Superoxide dismutase content in sperm correlates with motility recovery after thawing of cryopreserved human spermatozoa.

OBJECTIVE: To investigate the correlation between sperm superoxide dismutase (SOD) content and motility recovery after thawing of cryopreserved human sperm, based on the rationale that this antioxidant enzyme provides protection against reactive oxygen species-induced damage during cryopreservation. DESIGN: Prospective study. SETTING: Private infertility institute and university-based research laboratory. PATIENT(S): Forty-two consenting normozoospermic patients consulting for infertility. INTERVENTION(S): The SOD content was measured in sperm from unfractionated samples and in sperm recovered from the pellet fraction obtained after discontinuous density gradient centrifugation. MAIN OUTCOME MEASURE(S): Sperm motility was evaluated post-thaw in the two sets of samples and motility recovery was plotted against the sperm SOD content to determine their correlation. RESULT(S): There was a significant positive correlation between motility recovery after thawing and SOD content in sperm from the 90% gradient pellet containing highly purified mature sperm. There was also a significant negative correlation between motility after thawing and SOD content in the unfractionated sample. CONCLUSION(S): The positive correlation between post-thaw motility recovery and SOD content in mature spermatozoa provides a good predictor of post-thaw motility recovery after cryopreservation.

Effect of thawing temperature on the motility recovery of cryopreserved human spermatozoa.

OBJECTIVE: To investigate the effects of thawing temperature on sperm function after cryopreservation. The technical aspects of sperm cryopreservation have significantly improved over the last few decades. However, a standard protocol designed to optimize sperm motility recovery after thawing has not yet been established. DESIGN: Prospective study. SETTING: Private infertility institute and university-based research laboratory. PATIENT(S): Eighty consenting normozoospermic patients consulting for infertility. INTERVENTION(S): Spermatozoa from donor semen samples were thawed at different temperatures. MAIN OUTCOME MEASURE(S): Sperm motility, viability, adenosine-5'-triphosphate (ATP) content, acrosomal status, and DNA integrity were evaluated as a function of thawing temperature in cryopreserved human sperm samples. RESULT(S): Thawing at 40 degrees C resulted in a statistically significant increase in sperm motility recovery compared with thawing at temperatures between 20 degrees C and 37 degrees C. There were no statistically significant differences in sperm viability, acrosomal status, ATP content, and DNA integrity after thawing at 40 degrees C compared with thawing at temperatures between 20 degrees C and 37 degrees C. CONCLUSION(S): Sperm thawing at 40 degrees C could be safely used to improve motility recovery after sperm cryopreservation.

High cholesterol content and decreased membrane fluidity in human spermatozoa are associated with protein tyrosine phosphorylation and functional deficiencies.

Poor-quality sperm show reduced capacity to undergo capacitation-induced protein tyrosine phosphorylation and hyperactivation. Given that these deficiencies can be overcome by membrane-permeant stimulators of the cAMP-dependent kinase system, we hypothesize that the main defect underlying these deficiencies resides on the sperm plasma membrane. Spermatozoa from semen samples obtained from 15 consenting healthy donors were separated in 2 subpopulations, L45 (first interface) and L90 (pellet), using a 45:65:90 ISolate gradient centrifugation method. These sperm fractions were studied before and after a 6-hour capacitating incubation for sperm motion parameters (computer-assisted analysis), including hyperactivation, protein tyrosine phosphorylation (immunofluorescence), membrane fluidity (Laurdan fluorescence), and sterol and phospholipid content (high-performance thin-layer chromatography). In summary, data indicate that L45 (poor-motility) spermatozoa present an excess of cholesterol and desmosterol, which impairs the normal increase in membrane fluidity during capacitation and its consequent activation of protein tyrosine phosphorylation and hypermotility. Therefore, a defect in membrane composition and dynamics is underlying human sperm biochemical and functional deficiencies related to inadequate capacitation.

Capacitation-associated changes in membrane fluidity in asthenozoospermic human spermatozoa.

The fertilizing potential of human spermatozoa relies on their ability to capacitate as they travel through the female reproductive tract. During this process, cholesterol is released from the plasma membrane, altering its architecture and dynamics. Using ISolate gradients, we obtained high (L90)- and low (L45)-quality spermatozoa from asthenozoospermic human semen samples. We tested the hypothesis that the lower fertilizing ability of asthenozoospermic L90 cells could be related to a lower ability to increase their membrane fluidity during capacitation. We assessed two sets of fluorescent probes: (i) DPH, TMA-DPH and PA-DPH which senses the hydrophobic core, cytosolic and exofacial leaflets of the bilayer, respectively and (ii) Laurdan, sensitive to the amount of water molecules intercalated between lipid moieties of the membrane (membrane hydration). Before capacitation, membrane fluidity of asthenozoospermic sperm populations was similar to the corresponding fractions of normozoospermic cells when evaluated with DPH, TMA-DPH or PA-DPH. Asthenozoospermic whole samples displayed lower plasma membrane hydration than normozoospermic cells as evidenced with Laurdan. After capacitation, asthenozoospermic L45 and L90 cells failed to increase their membrane fluidity in opposition to normozoospermic cells. Interestingly, membrane hydration significantly correlated with the main sperm motion parameters analysed, being a low membrane hydration associated with poor sperm movement. These results show that low-motility spermatozoa are unable to respond to capacitation with the necessary changes in membrane fluidity. This defect in sperm plasma membrane rheology may be responsible for their poor functional quality and low fertilizing ability.

Capacitation-associated protein tyrosine phosphorylation and membrane fluidity changes are impaired in the spermatozoa of asthenozoospermic patients.

Sperm protein tyrosine phosphorylation has been associated with capacitation, motility changes, zona binding, and fertilizing ability. We previously demonstrated that gradient-isolated human sperm subpopulations differ in their plasma membrane composition, their ability to phosphorylate proteins in tyrosine residues, and their capacity to undergo hyperactivation. In this study, we have characterized capacitation-associated changes in protein tyrosine phosphorylation and membrane fluidity in spermatozoa of asthenozoospermic and normozoospermic patients consulting for infertility. Semen samples were studied at baseline and after a capacitating incubation with or without the addition of a permeable cAMP analog and a phosphodiesterase inhibitor. Basic sperm and computer-assisted motion parameters, hyperactivation, protein tyrosine phosphorylation (immunofluorescence and Western blot), and membrane fluidity (fluorescent Laurdan probe) were the main study parameters. In comparison with normozoospermic and proven-fertile donor semen, asthenozoospermic samples showed lower motility, velocity, and amplitude of lateral head displacement, both originally and after a 6-h capacitating incubation. Unlike those in normal samples, asthenozoospermic spermatozoa were unable to increase protein tyrosine phosphorylation during capacitation. Such impairment, however, was overcome when they were incubated with a membrane-permeable cAMP analog and a phosphodiesterase inhibitor, indicating a possible membrane defect. Confirming this hypothesis, plasma membranes of asthenozoospermic sperm showed decreased fluidity (increased Laurdan polarization), even after a capacitating incubation. In conclusion, spermatozoa from functional asthenozoospermic samples may owe their poor motility, and their inability to properly capacitate and develop hyperactivation, to an impairment in the tyrosine phosphorylation of critical proteins caused by decreased membrane fluidity. These findings suggest a molecular pathogenetic mechanism for a common seminal pathology associated with male infertility.

Calcium requirements for human sperm function in vitro.

OBJECTIVE: To determine extracellular calcium (Ca(2+)) requirements for the maintenance of human sperm function in vitro. DESIGN: Prospective study. SETTING: Basic research laboratory. PATIENT(S): Normozoospermic volunteers provided fresh semen samples; follicular fluid (human FF) and oocytes were collected from women undergoing IVF-ET. INTERVENTION(S): Spermatozoa were incubated for /=0.1 mM of Ca(2+) were able to undergo the AR when exposed to human FF in the presence of 2.5 mM of Ca(2+). Calcium concentrations of >/=0.22 mM were sufficient to reach protein tyrosine phosphorylation levels and hyperactivated motility values similar to those of controls. Higher Ca(2+) concentrations (>/=0.58 mM) were required to produce maximum human FF-induced AR in previously capacitated cells and to obtain an adequate sperm-ZP binding. CONCLUSION(S): Different steps of the fertilization process have distinctive Ca(2+) requirements. Whereas 0.22 mM of Ca(2+) is sufficient for the development of some capacitation-related events, human FF-induced AR and sperm-ZP interaction require 0.58 mM of this cation.