Spermatozoa acquire their fertilizing capacity during a complex maturation process that occurs in the epididymis. This process involves a substantial molecular remodeling at the surface of the gamete. Epididymis is divided into three regions (the caput, corpus, and cauda) or into 19 intraregional segments based on histology. Most studies carried out on epididymal maturation have been performed on sperm samples or tissue extracts. Here, we used MALDI imaging mass spectrometry in the positive and negative ion modes combined with spatial segmentation and automated metabolite annotation to study the precise localization of metabolites directly in the rat epididymis. The spatial segmentation revealed that the rat epididymis could be divided into several molecular clusters different from the 19 intraregional segments. The discriminative m/z values that contributed the most to each molecular cluster were then annotated and corresponded mainly to phosphatidylcholines, sphingolipids, glycerophosphates, triacylglycerols, plasmalogens, phosphatidylethanolamines, and lysophosphatidylcholines. A substantial remodeling of lipid composition during epididymal maturation was observed. It was characterized in particular by an increase in the number of sphingolipids and plasmalogens and a decrease in the proportion of triacylglycerols annotated from caput to cauda. Ion images reveal that molecules belonging to the same family can have very different localizations along the epididymis. For some of them, annotation was confirmed by on‐tissue MS/MS experiments. A 3D model of the epididymis head was reconstructed from 61 sections analyzed with a lateral resolution of 50 μm and can be used to obtain information on the localization of a given analyte in the whole volume of the tissue.

中文翻译：

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MALDI成像质谱法对大鼠附睾精子成熟的空间分割和代谢物注释

精子在附睾中发生的复杂成熟过程中获得其受精能力。该过程涉及配子表面的大量分子重塑。附睾根据组织学分为三个区域（头、体和尾）或 19 个区域内段。大多数关于附睾成熟的研究都是在精子样本或组织提取物上进行的。在这里，我们在正负离子模式下使用 MALDI 成像质谱仪，结合空间分割和自动代谢物注释，直接研究代谢物在大鼠附睾中的精确定位。空间分割显示，大鼠附睾可分为不同于19个区域内片段的几个分子簇。歧视性的m/z然后注释对每个分子簇贡献最大的值，并主要对应于磷脂酰胆碱、鞘脂、甘油磷酸酯、三酰甘油、缩醛磷脂、磷脂酰乙醇胺和溶血磷脂酰胆碱。观察到附睾成熟过程中脂质成分的大量重塑。它的特点是鞘脂和缩醛磷脂的数量增加，而从头到尾注释的三酰基甘油的比例减少。离子图像显示属于同一家族的分子沿着附睾具有非常不同的定位。对于其中一些，通过组织上 MS/MS 实验确认了注释。