High-resolution helium ion microscopy of epididymal epithelial cells and their interaction with spermatozoa

Abstract

We examined the rat and mouse epididymis using helium ion microscopy (HIM), a novel imaging technology that uses a scanning beam of He+ ions to produce nanometer resolution images of uncoated biological samples. Various tissue fixation, sectioning and dehydration methodswere evaluatedfor theirability to preservetissuearchitecture.Thecaudaepididymidiswas luminallyperfusedin vivo to removemost spermatozoa and the apical surface of the epithelial lining was exposed. Fixed epididymis samples were then subjected to critical point drying (CPD) and HIM. Apical stereocilia in principal cells and smaller apical membrane extensions in clear cellswere clearly distinguishable in both rat and mouse epididymis using this technology. After perfusion with an activating solution containing CPT-cAMP, a permeant analog of cAMP, clear cells exhibited an increase in the number and size of membrane ruffles or microplicae. In contrast, principal cells did not exhibit detectable structural modifications. High-resolutionHIMimaging clearly showed the ultrastructure of residual spermcells, including the presence ofconcentric ringsonthe midpiece, and of cytoplasmic droplets in some spermatozoa.Close epithelium-sperm interactionswere also detected.We found a number of sperm cells whose heads were anchored within the epididymal epithelium. In certain cases, the surface of the spermcytoplasmic droplet was covered with vesicle-like structureswhose size is consistentwith that of epididymosomes. In conclusion,we describe here the first application ofHIMtechnology to the study of the structure andmorphology of the rodent epididymis. HIMtechnology represents amajor imaging breakthrough that can be successfully applied to study the epididymis and spermatozoa, with the goal of advancing our understanding of their structure and function.