The proacrosin binding protein, sp32, is tyrosine phosphorylated during capacitation of pig sperm
The proacrosin binding protein, sp32, is tyrosine phosphorylated during capacitation of pig sperm
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DOI：
10.2164/jandrol.04163
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发表时间：
2005-07-01
影响因子：
--
通讯作者：
Bailey, JL
中科院分区：
文献类型：
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作者：
Dubé, C;Leclerc, P;Bailey, JL
哺乳动物的精子必须经过获能，即在雌性生殖道或体外的一个准备阶段，才能受精。我们之前已经描述了一种Mr 32000酪氨酸磷酸化蛋白“p32”，它出现在猪精子的获能过程中。p32的身份仍然未知；在容能过程中是否以及如何参与尚不清楚。本研究的目的是通过蛋白质组学技术鉴定p32。在非还原和还原条件下先后分离的蛋白质，Western blotting显示只有在使能条件下孵育精子时才出现酪氨酸磷酸化的p32。通过质谱/质谱法对该位点进行了测序，并鉴定为“sp32”，一种与原顶胞素成熟有关的蛋白质。用抗sp32抗体探测相同的膜，表明sp32在非容能和容能条件下都存在，并且显示出与p32完全相同的位点。抗磷酸酪氨酸或抗sp32抗体的免疫沉淀证实了这些结果。使用抗磷酸酪氨酸抗体或抗sp32抗体的间接免疫荧光显示有能力精子的标记相似，支持p32是sp32的酪氨酸磷酸化形式的假设。经离子载体诱导顶体反应后，顶体上的抗sp32和抗磷酸酪氨酸标记消失。这些结果表明，（前）交叉蛋白结合蛋白sp32是酪氨酸磷酸化蛋白p32，与获能相关。我们现在将重点关注酪氨酸磷酸化在受精相关事件中对sp32功能的意义。
Mammalian sperm must undergo capacitation, a preparation period in the female reproductive tract or in vitro, in order to fertilize. We have previously described a Mr 32000 tyrosine phosphorylated protein, "p32," that appears in pig sperm during capacitation. The identity of p32 remains unknown; if and how it is involved during capacitation is not understood. The objective of the present study was to identify p32 by proteomic techniques. Western blotting of proteins separated successively under nonreducing and then reducing conditions showed the appearance of the tyrosine phosphorylated p32 only when sperm were incubated in capacitating conditions. The spot was sequenced by mass spectrometry/mass spectrometry and identified as "sp32," a protein implicated in proacrosin maturation. The same membranes probed with anti-sp32 antibody demonstrated that sp32 is present in both noncapacitating and capacitating conditions and revealed exactly the same spot as p32. Immunoprecipitation with either anti-phosphotyrosine or anti-sp32 antibody corroborated these results. Indirect immunofluorescence with anti-phosphotyrosine antibody or anti-sp32 antibody show similar labeling of capacitated sperm, supporting the hypothesis that p32 is a tyrosine phosphorylated form of sp32. After ionophore treatment to induce the acrosome reaction, anti-sp32 and anti-phosphotyrosine labeling on the acrosome disappeared. These results demonstrate that sp32, a (pro)acrosin binding protein, is the p32, a tyrosine phosphorylated protein related to capacitation. We will now focus on the significance of tyrosine phosphorylation on sp32 function during fertilization-related events.