Watanabe Hitomi, Kohaya Natsuki, Kamoshita Maki, Fujiwara Katsuyoshi, Matsumura Kazuaki, Hyon Suong-Hyu, Ito Junya, Kashiwazaki Naomi
Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan.
Graduate School of Veterinary Sciences, Azabu University, Sagamihara, Kanagawa, Japan.
PLoS One. 2013 Dec 23;8(12):e83613. doi: 10.1371/journal.pone.0083613. eCollection 2013.
In cryopreservation of mammalian germ cells, unfertilized oocytes are one of the most available stages because these cryopreserved oocytes can be used for assisted reproductive technologies, including in vitro fertilization (IVF) and intracytoplasmic sperm injection. However, it has been generally reported that the fertility and developmental ability of the oocytes are reduced by cryopreservation. Therefore further improvement will be required. Very recently, a new cryoprotective agent (CPA), called as carboxylated ε-poly-L-lysine (COOH-PLL), has been developed to reduce physical and physiological damage by cryopreservation in mammalian stem cells. However, it is unclear the effect of COOH-PLL on fertility and developmental ability of vitrified oocytes. In this study, we used COOH-PLL as a CPA with ethylene glycol (EG) for vitrification of mouse oocytes. Cumulus-oocyte complexes (COCs) were collected from ICR mice and then vitrified with Cryotop using different concentration of COOH-PLL and EG. A combined treatment with COOH-PLL and EG showed high survival rate (more than 90%) of vitrified-warmed COCs after in vitro fertilization. In addition, the fertility and developmental ability of COCs vitrified with E20P10 [EG 20% (v/v) and COOH-PLL 10% (w/v)] or E15P15 group (EG 15% and COOH-PLL 15%) were significantly higher than those with E10P20 (EG10% and COOH-PLL 20%) or P30 group (PLL30%). The vitrified COCs in E20P10 group developed to term at a high success rate (46.2%) and it was significantly higher than that in control (E30) group (34.8%). Our present study demonstrated for the first time that COOH-PLL is effective for vitrification of mouse oocytes.
在哺乳动物生殖细胞的冷冻保存中，未受精的卵母细胞是最容易获取的阶段之一，因为这些冷冻保存的卵母细胞可用于辅助生殖技术，包括体外受精（IVF）和胞浆内单精子注射。然而，普遍报道称卵母细胞的生育能力和发育能力会因冷冻保存而降低。因此，还需要进一步改进。最近，一种名为羧化ε-聚-L-赖氨酸（COOH-PLL）的新型冷冻保护剂已被开发出来，用于减少冷冻保存对哺乳动物干细胞造成的物理和生理损伤。然而，COOH-PLL对玻璃化卵母细胞的生育能力和发育能力的影响尚不清楚。在本研究中，我们将COOH-PLL与乙二醇（EG）作为冷冻保护剂用于小鼠卵母细胞的玻璃化。从ICR小鼠中收集卵丘-卵母细胞复合体（COCs），然后使用不同浓度的COOH-PLL和EG通过Cryotop进行玻璃化。在体外受精后，COOH-PLL和EG的联合处理显示玻璃化-复温后的COCs具有较高的存活率（超过90%）。此外，用E20P10组（EG 20%（v/v）和COOH-PLL 10%（w/v））或E15P15组（EG 15%和COOH-PLL 15%）玻璃化的COCs的生育能力和发育能力显著高于用E10P20组（EG10%和COOH-PLL 20%）或P30组（PLL30%）玻璃化的COCs。E20P10组玻璃化的COCs以高成功率（46.2%）发育至足月，且显著高于对照组（E30）（34.8%）。我们目前的研究首次证明COOH-PLL对小鼠卵母细胞的玻璃化是有效的。
