Laboratorio de Estudios Reproductivos, Area of Clinical Biochemistry, School of Biochemical and Pharmaceutical Sciences, National University of Rosario, Suipacha 531, 2000 Rosario, Argentina.
Hum Reprod. 2010 Jun;25(6):1504-12. doi: 10.1093/humrep/deq063. Epub 2010 Mar 25.
Spermatozoa acquire the ability to fertilize an oocyte when they become capacitated. Capacitation takes place when sperm pass through the female reproductive tract, interacting with female fluids. Both tyrosine phosphorylation of sperm proteins and the ability to respond to acrosome reaction (AR) inducers have been associated with sperm capacitation. Recent data indicate that conditioned media (CM) from human oviductal tissue culture decrease sperm affinity for the zona pellucida in vitro. Since capacitation enables the sperm-oocyte interaction, the aim of the present study was to investigate the effect of CM on events related to sperm capacitation and to assess whether these effects were permanent.
Oviductal tissue was obtained from premenopausal patients (scheduled for hysterectomies because of uterine fibromyoma). The tissues were cultured as explants and CM were collected. Explant viability was assessed as tissue DNA integrity. Normozoospermic semen samples were obtained from healthy donors. Motile spermatozoa were incubated under capacitating conditions with or without increasing protein concentrations of CM for 6 or 22 h. Human follicular fluid-induced AR was detected by the Pisum sativum technique. Tyrosine phosphorylated proteins were detected with a monoclonal anti-phosphotyrosine antibody.
The incubation of spermatozoa in the presence of increasing concentrations of conditioned medium (CM) proteins caused a dose-dependent decrease in both tyrosine phosphorylation of sperm proteins and in the level of AR induction. When CM was removed from the sperm incubation media, the effects were reversed. Heat-inactivated CM did not affect either tyrosine phosphorylation or the induction of AR.
The present data suggest that proteins secreted from human oviductal tissue are able to inhibit events associated with sperm capacitation in vitro.
精子在获能时获得受精卵子的能力。当精子通过女性生殖道并与女性体液相互作用时，就会发生获能。精子蛋白的酪氨酸磷酸化和对顶体反应（AR）诱导剂的反应能力都与精子获能有关。最近的数据表明，人输卵管组织培养的条件培养基（CM）可降低精子在体外对透明带的亲和力。由于获能使精子-卵子相互作用成为可能，因此本研究的目的是研究 CM 对与精子获能相关的事件的影响，并评估这些影响是否是永久性的。
从绝经前患者（因子宫肌瘤而行子宫切除术）获得输卵管组织。将组织作为外植体培养并收集 CM。通过组织 DNA 完整性评估外植体的活力。从健康供体中获得正常形态的精液样本。在有或没有增加 CM 蛋白浓度的情况下，将活动精子在获能条件下孵育 6 或 22 小时。通过豌豆 sativum 技术检测人卵泡液诱导的 AR。用单克隆抗磷酸酪氨酸抗体检测酪氨酸磷酸化蛋白。
在含有逐渐增加浓度的条件培养基（CM）蛋白的精子孵育液中孵育精子，会导致精子蛋白的酪氨酸磷酸化和 AR 诱导水平呈剂量依赖性下降。当 CM 从精子孵育液中去除时，效果会逆转。热失活的 CM 不会影响酪氨酸磷酸化或 AR 的诱导。
本研究数据表明，人输卵管组织分泌的蛋白质能够抑制体外与精子获能相关的事件。
