Johnson L R, Foster J A, Haig-Ladewig L, VanScoy H, Rubin C S, Moss S B, Gerton G L
Center for Research on Reproduction and Women's Health, University of Pennsylvania Medical Center, Philadelphia 19104-6080, USA.
Dev Biol. 1997 Dec 15;192(2):340-50. doi: 10.1006/dbio.1997.8767.
The assembly of the mammalian sperm flagellum is a complex developmental event requiring the sequential activation of genes encoding the component parts and the coordinated assembly of these proteins during the differentiation of the haploid spermatid. In this study, the mechanism underlying the assembly of the fibrous sheath surrounding the axoneme was examined. The subject of the study was the major fibrous sheath protein of the mouse sperm flagellum, AKAP82, a member of the A Kinase Anchor Protein (AKAP) family of polypeptides that bind the regulatory (RII) subunit of protein kinase A (PK-A). Immunoelectron microscopy demonstrated that AKAP82 is present throughout the transverse ribs and longitudinal columns of the fibrous sheath. Since AKAP82 is initially synthesized as a precursor (pro-AKAP82) during spermiogenesis, an antiserum was raised against a peptide from the processed region of pro-AKAP82 (M(r) 97,000). In immunoblotting experiments, the antibody detected pro-AKAP82 in condensing spermatids but not in epididymal sperm. In addition, two other immunoreactive proteins of M(r) 109,000 (p109) and M(r) 26,000 (p26, representing the "pro" domain of the precursor) were present in epididymal sperm. Alkaline phosphatase treatment of epididymal sperm proteins demonstrated that p109 was a phosphorylated form of pro-AKAP82 that remained in sperm. By immunofluorescence, pro-AKAP82 was localized to the entire length of the principal piece in testicular sperm, while in epididymal sperm p109 and p26 were present only in the proximal portion of the principal piece. Pro-AKAP82 was solubilized when germ cells were extracted with Triton X-100. However, in sperm, both AKAP82 and p109 were almost totally resistant to these extraction conditions and remained in the particulate fraction even after extraction with Triton and dithiothreitol. Similar to pro-AKAP82, the RII subunit of PK-A was present in the Triton X-100-soluble fraction of developing germ cells. In sperm, much of the RII also became particulate, consistent with the hypothesis that AKAP82 anchors RII in the flagellum. These data indicate that pro-AKAP82 is synthesized in the cell body, transported down the axoneme to its site of assembly in the fibrous sheath, and then proteolytically clipped to form mature AKAP82.
哺乳动物精子鞭毛的组装是一个复杂的发育过程，需要在单倍体精子细胞分化过程中依次激活编码各组成部分的基因，并使这些蛋白质进行协调组装。在本研究中，对围绕轴丝的纤维鞘组装的潜在机制进行了研究。研究对象是小鼠精子鞭毛的主要纤维鞘蛋白AKAP82，它是A激酶锚定蛋白（AKAP）家族多肽的成员，该家族多肽可结合蛋白激酶A（PK-A）的调节（RII）亚基。免疫电子显微镜显示，AKAP82存在于纤维鞘的整个横向肋条和纵向柱中。由于AKAP82在精子发生过程中最初是以前体（pro-AKAP82）形式合成的，因此制备了一种针对pro-AKAP82加工区域（分子量97,000）的肽段的抗血清。在免疫印迹实验中，该抗体在浓缩精子细胞中检测到pro-AKAP82，但在附睾精子中未检测到。此外，在附睾精子中还存在另外两种免疫反应性蛋白，分子量分别为109,000（p109）和26,000（p26，代表前体的“pro”结构域）。对附睾精子蛋白进行碱性磷酸酶处理表明，p109是留在精子中的pro-AKAP82的磷酸化形式。通过免疫荧光检测，pro-AKAP82定位于睾丸精子主段的全长，而在附睾精子中，p109和p26仅存在于主段的近端部分。当用Triton X-100提取生殖细胞时，pro-AKAP82可被溶解。然而，在精子中，AKAP82和p109几乎完全抵抗这些提取条件，即使在用Triton和二硫苏糖醇提取后仍保留在颗粒部分。与pro-AKAP82类似，PK-A的RII亚基存在于发育中的生殖细胞的Triton X-100可溶部分。在精子中，许多RII也变成颗粒状，这与AKAP82将RII锚定在鞭毛中的假设一致。这些数据表明，pro-AKAP82在细胞体中合成，沿轴丝运输到其在纤维鞘中的组装位点，然后经蛋白水解剪切形成成熟的AKAP82。
