SEPT12 phosphorylation results in loss of the septin ring/sperm annulus, defective sperm motility and poor male fertility

<div><p>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 <i>in vivo</i> 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 <i>in vivo</i> genetic evidence characterizing importance of septin phosphorylation in the assembly, cellular function and physiological significance of septins.</p></div

The annulus was absent in SEPT12^S196E/S196E spermatozoa.

<p>(A) Schematic representation of mouse spermatozoa. The annulus (An) is located between the mitochondria (Mi) in the midpiece and the fibrous sheath (FS) in the principal piece of the sperm tail. Ax, axoneme. (B, C) Immunofluorescence images of SEPT12 (B, green) or SEPT4 (C, green) from WT and SEPT12 KI spermatozoa. The middle piece was visualized through mitochondrial staining (Mito-Tracker, red). Nuclear staining (DAPI, blue) and bright-field images are also shown. Annular regions are indicated with arrowheads. (D) Quantitative representation of the sperm tail defect in the annular region from WT and SEPT12 KI mice (Each genotype, N = 5). The data are presented as the means ± SEM. ***P<0.001. (E) Electron microscopy analysis of WT and SEPT12 homozygous KI spermatozoa. In WT spermatozoa, the annulus (An) was observed between the mitochondria (Mi) and fibrous sheath (FS). In contrast, the annulus was completely lost in SEPT12 homozygous KI spermatozoa. The sperm were isolated form the epididymal cauda. Ax, axoneme. Scale bar: 1 μm.</p

The SEPT12 Ser198 residue was phosphorylated by PKA.

<p>(A) The Ser198 phosphorylation of SEPT12 (p-Ser198) was gradually elevated through increasing the dosage of cAMP treatment. GFP-SEPT12 was overexpressed in 293T cells, followed by treatment with 0, 1, and 5 mM cAMP, and the expression of phosphorylated Ser198 and SEPT12 was detected using anti-phospho-Ser198 and anti-GFP antibodies, respectively. (B) GFP-SEPT12 was overexpressed in 293T and NT2/D1 cells, followed by treatment with 50 μM H89, and the expression of phosphorylated Ser198, SEPT12 and actin was detected using anti-phospho-Ser198, anti-GFP and anti-Actin antibodies, respectively. (C) GFP-SEPT12 and GFP-SEPT12S198A, with or without HA-PKACA2, were transfected into 293T cells and NT2/D1 cells as indicated, and the expression of phosphorylated Ser198, SEPT12 and PKACA2 was detected using anti-phospho-Ser198, anti-GFP and anti-HA antibodies, respectively. (D) Co-immunoprecipitation of GFP-SEPT12 with HA-PKACA2. 293T Cells or NT2/D1 cells were transfected with GFP-SEPT12 and HA-PKACA2, and the lysates were immunoprecipitated using an anti-GFP antibody. Immunoblotting was performed using anti-GFP and anti-HA antibodies. (E) SEPT12 and PKACA2 showed partial co-localization. GFP-SEPT12 and HA-PKACA2 were co-expressed in NT2/D1 cells, and immunofluorescence was detected using an anti-HA antibody. Scale bar, 10 μm.</p

Working models for SEPT12 phosphorylation during sperm annulus biogenesis.

<p>SEPT12 filament is comprised of SEPT12-7-6-2-2-6-7-12 and SEPT12-7-6-4-4-6-7-12 octamers. Phosphorylation on SEPT12 Ser198 through PKA dissociates SEPT12 from the SEPT7-6-2 and SEPT7-6-4 complexes but does not disrupt the SEPT12-SEPT12 interaction. In WT mice, sperm annulus is assembled by SEPT12 filament at sperm neck and migrated along axoneme toward mid-principal piece junction. Disorganization of SEPT12 filament in phospho-SEPT12 KI mice fails to establishment of sperm annulus and lead to sperm deformation. The dotted arrows indicate the elongation of the filament with the addition of the SEPT octamers. NC, the N- and C-termini of the septin proteins. G, the GTP-binding domain of septins.</p

PKA disrupts SEPT12 filament formation via Ser198.

<p>(A, B) GFP-SEPT12 filaments were gradually decreased, and the aggregates were gradually increased in a cAMP dose-dependent manner. GFP-SEPT12 was overexpressed in 293T cells with 0, 1, 5, and 10 mM cAMP treatment, and cells with GFP-filament fibers (A) and GFP aggregates (B) were counted. (C, D) PKACA2 regulates SEPT12-organized structures through Ser198. GFP-SEPT12 or GFP-SEPT12S198A with or without HA-PKACA2 was overexpressed in 293T cells, and cells with GFP-filament fibers (C) and GFP aggregates (D) were counted. Each quantification bar was based on the observation of more than 500 cells. The data are represented as the means ± SEM (n = 3). ** P < 0.01, and *** P < 0.001.</p

The expression patterns of mimetic phosphorylated Ser198 of SEPT12.

<p>(A, B) The GFP-SEPT12WT, GFP-SEPT12S198A, GFP-SEPT12S198D, and GFP-SEPT12S198E plasmids were transfected into NT2/D1 cells. (A) Different patterns of GFP-SEPT12 are shown. Scale bar, 10 μm. (B) The percentage of NT2/D1 cells with GFP filament fibers. (C) Effect of mimetic Ser198 phosphorylation of SEPT12 on SEPT12 filament formation. A total of 1 μg of the GFP-SEPT12WT plasmid was mixed with 0, 1, 2 or 4 μg of the GFP-SEPT12S198E plasmid and transfected into NT2/D1 cells. In B and C, the quantification bar was based on the observation of more than 100 cells for each experiment, and cells with GFP filament fibers were counted. The data are represented as the means ± SEM (n = 3). * P < 0.05, and *** P < 0.001.</p

Mimetic phosphorylated Ser198 of SEPT12 disrupts SEPT12-7-6-2 complex.

<p>(A) The 3D-structure of the SEPT12-SEPT7 complex model shows that phosphorylated Ser198 (p-S198) of SEPT12 (shown in pink) is located at the dimeric G-interface. The structure of SEPT12 was built based on SEPT7 (PDB code: 3T5D) using the homology modeling method on the Swiss-model server (<a href="http://swissmodel.expasy.org/" target="_blank">http://swissmodel.expasy.org/</a>). (B) Myc-SEPT6 and FLAG-SEPT7 were co-transfected with various GFP-SEPT12 plasmids into NT2/D1 cells, and the cell lysates were immunoprecipitated using an anti-GFP antibody. The expression of SEPT2, 6, 7 and SEPT12 was detected using anti-SEPT2, anti-Myc, anti-FLAG and anti-GFP antibodies, respectively. (C) His-SEPT7 pull-down assay in WT and SEPT12 KI testis. His-SEPT7 recombinant protein was incubated with Ni-NTA beads followed by testicular lysate from WT and SEPT12^S196E/S196E mice. The pull-down protein was collected and subjected to western blotting for SEPT12, SEPT2 and SEPT6. The level of His-SEPT7 was detected using anti-His antibody. For the control group, WT testicular lysate was incubated with BSA instead of His-SEPT7.</p

Male fertility of SEPT12^S196E heterozygous and homozygous mice.

<p>Male fertility of SEPT12^S196E heterozygous and homozygous mice.</p

SEPT12^S196E/S196E mice displayed abnormal sperm morphology and motility.

<p>(A) Comparison of amino acid sequences flanking the Ser198 analogous sites of SEPT12 in various species using the ClustalW2 program at EMBL-EBI. The bracketed amino acid residues are analogous sites of Ser198. (B) Schematic illustration of the strategy for generating the SEPT12^S196E KI allele in embryonic stem cells. (C) Gross morphology of the testis and epididymis from WT and SEPT12 KI mice. Scale bar, 1 cm per unit. (D) Hematoxylin and eosin staining of testicular sections of stage XII tubule (top) and epididymal (bottom) sections from WT and SEPT12 KI mice. Scale bar, 100 μm. (E) Quantitative representation of testis/body weight, sperm counts, abnormal sperm morphology and sperm motility from WT and SEPT12 KI mice (Each genotype, N = 5). The sperm were isolated form the vas deferens. The data are represented as the means ± SEM. ***P<0.001.</p

Expression patterns of SEPT12 human male germ cells with the c.474G/G (wild) and c.474A/A genotypes.

<p>(A.) Detection of SEPT12 signals during human spermiogenesis. (a.–a′): Round Spermatids (RS), (b.–b′) Elongating Spermatids (ES) and Mature Sperm (MS). Left: SEPT12 signal (green); Right: merge of SEPT12 (green) and DAPI (light blue) signals. (B.) Varied type of spermatozoa isolated from cases with the c.474A/A genotype. Left: SEPT12 signal (green); Right: merge of SEPT12 (green) and DAPI (light blue) signals (Magnification: ×1,000).</p