ANKEF1 is a key axonemal component essential for murine sperm motility and male fertility
- Academy for Advanced Interdisciplinary Studies, Peking University, China
- National Institute of Biological Sciences (NIBS), China
- State Key Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, China
- The School of Public Health, Xinxiang Medical University, China
- Tsinghua Institute of Multidisciplinary Biomedical Research, Tsinghua University, China
Figures
Figure 1 with 1 supplement
Ankef1 is critical for male reproductive function.
(A) Relative expression of Ankef1 mRNA in different tissues of adult mouse. Expression levels were normalized to Gapdh mRNA and are presented relative to the gut (set as 1). Different letters above the bars indicate statistically significant differences (p < 0.05). Data are presented as mean ± SEM (n = 3 biological replicates). Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (B) Relative expression of Ankef1 mRNA in testes at various postnatal days. Expression levels were normalized to Gapdh mRNA and are presented relative to the postnatal day 18 (P18, set as 1). Different letters above the bars indicate statistically significant differences (p < 0.05). Data are presented as mean ± SEM (n = 3 biological replicates). Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test. (C) CRISPR/Cas9 targeting scheme of mouse Ankef1 and genotyping of Ankef1 KO mouse. Ankef1-WT-F+Ankef1-screen-R (for WT) and Ankef1-screen-F+Ankef1-screen-R (for KO). nc, negative control (ddH2O). (D, E) Sperm count and percentage of normal sperm of cauda epididymal. Data are presented as mean ± SEM (n = 5 biological replicates). Statistical significance was determined by unpaired two-tailed Student’s t-test (ns, not significant). (F) Testis-to-body weight ratio of adult control and Ankef1 KO mouse (n = 7). Data are presented as mean ± SEM (n = 7 biological replicates). Statistical significance was determined by an unpaired two-tailed Student’s t-test (ns, not significant). (G) Hematoxylin and eosin (H&E) staining of mouse testis and epididymis. Coomassie Brilliant Blue R-250 staining of spermatozoa from control and Ankef1 KO male mouse. No significant abnormality was found in Ankef1 KO male mouse. No overt abnormalities were found in Ankef1 KO mouse. P, pachytene; ES, elongated sperm; RS, round sperm; SG, spermatogonia; ST, Sertoli cell.
Figure 1—figure supplement 1
ANKEF1 is conserved between human and other vertebrate model organisms.
(A) Sequence alignment of ANKEF1 proteins from several species. Sequences were derived from NCBI and compared with SnapGene (Version = 4.3.6). (B) Percent identity matrices of Ankef1 proteins across several common vertebrate model organisms. (C) Gross morphology of adult control and Ankef1 KO testes and epididymis. (D) The body size was similar between Ankef1+/− and Ankef1−/− mice.
Figure 2
Evaluation of in vitro fertilization (IVF) capacity of Ankef1 KO sperm.
(A–C) Fertilization rate of IVF using control and Ankef1 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 hr of incubation, both the control and Ankef1 KO sperm could penetrate cumulus oophorus as indicated by the red arrow and have the ability to bind to the zona pellucida. (E, 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. A black arrow indicates the intact or disappeared acrosome. Data are mean ± SEM. Sample sizes (biological replicates) were Ankef1+/− mice, n = 4 per group; Ankef1−/− mice, n = 3 per group. Within each genotype, the DMSO and A23187 groups were compared by an unpaired two-tailed t-test (ns, not significant).
Figure 3 with 2 supplements
Sperm motility of Ankef1 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 Ankef1 KO mouse. (B) Proportions of sperm at different velocity levels in control and Ankef1 KO mouse. (C) Knockout mouse had lower motile sperm (total motor capacity) and progressive motile sperm (progressive motor capacity) than control. (D) Trajectories of sperm per second. The meanings of different colors are shown in the graph. (E) Impaired migration of Ankef1 KO sperm from uterus into oviducts. The black arrow indicates sperm. (F) Uterine sperm counts after mating. (G) Oviduct sperm counts after mating. For both panels, data (mean ± SEM) compare control and Ankef1 KO mice, analyzed by an unpaired two-tailed t-test (uterus, n = 6 biological replicates, **p < 0.01; oviducts, n = 3 biological replicates, ***p < 0.001).
Figure 3—video 1
Sperm from Ankef1+/− mouse.
Video recording of sperm from Ankef1+/− mouse from the Computer Assisted Sperm Analyzer system (Version 12 CEROS, Hamilton Thorne Research). Capture rate was set at 60 frames/s.
Figure 3—video 2
Sperm from Ankef1−/− mouse.
Video recording of sperm from Ankef1−/− mouse from the Computer Assisted Sperm Analyzer system (Version 12 CEROS, Hamilton Thorne Research). Capture rate was set at 60 frames/s.
Figure 4 with 3 supplements
ANKEF1 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. ANKEF1-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 ANKEF1-Flag mouse using anti-Flag antibody (red: anti-Flag signal; Hoechst: blue). (D) Fractionation of sperm proteins using different lysis buffers. ANKEF1-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) Transmission electron microscopy (TEM) of sperm tails from control and Ankef1 KO mice. Cross-sections of the midpiece, principal piece, and end piece were examined. Red dashed boxes highlight regions of interest, and the magnified views of these boxed areas are shown in the upper right corner of each image. In three independent experiments, 20 sperm cross-sections per mouse were analyzed for each group, with consistent results observed.
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Figure 4—source data 1
PDF file containing original western blots for Figure 4A with relevant bands.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4A.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig4-data2-v1.zip
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Figure 4—source data 3
PDF file containing original western blots for Figure 4B with relevant bands.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig4-data3-v1.zip
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Figure 4—source data 4
Original files for western blot analysis displayed in Figure 4B.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig4-data4-v1.zip
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Figure 4—source data 5
PDF file containing original western blots for Figure 4D with relevant bands.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig4-data5-v1.zip
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Figure 4—source data 6
Original files for western blot analysis displayed in Figure 4D.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig4-data6-v1.zip
Figure 4—figure supplement 1
Generation and fertility assessment of Ankef1-Flag mice.
(A) The generation of Flag-tagged mouse and sperm head and tail separation. Schematic of Flag-tagged alleles of endogenous Ankef1 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. Data are presented as mean ± SEM (n = 3 biological replicates). Statistical significance was determined by an unpaired two-tailed Student’s t-test (ns, not significant). (D) Sperm head and tail were separated by repeated freeze–thaw and stained with Coomassie Brilliant Blue R-250.
Figure 4—figure supplement 2
ANKEF1 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 ANKEF1 protein was detected in the cilia of the respiratory tract in ANKEF1-Flag mice, and it exhibited a similar pattern to the α-tubulin signal. The scale bar represents 25 μm.
Figure 4—figure supplement 3
The deformed doublet microtubule (DMT) in the transmission electron microscopy (TEM) results.
(A) The presence of both normal and deformed DMTs in the TEM and STA results is shown. (B) The deformed DMTs in the TEM results are all marked by red dashed boxes, and the TEM results presented in (A) are all cropped from (B).
Figure 5 with 1 supplement
ANKEF1 is a component of nexin–dynein regulatory complex (N-DRC) in sperm flagella.
(A) Identification of sperm proteins in liquid chromatography–mass spectrometry (LC–MS/MS) analysis. Black star indicates N-DRC components. (B, C) Individual DRC components were coexpressed in HEK293T cells. Immunoprecipitation of ANKEF1-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 ANKEF1-Flag. (D) Schematic of various truncated ANKEF1 vectors. Flag-tag was linked posterior to the C-terminal of ANKEF1. Green and yellow boxes show the ANK domain and EF-hand domain of ANKEF1, respectively. Light yellow boxes indicate Flag tag. (E, F) The interaction between various truncated ANKEF1-Flag and TCTE1-MYC or GAS8-MYC was 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 ANKEF1 with GAS8 and TCTE1.
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Figure 5—source data 1
PDF file containing original western blots for Figure 5B with relevant bands and treatments.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-data1-v1.zip
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Figure 5—source data 2
Original files for western blot analysis displayed in Figure 5B.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-data2-v1.zip
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Figure 5—source data 3
PDF file containing original western blots for Figure 5C with relevant bands and treatments.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-data3-v1.zip
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Figure 5—source data 4
Original files for western blot analysis displayed in Figure 5C.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-data4-v1.zip
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Figure 5—source data 5
PDF file containing original western blots for Figure 5E with relevant bands and treatments.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-data5-v1.zip
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Figure 5—source data 6
Original files for western blot analysis displayed in Figure 5E.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-data6-v1.zip
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Figure 5—source data 7
PDF file containing original western blots for Figure 5F with relevant bands and treatments.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-data7-v1.zip
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Figure 5—source data 8
Original files for western blot analysis displayed in Figure 5F.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-data8-v1.zip
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Figure 5—source data 9
PDF file containing original western blots for Figure 5G with relevant bands and treatments.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-data9-v1.zip
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Figure 5—source data 10
Original files for western blot analysis displayed in Figure 5G.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-data10-v1.zip
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Figure 5—source data 11
Original files for Figure 5A.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-data11-v1.xls
Figure 5—figure supplement 1
Identify the interaction of ANKEF1 and other nexin–dynein regulatory complex (N-DRC) components.
(A) Co-IP followed by WB analysis was performed using lysates collected from HEK293T cells transfected with Flag-tagged ANKEF1. Immunoprecipitated proteins by anti-MYC antibody were analyzed by WB using anti-Flag antibodies. (B) HEK293T cells transiently expressing ANKEF1-Flag and DRC-MYC were stained with Flag (red) and MYC (white) to visualize ANKEF1 and DRC, respectively. DAPI (blue) was used to visualize the nuclei.
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Figure 5—figure supplement 1—source data 1
PDF files containing the original western blots, indicating relevant bands, for the co-immunoprecipitation of ANKEF1-Flag with DRC1.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Original file of Western blot analysis of co-immunoprecipitation of ANKEF1-Flag and DRC1.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data2-v1.zip
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Figure 5—figure supplement 1—source data 3
PDF files containing the original western blots, indicating relevant bands, for the co-immunoprecipitation of ANKEF1-Flag with DRC2.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data3-v1.zip
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Figure 5—figure supplement 1—source data 4
Original file of Western blot analysis of co-immunoprecipitation of ANKEF1-Flag and DRC2.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data4-v1.zip
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Figure 5—figure supplement 1—source data 5
PDF files containing the original western blots, indicating relevant bands, for the co-immunoprecipitation of ANKEF1-Flag with LRRC48.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data5-v1.zip
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Figure 5—figure supplement 1—source data 6
Original file of Western blot analysis of co-immunoprecipitation of ANKEF1-Flag and LRRC48.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data6-v1.zip
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Figure 5—figure supplement 1—source data 7
PDF files containing the original western blots, indicating relevant bands, for the co-immunoprecipitation of ANKEF1-Flag with EFCAB2.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data7-v1.zip
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Figure 5—figure supplement 1—source data 8
Original file of Western blot analysis of co-immunoprecipitation of ANKEF1-Flag and EFCAB2.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data8-v1.zip
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Figure 5—figure supplement 1—source data 9
PDF files containing the original western blots, indicating relevant bands, for the co-immunoprecipitation of ANKEF1-Flag with IQCG.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data9-v1.zip
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Figure 5—figure supplement 1—source data 10
Original file of Western blot analysis of co-immunoprecipitation of ANKEF1-Flag and IQCG.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data10-v1.zip
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Figure 5—figure supplement 1—source data 11
PDF files containing the original western blots, indicating relevant bands, for the co-immunoprecipitation of ANKEF1-Flag with IQCD.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data11-v1.zip
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Figure 5—figure supplement 1—source data 12
Original file of Western blot analysis of co-immunoprecipitation of ANKEF1-Flag and IQCD.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data12-v1.zip
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Figure 5—figure supplement 1—source data 13
PDF files containing the original western blots, indicating relevant bands, for the co-immunoprecipitation of ANKEF1-Flag with DRC7.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data13-v1.zip
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Figure 5—figure supplement 1—source data 14
Original file of Western blot analysis of co-immunoprecipitation of ANKEF1-Flag and DRC7.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data14-v1.zip
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Figure 5—figure supplement 1—source data 15
PDF files containing the original western blots, indicating relevant bands, for the co-immunoprecipitation of ANKEF1-Flag with IQCA1.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data15-v1.zip
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Figure 5—figure supplement 1—source data 16
Original file of Western blot analysis of co-immunoprecipitation of ANKEF1-Flag and IQCA1.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig5-figsupp1-data16-v1.zip
Figure 6 with 2 supplements
Absence of ANKEF1 does not affect energy metabolism.
(A) The differentially expressed proteins of Ankef1+/– and Ankef1−/− were identified by 4D-SmartDIA. (B) Heatmap of relative protein abundance changes between control and knockout mouse sperm. (C) Differences in the expression of nexin–dynein regulatory complex (N-DRC) protein components identified by mass spectra. (D) Gene Set Enrichment Analysis (GSEA) of glycolysis and gluconeogenesis. (E) Measured levels of ATP between wild-type and Ankef1 null sperm. Data are presented as mean ± SEM (n = 3 biological replicates). Statistical significance was determined by an unpaired two-tailed Student’s t-test (*p < 0.05, ns, not significant).
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Figure 6—source data 1
Source data files for panels B and D of Figure 6 provide the whole-sperm proteomic profiling results from ANKEF1-knockout and control mice.
- https://cdn.elifesciences.org/articles/105321/elife-105321-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
The mitochondrial membrane potential and reactive oxygen species (ROS) levels of Ankef1 null sperm were normal.
(A) Mitochondrial activities assessed by fluorescence of tetramethylrhodamine methyl ester (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. (C) ROS levels assessed by fluorescence of DCFH-DA, FITC filter. The higher the concentration of ROS, the higher the fluorescence intensity. (D) Graphs show semi-quantitative of FITC fluorescence intensity. Data are presented as mean ± SEM (n = 3 biological replicates). Statistical significance was determined by an unpaired two-tailed Student’s t-test (ns, not significant).
Figure 6—figure supplement 2
Immunofluorescence results of ANKEF1-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).
Figure 7 with 3 supplements
The overall structure of Ankef1-KO mouse sperm doublet microtubule (DMT).
(A) Side view and top sectional view of WT/Ankef1−/− mouse sperm axoneme are shown in the tomogram slices. DMT and outer dynein arm (ODA) are marked with white dashed lines and white arrows, respectively. (B) The cryo-EM map of Ankef1−/− mouse sperm DMT with an 8 nm repeat was obtained by sub-tomogram analysis. (C) Loss of density in Ankef1−/− DMT structure. The transverse sectional view of DMT is shown. The lost density (khaki color) was obtained by subtracting the density map of Ankef1−/− DMT from that of the WT DMT. (D) The model of 16 nm-repeats WT DMT (PDB: 8I7O) was fitted in the 8 nm repeat WT DMT map and Ankef1−/− DMT map. The 8 nm repeats DMT density map was obtained by summing two 16 nm repeats DMTs that were staggered 8 nm apart from each other.
Figure 7—figure supplement 1
Cryo-focused ion beam (cryo-FIB) milling and the half-map Fourier Shell Correlation (FSC) of the Ankef1−/− 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 Ankef1−/− doublet microtubule (DMT) is shown. 1 Å = 0.1 nm.
Figure 7—figure supplement 2
The data processing of ANKEF1-KO mouse sperm doublet microtubule (DMT).
The pixel sizes at different binning levels are indicated in angstroms per pixel (Å/px for short).
Figure 7—figure supplement 3
The states of doublet microtubule (DMT) particles in sperm of Ankef1-KO mouse.
(A, C) Tomogram slices of WT and Ankef1-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, D) Comparison of DMT particle states between WT and Ankef1-KO in Dynamo. The visual angles of the DMT particles shown in (B, D) show that the DMT fibers within the white box in (A, B) are divided equally into 10 slices along the direction of the white arrow, respectively. The DMT particle shapes of WT and Ankef1-KO are marked by white dashed lines on the right of (B, D). The white arrow in (D) identifies the junction of A- 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.
Figure 8
Localization of ANKEF1 in sperm axoneme.
(A) Localization of nexin–dynein regulatory complex (N-DRC) in 96 nm repeats doublet microtubule (DMT) of mammalian sperm (EMD-50664). (B) Comparison of AlphaFold predicted mouse ANKEF1 structures with known bovine ANKEF1 structures (PDB: 9FQR). (C) Localization of ANKEF1 in 96 nm repeats DMT map (EMD-50664). (D) Structure and composition of N-DRC in 96 nm repeats DMT of mammalian sperm (EMD-50664). (E) The relationship between ANKEF1 and its interacting proteins as shown in the electron microscope density map (EMD-50664). (F) The relationship between ANKEF1 and its interacting proteins as shown in 96 nm repeats DMT model of mammalian sperm (PDB: 9FQR). (G) Localization of ANKEF1 in the in situ mouse sperm 96 nm repeats DMT map (EMD-27444).
Figure 9
The functional model of ANKEF1.
ANKEF1, as a component of the nexin–dynein regulatory complex (N-DRC) connecting adjacent doublet microtubules (DMTs), enhances the elasticity of the N-DRC. During sperm movement, the flagellar beating causes adjacent DMT to move toward each other, and the intact N-DRC structure provides good cushioning. In ANKEF1-knockout mice, the absence of ANKEF1 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.
Tables
Table 1
Knockout of Ankef1 causes male infertility in mice.
| Females | Plugs | Litters | Mean litter size | |
|---|---|---|---|---|
| WT (n = 6) | 12 | 11 | 10 | 8 |
| Ankef1+/− (n = 6) | 12 | 12 | 10 | 8.5 |
| Ankef1−/− (n = 6) | 12 | 10 | 0 | 0 |
Additional files
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Supplementary file 1
Primer sequences for genotyping and RT-qPCR analysis of Ankef1 knockout mice.
- https://cdn.elifesciences.org/articles/105321/elife-105321-supp1-v1.xlsx
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Supplementary file 2
Antibodies used in this study.
- https://cdn.elifesciences.org/articles/105321/elife-105321-supp2-v1.xlsx
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Supplementary file 3
Primer sequences for vector construction in co-immunoprecipitation (co-IP) assays.
- https://cdn.elifesciences.org/articles/105321/elife-105321-supp3-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/105321/elife-105321-mdarchecklist1-v1.docx
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ANKEF1 is a key axonemal component essential for murine sperm motility and male fertility
eLife 14:RP105321.
https://doi.org/10.7554/eLife.105321.4
