXEL,目录号:61-7347-30)
14. C57BL/6N 和 C57BL/6J 雌性小鼠(Japan SLC, Inc.):4-6 周龄和≥10 周龄(7-9 周龄除外,因为它们不适合超排卵)
15. eCG (ASKA Animal Health Co., Ltd.,SEROTROPIN ® ,1,000 IU × 10 瓶,目录号:无应用),4°C 储存
16. AIS plus eCG (Kyudo Co., Ltd.,CARD HyperOva ® ,1 mL,目录号:F021),-20°C 储存
17. hCG (ASKA Animal Health Co., Ltd.,Gonadotropin for animal,3,000 IU × 5 小瓶,目录号:No application),4°C 储存
18. 盐水(Otsuka Pharmaceutical Co.,Ltd.,20 mL×50小瓶,目录号:不适用):室温储存
19. CARD mHTF培养基(Kyudo Co.,Ltd.,2 mL,目录号:GA017),4°C保存
20. M2 培养基(Merck, EmbryoMax ® M2 Medium,目录号:MR-015-D),-20°C 储存
21. 透明质酸酶(Sigma-Aldrich,目录号:H3506-100MG),储存于-20°C
22. 液体石蜡( Nacalai Tesque ,Inc.,特制试剂,目录号:26137-85):室温避光,避光保存
23. 70%乙醇(Yoshida Pharmaceutical Company, Ecosyoueta消毒液,目录号:14987288980046)
24. 铝箔(AXEL,目录号:6-713-01)


设备


1. 防护设备(例如,口罩、护目镜和实验室外套)
2. CO 2培养箱(ASTEC Co., Ltd.,型号:SCA-165DS)
3. 立体显微镜(尼康,型号:SMZ645)
4. 干热灭菌器(松下,型号:MOV-212S-PJ)
5. 精密天平(AXEL,型号:1-1726-01)
6. 安瓿刀(AXEL,型号:5-124-22)
7. 酒精灯(AXEL,型号:6-487-01)
8. 移液器控制器(FALCON,型号:357469)
9. P-1000 移液器(GILSON,型号:F120602)
10. P-200 移液器(GILSON,型号:F123601)
11. 微量移液器(Eppendorf,型号:4920000024)
12. 吸管(Drummond,型号:2-040-000)
13. 过滤器(Millipore,型号:SLGPR33RS)
14. 安瓿玻璃切割机(AXEL,型号:5-124-22)
15. 大直剪(夏目制作所,型号:B-3)
16. 小直剪(夏目制作所,型号:B-12)
17. 弯头镊子(AXEL,型号:6-531-19)
18. 镊子(AXEL,型号:2-529-12)
19. 精密镊子(DUMONT,型号:NO.5-INOX)
20. 针(AXEL,型号:2-013-01)
21. 石蜡液分配器( Nichiryo Co., Ltd.,型号:00-DP-2B)
22. 机械计数计数器(AXEL,型号:63-1584-34)
23. 塑料笼子( Clea Japan, Inc.,型号:CL-0103-2 Mouse T PX)


软件


1. Microsoft Excel(微软公司)


程序


A. 动物
在以下特定的无病原体条件下培育C57BL /6 品系(每笼 2-4 只小鼠) :在 23 ± 1 °C 控制温度下,12 小时明暗循环(8:00 亮灯,20 点熄灯) :00),并随意获得食物和水。


B. 心电图(7.5 IU/100 µL)的制备
1. 打开三个装有eCG粉末和两瓶生理盐水的小瓶。
2. 使用带有 1,000 µL 吸头的 P-1000 移液器向每个eCG小瓶中加入 2 mL 盐水,通过移液器混合,然后将溶液(总体积为 6 mL)转移到 50 mL 管中。
3 、重复步骤B2两次,收集所有心电图,即将心电图粉末(3,000 IU)溶解在18毫升生理盐水中。
4. 用移液器控制器和 10 mL 塑料移液管将 22 mL 生理盐水加入 50 mL 试管中,以获得心电图工作溶液,即将心电图粉末(3,000 IU)溶解在 40 mL 生理盐水中。
5. 在 1.5 mL 微量离心管中制备 1.2 mL 等分试样(每只小鼠 7.5 IU/100 µL),并在 -20 ° C下储存直至使用,最长可保存 6 个月。
6.需要时,将 eCG溶液在室温下完全解冻,并在使用前充分混合。


C. hCG的制备(7.5 IU/100 µL)
1. 打开一个装有hCG粉末和两瓶生理盐水的小瓶。
2.使用带有新的 1,000 µL 吸头的 P-1000 移液器 将2 mL 生理盐水加入hCG小瓶中,通过移液器混合,然后将溶液转移到新的 50 mL 管中。
3. 重复步骤C2两次,收集所有的hCG ,即将hCG粉末(3,000 IU)溶解在6 mL生理盐水中。
4. 使用移液器控制器和新的 10 mL 塑料移液管将 34 mL 生理盐水加入 50 mL 管中,以获得hCG工作溶液,即将hCG粉末(3,000 IU)溶解在 40 mL 生理盐水中。
5.在 1.5 mL 微量离心管中 制备 1.2 mL 等分试样(每只小鼠 7.5 IU/100 μL ),并在 -20°C 下储存直至使用,最长可保存 6 个月。
6.需要时,将 hCG溶液在室温下完全解冻,并在使用前充分混合。


D. 含有透明质酸酶的M2培养基的制备
1. 量取 0.5 mg 透明质酸酶并添加到新的 50 mL 试管中。
2. 解冻 M2 培养基,向含有透明质酸酶(1% 溶液)的试管中加入 50 mL。
3. 轻轻颠倒充分混合。
4. 在 1.5 mL 微量离心管中制备 1 mL 等分试样,并在 -20°C 下储存直至使用。
5. 需要时,将溶液在室温下完全解冻,并在使用前充分混合。
注意:透明质酸酶是无菌的,但未经过滤,因为该试剂是在干净的工作台上调整的。


E. 用于鸡蛋处理的玻璃移液器的制备(见视频 1)
1. 使用安瓿玻璃切割器在中间切割玻璃移液器(图 1A)。
2. 用酒精灯的火焰加热玻璃移液管的切割边缘使其光滑。
3. 用一只手握住玻璃吸管的两侧和一对 c弯头镊子。
4. 加热移液器的中部直到它稍微变软(图1B),从火焰中取出,立即将两端拉平。
5. 剪掉移液器的多余部分,加热,轻轻抚平吸头。光滑的尖端不太可能损坏鸡蛋和盘子。
6. 在立体显微镜下检查吸头(图 1C)并选择内径约为 150–200 µm 的玻璃移液器。
7. 用铝箔包裹玻璃移液器(图 1D),干热灭菌(180 °C,30 分钟),并在室温下储存在新的15 mL 管中。
注意:具有适当内径的移液器可以轻松处理鸡蛋。一次实验应准备多支玻璃移液器,操作不便时更换新移液器 (例如,卵丘细胞粘附在尖端)。




视频 1. 制作玻璃移液器。
https://os.bio-protocol.org/doc/upprotocol/p4557/Abstract10_0_20220307101956355/Movie%201.mp4 




图 1. 制作玻璃移液器。 
(A) 切割玻璃移液器。 (B) 加热和软化玻璃吸管。 插入,拉玻璃吸管。 (C) 玻璃移液器的尖端。 插入,移液器吸头的放大视图。 (D) 包裹玻璃吸管。


F. 超排卵
1. 在室温下解冻 eCG或CARD HyperOva ®溶液并充分混合。
2. 用注射器吸取所需量的eCG或HyperOva ® (每只小鼠100 μL ) ;例如,为 10 只雌性小鼠吸出 1 mL 的eCG或HyperOva ® (图 2A)。
3. 握住鼠标,将 100 µL eCG或HyperOva ®注入腹腔(图 2B 和 2C)。
4. 将老鼠放回笼子。
5.注射 eCG或HyperOva ®后 48 小时,注射 100 µL hCG 以相同的方式(图2A、2B和2C)。
6. 将老鼠放回笼子。
注意:错误的剂量或激素注射时间不足会减少正常排卵的卵数。




图e 2. 超排卵程序和卵子收集。 
(A) 填充注射器。 (B) 雌性小鼠的腹腔注射。 (C) 超排卵试剂注射和卵子收集的时间表。 AIS,抗抑制素血清;心电图, 马绒毛膜促性腺激素; hCG ,人绒毛膜促性腺激素。


G. 菜肴的准备
1. 在空的 35 mm 培养皿中加入石蜡液(图 3A 和 3B)。
2. 将四滴和六滴 CARD mHTF培养基(每滴 100 µL)放入两个 60 mm 培养皿中,并用石蜡液覆盖这些滴(图 3A、3C、3D 和 3E)。
3.在收集卵-积云复合物之前,将 这些培养皿在37 °C和 5% CO 2的 CO 2培养箱中孵育至少 30 分钟(图 3F)。
4. 解冻含有透明质酸酶的 M2 培养基,将四滴(每滴 200 µL)置于新的 60 mm 培养皿中(图 3G 和 3H)。
注意:在洗鸡蛋之前准备好这个。无需用石蜡液覆盖 M2 培养基滴并将其放入 CO 2培养箱中。由于 M2 培养基是一种 4-(2-羟乙基)-1-哌嗪乙磺酸基培养基,它可以在 CO 2培养箱外的室温下长期使用(Carter 等,1993)。




图e 3. 本实验中使用的菜肴。 
(A) 分配石蜡液。 (B) 为孤立的输卵管保留盘子。 (C, E) 用于收集卵积云复合物的盘子。 (D) 洗鸡蛋的盘子。 (F) CO 2培养箱。 (G,H)从鸡蛋中去除积云细胞的盘子。


H. 卵-积云复合体的集合
1. 将剪刀和镊子放在工作台上(图 4A)并戴上手套(必要时戴上其他防护设备)。
2.注射 hCG后14-16小时,处死雌性小鼠(图 2C)。
3. 用70%乙醇消毒腹部,用大剪刀剪开小鼠腹部皮肤。
4. 拉动切割皮肤的两侧并进入小鼠腹膜。
5. 用小剪刀剪开腹膜。
6. 通过移动肠道和内脏的位置来定位V形子宫、输卵管和卵巢。
7. 用一对精密镊子夹住输卵管连接处,切开子宫输卵管和峡部壶腹连接处,将输卵管与子宫分开(图 5A 和 5B)。
8. 通过去除脂肪组织、血液和组织液来清洁输卵管,并将输卵管转移到从 CO 2培养箱中移出的培养皿中。
注意:使用保存盘将收集盘的暴露时间减少到室温。
9. 将收集盘从 CO 2培养箱移到工作台上,使用一对精密镊子将输卵管转移到收集盘上的液体石蜡中,并将输卵管放在该盘的底部。
10. 在立体显微镜下(视频 2 和图 4B )使用针头从壶腹取出卵-积云复合物,并将卵-积云复合物添加到收集盘上的一滴 CARD mHTF培养基中(图 6A 和 6B)。超排卵处理平均每只雌性小鼠诱导 20-50 个卵子( Shindo et al ., 2021),一滴包括 <100 个鸡蛋。
11. 将收集盘移至 CO 2培养箱。
12.对所有雌性小鼠 重复步骤H2-H 11。
注意:准备与超排卵小鼠数量相对应的 CARD mHTF介质滴。




图e 4. 本实验中使用的工具。 
(A) 用于腹部探查的解剖装置。 (B) 立体显微镜。用于卵-积云复合体的插入、处理装置。 (C) 用于鸡蛋转移的嘴移液装置。 (D)带有吸气介质的玻璃移液器。箭头,介质的液体表面。




图 5。 卵巢、输卵管和子宫的图片。
( A)卵巢、输卵管和子宫的序列视图。每个连接点由虚线表示。 (B)输卵管的分离视图。箭头表示壶腹。




视频 2. 从壶腹中取出卵积云复合体。
https://os.bio-protocol.org/doc/upprotocol/p4557/Abstract10_0_20220307100233534/Movie%202.mp4 


I. 去除卵丘细胞并计数卵子
1. 将收集和洗涤的盘子从 CO 2培养箱移到工作台上。
2. 使用口腔移液器吸出含有透明质酸酶的 M2 培养基(图 4C),并将卵-积云复合物从收集盘上的 CARD mHTF培养基滴转移到去除盘上带有透明质酸酶的 M2 培养基滴中。
注意:吸出含有透明质酸酶的 M2 培养基,直到玻璃移液器的较宽部分,以便于处理鸡蛋(图4D)。
3. 嘴移液从卵丘复合物中去除卵丘细胞,并将卵转移到洗涤盘上的 CARD mHTF介质滴中(图 6C)。
4. 在洗涤盘上的三滴 CARD mHTF培养基之间移动鸡蛋来清洗鸡蛋。
5. 重复步骤I2-I4 。
6. 使用机械计数计数器计算每滴正常鸡蛋的数量,并将结果记录在 Microsoft Excel 中。正常和异常的鸡蛋很容易通过形态来区分(图 6D 和 6E)。
注意:如果正常卵子数量减少,请检查所用溶液的有效期、超排卵时间以及小鼠的年龄是否适合超排卵。




图6.鸡蛋操作。 
(A) 从壶腹中取出卵积云复合体。 这一滴包含 47 个鸡蛋。 (B) 透明质酸酶处理前的卵丘复合物。 (C) 透明质酸酶处理后的鸡蛋。 (D) 正常鸡蛋。 (E) 异常鸡蛋。


数据分析


使用 Microsoft Excel( Shindo )执行学生t检验或 Mann-Whitney 的U检验 等人,2021)。


笔记


1. 将两倍量(每只小鼠 200 μL)的超排卵试剂注射到体重为 35 克或更多的小鼠中。
2. 如果您需要计算每只老鼠的鸡蛋数量,请为每只老鼠涂抹一滴。
3. 减少鸡蛋暴露于室温的时间 以避免在进行 ICSI 时降低开发效率。
4. 如有必要,用显微镜相机拍照。


确认致谢


这项工作得到了 JSPS KAKENHI(授权号:JP19K09682 和 JP19H01067)和国家儿童健康中心的部分支持。 发展(2021C-27)。该协议改编自两篇原创文章( Shindo 等人,2019 年和 2021 年)。我们要感谢意得辑 ( www.editage.com ) 的英文编辑。


利益争夺


作者声明没有竞争利益。


伦理


所有动物实验均按照经批准的方案进行 国家儿童健康与发展研究所机构动物护理和使用委员会 (实验编号,A2005-007 和 A2020-005)。


参考文献


1. 卡特,DA(1993)。为受精的单细胞小鼠卵子制备培养基。 方法 Mol Biol 18:141-1 43 。
2. Shindo , M., Inui, M., Kang, W., Tamano , M., Tingwei , C., Takada, S., Hibino, T., Yoshida, M., Yoshida, K., Okada, H.,等人。 (2019)。精液基因簇的缺失加强了 SVS2 在男性生育力中的关键作用。 国际分子科学杂志20(18):4557。
3. Shindo , M.、Tsumura, H.、Miyado, K.、Kang, W.、Kawano, N.、Yoshida, T.、 Fukami , M. 和 Miyado, M. (2021)。 C57BL/6N 和 C57BL/6J 小鼠亚株对超排卵的反应相似。 MicroPubl Biol 2021。
4. Takeo, T. 和Nakagata , N. (2015)。使用抑制素抗血清和马绒毛膜促性腺激素联合给药的超排卵增加了 C57BL/6 雌性小鼠的排卵卵母细胞数量。 公共科学图书馆一10:e0128330 。
5. Wang, H.、Herath, CB、Xia, G.、Watanabe, G. 和 Taya, K. (2001)。施用抑制素中和抗血清后小鼠的超排卵、受精和体外胚胎发育。 复制122(5):809-816。


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免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Shindo, M., Miyado, K., Kang, W., Fukami, M. and Miyado, M. (2022). Efficient Superovulation and Egg Collection from Mice. Bio-protocol 12(11): e4439. DOI: 10.21769/BioProtoc.4439.
  2. Shindo, M., Tsumura, H., Miyado, K., Kang, W., Kawano, N., Yoshida, T., Fukami, M. and Miyado, M. (2021). Similar responsiveness between C57BL/6N and C57BL/6J mouse substrains to superovulation. MicroPubl Biol 2021.
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