Investigating the role of murine epididymosomes and uterosomes in GPI-linked protein transfer to sperm using SPAM1 as a model
Investigating the role of murine epididymosomes and uterosomes in GPI-linked protein transfer to sperm using SPAM1 as a model
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DOI：
10.1002/mrd.20907
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发表时间：
2008-11-01
影响因子：
2.5
通讯作者：
Martin-Deleon, Patricia A.
中科院分区：
文献类型：
--
作者：
Griffiths, Genevieve S.;Galileo, Deni S.;Martin-Deleon, Patricia A.
精液中的糖基磷脂酰肌醇(GPI)连接蛋白的摄取发生在雄性生殖道和发情期雌性生殖道。在男性中，这归因于分泌到附睾腺和前列腺中的膜性小泡。虽然附睾体的特征已经被描述，但还没有关于女性腔液中存在小泡的报道。在这里，我们报道了小鼠发情雌性生殖液中存在小泡，被描述为“子宫小体”；并使用精子黏附分子1(SPAM1/PH-20)，一种在男性和女性生殖液中发现的众所周知的透明质酸酶作为模型，研究小泡介导的GPI连接蛋白到精子的转移。附睾液(ELF)和子宫腔积液(ULF)超速离心后分离出的附睾体和子宫体，经电子显微镜观察，直径接近10~70 nm，接近15~50 nm。子宫小体的结构完整性通过其对低渗和冻融应激的抗性得到证实；免疫金标记法将SPAM1定位于其外膜表面，附睾体也是如此。SPAM1是在附睾体和子宫体孵育过程中被尾部精子获得的；当GPI锚被酶切割时，摄取被取消。在荧光标记的小泡中孵育后，通过共聚焦和透射电子显微镜(TEM)分析精子，发现SPAM1通常位于鞭毛顶体和中段的膜上。高倍电子显微镜图像显示，小泡并列在精子质膜上，潜在地转移了SPAM1。综上所述，这些结果暗示囊泡对接是囊泡介导的GPI连接蛋白从小鼠生殖液转移到精子的机制。
Sperm uptake of glycosyl phosphatidylinositol (GPI)-linked proteins from luminal fluids has been shown to occur in male and estrous female reproductive tracts. In males, this is attributed to membranous vesicles secreted into the epididymis and prostate. While epididymosomes have been characterized, there have been no reports of the presence of vesicles in female luminal fluids. Here we report the presence of vesicles, characterized as "uterosomes," in the murine estrous female reproductive fluid; and use Sperm Adhesion Molecule 1 (SPAM1/PH-20), a well-known hyaluronidase found in male and female fluids, as a model to investigate vesicle-mediated GPI-linked protein transfer to sperm. Epididymosomes and uterosomes isolated after ultracentrifugation of epididymal (ELF) and uterine luminal fluid (ULF) were analyzed by electron microscopy and shown to be similar to 10-70 and similar to 15-50 nm in diameter. The structural integrity of uterosomes was confirmed by their resistance to hypo-osmotic and freeze/thaw stresses; and immunogold labeling localized SPAM1 to their outer membrane surface, as was the case for epididymosomes. SPAM1 was acquired by caudal sperm during incubation in epididymosomes and uterosomes; uptake was abolished when the GPI anchor was enzymatically cleaved. Sperm analyzed by confocal and transmission electron microscopy (TEM) after incubation in fluorescently labeled vesicles revealed the label on the membrane over the acrosome and midpiece of the flagella, where SPAM1 normally resides. High magnification TEM images demonstrated vesicles juxtaposed to the sperm plasma membrane potentially transferring SPAM1. Taken together, these results implicate vesicular docking as the mechanism of vesicle-mediated GPI-linked protein transfer to sperm from murine reproductive fluids.