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  • Title: Rapid screening of cryopreservation protocols for murine prepubertal testicular tissue by histology and PCNA immunostaining.
    Author: Milazzo JP, Travers A, Bironneau A, Safsaf A, Gruel E, Arnoult C, Macé B, Boyer O, Rives N.
    Journal: J Androl; 2010; 31(6):617-30. PubMed ID: 20203335.
    Abstract:
    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.
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