E. de Lamirande

CAPACITATION-ASSOCIATED PRODUCTION OF SUPEROXIDE ANION BY HUMAN SPERMATOZOA

Previous results demonstrating the role of the superoxide anion in human sperm capacitation indirectly suggest that the superoxide anion is not only essential to this process, but is also generated by spermatozoa themselves. Our aim was to provide direct evidence for production of superoxide anion by capacitating spermatozoa. Three chemiluminescence probes were tested to detect the superoxide anion. Luminol- and lucigenin-amplified chemiluminescence of capacitating spermatozoa did not differ from that of background level. However, significant SOD-inhibitable chemiluminescence was observed when the Cypridina luciferin analog, MCLA, was used as probe. The chemiluminescence associated to capacitation of spermatozoa incubated in Hams F-10 supplemented with fetal cord serum ultrafiltrate was 1270 +/- 77 mV/10 s (with 8 x 10(6) cells/ml; n = 37), and corresponded to levels of sperm hyperactivation (12 +/- 1%) and capacitation (17 +/- 2%) that were significantly different from those of control spermatozoa (4.9 +/- 0.8% and 6 +/- 1%, respectively). MCLA was cell impermeant and superoxide specific. The level of capacitation-associated chemiluminescence was directly related to sperm concentration up to 30 x 10(6) cells/ml. Sperm production of superoxide anion started at the beginning of incubation in capacitating conditions and was sustained over more than 4 h. This is the first direct evidence for the generation, by human spermatozoa, of a sustained level of superoxide anion that is associated with the progressive development of hyperactivation and capacitation.

MORPHOLOGICAL AND BIOCHEMICAL CHANGES FOLLOWING ACUTE UNILATERAL TESTICULAR TORSION IN PREPUBERTAL RATS

PURPOSE The role played by reactive oxygen species in the effects of testicular torsion and torsion/detorsion on ipsilateral and contralateral testis was investigated. MATERIALS AND METHODS Prepubertal rats were submitted to unilateral testicular torsion alone or followed by detorsion for up to 1 week. Morphology and biochemical parameters (thiobarbituric reactive substances, superoxide dismutase-like and catalase-like activities) were evaluated. RESULTS Torsion (1 or 2 h) alone induced time-dependent morphological damages that worsened progressively after detorsion in ipsilateral testis but had no effect on contralateral testis. The levels of antioxidants against the superoxide anion and hydrogen peroxide decreased in ipsilateral but not in contralateral testis. However, the level of thiobarbituric reactive substances (an indicator of lipid perioxidation) decreased after testicular torsion or torsion/detorsion in both testes. CONCLUSION These data emphasize that oxidative stress may play a role in testicular damage caused by torsion/detorsion and that biochemical indicators of oxidative stress are more sensitive than histological techniques in detecting modifications in the contralateral testis.

Levels of semenogelin in human spermatozoa decrease during capacitation: involvement of reactive oxygen species and zinc

BACKGROUND Semenogelin (Sg), the main protein of human semen coagulum, prevents sperm capacitation. The objective of this study was to examine the role of Sg and its mechanism of action. METHODS AND RESULTS Sg blocked sperm capacitation triggered by various stimuli, via inhibition of superoxide anion (O(2)*-; luminescence assay) and nitric oxide (NO*; tested using diaminofluorescein) generation. Triton-soluble and -insoluble sperm fractions contained Sg and Sg peptides (immunoblotting), the level of which decreased with initiation of capacitation. This drop was prevented by superoxide dismutase and NO* synthase inhibitor and was reproduced by addition of O(2)*- and NO*. Zinc (Zn(2+)) blocked and a zinc chelator (TPEN) promoted the decline in Sg levels. There was a decreased labelling of Sg on the head in capacitating spermatozoa with the two fixation techniques tested (immunocytochemistry). Reactive oxygen species (ROS) (O(2)*- and NO*) caused, these changes, and zinc prevented them. Spermatozoa quickly internalized Sg upon incubation and Sg was then rapidly degraded in a zinc-inhibitable manner. CONCLUSIONS Sg blocked capacitation mainly via inhibition of ROS generation. Spermatozoa appeared permeable to Sg and processed Sg in a zinc-inhibitable fashion. ROS themselves could promote sperm disposal of Sg which maybe one of the mechanisms that allows initiation of capacitation.