Paranko J, Yagi A, Kuusisto M
Department of Anatomy, University of Turku, Finland.
Anat Rec. 1994 Dec;240(4):516-27. doi: 10.1002/ar.1092400409.
Presence of immunocytochemically detectable actin in the rat and mouse sperm head has been enigmatic for years. In this study, we demonstrate actin in the perinuclear theca and show that the detection of actin epitopes in the rat and mouse epididymal spermatozoa can effectively be enhanced by pre-extraction of sperm cells with SDS.
The study with one monoclonal and one polyclonal anti-actin antibody was carried out at conventional and confocal fluorescence and electron microscope level, and by immunoblotting of proteins isolated from the head and tail fractions.
In the head of the control methanol-acetone fixed rat spermatozoa, the polyclonal antibody gave a stronger immunostaining in the postacrosomal area and in the perforatorium than the monoclonal antibody. In the mouse sperm head, the monoclonal antibody labeled the ventral edge of the postacrosomal area and slightly the perforatorium, whereas the polyclonal antibody stained the entire perinuclear space. In the SDS-extracted spermatozoa, an intense postacrosomal and perforatorial labeling was obtained with both antibodies but, in particular in the rat spermatozoa, the middle lateral portion of the postacrosomal segment remained unlabeled. Sonication seemed to cause structural modifications which specifically impeded staining with the monoclonal antibody. Both antibodies detected actin in the basal plate and the monoclonal antibody in the neck. Amorphous matrix of the connecting piece showed immunogold labeling. In the tail, the monoclonal antibody recognized actin and a relatively basic 53 kDa polypeptide, whereas the polyclonal antibody reacted with several protein bands. SDS-soluble actin of the tail was addressed to the midpiece and the SDS-insoluble 53 kDa protein profoundly to the outer dense fibers of the principal piece.
Intense labeling of actin in the SDS-extracted rat and mouse spermatozoa was presumably due to the generated demasking of actin epitopes embedded in the perinuclear cytoplasm. The results are important in confirming that actin in the rat and mouse sperm head is not lost during spermiogenesis but apparently contributes to the three-dimensional packing of the mature perinuclear cytoplasm. This study further demonstrates the importance of the methods used in sample preparation and advantages of confocal microscopy when attempting to detect cytoskeletal proteins which, as in spermatozoa, may occur in small quantities.
多年来，大鼠和小鼠精子头部免疫细胞化学可检测到的肌动蛋白的存在一直是个谜。在本研究中，我们证实了核周膜中有肌动蛋白，并表明用十二烷基硫酸钠（SDS）对精子细胞进行预提取可有效增强对大鼠和小鼠附睾精子中肌动蛋白表位的检测。
使用一种单克隆抗肌动蛋白抗体和一种多克隆抗肌动蛋白抗体，在传统和共聚焦荧光显微镜及电子显微镜水平上进行研究，并对从头部和尾部组分中分离的蛋白质进行免疫印迹分析。
在对照的甲醇 - 丙酮固定的大鼠精子头部，多克隆抗体在顶体后区域和穿孔器中的免疫染色比单克隆抗体更强。在小鼠精子头部，单克隆抗体标记顶体后区域的腹侧边缘并轻微标记穿孔器，而多克隆抗体则对整个核周空间进行染色。在经SDS提取的精子中，两种抗体均获得强烈的顶体后和穿孔器标记，但特别是在大鼠精子中，顶体后段的中外侧部分未被标记。超声处理似乎导致结构改变，特别阻碍了单克隆抗体的染色。两种抗体均在基板中检测到肌动蛋白，单克隆抗体在颈部检测到肌动蛋白。连接段的无定形基质显示免疫金标记。在尾部，单克隆抗体识别肌动蛋白和一种相对碱性的53 kDa多肽，而多克隆抗体与几条蛋白带发生反应。尾部的SDS可溶性肌动蛋白定位于线粒体鞘，而SDS不溶性53 kDa蛋白则主要定位于主段的外周致密纤维。
经SDS提取的大鼠和小鼠精子中肌动蛋白的强烈标记可能是由于核周细胞质中嵌入的肌动蛋白表位产生的去掩蔽作用。这些结果对于证实大鼠和小鼠精子头部的肌动蛋白在精子发生过程中并未丢失，但显然有助于成熟核周细胞质的三维包装具有重要意义。本研究进一步证明了样品制备中所用方法的重要性以及共聚焦显微镜在试图检测如精子中可能少量存在的细胞骨架蛋白时的优势。
