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Studies were conducted to determine the efficiency of decondensation protocols. Sperm obtained from seven normal donors was immediately washed after liquefaction and then decondensed using the method of West or al. (1989) and my original protocol. My optimized protocol entailed mixing 1 ml aliquots of semen with 4 ml phosphate buffered saline (PBS) Following centrifugation, pellets were resuspended in 1 ml PBS containing 6 mM EDTA. After centrifugation, pellets were resuspended in 1 ml PBS containing 2 mM dithiothreitol at 37degrees C for 45 min. Following mixing with 2 ml PBS and centrifugation, pellets were resuspended by vortexing. While vortexing, 5 ml of fixative were gently added. Slide preparation was accomplished using the smear method and it was stored at 4degrees C. When comparing these protocols, the degree of sperm decondensation and head swelling was monitored by measuring nuclear length, area, perimeter, and degree of roundness using FISH analysis software. Apparent copy number for chromosome 1 and, separately, for the sex chromosomes was determined by FISH using satellite DNA probes for loci DIZ1, DXZ1 and DYZ3. Sperm treated by my decondensation protocol showed significant increases (p<0.05) in length, area, perimeter, and degree of roundness. There was a significant decrease (p<0.05) in the frequency of nuclei displaying no signal but no change in the frequency of nuclei with two signals in samples decondensed by my protocol.. My data suggested that decondensation using my original protocol may lower the frequency of cells with spurious 'nullisomy' due to hybridization failure without inducing spurious 'disomy' resulting from increased distances between split signals. |
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