One of the first events of mammalian sperm capacitation is the activation of the soluble adenyl cyclase/cAMP/protein kinase A (SACY/cAMP/PKA) pathway. Here, we evaluated whether the increase in PKA activity at the onset of human sperm capacitation is responsible for the activation of the sperm proteasome and whether this activation is required for capacitation progress. Viable human sperm were incubated with inhibitors of the SACY/cAMP/PKA pathway. The chymotrypsin-like activity of the sperm proteasome was evaluated using a fluorogenic substrate. Sperm capacitation status was evaluated using the chlortetracycline assay and tyrosine phosphorylation. To determine whether proteasomal subunits were phosphorylated by PKA, the proteasome was immunoprecipitated and tested on a western blot using an antibody against phosphorylated PKA substrates. Immunofluorescence microscopy analysis and co-immunoprecipitation (IPP) were used to investigate an association between the catalytic subunit alpha of PKA (PKA-Cα) and the proteasome. The chymotrypsin-like activity of the sperm proteasome significantly increased after 5 min of capacitation (P < 0.001) and remained high for the remaining incubation time. Treatment with H89, KT5720 or KH7 significantly decreased the chymotrypsin-like activity of the proteasome (P < 0.001). IPP experiments indicated that PKA inhibition significantly modified phosphorylation of proteasome subunits. In addition, PKA-Cα colocalized with the proteasome in the equatorial segment and in the connecting piece, and co-immunoprecipitated with the proteasome. This is the first demonstration of sperm proteasome activity being directly regulated by SACY/PKA-Cα. This novel discovery extends our current knowledge of sperm physiology and may be used to manage sperm capacitation during assisted reproductive technology procedures.

中文翻译：

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蛋白酶体的胰凝乳蛋白酶样活性的激活受可溶性腺苷酸环化酶/ cAMP /蛋白激酶A途径的调节，是人类精子获能所必需的。

哺乳动物精子获能的首批事件之一是可溶性腺苷酸环化酶/ cAMP /蛋白激酶A（SACY / cAMP / PKA）途径的激活。在这里，我们评估了在人类精子获能开始时PKA活性的增加是否是精子蛋白酶体活化的原因，以及这种获能是否需要进行获能。将活的人精子与SACY / cAMP / PKA途径的抑制剂一起孵育。使用荧光底物评估了精子蛋白酶体的胰凝乳蛋白酶样活性。用金霉素测定和酪氨酸磷酸化评估精子获能状态。为了确定蛋白酶体亚基是否被PKA磷酸化，将蛋白酶体免疫沉淀，并使用抗磷酸化PKA底物的抗体在Western blot上进行测试。免疫荧光显微镜分析和免疫共沉淀（IPP）用于研究PKA的催化亚基α（PKA-Cα）与蛋白酶体之间的联系。获能5分钟后，精子蛋白酶体的胰凝乳蛋白酶样活性显着增加（P <0.001），并在剩余的孵育时间内保持较高水平。用H89，KT5720或KH7处理可显着降低蛋白酶体的胰凝乳蛋白酶样活性（P <0.001）。IPP实验表明，PKA抑制作用显着改变了蛋白酶体亚基的磷酸化。另外，PKA-Cα与蛋白酶体在赤道节段和连接片段中共定位，并与蛋白酶体共免疫沉淀。这是SACY /PKA-Cα直接调节精子蛋白酶体活性的第一个证明。