Spermatozoa acquire their fertilizing capacity during a complex maturation process that occurs in the epididymis. This process involves a substantial remodeling of the proteins and lipids located at the surface of the gamete. Epididymis can be divided into three regions (the caput, corpus, and cauda) or into 19 intra-regional segments based on histology. Most studies carried out on the evolution of the lipid composition during epididymal maturation has been performed on sperm samples or tissue extracts.
Here, we used MSI to study the precise localization of lipids directly in the rat epididymis tissue. The spatial segmentation of molecular information provided by MSI revealed that the rat epididymis could be divided into 21 molecular clusters different from the 19 intra-regional segments. The discriminative m/zvalues that contributed the most to each molecular cluster were then annotated and corresponded mainly to phosphatidylcholines, phosphatidylethanolamines, triacylglycerols, plasmalogens, phosphosphingomyelins, glycerophosphates, lysophosphatidylethanolamines and lysophosphatidylcholines.
Although phosphatidylcholines, triacylglycerols, phosphatidylethanolamines were predominantly detected in the epididymis caput, an important increase in the number of plasmalogens, phosphosphingomyelins and lysophosphatidylcholines annotated in the cauda was observed. MALDI images reveal that molecules belonging to the same family can have 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.
Work is currently ongoing to integrate our high quality in-situ lipid-distribution dataset with quantitative proteomes obtained for the three main epididymal regions, with the aim to identify the key enzymes involved in lipid metabolism within the organ. This pioneering work opens new perspectives for elucidating the role of lipid metabolism in sperm maturation during its epididymal transit.