The ultrastructural analysis of spermatozoa morphology is critically important to understand male fertility and the aetiology of infertility, including evolutionary processes [1]. Modern scanning electron microscopy (SEM) provides visualization of external and internal (using an automated ultra-microtome or focused ion beam to serially remove thin layers from the surface of the resin embedded sample) with nanometre resolution. To utilize this possibility, the protocols of sample preparation should be revised to provide the ultimate preservation of the ultrastructural features.
We have developed and validated two protocols of sample preparation to examine of the external morphology of sperm (with preservation of the plasma membrane and with its removal) and two high-pressure freezing and freeze-substitution based protocols for examination of the internal morphology of sperm [2].
The developed advanced sample preparation protocols and visualization using a high-resolution FEG-SEM (ThermoFisher Elstar G4 operating at accelerating voltage of 2 kV) 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.
We believe this is the first example of three-dimensional ultrastructural analyses of the sperm using FIB-SEM tomography. This technique provides the full-volume reconstruction of cells and can dramatically improve the understanding of the structure-function relationship in sperm, including allowing accurate three-dimensional topology of relevance to sperm motility research and ultimately the understanding of sperm competition. We believe the developed protocols have the potential to accelerate discovery in the relationship between sperm structure and function, including the analysis of the consequences of genetic and environmental factors.