三种冷冻保护剂对新生小鼠睾丸组织冷冻效果的比较李淼1郁丽丽.DOCVIP

三种冷冻保护剂对新生小鼠睾丸组织冷冻效果的比较 李淼1，郁丽丽2，张艺宝3，强苏静2，刘丽均2，徐平1,2 中国科学院上海生命科学研究院，上海201615；2.上海斯莱克实验动物有限责任公司，上海201615；3.南京农业大学动物医学系，南京210095） 【摘要】目的探讨三种不同冷冻保护剂对新生C57BL/6J小鼠睾丸冷冻的效果。方法5日龄C57BL/6J雄鼠睾丸组织，分别用3种不同冷冻保护剂（二甲基亚砜DMSO、丙二醇PROH、EFS）进行玻璃化冷冻。复苏冷冻后的睾丸组织，每组一部分进行组织学分析，另一部分进行组织移植，移植后检测关键基因表达水平以及卵胞质内单精注射，并设相应对照组。结果3个实验组睾丸组织形态改变与对照组相比均具有显著性差异（P＜0.01），其中EFS组分值最低即改变最小；原位移植8周后组织块生长发育，存活率分别为DMSO组16.7%、PROH组13.3%、EFS组23.3%，存在成熟精子；睾丸组织特异性基因pgk-2和TESK1正常表达；卵胞质内单精注射表明移植后睾丸内精子均具有受精能力，胚胎移植后可得到正常子代小鼠（DMSO：PROH：EFS=5：2：8）。结论3种冷冻保护剂均能良好保存睾丸组织结构和功能。其中，EFS方法保存的睾丸组织形态改变最小、功能完善更适于睾丸组织的冷冻。 【关键词】小鼠睾丸；冷冻保护剂；冷冻效果 Assessment of three cryoprotectants for mouse immature testicular tissue LI Miao1，YU Lili2，ZHANG Yibao3，QIANG Sujing2，LIU Lijun2，XU Ping1, 2 Shanghai Institutes for Biological Sciences, Chinese Academy of Science，Shanghai 201615；2.Shanghai SLAC Laboratory Animal Ltd. Co.，Shanghai 201615；3.Veterinary college of Nanjing Agriculture University, Nanjing 210095) 【Abstract】ObjectiveTo compare the freezing effect of three cryoprotectants usevitrification-cryopreservation methods on immature C57BL/6J mouse testicular tissues.MethodTestis from five-day-old mouse were cryopreserved using DMSO、PROH and EFS cryopreservation solution and thawed respectively. Cryodamage of seminiferous cords was HE stained and semi-quantitatively determined, establishing a scoring of alterations. The function of testicular tissues after xenografting was evaluated by Real Time quantitive PCR and assisted reproduction. ResultsThe morphology of the fresh and frozen–thawed samples was significant different(P﹤0.05). After eight-week xenografting, mature sperms were achieved from all tissues and both methods resulted in pregnancies.The number of progenies from DMSO, PROH and EFS were 5, 2 and 8 respectively, after micromanipulation and embryos implanting. Moreover, the tissue-specific genes were expressed normally. ConclusionsThe threefreezing protocols are able to maintain mouse immature testicular tissue architecture and function