Figure 2. Evidence that spermatozoa release MSP. (A) Western blot. Males and mated females contain MSP, but unmated females do not. Minor N- and C-terminal fragments (*N and *C) result from scission of MSP during the boiling step of lysate preparation (data not shown). The male lysate was overexposed to visualize *N (center lane, 15 seconds exposure time). (B,C) Detection of MSP (red) in the proximal gonad arm of mated females (left panels in B). MSP extends beyond the distal constriction (arrowhead) of the spermatheca (sp). A sharp boundary in staining intensity is observed between the -1 and -2 oocytes (arrow). DNA (blue) is shown in the merged images (lower panels in B). No MSP staining is seen in unmated females (right panels in B). The unmated control was overexposed to visualize the outline of the gonad. The relative fluorescence intensity of the MSP signal is shown in C. (D) The distance that the MSP signal extends from spermatozoa in mated females (*P<0.001, error bars represent s.d.). (E) The relative intensity of the MSP signal (fold above background) in the proximal gonad. *<0.02, when compared to all the other measurements shown. P>0.15, when compared with the other
emo-1(
oz1) mated female values, but P<0.05, when compared with the unmated female controls. (F) Punctate distribution of extracellular MSP. Projections of confocal 3D data stacks from mated females prepared by gonad dissection (left panel, MSP is red) or whole-mount fixation (right panel, MSP is pink and DNA is red). Large MSP puncta (arrows) are outside spermatozoa (s) in both the spermatheca (left panel, sp) and the uterus (u). More diffuse MSP fills the spermatheca (left panel) and extracellular spaces surrounding embryos (e, right panel). No MSP is observed in the distal gonad (dg). (G) MSP puncta (arrows) in close proximity to spermatozoa (s) in the uterus, detected by wide-field microscopy. Note the extended pseudopod (ps, bottom panels) and the sperm DNA (blue). Scale bars: B,C, 10 um; F, 5 um; G, 10 um.