EJACULATED AND EPIDIDYMAL MOUSE SPERMATOZOA ARE DIFFERENT IN THEIR SUSCEPTIBILITY TO NUCLEASE- DEPENDENT DNA DAMAGE AND NUCLEASE ACTIVITY

Abstract

Non-conventional sperm preservation methods, such as freeze-drying or freezing without cryoprotection are successful in preservation of epididymal mouse sperm. However, when ejaculated spermatozoa retrieved from the uteri were preserved in the same way, the proportion of fetuses after ICSI remained below 10%, much lower than obtained with epididymal sperm (up to 70%). Chromosome analysis of the zygotes produced with preserved ejaculated sperm revealed severe DNA damage in paternal complements. The major difference between isolated epididymal sperm and ejaculated sperm retrieved from the uteri is that the former are collected from the cauda epididymides as a dense sperm mass with minute amount of epididymal fluid while the latter are released from the uteri as a suspension consisting of ejaculate and other uterine components. We speculated that some factor/s present in the uterus after mating is responsible for the observed chromosome damage. To test this we exposed epididymal sperm to the content of the uterus from the female mated with a vasectomized male. When exposed sperm were injected into the oocytes, only 47% (43/76) of zygotes were chromosomally normal. When these sperm were frozen without cryoprotection prior to ICSI the damage was more severe (16%, 12/73 normal). There were no differences in the proportion of normal chromosomes after ICSI with non-exposed fresh and frozen sperm (83%, 66/80 vs. 79%, 54/68, P>0.05) indicating that rapid freezing per se does not impair chromosome integrity. When sperm exposed to the uterine content were subsequently washed and resuspended in a fresh medium prior to freezing and ICSI, sperm chromosome integrity was maintained (72–89% in all groups). To test which component of the uterine content caused paternal chromosome breaks we exposed epididymal sperm to either “pure uterine content” (the content of the uterus from a female in estrus) or to the content of the seminal vesicle, and freeze without cryoprotection. Sperm exposed to ‘pure uterine content’ yielded chromosomally normal zygotes (93%, 28/30). Sperm exposed to seminal vesicle resulted in pronuclei stage arrest in 62% (83/133) of the injected oocytes. Almost all (98%, 45/46) of the oocytes that developed farther contained broken paternal chromosomes, and the damage was extremely severe. Comet assay verified chromosome analysis results and confirmed lack of DNA damage in sperm exposed to ‘pure uterine content’ and severe damage in sperm exposed to seminal vesicle. When extracts prepared from epididymal and ejaculated sperm and their associated fluids were used to digest plasmid DNA, more prominent degradation was noted with ejaculated sperm. The results imply that there are intrinsic differences between the epididymal and ejaculated mouse sperm preparations in their susceptibility to nuclease-dependent DNA damage and in their nuclease activity. [This material is based on work supported by NIH HD048446 and HD048845 grants to MAW]. (platform)