Figure 1. CatSper1 is specifically processed during in vitro capacitation.

(A–B) CatSper1 undergoes post-translational modification during spermiogenesis and epididymal maturation. (A) A gradual decrease in electrophoretic mobility of CatSper1 is observed by western blot analysis. (B) Mouse CatSper1 is an O-glycosylated protein. Apparent molecular weights of CatSper1 proteins were analyzed by immunoblotting recombinant CatSper1 expressed in 293 T cells (CS1) and native CatSper1 from whole cell lysate (WCL) of testis and sperm (non-capacitated, 0 min; capacitated, 90 min) compared with those of CatSper in sperm membrane fraction treated with or without O-glycosidase (O-Gly). The dotted line indicates different exposure time of the same membrane. (C) CatSper resides in lipid rafts subdomains of the plasma membrane in mature sperm. Solubilized sperm proteins were fractionized by discontinuous sucrose density gradient (5, 30, and 40%) centrifugation. (D) CatSper1 is degraded during the late stage of capacitation. Protein expression levels of CatSper1 and caveolin-1, but not CatSper3, CatSperε, or carbonic anhydrase 4 (CAIV) are altered by in vitro capacitation. (E–G) CatSper1 is cleaved within the N-terminal domain (NTD). (E) A cartoon of full-length (FL, top) and N-terminal truncated (ND, bottom) recombinant CatSper1 protein expressed in the study (left). Both proteins are tagged with HA at their respective C-termini (orange). The CatSper1 antibody used in this study is raised against the 1–150 aa region of CatSper1 (blue, Ren et al., 2001). Detection of recombinant FL-CatSper1 and ND-CatSper1 expressed in 293 T cells (right). (F) A cartoon of the experimental scheme to test NTD truncation of CatSper1. FL-CatSper1 and ND-CatSper1 expressed in 293 T cells were solubilized and pulled-down using agarose resin conjugated with HA antibody. The enriched recombinant proteins were incubated with solubilized sperm lysates at 37°C for 0, 10, 30, and 60 min and subjected to immunoblot. (G) FL-CatSper1 is cleaved at NTD by solubilized sperm lysate. FL-CatSper1 (gray arrow) decreases while truncated form (red arrow) increases by incubation with solubilized sperm lysates (top). ND-CatSper1 proteins remain largely unchanged under the same conditions (bottom). The right panel shows quantification of the protein levels by measuring the band intensity of target proteins at each time point, normalized by total FL-CatSper1 at 0 min points (n = 3; intact, gray bars; cleaved, red bars) or ND-CatSper1 (n = 4; black dots). The sum of intact and cleaved FL-CatSper1 levels was used for total FL-CatSper1. Statistical analyses were performed between relative levels at each time point within each protein. Different letters indicate the significant difference. Data is represented as mean ± SEM. See also Figure 1—source data 1. Immunoblotting was performed with the HA antibody (E and G). (H) Capacitation-associated CatSper1 degradation is regulated by phosphorylation. CatSper1 degradation is accelerated by PKA inhibition. A PKA inhibitor, H89 (50 μM), enhances capacitation-associated CatSper1 degradation. A protein phophatase1 inhibitor, calyculin A (Caly A, 0.1 μM), prevents the CatSper1 degradation during sperm capacitation in vitro. (I–J) Ca²⁺ influx accelerates CatSper1 degradation. (I) CatSper activation during capacitation and Ca²⁺ ionophore treatment (A23187, 10 μM) facilitates the CatSper1 cleavage. (J) Representative immunofluorescence images of CatSper1 in the spermatozoa incubated under the conditions used in (I). The extent of CatSper1 degradation is heterogeneous in the capacitated sperm cells (bottom) compared with A23187-treated uncapacitated sperm cells (middle). (K) Capacitation-associated CatSper1 degradation is blocked by calpain inhibitors (I, II, and III). 20 μM of each calpain inhibitor was treated with sperm during capacitation. Blots shown here are representative of three independent experiments.

Figure 1—figure supplement 1. Molecular mechanism for CatSper1 down-regulation during sperm capacitation.

(A) CatSper1 is not a phosphoprotein. Treatment of sonication-solubilized cauda sperm (1 × 10⁶ cells) with protein phosphatase 1 (PP1, 0.1 unit), protein tyrosine phosphatase (PTP1B, 5 units), and PTP inhibitor (VO₄^3-, 1 mM) does not change the molecular weight of CatSper1. (B) N-glycosidase, PNGase F, does not change the electrophoretic mobility of CatSper1 from sperm membrane fraction. (C) CatSper complex in the flagellar membrane is compartmentalized and protected from a protease. The membrane fraction from cauda sperm cells were incubated with 0.1% Triton X-100 (Tx) in PBS at 4°C for 0, 5, 10, 15, and 20 min. The addition of Triton X-100 results in cleavage within the CatSper1 N-terminus. N-terminal truncations of CatSper1 were not observed in sperm directly lysed in the sampling buffer. (D) Inhibition of PKA-anchoring to AKAP by st-HT31 peptide (10 μM) accelerates CatSper1 degradation in capacitating sperm. (E) CatSper1 processing involves two distinct signaling pathways. Treatment with Ca²⁺ ionophore A23187 (10 μM, blue) accelerates CatSper1 processing in the presence of PKA inhibitor, H89 (50 μM, gray) in non-capacitating spermatozoa (top, NC). Chelating intracellular Ca²⁺ by loading sperm cells with BAPTA-AM (10 μM, brown) does not affect CatSper1 processing regardless of PKA inhibition under capacitating conditions (bottom, Cap). (F) A model proposes the molecular mechanism of full-length CatSper1 downregulation in capacitated sperm cells. Increase in intracellular pH activates the CatSper channel in sperm undergoing capacitation CatSper1-degrading protease X is activated by the CatSper-mediated rise in intracellular Ca²⁺ and cleaves the -terminal domain of CatSper1. We speculate that the activity of the Ca²⁺-dependent protease X can be further modulated by PKA phosphorylation cascades (i.e. dephosphorylation by PP1/PP2A); Ca²⁺ suppresses PKA activation (dotted lines). The cleaved N-terminal fragment from CatSper1 is subject to degradation. The CatSper channel containing the N-terminal truncated CatSper1 might become inactive. (G) A representative immunofluorescence image of conglomerated sperm in the uterus. The uterus was collected after 8 hr coitus, subjected to clearing, and immunostained. Sperm cells with intact or processed Catsper1 are visible in the uterus. Blots shown here are representative of three independent experiments.