Purification and characterization of sperm maturation antigens and their stabilizing factors

精子成熟抗原及其稳定因子的纯化和表征

• 批准号: 62570011
• 负责人: TOSHIMORI Kiyotaka
• 金额: $ 1.02万
• 依托单位: Miyazaki Medical College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1987
• 资助国家: 日本
• 起止时间: 1987 至 1988
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-62570011/
• 关键词: Monoclonal antibody; Epididymis; Sperm maturation antigen; Antigen stabilizing factor; Receptor; マウス; 哺乳動物; モノクローナル抗体

Mammalian spermatozoa functionally mature during epididymal passage. This maturation is called sperm maturation in the epididymis. A monoclonal antibody (MAB) T21 (class IgM) from a hybridoma cell line that i established was employed to systematically analyze the phenomenon. Following results were obtained during the grant period.1. MAB T21 recognized a sperm maturation antigen, a sialoglycoprotein of about 54,000 dalton (sgp54).2, Due to masking of the epitope (cryptodeterminant) by sialic acid, it was unsuccessful to purify the sgp54 utilizing antigen-antibody affinity chromatography. Better purification was obtained by combination of WGA- or lentil-lection affinity and ion-exchange chromatography.3, Sialidase treatment demonstrated that the epididymal epididymal epithelial cells (clear cells) secreted sgp54 which was carried by small vesicles and membranous structures into the epididymal lumen, and then it attached to the immature spermatozoa. Binding of sgp54 to spermatozoa was inf … More luenced by a factor, possibly an antigen stabilizing factor (ASF). The molecular weight of ASF was estimated 50,000-60,000. The amount of the factor in the epididymal fluid seemed to be poor. Purification protocol of the ASF was improved using combination of column-chromatography technique. Assay protocol was improved, but the immunoreactivity of the sperm after indirect immunofluorescence technique must be judged by human eyes. This judging step was a time-comsuming work, and one might criticize the judging way was not objective. I think that more objective method is to be developed to get confirmative data.4, In vitro co-culture of immature sperm with sgp54 suggested that the sgp54-receptor existed on the immature sperm plasma membrane. The receptor was supposed to be synthesized during spermatogenesis. Interestingly, the rat and hamster mature spermatozoa also had the sgp54 receptor, even though these animals did not have sgp54 molecules. Such a kind of receptor will be present in mammalian sperm surface to attach to their own maturation molecule. Less

哺乳动物精子在附睾通道中功能成熟。这种成熟称为附睾中的精子成熟。利用所建立的杂交瘤细胞系的单克隆抗体（MAB） T21 （IgM类）对该现象进行了系统分析。在资助期间获得了以下结果：MAB T21识别出一种精子成熟抗原，一种约54,000道尔顿（sgp54）的唾液糖蛋白。2、由于表位（隐决定因子）被唾液酸掩盖，使用抗原-抗体亲和层析纯化sgp54未能成功。采用WGA或扁豆选择亲和和离子交换色谱相结合的方法纯化效果较好。3、唾液酸酶处理表明附睾附睾上皮细胞（透明细胞）分泌sgp54， sgp54通过小囊泡和膜状结构携带进入附睾管腔，然后附着在未成熟精子上。sgp54与精子的结合受到一个因素的影响，可能是抗原稳定因子（ASF）。ASF的分子量估计在50,000-60,000之间。附睾液中该因子的含量似乎很低。采用柱层析相结合的方法改进了ASF的纯化方案。虽然对检测方案进行了改进，但间接免疫荧光法检测精子的免疫反应性必须靠人眼判断。这个判断步骤是一项耗时的工作，有人可能会批评这种判断方式不客观。我认为需要开发更客观的方法来获得确证数据。4、未成熟精子与sgp54体外共培养表明，sgp54受体存在于未成熟精子的质膜上。这种受体应该是在精子形成过程中合成的。有趣的是，大鼠和仓鼠的成熟精子也有sgp54受体，尽管这些动物没有sgp54分子。这种受体存在于哺乳动物精子表面，附着在自身的成熟分子上。少

项目成果

Toshimori,K.;Oura,C.: Proc.Kagoshima Int.Symposium Glyoconjugates in Medicine. 1. 108-112 (1988)

Toshimori,K.；Oura,C.：Proc.Kagoshima Int.Symposium Glyoconjugates in Medicine。

Toshimori,K.;Araki,S.;Oura,C.: Biology of Reproduction. (1989)

Toshimori，K.；Araki，S.；Oura，C.：生殖生物学。

Toshimori K.; Oura C.: "Secreted sialic acid influences the sperm surface antigen expression during epididymal passage in the mouse." Proc. Kagoshima Int. Symposium Glycoconjugates in Medicine. 1. 108-112 (1988)

俊森K.；