Ankrd5 is critical for male reproductive function.

(A and B) Relative expression of Ankrd5 mRNA in different tissues of adult mouse and testes at various postnatal days. The Ankrd5 mRNA expression levels were normalized to the expression of Gapdh mRNA (n=3). (C) CRISPR/Cas9 targeting scheme of mouse Ankrd5 and genotyping of Ankrd5 KO mouse. Ankrd5-WT-F + Ankrd5-screen-R (for WT) and Ankrd5-screen-F + Ankrd5-screen-R (for KO). nc, negative control (ddH2O). (D and E) Sperm count and percentage of normal sperm of cauda epididymal from control and Ankrd5 KO mouse (n=5). (F) Testis to body weight ratio of adult control and Ankrd5 KO mouse (n=7). (G) Hematoxylin and eosin (H&E) staining of mouse testis and epididymis. Coomassie Brilliant Blue R-250 staining of spermatozoa from control and Ankrd5 KO male mouse. No significant abnormality was found in Ankrd5 KO male mouse. No overt abnormalities were found in Ankrd5 KO mouse. P, pachytene; ES, elongated sperm; RS, round sperm; SG, spermatogonia; ST, Sertoli cell. All values in this figure are shown as the mean ± SE.

Knockout of Ankrd5 causes male infertility of mouse Male mouse

Evaluation of in vitro fertilization capacity of Ankrd5 KO sperm.

(A-C) Fertilization rate of IVF using control and Ankrd5 KO spermatozoa. Three types of oocytes (cumulus-intact, cumulus-free, and zona pellucida-free) were used for IVF. (D) Egg observation after IVF. After 4 hours of incubation, both of control and Ankrd5 KO sperm could penetrate cumulus oophorus as indicated by the red arrow and have the ability to bind to the zona pellucida. (E and F) Sperm were incubated in capacitation medium treated with A23187 (dissolved in DMSO) and DMSO (dissolvent control group) and stained with Coomassie Brilliant Blue R-250. Black arrow indicates the intact or disappeared acrosome; Values represent mean ± SE (n=3).

Sperm motility of Ankrd5 KO male mouse.

(A) Average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), straightness (STR), and linearity (LIN) of sperm from control and Ankrd5 KO mouse. **P < 0.01, ***P < 0.001, Student’s t test; error bars represent SE (n = 3). (B) Proportions of sperm at different velocity levels in control and Ankrd5 KO mouse. **P < 0.01, ***P < 0.001, Student’s t test; error bars represent SE (n = 3). (C) Knockout mouse had lower motile sperm (total motor capacity) and progressive motile sperm (progressive motor capacity) than control. **P < 0.01, ***P < 0.001, Student’s t test; error bars represent SE (n = 3). (D) Trajectories of sperm per second. The meanings of different colors are shown in the graph. (E) Impaired migration of Ankrd5 KO sperm from uterus into oviducts. The black arrow indicates sperm. (F and G) Numbers of sperm from control and Ankrd5 KO mouse in female uterus and oviducts after mating. **P < 0.01, ***P <0.001, Student’s t test; error bars represent SE (n = 3).

ANKRD5 is located in the midpiece of sperm axoneme.

(A) Immunoblotting analysis of various mouse tissues. GAPDH was used as the loading control. (B) Head and tail separation of mouse spermatozoa. ANKRD5-FLAG was detected in the tail fraction. PRM2 was used as a marker for sperm head. BASIGIN, AKAP4 and acetylated tubulin were detected as a marker for tails. (C) Immunofluorescence staining results of spermatozoa from wild-type and ANKRD5-FLAG mouse using anti-FLAG antibody (red: anti-FLAG signal; Hoechst: blue). (D) Fractionation of sperm proteins using different lysis buffers. ANKRD5-FLAG was found in the SDS-soluble fraction that contains axonemal proteins. BASIGIN, acetylated tubulin, and AKAP4 were detected as a marker for Triton-soluble, SDS-soluble, and SDS-resistant fractions, respectively. (E) Ultrastructure of sperm tails in control and Ankrd5 KO mouse. Cross-sections of midpiece, principal piece and end piece were observed using transmission electron microscopy.

ANKRD5 is a component of N-DRC in sperm flagella.

(A) Identification of sperm proteins in LC-MS/MS analysis. Black star indicates N-DRC components. (B and C) Individual DRC components were coexpressed in HEK293T cells. Immunoprecipitation of ANKRD5-FLAG resulted in the co-precipitation of GAS8-MYC and TCTE1-MYC. Similarly, immunoprecipitation of GAS8-MYC and TCTE1-MYC also led to the co-precipitation of ANKRD5-FLAG. (D) Schematic of various truncated ANKRD5 vectors. FLAG-tag was linked posterior to the C-terminal of ANKRD5. Green and yellow boxes show the ANK domain and EF-Hand domain of ANKRD5, respectively. Light yellow boxes indicate FLAG tag. (E and F) The interaction between various truncated ANKRD5-FLAG and TCTE1-MYC or GAS8-MYC were confirmed by co-IP followed by WB analysis using anti-FLAG, and anti-MYC antibodies. (G) Effect of calcium ion and EDTA treatment on the interaction of ANKRD5 with GAS8 and TCTE1.

Absence of ANKRD5 does not affect energy metabolism.

(A) The differentially expressed proteins of Ankrd5+/– and Ankrd5+/- were identified by 4D-SmartDIA. (B) Heatmap of relative protein abundance changes between control and knockout mouse sperm. (C) Differences in the expression of N-DRC protein components identified by mass spectra. *P < 0.05, Student’s t test; error bars represent SE (n = 3). (D) GSEA analysis of glycolysis and gluconeogenesis. (E) Measured levels of ATP between wild-type and Ankrd5 null sperm. Student’s t test; error bars represent SE (n = 3).

The overall structure of Ankrd5-KO mouse sperm DMT.

(A) Side view and top sectional view of WT/Ankrd5-/- mouse sperm axoneme are shown in the tomogram slices. DMT and ODA are marked with white dashed lines and white arrows, respectively. (B) The cryo-EM map of Ankrd5-/- mouse sperm DMT with an 8 nm repeat was obtained by sub-tomogram analysis. (C) Loss of density in Ankrd5-/- DMT structure. The transverse sectional view of DMT is shown. The lost density (khaki color) was obtained by subtracting the density map of Ankrd5-/- DMT from that of the WT DMT. (D) The model of 16nm-repeats WT DMT (PDB: 8I7O) was fitted in the 8nm repeat WT DMT map and Ankrd5-/- DMT map. The 8nm repeats DMT density map was obtained by summing two 16nm repeats DMTs that were staggered 8nm apart from each other.

Localization of ANKRD5 in sperm axoneme.

(A) Localization of N-DRC in 96nm repeats DMT of mammalian sperm (EMD-50664). (B) Comparison of AlphaFold predicted mouse ANKRD5 structures with known bovine ANKRD5 structures (PDB: 9FQR). (C) Localization of ANKRD5 in 96nm repeats DMT map (EMD-50664). (D) Structure and composition of N-DRC in 96nm repeats DMT of mammalian sperm (EMD-50664). (E) The relationship between ANKRD5 and its interacting proteins as shown in the electron microscope density map (EMD-50664). (F) The relationship between ANKRD5 and its interacting proteins as shown in 96nm repeats DMT model of mammalian sperm (PDB: 9FQR). (G) Localization of ANKRD5 in the in-situ mouse sprem 96nm repeats DMT map (EMD-27444).

The functional model of ANKRD5. ANKRD5, as a component of the N-DRC connecting adjacent DMTs, enhances the elasticity of the N-DRC.

During sperm movement, the flagellar beating causes adjacent DMT to move towards each other, and the intact N-DRC structure provides good cushioning. In ANKRD5 -knockout mice, the absence of ANKRD5 weakens the N-DRC’s buffering capacity. This subjects the axoneme, especially during intense movement, to greater mechanical stress, leading to the deformation of B tube and impaired sperm motility.

ANKRD5 is conserved between human and other vertebrate model organisms.

(A) Sequence alignment of ANKRD5 proteins from several species. Sequences were derived from NCBI and compared with SnapGene (Version=4.3.6). (B) Percent identity matrices of Ankrd5 proteins across several common vertebrate model organisms. (C) Gross morphology of adult control and Ankrd5 KO testes and epididymis. (D) The body size was similar between Ankrd5+/- and Ankrd5-/- mice.

The generation of FLAG-tagged mouse and sperm head and tail separation.

Schematic of FLAG-tagged alleles of endogenous Ankrd5 generated using CRISPR/Cas9. (B) Hematoxylin and eosin (H&E) staining of FLAG-tagged mouse testis. No overt abnormalities were found. P, pachytene; ES, elongated sperm; RS, round sperm; SG, spermatogonia; ST, Sertoli cell. (C) There was no significant difference in litter size between WT and FLAG-tagged mouse. Values represent mean ± SE (n=3). (D) Sperm head and tail were separated by repeated freeze-thaw and stained with Coomassie Brilliant Blue R-250.

ANKRD5 was expressed in mouse respiratory cilia.

Mouse respiratory cilia were double-stained with anti-α-tubulin antibody (red) and anti-FLAG antibody (green), nuclei were stained with DAPI (blue). Expression of ANKRD5 protein was detected in the cilia of the respiratory tract in ANKRD5-FLAG mice, and it exhibited a similar pattern to the α-tubulin signal. The scale bar represents 25μm.

Identify the interaction of ANKRD5 and other N-DRC components.

(A) Co-IP followed by WB analysis were performed using lysates collected from HEK293T cells transfected with FLAG tagged ANKRD5. Immunoprecipitated proteins by anti-MYC antibody were analyzed by WB using anti-FLAG antibodies. (B) HEK293T cells transiently expressing ANKRD5-FLAG and DRC-MYC were stained with FLAG (red) and MYC (white) to visualize ANKRD5 and DRC, respectively. DAPI (blue) was used to visualize the nuclei.

The mitochondrial membrane potential and ROS levels of Ankrd5 null sperm was normal.

(A) Mitochondrial activities assessed by fluorescence of TMRM, RFP filter. The higher the potential, the higher the concentration of TMRM in mitochondria, resulting in an increased fluorescence intensity. (B) Graphs show semi-quantitative of TMRM fluorescence intensity. Values represent mean ± SE (n=3). (C) ROS levels assessed by fluorescence of DCFH-DA, FITC filter. The higher the concentration of ROS, the higher fluorescence intensity. (D) Graphs show semi-quantitative of FITC fluorescence intensity. Values represent mean ± SE (n=3).

Immunofluorescence results of ANKRD5-null sperm and control.

DRC11 serves as a marker protein for N-DRC (nexin-dynein regulatory complex), NME5 as a marker for RS (radial spoke), DNALI1 as a marker for IDA (inner dynein arm), and DNAI1 as a marker for ODA (outer dynein arm).

Cryo-FIB milling and the half-map Fourier Shell Correlation (FSC) of the Ankrd5-/- mouse sperm axoneme.

(A) Inspection of frozen sperm on the grid after FIB milling. The thin lamellae were used for data collection. (B) The half-map Fourier Shell Correlation (FSC) plot of Ankrd5-/- DMT is shown.1 Å = 0.1 nm

The data processing of ANKRD5-KO mouse sperm DMT.

The pixel sizes at different binning levels are indicated in angstroms per pixel (Å/px for short).

The states of DMT particles in sperm of Ankrd5-KO mouse.

(A) and (C) Tomogram slices of WT and Ankrd5-KO in Dynamo (The data for WT mouse sperm was EMPIARC-200007). DMT and RS are marked with white dashed lines and white arrows, respectively. (B) and (D) Comparison of DMT particle states between WT and Ankrd5-KO in Dynamo. The visual angles of the DMT particles shown in (B) and (D) show that the DMT fibers within the white box in (A) and (B) are divided equally into 10 slices along the direction of the white arrow, respectively. The DMT particle shapes of WT and Ankrd5-KO are marked by white dashed lines on the right of (B) and (D). The white arrow in (D) identifies the junction of A-tube and B-tube that is suspected to be disconnected. (E) Deformed particles discarded in 3D classification and final aligned DMT artifacts. (F) 3D classification of attempted RS locations.