Electrophoretic Gel Analysis of Hoechst 33342 Stained and Flow Cytometrically Sorted Bovine Sperm Membrane Proteins
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Little information is available concerning the influence that Hoechst 33342 staining and flow cytometric sorting procedures have on the sperm membrane. The objective of this study was to investigate whether the current methods used to stain and flow-separate sperm into separate populations affect the membrane protein composition. Bovine sperm were diluted in HEPES-PVA buffer and incubated unstained (US) or stained with Hoechst 33342 (S) at 5 g per 10 10 6 sperm for 1 h at 35C. As a control (C), sperm were washed once in HEPES-PVA buffer with no incubation. Stained sperm were also sorted (S sort ) using an EPICS V/750 series flow cytometer/cell sorter, modified for sperm sorting. Sperm membranes from the four treatments were harvested utilizing deoxycholic acid (Na salt) treatment. Membrane proteins harvested from 30 10 6 sperm were lyophilized and separated by one- and two-dimensional gel electrophoresis. Membrane protein banding profiles from one-dimensional separation for the four groups showed three variations. A 97 kDa membrane protein seen in C, US and S sperm membranes was absent in S sort sperm. A doublet protein of seminal plasma origin (110 kDa), appeared as two distinct bands in US and S, but was modified in C and S sort . Another doublet protein of seminal plasma origin (180 kDa), was present in C, US and S samples but not in S sort sperm membranes. Two-dimensional electrophoretic protein maps illustrated a loss of seminal plasma proteins, of various molecular weights and pH values, in C and possibly S sort , which were present in US and S: 66 kDa, pH 5.5; 45 kDa, pH 5.5; 30 kDa, pH 4.5-5.5 proteins. Additionally, there were seminal plasma proteins of low molecular weight (< 29 kDa, pH 6.0, 6.3 and 6.9) which appeared to remain adsorbed to the sperm surface. A protein at 41 kDa, pH 6.1 was present in C, but absent in US and S maps, and a decrease in the amount of a 51 kDa, pH 6.5 protein was observed. The appearance of two proteins in US and S maps (66 kDa, pH 6.1 and 29 kDa, pH 6.8), were not seen in C protein maps. Proteins present on the sperm surface appear to change during the process of staining and sorting. These membrane changes may influence sperm function and thus sperm-oocyte interactions. 1996 Blackwell Wissenschafts-Verlag, Berlin.
