Figure 7.

OVS deliver elevated levels of PMCA1 to capacitated, compared to uncapacitated, sperm following co-incubation with OLF/OVS. (A) Uncapacitated (uncap) and capacitated (cap) sperm were co-incubated with OLF or PBS (control) for 3 h and after washing processed for indirect IF. Flow cytometry was performed after validating the specificity of the anti-PMCA1 antibody, using the IgG isotype (Supplemental Fig. S2). (a) For uncap sperm in PBS (control), only 12.7% showed fluorescence intensity (FI) at the highest level (marked region), while in OLF there was 27% (a > 2-fold increase) (b). (c) For OLF with cap sperm, 67.7% of the cells had FI in the marked region, >2-fold increase, compared to uncap in OLF. (B) The average frequencies of sperm with the highest FIs in three experiments showed significant differences between co-incubation in PBS and OLF (P < 0.05) for uncap, and for cap and uncap in OLF (P = 0.001). (C) Confocal images of cap and uncap sperm immuno-labeled for PMCA1 following co-incubation in PBS (a, d) and OLF (b, e), IgG isotype control is seen in (c). The PMCA1 signal is most intense in (b, e). The sperm heads are stained with DAPI in the middle panel followed by the merged image. (f) Analysis of ~200 sperm/group show significant (χ² = 5.33; P = 0.02) differences between the number of immuno-labeled cap and uncap cells in OLF, while highly significantly more positive cells were seen for uncap (χ² = 112.58; P < 0.001) and cap (χ² = 789.95; P < 0.001) in OLF vs PBS. (Original mag. = ×630).