A cyclic adenosine 3′,5′-monophosphate stimulates phospholipase Cγ1-calcium signaling via the activation of tyrosine kinase in boar spermatozoa
A cyclic adenosine 3′,5′-monophosphate stimulates phospholipase Cγ1-calcium signaling via the activation of tyrosine kinase in boar spermatozoa
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DOI：
10.2164/jandrol.05053
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发表时间：
2005-11-01
影响因子：
--
通讯作者：
Miyake, M
中科院分区：
文献类型：
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作者：
Harayama, H;Murase, T;Miyake, M
本研究的目的是揭示猪精子细胞内信号转导的下游部分，该信号是由环磷酸腺苷(CAMP)依赖的酪氨酸激酶，包括脾酪氨酸激酶(SYK)介导的。将射出的精子与cBiMPS(一种细胞渗透性cAMP类似物，0.1 mM)在38.5℃孵育180分钟，然后进行Western印迹和间接免疫荧光。精子与cBiMPS孵育可诱导连接段和主体段SYK连接区的酪氨酸磷酸化(这是与磷脂酶C L结合所必需的)，但加入H-(一种蛋白激酶A抑制剂，0.01-0.1 mM)后，这种酪氨酸磷酸化作用被取消。此外，在相同的精子片段中，依赖cAMP的酪氨酸磷酸化也被诱导在PLC-Gamma 1的关键调控残基上，但这种作用被加入除草剂除草剂A(一种酪氨酸酶抑制剂，5 mU M)所抑制。这些结果表明，精子cAMP依赖的酪氨酸激酶，包括SYK，与PLC-Gamma 1的激活有关。间接免疫荧光清楚地检测到连接部分的肌醇1，4，5-三磷酸(IP3)受体和钙网蛋白，表明内部存在钙储存。用细胞透性钙指示剂Fluo-3/AM进行细胞成像显示，精子与cBiMPS孵育后，中段细胞内游离钙增加，但加入U-73122(0.02 mM的磷脂酶C抑制剂)后，细胞内游离钙增加。根据我们的发现，我们认为猪精子的连接件具有PLC-Gamma 1-IP3受体-钙信号，该信号由cAMP触发，由PKA和包括SYK在内的除草剂敏感的酪氨酸激酶介导。
The aim of this study was to reveal a downstream part of the intracellular signaling that is mediated by cyclic adenosine monophosphate (cAMP)-dependent tyrosine kinases, including spleen tyrosine (Y) kinase (SYK), in boar spermatozoa. Ejaculated spermatozoa were incubated with cBiMPS (a cell-permeable cAMP analog; 0.1 mM) at 38.5 degrees C for 180 minutes and then used for Western blot and indirect immunofluorescence. Incubation of spermatozoa with cBiMPS induced tyrosine phosphorylation at the linker region of SYK (which was essential to binding to phospholipase C [PLC]gamma l) in the connecting and principal pieces, but the tyrosine phosphorylation was abolished by the addition of H-89 (a protein kinase A [PKA] inhibitor; 0.01-0.1 mM). Moreover, the cAMP-dependent tyrosine phosphorylation was also induced at the key regulatory residue of PLC gamma 1 in the same segments of spermatozoa, but it was inhibited by the addition of herbimycin A (a tyrosine kinase inhibitor; 5 mu M). These results suggest that the sperm cAMP-dependent tyrosine kinases, including SYK, are linked to the activation of PLC gamma 1. Indirect immunofluorescence clearly detected both inositol 1, 4,5-trisphosphate (IP3) receptor and calreticulin in the connecting piece, indicating the presence of internal calcium store. Cell imaging with fluo-3/AM (a cell-permeable Ca2+ indicator) showed that incubation of spermatozoa with cBiMPS increased intracellular free calcium in the middle piece, but that it was reduced by the addition of U-73122 (a PLC inhibitor; 0.02 mM). Based on our findings, we conclude that the connecting piece of boar spermatozoa possesses the PLC gamma 1-IP3 receptor-calcium signaling that is triggered by cAMP and mediated by PKA and herbimycin A-sensitive tyrosine kinases, including SYK.