Division of Reproductive Biotechnology and Innovation, Center for Animal Resources and Development, Institute of Resource Development and Analysis, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto, 860-0811, Japan.
Division of Reproductive Engineering, Center for Animal Resources and Development, Institute of Resource Development and Analysis, Kumamoto University, 2-2-1 Honjo, Chuo-ku, Kumamoto 860-0811, Kumamoto, 860-0811, Japan.
Theriogenology. 2023 Mar 15;199:69-76. doi: 10.1016/j.theriogenology.2023.01.016. Epub 2023 Jan 18.
Previous studies have observed the fertilization process in rats using whole-mount preparation at different time-points after insemination. However, very few reports have described the various events during the fertilization process using an inverted microscope without whole-mount. Moreover, to the best of our knowledge, no reports have described the observation of changes in sperm motility associated with sperm penetration into oocytes. In this study, in vitro fertilization was performed using frozen-thawed sperm in various rat strains (SD, Wistar, LE, F344, and BN) and oocytes from the SD strain, and the process of sperm penetration into the oocytes and the subsequent development were observed. The sperm motility was assessed, and the correlation between the process of sperm penetration into the oocytes and sperm motility over time was examined. The motility of frozen sperm from the SD, Wistar, LE, and F344 increased at 2-3 h after thawing, at which time the sperm attached themselves to the zona pellucida. Sperm penetration into the zona pellucida occurred after 3-5 h, and pronuclei were formed in the cytoplasm of oocytes 5-9 h after insemination. The fertilities of frozen-thawed sperm from the SD, Wistar, LE, and F344 were 92.7%, 90.0%, 90.7%, and 68.7%, respectively. However, no increase in motility was observed after thawing of frozen sperm from the BN, and the fertility was only 21%. In addition, very few polyspermic oocytes were observed with use of frozen-thawed sperm of all strains. In summary, rats are suitable animals for the observation of sperm penetration into the oocytes, and we determined the timing of fertilization events in IVF using frozen-thawed rat sperm.
先前的研究已经在不同授精后时间点使用整体制备观察了大鼠的受精过程。然而，很少有报道描述了在没有整体制备的情况下使用倒置显微镜进行受精过程中的各种事件。此外，据我们所知，没有报道描述了与精子穿透卵母细胞相关的精子运动变化的观察。在这项研究中，使用不同大鼠品系（SD、Wistar、LE、F344 和 BN）的冷冻解冻精子和 SD 品系的卵母细胞进行了体外受精，观察了精子穿透卵母细胞的过程和随后的发育。评估了精子的运动能力，并检查了精子穿透卵母细胞的过程与精子运动能力随时间的相关性。解冻后 2-3 小时，SD、Wistar、LE 和 F344 的冷冻精子的运动能力增加，此时精子附着在透明带。解冻后 3-5 小时发生精子穿透透明带，授精后 5-9 小时卵母细胞质中形成原核。SD、Wistar、LE 和 F344 的冷冻解冻精子的受精率分别为 92.7%、90.0%、90.7%和 68.7%。然而，BN 的冷冻解冻精子解冻后没有观察到运动能力的增加，受精率仅为 21%。此外，所有品系的冷冻解冻精子很少观察到多精受精的卵母细胞。总之，大鼠是观察精子穿透卵母细胞的合适动物，我们确定了使用冷冻解冻大鼠精子进行 IVF 的受精事件的时间。
