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

Authors

  • J. P. Milazzo,

    1. EA 4308 “Spermatogenesis and Male Gamete Quality,” Reproductive Biology Laboratory—CECOS, Rouen University Hospital, Institute for Biomedical Research, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 27, University of Rouen, Rouen, France
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  • A. Travers,

    1. EA 4308 “Spermatogenesis and Male Gamete Quality,” Reproductive Biology Laboratory—CECOS, Rouen University Hospital, Institute for Biomedical Research, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 27, University of Rouen, Rouen, France
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  • A. Bironneau,

    1. EA 4308 “Spermatogenesis and Male Gamete Quality,” Reproductive Biology Laboratory—CECOS, Rouen University Hospital, Institute for Biomedical Research, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 27, University of Rouen, Rouen, France
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  • A. Safsaf,

    1. EA 4308 “Spermatogenesis and Male Gamete Quality,” Reproductive Biology Laboratory—CECOS, Rouen University Hospital, Institute for Biomedical Research, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 27, University of Rouen, Rouen, France
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  • E. Gruel,

    1. EA 4308 “Spermatogenesis and Male Gamete Quality,” Reproductive Biology Laboratory—CECOS, Rouen University Hospital, Institute for Biomedical Research, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 27, University of Rouen, Rouen, France
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  • C. Arnoult,

    1. Institut National de la Santé et de la Recherche Médicale, Unité 905, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides, Institute for Biomedical Research, University of Rouen and Department of Immunology, Rouen University Hospital, Rouen, France
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  • B. Macé,

    1. EA 4308 “Spermatogenesis and Male Gamete Quality,” Reproductive Biology Laboratory—CECOS, Rouen University Hospital, Institute for Biomedical Research, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 27, University of Rouen, Rouen, France
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  • O. Boyer,

    1. Institut National de la Santé et de la Recherche Médicale, Unité 905, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides, Institute for Biomedical Research, University of Rouen and Department of Immunology, Rouen University Hospital, Rouen, France
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  • Dr N. Rives

    Corresponding author
    1. EA 4308 “Spermatogenesis and Male Gamete Quality,” Reproductive Biology Laboratory—CECOS, Rouen University Hospital, Institute for Biomedical Research, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides 27, University of Rouen, Rouen, France
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Reproductive Biology Laboratory—CECOS, Rouen University Hospital, 1 rue de Germont, 76031 Rouen Cedex, France (e-mail: nathalie.rives@chu-rouen.fr).

Abstract

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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