Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes.
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 degrees C) or for 1-7 days at 4 degrees C before freeze-drying. Freeze-dried spermatozoa were maintained for up to 1 year at 4 degrees 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.
Kusakabe
H
H
Department of Biological Sciences, Asahikawa Medical College, 2-1-1-1 Midorigaoka-higashi, Asahikawa 078-8510, Japan. hkusa55@asahikawa-med.ac.jp
Yanagimachi
R
R
Kamiguchi
Y
Y
D000818:Animals; D001639:Bicarbonates; D001755:Blastocyst / Q000502:physiology; D002478:Cells, Cultured; D002871:Chromosome Banding; D033481:Chromosomes, Mammalian / Q000648:ultrastructure*; D004533:Egtazic Acid; D010064:Embryo Implantation; D004622:Embryo, Mammalian / Q000502:physiology; D047108:Embryonic Development; D005260:Female; D005612:Freeze Drying; D006801:Humans; D008297:Male; D051379:Mice; D009865:Oocytes; D020554:Sperm Injections, Intracytoplasmic; D013094:Spermatozoa / Q000648:ultrastructure*; D014325:Tromethamine
D001639:Bicarbonates; D014325:Tromethamine; C091201:Tribonate; D004533:Egtazic Acid