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5'-Nucleotidase is involved in sperm capacitation via the cAMP-adenosine pathway and in sperm motility via direct adenosine production from AMP. Since these functions are reduced in varicocele, the aim of this study was to investigate whether the enzyme levels were altered in sperm from varicocele patients. The mean (SD) international units (IU) of 5'-nucleotidase activity in seminal plasma from 35 varicocele III patients was 0.16(0.09) IU ml(-1) vs 0.35(0.13) IU ml(-1) in 53 controls, this decrease being statistically significant at p < or = 0.001. A significant decrease in activity, expressed as international units per mg of protein concentration in spermatozoa homogenates, was also observed with spermatozoa: 0.0018(0.0017) IU mg(-1) in varicocele III vs 0.0081(0.0060) IU mg(-1) in controls, at p < or = 0.001. Compared to controls, the activity decrease observed both in spermatozoa and seminal plasma from 45 men with varicocele I was not statistically significant at p < or = 0.05. To determine the diagnostic value of 5'-nucleotidase in assessing sperm fertility in varicocele III, we used the likelihood ratios method and best cut-offs were identified in receiver operating characteristic curves. With a prevalence of 36%, the post-test probability of infertility was 91% in spermatozoa and 78% in seminal plasma. The cut-off values of 5'-nucleotidase activity discriminating for fertile/unfertile semen were 0.2 IU ml(-1) in seminal plasma and 0.003 IU mg(-1) of protein in spermatozoa. Overall, determination of 5'-nucleotidase activity, especially in spermatozoa, can be useful to characterize different varicocele degrees as well as the sperm fertility potential.

Fourier transform infrared spectroscopy was used to investigate the secondary structure of boar sperm proacrosin at p2H 5.5, to determine the structural changes following protein autoactivation to beta-acrosin at p2H 8.0 and to study the effect of suramin binding on the protein secondary structure. At p2H 5.5, proacrosin contents of alpha-helix, beta-sheet, turns, and unordered structures were estimated to be 9, 49-51, 16-18, and 24%, respectively. At p2H 8.0, the protein was partially insoluble; spectral analysis of the soluble fraction, which contained beta-acrosin, showed an overall secondary structure quite similar to that of proacrosin at p2H 5.5. However, p2H 8.0 spectra of the soluble protein (beta-acrosin), together with the thermal denaturation experiments, indicated that, compared to proacrosin, beta-acrosin showed an increased content of beta-sheets exposed to the solvent as well as a different tertiary structure. Proacrosin/suramin interaction at p2H 5.5 resulted in the formation of soluble and insoluble complexes and the relevant infrared spectra showed only minor differences with respect to the native proacrosin. However, the thermal denaturation curves revealed that suramin induced a destabilization of proacrosin structure. The data also indicated that suramin could modify the interaction characteristics of proacrosin aspartyl and glutamyl residues, thus suggesting competition of suramin with these two residues for ionic interactions.

A polyclonal rabbit antibody against 5'-nucleotidase purified from bull seminal plasma was used to localize the antigen on bovine spermatozoa. Spermatozoa taken from the ampulla of the vas deferens showed strong immunofluorescence at the anterior rim of the head portion. Evaluation of spermatozoa prepared from different segments of the seminal pathway indicated the presence of the antigen already in rete testis and epididymal spermatozoa. On cryostat sections of testis tissue a positive immunoreaction was found in the anterior head portion of elongated spermatids, but not in earlier forms of sperm development. This distribution corresponded with the enzyme activity and results of Western blotting in extracts of testicular and epididymal spermatozoa. Immunoelectron microscopy of ampullary spermatozoa using antibody detection with gold-labelled anti-rabbit IgG showed a clear-cut labelling of the plasma membrane in the acrosome region. Treatment of ampullary spermatozoa with 0.1% Triton X-100 did not completely remove the immunoreactive material from the acrosome, showing a very stable linkage of the protein to the plasma membrane. Treatment with phospholipase C from Bacillus thuringiensis, however, removed immunoreactive material from the plasma membrane, indicating its binding by a phosphoinositol anchor. Our findings show that endogenous 5'-nucleotidase is present on the plasma membrane covering the anterior head portion of bovine spermatozoa and indicate specialized functions during the acrosomal reaction. Soluble enzyme derived from seminal vesicle secretion covers the whole sperm surface during emission, but is not covalently bound. It provides generalized enzyme activity to the sperm surface in addition to the specialized area of the sperm head.