95 IN VITRO MATURATION AND FERTILIZATION OF OOCYTES FROM OVARIAN TISSUES CRYOPRESERVED AND XENOGRAFTED INTO NUDE MICE
95 IN VITRO MATURATION AND FERTILIZATION OF OOCYTES FROM OVARIAN TISSUES CRYOPRESERVED AND XENOGRAFTED INTO NUDE MICE
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95 来自冷冻保存并异种移植到裸鼠体内的卵巢组织的卵母细胞的体外成熟和受精
DOI：
10.1071/rdv22n1ab95
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发表时间：
2010
期刊：
影响因子：
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通讯作者：
H. Kaneko
中科院分区：
文献类型：
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作者：
K. Kikuchi;N. Kashiwazaki;M. Nakai;J. Noguchi;J. Ito;H. Kaneko
原始卵泡作为卵巢卵泡的储存库，是用于医疗、农业和动物学目的的卵母细胞的潜在来源。卵巢异种移植似乎有利于原始卵泡(原始卵母细胞)中的卵母细胞成熟，有利于家畜或濒危动物的保护和繁殖。我们已经从猪原始卵母细胞异种移植到裸鼠体内产生了可存活的胚胎(Kaneko等人。2006年再生产)。卵巢组织冷冻保存后的异种移植将是一种非常有效的工具。最近，Moniruzzaman et al.2009年Therigenology)报道，卵巢组织中的卵泡在冷冻保存和异种移植后能够发育到腔前阶段，但无法获得卵母细胞。在本研究中，我们对不同时间的冷冻保护剂乙二醇(EG)中的组织进行了玻璃化冷冻，并评估了收集卵母细胞的可能性以及它们的成熟和受精能力。取20日龄左右的仔猪卵巢组织，切成约1.0~2.0 mm的立方体。在IVC-PyrLac中的4%EG中平衡后(Kikuchi等人)。2002年的Biol.Reprod。)以玻璃化液(35%EG、5%聚乙烯吡咯烷酮、0.3M海藻糖在BS中)浸泡15min后，分别加入约4μ的L玻璃化液，浸泡45min或7min，滴入液氮中。在LN2中储存后，将微滴在37°C的加温溶液(BS中0.4M海藻糖)中转移2分钟，然后连续转移2分钟，生成BS中的0.2M、0.1M或0.05M海藻糖(Kaneko等人)。2006年再生产)，将20~30块组织移植到去卵巢裸鼠的肾被膜内。用猪FSH(62.5U·L~(-1)渗透压)处理宿主小鼠12天，观察移植卵巢的存活情况。当移植物中有明显的有腔卵泡时，使用外科刀片收集卵母细胞。然后它们在体外成熟并受精(Kikuchi等人)。2002年的Biol.报告。)。在移植后62~125d，45-S组和7-min组的12只小鼠中有6只获得了含有有腔卵泡的卵巢。分别采集39个和49个完全成熟的卵母细胞进行培养，按第一极体排出计算的成熟率分别为18%(7/39)和33%(16/49)。精子穿透卵母细胞的符合率分别为83%(5/6)和88%(14/16)。受精后10h，所有卵母细胞均形成雄核和雌核。经卡方检验，无论有无Yates‘s校正，两组浸润率差异均无统计学意义。总之，可以从冷冻保存的原始卵泡中收集完全发育成熟的猪卵母细胞，并将其移植到裸鼠体内。玻璃化冷冻前的浸泡时间不会影响卵母细胞的成熟或受精能力。
Primordial follicles act as stores of ovarian follicles and are potential sources of oocytes for medical, agricultural, and zoological purposes. Ovarian xenografting seems to be advantageous for maturing the oocytes in primordial follicles (primordial oocytes) and useful for the conservation and reproduction of domestic or endangered animals. We have generated viable embryos from porcine primordial oocytes xenografted into nude mice (Kaneko et al. 2006 Reproduction). Xenografting of ovarian tissues after cryopreservation would be a very powerful tool for this purpose. Recently, Moniruzzaman et al. 2009 Theriogenology) reported that follicles were able to develop to the pre-antral stage in ovarian tissues after cryopreservation and xenografting, but that oocytes were not obtainable from them. In the present study, we vitrified the tissue after different immersion periods in a cryoprotectant, ethylene glycol (EG), and evaluated the possibility of oocyte collection, and also their maturation and fertilization abilities. Ovarian tissue from piglets approximately 20 days old was minced into cubes of about 1.0 to 2.0 mm. After equilibration in 4% EG in IVC-PyrLac (Kikuchi et al. 2002 Biol. Reprod.) as a base solution (BS) for 15 min, they were immersed in vitrification solution (35% EG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BS) for 45 s or 7 min (45-s and 7-min immersion groups, respectively), then dropped with about 4 μL of vitrification solution into liquid nitrogen (LN2). After storage in LN2, microdroplets were transferred in warming solution (0.4 M trehalose in BS) at 37°C for 2 min, then consecutively transferred for 2-min periods into 0.2 M, 0.1 M, or 0.05 M trehalose in BS.As described previously (Kaneko et al. 2006 Reproduction), 20 to 30 pieces of tissue were grafted into kidney capsules of ovariectomized nude mice. The host mice were treated with porcine FSH (62.5 U mL-1 in osmotic pomp) for 12 days before assessment of the survival of the ovarian grafts. When antral follicles were evident in the grafts, oocytes were collected using a surgical blade. They were then matured and fertilized in vitro (Kikuchi et al. 2002 Biol. Reprod.). Ovaries containing antral follicles were obtained between 62 and 125 days after grafting from 6 out of 12 mice in the both 45-s and 7-min groups. When a total of 39 and 49 fully grown oocytes, respectively, had been collected from these groups and cultured, the maturation rates calculated on the basis of 1st polar body extrusion were 18% (7/39) and 33% (16/49), respectively. The corresponding rates for sperm-penetrated oocytes were 83% (5/6) and 88% (14/16), respectively. All the oocytes formed male and female pronuclei at 10 h after insemination. The rates in the 2 immersion groups did not differ significantly by chi-square test, with or without Yates’ correction. In conclusion, fully grown porcine oocytes can be collected from primordial follicles that have been cryopreserved and xenografted into nude mice. The period of immersion before vitrification may not affect oocyte maturation or fertilization ability.