PDF《spermatozoa》

REVIEW ARTICLE Chromosomal integrity and DNA damage in freeze-dried spermatozoa Hirokazu Kusakabe Received:

10 April

2011 / Accepted:

12 May

2011 / Published online:

1 June

2011 ? Japan Society for Reproductive Medicine

2011 Abstract Freeze-drying technology may one day be used to preserve mammalian spermatozoa inde?nitely without cryopreservation.

Freeze-dried mouse spermatozoa stored below 4°C for up to

1 year have maintained the ability to fertilize oocytes and support normal development. The maximum storage period for spermatozoa increases at lower storage temperatures. Freeze-drying, per se, may reduce the integrity of chromosomes in freeze-dried mouse spermatozoa, but induction of chromosomal damage is suppressed if spermatozoa are incubated with divalent cation chelating agents prior to freeze-drying. Neverthe- less, chromosomal damage does accumulate in spermato- zoa stored at temperatures above 4°C. Currently, no established methods or strategies can prevent or reduce damage accumulation, and damage accumulation during storage is a serious obstacle to advances in freeze-drying technology. Chromosomal integrity of freeze-dried human spermatozoa have roughly background levels of chromo- somal damage after storage at 4°C for

1 month, but whe- ther these spermatozoa can produce healthy newborns is unknown. The safety of using freeze-dried human sper- matozoa must be evaluated based on the risks of heritable chromosome and DNA damage that accumulates during storage. Keywords Chromosome ? Cryopreservation ? DNA damage ? Freeze-drying ? Spermatozoa Introduction Cryopreservation with liquid nitrogen and storage at very low temperatures (-80°C) are used for many cell and tissue types and many purposes in a wide range of biological ?elds because these samples maintain many important characters and their genetic material is largely unaltered during cryo- preservation and cryostorage. Nevertheless, sperm preser- vation methods that do not require liquid nitrogen-based cryopreservation are needed for the following reasons. (1) Liquid nitrogen is not readily available in many countries and places (e.g., many developing countries, the Paci?c Islands, space stations). (2) These cryopreserved samples are often destroyed or damaged because low-temperature stor- age facilities fail due to human errors or loss of power. (3) These samples may be contaminated by pathogenic viruses that are stored in the same cryostorage facilities [1]. Although incidents of cross-contamination are rare in cryo- banks, it is dif?cult to make sure that it has not occurred yet [2]. For these reasons, advances in sperm preservation techniques that do not require liquid nitrogen or deep-freezer storage may contribute to the safe preservation of the gen- ome resourcesof mammalian species.(4) Potentially, freeze- dried spermatozoa may be transported anywhere without any refrigerants, such as dry ice [3, 4]. This review focuses on freeze-drying of mammalian spermatozoa, and particularly mouse and human sperma- tozoa. Recent progress and persistent problems associated with the methods used to maintain the integrity of DNA and chromosomes of the freeze-dried spermatozoa are discussed. H. Kusakabe (&

) Department of Biological Sciences, Asahikawa Medical University, 2-1-1-1 Midorigaoka-higashi, Asahikawa 078-8510, Japan e-mail: hkusa55@asahikawa-med.ac.jp

123 Reprod Med Biol (2011) 10:199C210 DOI 10.1007/s12522-011-0092-7 Participation of motionless spermatozoa in fertilization A method for freeze-drying spermatozoa was published approximately a half century ago. More recently, Polge et al. [5] reported that the majority of freeze-dried fowl spermatozoa were motile after rehydration, and in 1976, Larson and Graham [6] reported that some freeze-dried bull spermatozoa were motile after rehydration. Moreover, even motionless spermatozoa can fertilize oocytes and support normal development with advances in intracyto- plasmic sperm injection (ICSI) [7]. These advances in ICSI led us to consider simple methods for mammalian sper- matozoa preservation that do not require cryoprotectants [8] and the retrieval of sperm from frozen cadavers [9, 10]. Freeze-dried spermatozoa need not be motile after rehy- dration;