Qin Yumin, Feng Shenjiong, Zheng Min, Liu Xiaojuan, Zhao Jianping, Zhao Qintao, Ye Junhua, Mi Jidong, Zhong Yougang
College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
Beijing SINOGENE Biotechnology Co., Ltd., Beijing 102200, China.
Life (Basel). 2022 Nov 3;12(11):1778. doi: 10.3390/life12111778.
Gene-edited dogs are promising models for biomedical research because they have hundreds of genetic diseases that are similar to humans. A common method for producing gene-edited dogs is assisted reproductive technology (ART) using in vivo oocytes or embryos, but it is much more inefficient and has a higher cost. ART for dogs has lagged mostly because of the lack of an efficient in vitro maturation system. Because early maturation of canine oocytes occurs in follicles with extremely high concentrations of progesterone (P4), we hypothesize that P4 has an important role during maturation. In this study, we obtained ovaries of female dogs and collected cumulus−oocyte complexes, which were cultured in vitro in microdrops containing different P4 concentrations (0, 10, 40, 100 or 200 µg/mL). We found that 40 µg/mL P4 produced the highest oocyte maturation rate (29.7% ± 7.1%, p < 0.05). We also evaluated the quality of in vitro matured oocytes by in vitro fertilization and single-cell RNA sequencing, and both indicated an improvement in oocyte developmental potential. In conclusion, we successfully obtained the first live dogs using in vitro matured oocytes by adding P4 to optimize the in vitro maturation system of canine oocytes, and established a new and low-cost method to produce dogs via in vitro maturation and in vitro fertilization.
基因编辑犬是生物医学研究中很有前景的模型，因为它们患有数百种与人类相似的遗传疾病。生产基因编辑犬的一种常见方法是使用体内卵母细胞或胚胎的辅助生殖技术（ART），但这种方法效率低得多且成本更高。犬类ART大多滞后是因为缺乏有效的体外成熟系统。由于犬类卵母细胞的早期成熟发生在孕酮（P4）浓度极高的卵泡中，我们推测P4在成熟过程中起重要作用。在本研究中，我们获取了雌性犬的卵巢并收集了卵丘-卵母细胞复合体，将其在含有不同P4浓度（0、10、40、100或200µg/mL）的微滴中进行体外培养。我们发现40µg/mL的P4产生了最高的卵母细胞成熟率（29.7%±7.1%，p<0.05）。我们还通过体外受精和单细胞RNA测序评估了体外成熟卵母细胞的质量，两者均表明卵母细胞发育潜力有所提高。总之，我们通过添加P4优化犬类卵母细胞的体外成熟系统，成功地使用体外成熟的卵母细胞获得了首批活体犬，并建立了一种通过体外成熟和体外受精生产犬的新的低成本方法。
