Sperm motility loss and FM4-64 fluorescence dynamics in acrosome-reacted transgenic EGFP-DsRed2 sperm.

A) Representative time series of transgenic EGFP-DsRed2 sperm attached to concanavalin A-coated coverslips, with AE induced by 100 μM progesterone. White squares indicate cells with spontaneous AE (prior to induction), while pink squares highlight cells with progesterone-induced AE. A schematic representation of AE in this transgenic model is shown on the right side of the panel. Scale bar = 20 μm. B) Representative time series of transgenic EGFP-DsRed2 sperm that have already experienced AE, attached to laminin-coated coverslips. The upper panel displays a cell with motility after AE, and the lower panel shows an immotile cell. DsRed2 is presented in orange, and EGFP in green. Scale bar = 10 μm. C) Quantification of motile and immotile acrosome-reacted sperm (EGFP-). A total of 235 cells were counted across at least three independent experiments. D) Representative time series of transgenic EGFP-DsRed2 sperm stained with 10 μM FM4-64 and attached to concanavalin A-coated coverslips, with AE induced by 100 μM progesterone. White squares indicate cells exhibiting patterns I, II, or III after progesterone induction. Scale bar = 20 μm. A schematic representation of AE in this transgenic model stained with FM4-64 is shown on the right side of the panel. E-G) Representative images of capacitated transgenic EGFP-DsRed2 sperm stained with 10 μM FM4-64. Panel E) displays an acrosome-intact, motile sperm (Pattern I), F) shows an acrosome-reacted sperm with motility and low FM4-64 midpiece fluorescence (Pattern II), and G) presents an acrosome-reacted sperm with no motility and high FM4-64 midpiece fluorescence (Pattern III). In all three cases, cells were induced with 100 μM progesterone. Scale bar = 10 μm. Enlarged images of each pattern are shown on the right panel. Scale bar = 2 μm. Representative images from at least five independent experiments are displayed.

Midpiece contraction coincides with the onset of AE.

A) Left panel displays a wide-field fluorescence image of capacitated CD1 sperm membrane stained with 0.5 μM FM4-64, while the right panel shows its super-resolution SRRF reconstruction. Scale bar = 1 μm. B) Representative time series of sperm midpiece with no AE (no reac), spontaneous exocytosis (spont), progesterone (prog, 100 μM) and ionomycin-induced (iono, 10 μM) exocytosis, respectively. Following acquisition, images were analyzed using SRRF. Insets in the sperm head show wide-field images of AE. The midpiece diameter value is displayed in the bottom left corner for each time point. Scale bar = 1 μm. C) Quantification of midpiece diameter changes for each experimental group across time. Data are presented as a percentage of the initial diameter value before induction for each cell. D-E) Quantification of FM4-64 fluorescence in the sperm head and midpiece, respectively, for each experimental group across time. Data are presented as times of increases compared to initial fluorescence before AE induction. *p<0.05; #p<0.01; $p<0.001 and &p<0.0001 compared to the non-reacted group. A nonparametric Kruskal-Wallis test was performed in combination with Dunn’s multiple comparisons test. Representative images of at least 5 independent experiments are shown, with 36 cells analyzed. F) Comparison of the midpiece architecture in acrosome-intact (AI, upper panel) and acrosome-reacted (AR, lower panel) sperm using scanning electron microscopy (SEM). Representatives images are shown, middle panels show quantification of these images whereas the left panel shows the quantification of all replicates. Data is presented as mean ± SEM, Kruskal-Wallis test was employed, p = 0.013, (AI n=85, AR n=72). Scale bar = 2 μm. G) Capacitated transgenic EGFP-DsRed2 sperm were induced by 100 µM progesterone. Cells were fixed and immunostained against -GLUT3 in order to see the plasma membrane in the midpiece. Representative images of at least 2 independent experiments are shown. Left panel shows quantification of midpiece diameter in acrosome-intact and acrosome-reacted EGFP-DsRed2 sperm. Data is presented as mean ± sem. A nonparametric Mann Whitney test was performed, ****p<0.001 (AI n=84, AR n=47). Scale bar = 5 μm.

Contraction initiation preferentially occurs near the head-midpiece junction.

A) Schematic diagram illustrating the generation of super-resolution kymographs from SRRF-processed images. Crosslines are drawn every 2.5 μm through the sperm midpiece, and the Image-J Kymograph builder plug-in is used to create kymographs. The x-axis represents time, and the y-axis shows diameter changes. For wide-field images, a line along the midpiece is drawn to create fluorescence kymographs, with the y-axis representing midpiece length. Three sections of the midpiece are defined: proximal [0-7 μm], central [7-14 μm], and distal [14-21 μm]. B) Relative frequency graph displaying the distribution of the initiation sites for midpiece contractions in sperm with no AE (no reac), spontaneous exocytosis (spont), progesterone-induced (prog, 100 μM) exocytosis, and ionomycin-induced (iono, 10 μM) exocytosis, respectively. The x-axis indicates the midpiece section where the contraction begins: proximal (P), central (C), or distal (D). A chi2 test was performed using the R language environment. C-D) Representative contraction kymographs and diameter measurements for progesterone-induced (100 μM) AE with one or two contraction initiation sites, respectively. In contraction kymographs, yellow lines demarcate midpiece sections, and colored spots indicate where super-resolution kymographs were created. Both kymograph and diameter measurement graphs display a dotted vertical line marking the induction point. For C, horizontal scale bar = 5 min and vertical scale bar = 1 μm and for D, horizontal scale bar = 3 min and vertical scale bar = 1 μm. Data from at least 5 independent experiments are shown, with 36 cells analyzed.

Midpiece contraction is driven by [Ca2+]i changes.

A) The representative time series demonstrates [Ca2+]i and AE dynamics. Capacitated F1 sperm, loaded with Fluo4 AM, were immobilized on concanavalin A-coated coverslips and incubated in a recording medium (rec) containing 10 μM FM4-64. Rec was added as indicated by arrowheads. Scale bar = 10 μm. Beneath each frame in the Fluo4 (green) and FM4-64 (red) images, a color code displays the normalized intensity of the fluorescence signal (scale bar on the right of the panel). B) Kymograph-like analysis of the midpiece of 20 sperm following the addition of recording medium. Each row depicting the [Ca2+]i (upper) and membrane (lower) dynamics of a single cell over time. A white dotted line indicates the moment of addition. The images presented are representative of at least five independent experiments. C) The representative time series demonstrates [Ca2+]i and AE dynamics. Progesterone (prog, 100 μM) was added as indicated by arrowheads. Scale bar = 10 μm. Beneath each frame in the Fluo4 (green) and FM4-64 (red) images, a color code displays the normalized intensity of the fluorescence signal (scale bar on the right of the panel). D) Kymograph-like analysis of the midpiece of 20 sperm following the addition of prog. Each row depicting the [Ca2+]i (upper) and membrane (lower) dynamics of a single cell over time. A white dotted line indicates the moment of addition. The images presented are representative of at least five independent experiments. Consistently, a [Ca2+]i transient precedes contraction, which is proportional to the increase in FM4-64 fluorescence, as shown in Figure S2D. E) Representative time series of [Ca2+]i and midpiece contraction dynamics. Capacitated CD1 sperm were loaded with Fluo4 AM, immobilized on concanavalin A-coated coverslips, and incubated in a recording medium containing 0.5 μM FM4-64. AE was induced with 100 μM progesterone (prog, arrowhead). Fluo4 images are widefield images, while FM4-64 images are SRRF-processed (super-resolution). Scale bar = 1 μm. F) 3D kymographs of [Ca2+]i (left) and contraction (right) dynamics. Data are normalized to the mean of the frames before the induction of AE. Representative images from at least 5 independent experiments are shown, with 36 cells analyzed.

The flagellar membrane approaches the actin cytoskeleton in the midpiece of the sperm flagellum during midpiece contraction and AE.

A-B) Representative time series of plasma membrane and actin cytoskeleton colocalization in the midpiece in the absence of AE (A) and during progesterone-induced AE (B, prog, 100 μM; FM4-64 shown in red, SiR-actin shown in cyan). Capacitated CD1 sperm were loaded with 100 nM SiR-actin, immobilized on concanavalin A-coated coverslips, and incubated in a recording medium containing 0.5 μM FM4-64. Scale bar = 1 μm. Representative images from at least 5 independent experiments are shown, with 36 cells analyzed. C) Manders’ colocalization coefficients for acrosome-intact and acrosome-reacted cells in the midpiece. M1 was assigned to FM4-64, and M2 to SiR-actin. Data are presented as mean ± SEM. ****p<0.0001 represents statistical significance. Paired t test was performed. D-G) Representative images of sperm midpiece stained with the acrosome marker PNA (left panel, upper right insets, epifluorescence) and phalloidin (actin filaments, STORM) for acrosome-intact (D-E) and acrosome-reacted (F-G) cells. The left panel displays a longitudinal section of the midpiece (Scale bar = 10 μm), while the right panel illustrates the radial distribution (Scale bar = 0.2 μm). E and G) Schematics of the analyzed actin double helix parameters in the midpiece: helical pitch (l, distance between turns of the helix, left panel), helical pitch frequency (f0, number of turns the helix makes per 1 μm), and radial distribution (R, radius of the double helix, right panel). Representative images from at least 3 independent experiments are shown. Four acrosome-intact cells and seven acrosome-reacted cells were analyzed.

Occurrence of midpiece contraction in sperm located within the perivitelline space.

Representative images of IVF experiments using EGFP-DsRed2 sperm. Oocyte-sperm complexes were stained with 10 μg/ml Hoechst and 10 μM FM4-64. A) Representative time series of DIC images showing a sperm moving within the perivitelline space. Scale bar in right panel = 20 μm, scale bar in left panel = 10 μm. B) DIC, Hoechst, EGFP, DsRed2*, and FM4-64 images are shown for the case depicted in Figure 6A, note that, as the sperm is moving, it is located in a different position in the perivitelline space. The area depicted in the upper panel is shown in higher magnification in the lower panel. Scale bar in upper panel = 20 μm, scale bar in lower panel = 10 μm. C-D) DIC, Hoechst, EGFP, DsRed2*, and FM4-64 images are shown for a (C) sperm that have passed through the ZP, displaying AE with an initially non-contracted midpiece. After 20 minutes, as shown in (D), the midpiece becomes contracted. The area depicted in the upper panel is shown in higher magnification in the lower panel. Scale bar in upper panel = 20 μm, scale bar in lower panel = 10 μm. Representative images from at least 6 independent experiments are shown. A total of 23 oocytes and 69 sperm were analyzed.

Contraction of the midpiece occurs after sperm-egg fusion.

A) Schematic representation of the acquisition settings for the sperm-oocyte fusion assay. Images were taken every 7 μm along the z-axis. B) DIC image of a sperm-oocyte complex, with the area depicted in higher magnification in panel C. Scale bar = 20 μm. C) Representative time series of sperm-oocyte fusion assay experiments using EGFP-DsRed2 sperm. Oocytes were stained with 1 μg/ml Hoechst and 10 μM FM4-64. DIC, Hoechst, EGFP, DsRed2*, and FM4-64 images are shown over time. Scale bar = 10 μm. Note that midpiece contraction occurs after sperm-egg fusion and is proportional to the increase in FM4-64 fluorescence, as shown in Figure S2D, highlighting its potential importance in the fertilization process. Representative images from at least 4 independent experiments are displayed.

Midpiece contraction occurs in sperm-egg fusion after a decrease in [Ca2+]i.

A) Representative time series of sperm-oocyte fusion assay experiments using wild-type sperm loaded with 1 μM Fluo-4. Oocytes were stained with 1 μg/ml Hoechst and 10 μM FM4-64. DIC, Hoechst, Fluo-4, and FM4-64 images are shown over time. Scale bar = 10 μm. The color code below each frame in the Hoechst (shown in blue), Fluo-4 (shown in green), and FM4-64 (shown in red) images indicates the normalized intensity of the fluorescence signal (scale bar on the right of the panel). B-F) Quantification of sperm showing midpiece contraction (B, indicated by increased FM4-64 fluorescence), midpiece folding (C), midpiece unfolding (D), Fluo-4 fluorescence dynamics in the head (E), and different patterns in the midpiece (F) during fusion. Data are presented as the mean ± SEM of the percentage of sperm counted for each experiment. Representative images and data from at least 3 independent experiments are shown. A total of 74 oocytes and 136 sperm were analyzed. Note that midpiece contraction occurs in sperm-egg fusion after a decrease in Fluo-4 fluorescence.

Proposed model of the structural reorganization of the sperm actin cytoskeleton during key events of fertilization.

The double helix actin network surrounding the mitochondrial sheath of the midpiece undergoes structural changes prior to the motility cessation. This structural modification is accompanied by a decrease in diameter of the midpiece and is driven by intracellular calcium changes that occur concomitant with a reorganization of the actin helicoidal cortex. Although midpiece contraction may occur in a subset of cells that undergo AE (A and B), the midpiece contraction occurs prior to motility cessation observed after sperm-egg fusion (C and D).