SPAM1 (PH-20) protein and mRNA expression in the epididymides of humans and macaques: utilizing laser microdissection/RT-PCR.
SPAM1 (PH-20) protein and mRNA expression in the epididymides of humans and macaques: utilizing laser microdissection/RT-PCR.
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DOI：
10.1186/1477-7827-1-54
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发表时间：
2003-08-06
期刊：
影响因子：
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通讯作者：
Martin-DeLeon, Patricia A
中科院分区：
文献类型：
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作者：
Evans, Eric A;Zhang, Hong;Martin-DeLeon, Patricia A
背景技术：精子粘附分子1（SPAM 1）是一种重要的精子表面透明质酸酶，在哺乳动物受精过程中至少具有三种功能。以前，我们的实验室报告说，在小鼠中，除了在睾丸中的表达，Spam 1是在附睾中合成的，在那里它被发现在所有三个区域的上皮细胞的主细胞的膜囊泡。由于SPAM 1是广泛保守的哺乳动物之间的研究的目的是，以确定其在附睾中的表达模式是保守的啮齿动物和灵长类动物。方法：我们使用激光显微切割（LM）/RT-PCR冷冻和石蜡包埋的人类（n = 3）和猕猴（n = 2）的附睾切片以及原位转录杂交，以确定是否转录本存在于附睾上皮。Western分析和免疫组化检测并证实蛋白表达，透明质酸底物凝胶电泳分析其透明质酸酶活性。也进行了计算机分析的近端启动子的SPAM 1，以确定相关的推定的转录结合位点的雄激素receptor.RESULTS：我们表明，mRNA独特的SPAM 1是存在于主细胞的附睾上皮在所有个人的两个物种的研究。SPAM 1蛋白存在于附睾的所有三个区域以及输精管中，并且与转录本类似地定位。SPAM 1显示在pH 7.0下具有透明质酸酶活性。在SPAM 1的近端启动子被发现推定的附睾转录因子结合位点，包括雄激素受体元件（战神），与附睾expressions.CONCLUSIONS一致：这些研究结果使我们能够得出结论，附睾SPAM 1是保守的至少两个哺乳动物类，啮齿动物和灵长类动物。这种表达的保守性表明该蛋白可能在精子成熟中发挥重要作用。
BACKGROUND: The Sperm Adhesion Molecule 1 (SPAM1) is an important sperm surface hyaluronidase with at least three functions in mammalian fertilization. Previously our laboratory reported that in the mouse, in addition to its expression in the testis, Spam1 is synthesized in the epididymis where it is found in membranous vesicles in the principal cells of the epithelium in all three regions. Since SPAM1 is widely conserved among mammals the aim of the study was to determine if its expression pattern in the epididymis is conserved in rodents and primates.METHODS: We used laser microdissection (LM)/RT-PCR on frozen and paraffin-embedded epididymal sections of humans (n = 3) and macaques (n = 2) as well as in situ transcript hybridization to determine if transcripts are present in the epididymal epithelium. Western analysis and immunohistochemistry were used to detect and confirm the protein expression, and hyaluronic acid substrate gel electrophoresis analyzed its hyaluronidase activity. An in silico analysis of the proximal promoter of SPAM1 was also performed to identify relevant putative transcription binding sites for the androgen receptor.RESULTS: We demonstrate that mRNA unique to SPAM1 is present in the principal cells of the epididymal epithelium in all individuals of both species studied. SPAM1 protein is present in all three regions of the epididymis, as well as the vas deferens, and is localized similarly to the transcripts. SPAM1 was shown to have hyaluronidase activity at pH 7.0. In the proximal promoter of SPAM1 were uncovered putative epididymal transcription factor binding sites including androgen receptor elements (AREs), consistent with epididymal expression.CONCLUSIONS: These findings allow us to conclude that epididymal SPAM1 is conserved in at least two mammalian classes, rodents and primates. This conservation of expression suggests that the protein is likely to play an important function, possibly in sperm maturation.