Two distinct Ca(2+) signaling pathways modulate sperm flagellar beating patterns in mice

Abstract

Hyperactivation, a swimming pattern of mammalian sperm in the oviduct, is essential for fertilization. It is characterized by asymmetrical flagellar beating and an increase of cytoplasmic Ca(2+). We observed that some mouse sperm swimming in the oviduct produce high-amplitude pro-hook bends (bends in the direction of the hook on the head), whereas other sperm produce high-amplitude anti-hook bends. Switching direction of the major bends could serve to redirect sperm toward oocytes. We hypothesized that different Ca(2+) signaling pathways produce high-amplitude pro-hook and anti-hook bends. In vitro, sperm that hyperactivated during capacitation (because of activation of CATSPER plasma membrane Ca(2+) channels) developed high-amplitude pro-hook bends. The CATSPER activators procaine and 4-aminopyridine (4-AP) also induced high-amplitude pro-hook bends. Thimerosal, which triggers a Ca(2+) release from internal stores, induced high-amplitude anti-hook bends. Activation of CATSPER channels is facilitated by a pH rise, so both Ca(2+) and pH responses to treatments with 4-AP and thimerosal were monitored. Thimerosal triggered a Ca(2+) increase that initiated at the base of the flagellum, whereas 4-AP initiated a rise in the proximal principal piece. Only 4-AP triggered a flagellar pH rise. Proteins were extracted from sperm for examination of phosphorylation patterns induced by Ca(2+) signaling. Procaine and 4-AP induced phosphorylation of proteins on threonine and serine, whereas thimerosal primarily induced dephosphorylation of proteins. Tyrosine phosphorylation was unaffected. We concluded that hyperactivation, which is associated with capacitation, can be modulated by release of Ca(2+) from intracellular stores to reverse the direction of the dominant flagellar bend and, thus, redirect sperm.

FIG. 1

Flagellar beating patterns in mouse sperm. Images are individual frames taken from the Supplemental Movies A–E. The upper row of images (A–E) shows the maximal pro-hook flagellar bends after various treatments, whereas the lower row of images (F–J) shows the maximal anti-hook flagellar bends. A and F) Sperm hyperactivated under capacitating conditions. B and G) Uncapacitated sperm treated with 5 mM procaine. C and H) Uncapacitated sperm treated with 4 mM 4-AP. D and I) Uncapacitated sperm treated with 100 μM thimerosal. E and J) Control (uncapacitated sperm treated with medium).

FIG. 2

Percentage of motile sperm swimming in dominant pro- and anti-hook beating patterns after each treatment. Data are from three males and are presented as the mean ± SEM. For all treatments, an asterisk (*) indicates a significant difference (P < 0.01) between the percentage of dominant pro-hook and anti-hook patterns for each treatment. Cap, capacitated sperm; Pro, 5 mM procaine; 4-AP, 4 mM 4-AP; Thi, 100 μM thimerosal; Con, control sperm.

FIG. 3

Percentage of motile sperm swimming in dominant pro- and anti-hook beating patterns after treatment with a combination of pharmacological agents. A) Sperm were treated with 100 μM thimerosal after achieving maximal response to 5 mM procaine or 4 mM 4-AP or after incubation under capacitating conditions for 2 h. B) Sperm were treated with 5 mM procaine or 4 mM 4-AP after achieving maximal response by 100 μM thimerosal. Data are from three males and are presented as the mean ± SEM. An asterisk (*) indicates a significant difference (P < 0.01) between the percentage of dominant pro-hook and anti-hook patterns for each treatment.

FIG. 4

A) Pseudocolor images of fluorescence of the Ca²⁺ indicator Fluo-4 in sperm treated with 4 mM 4-AP in the presence of 2 mM Ca²⁺ (a) or sperm treated with medium as a control (b), or with 100 μM thimerosal in the presence of 30 μM Ca²⁺ (c) or sperm treated with medium as a control (d). Images are colorized individual frames taken from Supplemental Movies F, G, H, and I, respectively. Elapsed time is indicated above the images, and arrows indicate time of addition. The base of the flagellum is indicated by triangles, and the cytoplasmic droplet is indicated by arrowheads. Warmer colors indicate higher Ca²⁺. B) Pseudocolor images of fluorescence of pH indicator BCECF in sperm treated with 4 mM 4-AP (a), 100 μM thimerosal (b), or medium control (c). Images are colorized individual frames taken from Supplemental Movies J, K, and L, respectively. Elapsed time is indicated above the images, and arrows indicate time of addition. In a, the arrowheads indicate the increased signal in the principal piece; in b and c, the arrowheads indicate the proximal principal piece. Warmer colors indicate higher pH.

FIG. 5

The fluorescence of the pH indicator BCECF in sperm populations monitored using a microplate reader. Arrow indicates time of addition. Data are from three males and are presented as the mean ± SEM.

FIG. 6

Percentage of acrosome-reacted sperm in response to treatments with procaine, 4-AP, thimerosal, and medium control. The positive control is A23187, a Ca²⁺ ionophore. Data are from three males and are presented as the mean ± SEM. An asterisk (*) indicates a significant difference (P < 0.01) from the medium (negative) control. Con, control sperm; Thi, 100 μM thimerosal; Pro, 5 mM procaine; A23, 20 μM A23187; 4-AP, 4 mM 4-AP.

FIG. 7

One-dimensional electrophoresis followed by Western blot detection of proteins phosphorylated on tyrosine after various treatments. Con, control sperm; Thi, 100 μM thimerosal; Pro, 5 mM procaine; 4-AP, 4 mM 4-AP; Cap, capacitated sperm.

FIG. 8

Two-dimensional electrophoresis followed by Western blot detection of proteins phosphorylated on serine and threonine after various treatments. A) Capacitated sperm. B) 5 mM procaine. C) 4 mM 4-AP. D) 100 μM thimerosal (circles indicate major reductions in phosphorylation compared with E). E) Control sperm.

FIG. 9

Immunofluorescent localization of proteins phosphorylated on serine and threonine after various treatments. Immunofluorescence images are shown on the left and corresponding bright-field images on the right. For phosphothreonine images, the principal piece of sperm is indicated by triangles, and the head is indicated by arrowheads. For phosphoserine images, the base of the flagellum is indicated by triangles. A) 5 mM Procaine. B) 4 mM 4-AP. C) 100 μM thimerosal. D) Medium control. Bar = 10 μm.