title: Comparison of conditions for cryopreservation of testicular tissue from immature mice.
abstract: BACKGROUND
  Cryopreservation of immature testicular tissue could be considered as a major step in fertility preservation for young boys with cancer. In the present study, eight different freezing protocols were evaluated in immature mice testis.
  
  
  METHODS
  Testis from six-day-old mice were frozen using either 1,2-propanediol (PROH) or dimethylsulphoxide (DMSO: D) at 1.5 M. Different cooling rate curves were tested: (i) controlled slow protocol with seeding (CS+) or (ii) without seeding (CS-), (iii) controlled rapid protocol and (iv) non-controlled protocol. Cryodamage of seminiferous cords was semi-quantitatively determined, establishing a scoring of alterations. Cell viability and apoptosis induction were assessed on testicular cell suspensions immediately after digestion (D0) and after a 20-h culture period (D1). Cells recovered after digestion of 100 mg tissue and the rate of living and non-apoptotic cells were quantified at D0 and D1. A long-term culture (9 days) of testis pieces was carried out for the protocol offering the best survival. Testosterone production, intratubular cell proliferation and tubule growth were assessed.
  
  
  RESULTS
  DMSO produced optimal results in the different cooling rate curves tested when compared with PROH. Optimal results were obtained for the DCS- procedure (P < 0.05). Testosterone production, tubule growth and cell proliferation of post-thaw pieces were similar to fresh samples.
  
  
  CONCLUSIONS
  Testis freezing with 1.5 M DMSO in a CS- procedure was found to maintain not only immature testicular tissue architecture, but also viability of testicular cells, endocrine and partial exocrine functions of the testis. Semi-quantitative evaluation of seminiferous cord cryodamage can be effectively used to rapidly screen optimal freezing conditions and as a possible quality control in a human application.
authors.affiliation: Reproductive Biology Laboratory-CECOS, Rouen University Hospital, Institute for Biomedical Research, University of Rouen, 1 rue de Germont, 76031 Rouen Cedex, France.
authors.forename: J P
authors.initials: JP
authors.lastname: Milazzo
authors.forename: L
authors.initials: L
authors.lastname: Vaudreuil
authors.forename: B
authors.initials: B
authors.lastname: Cauliez
authors.forename: E
authors.initials: E
authors.lastname: Gruel
authors.forename: L
authors.initials: L
authors.lastname: Massé
authors.forename: N
authors.initials: N
authors.lastname: Mousset-Siméon
authors.forename: B
authors.initials: B
authors.lastname: Macé
authors.forename: N
authors.initials: N
authors.lastname: Rives
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D002452:Cell Count; D002470:Cell Survival; D015925:Cryopreservation; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D013737:Testis / Q000033:anatomy & histology / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D013739:Testosterone / Q000096:biosynthesis
chemical_list: D018809:Proliferating Cell Nuclear Antigen; D013739:Testosterone