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Heads you win, tails you lose: filtration processing for the microscopical examination of sperm heads

Summary

The sperm head plays a key role in many fertilisation events and determining the precise location of molecules within the head region is important in mechanistically dissecting the fertilisation process. Such molecules may be present in low copy number and many sperm head profiles must be examined to localise them to particular subcellular structures with confidence.

Filtration has traditionally been used for the purpose of concentrating biological material, such as free‐living cells, spores, and subcellular fractions, and little attempt has been made to extend the procedure to encompass the entire processing schedule, mainly due to the incompatibility of intermediate dehydrating solvents with membrane filters. The novel and simple technique of filtration processing that we describe produced a dense mat of cells, with several sperm heads being visible in coronal orientation in a high‐power field at the light microscopic level, and allowed positive immunocytochemical staining to be identified with confidence. This new technique exploits the low viscosity of LR White acrylic resin to allow the entire processing procedure to be undertaken in the filtration apparatus. In contrast, conventional techniques for preparing free‐living cells, namely pre‐embedding in a supportive matrix prior to processing, and centrifugation at each stage of the processing procedure, proved suboptimal, partly due to the final concentration that could be achieved, but mainly due to the random orientation of cells that these techniques afforded.

Lay description

Conventional approaches for processing free‐living cells into resin for subsequent sectioning and examination by light and electron microscopy involve pre‐embedding in a supportive matrix or repeated centrifugation during the processing procedure to prevent loss of material. Here we describe a novel, simple and effective technique for processing sperm cells into resin by conducting the entire process in the filtration apparatus. This technique proved to be far superior to conventional procedures in terms of cell concentration. As a consequence, the heads of several sperm could be clearly seen in coronal orientation by light microscopy, and positive immunocytochemical staining of the sperm heads could be identified with confidence at the light microscopic level. This technique can, in principle, be applied to other samples of free‐living cells, spores, or cellular components, and may be of particular value where only small amounts of material are available for study.

Authors:   I. A. BREWIS, M. A. WATKINSON, C. J. VON RUHLAND
Journal:   Journal of Microscopy
Year:   2016
Pages:   n/a
DOI:   10.1111/jmi.12505
Publication date:   24-Nov-2016
Facts, background information, dossiers
  • Matrix
  • light
  • fertilisation
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