FIGURE 4.

Capacitation-associated tyrosine phosphorylation of murine sperm is altered in the presence of DEHP. (A) Representative western blot image shows the time course of protein tyrosine phosphorylation under capacitating conditions in the presence or absence of 10 μM DEHP. DEHP was added to the media immediately before the start of the capacitation process. Sperm lysates were obtained at the indicated times (30, 60, 90, and 120 min) and subjected to SDS-PAGE immunoblotting. Tyrosine phosphorylation was detected with a monoclonal phospho-tyrosine (PY) antibody. Acetylated-tubulin (Ac-Tubulin) was used as a loading control. (B) Levels of relative tyrosine phosphorylation obtained as total densities extracted from (A) and normalized to the densities of the loading control. Each data point represents the mean of one of the three independent experiments. (C) Immunofluorescent localization of tyrosine phosphorylated proteins as visualized by PY antibody. Increased phosphorylation detected after 60 min of capacitation in the mid-piece region of spermatozoa in DEHP- treatment group (right panels) as compared to control untreated spermatozoa (left panels). Lower panels represent insets from the corresponding region of interests indicated on the upper panels by dashed rectangular. (D) A representative flow cytometry data showing an increase in global tyrosine phosphorylation in 10 μM DEHP- treated spermatozoa (red) at 60 min of capacitation compared to the vehicle control (blue). Tyrosine phosphoproteins were detected using a CF 647 dye conjugated to an anti-PY antibody. Inset: fold increase in mean fluorescent intensity normalized to mode as detected by the flow cytometer compared to control conditions. Data are means ± S.E.M. ** indicates statistical significance (P < 0.01) between control spermatozoa and spermatozoa exposed to 10 μM DEHP. *** indicates statistical significance (P < 0.001).