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

为了研究与受精相关的精子抗原的分子性质，利用能化的人精子免疫BALB/c，制备了具有较强受精阻断活性的单抗（1G1）。MAb 1G1强烈抑制人类精子进入无带仓鼠卵。免疫荧光染色时，它与顶体反应的人精子的顶端部分发生反应，但与顶体反应前的活精子不发生反应。在石蜡包埋的睾丸切片中，用MAb 1G1染色圆形精子、精母细胞和精子。精原细胞、支持细胞、间质细胞和其他体细胞组织未染色。猴子、公牛、老鼠和野猪的精子没有被染色。因此，精子的染色具有物种特异性。免疫印迹法检测到与单抗1G1对应的抗原在27kDa处出现条带。周期性酸处理不破坏抗原组分的反应性。因此，人们推测抗原分子可能是一种肽。用MAb 1G1筛选人睾丸cDNA文库后，分离出一个编码抗原的cDNA克隆。根据cDNA克隆的核苷酸序列，他们发现该cDNA含有人类多泛素DNA。这些结果提示泛素可能在人类受精中起重要作用。

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.