摘要
目的:探究比较目前常见主流睾丸组织冷冻方法学,即玻璃化冷冻与慢速程序化冷冻,筛选适宜的睾丸组织冷冻方法。方法:分别收集3周龄小鼠及<2岁龄青春期前食蟹猴的睾丸组织,将其剪碎至6~26 mm^(3),均分为3组:新鲜组、玻璃化冷冻组和慢速程序化冷冻组,在冷冻5~7 d后复苏冷冻睾丸组织,使用HE染色、免疫荧光染色、TUNEL染色和Western印迹对冷冻-复苏睾丸组织进行评估。结果:①在C57BL6/J小鼠睾丸组织中,慢速程序化冷冻组精原干细胞标志物UCHL1表达量高于玻璃化冷冻组,凋亡细胞(TUNEL+细胞)含量低于玻璃化冷冻组。②食蟹猴睾丸组织中,慢速程序化冷冻组精原干细胞标志物UCHL1、细胞增殖标志物PCNA的表达量高于玻璃化冷冻组。结论:玻璃化冷冻和慢速程序化冷冻均可成功保存青春期前未成熟的小鼠及食蟹猴睾丸组织。但是在玻璃化冷冻保存中,对于冻存体积为6~26 mm^(3)的睾丸组织存在更多冷冻损伤区域,可能影响睾丸组织中精原干细胞的冷冻储存效果。
Objective:To explore and compare the current mainstream testicular tissue freezing methods,namely vitrification and controlled slow freezing,and optimize the best testicular tissue freezing methods.Methods:Testicular tissues of 3-week-old mice and<2-year old prepubertal cynomophage monkeys were collected and cut to 6-26 mm^(3),and divided into three groups:Fresh group,vitrification group and controlled slow freezing group were resuscitated after 5-7 days of freezing.HE staining,immunofluorescence staining,TUNEL staining and Western blot were used to evaluate the frozen-thawed testicular tissue.Results:1.In the testes of C57BL6/J mice,the expression level of spermatogonial stem cell marker UCHL1 in the controlled slow freezing group was higher than that in the vitrification group,and the content of apoptotic cells(TUNEL+cells)was lower than that in the vitrification group.2.In the testicular tissue of cynomolgus monkeys,the expression levels of spermatogonial stem cell markers UCHL1 and cell proliferation marker PCNA in the CSF group were higher than those in the vitrification group.Conclusion:Both vitrification and CSF can successfully preserve the testes of immature mice and cynomolgus monkeys before puberty.However,in the vitrification,there are more frozen damaged areas in the testicular tissue with the frozen volume of 6-26mm^(3),which may affect the cryopreservation of spermatogonial stem cells in the testicular tissue.
作者
马朦惠
苏文龙
吕林艳
陈海城
方丛
孙鹏
刘贵华
MA Meng-hui;SU Weng-long;LU Lin-yan;CHEN Hai-chneg;FANG Cong;SUN Peng;LIU Gui-hua(Center of Reproductive Medicine,Sixth Affiliated Hospital of Sun Yat-sen University;Biomedical Innovation Center,The Sixth Affiliated Hospital,Sun Yat-sen University;Guangdong Provincial Engineering and Technology Research Center for Fertility Preservation,Guangzhou,Guangdong 510000,China)
出处
《中华男科学杂志》
CAS
CSCD
北大核心
2023年第9期776-782,共7页
National Journal of Andrology
基金
国家自然科学基金(81971759,82171604)
关键词
玻璃化冷冻
慢速程序化冷冻
男性生育保存
精原干细胞
vitrification
controlled slow freezing
male fertility preservation
spermatogonial stem cell
