Denis Korneev (1,2,3), D. Jo Merriner (1), Gediminas Gervinskas (2), Alex de Marco (2,4), Moira K. O’Bryan (1,3)
Frontiers in Cell and Developmental Biology, April 2021. DOI: 10.3389/fcell.2021.672592

Keywords

sperm; electron microscopy; high-pressure freezing; focused ion beam; male infertility; male fertility

Abstract

The analysis of spermatozoa morphology is fundamental to understand male fertility and the etiology of infertility. Traditionally scanning electron microscopy (SEM) has been used to define surface topology. Recently, however, it has become a critical tool for three-dimensional analysis of internal cellular ultrastructure. Modern SEM provides nanometer-scale resolution, but the meaningfulness of such information is proportional to the quality of the sample preservation. In this study, we demonstrate that sperm quickly and robustly adhere to gold-coated surfaces. Leveraging this property, we developed three step-by-step protocols fulfilling different needs for sperm imaging: chemically fixed monolayers for SEM examination of the external morphology, and two high-pressure freezing-based protocols for fast SEM examination of full cell internal morphology and focused ion-beam SEM tomography. These analyses allow previously unappreciated insights into mouse sperm ultrastructure, including the identification of novel structures within the fibrous sheath and domain-specific interactions between the plasma membrane and exosome-like structures.

How Our Software Was Used

Dragonfly was used to process and visualize FIB-SEM data stacks.

Author Affiliation

(1) School of Biological Sciences, Monash University, Melbourne, VIC, Australia.
(2) Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, VIC, Australia.
(3) Faculty of Science, School of BioSciences, University of Melbourne, Melbourne, VIC, Australia.
(4) ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, Melbourne, VIC, Australia.