Sequence Analysis of the 24-kDa Antigen from Cauda Spermatozoa
To obtain clues to the functionality of the 24-kDa form of 2D6 antigen, it was subjected to a primary sequence analysis followed by database searches to identify potential orthologues in other tissues. For this purpose, the reduced 24-kDa subunit was excised from nonreducing/reducing two-dimensional PAGE gels and trypsinized in situ, and released peptides were subjected to mass spectrometry website
Uptake of 2D6-Reactive Antigens from CEP: Cell and Species Specificity of Binding
To investigate the specificity of binding of secreted 2D6 antigens to the sperm plasma membrane and the conditions that regulate it, immature spermatozoa from the proximal caput epididymidis were resuspended in CEP(1 mg/ml) diluted in PBS, pH 7.4, and incubated in vitro. As shown in Figure 11A, a significant amount of 2D6-reactive antigens had bound to spermatozoa after 60 min and were localized to the tail (Fig. 11B). Prior dialysis of CEP against PBS had no significant effect on binding (Fig. 11A), nor had the presence of 20 mM DTT or 0.5 M KCl (results not shown). Uptake was also cell-type specific, as neither erythrocytes nor lymphocytes stained with 2D6 McAb after incubation in CEP (Fig. 12, A-D). However, when mouse proximal caput spermatozoa were incubated with rat CEP, they became 2D6 reactive (Fig. 12, E-H). Similar results were obtained with hamster caput spermatozoa (results not shown).
FIG. 11. A) FACS analysis of immature spermatozoa from the proximal caput epididymidis after incubation in CEP. Prior dialysis of CEP has no effect on uptake of 2D6 antigen. B) Immature spermatozoa incubated in CEP stain positively with 2D6 McAb over the tail with a patchy distribution similar to those matured in vivo in the cauda region, a-d X 212. a and b) Proximal caput sperm stained with 2D6 McAb/FITC-RAM. A weak reaction is present on the head of 25-30% of sperm. c and d) Proximal caput sperm after incubation in CEP for 60 min. Left-hand side, phase contrast. Right-hand side, fluorescence.
FIG. 12. Absorption of 2D6 antigen from CEP is cell-type specific. Proximal caput spermatozoa, red blood cells, and lymphocytes (rat) were incubated in CEP for 60 min and stained with 2D6 McAb/FITC-RAM. A) Cells incubated in CEP + FITC-RAM. B) Cells incubated in CEP + 2D6 McAb + FITC-RAM. C) Cells incubated with FITC-RAM only. D) Cells not incubated in CEP but stained with 2D6 McAb + FITC-RAM. Immature mouse spermatozoa incubated in rat CEP also absorbed 2D6 antigen. E and F) Mouse proximal caput sperm before incubation in CEP. G and H) Mouse proximal caput spermatozoa after incubation in CEP for 60 min. Left-hand side, phase contrast. Right-hand side, fluorescence. E, F, G, and H: X 262.