Dynamic relocation of PKA regulatory subunits during sperm capacitation: Linking PKA to the CatSper signaling complex

Abstract

To fertilize an oocyte, mammalian spermatozoa must undergo a maturation step known as capacitation that takes place after ejaculation. Protein kinase A (PKA) plays a fundamental role in capacitation in all mammalian species. Before capacitation, PKA is maintained in an inactive state where the catalytic subunits are bound to a dimer of inhibitory regulatory subunits. A key element in the regulation of PKA lies in its intracellular compartmentalization achieved by docking at A-kinase anchoring proteins (AKAP). Despite the crucial role of the modulation of local PKA activity in fertilization, its localization and mechanism of compartmentalization are not well understood. Here, we approach this problem using quantitative laser scanning microscopy and superresolution imaging to dissect the interactions and relocalization of PKA subunits during capacitation. We find that in the resting state, both catalytic and regulatory subunits colocalize close to the axoneme. Upon capacitation, the PKA regulatory subunits, but not the catalytic subunits, relocate to a quadrilateral structure along the flagellum principal piece, in the vicinity of AKAP4 and the CatSper channel signaling complex. Furthermore, this quadrilateral localization of PKA regulatory subunits disappears in sperm from CatSper1 knockout mice. The sharp difference between the localization of PKA regulatory subunits in capacitated vs. noncapacitated sperm cells demonstrates its suitability as a biomarker for identifying capacitation, an enduring problem in the study of sperm physiology.

Keywords: Cdc42; DNA-PAINT; fertilization; protein kinase A (PKA); superresolution.

Fig. 1.

Pairwise two-color imaging of PKA-C, PKA-RII, and AKAP4 in capacitated and noncapacitated mouse sperm cells as obtained by immunofluorescence with LSM. In all images, the sperm head is on the right end, and the first fluorescent region of the flagellum (on the right side) is the mid-piece. In each section, the Lower Left panel shows a detailed zoom of the image. The Lower Right panel shows the intensity profile of the region shown in the Lower Left, across a plane that crosses the center of the flagellum. (A) Representative immunofluorescence of PKA-C and PKA-RII in a noncapacitated cell. The intensity cross-section exhibits a single-peak Gaussian-like profile for both labels. (B) PKA-C and PKA-RII in a cell stimulated with capacitation-inducing medium. The intensity cross-section appears as a single peak for PKA-C and as a double-peak distribution for PKA-RII. (C) AKAP4 and PKA-RII in a noncapacitated cell. The intensity cross-section appears as a single peak for PKA-RII and a double-peak distribution for AKAP4. (D) AKAP4 and PKA-RII in a cell stimulated with capacitation-inducing medium. The intensity cross-section profile shows two peaks for AKAP4 and PKA-RII. Scale bars for the Upper and Lower panels are 5 µm and 0.5 µm, respectively.

Fig. 2.

Quantification of the width of the distributions of PKA-RII, PKA-C, and AKAP4 in the sperm principal piece. (A) Sketch showing how the FWHM are computed in single-peak and double-peak distributions. (B) Box plots for the widths of PKA-RII, PKA-C, and AKAP4 in noncapacitated cells (NC, magenta) and in cells incubated in capacitation-inducing media (CAP, blue). The whiskers indicate 5th and 95th percentiles. (C) Scatter plot of AKAP4 width vs. PKA-RII width in noncapacitating and capacitating media. The dashed line indicates both parameters are equal. A correlation is observed between the two widths in a subset of the cells in capacitation-inducing medium. This subset likely corresponds to capacitated cells. (D) Scatter plot of PKA-C width vs. PKA-RII width in noncapacitating and capacitating media. The dashed line indicates both parameters are equal. A correlation is observed between the two widths in the cells incubated in noncapacitating medium and in a subset of the cells in capacitation-inducing medium. This subset likely corresponds to noncapacitated cells.

Fig. 3.

DNA-PAINT 3D superresolution imaging of PKA-C, PKA-RII, and AKAP4 in the sperm principal piece. (A) Representative superresolution images of PKA-C and PKA-RII in a noncapacitated cell. (B) PKA-C and PKA-RII in a cell stimulated with capacitating medium. (C) AKAP4 and PKA-RII in a noncapacitated cell. (D) AKAP4 and PKA-RII in a cell stimulated with the capacitation-inducing medium. The Upper panels show individual cross sections (yz plane) for each channel and a merged image of both cross sections. The Lower panels show the longitudinal view of the superresolution images along the principal piece (xy plane). In all images, PKA-C is shown in red, PKA-RII in cyan, and AKAP4 in yellow. Scale bars for the Upper and Lower panels are 200 and 500 nm, respectively.

Fig. 4.

Representative DNA-PAINT 3D superresolution image of PKA-R and Cdc42 in the sperm principal piece of a capacitated cell. The Upper panels show individual cross sections (yz plane) for each channel and merged images of both cross sections. The Lower panel shows the longitudinal view of the superresolution two-color image along the principal piece (xy plane). Scale bars for the Upper and Lower panels are 200 and 500 nm, respectively.

Fig. 5.

Two-color imaging of AKAP4 and PKA-RII in CatSper1 KO and HET mouse sperm cells that were incubated in capacitation-inducing media. (A) Image of the HET cell. (B) Intensity profiles of the cross-section of the HET cell showing that both AKAP4 (yellow) and PKA-RII (cyan) exhibit two peaks. (C) Image of CatSper1 KO cell. (D) Intensity profile of the cross-section of CatSper1 KO cell showing that AKAP4 exhibits two peaks but not PKA-RII. (E) Quantification of the widths of PKA-RII and AKAP4 in capacitated CatSper1 KO and HET cells, incubated in capacitation-inducing media. (Scale bars are 5 µm.)