A CUG-initiated CATSPERθ functions in the CatSper channel assembly and serves as a checkpoint for flagellar trafficking

Calcium signaling is critical for successful fertilization. In spermatozoa, calcium influx into the sperm flagella mediated by the sperm specific CatSper calcium channel is necessary for hyperactivated motility and male fertility. CatSper is a macromolecular complex and is repeatedly arranged in zigzag rows within four linear nanodomains along the sperm flagella. Here, we report that the Tmem249-encoded transmembrane domain containing protein, CATSPERθ, is essential for the CatSper channel assembly during sperm tail formation. CATSPERθ facilitates the channel assembly by serving as a scaffold for a pore forming subunit CATSPER4. CATSPERθ is specifically localized at the interface of a CatSper dimer and can self-interact, suggesting its potential role in CatSper dimer formation. Male mice lacking CATSPERθ are infertile because the sperm lack the entire CatSper channel from sperm flagella, rendering sperm unable to hyperactivate, regardless of their normal expression in the testis. In contrast, genetic abrogation of any of the other CatSper transmembrane subunits results in loss of CATSPERθ protein in the spermatid cells during spermatogenesis. CATSPERθ might acts as a checkpoint for the properly assembled CatSper channel complex to traffic to sperm flagella. This study provides insights into the CatSper channel assembly and elucidates the physiological role of CATSPERθ in sperm motility and male fertility.


Recombinant protein expression in mammalian cells and immunoprecipitation.
Different mouse Catsperq ORF and truncations were subcloned into phCMV3 backbone. CATSPER4 and truncations were subcloned into pcDNA3.1 backbone. HEK293T cells were transiently transfected with indicated plasmids with Lipofectamine 2000 (Invitrogen), following the manufacturer's instruction. Transfected cells were used for co-IP experiments. Briefly, after transfection for 18-20h, cells were lysed with 1% Triton X-100 in PBS containing EDTA-free protease inhibitor cocktail (Roche) by rocking at 4°C for 1h and centrifuged at 18,000 x g for 30 min at 4°C. Solubilized proteins in the supernatant were mixed with anti-HA magnetic beads (Pierce, 88836) at 4°C overnight and washed with 0.1% Triton PBS for three times. Co-IP products were eluted with 2x LDS sampling buffer supplemented with 50 mM dithiothreitol (DTT) and denatured at 75°C for 10 min. Primary antibodies used for the western blotting were rabbit monoclonal anti-HA (CST, #3724), and rabbit polyclonal anti-Flag (CST, #14793). For secondary antibodies, anti-rabbit IgG-HRP (Jackson ImmunoResearch) were used.
Preparation of whole sperm lysate and solubilized protein extracts. Mouse epididymal spermatozoa washed in PBS were directly lysed in 2x SDS sample buffer. Then the whole sperm lysates were centrifuged at 18,000 x g, 4°C for 10 min. After adjusting DTT to 50 mM, supernatant was denatured at 75°C for 10 min before loading to gel.
Mouse sperm preparation and in vitro capacitation. Epididymal spermatozoa from adult male mice were collected by swim-out from caudal epididymis in M2 medium (EMD Millipore, MR-015-D). Collected sperm were incubated in human tubular fluid HTF medium (EMD Millipore, MR-070-D) at 2x10 6 cells/ml concentration to induce capacitation at 37°C, 5% CO2 for 90 min.
Structured illumination microscopy. Structured illumination microscopy (SIM) imaging was performed with Zeiss LSM710 Elyra P1 using alpha Plan-APO 100X/1.46 oil objective lens. Samples were prepared as described in Sperm Immunocytochemistry. z stack images were acquired with 200 nm intervals and each section was taken using 5 grid rotations with a 51 nm SIM grating period and a laser at 561 nm wavelength. Raw images were processed and rendered using Zen 2012 SP2 software (Carl Zeiss).

Motility analysis.
Cauda epididymal spermatozoa were dispersed in a drop of TYH medium and incubated for 10 min and 2 h at 37°C under 5% CO2. Spermatozoa were collected from the top of the drop and analyzed with CEROS II (software version 1.11.9; Hamilton Thorne Biosciences) sperm analysis system. Sperm motility (%) and progressive motility (%) were quantified, and motion parameters including straight line velocity (VSL), average path velocity (VAP) and curvilinear velocity (VCL) were measured. Spermatozoa were considered progressively motile when VSL/VAP ≥ 0.8 and VAP ≥ 50 μm/s.

Antibodies and Reagents.
Rabbit polyclonal antibodies specific to mouse CATSPER1, 3, 4, b, d, z and EFCAB9 were described previously (1-5). To produce antibody recognizing mouse CATSPERq, peptide corresponding to mouse CATSPERq (229-242, TQVYTKSSVNDLDV) was synthesized and conjugated to KLH carrier protein (GenScript). Antisera from the immunized rabbits were affinity-purified using the peptide immobilized SulfoLink Coupling Resin (Thermo, 20401). Anti-HA (CST, #3724), anti-Flag (CST, #14793), anti-acetylated Tubulin (Sigma, T7451). Fitting of CatSper cryo-EM and cryo-ET. The single particle cryo-EM structure and corresponding atomic model of isolated CatSper complex (Lin et al) were fitted as a rigid body into the cryo-ET structure of the in situ CatSper complex (Zhao, et al) using the "fit-in-map" functionality in UCSF chimera. The protein density map of CATSPERq was isolated by "volume zone" functionality in Chimera X-1.3.

Motility correlative Imaging.
Live sperm motility was recorded as in Flagellar waveform analysis, but with photo-etched coverslips (Electron Microscopy Sciences #72265-12). Immediately after video recording, the coverslip was used for immunostaining CATSPERq using the same method as in Sperm immunocytochemistry. Images photographed at the focus of the sperm and the focus of the engraved coordinates on the coverslip from the same field were merged. The cells on the merged images were backtracked to the video to identify the sperm motility.