Endoh Keisuke, Mochida Keiji, Ogonuki Narumi, Ohkawa Mika, Shinmen Akie, Ito Masao, Kashiwazaki Naomi, Ogura Atsuo
RIKEN Bioresource Center, Tsukuba, Japan.
J Reprod Dev. 2007 Dec;53(6):1199-206. doi: 10.1262/jrd.19058. Epub 2007 Sep 4.
Assessment of the developmental ability of oocytes following freezing and thawing is an important step for optimizing oocyte cryopreservation techniques. However, the in vitro fertilization of frozen-thawed mouse oocytes is often inefficient because of incomplete capacitation of spermatozoa in the absence of surrounding cumulus cells. This study was undertaken to determine whether the oocyte cryopreservation efficiency of different strains of mice could be assessed from the development of oocytes following parthenogenetic activation and intracytoplasmic sperm injection (ICSI). Oocytes were collected from hybrid (C57BL/6 x DBA/2) F1 or inbred (C57BL/6J, C3H/HeN, DBA/2J and BALB/cA) strains and were vitrified in a solution containing ethylene glycol, DMSO, Ficoll and sucrose. In the first series of experiments, oocytes were activated parthenogenetically by Sr(2+) treatment after warming. The oocytes from the inbred strains, but not those of the F1 hybrid, were diploidized by cytochalasin treatment to obtain a sufficient number of blastocysts. In all strains tested, parthenogenetic embryos derived from vitrified oocytes developed into blastocysts at rates between 23 and 68%. In the second series of experiments, vitrified oocytes from each strain were injected with homologous spermatozoa after warming. Normal offspring were obtained from all strains at rates between 5 and 26% per embryo transferred. Thus, the feasibility of oocyte cryopreservation protocols can be assessed easily by in vitro development of parthenogenetic embryos or by in vivo development of ICSI embryos. Moreover, the oocytes of these four major inbred strains of mice can be cryopreserved safely for production of offspring.
评估冻融后卵母细胞的发育能力是优化卵母细胞冷冻保存技术的重要一步。然而,由于在没有周围卵丘细胞的情况下精子获能不完全,冻融后的小鼠卵母细胞体外受精往往效率低下。本研究旨在确定是否可以通过孤雌激活和胞浆内单精子注射(ICSI)后卵母细胞的发育情况来评估不同品系小鼠的卵母细胞冷冻保存效率。从杂交(C57BL/6×DBA/2)F1或近交(C57BL/6J、C3H/HeN、DBA/2J和BALB/cA)品系中收集卵母细胞,并在含有乙二醇、二甲基亚砜、聚蔗糖和蔗糖的溶液中进行玻璃化冷冻。在第一系列实验中,解冻后的卵母细胞通过Sr(2+)处理进行孤雌激活。近交品系的卵母细胞经细胞松弛素处理后进行二倍体化,以获得足够数量的囊胚,而F1杂交品系的卵母细胞则不需要。在所有测试品系中,来自玻璃化冷冻卵母细胞的孤雌胚胎发育成囊胚的比率在23%至68%之间。在第二系列实验中,解冻后向每个品系的玻璃化冷冻卵母细胞中注射同源精子。每个移植胚胎产生正常后代的比率在5%至26%之间,所有品系均获得了正常后代。因此,通过孤雌胚胎的体外发育或ICSI胚胎的体内发育,可以轻松评估卵母细胞冷冻保存方案的可行性。此外,这四种主要近交品系小鼠的卵母细胞可以安全地冷冻保存以用于后代生产。