| Abstrakt: |
Despite the large numbers of spermatozoa present in an ejaculate, only minorities are able to meet the stringent requirements needed to fertilise an oocyte. It has been reported that DNA damage of sperm does not impair in vitro fertilization of the oocyte, but the embryonic development is highly influenced by the degree of DNA damage. To our knowledge it has not been determined if the selection processes for mammalian fertilization in vivo may screen and somehow determine the physical quality of DNA. In order to analyze if there are some in vivo selection mechanism for intact DNA spermatozoa, we have analyzed, using COMET assay, the level of DNA damage in spermatozoa recovered from epididymal caudae of CD1 mice, from the uterine horns and from oviducts of CD1 females 2 hours after mating. Spermatozoa were mixed with low melting agarose, dropped onto a slide, incubated at room temeprature for 60 min in lysis buffer (2,5M NaCl, 100 mM EDTA, 10 mM Tris-HCl, 40 mM Dithiotreitol, 2% Triton X-100). Then, proteinase K was added (200 mcg/ml) and samples were further incubated at 37°C for 240 min. After three washes, electrophoresis was performed by submerging slides in a horizontal gel electrophoresis tank under 0,5% of TBE buffer and a current of 25V, 300mA, 20 min. For each sample, 100–200 cells were analyzed using a COMET analysis software. The percentage of tail DNA and the COMET moment was calculated for each cell. One way ANOVA was used to compare the results form each group. DNA damage in spermatozoa recovered from the uterus showed a significant increase (Tail DNA 35.35%; P<0.001, Tail Moment 31.03%, p<0.05) compared with the sperm obtained from the epididymal caudae (Tail DNA 21.81%, Tail Moment 12.65%, p<0.05). COMET values decrease in the spermatozoa recovered from the oviduct (Tail DNA 22.03%; P<0.001, Tail Moment 7.35%, p<0.05) compared with the sperm obtained from the uterus. Our result could be explained either by the existence of two sub-populations of ejaculated sperm in mice or by the existence of an in vivo selection mechanism for spermatozoa without DNA damage. The in vitro identification of these competent cells would become ideal for the selection of spermatozoa in assisted reproductive technologies. (platform) |
