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Fig. 3. Localization of exported MSP and specificity of release. (A) Localization
of MSP at the surface of the –1 oocyte. Single confocal sections at the
indicated level of a 3D data stack through a mated female gonad stained for
MSP (red) and RME-2 yolk receptor (green). No spermatozoa were seen in the
indicated region (line) of the spermatheca (sp), thus the staining observed is
extracellular to spermatozoa. A projection of the entire stack is presented as
Movie 3 in the supplementary material. (B) Single angle views of a 3D
reconstruction of the data stack represented in A. The image is cut to show
surface and interior views of the –1 oocyte, at the indicated angles.
Overlap between the MSP (red) and RME-2 (green) signals is yellow. Note the
oocyte surface is slightly compressed where it abuts the spermatheca. The
entire reconstruction is presented as Movie 4 in the supplementary material.
(C) Intact and viable spermatozoa release MSP, as shown by a mated female
stained for DNA (blue), MitoTracker (red) and MSP (green). Note, the
MitoTracker staining is limited to the spermatozoa (s), but the MSP staining
extends at least 50 µm from the most distal spermatozoa (arrowhead). (D)
MSP localizes to extracellular puncta and apparent buds at the spermatozoa
surface. Projections of confocal 3D data stacks from mated females stained in
wholemount for MSP (red) and MSD proteins (green), with overlap in yellow.
Images are superimposed on the DIC channel, showing spermatozoa (s) in the
uterus. Puncta (arrows) and surface blebs (arrowheads) contain MSP, but not
MSD proteins. A 3D projection of similar data is presented in Movie 5 in the
supplementary material. (E) Budding generates MSP puncta. Single angle views
of a 3D reconstruction of MSP (red) and MSD (green) staining, with overlap in
yellow. The image is cut to show interior and surface views of the
spermatozoa. Apparent sites of budding contain MSP but not MSD. The entire
reconstruction is presented as Movie 6 in the supplementary material. Scale
bars: 20 µm.
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