Journal of Andrology
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Published-Ahead-of-Print March 4, 2010, DOI:10.2164/jandrol.109.009324

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Rapid Screening of Cryopreservation Protocols for Murine Prepubertal Testicular Tissue by Histology and PCNA Immunostaining

Jean-Pierre Milazzo , Albanne Travers , Amandine Bironneau , Atmane Safsaf , Christophe Arnoult , Bertrand Macé , Olivier Boyer , and Nathalie Rives *

* To whom correspondence should be addressed. E-mail: nathalie.rives{at}chu-rouen.fr.

Numerous parameters have to be tested to identify optimal conditions for prepubertal testicular tissue banking. Our study evaluated nineteen different cryopreservation conditions of immature testicular tissue using a rapid screening method. Immature mice testis were cryopreserved using either 1,2-propanediol (PROH) or dimethyl-sulphoxide (DMSO) at a concentration of 0.75 or 1.5M using a controlled slow cooling rate protocol with (S+) or without seeding (S-). Equilibration was performed either at room temperature (RT) or at 4°C during 15 (15') or 30 minutes (30'). Seminiferous cord cryodamage was determined by scoring morphological alterations. Cell proliferation ability was evaluated using proliferating cell nuclear antigen (PCNA) antibody. Testes cryopreserved with optimal conditions were grafted into immunodeficient mice. Highest proportions of PCNA-positive nuclei and lowest morphological alterations were observed with DMSO 1.5M. Tissues were more altered with the conditions using 0.75M DMSO or PROH. Complete germ cell maturation was observed after allografting of testicular pieces previously frozen with DMSO1.5M S- 30'. DMSO1.5M (S+ or S-) protocol proved not solely to preserve prepubertal mice testicular tissue architecture but also germ cell and Sertoli cell proliferation potential. Allografting of thawed testis fragments into immunodeficient mice confirmed that DMSO1.5M S- 30' protocol maintained testicular somatic and germ cell functions. Post-thaw histological evaluation and PCNA immunostaining are useful to rapidly test numerous freezing-thawing parameters. They may also be efficient tools to control human prepubertal frozen testes quality, within the context of a clinical application.


Key words: Cryopreservation • Spermatogenesis






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