Two-dimensional polyacrylamide gel electrophoresis of membrane proteins from flow cytometrically sorted ram sperm

Leahy, T., Marti, J. I., Crossett, B., Evan, G. and Maxwell, W. M. C. (2011) Two-dimensional polyacrylamide gel electrophoresis of membrane proteins from flow cytometrically sorted ram sperm. Theriogenology, 75 5: 962-971. doi:10.1016/j.theriogenology.2010.11.003


Author Leahy, T.
Marti, J. I.
Crossett, B.
Evan, G.
Maxwell, W. M. C.
Title Two-dimensional polyacrylamide gel electrophoresis of membrane proteins from flow cytometrically sorted ram sperm
Journal name Theriogenology   Check publisher's open access policy
ISSN 0093-691X
1879-3231
Publication date 2011-03-15
Year available 2010
Sub-type Article (original research)
DOI 10.1016/j.theriogenology.2010.11.003
Open Access Status Not yet assessed
Volume 75
Issue 5
Start page 962
End page 971
Total pages 10
Place of publication Philadelphia, PA , U.S.A.
Publisher Elsevier
Language eng
Formatted abstract
Membrane proteins orchestrate key events required for participation of sperm in fertilisation. These proteins may be removed or altered due to the mechanical and dilution stressors associated with sex-sorting of sperm. Ram sperm were incubated with Hoechst 33342 and flow-sorted. Sex-selected (viable, orientated) and waste (separated into non-viable or non-orientated) sperm populations were collected, or sperm were not sorted. Sperm membrane proteins were extracted and characterised by one- and two-dimensional PAGE. Densiometric analysis of protein bands separated by one-dimensional PAGE showed proteins of 30 and 28 kDa as doublet bands on non-sorted sperm, and single bands on sex-sorted sperm, and the proportion of a 14 kDa protein was 3-fold higher in non-sorted compared to sorted sperm. Proteins in the 14 kDa band were identified by mass spectroscopy as a bovine Fibronectin type-2 protein (Fn-2), cytochrome oxidase 5a (Cox5a) and a sperm membrane associated protein (SLLP1). The abundance of these proteins in the two-dimensional gels was lowest in the sorted sperm population identified as viable during sorting (orientated and non-orientated sperm) and highest in the non-viable sperm population (P < 0.001). We concluded that the membrane protein profile was different for sex-sorted compared with non-sorted sperm, due to the selection of plasma membrane-intact cells in the flow-sorted population. This provided further evidence that sex-sorting selected a homogenous population of sperm with superior function to non-sorted sperm. Furthermore, this was apparently the first time sperm membrane acrosome associated protein was reported in ram sperm, and it was demonstrated that seminal plasma proteins remained on the sperm membrane after sex-sorting.
Keyword SLLP1
sp32
Fibronectin Type 2
Sex pre-selection
Sex-sorting
Sheep
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes 'The attached [author] copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. Available online 31 December 2010.

Document type: Journal Article
Sub-type: Article (original research)
Collections: Non HERDC
School of Veterinary Science Publications
 
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Created: Tue, 05 Apr 2011, 00:11:28 EST by Dr Tamara Leahy on behalf of School of Veterinary Science