Buffone Mariano G, Calamera Juan C, Verstraeten Sandra V, Doncel Gustavo F
Laboratorio de Estudios en Reproducción (LER), Buenos Aires, Argentina.
Reproduction. 2005 Jun;129(6):697-705. doi: 10.1530/rep.1.00584.
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.
精子蛋白酪氨酸磷酸化与获能、运动变化、透明带结合及受精能力有关。我们之前证明，梯度分离的人类精子亚群在质膜组成、酪氨酸残基蛋白磷酸化能力以及超活化能力方面存在差异。在本研究中，我们对因不育前来咨询的弱精子症患者和正常精子症患者精子中与获能相关的蛋白酪氨酸磷酸化和膜流动性变化进行了表征。在基线时以及在添加或不添加可渗透的环磷酸腺苷（cAMP）类似物和磷酸二酯酶抑制剂的获能孵育后，对精液样本进行研究。基本精子参数和计算机辅助运动参数、超活化、蛋白酪氨酸磷酸化（免疫荧光和蛋白质印迹法）以及膜流动性（荧光劳丹探针）是主要研究参数。与正常精子症和已证实有生育能力的供体精液相比，弱精子症样本最初以及在6小时获能孵育后，其活力、速度和头部侧向位移幅度均较低。与正常样本不同，弱精子症精子在获能过程中无法增加蛋白酪氨酸磷酸化。然而，当它们与膜可渗透的cAMP类似物和磷酸二酯酶抑制剂一起孵育时，这种损伤得以克服，这表明可能存在膜缺陷。证实这一假设的是，即使在获能孵育后，弱精子症精子的质膜流动性仍降低（劳丹极化增加）。总之，功能性弱精子症样本中的精子活力差、无法正常获能和产生超活化，可能是由于膜流动性降低导致关键蛋白酪氨酸磷酸化受损所致。这些发现提示了一种与男性不育相关的常见精液病理学的分子发病机制。
