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.

[1]  E. Baldi,et al.  Tyrosine Phosphorylation of the A Kinase Anchoring Protein 3 (AKAP3) and Soluble Adenylate Cyclase Are Involved in the Increase of Human Sperm Motility by Bicarbonate1 , 2005, Biology of reproduction.

[2]  M. Ensslin,et al.  Mammalian fertilization , 2004, Current Biology.

[3]  E. Baldi,et al.  Increased phosphorylation of AKAP by inhibition of phosphatidylinositol 3-kinase enhances human sperm motility through tail recruitment of protein kinase A , 2004, Journal of Cell Science.

[4]  E. Gratton,et al.  Membrane lipid domains and dynamics as detected by Laurdan fluorescence , 1995, Journal of Fluorescence.

[5]  G. Doncel,et al.  Human sperm subpopulations: relationship between functional quality and protein tyrosine phosphorylation. , 2004, Human reproduction.

[6]  G. Doncel,et al.  Superoxide dismutase content and fatty acid composition in subsets of human spermatozoa from normozoospermic, asthenozoospermic, and polyzoospermic semen samples , 2003, Molecular reproduction and development.

[7]  M. Bajpai,et al.  Involvement of tyrosine kinase and cAMP-dependent kinase cross-talk in the regulation of human sperm motility. , 2003, Reproduction.

[8]  A. Campana,et al.  Localization of Tyrosine Phosphorylated Proteins in Human Sperm and Relation to Capacitation and Zona Pellucida Binding1 , 2003, Biology of reproduction.

[9]  G. Doncel,et al.  Incidence of sperm-tail tyrosine phosphorylation and hyperactivated motility in normozoospermic and asthenozoospermic human sperm samples. , 2003, Biocell : official journal of the Sociedades Latinoamericanas de Microscopia Electronica ... et. al.

[10]  M. Bajpai,et al.  EFFECT OF TYROSINE KINASE INHIBITORS ON TYROSINE PHOSPHORYLATION AND MOTILITY PARAMETERS IN HUMAN SPERM , 2003, Archives of andrology.

[11]  J. D. Bell,et al.  Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order. , 2002, Biochimica et biophysica acta.

[12]  Ignacio A. Demarco,et al.  Novel signaling pathways involved in sperm acquisition of fertilizing capacity. , 2002, Journal of reproductive immunology.

[13]  G. Arnaldi,et al.  Laurdan fluorescence: a simple method to evaluate sperm plasma membrane alterations. , 2001, Fertility and sterility.

[14]  E. Baldi,et al.  Phosphatidylinositol 3-kinase inhibition enhances human sperm motility. , 2001, Human reproduction.

[15]  C. V. D. van de Lest,et al.  Capacitation dependent activation of tyrosine phosphorylation generates two sperm head plasma membrane proteins with high primary binding affinity for the zona pellucida , 2001, Molecular reproduction and development.

[16]  B. Gadella,et al.  The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane. , 2000, Development.

[17]  A. Travis,et al.  Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation. , 1999, Molecular human reproduction.

[18]  G. Kopf,et al.  Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation. , 1999, Developmental biology.

[19]  B. Gadella,et al.  Dynamics in the membrane organization of the mammalian sperm cell and functionality in fertilization. , 1999, The Veterinary quarterly.

[20]  G. Weinbauer,et al.  Responses of monkey epididymal sperm of different maturational status to second messengers mediating protein tyrosine phosphorylation, acrosome reaction, and motility , 1999, Molecular reproduction and development.

[21]  M. Okuno,et al.  Role of tyrosine phosphorylation of flagellar proteins in hamster sperm hyperactivation. , 1999, Biology of reproduction.

[22]  G. Kopf,et al.  Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm. , 1999, Biology of reproduction.

[23]  M. Mahony,et al.  Modulation of sperm tail protein tyrosine phosphorylation by pentoxifylline and its correlation with hyperactivated motility. , 1999, Fertility and sterility.

[24]  B. Pukazhenthi,et al.  Regulation of sperm function by protein tyrosine phosphorylation in diverse wild felid species. , 1998, Journal of andrology.

[25]  M. Leisner,et al.  Ultrastructural pathology of the sperm flagellum: association between flagellar pathology and fertility prognosis in severely asthenozoospermic men. , 1998, Human reproduction.

[26]  S. Mortimer,et al.  Effect of seminal plasma on capacitation and hyperactivation in human spermatozoa. , 1998, Human reproduction.

[27]  N. Cross,et al.  Role of cholesterol in sperm capacitation. , 1998, Biology of reproduction.

[28]  G. Kopf,et al.  Regulation of protein phosphorylation during sperm capacitation. , 1998, Biology of reproduction.

[29]  R. Aitken,et al.  A novel signal transduction cascade in capacitating human spermatozoa characterised by a redox-regulated, cAMP-mediated induction of tyrosine phosphorylation. , 1998, Journal of cell science.

[30]  S. Vijayaraghavan,et al.  Protein Kinase A-anchoring Inhibitor Peptides Arrest Mammalian Sperm Motility* , 1997, The Journal of Biological Chemistry.

[31]  G. Gerton,et al.  Regulation of protein tyrosine phosphorylation in human sperm by a calcium/calmodulin-dependent mechanism: identification of A kinase anchor proteins as major substrates for tyrosine phosphorylation. , 1996, Developmental biology.

[32]  C. Gagnon,et al.  Cyclic adenosine 3',5'monophosphate-dependent regulation of protein tyrosine phosphorylation in relation to human sperm capacitation and motility. , 1996, Biology of reproduction.

[33]  G S Kopf,et al.  Capacitation of mouse spermatozoa. II. Protein tyrosine phosphorylation and capacitation are regulated by a cAMP-dependent pathway. , 1995, Development.

[34]  G. Kopf,et al.  Capacitation of mouse spermatozoa. I. Correlation between the capacitation state and protein tyrosine phosphorylation. , 1995, Development.

[35]  D. Buckingham,et al.  Relationships between biochemical markers for residual sperm cytoplasm, reactive oxygen species generation, and the presence of leukocytes and precursor germ cells in human sperm suspensions , 1994, Molecular reproduction and development.

[36]  M. A. Swan,et al.  Effects of the lipid peroxidation product (E)-4-hydroxy-2-nonenal on ram sperm function. , 1993, Journal of reproduction and fertility.

[37]  R. Aitken,et al.  Analysis of lipid peroxidation mechanisms in human spermatozoa , 1993, Molecular reproduction and development.

[38]  J. Overstreet,et al.  Glycoconjugates of the human sperm surface: distribution and alterations that accompany capacitation in vitro. , 1987, Gamete research.

[39]  C. Lombard,et al.  Sperm morphologic features as a prognostic factor in in vitro fertilization. , 1986, Fertility and sterility.

[40]  L J Burkman,et al.  Characterization of hyperactivated motility by human spermatozoa during capacitation: comparison of fertile and oligozoospermic sperm populations. , 1984, Archives of andrology.

[41]  M. M. Lees,et al.  An analysis of sperm function in cases of unexplained infertility: conventional criteria, movement characteristics, and fertilizing capacity. , 1982, Fertility and sterility.

[42]  B. K. Davis.  Timing of fertilization in mammals: sperm cholesterol/phospholipid ratio as a determinant of the capacitation interval. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[43]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.