Identification and characterization of cystatin-related epididymal spermatogenic protein in human spermatozoa: localization in the equatorial segment.
Identification and characterization of cystatin-related epididymal spermatogenic protein in human spermatozoa: localization in the equatorial segment.
复制标题
人类精子中半胱氨酸蛋白酶抑制剂相关附睾生精蛋白的鉴定和表征：定位于赤道段。
DOI：
10.1095/biolreprod.102.003970
复制
发表时间：
2002
影响因子：
3.6
通讯作者：
Cornwall,GailA
中科院分区：
文献类型：
--
作者：
Wassler,Michael;Syntin,Patrick;Sutton-Walsh,HG;Hsia,Nelson;Hardy,DanielM;Cornwall,GailA
我们早期的小鼠研究表明，胱抑素相关的附睾生精（克雷斯）蛋白在睾丸中的精子细胞中高度表达，并存在于小鼠精子顶体中，表明在精子功能，受精，或两者的特定作用。然而，人类的CRES基因是否与小鼠的基因相似并在生殖细胞中表达尚未确定。北方杂交和原位杂交结果表明，CREs在人睾丸中高度表达，尤其是在圆形精子细胞中。逆转录-聚合酶链反应检测附睾组织中克雷斯mRNA的表达。从人睾丸和射精精子制备的蛋白质裂解物的蛋白质印迹分析表明，主要的19 kDa的蛋白质和次要的14 kDa的蛋白质。然而，相反的顶体定位的克雷斯蛋白在小鼠精子中，间接免疫荧光的人精子处理与甲醇/乙酸使用抗人克雷斯抗体显示，克雷斯严格定位于赤道段。此外，在获能和顶体反应的精子中观察到相同的染色。为了确定克雷斯是否与质膜结合，在获能和顶体反应后将活精子与克雷斯抗体孵育。只有顶体反应的精子表现出微弱但特异的赤道染色。综上所述，这些研究表明，克雷斯蛋白存在于精子赤道段，并在受精过程中变得可接近细胞外环境。
Our earlier studies in mouse have shown that the cystatin-related epididymal spermatogenic (CRES) protein is highly expressed in elongating spermatids in the testis and is present in mouse sperm acrosomes, suggesting specific roles in sperm function, fertilization, or both. However, whether the humanCRESgene is similar to that of the mouse and is expressed in germ cells has not yet been determined. Therefore, the present study was undertaken to characterize the human ortholog of mouseCres.Northern blot and in situ hybridization experiments showed thatCRESis highly expressed in the human testis, specifically within clusters of round spermatids. Furthermore, reverse transcription-polymerase chain reaction detected CRES mRNA in the epididymis. Western blot analysis of protein lysates prepared from human testis and ejaculated spermatozoa showed a predominant 19-kDa protein and a minor 14-kDa protein. However, in contrast to the acrosomal localization of CRES protein in mouse spermatozoa, indirect immunofluorescence of human spermatozoa treated with methanol/acetic acid using anti-human CRES antibodies revealed that CRES was strictly localized to the equatorial segment. Furthermore, the same staining was observed in both capacitated and acrosome-reacted spermatozoa. To determine whether CRES was associated with the plasma membrane, live spermatozoa were incubated with CRES antibody after capacitation and acrosome reaction. Only acrosome-reacted spermatozoa showed a weak but specific equatorial staining. Taken together, these studies show that CRES protein is present in the sperm equatorial segment and becomes accessible to the extracellular environment during fertilization.