Hum. Reprod. Advance Access originally published online on November 6, 2007
Human Reproduction 2008 23(1):17-28; doi:10.1093/humrep/dem355
Comparison of conditions for cryopreservation of testicular tissue from immature mice
1 Reproductive Biology Laboratory—CECOS, Rouen University Hospital, Institute for Biomedical Research, University of Rouen, 1 rue de Germont, 76031 Rouen Cedex, France 2 Reproductive Biology Laboratory—CECOS, Rouen University Hospital & CIC Inserm 0204, Institute for Biomedical Research, University of Rouen, 76031 Rouen Cedex, France 3 Biochemistry Laboratory, Rouen University Hospital, Institute for Biomedical Research, University of Rouen, 76031 Rouen Cedex, France
4 Correspondence address. Tel: +2-32-888225; Fax: +2-35-982007; E-mail: nathalie.rives{at}chu-rouen.fr
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.
Key words: cryopreservation/male germ cell/immature testicular tissue
Submitted on February 28, 2007; resubmitted on September 21, 2007; accepted on October 8, 2007.
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