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

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 (S2). Equilibration was performed either at room temperature or at 4uC 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, S2, 30 minutes. The 1.5 M DMSO, S+ or S2 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, S2, 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.

Assessment of the optimal vitrification protocol for pre-pubertal mice testes leading to successful in vitro production of flagellated spermatozoa

“…In this study, vitrification protocols (SSV 1 , SSV 2 , OPS, SDV and mSSV 1 ) were compared with our conventional CSF (Milazzo et al, 2008(Milazzo et al, , 2010Travers et al, 2011) in terms of morphological alterations, cell death assessment, germ cell proliferation ability and differentiation. The mSSV 1 protocol using DMSO, EG and sucrose with modified post-thaw conditions retained not only the peripheral morphological integrity of the tissue but also better in vitro endocrine and exocrine functions, closer to the FT in comparison with CSF.…”

“…For CSF, the testes samples were frozen as previously described by our laboratory (Milazzo et al, 2008(Milazzo et al, , 2010. Briefly, the freezing media consisted of L15 supplemented with 1.5 M of DMSO, 0.05 M of sucrose and 10% (v/v) FCS.…”

What is the best cryopreservation protocol for human testicular tissue banking?

“…PCNA is a central component of the DNA replication machinery and is also involved in many cell processes, such as DNA recombination and repair, chromosome assembly and cell cycle progression via its protein-protein interactions. Therefore, it has been promoted as a good predictive marker for cryoinjury (Milazzo et al, 2010). The PCNA index was estimated as the percentage of PCNA-positive germ cells located at the basement membrane.…”

“…At present, controlled slow freezing (CSF) using dimethylsulphoxide (DMSO) as a cryoprotectant is the method used to cryopreserve immature testicular tissue in animal models Shinohara et al, 2002;Luetjens et al, 2008;Milazzo et al, 2010). In rodents, microinsemination using sperm retrieved from grafted cryopreserved prepubertal testicular tissue has even led to the birth of healthy offspring (Shinohara et al, 2002).…”

New Approaches of Ovarian Tissue Cryopreservation from Domestic Animal Species

“…Cryopreservation of testicular tissue has been studied since about ten years in animals (Jezek et al, 2001;Jahnukainen et al, 2007;Milazzo et al, 2008;Zeng et al, 2009;Abrishami et al, 2010;Milazzo et al, 2010;Curaba et al, 2011) and human (Bahadur and Ralph, 1999;Bahadur et al, 2000;Guerin, 2005;Revel and Mejia, 2010). It is the only available solution for pre-pubertal boys who must receive a gonadotoxic treatment (eg.…”