Hum. Reprod. Advance Access published online on December 1, 2007
Human Reproduction, doi:10.1093/humrep/dem252
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Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes
1 Department of Biological Sciences, Asahikawa Medical College, 2-1-1-1 Midorigaoka-higashi, Asahikawa 078-8510, Japan 2 Institute for Biogenesis Research, University of Hawaii Medical School, 1960 East-West Road, Honolulu, Hawaii 96822, USA
3 Correspondence address. E-mail: hkusa55{at}asahikawa-med.ac.jp
BACKGROUND: Although mouse spermatozoa can be freeze-dried without losing their reproductive capacity, the technique needs further improvements to reduce the incidence of chromosomal damage to spermatozoa. Effects of freeze-drying on human spermatozoa are unknown.
METHODS: Mouse spermatozoa were suspended in a Tris-buffered EGTA solution briefly (10 min at 37°C) or for 1–7 days at 4°C before freeze-drying. Freeze-dried spermatozoa were maintained for up to 1 year at 4°C before injection. Sperm chromosomes were examined during the first mitosis (cleavage) of zygotes. The ability of sperm to support embryo development was assessed by examining mid-gestation fetuses (Day 14) after transfer of 2-cell embryos to surrogate mothers. Chromosome integrity of freeze-dried human spermatozoa was examined by injecting individual spermatozoa into mouse oocytes which were previously enucleated.
RESULTS: When mouse spermatozoa were freeze-dried immediately after suspension in Tris-buffered EGTA solution, only c.40% had normal chromosomes. When the mouse spermatozoa were kept in the same solution for 3–7 days before freeze-drying, 85–95% had normal chromosomes and they were able to support embryo development better than those which were in the solution briefly (P < 0.05). Freeze-dried human spermatozoa well maintained their chromosomes regardless of the duration of pre-freeze-drying incubation of spermatozoa in the Tris-buffered EGTA solution.
CONCLUSIONS: Prior incubation of mouse spermatozoa in Tris-buffered EGTA solution for several days makes sperm chromosomes more resistant to freeze-drying. As the consequence, spermatozoa freeze-dried this way support embryo development better than those exposed to Tris-buffered EGTA solution only briefly. Freeze-dried human spermatozoa well maintained their chromosomes without pre-freeze-drying incubation in Tris-buffered EGTA solution.
Key words: ICSI/freeze-drying/human sperm/mouse sperm/chromosome
Submitted on January 9, 2007; resubmitted on June 28, 2007; accepted on July 11, 2007.