title: Rapid screening of cryopreservation protocols for murine prepubertal testicular tissue by histology and PCNA immunostaining.
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.
authors.affiliation: Reproductive Biology Laboratory-CECOS, Institute for Biomedical Research, Rouen University Hospital, 1 rue de Germont, Rouen Cedex, France.
authors.forename: J P
authors.initials: JP
authors.lastname: Milazzo
authors.forename: A
authors.initials: A
authors.lastname: Travers
authors.forename: A
authors.initials: A
authors.lastname: Bironneau
authors.forename: A
authors.initials: A
authors.lastname: Safsaf
authors.forename: E
authors.initials: E
authors.lastname: Gruel
authors.forename: C
authors.initials: C
authors.lastname: Arnoult
authors.forename: B
authors.initials: B
authors.lastname: Macé
authors.forename: O
authors.initials: O
authors.lastname: Boyer
authors.forename: N
authors.initials: N
authors.lastname: Rives
mesh_terms: D000818:Animals; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000008:administration & dosage; D008297:Male; D051379:Mice; D008819:Mice, Nude; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis / Q000378:metabolism*; D019946:Propylene Glycol / Q000494:pharmacology; D013737:Testis / Q000166:cytology* / Q000187:drug effects / Q000502:physiology
chemical_list: D003451:Cryoprotective Agents; D018809:Proliferating Cell Nuclear Antigen; D019946:Propylene Glycol; D004121:Dimethyl Sulfoxide