Immunofluorescence study of actin, acrosin, dynein, tubulin and hyaluronidase and their impact on in-vitro fertilization
Using polyclonal antibodies, the distribution of actin, acrosin, dynein, tubulin and hyaluronidase has been examined by indirect immunofluorescence in sperm preparations from fertile donors and in-vitro fertilization (TVF) patients. After recording sperm parameters in native semen, spermatozoa were washed free of seminal plasma using either the swimup or the Percoll filtration technique. Prior to insemination, aliquots of the washed sperm suspensions were prepared for antibody staining.
... y staining. Spermatozoa from fertile donors were analysed in order to establish the specific fluorescence patterns of each antibody and the threshold scores of normality. Immunofluorescence scores obtained from FVF patients were then analysed with respect to FVF outcome. For each tested protein, the number of normal samples were significantly lower in the group which did not fertilize and fertilization rates were significantly reduced when any of the tested proteins were scored as pathological. Normal fluorescence scores were correlated with morphology, motflity, velocity and to a lesser extent with sperm concentration in native semen. On the basis of receiver-operating characteristic curves, likelihood ratios and Cohen's kappa values, the presence of acrosin and tubulin yields the most useful information on sperm functional and structural status and on its fertilizing ability.