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Isolation and partial characterization of the outer dense fibres and fibrous sheath from the sperm tail of a marsupial: the brushtail possum (Trichosurus vulpecula)

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dc.contributor.authorRicci, M.
dc.contributor.authorBreed, W.
dc.date.issued2001
dc.descriptionCopyright © 2001 Society for Reproduction and Fertility
dc.description.abstractThe 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.
dc.identifier.citationReproduction, 2001; 121(3):373-388
dc.identifier.doi10.1530/rep.0.1210373
dc.identifier.issn1470-1626
dc.identifier.issn1741-7899
dc.identifier.orcidBreed, W. [0000-0003-1739-6593]
dc.identifier.urihttp://hdl.handle.net/2440/32655
dc.language.isoen
dc.provenanceThis journal supersedes Journal of Reproduction and Fertility and Reviews of Reproduction.
dc.publisherJournals of Reproduction Fertility Ltd
dc.source.urihttps://doi.org/10.1530/rep.0.1210373
dc.subjectEpididymis
dc.subjectSperm Tail
dc.subjectCytoskeleton
dc.subjectAnimals
dc.subjectOpossums
dc.subjectMercaptoethanol
dc.subjectSodium Dodecyl Sulfate
dc.subjectMicroscopy, Immunoelectron
dc.subjectFluorescent Antibody Technique, Indirect
dc.subjectBlotting, Western
dc.subjectCentrifugation, Density Gradient
dc.subjectImmunohistochemistry
dc.subjectSolubility
dc.subjectMale
dc.titleIsolation and partial characterization of the outer dense fibres and fibrous sheath from the sperm tail of a marsupial: the brushtail possum (Trichosurus vulpecula)
dc.typeJournal article
pubs.publication-statusPublished

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