Beecher K L, Homyk M, Lee C G, Herr J C
Department of Anatomy and Cell Biology, University of Virginia, Charlottesville 22908.
Biol Reprod. 1993 Jan;48(1):154-64. doi: 10.1095/biolreprod48.1.154.
This study characterizes a common antigen recognized by two monoclonal antibodies (mAbs) that immunoreact with the principal piece of the human sperm flagellum. By means of immunofluorescence microscopy, mAbs S69 (IgM) and S70 (IgG1) (WHO Task-force nomenclature) were observed to bind to the principal piece of methanol- or detergent-permeabilized human spermatozoa, but did not react with live swimming spermatozoa as assessed by immunofluorescence microscopy. Faint immunofluorescence was also seen on the connecting piece in approximately 40% of the spermatozoa. Immunoreactivity in both regions was resistant to sequential extraction with Triton X-100, sodium thiocyanate, and urea. Pre-embedding electron microscopic immunogold labeling of ejaculated spermatozoa with mAb S69 showed gold particles located on the fibrous sheath. Immunoreactive peptides of 68, 53, and 45 kDa were recognized by both S69 and S70 mAbs on immunoblots of nonreduced human sperm extracts, while a 68-kDa band and a strongly immunoreactive triplet from 54 to 51 kDa were recognized in reduced sperm extracts. Human fibrous sheaths were isolated by differential solubilization and centrifugation and characterized by transmission electron microscopy. The 68-kDa and 54-51-kDa bands were enriched and found to be major polypeptides in the isolated fibrous sheath fraction. These results suggest that the S69/S70 antigen, which we term SP (sperm protein) (68 kDa, 54-51 kDa), is a component of the human fibrous sheath.
本研究鉴定了一种共同抗原，该抗原可被两种单克隆抗体（mAb）识别，这两种单克隆抗体可与人精子鞭毛主段发生免疫反应。通过免疫荧光显微镜观察发现，mAb S69（IgM）和S70（IgG1）（世界卫生组织工作组命名法）可与经甲醇或去污剂通透处理的人精子主段结合，但通过免疫荧光显微镜评估发现，它们不与活的游动精子发生反应。在大约40%的精子中，连接段也可见微弱的免疫荧光。这两个区域的免疫反应性对用Triton X-100、硫氰酸钠和尿素进行的顺序提取具有抗性。用mAb S69对射出精子进行预包埋电子显微镜免疫金标记，结果显示金颗粒位于纤维鞘上。在未还原的人精子提取物免疫印迹上，68 kDa、53 kDa和45 kDa的免疫反应性肽段可被S69和S70两种单克隆抗体识别，而在还原的精子提取物中可识别出一条68 kDa的条带和一个从54 kDa到51 kDa的强免疫反应性三联体。通过差速溶解和离心分离出人纤维鞘，并通过透射电子显微镜对其进行表征。68 kDa和54 - 51 kDa的条带得到富集，并被发现是分离的纤维鞘组分中的主要多肽。这些结果表明，我们命名为SP（精子蛋白）（68 kDa，54 - 51 kDa）的S69/S70抗原是人纤维鞘的一个组成部分。
