Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes
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This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Human Reproduction following peer review. The definitive publisher-authenticated version Kusakabe, H. ; Yanagimachi, R. ; Kamiguchi, Y., Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes, Human Reproduction 23(2), FEB 2008, pp. 233-239, is available online at: http://humrep.oxfordjournals.org/cgi/content/abstract/23/2/233authorBACKGROUND: Although mouse spermatozoa can be freeze-dried without losing their reproductive capacity, the technique needs further improvements to reduce the incidence of chromosomal damages to spermatozoa. Effects of freeze-drying on human spermatozoa are unknown. METHODS: Mouse spermatozoa were suspended in a Tris-buffered EGTA solution briefly (about 10 min at 37℃) or for 1-7 days at 4℃ before freeze-drying. Freeze-dried spermatozoa were maintained for up to 1 year at 4℃ before injection. Sperm chromosomes were examined during the first mitosis (cleavage) of zygotes. Sperm’s ability to support embryo development was assessed by examining mid-gestation fetuses 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 about 40% had normal chromosomes. When the 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. 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.
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