BSTRACTMale infertility has become a worldwide health problem, but the etiologies of most cases are still unknown. SEPT12, a GTP-binding protein, is involved in male fertility. Two SEPT12 mutations (SEPT12 T89M and SEPT12 D197N ) have been identified in infertile men who have a defective sperm annulus with a bent tail. The function of SEPT12 in the sperm annulus is still unclear. Here, we found that SEPT12 formed a filamentous structure with SEPT7, SEPT 6, SEPT2 and SEPT4 at the sperm annulus. The SEPT12-based septin core complex was assembled as octameric filaments comprising the SEPT proteins 12-7-6-2-2-6-7-12 or 12-7-6-4-4-6-7-12. In addition, the GTP-binding domain of SEPT12 was crucial for its interaction with SEPT7, and the N-and C-termini of SEPT12 were required for the interaction of SEPT12 with itself to polymerize octamers into filaments. Mutant mice carrying the SEPT12 D197N mutation, which disrupts SEPT12 filament formation, showed a disorganized sperm annulus, bent tail, reduced motility and loss of the SEPT ring structure at the sperm annulus. These phenotypes were also observed in an infertile man carrying SEPT12 D197N .Taken together, our results demonstrate the molecular architecture of SEPT12 filaments at the sperm annulus, their mechanical support of sperm motility, and their correlation with male infertility.

Septins are critical for numerous cellular processes through the formation of heteromeric filaments and rings indicating the importance of structural regulators in septin assembly. Several posttranslational modifications (PTMs) mediate the dynamics of septin filaments in yeast. However, little is known about the role of PTMs in regulating mammalian septin assembly, and the in vivo significance of PTMs on mammalian septin assembly and function remains unknown. Here, we showed that SEPT12 was phosphorylated on Ser198 using mass spectrometry, and we generated SEPT12 phosphomimetic knock-in (KI) mice to study its biological significance. The homozygous KI mice displayed poor male fertility due to deformed sperm with defective motility and loss of annulus, a septin-based ring structure. Immunohistochemistry of KI testicular sections suggested that SEPT12 phosphorylation inhibits septin ring assembly during annulus biogenesis. We also observed that SEPT12 was phosphorylated via PKA, and its phosphorylation interfered with SEPT12 polymerization into complexes and filaments. Collectively, our data indicate that SEPT12 phosphorylation inhibits SEPT12 filament formation, leading to loss of the sperm annulus/septin ring and poor male fertility. Thus, we provide the first in vivo genetic evidence characterizing importance of septin phosphorylation in the assembly, cellular function and physiological significance of septins.

Septins are GTP-binding proteins that form heteromeric filaments for proper cell growth and migration. Among the septins, septin7 (SEPT7) is an important component of all septin filaments. Here we show that protein kinase A (PKA) phosphorylates SEPT7 at Thr197, thus disrupting septin filament dynamics and ciliogenesis. The Thr197 residue of SEPT7, a PKA phosphorylating site, was conserved among different species. Treatment with cAMP or overexpression of PKA catalytic subunit (PKACA2) induced SEPT7 phosphorylation, followed by disruption of septin filament formation. Constitutive phosphorylation of SEPT7 at Thr197 reduced SEPT7‒SEPT7 interaction, but did not affect SEPT7‒SEPT6‒SEPT2 or SEPT4 interaction. Moreover, we noted that SEPT7 interacted with PKACA2 via its GTP-binding domain. Furthermore, PKA-mediated SEPT7 phosphorylation disrupted primary cilia formation. Thus, our data uncover the novel biological function of SEPT7 phosphorylation in septin filament polymerization and primary cilia formation.

The sperm annulus, a septin-based ring structure, is important for reproductive physiology. It is composed of SEPT12-based septin core complex followed by assembling as octameric filament. In clinical examinations, mutations of Septin12 result in male infertility, immotile sperm, as well as sperm with defective annuli. The dynamic assembly of septin filaments is regulated by several posttranslational modifications, including sumoylation, acetylation, and phosphorylation. Here, we briefly review the biological significance and the regulation of SEPT12 phosphorylation in the mammalian sperm physiology. During mammalian spermiogenesis, the phosphorylation of SEPT12 on Ser198 residue is important in regulating mammalian annulus architectures. SEPT12 phosphomimetic knock-in mice displayed poor male fertility due to weak sperm motility and loss of the sperm annulus. SEPT12 is phosphorylated via Protein kinase A (PKA), and its phosphorylation interfered with SEPT12 polymerization into complexes and filaments. Taken together, the phosphorylation status of SEPT12 is crucial for its function in regulating the mammalian sperm physiology. This article is protected by copyright. All rights reserved.

The connecting pieces of the sperm neck link the flagellum and the sperm head, and they are important for initiating flagellar beating. The connecting pieces are important building blocks for the sperm neck; however, the mechanism of connecting piece assembly is poorly understood. In the present study, we explored the role of septins in sperm motility and found that Sept12D197N knock-in (KI) mice produce acephalic and immotile spermatozoa. Electron microscopy analysis showed defective connecting pieces in sperm from KI mice, indicating that SEPT12 is required for the establishment of connecting pieces. We also found that SEPT12 formed a complex with SEPT1, SEPT2, SEPT10 and SEPT11 at the sperm neck and that the D197N mutation disrupted the complex, suggesting that the SEPT12 complex is involved in the assembly of connecting pieces. Additionally, we found that SEPT12 interacted and colocalized with γ-tubulin in elongating spermatids, implying that SEPT12 and pericentriolar materials jointly contribute to the formation of connecting pieces. Collectively, our findings suggest that SEPT12 is required for the formation of striated columns, and the capitulum and for maintaining the stability of the sperm head–tail junction.