Evidence for the Involvement of Calmodulin in Mouse Sperm Capacitation1
Evidence for the Involvement of Calmodulin in Mouse Sperm Capacitation1
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DOI：
10.1095/biolreprod62.5.1231
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发表时间：
2000-05
期刊：
影响因子：
--
通讯作者：
Y. Si;P. Olds‐Clarke
中科院分区：
文献类型：
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作者：
Y. Si;P. Olds‐Clarke
摘要虽然Ca~(2+)在哺乳动物精子获能过程中起着重要作用，但其下游靶点尚未明确。本研究的目的是使用钙调素(CaM)拮抗剂W7和钙咪唑(CZ)来研究钙离子特异性结合蛋白CaM在获能中的可能作用。通过金霉素染色后的B型和溶血磷脂酰胆碱(LPC)对精子顶体反应(AR)的能力来评价与获能相关的精子膜变化。W7或CZ对B型精子百分率有明显的抑制作用，且呈浓度依赖关系。在100μM W7或10μM CZ时，这些抑制剂还显著降低了精子经历LPC诱导的AR的能力。外源性cAMP类似物可克服对B型和LPC诱发的AR的抑制。用100μM W7处理精子后，其体外受精能力也显著下降。在100μM时，W5，一种效力较低的脱氯W7类似物，对B型、LPC诱导的AR或受精能力没有影响。100μMW7(相对于100μMW5)和10μM CZ对精子存活率和蛋白酪氨酸磷酸化无显著影响，但活动率和高活动率显著降低。拮抗剂抑制的精子活力可通过对照组稀释液恢复，但不能通过cAMP类似物恢复。这些结果表明，CaM在cAMP的上游参与了对小鼠精子获能重要的膜变化的调节，并且这一途径至少部分地与控制酪氨酸磷酸化和过度激活的途径分开。
Abstract Although Ca2+ is of fundamental importance in mammalian sperm capacitation, its downstream targets have not been definitively demonstrated. The purpose of this study was to use the calmodulin (CaM) antagonists W7 and calmidazolium (CZ) to investigate the possible role of CaM, a Ca2+-specific binding protein, in capacitation. Sperm membrane changes associated with capacitation were assessed by the B pattern after chlortetracycline staining and by the ability to undergo the acrosome reaction (AR) in response to lysophosphatidylcholine (LPC). The percentage of B pattern sperm was significantly inhibited by W7 or CZ in a concentration-dependent manner. At 100 μM W7 or 10 μM CZ, these inhibitors also significantly reduced the sperm's ability to undergo the LPC-induced AR. Inhibition of the B pattern and the LPC-induced AR was overcome by exogenous cAMP analogues. Treatment of the sperm with 100 μM W7 also resulted in a significant decrease in their ability to fertilize eggs in vitro. At 100 μM, W5, a less potent dechlorinated W7 analogue, had no effect on the B pattern, LPC-induced AR, or fertilization competence. Sperm viability and protein tyrosine phosphorylation were not substantially affected by 100 μM W7 (relative to 100 μM W5) or 10 μM CZ; however, the percentages of motile and hyperactivated sperm were significantly reduced. The antagonist-inhibited sperm motility was restored by dilution in control medium, but not by cAMP analogues. These results suggest that CaM participates in the regulation of membrane changes important for mouse sperm capacitation, at a point upstream from cAMP, and that this pathway is at least partially separable from pathways controlling tyrosine phosphorylation and hyperactivation.