CRIS—A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending
Figure 6
CRIS controls the development of flagellar bending.
(A) Electron micrograph (cross section) of the midpiece (left) and principal piece (right) of the sperm flagellum from wild-type (+/+, top) and Cris-deficient (−/−, bottom) mice. (B) Minimal projection of a cross-section through the midpiece of the sperm flagellum from Cris-deficient (−/−) mice. The corresponding movie (Movie S1) can be found in the supplementary information. (C) Analysis of sperm motility. Velocity of straight line path (VSL) has been determined from the corresponding movies (Movie S2, S3). Data are presented as mean ± s.d. (D) Analysis of PKA- and tyrosine kinase-dependent protein phosphorylation under capacitating conditions and non-capacitating conditions. Per conditions, 1×106 cells were used. Immunoblots have been probed with PKA and tyrosine kinase substrate-specific (4G10) antibodies. Loading control: β-tubulin. (E) Ca2+ imaging of mouse sperm loaded with caged 2-Diazo and Fluo-8. 2-Diazo was released using a UV flash. Shown is a representative trace of a wild-type sperm. Fluorescence has been background-subtracted and normalized to the value before the flash. (F) Flagellar waveform analysis of sperm from Cris-deficient mice loaded with caged 2-Diazo. Representative analysis showing the asymmetry index before and after the UV flash. Corresponding images of the flagellar waveform before and after the UV flash (yellow arrow) are included. (G) Mean values of the asymmetry index for wild-type (blue) and mutant sperm (black). Data are plotted as mean ± s.e.m. Corresponding movies (Movie S4, S5) can be found in the supplementary information (+/+: n = 24, −/−: n = 22). (H) Relative change of asymmetry index for wild-type (blue) and mutant sperm (black). Values after the UV flash were normalized to the values before the flash. Individual data are plotted as circles, the corresponding mean is represented by a red line, and the s.e.m. is indicated with a box, p = 0.0075.