CatSper ion channels and CatSper3 and CatSper4 localization. (a) Simplified homology tree of voltage-gated ligand ion channels. One representative member of each channel family was chosen and aligned by using an identity matrix with ClustalW (Ver. 1.4). (b) In situ hybridization using CatSper3- and CatSper4-specific probes shows mRNA in specific stages within the testis. CatSper mRNAs were not detected in some stages of spermatogenic cells along the testis seminiferous epithelium, in contrast to the testis sections stained with sense probes. (c) Immunostaining of mouse epididymal sperm with anti-CatSper4 antibody. Labeling of the sperm head is nonspecific, as shown by comparison with the CatSper4^−/− sperm; CatSper4 is specifically labeled only in the principal piece of the tail (arrowheads).

I_CatSper was absent in CatSper^−/− sperm. (a) Sperm (WT) whole-cell current evoked by a 1-s ramp from −100 to +100 mV, holding potential (HP) = 0 mV. The Na⁺-peak current recorded at −100 mV in DVF conditions was −970 pA and decreased to −13 pA in 2 mM Ca²⁺. (b) Monovalent current through CatSper channels (DVF solution) was absent in CatSper^−/− sperm cells. (c) Average of the Na⁺ current in DVF solution measured from WT and CatSper^−/− sperm cells.

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.

Interactions between CatSper proteins. (a) Epitope-tagged CatSper2 (FLAG-CS2), CatSper3 (HA-CS3), or CatSper4 (FLAG-CS4) were transfected into an MZ8 cell line stably expressing CatSper1 (CS1). FLAG-CatSper2, HA-CatSper3, and FLAG-CatSper4 were detected in the respective cell lysates (Left). After immunoprecipitation with anti-HA and -FLAG antibodies, immune complexes were probed with anti-CatSper1 antibody (Right). Negative controls were lysates from CatSper1 cells transfected with FLAG-GIRK4 or HA-TRPV6. (b) Proteins solubilized from testis microsomes were immunoprecipitated with specific anti-CatSper antibodies. Anti-CatSper3 and -CatSper4 pulled down CatSper3 and CatSper4 from WT testes but not from homozygous mutant testes (Left). These antibodies also pulled down their respective proteins from CatSper1^−/− mice (Center). Anti-CatSper1 coimmunoprecipitated CatSper3 and CatSper4 in the same protein complexes from WT but not from CatSper1^−/− testes (Right). Bottom Left shows CatSper1 in preparations used for immunoprecipitations; Bottom Right shows immunoprecipitation input control with anti-Na, K-ATPase, a plasma membrane protein.