ESTABLISHMENT AND CHARACTERIZATION OF HUMAN SPERM SPECIFIC MONOCLONAL ANTIBODY 1G1 WITH INHIBITORY EFFECT OF SPERM-ZONA PELLUCIDA INTERACTION

具有抑制精子-透明带相互作用作用的人精子特异性单克隆抗体1G1的建立和表征

• 批准号: 04671026
• 负责人: KOYAMA Koji
• 金额: $ 1.34万
• 依托单位: HYOGO COLLEGE OF MEDICINE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04671026/
• 关键词: monoclonal antibody; fertilization; ubiquitin; human sperm; 精子先体; ユビキチン; 精子不動化; 精子付着精漿抗原; 遺伝子クローニング

The MAb(1G1) possessing a strong fertilization-blocking activity was prepared by immunizing BALB/c with capacitated human sperm in order to study the molecular nature of sperm antigens relevant to fertilization. The MAb 1G1 strongly inhibited human sperm penetration into zona-free hamster eggs. It reacted to the apical portion of acrosome-reacted human sperm, but did not react to the live sperm before acrosome reaction, when the immunofluorescence staining were performed. In paraffin-embedded testis sections, the round spermatids, spermatocytes and spermatozoa were stained with the MAb 1G1. The spermatogonia, Sertoli cells, Leydig cells, and other somatic tissues were not stained. The sperm of monkeys, bulls, mice and boars were not stained. Therefore, the staining of sperm was species specific. The antigen corresponding to the MAb 1G1 showed a band at a 27kDa by immunoblotting. The reactivity of the antigenic components were not destroyed by periodic acid treatment. As a result, it was postulated that the antigenic molecule might be a peptide. After screening the human testis cDNA library with the MAb 1G1, we isolated a cDNA clone encoding the antigen. According to the nucleotide sequences of th cDNA clone, they showed that the cDNA contained human poly-ubiquitin DNA.These results suggests that ubiquitin might play an important role in human fertilization.

为了研究与受精相关的精子抗原的分子性质，用获能人精子免疫BALB/c，制备了具有较强受精阻断活性的单抗(1G1)。单抗1G1强烈抑制人类精子对去透明带仓鼠卵子的渗透。免疫荧光染色时，它与顶体反应的人精子顶端部分反应，但与顶体反应前的活精子不反应。在石蜡包埋的睾丸切片中，圆形精子细胞、精母细胞和精子均被单抗1G1染色。精原细胞、支持细胞、间质细胞等体细胞未见染色。猴子、公牛、小鼠和公猪的精子未被染色。因此，精子的染色具有物种特异性。免疫印迹显示，与单抗1G1对应的抗原在27 kDa处有一条带。高碘酸处理不破坏抗原组分的反应性。因此，推测该抗原分子可能是一种多肽。在用单抗1G1筛选人睾丸cDNA文库后，我们分离到了编码该抗原的cDNA克隆。根据TH克隆的核苷酸序列分析表明，该克隆含有人多泛素DNA，提示泛素可能在人类受精过程中发挥重要作用。

项目成果

Milena-Dubaua Mikalava et al: "Identification and chracterization of a 27KD acrosome protein of human sperm defined by monoclonal antibody with fertilization-blocking effect" Journal of Reproductive Immunology. (印刷中). (1994)

Milena-Dubaua Mikalava 等人：“具有受精阻断作用的单克隆抗体定义的人类精子 27KD 顶体蛋白的鉴定和表征”《生殖免疫学杂志》（1994 年出版）。

Milena Dubova‐Mihailova et al: "Identification and characten zation of a 27KD acrosome protein of human speum defined by amonoclonal antibocly with ferlilization‐blochaig effect" Journal of Reproductive Lmmnology. (印刷中). (1994)

Milena Dubova-Mihailova 等人：“具有受精-blochaig 效应的单克隆抗体定义的人痰 27KD 顶体蛋白的鉴定和表征”《生殖免疫学杂志》（1994 年）。

Milena Dubova-Mihailoval), Shinji Komori, Kinu Kameda, Yoshiyuki Tsuji, Koji Koyama, and Shinzo Isojima: "Identificcation and characterization of a 27 kD acrosome protein of human sperm difined by a monoclonal antibody with fertilization-blocking effect"

Milena Dubova-Mihailoval)、Shinji Komori、Kinu Kameda、Yoshiyuki Tsuji、Koji Koyama 和 Shinzo Isojima：“具有受精阻断作用的单克隆抗体对人类精子 27 kD 顶体蛋白的鉴定和表征”