Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity
Sperm motility development in the epididymis is associated with decreased glycogen synthase kinase-3 and protein phosphatase 1 activity
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DOI：
10.1095/biolreprod54.3.709
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发表时间：
1996-03-01
影响因子：
3.6
通讯作者：
Greengard, P
中科院分区：
文献类型：
--
作者：
Vijayaraghavan, S;Stephens, DT;Greengard, P
不活动的牛头附睾精子所含的蛋白质磷酸酶活性水平是成熟活动尾部精子的两倍。比较冈田酸(OA)和花盏花素A(CA)对内源性磷酸酶活性的抑制情况，发现了一种与1型蛋白磷酸酶(PP1)优势一致的模式。免疫印迹分析证实PP1(Gamma2)(PP1的睾丸特异性亚型)是精子中唯一的PP1亚型，几乎没有蛋白磷酸酶2A(PP2A)。此外，在已知的PP1抑制剂，即DARPP-32、抑制物1(I1)和抑制物2(I2)中，仅在精子中检测到I2样活性。从精子中提纯的热稳定性类I2活性对PP1的抑制可被纯化的糖原合成酶-3(GSK-3)逆转。此外，精子提取物含有PP1和I2的非活性复合体(称为PP1I)，也可以被纯化的GSK-3激活。对纯化的PP1I的激活证明精子中存在GSK-3，定量结果显示静止的头状精子的GSK-3活性比活动的尾状精子高6倍。免疫印迹分析证实了GSK-3在精子中的表达，并揭示了α和β亚型的存在。我们的发现表明，在静止的精子中测量到的较高的PP1活性，可能是由于较高的GSK-3活性，负责抑制运动。观察到微摩尔水平的磷酸酶抑制剂OA和纳摩尔水平的磷酸酶抑制剂CA均能诱导完全静止的牛头附属器精子运动，并能刺激成熟尾部精子的运动活动，从而支持了这一结论。用这些抑制剂处理后，精液中cAMP、pH和钙的水平没有改变。这些结果为精子活力的发育和调节提供了生化基础，并可能在PP1/I2/GSK-3系统中发挥生理作用。
Immotile bovine caput epididymal sperm contain levels of protein phosphatase activity twofold higher than do mature motile caudal sperm. Comparison of the inhibition profiles of endogenous phosphatase activities detected by okadaic acid (OA) and calyculin A (CA) revealed a pattern consistent with the predominance of a type 1 protein phosphatase (PP1). Immunoblot analysis identified PP1(gamma 2) (the testis-specific isoform of PP1) as the only PP1 isoform in sperm and showed little protein phosphatase 2A (PP2A). In addition, of the known PP1 inhibitors, i.e., DARPP-32, inhibitor 1 (I1), and inhibitor 2 (I2), only I2-like activity was detected in sperm. Inhibition of PP1 by the heat-stable I2-like activity purified from sperm could be reversed with purified glycogen synthase kinase-3 (GSK-3). Furthermore, sperm extracts contain an inactive complex of PP1 and I2 (termed PP1I) that could also be activated by purified GSK-3. The presence of GSK-3 in sperm was demonstrated by activation of purified PP1I, and quantitation revealed that immotile caput sperm contained sixfold higher GSK-3 activity than motile caudal sperm. Immunoblot analysis confirmed the expression of GSK-3 in sperm and revealed the occurrence of both the a and beta isoforms.Our findings suggest that the higher PP1 activity measured in immotile sperm, presumably due to higher GSK-3 activity, is responsible for holding motility in check. This conclusion was supported by the observation that the phosphatase inhibitors OA and CA, at micromolar and nanomolar levels, respectively, were able to induce motility in completely immotile bovine caput epididymal sperm and to stimulate the kinetic activity of mature caudal sperm. The intrasperm levels of cAMP, pH, and calcium were unaltered by treatment with these inhibitors. The results suggest a biochemical basis for the development and regulation of sperm motility and a possible physiological role for the PP1/I2/GSK-3 system.