Haoting Wang

Fertilization requires gamete recognition and membrane fusion, yet the molecular basis of this process in vertebrates remains unknown. Here we identify SPARK (sperm protein assembly and receptor-binding key), a conserved multi-protein complex that integrates all known sperm fertilization factors, including TMEM81–IZUMO1–SPACA6 and DCST1/2, together with two newly identified components, TMDD1 and FAM187A. SPARK subunits are mutually dependent for stability in mature sperm, and disruption of any single component causes male sterility in zebrafish and mice. Incubating zebrafish sperm with soluble egg receptor Bouncer partially rescues fertilization of Bouncer-deficient eggs in a SPARK-dependent manner, consistent with egg receptor binding priming the complex for fusion. Thus, we propose SPARK as a con-served molecular machine that couples gamete recognition to membrane fusion.

Sperm motility is essential for male fertility and is tightly controlled by signaling events in the flagellum. Slc9c1 encodes a sperm-specific Na ⁺ /H ⁺ exchanger (sNHE/SLC9C1) that localizes to the flagellum and is indispensable for sperm motility and male fertility. SLC9C1 is unique among Na ⁺ /H ⁺ exchangers in that it possesses a voltage-sensing domain (VSD), the physiological function of which remains poorly understood in mammals. Here, by analyzing coevolving genes with Slc9c1 , we identified Tmem217 , which encodes a transmembrane protein that is localized in the sperm flagellum. Knockout (KO) of Tmem217 in mice resulted in sperm motility defects and male infertility, phenocopying Slc9c1 KO mice. Coimmunoprecipitation and structural prediction analyses indicated that TMEM217 binds to SLC9C1 via its VSD. Further analyses indicated that the amounts of SLC9C1 and its associated protein, soluble adenylyl cyclase (sAC), were lost in mature Tmem217 KO spermatozoa, leading to disrupted 3′,5′-cyclic monophosphate (cAMP) signaling pathways. Remarkably, cAMP analogs restored the impaired motility and fertilizing ability of Tmem217 KO spermatozoa in vitro, validating the essential role of TMEM217 in regulating cAMP production. Our findings indicate that the association of TMEM217 with SLC9C1 via its VSD is critical for the proper organization and function of the SLC9C1–sAC–cAMP axis in mature spermatozoa.

Radial spokes (RSs) are conserved multimolecular structures attached to the axonemal microtubule doublets and are essential for the motility control of both cilia and sperm flagella. CFAP91, an RS3 protein, is implicated in human male infertility, yet its molecular function remains poorly understood. Here, we demonstrate that Cfap91 knockout (KO) mice exhibit impaired sperm flagellum formation and male infertility. Using a transgenic rescue model expressing FLAG- and BioID2-tagged CFAP91, we reveal that CFAP91 immunoprecipitates with RS3 proteins CFAP251 and LRRC23, whose localization is disrupted in Cfap91 KO sperm flagella. In addition, proximity labeling in mature spermatozoa identifies EFCAB5 as a sperm-specific CFAP91-proximal component. We show that Efcab5 KO males exhibit reduced sperm motility and fertility. Our findings establish CFAP91 as an essential scaffolder of RS3 assembly and EFCAB5 as a sperm-specialized movement regulator, advancing understanding of axonemal specialization in mammalian spermatozoa and its relevance to male infertility.

Sperm heads contain not only the nucleus but also the acrosome which is a distinctive cap-like structure located anterior to the nucleus and is derived from the Golgi apparatus. The Golgi Associated RAB2 Interactors (GARINs; also known as FAM71) protein family shows predominant expression in the testis and all possess a RAB2-binding domain which confers binding affinity to RAB2, a small GTPase that is responsible for membrane transport and vesicle trafficking. Our previous study showed that GARIN1A and GARIN1B are important for acrosome biogenesis and that GARIN1B is indispensable for male fertility in mice. Here, we generated KO mice of other Garins, namely Garin2, Garin3, Garin4, Garin5a, and Garin5b (Garin2-5b). Using computer-assisted morphological analysis, we found that the loss of each Garin2-5b resulted in aberrant sperm head morphogenesis. While the fertilities of Garin2^-/- and Garin4^-/- males are normal, Garin5a^-/- and Garin5b^-/- males are subfertile, and Garin3^-/- males are infertile. Further analysis revealed that Garin3^-/- males exhibited abnormal acrosomal morphology, but not as severely as Garin1b^-/- males; instead, the amounts of membrane proteins, particularly ADAM family proteins, decreased in Garin3 KO spermatozoa. Moreover, only Garin4 KO mice exhibit vacuoles in the sperm head. These results indicate that GARINs assure correct head morphogenesis and some members of the GARIN family function distinctively in male fertility.