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IVF小鼠体外受精实验

王智浩王龙飞 |  2023-12-08  | DOI: 10.21769/BioProtoc.v1374
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视频简介IVF实验在小鼠模型中为研究生殖调控基因、激素对受精和胚胎发育的影响提供了理想的平台。这项技术不仅在解决生育问题上具有重要意义,同时也为深入理解哺乳动物生殖生物学提供了丰富的实验数据,为相关疾病的研究和治疗奠定了基础。
本视频作者 王智浩 负责了本视频的策划、文档撰写、视频拍摄、剪辑、校审和统筹工作,王龙飞 负责了本视频的实验设计、出镜演示和审阅工作。本实验方案源自Manipulating the Mouse Embryo: A Laboratory Manual (Fourth Edition) 一书,同时参考了相关文献,最终根据作者的实验经验复现、改进而成。本实验方案成功率较高,胚胎受精率较高,可以满足后续诸如胚胎移植等实验的需求,是作者根据经验总结改进形成的较为成熟且操作简便的体系和方法。
  • 视频介绍

一、视频摘要

In Vitro Fertilization (IVF) 是生殖生物学研究中一项重要的实验技术,具有诸多优势,如操作简便、结果可控、可用于研究生育障碍等。该实验在小鼠模型中为研究生殖调控基因、激素对受精和胚胎发育的影响等研究提供了理想的平台。本实验方案源自Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),同时参考相关文献,最终根据作者的实验经验复现、改进而成。本方案成功率较高,胚胎受精率较高,可以满足后续诸如胚胎移植等实验的需求,是较为成熟且操作简便的体系和方法。

In Vitro Fertilization (IVF) is a key technique in reproductive biology, valued for its simplicity and applicability to fertility disorder research in mouse models. Derived from reputable sources and refined through experience, our protocol ensures high success and efficiency, making it a user-friendly system for experiments, including embryo transplantation.

 

 

二、关键词

体外受精技术;小鼠;胚胎移植;

IVF; Mouse; Embryo Transfer

 

 

三、实验样品信息,试剂、耗材或仪器

1. 样品信息

动物:

3-6月龄成年C57/BL6J雄鼠,1-2

8周龄雌性供体C57/BL6J小鼠,若干只

Animals:

Male C57/BL6J mice, 3-6 months old, 1-2 individuals

Female C57/BL6J mice, 8 weeks old, several individuals

 

 

2. 试剂、耗材和仪器

试剂:

1.     注射用血促性素(PMSG, Pregnant Mare Serum Gonadotropin(宁波第二激素厂, cat. G024)

2.     注射用绒促性素(hCG, human Chorionic Gonadotropin,(宁波第二激素厂, cat. GN026)

3.     矿物油(Sigmacat. M8410

4.     HTF受精液(易核,cat. M1130

5.     KSOM培养基(易核,cat. M1430

6.     M2培养基(Sigma-Aldrichcat. M7167

材料:

1.     35mm60mm细胞培养皿

2.     胚胎移植口吸管(北京吉田生物科技有限公司,规格:1.0×500mm

3.     各规格吸头

4.     1mL无菌注射器

5.     50mL离心管

6.     3mL无菌巴斯德吸管

7.     铝箔纸

3. 仪器和软件

仪器设备:

生物安全柜、CO2培养箱、体式显微镜、手术剪若干、显微镊若干、酒精灯、玻璃恒温板

Materials:

1.     35mm, 60mm cell culture dishes

2.     Embryo transfer pipette (Beijing Jitai Biotechnology Co., Ltd., specifications: 1.0×500mm)

3.     Various sizes of pipette tips

4.     1mL sterile syringe

5.     50mL centrifuge tubes

6.     3mL sterile Pasteur pipette

7.     Aluminum foil

Reagents:

1.     Pregnant Mare Serum Gonadotropin (PMSG) for injection (Ningbo Second Hormone Factory, cat. G024)

2.     Human Chorionic Gonadotropin (hCG) for injection (Ningbo Second Hormone Factory, cat. GN026)

3.     Mineral oil (Sigma, cat. M8410)

4.     HTF fertilization medium (易核, cat. M1130)

5.     KSOM culture medium (易核, cat. M1430)

6.     M2 culture medium (Sigma-Aldrich, cat. M7167)

Equipment:

Biological safety cabinet, CO2 incubator, inverted microscope, surgical scissors, fine forceps, alcohol lamp

 

 

四、实验操作

主要步骤:

1. 小鼠超数排卵

2. 实验前准备

3. 精子采集与获能

4. 卵母细胞采集

5. 体外受精

6. 原核形态观察

Main Steps:

1. Superovulation in mice

2. Experimental setup

3. Sperm collection and capacitation

4. Oocyte retrieval

5. In vitro fertilization

6. Prokaryotic morphological observation

 

 

 

五、注意事项

注意事项:

1. 培养液需在培养箱中平衡过夜;

2. 矿物油液封时,没过液滴不要弄到皿盖,否则会影响气体交换。

3. HTF中加入1%谷胱甘肽,可提高发育效率。

4. 精子,卵子对环境敏感,每次操作时间最好控制在15min以内。

Guidelines:

1. Allow the culture medium to equilibrate overnight in the incubator.

2. When sealing with mineral oil, ensure that droplets do not reach the lid of the dish to avoid interference with gas exchange.

3. Add 1% glutathione to the HTF medium to enhance developmental efficiency.

4. Both sperm and eggs are sensitive to the environment; aim to complete each operation within a time frame of 15 minutes to maintain optimal conditions.

 

 

六、结果分析(可选)

可能出现问题的解决方案:

1. 超排效果不理想:为达到更好的超排受精效果,应挑选处于发情间期青春期雌鼠;激素配制时,选用无菌生理盐水避光稀释,1ml分装,-20°C冻存,避免反复冻融影响激素作用,从而影响超排效果。根据体重腹腔注射激素,每20g体重注射8U,过高会造成卵巢囊肿,过低则会导致无法完全发挥超数排卵的潜力。

2. 精子质量差,受精率低:划开附睾尾时,有丝带状出现;培养箱培养十分钟后观察液体成乳白色,镜检精子活动性好;获能液中培养1.5h-2h;添加精子时,将精子添加在卵团对侧,模拟体内环境,竞争出质量较好的精子。

3. 胚胎发育率低:HTF培养液适用于精子获能,体外受精及胚胎发育至2cell,但HTF容易导致2cell阻滞,需在受精完成后将胚胎转移到KSOM培养液。由于胚胎在各个阶段的所需要的营养成分不同,需要在4cell时更换KSOM培养液,以及时补充营养;同时每个液滴不要超过30个胚胎,保证营养充分。另外,胚胎对环境敏感,要时刻注意培养箱环境处于37°C5%CO2状态下,且操作迅速,防止环境失衡。KSOM培养液中含有酚红指示剂,可根据颜色判断酸碱环境,越碱越紫。取出观察时也应位于37°C热台上,每次时间不超过5min

 

Possible Issues and Solutions:

1. Suboptimal superovulation: To improve superovulation for fertilization in female mice, it is recommended to select adolescent mice in estrus. It is also important to use sterile saline for hormone dilution, aliquot 1ml, and store at -20°C to prevent repeated freezing and thawing, which can affect hormone efficacy and superovulation results. Administer hormones intraperitoneally based on weight, injecting 8 units per 20 grams; exceeding this amount may result in ovarian cysts, while administering too little may hinder optimal superovulation potential.

2. Poor sperm quality and low fertilization rate: When cutting the epididymal tail, observe the appearance of a ribbon-like structure. After 10 minutes of incubation in the culture dish, the liquid should turn milky white, indicating good sperm motility under microscopic examination. Incubate in capacitation medium for 1.5-2 hours. When adding sperm, place them on the opposite side of the egg cluster to mimic the in vivo environment, promoting competition and selecting for higher-quality sperm.

3. Low embryo development rate: HTF medium is suitable for sperm capacitation, in vitro fertilization, and embryo development up to the 2-cell stage. However, HTF may lead to 2-cell blockage, so transfer embryos to KSOM medium after fertilization. Since embryos require different nutrients at each stage, switch to KSOM medium at the 4-cell stage for timely nutritional supplementation. Ensure that each droplet contains no more than 30 embryos for sufficient nutrition. Embryos are sensitive to environmental conditions, so maintain the incubator at 37°C, 5% CO2, and work quickly to prevent imbalances. KSOM medium contains phenol red as an indicator, with a more purple color indicating alkalinity. When observing, place it on a 37°C heating stage and limit each session to 5 minutes.

 

 

七、参考文献(可选)

1.      田风林, 宋学功 & 邓树义. (2020). 不同剂量生殖激素对小鼠超排效果的影响. 中国奶牛 (03), 27-29. doi:10.19305/j.cnki.11-3009/s.2020.03.008.

2.      杨伟伟, 顾美儿, 桂飞, 宋晓明, 孙筱品, 唐蔚, 吴宝金. (2022). 小鼠体外受精. // 实验动物胚胎操作实验手册. Bio-101: e1010947. DOI: 10.21769/BioProtoc.1010947.

3.      Nagy, A. (2002). Manipulating the Mouse Embryo: A Laboratory Manual (3rd edition). Cold Spring Harbor Laboratory Press.

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