三种冷冻保护剂对新生小鼠睾丸组织冷冻效果的比较

Assessment of three cryoprotectants for mouse immature testicular tissue

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<dimethyl sulfoxide,DMSO>、丙二醇<propylene Glycol,PROH>、EFS20/40)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。

Objective To compare the freezing effect of three eryoprotectants use vitrification-cryopreservation methods on immature C57BL/6J mouse testicu]ar tissues. Method Testis from five-day-old mouse were cryopreserved using Dimethyl Sulfoxide、Propylene Glycol and EFS cryopreservation solutions and thawed respectively. Cryodamage of seminiferous cords was H＆E stained and semi-quantitatlvely determined, establishing a scoring of alterations. The function of testicular tissues after xenografting was evaluated by Real Time quantitive PCR and assisted reproduction. Results The morphology of the fresh and frozen-thawed samples was significant difference （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 micromanlpulation and embryos implanting. Moreover, the tissue-specific genes were expressed normally. Conclusions The threefreezing protocols are able to maintain mouse immature testieular tissue architecture and functionality. The preference is EFS cryoprotactants solution.