Hyperactivated sperm motility driven by CatSper2 is required for fertilization

Abstract

Elevations of sperm Ca2+ seem to be responsible for an asymmetric form of motility called hyperactivation, which is first seen near the time of fertilization. The mechanism by which intracellular Ca2+ concentrations increase remains unknown despite considerable investigation. Although several prototypical voltage-gated calcium channels are present in spermatozoa, they are not essential for motility. Furthermore, the forward velocity and percentage of motility of spermatozoa are associated with infertility, but their importance relative to hyperactivation also remains unknown. We show here that disruption of the gene for a recently described sperm-specific voltage-gated cation channel, CatSper2, fails to significantly alter sperm production, protein tyrosine phosphorylation that is associated with capacitation, induction of the acrosome reaction, forward velocity, or percentage of motility, yet CatSper2-/- males are completely infertile. The defect that we identify in the null sperm cells is a failure to acquire hyperactivated motility, which seems to render spermatozoa incapable of generating the "power" needed for penetration of the extracellular matrix of the egg. A loss of power is suggested also by experiments in which the viscosity of the medium was increased after incubation of spermatozoa in normal capacitating conditions. In high-viscosity medium, CatSper2-null spermatozoa lost the ability to swim forward, whereas wild-type cells continued to move forward. Thus, CatSper2 is responsible for driving hyperactivated motility, and, even with typical sperm forward velocities, fertilization is not possible in the absence of this highly active form of motility.

Fig. 1.

Targeted disruption of the mouse CatSper2 gene. (a) A schematic representation of a portion of the endogenous CatSper2 gene, the targeting vector, and the expected recombinant mutation. Exons are indicated by the numbered bars. The gene-specific probes (P5 and P3), restriction sites used for Southern blot analysis of homologous recombination (SphI for P5 and BamHI and SacI for P3), and expected restriction fragments are shown. The underlined restriction sites originate from the targeting vector. Neomycin (N) and thymidine kinase (TK) selection cassettes are shown. WT, wild type. (b Upper) Southern blot analysis of SM1 genomic DNA showing restriction fragments with the predicted sizes (kb) using both gene-specific probes. Data are representative of three independent clones. (b Lower) Mouse genotyping by PCR with genotype-specific primers showing the expected products. (c) SDS polyacrylamide gel electrophoresis (4–20% gradient) of mouse sperm total protein (1 million cells per lane) stained with Coomassie blue (Left) and the same samples (electrophoresed on an 8% SDS polyacrylamide gel) probed with the CatSper2 C-terminal antibody (Right), demonstrating the absence of CatSper2 in homozygous null (–/–) mice. Relative migration of protein standards (M_r × 10³) is shown. (d) Immunocytochemistry of methanol-fixed mouse sperm with the C-terminal CatSper2 antibody. In b–d, the genotypes are wild type (+/+), heterozygote (+/–), and null (–/–).

Fig. 2.

CatSper2 inactivation results in complete male infertility under normal breeding and in vitro conditions. (a) Failure of fertilization after normal matings. Numbers above the bars indicate the number of animals mated. (b) In vitro fertility of CatSper2 homozygous null male mice (n = 3 animals for each genotype). The numbers above the bars indicate the total number of eggs examined.

Fig. 3.

Incubation under capacitating conditions results in normal protein tyrosine phosphorylation patterns in both wild-type (WT) and CatSper2-null spermatozoa. (Upper) Anti-phosphotyrosine immunoblot of spermatozoa from CatSper2 wild-type and null males that were incubated for t = 0 and t = 90 min in conditions that promote capacitation. Relative migration of protein standards (M × 10³ r) is shown. (Lower) The same immunoblot probed with an anti-tubulin antibody as a loading control after removing the anti-phosphotyrosine and secondary antibodies.

Fig. 4.

CatSper2 wild-type (WT) and null sperm cell motility parameters. (a–c) Average forward velocity of WT and null spermatozoa at 5 min and 90 min after swim-out. (d) Average percentage of motility of WT and null spermatozoa. The data shown in a–d are the sperm cell population means ± SEM (n = 6 animals for each genotype). (e–h) Plots of sperm cell track velocity (VCL) vs. straightness of trajectory (STR), defined as the linear velocity (VSL) divided by the path velocity (VAP), multiplied by 100. Higher values indicate a straighter trajectory. Each data point represents an individual sperm cell (n = 240 from six animals for each genotype). The different symbols represent spermatozoa from different animals. The boxes define regions containing high-velocity nonlinear trajectory spermatozoa delimited by the 90th percentile track velocity and 10th percentile STR values from the noncapacitated wild-type spermatozoa at t = 5 min. All spermatozoa within these boxes were hyperactivated.

Fig. 5.

CatSper2 null spermatozoa can fertilize eggs without an extracellular matrix but cannot penetrate a viscous environment. (a) Removal of the extracellular matrix of the egg allows CatSper2 null mice to fertilize eggs in vitro (n = 3 animals for each genotype). Numbers above the bars indicate the total number of eggs examined. ZP, zona pellucida. (b) Increased viscosity of the medium does not block wild-type (WT) sperm from swimming but does block the motility of CatSper2 null spermatozoa after incubation in conditions that promote capacitation. The values indicated are expressed as the mean ± SD sperm cell path velocities of the various samples. The open bars represent normal medium, and the filled bars represent medium containing 0.75% (wt/vol) long chain polyacrylamide.

Fig. 6.

A hypothetical model for initiation of sperm cell hyperactivated motility. Undefined female factors are proposed to initiate a signaling mechanism that opens that CatSper channel(s), allowing calcium entry into the sperm cell flagellum. Calcium then interacts with components of the axoneme to modify the flagellar beat from a symmetric to an asymmetric form.