Milazzo J P, Travers A, Bironneau A, Safsaf A, Gruel E, Arnoult C, Macé B, Boyer O, Rives N
Reproductive Biology Laboratory-CECOS, Institute for Biomedical Research, Rouen University Hospital, 1 rue de Germont, Rouen Cedex, France.
J Androl. 2010 Nov-Dec;31(6):617-30. doi: 10.2164/jandrol.109.009324. Epub 2010 Mar 4.
Numerous parameters have to be tested to identify optimal conditions for prepubertal testicular tissue banking. Our study evaluated 19 different cryopreservation conditions for immature testicular tissue using a rapid screening method. Immature mice testes were cryopreserved using either 1,2-propanediol (PROH) or dimethyl sulfoxide (DMSO) at a concentration of 0.75 or 1.5 M using a controlled slow-cooling rate protocol with (S+) or without seeding (S+). Equilibration was performed either at room temperature or at 4°C for 15 or 30 minutes. Seminiferous cord cryodamage was determined by scoring morphologic alterations. Cell proliferation ability was evaluated using a proliferating cell nuclear antigen (PCNA) antibody. Testes cryopreserved with optimal conditions were grafted into immunodeficient mice. The highest proportions of PCNA-positive nuclei and lowest morphologic alterations were observed with 1.5 M DMSO. Tissues were more altered with 0.75 M DMSO or PROH. Complete germ cell maturation was observed after allografting of testicular pieces previously frozen with 1.5 M DMSO, S+, 30 minutes. The 1.5 M DMSO, S+ or S+ protocol preserved prepubertal mice testicular tissue architecture and germ cell and Sertoli cell proliferation potential. Allografting of thawed testis fragments into immunodeficient mice confirmed that the 1.5 M DMSO, S+, 30 minutes protocol maintained testicular somatic and germ cell functions. Postthaw histologic evaluation and PCNA immunostaining are useful to rapidly test numerous freeze-thaw parameters. They may also be efficient tools to control human prepubertal frozen testis quality, within the context of a clinical application.
为确定青春期前睾丸组织冻存的最佳条件，必须对众多参数进行测试。我们的研究使用快速筛选方法评估了19种不同的未成熟睾丸组织冷冻保存条件。使用1,2 - 丙二醇（PROH）或二甲基亚砜（DMSO），浓度为0.75或1.5 M，采用控制慢冷速率方案，有（S +）或无接种（S -），对未成熟小鼠睾丸进行冷冻保存。在室温或4°C下平衡15或30分钟。通过对形态学改变进行评分来确定生精索冷冻损伤。使用增殖细胞核抗原（PCNA）抗体评估细胞增殖能力。将在最佳条件下冷冻保存的睾丸移植到免疫缺陷小鼠体内。使用1.5 M DMSO时观察到PCNA阳性细胞核比例最高且形态学改变最少。使用0.75 M DMSO或PROH时组织改变更多。在用1.5 M DMSO、S +、30分钟预先冷冻的睾丸组织片段同种异体移植后观察到完全的生殖细胞成熟。1.5 M DMSO、S +或S -方案保留了青春期前小鼠睾丸组织结构以及生殖细胞和支持细胞的增殖潜能。将解冻后的睾丸片段移植到免疫缺陷小鼠体内证实，1.5 M DMSO、S +、30分钟方案维持了睾丸体细胞和生殖细胞功能。解冻后的组织学评估和PCNA免疫染色有助于快速测试众多冻融参数。在临床应用背景下，它们也可能是控制人类青春期前冷冻睾丸质量的有效工具。
