利用辅助生殖技术挽救基因修饰小鼠的实例分析

doi:10.12300/j.issn.1674-5817.2024.107

摘要/Abstract

摘要：

目的利用辅助生殖技术挽救生殖障碍的基因修饰小鼠品系，为完善珍贵实验小鼠品系的拯救技术更新提供参考。方法对28种不同品系的9～18月龄不育雄性小鼠实施体外受精-胚胎移植（in vitro fertilization-embryo transfer，IVF-ET）技术，即通过观察不育雄性小鼠的精子密度、精子活动等指标，选择活力较强的精子用于IVF-ET实验，在仔鼠出生后统计受精率、异形卵率和仔鼠出生率。对12种不同品系的8～18月龄不孕雌性小鼠实施优化的卵巢移植技术，即选取相同背景的6周龄雌性小鼠作为受体小鼠，摘除受体小鼠一侧完整的卵巢并结扎其另一侧输卵管，然后分离出供体小鼠一侧卵巢，将其原位植入受体小鼠摘除卵巢的一侧。术后21 d将受体小鼠与相同背景的8周龄野生型雄性小鼠合笼繁育，待仔鼠出生后，统计卵巢移植受体妊娠率、受体活产率等数据。结果利用IVF-ET技术成功挽救28种小鼠品系，雄性小鼠最大年龄为18月龄。首轮IVF-ET实验成功率为89.29%（25/28）。生殖障碍雄性小鼠IVF的平均受精率为（51.01±14.97）%，异形卵率为（9.03±5.28）%，仔鼠出生率（18.60±7.03）%。40只卵巢移植受体小鼠中39只存活，卵巢移植受体妊娠率为33.33%（13/39），卵巢移植后受体活产率为17.95%（7/39）。采用卵巢移植技术成功挽救4种小鼠品系，其中雌性小鼠最大年龄为18月龄；另外，8种品系因最终未获得存活至性成熟的子代小鼠而未被挽救。结论针对不同原因引起的生殖障碍雄性小鼠，特别是对于错过最佳繁育年龄的小鼠，IVF-ET技术可有效进行品系挽救，获得子代小鼠。针对老龄雌性小鼠的品系挽救，卵巢移植技术可作为重要备选方案，但成功率相对IVF-ET技术较低，存在一定实验风险。

关键词: 基因修饰小鼠, 中老龄, 生殖障碍, 体外受精-胚胎移植, 卵巢移植

Abstract:

Objective The utilization of assisted reproductive technology to rescue genetically modified mouse strains with reproductive disorders provides a reference for improving techniques to preserve valuable experimental mouse strains. Methods In vitro fertilization-embryo transfer (IVF-ET) technology was performed on 28 strains of infertile male mice aged 9-18 months. Several indicators such as sperm density and sperm motility in infertile male mice were assessed to select the most viable sperm for IVF-ET experiments. Fertility rate, abnormal egg rate, and birth rate were recorded after the birth of the pups. An optimized ovarian transplantation procedure was applied to 12 strains of infertile female mice aged 8-18 months. 6-week-old female mice with the same genetic background were selected as recipients. One intact ovary was removed from each recipient mouse, and the contralateral oviduct was ligated. An ovary from a donor mouse was isolated and transplanted orthotopically into the side where the ovary had been removed in the recipient mouse. Twenty-one days post-surgery, recipient mice were co-housed with 8-week-old wild type male mice of the same genetic background for breeding. Data such as the pregnancy rate and live birth rate of the recipients were recorded after the birth of the pups. Results IVF-ET successfully rescued 28 mouse strains, with the oldest male mice being 18 months old. The success rate of the first round of IVF-ET experiments was 89.29% (25/28). The average fertility rate of IVF in infertile male mice was (51.01±14.97)%, the abnormal egg rate was (9.03±5.28)%, and the birth rate of offspring mice was (18.60±7.03)%. 39 out of 40 ovarian transplant recipient mice survived, with a pregnancy rate of 33.33% (13/39) for ovarian transplant recipients, and a live birth rate of 17.95% (7/39). Four mouse strains were successfully rescued using optimized ovarian transplantation technology, with the oldest female mice being 18 months old. 8 strains were not rescued as they failed to produce offspring that survived to sexual maturity. Conclusion IVF-ET is an effective approach for rescuing mice with reproductive disorders caused by different reasons, especially for those beyond the optimal breeding age. Ovarian transplantation technology can also be used as an alternative for aged female mice. But its success rate is relatively lower than that of IVF-ET, and carries a higher experimental risk.

Key words: Genetically modified mouse, Middle-aged and aged, Reproductive disorders, In vitro fertilization-embryo transfer, Ovarian transplantation

中图分类号:

Q95-33

王芊芊,陶斯珏,卫振,等. 利用辅助生殖技术挽救基因修饰小鼠的实例分析[J]. 实验动物与比较医学, 2025, 45(1): 79-86. DOI: 10.12300/j.issn.1674-5817.2024.107.

WANG Qianqian,TAO Sijue,WEI Zhen,et al. A Case Study of Using Assisted Reproductive Technology to Rescue Genetically Modified Mice with Reproductive Disorder Phenotypes[J]. Laboratory Animal and Comparative Medicine, 2025, 45(1): 79-86. DOI: 10.12300/j.issn.1674-5817.2024.107.

图/表 5

图1 雄性小鼠采集精子团及获能1 h后的精子状态注：A，采集的雄性小鼠一侧附睾尾与一段输精管；B，将附睾尾中采集的精子团放入精子获能液滴中；C，2只不同品系雄性小鼠的精子在获能1 h后的精子状态，左侧培养液滴中的精子稀薄，右侧培养液滴中精子散开均匀。

Figure 1 Sperm masses collected from male mice and sperm status after 1 h of capacitationNote：A， The unilateral cauda epididymidis and a segment of the vas deferens from a male mouse； B， Sperm masses collected from the cauda epididymidis placed into droplets of sperm capacitation medium； C， The sperm status of two different strains of male mice after 1 h of capacitation： in the left culture droplet， the sperm were sparse， while in the right culture droplet， they were evenly dispersed.

图2 小鼠卵巢移植手术注：A，在受体小鼠一侧输卵管的两处位置进行结扎；B，摘取供体小鼠一侧完整的卵巢组织；C，固定受体小鼠另一侧卵巢和输卵管；D，将供体小鼠一侧完整的卵巢组织移植入摘除了受体小鼠卵巢的囊膜内，并缝合囊膜。

Figure 2 Ovarian transplantation surgery in miceNote： A， Ligation was performed at two sites on the oviduct of one side of the recipient mouse； B， An intact ovary from one side of the donor mouse was harvested； C， The ovary and oviduct on the other side of the recipient were stabilized； D， The intact ovary from the donor was transplanted into the ovarian capsule of the recipient at the site where the ovary had been removed， followed by suturing of the capsule.

表1 经体外受精-胚胎移植挽救的不育基因修饰小鼠的异形卵率、受精率及仔鼠出生率 (xˉ±s)

Table 1 Abnormal egg rate, fertility rate and birth rate of infertile genetically modified mice rescued by in vitro fertilization-embryo transfer

不育雄性小鼠月龄/月 Age of Infertile male mice/month	动物数量 n	异形卵率/% Abnormal egg rate/%	受精率/% Fertility rate/%	仔鼠出生率/% Birth rate/%
9	3	4.35±2.21	48.99±28.09	19.87±13.93
10	4	11.55±8.29	62.34±11.32	16.84±5.43
11	4	9.38±2.35	60.85±10.46	17.41±10.46
12	4	7.14±1.12	48.87±13.72	19.04±5.31
13	4	9.50±6.44	42.21±5.86	19.68±1.92
14	4	13.15±7.72	53.07±12.79	16.51±4.60
15	1	6.13	36.79	12.82
16	1	5.04	43.17	15.00
17	1	9.09	47.20	20.00
18	2	8.31±3.53	42.45±27.12	27.66±10.50

图3 青年组与中老龄组受精率、异形卵率和仔鼠出生率的比较注：A，青年组与中老年组小鼠体外受精后的受精率比较；B，青年组与中老年组小鼠体外受精后的异形卵率比较；C，青年组与中老年组小鼠体外受精-胚胎移植后的仔鼠出生率比较。*P＜0.05，***P＜0.001，ns，差异无统计学意义；D，雄性小鼠年龄与受精率的关系。

Figure 3 Comparison between the young group and the middle-aged and aged group in their fertility rates， abnormal egg rates， and birth ratesNote： A， Comparison of in vitro fertilization （IVF） fertilization rates between the young group and the middle-aged and aged group； B， Comparison of IVF abnormal egg rates between the young group and the middle-aged and aged group； C， Comparison of IVF-embryo transfer （ET） birth rate between the young group and the middle-aged and aged group. *P<0.05， ***P<0.001； ns， differences were not statistically significant； D， Correlation between male mice age and fertility rate.

表2 经卵巢移植成功挽救的不育基因修饰小鼠的供体小鼠月龄、妊娠率和活产率

Table 2 Age of donor mice, pregnancy rate and live birth rate of infertile genetically modified mice successfully rescued through ovarian transplantation

供体雌性小鼠月龄/月 Age of donor mice/month	左侧卵巢移植的受体小鼠数/只 The number of recipients for donor’s left ovarian transplantation	右侧卵巢移植的受体小鼠数/只 The number of recipients for donor’s right ovarian transplantation	妊娠率/% Pregnancy rate/%	活产率/% Live birth rate/%
10	1	1	100.00 （2/2）	100.00 （2/2）
15	1	1	50.00 （1/2）	100.00 （1/1）
15	1	2	66.67 （2/3）	100.00 （2/2）
18	1	1	100.00 （2/2）	100.00 （2/2）

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