Oviductosome-Sperm Membrane Interaction in Cargo Delivery DETECTION OF FUSION AND UNDERLYING MOLECULAR PLAYERS USING THREE-DIMENSIONAL SUPER-RESOLUTION STRUCTURED ILLUMINATION MICROSCOPY (SR-SIM)
Oviductosome-Sperm Membrane Interaction in Cargo Delivery DETECTION OF FUSION AND UNDERLYING MOLECULAR PLAYERS USING THREE-DIMENSIONAL SUPER-RESOLUTION STRUCTURED ILLUMINATION MICROSCOPY (SR-SIM)
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DOI：
10.1074/jbc.m114.633156
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发表时间：
2015-07-17
影响因子：
4.8
通讯作者：
Martin-DeLeon, Patricia A.
中科院分区：
文献类型：
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作者：
Al-Dossary, Amal A.;Bathala, Pradeepthi;Martin-DeLeon, Patricia A.
输卵管小体(OVS)、外切体/微囊(OVS)向精子输送钙离子外排泵，质膜钙离子ATPase 4(PMCA4)，在精子受精能力中可能发挥重要作用(Al-Dossary，A.A.，Strehler，E.，和Martin-DeLeon，P.A.(2013)PLOS One 8，e80181)。目前尚不清楚外切体/微囊如何将PMCA4等跨膜蛋白运送到精子。在这里，我们定义了一种新的实验方法，用于在纳米级评估OVS与精子的相互作用，使用亲脂染料(FM4-64FX)和三维SR/SIM，与传统的共聚焦显微镜相比，其体积分辨率提高了8倍。共孵化分析检测到预先标记的OVS与精子融合，主要是在头部和中段。免疫荧光显示PMCA4a在WT和Pmca4KO精子的输卵管胞体内分布，内源性PMCA4a分布在顶体内膜上。透射免疫电子显微镜证实融合，卵母细胞内可见免疫金粒，精子膜上可见融合柄。免疫荧光与αv整合素亚单位共定位于OVS，该亚单位与CD9一起主要位于精子头部和中段。在获能和顶体反应的精子中，通过抗体、外源配体(玻璃连蛋白和纤维连接蛋白)及其RGD型识别基序阻断整合素/配体的相互作用，融合受到显著抑制(p<；0.001)。我们的结果提供了证据，包括精子和卵子上的αvβ3和α5β1整合素的受体/配体相互作用，促进了卵子在将跨膜蛋白输送到精子中的融合。已发现的机制可能还涉及前列腺体、附睾体和子宫体的货物递送。
Oviductosomes ((OVS), exosomes/microvesicles), which deliver the Ca2(+) efflux pump, plasma membrane Ca2(+) ATPase 4 (PMCA4), to sperm are likely to play an important role in sperm fertilizing ability (Al-Dossary, A. A., Strehler, E. E., and Martin-DeLeon, P. A. (2013) PloS one 8, e80181). It is unknown how exosomes/microvesicles deliver transmembrane proteins such as PMCA4 to sperm. Here we define a novel experimental approach for the assessment of the interaction of OVS with sperm at a nanoscale level, using a lipophilic dye (FM4-64FX) and three-dimensional SR/SIM, which has an 8-fold increase in volumetric resolution, compared with conventional confocal microscopy. Coincubation assays detected fusion of prelabeled OVS with sperm, primarily over the head and midpiece. Immunofluorescence revealed oviductosomal delivery of PMCA4a to WT and Pmca4 KO sperm, and also endogenous PMCA4a on the inner acrosomal membrane. Fusion was confirmed by transmission immunoelectron microscopy, showing immunogold particles in OVS, and fusion stalks on sperm membrane. Immunofluorescence colocalized OVS with the alpha v integrin subunit which, along with CD9, resides primarily on the sperm head and midpiece. In capacitated and acrosome reacted sperm, fusion was significantly (p < 0.001) inhibited by blocking integrin/ligand interactions via antibodies, exogenous ligands (vitronectin and fibronectin), and their RGD recognition motif. Our results provide evidence that receptor/ligand interactions, involving alpha v beta 3 and alpha 5 beta 1integrins on sperm and OVS, facilitate fusion of OVS in the delivery of transmembrane proteins to sperm. The mechanism uncovered is likely to be also involved in cargo delivery of prostasomes, epididymosomes, and uterosomes.