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. Author manuscript; available in PMC: 2010 Mar 1.
Published in final edited form as: J Cell Physiol. 2009 Mar;218(3):522–536. doi: 10.1002/jcp.21624

Fig. 2.

Fig. 2

Localization of sterols in murine and human sperm. (A) Schematic showing the recombinant PFO peptides used for localization studies. Constructs were made producing the sterol-binding D4 domain of PFO fused with EGFP (PFO-D4; amino acids 363-472). A non-functional control construct was also made in which the sterol-binding region of the D4 domain was truncated (PFO-D4-T; Δ429). (B) Live murine epididymal sperm labeled with PFO-D4. Panels show an epifluorescence and corresponding DIC image of a live swimming sperm (note the movement of the tail) labeled using PFO-D4. There was clear segregation of the PFO-D4 fluorescence to the APM. There was no change in localization in dead cells. (C) Live murine sperm labeled with PFO-D4-T. Panels show an epifluorescence and corresponding DIC image of a live swimming sperm (again, note the movement of the tail) labeled using PFO-D4-T. There was no binding of this EGFP-conjugated non-functional truncated mutant protein confirming the binding specificity of PFO-D4. (D) Live human ejaculated sperm labeled with PFO-D4. Panels show an epifluorescence and corresponding DIC image of a live swimming sperm. Segregation of the PFO-D4 fluorescence to the APM was conserved in human sperm. (E) Fluorescence localization of filipin-sterol complexes in a fixed murine sperm. Panels show an epifluorescence and corresponding DIC image; there is sterol-enrichment in the APM domain of sperm in agreement with the observation in live cells. All panels show representative data obtained from at least 3 separate trials, with multiple cells observed per trial.