Visconti P E, Johnson L R, Oyaski M, Fornés M, Moss S B, Gerton G L, Kopf G S
Department of Obstetrics and Gynecology, University of Pennsylvania Medical Center, Philadelphia 19104-6080, USA.
Dev Biol. 1997 Dec 15;192(2):351-63. doi: 10.1006/dbio.1997.8768.
The molecular basis of mammalian sperm capacitation, defined as those biochemical and functional changes that render the sperm competent to fertilize the egg, is poorly understood. This extratesticular maturational process is accompanied by the activation of a unique signal transduction pathway involving the cAMP-dependent up-regulation of protein tyrosine phosphorylation presumably through the activation of protein kinase A (PK-A). We demonstrate in this report that capacitation of cauda epididymal mouse sperm in vitro was accompanied by a time-dependent increase in PK-A activity. This increase in PK-A activity did not occur in a medium that does not support capacitation. While PK-A catalytic and RI/RII regulatory subunits, as well as PK-A enzyme activity, were found in both the Triton X-100-soluble and -insoluble fractions of the sperm, the increase in PK-A activity accompanying capacitation was associated with enzyme activity found in the soluble fraction. Moreover, the regulatory and catalytic subunits of PK-A were observed by indirect immunofluorescence to be present throughout the head, midpiece, and principal piece of the sperm. Thus, PK-A appears to be functional in multiple compartments of this highly differentiated cell. A fraction of the Triton X-100-insoluble PK-A is presumably tethered by AKAP82, the major protein of the fibrous sheath of the sperm flagellum which anchors and compartmentalizes PK-A to the cytoskeleton via the RII subunit of PK-A. Using various recombinant truncated AKAP82 constructs in a gel overlay assay, the RII subunit-binding domain of this protein was mapped to a 57-amino-acid residue region at its N-terminus. Computer analysis revealed a 14-amino-acid region that resembled the RII-binding domains of other A Kinase Anchor Proteins. A synthetic peptide corresponding to this domain inhibited AKAP82-RII binding in a gel overlay assay, providing further support that AKAP82 is an anchoring protein for the subcellular localization of PK-A in the mouse sperm fibrous sheath. This work, along with previous findings that cAMP is a key intermediary second messenger in regulating protein tyrosine phosphorylation and capacitation, further supports the importance of PK-A in these processes and necessitates a further understanding of the contribution of both the soluble and insoluble forms of PK-A, as well as AKAP82, to sperm function.
哺乳动物精子获能的分子基础，即那些使精子具备使卵子受精能力的生化和功能变化，目前仍知之甚少。这个睾丸外成熟过程伴随着一条独特信号转导通路的激活，该通路涉及可能通过蛋白激酶A（PK-A）的激活而导致的蛋白酪氨酸磷酸化的cAMP依赖性上调。我们在本报告中证明，体外附睾尾部小鼠精子的获能伴随着PK-A活性随时间的增加。在不支持获能的培养基中未出现PK-A活性的这种增加。虽然在精子的Triton X-100可溶性和不溶性部分均发现了PK-A催化亚基和RI/RII调节亚基以及PK-A酶活性，但获能时PK-A活性的增加与可溶性部分中的酶活性相关。此外，通过间接免疫荧光观察到PK-A的调节亚基和催化亚基存在于精子的整个头部、中段和主段。因此，PK-A似乎在这个高度分化细胞的多个区室中发挥作用。Triton X-100不溶性PK-A的一部分可能由AKAP82锚定，AKAP82是精子鞭毛纤维鞘的主要蛋白质，它通过PK-A的RII亚基将PK-A锚定并分隔到细胞骨架上。在凝胶覆盖试验中使用各种重组截短的AKAP82构建体，该蛋白的RII亚基结合域被定位到其N端的一个57个氨基酸残基区域。计算机分析揭示了一个14个氨基酸的区域，其类似于其他A激酶锚定蛋白的RII结合域。在凝胶覆盖试验中，与该结构域对应的合成肽抑制了AKAP82-RII结合，进一步支持了AKAP82是PK-A在小鼠精子纤维鞘中亚细胞定位的锚定蛋白。这项工作，连同先前关于cAMP是调节蛋白酪氨酸磷酸化和获能的关键中间第二信使的发现，进一步支持了PK-A在这些过程中的重要性，并且有必要进一步了解PK-A的可溶性和不溶性形式以及AKAP82对精子功能的贡献。
