Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection
Production of normal offspring from mouse oocytes injected with spermatozoa cryopreserved with or without cryoprotection
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DOI：
10.1530/jrf.0.1120011
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发表时间：
1998-01-01
期刊：
影响因子：
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通讯作者：
Yanagimachi, R
中科院分区：
文献类型：
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作者：
Wakayama, T;Whittingham, DG;Yanagimachi, R
将小鼠附睾精子悬浮于含或不含18%(w/v)棉子糖的不同生理溶液(CZB、PBS或等渗盐水)中，然后冷却至-20℃、-50℃或-196℃，保存1~28天。解冻后，用棉子糖冷冻的少数精子部分活动(约2%)，但在所有其他处理中，它们是静止的，通过区分活精子和死精子的染色来诊断为“死”。注射棉子糖冷冻精子的精子头(核)后，几乎所有的卵母细胞都能正常受精(95-100%)并发育到2-细胞期(-100%)。生理培养基间无差异。用CZB液冷冻的精子受精后，大多数卵母细胞发育到囊胚(80-94%)，但用PBS和等渗盐水冷冻后，受精卵的发育率显着降低，尤其是在不含棉籽糖的情况下(69%和70%对51%和50%)。所有处理均获得正常生育后代，但将精子保存在-196℃、加或不加棉籽糖和CZB加棉籽糖的等渗盐水中的后代明显减少。睾丸精子对冷冻非常敏感：大约50%的精子在CZB中冷冻到-196℃，解冻后有或没有棉子糖解体。注射完整(光镜下)睾丸精子头的卵母细胞几乎100%发育到2-细胞期，但囊胚的发育率明显低于对照组，尤其是不含棉籽糖的卵母细胞。这些数据表明，与保留完整精子的正常生理功能相比，冷冻保存精子核需要的条件不那么严格。当不能存活的精子的核被注射到卵母细胞中时，运动性和质膜完整性对于受精和产生活后代并不是必不可少的。
Epididymal mouse spermatozoa were suspended in various physiological solutions (CZB, PBS or isotonic saline) with or without 18% (w/v) raffinose before cooling to -20 degrees, -50 degrees or -196 degrees C and storage for 1-28 days. After thawing, a few spermatozoa frozen with raffinose were partially motile (about 2%) but in all other treatments they were immotile and diagnosed as 'dead' by staining that differentiates between live and dead spermatozoa. Almost all oocytes injected with sperm heads (nuclei) from spermatozoa frozen with and without raffinose were fertilized normally (95-100%) and developed to the two-cell stage (89-100%). No differences were found between the physiological media. The majority of oocytes fertilized with spermatozoa frozen in CZB medium developed to blastocysts (80-94%) but development was significantly reduced after fertilization with spermatozoa frozen in PBS and isotonic saline especially in the absence of raffinose (69 and 70% versus 51 and 50%). Normal fertile offspring were obtained in all treatments but there were significantly fewer offspring with spermatozoa stored at -196 degrees C in isotonic saline with or without raffinose and CZB with raffinose. Testicular spermatozoa were extremely sensitive to cryodamage: about 50% frozen to -196 degrees C in CZB with or without raffinose disintegrated after thawing. Almost 100% of oocytes injected with sperm heads from intact (at light microscope level) testicular spermatozoa developed to the two-cell stage but development to blastocysts was reduced significantly compared with that of controls especially those without raffinose. The data indicate that cryopreservation of sperm nuclei requires less stringent conditions than those for the retention of normal physiological function of intact spermatozoa. Motility and plasma membrane integrity are not essential for fertilization and the production of live offspring when nuclei of nonviable spermatozoa are injected into oocytes.