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Type: Journal article
Title: Isolation and partial characterization of the outer dense fibres and fibrous sheath from the sperm tail of a marsupial: the brushtail possum (Trichosurus vulpecula)
Author: Ricci, M.
Breed, W.
Citation: Reproduction, 2001; 121(3):373-388
Publisher: Journals of Reproduction Fertility Ltd
Issue Date: 2001
ISSN: 1470-1626
Abstract: The flagellum of a mammalian spermatozoon consists of a central axoneme surrounded by two cytoskeletal structures, the outer dense fibres and the fibrous sheath, which may aid in sperm motility or stability. In this study the outer dense fibres and fibrous sheath were isolated and partially characterized in a marsupial species, the brushtail possum (Trichosurus vulpecula). Spermatozoa from the cauda epididymidis were decapitated by sonication, and the head and tail fractions were separated by centrifugation over a 20, 40 and 60% (w/v) sucrose density gradient. After confirming sperm tail purity by Nomarski microscopy, the tails were incubated in either SDS-dithiothreitol to isolate the outer dense fibres or urea-dithiothreitol to isolate the fibrous sheaths. Purified outer dense fibres and fibrous sheaths were solubilized in SDS and beta-mercaptoethanol and proteins were separated by one-dimensional PAGE. Coomassie blue staining showed that the outer dense fibres were composed of seven major proteins (molecular masses: 73, 58, 55, 54, 52, 41 and 16 kDa), and the fibrous sheath was composed of 12 major proteins (molecular masses: 106, 76, 66, 62, 55, 53, 52, 46, 40, 30, 28 and 16 kDa). A polyclonal antibody to the fibrous sheath proteins showed strong crossreactivity with those of fibrous sheath from spermatozoa of several other marsupial species, as well as those from laboratory rats. Subsequent western blotting identified the immunoreactive 76 and 62 kDa proteins from all species, thus indicating their high conservation between species. No crossreactivity of the fibrous sheath antibody to any other cytoskeletal structures, including the outer dense fibres, mid-piece fibre network or connecting laminae, or to the acrosome or underlying subacrosomal material, was evident, indicating that the fibrous sheath proteins are localized to this structure alone. Further work is in progress to determine the extent of homology of these proteins to those in eutherian mammals.
Keywords: Epididymis
Sperm Tail
Sodium Dodecyl Sulfate
Microscopy, Immunoelectron
Fluorescent Antibody Technique, Indirect
Blotting, Western
Centrifugation, Density Gradient
Description: Copyright © 2001 Society for Reproduction and Fertility
Provenance: This journal supersedes Journal of Reproduction and Fertility and Reviews of Reproduction.
DOI: 10.1530/rep.0.1210373
Published version:
Appears in Collections:Anatomical Sciences publications
Aurora harvest
Environment Institute publications

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