CatSper3−/− and CatSper4−/− male mice are infertile, and their sperm fail to hyperactivate. (a) WT, CatSper3−/−, or CatSper4−/− males (four each) were mated to two WT females over 3 months. WT males fathered 14 litters, compared with no litters for CatSper3−/− and CatSper4−/− males. As expected for WT 129Sv mice, an average of approximately eight pups per litter were born. (b) Initially, the percentage of motile sperm from WT and mutant mice was comparable. Over 90 min, 50–70% of isolated sperm cells from WT mice remained motile, whereas ≈80% of spermatozoa from CatSper−/− mice lost their motility. (c) In standard computer-assisted sperm analysis measurements of path velocity (VAP, velocity of the averaged path), linear velocity, and track velocity, WT and CatSper-null sperm cells were initially similar, with significant differences by 90 min. (d) Mature spermatozoa from WT, CatSper3−/−, or CatSper4−/− mice have normal morphology. (e) Measurement of bending angle (inset, α) shows that capacitated WT sperm cells have larger ranges of motion than CatSper3−/− and CatSper4−/− sperm cells. Thus, mutant mice spermatozoa lack this aspect of hyperactivated motility.