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Relationship between fertilizing ability of frozen human spermatozoa and capacity for heparin binding and nuclear decondensation

Summary. Nuclear decondensation of spermatozoa induced by heparin, reduced glutathione (GSH) or a mixture of heparin and GSH was studied using frozenthawed human spermatozoa. The percentages of decondensed spermatozoa in controls and after treatment for 60 min with 30 µmol heparin l^–1, 5 mmol GSH l^–1, or heparin–GSH mixture were 1·5, 22·1, 4·3 and 37·6%, respectively. Most of the decondensed spermatozoa were eliminated by Percoll gradient centrifugation of samples previously treated with heparin or heparin–GSH mixture. However, comparable numbers of motile spermatozoa were recovered in the control and in each treated sample, demonstrating that a major proportion of motile spermatozoa was resistant to heparin (or heparin–GSH) effects on nuclear decondensation of spermatozoa.

Fertilization of hamster oocytes was attempted using spermatozoa recovered in the 90% Percoll fraction and resistant to heparin–GSH decondensing mixture. Although insemination used a constant number of motile spermatozoa, fertilization rates were higher after treatments with heparin and GSH alone than in control or heparin–GSH-treated samples. In addition the number of spermatozoa that attached to the oocyte plasma membrane was a sixth or a half for sperm pretreated with heparin–GSH or heparin alone, respectively compared with untreated values. However, there was no evidence for induced acrosomal reaction by heparin and GSH, at least at the concentrations used.

Qualitative analyses of heparin-binding sites were performed on untreated spermatozoa recovered in the 90% Percoll fraction by incubating spermatozoa in the presence of heparin covalently linked to albumin and coupled to colloidal gold (5 nm). Among this population of spermatozoa, 40·5% bound heparin–gold and labelling was mainly observed on the sperm head surface (88% of labelled spermatozoa) with (59·5%) or without (28·5%) tail labelling. Only a small proportion (23%) of spermatozoa that attached to the oocyte plasma membrane bound heparin–gold conjugate and only weak labelling was observed on the sperm head. Moreover, the proportion of spermatozoa that bound heparin–gold conjugate decreased (r = –0·77, P < 0·0001) in relation to increasing concentrations of motile spermatozoa in the sample.

Our results obtained with frozen–thawed spermatozoa show that (i) there is a close relationship between sperm motility and the inability of spermatozoa to bind heparin, (ii) a high decondensing capacity of human spermatozoa is not representative of a higher fertilizing competence, and (iii) most motile spermatozoa do not bind heparin on their membranes.

Keywords: heparin; glutathione; sperm nuclear decondensation; spermatozoa–egg interaction; human

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