Wakayama T, Whittingham D G, Yanagimachi R
Department of Anatomy and Reproductive Biology, University of Hawaii Medical School, Honolulu 96822, USA.
J Reprod Fertil. 1998 Jan;112(1):11-7. doi: 10.1530/jrf.0.1120011.
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.
附睾小鼠精子悬浮于各种生理溶液（CZB、PBS或等渗盐水）中，添加或不添加18%（w/v）棉子糖，然后冷却至-20℃、-50℃或-196℃并储存1 - 28天。解冻后，少数添加棉子糖冷冻的精子有部分活动能力（约2%），但在所有其他处理中精子均无活动能力，通过区分活精子和死精子的染色诊断为“死亡”。几乎所有注射了添加和未添加棉子糖冷冻精子头部（细胞核）的卵母细胞均正常受精（95 - 100%）并发育至二细胞期（89 - 100%）。在生理培养基之间未发现差异。在CZB培养基中冷冻的精子使大多数卵母细胞发育至囊胚（80 - 94%），但用PBS和等渗盐水中冷冻的精子受精后发育显著减少，尤其是在没有棉子糖的情况下（分别为69%和70%，而没有棉子糖时为51%和50%）。所有处理均获得了正常可育后代，但在添加或不添加棉子糖的等渗盐水中以及添加棉子糖的CZB中储存于-196℃的精子产生的后代明显较少。睾丸精子对冷冻损伤极为敏感：在添加或不添加棉子糖的CZB中冷冻至-196℃后，约50%的精子解冻后解体。几乎100%注射了完整（在光学显微镜水平）睾丸精子头部的卵母细胞发育至二细胞期，但与对照组相比，发育至囊胚的比例显著降低，尤其是那些没有棉子糖的对照组。数据表明，精子细胞核的冷冻保存所需条件不如完整精子保持正常生理功能所需条件严格。当将无活力精子的细胞核注入卵母细胞时，活力和质膜完整性对于受精和产生活后代并非必需。
