Comparison of detergent-solubilized membrane and soluble proteins from flow cytometrically sorted X- and Y-chromosome bearing porcine spermatozoa by high resolution 2-D electrophoresis.
The only known and measurable difference between X- and Y-chromosome bearing spermatozoa is the small difference in their DNA content. The X sperm in the human carry 2.8% more DNA than the Y sperm, while in domestic livestock this difference ranges from 3.0 to 4.2%. The only successful sperm separation method, flow cytometric sorting, is based on this difference in DNA content. Using this technique, X and Y sperm populations with purities greater than 90% can be obtained. The number of spermatozoa that can be sorted in a given time period, however, is too low for application of this technique in routine artificial insemination. Therefore, the search for a marker other than DNA to differentiate between X and Y sperm remains of interest in order to develop a method for large scale X and Y sperm separation. The aim of the present study was to investigate whether porcine X and Y sperm contain some difference in their plasma membrane proteins. The flow cytometric sorting of sperm enabled a direct comparison of the proteins of the X and Y sperm populations. High resolution two-dimensional (2-D) electrophoresis was used; however, adaptations were needed to enable its use for analysis of proteins of flow cytometrically sorted sperm, both in the sorting procedure, membrane protein solubilization, and in the 2-D electrophoresis. Up to 1000 protein spots per gel could be detected and quantified. Comparison of the 2-D protein patterns revealed differences in protein spots between sperm of two individual boars. However, no differences in protein spots between the X and Y sperm fractions were found. These results provide additional support for the view that X- and Y-chromosome bearing spermatozoa are phenotypically identical, and cast doubt on the likelihood that a surface marker can provide a base for X and Y sperm separation.
Reproduction Department, DLO-Institute for Animal Science and Health, Lelystad, The Netherlands.
P J
PJ
Hendriksen
G R
GR
Welch
J A
JA
Grootegoed
T
T
Van der Lende
L A
LA
Johnson
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002793:Cholic Acids / Q000737:chemistry*; D003902:Detergents / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005434:Flow Cytometry / Q000379:methods*; D008297:Male; D008565:Membrane Proteins / Q000032:analysis*; D013094:Spermatozoa / Q000378:metabolism*; D013552:Swine; D014960:X Chromosome; D014998:Y Chromosome
D002793:Cholic Acids; D003902:Detergents; D008565:Membrane Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate