New Insights Into Sperm Ultrastructure Through Enhanced Scanning Electron Microscopy

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.

Genetic mutation ofCep76results in male infertility due to abnormal sperm tail composition

“…Images were taken either on a Jeol 1400 Plus electron microscope at the Vera and Clive Ramaciotti Centre for Electron Microscopy (Monash University, Australia), or on a Talos L120C or a FEI Teneo VolumeScope at the Ian Holmes Imaging Center (The University of Melbourne). To view the mitochondria, annulus, and fibrous sheath structure of sperm via SEM, sperm were isolated from the cauda epididymis and incubated in 100 μl of 1× PBS for 30 min to strip the plasma membrane, then processed as outlined in reference ( 76 ).…”

CEP76 is a critical regulator of male germ cell transition zone function and tail composition

“…Images were taken either on a Jeol 1400 Plus electron microscope at the Vera and Clive Ramaciotti Centre for Electron Microscopy (Monash University, Australia), or a Talos L120C or a FEI Teneo VolumeScope at the Ian Holmes Imaging Center (The University of Melbourne). To view the mitochondria, annulus, and fibrous sheath structure of sperm via SEM, sperm were isolated from the cauda epididymis and incubated in 100 µl of 1 x PBS for 30 min to strip the plasma membrane then processed as outlined in [69].…”

AXDND1 is required to balance spermatogonial commitment and for sperm tail formation in mice and humans

“…Images were taken on a Jeol 1400 Plus electron microscope at the Vera and Clive Ramaciotti Centre for Electron Microscopy (Monash University, Australia), a Talos L120C or a FEI Teneo VolumeScope at the Ian Holmes Imaging Cente (Bio21 Institute, Parkville, Australia). To view the sperm tail ultrastructure, sperm were isolated from the cauda epididymis and incubated in 100 µl of 1 x PBS for 30 min to strip the plasma membrane as previously described (45). Samples were then diluted in 0.1 M sodium cacodylate buffer and settled onto gold coated glass.…”