PRIMATE SPERM CONTAIN PROTEIN PHOSPHATASE 1 BIOCHEMICAL MEDIATOR OF MOTILITY

灵长类精子含有蛋白质磷酸酶 1 活力生化介质

• 批准号: 6247234
• 负责人: GARY D SMITH
• 金额: $ 4.86万
• 依托单位: OREGON REGIONAL PRIMATE RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6247234
• 关键词: Primates; growth factor; human tissue; reproductive system

Sperm motility initiation, capacitation and hyperactivation are modulated by an interplay of intracellular Ca 2+, cyclic AMP and pH. Mechanisms by which these mediators alter sperm function have not been elucidated, but may involve reversible alterations in regulatory protein phosphorylation. Studies were designed to investigate the influence of the protein phosphatase (PP) inhibitor calyculin A (CL-A) on human sperm motility and to characterize the CL-A sensitive PP and its endogenous regulators in human and rhesus monkey sperm. CL-A (5-50 nM) treatment of human swim-up supernatants and pellets resulted in accelerated sperm velocity in both fractions and an increase in percent motility in pellet fractions. These results suggest that inhibition of PP1 or PP2A could be responsible for the motility stimulation, since these phosphatases are sensitive to nanomolar quantities of CL-A. PP activity in human (n=4) and rhesus monkey (n=4) sperm extracts were measured using [ 32P]phosphorylase-`, the preferred substrate for PP1 and PP2A, in the absence of divalent cations. Human (6.2 q 4.5 x10 -2 mU/10 6 sperm) and monkey (4.3 q 0.8 x 10 2 mU/10 6 sperm) extracts contained PP1 and/or PP2A activity. Sperm PP activity inhibition profiles by okadaic acid and CL-A were identical to those reported for PP1 derived from other tissue sources. Western blot analysis with antibodies against various isoforms of PP1 documented the presence of PP1 2 in human and monkey sperm. PP1 activity in most tissues is regulated by the heat-stable inhibitors I1 and I2. Sperm extracts contained inhibitor activity similar to I2, and glycogen synthase kinase-3 activity, which is involved in the activation of the inactive PP1-I2 complex. These results indicate, for the first time, that human and rhesus monkey sperm contain PP1, regulators of PP1 and that inhibition of PP1 activity by CL-A can enhance motility.

精子运动起始、获能和超活化是 受细胞内Ca 2+、cAMP和pH的相互作用调节。 这些介质改变精子功能的机制还不清楚。 阐明，但可能涉及可逆的改变，在调节 蛋白质磷酸化 研究旨在调查 蛋白磷酸酶（PP）抑制剂calyculin A（CL-A） 对人精子活力的影响，并表征CL-A敏感的PP， 其内源性调节剂在人类和恒河猴精子。 CL-a (5-50 nM）处理人上游上清液和沉淀， 在两种组分中，精子速度加快， 颗粒级分中的运动性百分比。 这些结果表明 PP 1或PP 2A的抑制可能与运动有关 刺激，因为这些磷酸酶对纳摩尔浓度敏感， CL-A的量。 人（n=4）和恒河猴中的PP活性 （n=4）精子提取物用[32 P]磷酸化酶-`， PP 1和PP 2A的优选底物，在不存在二价 阳离子。 人（6.2 q 4.5 x10 - 2 mU/10 6精子）和猴（4.3 q 0.8 × 10 2 mU/10 6精子）提取物含有PP 1和/或PP 2 A活性。 冈田酸和CL-A对精子PP活性的抑制曲线分别为： 与来自其他组织来源的PP 1报告的结果相同。 使用针对PP 1的各种亚型的抗体的蛋白质印迹分析 证明了人和猴精子中存在PP 1 2。 PP1 大多数组织中的活性由热稳定抑制剂I1调节 和I2。 精子提取物含有类似于I2的抑制剂活性， 和糖原合成酶激酶-3活性，这是参与 激活无活性的PP 1-I2复合物。 这些结果表明， 人类和恒河猴精子中首次含有PP 1， CL-A对PP 1活性的抑制作用， 增强运动性。