The sperm calcium channel CatSper plays a central role in successful fertilization as a primary Ca²⁺ gateway. Here, we applied cryo-electron tomography to visualize the higher-order organization of the native CatSper complex in intact mammalian sperm. The repeating CatSper units form long zigzag-rows along mouse and human sperm flagella. Above each tetrameric channel pore, most of the extracellular domains form a canopy that interconnects to a zigzag-shaped roof. Murine CatSper contains an additional wing-structure connected to the tetrameric channel. The intracellular domains link two neighboring channels to a diagonal array, suggesting a dimer formation. Fitting of an atomic model of isolated monomeric CatSper to the in situ map reveals supramolecular interactions and assembly of the CatSper complex. Loss of EFCAB9-CATSPERζ alters the architecture and interactions of the channels, resulting in fragmentation and misalignment of the zigzag-rows and disruption of flagellar movement in Efcab9^−/− sperm. This work offers unique insights into the structural basis for understanding CatSper regulation of sperm motility.

Although gamete fusion represents the central event in sexual reproduction, the required protein machinery is poorly defined. In sperm cells, Izumo1 and several Izumo1-associated proteins play an essential role for this process. However, so far, the mechanisms underlying transport and maturation of Izumo1 and its incorporation into high molecular weight complexes are incompletely defined. Here, we provide a detailed characterization of the C11orf94 protein, which we rename Frey, which provides a platform for the assembly of Izumo1 complexes. By retaining Izumo1 in the endoplasmic reticulum, Frey facilitates its incorporation into high molecular weight complexes. To fulfill its function, the unstable Frey protein is stabilized within the catalytic center of an intramembrane protease. Loss of Frey results in reduced assembly of Izumo1 complexes and male infertility due to impaired gamete fusion. Collectively, these findings provide mechanistic insights into the early biogenesis and functional relevance of Izumo1 complexes.

A new life starts with successful fertilization whereby one sperm from a pool of millions fertilizes the oocyte. Sperm motility is one key factor for this selection process, which depends on a coordinated flagellar movement. The flagellar beat cycle is regulated by Ca²⁺ entry via CatSper, cAMP, Mg²⁺, ADP and ATP. This study characterizes the effects of these parameters for 4D sperm motility, especially for flagellar movement and the conserved clockwise (CW) path chirality of murine sperm. Therefore, we use detergent-extracted mouse sperm and digital holographic microscopy (DHM) to show that a balanced ratio of ATP to Mg²⁺ in addition with 18 µM cAMP and 1 mM ADP is necessary for controlled flagellar movement, induction of rolling along the long axis and CW path chirality. Rolling along the sperm’s long axis, a proposed mechanism for sperm selection, is absent in sea urchin sperm, lacking flagellar fibrous sheath (FS) and outer-dense fibers (ODFs). In sperm lacking CABYR, a Ca²⁺-binding tyrosine-phosphorylation regulated protein located in the FS, the swim path chirality is preserved. We conclude that specific concentrations of ATP, ADP, cAMP and Mg²⁺ as well as a functional CABYR play an important role for sperm motility especially for path chirality.

Male reproduction depends on hormonally driven behaviors and numerous genes for testis development and spermatogenesis. Neuroplastin-deficient (Nptn^−/−) male mice cannot sire offspring. By immunohistochemistry, we characterized neuroplastin expression in the testis. Breeding, mating behavior, hormonal regulation, testicular development, and spermatogenesis were analyzed in cell-type specific neuroplastin mutant mice. Leydig, Sertoli, peritubular myoid, and germ cells express Np, but spermatogenesis and sperm number are not affected in Nptn^−/− males. Neuroplastin lack from CNS neurons or restricted to spermatogonia or Sertoli cells permitted reproduction. Normal luteinizing hormone (LH) and follicle-stimulating hormone (FSH) blood levels in Nptn^−/− males support undisturbed hormonal regulation in the brain. However, Nptn^−/− males lack mounting behavior accompanied by low testosterone blood levels. Testosterone rise from juvenile to adult blood levels is absent in Nptn^−/− males. LH-receptor stimulation raising intracellular Ca²⁺ in Leydig cells triggers testosterone production. Reduced Plasma Membrane Ca²⁺ ATPase 1 (PMCA1) in Nptn^−/− Leydig cells suggests that Nptn^−/− Leydig cells produce sufficient testosterone for testis and sperm development, but a lack of PMCA-Np complexes prevents the increase from reaching adult blood levels. Behavioral immaturity with low testosterone blood levels underlies infertility of Nptn^−/− males, revealing that Np is essential for reproduction.

Carbonic anhydrase II (CAII) and IV (CAIV) orchestrate pH regulation essential for male fertility through distinct cellular compartments, with CAII functioning intracellularly and CAIV GPI-anchored extracellularly in epididymal and vas deferens epithelia as well as on spermatozoa. These enzymes control pH environments required for sperm maturation and capacitation by regulating bicarbonate availability, which triggers soluble adenylyl cyclase/cAMP signaling, elevates intracellular pH (pH _i ), and enables motility activation. However, the specific contributions of CAII and CAIV to luminal pH and sperm pH _i regulation remain unclear. Here, we show that genetic ablation of either CAII or CAIV in mice impairs normal luminal acidification in the male reproductive tract and unexpectedly lowers the basal pH _i of sperm. This lower basal pH _i blunts subsequent intracellular alkalinization, thereby diminishing the activation of pH-sensitive CatSper Ca ²⁺ channels essential for hyperactivated motility. Mechanistically, we demonstrate that this basal pH _i reduction occurs independently of the CatSper channel itself, but it is phenocopied in K ⁺ channel Slo3 -deficient sperm, suggesting a homeostatic link between flagellar pH and membrane potential. Super-resolution imaging reveals that CAII is located within the flagellum near the CatSper channel in the principal piece, while CAIV is distributed along the entire flagellar membrane. Together, our findings demonstrate that CAII and CAIV employ spatially coordinated mechanisms to regulate pH. By maintaining an acidic luminal environment for sperm maturation and priming the subsequent alkalinization of sperm pH _i , these enzymes modulate downstream ion channel activity to govern sperm motility and fertility. One-sentence summary: Spatially coordinated carbonic anhydrases II and IV govern sperm motility by establishing the dual-compartment pH environments necessary to activate downstream flagellar ion channels.