Department of Biomedicine, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile.
Biol Reprod. 2011 Aug;85(2):357-66. doi: 10.1095/biolreprod.111.092254. Epub 2011 May 4.
The aim of this work was to determine whether laminin (Ln), an extracellular matrix protein, induces the intracellular events that may be involved in producing the acrosome reaction in human sperm. To this end, we evaluated the effect of Ln on tyrosine phosphorylation, intracellular calcium concentration, proteasome activity, and phosphorylation in human sperm. Aliquots of highly motile sperm selected with a Percoll gradient, were incubated with different concentrations of Ln (0-20 μg/ml) for different periods (0-18 h). The percentage of viable acrosome-reacted sperm was evaluated using fluorescein isothiocyanate-labeled Pisum sativum agglutinin and Hoechst 33258 DNA dye. Tyrosine phosphorylation was evaluated by Western blot analysis. The chymotrypsin-like activity of the proteasome was evaluated with a fluorogenic peptide, and intracellular calcium concentration was measured with fura-2. The results indicate that Ln stimulated the acrosome reaction of human sperm in a dose-dependent manner. This increase was drastically inhibited in the presence of herbimycin A, SU6656, and epoxomicin. In addition, Ln increased proteasome activity and phosphorylation; both events were inhibited by herbimycin A and SU6656. Finally, Ln induced an increase in intracellular calcium concentration, which was inhibited by SU6656 and epoxomicin. These results suggest that Ln is able to induce the acrosome reaction. This effect may be mediated by Src kinase and the proteasome, with the consequent induction of a calcium influx.
本研究旨在确定细胞外基质蛋白层粘连蛋白（Ln）是否诱导人精子顶体反应中可能涉及的细胞内事件。为此，我们评估了 Ln 对人精子酪氨酸磷酸化、细胞内钙离子浓度、蛋白酶体活性和磷酸化的影响。用 Percoll 梯度分离出高活力的精子，用不同浓度的 Ln（0-20μg/ml）孵育不同时间（0-18 小时）。用异硫氰酸荧光素标记的豌豆凝集素和 Hoechst 33258 DNA 染料评估活顶体反应精子的比例。通过 Western blot 分析评估酪氨酸磷酸化。用荧光肽评估蛋白酶体的糜蛋白酶样活性，用 fura-2 测量细胞内钙离子浓度。结果表明，Ln 以剂量依赖的方式刺激人精子的顶体反应。在存在 herbimycin A、SU6656 和 epoxomicin 的情况下，这种增加被明显抑制。此外，Ln 增加了蛋白酶体活性和磷酸化；这两种事件都被 herbimycin A 和 SU6656 抑制。最后，Ln 诱导细胞内钙离子浓度增加，该增加被 SU6656 和 epoxomicin 抑制。这些结果表明 Ln 能够诱导顶体反应。这种作用可能通过 Src 激酶和蛋白酶体介导，随后诱导钙离子内流。
