FBXO24 deletion causes abnormal accumulation of membraneless electron-dense granules in sperm flagella and male infertility
- Research Institute for Microbial Diseases, Osaka University, Japan
- Graduate School of Pharmaceutical Sciences, Osaka University, Japan
- Center for Infectious Disease Education and Research (CiDER), Osaka University, Japan
- The Institute of Medical Science, The University of Tokyo, Japan
- Center for Advanced Modalities and DDS (CAMaD), Osaka University, Japan
Figures
Figure 1 with 1 supplement
Fbxo24 is predominantly expressed in testes and essential for male fertility.
(A) RT-PCR of Fbxo24 in mouse adult tissues. Fbxo24 is predominantly expressed in the testis. Br: brain, Th: thymus, Lu: lung, He: heart, Li: liver, Sp: spleen, Ki: kidney, Te: testis, Ut: uterus, Ov: ovary, and N.C.: negative control (water). Actb was used as a control. (B) RT-PCR of Fbxo24 using RNAs obtained from mouse testes at various postnatal days. Actb was used as a control. Water was used as a negative control (N.C.). (C) Construction of expression vectors for FBXO24 with (wild-type [WT]) or without (ΔF) the F-box domain. (D) Fbxo24 (WT)-FLAG or Fbxo24 (ΔF)-FLAG was transiently expressed with Skp1-1D4 in HEK293T cells. Immunoprecipitation (IP) was performed using anti-1D4 antibody or anti-FLAG antibody. FBXO24-FLAG interacts with SKP1-1D4 via the F-box domain. α-Tubulin was used as a loading control. (E) Schematic for generating Fbxo24 knockout (KO) mice using the CRISPR/Cas9 system. White boxes indicate untranslated regions while black boxes indicate protein coding regions. The gRNAs used are shown. Fw and Rv indicate the forward and reverse primer used for genotyping, respectively. (F) Genotyping of obtained Fbxo24 mutant mice. Fw #1-Rv #1 primers for KO allele and Fw #2-Rv #2 primers for WT allele in (E) were used. N.C. indicates negative control (water). (G) Amplicons of the PCR product using Fw #1-Rv #1 primers were subjected to direct sequencing and the 11,575 bp deletion was confirmed in the KO allele. (H) The number of pups born per plug was counted to assess male fertility. Each WT or KO male was mated with three WT females for 3 months. Error bars are mean ± standard deviation. Statistical significance was assessed with a two-tailed Welch's t-test. ***p<0.001.
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Figure 1—source data 1
Uncropped and labeled gels for Figure 1A and B.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig1-data1-v1.zip
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Figure 1—source data 2
Raw unedited gels for Figure 1A and B.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig1-data2-v1.zip
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Figure 1—source data 3
Uncropped and labeled blots for Figure 1D.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig1-data3-v1.zip
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Figure 1—source data 4
Raw unedited blots for Figure 1D.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig1-data4-v1.zip
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Figure 1—source data 5
Uncropped and labeled gels for Figure 1F.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig1-data5-v1.zip
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Figure 1—source data 6
Raw unedited gels for Figure 1F.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig1-data6-v1.zip
Figure 1—figure supplement 1
Characterization of mouse and human FBXO24.
(A) Expression profile of mouse Fbxo24 in spermatogenic cells is shown. Expression profiling was obtained from the testis single-cell RNA-seq dataset (Hermann et al., 2018). Ud spg: undifferentiated spermatogonia, A1- A2 Spg: A1 and A2 differentiating spermatogonia, A3-B Spg: A3, A4, In, and B differentiating spermatogonia, Prele Sc: preleptotene spermatocytes, Le/Zy Sc: leptotene/zygotene spermatocytes, Pa Sc: pachytene spermatocytes, Di/Se Sc: diplotene/secondary spermatocytes, Early St: early round spermatids, Mid St: mid round spermatids, Late St: late round spermatids, SC: Sertoli cells, PTM: peritubular myoid cells, LC: Leydig cells, and EC: endothelial cells. (B) Expression of human FBXO24 in spermatogenic cells is shown. Expression profiling was obtained from the testis single-cell RNA-seq dataset (Hermann et al., 2018). Dif Spg: differentiating spermatogonia. (C) Pairwise sequence alignment of amino acids between mouse and human FBXO24. Dark blue highlight indicates identical amino acids. (D) The F-box domain of mouse and human FBXO24 was identified with a protein database search (Simple Modular Architecture Research Tool [SMART]; http://smart.embl-heidelberg.de/).
Figure 2 with 3 supplements
FBXO24 is essential for sperm flagellum formation and motility.
(A) The histology of seminiferous tubules at different stages. An asterisk indicates remaining sperm heads. (B) Morphology of mature spermatozoa obtained from cauda epididymis. White arrowhead indicates straight spermatozoa. Red arrowhead indicates bent spermatozoa. Black asterisk indicates coiled spermatozoa. Red asterisk indicates headless spermatozoa. (C) Stacked bar graph showing the frequency of sperm morphology classified as straight, bent, coiled, and headless. n = 3 independent experiments. (D) Percentages of motile spermatozoa were analyzed at 10 and 120 min after incubation in capacitation medium. (E) VAP (average path velocity) was analyzed at 10 and 120 min after incubation in capacitation medium. (F) VSL (straight line velocity) was analyzed at 10 and 120 min after incubation in capacitation medium. (G) VCL (curvilinear velocity) was analyzed at 10 and 120 min after incubation in capacitation medium. Error bars are mean ± standard deviation. Each dot indicates individual mouse. Statistical significance was assessed with a two-tailed Welch's t-test. *p<0.05, **p<0.01, ***p<0.001.
Figure 2—figure supplement 1
Histological analyses of Fbxo24 knockout (KO) testis and epididymis.
(A) Gross morphology of testes obtained from Fbxo24 heterozygous and KO males. (B) Test weight (mg)/body weight (mg) of Fbxo24 heterozygous and KO mice. (C) PAS-hematoxylin staining of testes and cauda epididymis sections. (D) Spermatozoa obtained from the cauda epididymis were stained for IZUMO1 (magenta). Nuclei were stained with Hoechst 33342 (blue). Error bars are mean ± standard deviation. Each dot indicates individual mouse. Statistical significance was assessed with a two-tailed Welch's t-test. n.s. = non-significant.
Figure 2—video 1
Sperm motility of Fbxo24 heterozygous mice.
Sperm motion was recorded after 120 min of incubation in TYH medium. Actual width of the image is 870 µm.
Figure 2—video 2
Sperm motility of Fbxo24 knockout (KO) mice.
Sperm motion was recorded after 120 min of incubation in TYH medium. Actual width of the image is 870 µm.
Figure 3 with 1 supplement
In vitro fertilizing ability and in vivo sperm migration.
(A) The fertilizing ability of spermatozoa was analyzed in vitro using cumulus-intact oocytes. (B) The fertilizing ability of spermatozoa was analyzed in vitro using cumulus-free oocytes. (C) The fertilizing ability of spermatozoa was analyzed in vitro using zona-free oocytes. (D) Uterus and oviducts of wild-type (WT) females mated with WT or Fbxo24 knockout (KO) males carrying Red Body Green Sperm (RBGS) transgene. Female reproductive tracts were dissected 4 hr after confirming a plug. Right figures are magnified images of the boxes indicated in the middle panels. (E) Immunoblotting of ADAM3 using testis and mature spermatozoa of Fbxo24 heterozygous or KO mice. (F) Intracytoplasmic sperm injection (ICSI) experiment. The number of two-cell stage embryos and pups developed from WT oocytes injected with WT or Fbxo24 KO spermatozoa. (G) Pups derived from WT or Fbxo24 KO spermatozoa. (H) Genotyping of pups obtained from Fbxo24 KO spermatozoa. N.C. indicates negative control (water). Error bars are mean ± standard deviation. Each dot indicates individual mouse. Statistical significance was assessed with a two-tailed Welch's t-test. *p<0.05, **p<0.01, ***p<0.001.
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Figure 3—source data 1
Uncropped and labeled blots for Figure 3E.
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Figure 3—source data 2
Raw unedited blots for Figure 3E.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig3-data2-v1.zip
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Figure 3—source data 3
Uncropped and labeled gels for Figure 3H.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig3-data3-v1.zip
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Figure 3—source data 4
Raw unedited gels for Figure 3H.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig3-data4-v1.zip
Figure 3—figure supplement 1
Lack of Fbxo24 impairs sperm viability and the acrosome reaction.
(A) Viability of cauda epididymal spermatozoa was assessed with propidium iodide (PI) at 10 and 120 min incubation in TYH medium. (B) The acrosome reaction rates of cauda epididymal spermatozoa were assessed using Red Body Green Sperm (RBGS) Tg mice after 10 and 120 min of incubation in TYH medium. To induce the acrosome reaction, Ca2+ ionophore A23187 was added to the medium after 120 min of incubation. (C) Mass spectrometry (MS) analyses of Triton X-100 soluble proteins obtained from mature spermatozoa. Quantitative value of identified SNARE-related proteins and PLCD4 were listed. (D) Immunodetection of STX2 in Fbxo24 wild-type (WT) and knockout (KO) spermatozoa. IZUMO1 was used as a loading control. (E) Immunodetection of LY6K and ADAM3 in Fbxo24 WT and KO spermatozoa. SLC2A3 was used as a loading control. Error bars are mean ± standard deviation. Each dot indicates individual mouse. Statistical significance was assessed with a two-tailed Welch's t-test. *p<0.05, **p<0.01, n.s. = non-significant.
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Figure 3—figure supplement 1—source data 1
Uncropped and labeled blots for Figure 3—figure supplement 1D and E.
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Figure 3—figure supplement 1—source data 2
Raw unedited blots for Figure 3—figure supplement 1D and E.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig3-figsupp1-data2-v1.zip
Figure 4 with 1 supplement
Numerous membraneless electron-dense granules were found in Fbxo24 knockout (KO) spermatozoa.
(A) Epididymal spermatozoa of Red Body Green Sperm (RBGS) Tg mice were stained with Hoechst 33342 (nuclei). Mitochondria were labeled with su9-DsRed2. White arrowheads indicate disorganized mitochondria. (B) Sperm mitochondrial sheath formation during spermiogenesis was observed by scanning electron microscopy (SEM). (C) Cross sections of spermatozoa in the cauda epididymis. Asterisks indicate electron-dense granules. (D) Longitudinal sections of spermatozoa in the cauda epididymis. An asterisk indicates electron-dense granules. (E) Percentages of morphologically abnormal mitochondria observed with transmission electron microscopy (TEM). The number of flagellar sections analyzed is shown above each bar. (F) Percentages of electron-dense granules observed in the midpiece cross sections with TEM. The number of flagellar sections analyzed is shown above each bar. Error bars are mean ± standard deviation. Each dot indicates individual mouse. Statistical significance was assessed with a two-tailed Welch's t-test. *p<0.05, **p<0.01.
Figure 4—figure supplement 1
Sperm flagellum ultrastructure was disorganized in Fbxo24 knockout (KO) mice.
(A) Localization of the annulus in cauda epididymal spermatozoa. Anti-SEPT4 antibody was used to visualize the annulus (green). Nuclei were detected with Hoechst 33342 (blue). The acrosome was detected with anti-IZUMO1 antibody (magenta). (B) Transmission electron microscopy (TEM) observation of the principal piece. A white arrowhead indicates a disruption of the axoneme and outer dense fiber (ODF). (C) Percentages of morphologically abnormal fibrous sheaths (FS) observed with TEM. The number of flagellar sections analyzed is shown above each bar. (D) Percentages of morphologically abnormal axonemes (AX) observed with TEM. The number of flagellar sections analyzed are shown above each bar. (E) Percentages of morphologically abnormal ODF observed with TEM. The number of flagellar sections analyzed are shown above each bar. (F) TEM observation of the principal piece. A red arrowhead indicates electron-dense granules. (G) Percentages of electron-dense granules observed in principal piece cross sections. The number of flagellar sections analyzed are shown above each bar. Error bars are mean ± standard deviation. Each dot indicates individual mouse. Statistical significance was assessed with a two-tailed Welch's t-test. *p<0.05, ***p<0.001.
Figure 5 with 2 supplements
IPO5 and KPNB1 accumulate in Fbxo24 knockout (KO) spermatozoa.
(A) Mass spectrometry analyses of mature spermatozoa. Significantly upregulated proteins are shown with red dots whereas significantly downregulated proteins are shown with blue dots. (B) Immunoblotting analysis was performed using proteins extracted from testes or mature spermatozoa. IPO5 was detected using rabbit anti-IPO5 polyclonal antibody. KPNA2 and IZUMO1 were detected as negative control and loading control, respectively. (C) Spermatozoa obtained from the cauda epididymis were stained for IPO5 (magenta) using rabbit anti-IPO5 polyclonal antibody. Nuclei were stained with Hoechst 33342 (blue). (D) Immunoprecipitation (IP) of FBXO24-FLAG from Fbxo24-FLAG Tg testes. FBXO24 could interact with IPO5. IPO5 was detected using mouse anti-IPO5 monoclonal antibody. IPO5 band is slightly larger after IP likely due to different protein composition in the sample. β-Actin was used as a loading control. A picture with high contrast was shown for the input FLAG band. (E) IP of FBXO24-FLAG using HEK293T cells. FBXO24 could interact with IPO5 but not with KPNB1. IPO5 was detected using mouse anti-IPO5 monoclonal antibody. β-Actin was used as a loading control. (F) A ubiquitination assay of IPO5 using HEK293T cells. IPO5-PA was immunoprecipitated and the level of ubiquitination was analyzed using anti-HA antibody. β-Actin was used as a loading control.
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Figure 5—source data 1
Uncropped and labeled blots for Figure 5B, D, E, and F.
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Figure 5—source data 2
Raw unedited blots for Figure 5B, D, E, and F.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig5-data2-v1.zip
Figure 5—figure supplement 1
No clear differences were found in the amount and localization of ribonucleoprotein (RNP) granule-related proteins between control and Fbxo24 knockout (KO) testes.
(A) Immunodetection of ADAD1, ADAD2, MILI, MIWI, RNF-17, YTHDC2, TSKS, and TSSK1 in Fbxo24 heterozygous and KO testes. β-Actin was used as a loading control. (B) Immunofluorescence observation of MIWI (green) in wild-type (WT) and Fbxo24 KO testes. Acrosomes were stained with PNA (red) and nuclei were stained with Hoechst 33342 (white). (C) Immunofluorescence observation of TSKS (green) in WT and Fbxo24 KO testes. Acrosomes were stained with PNA (red) and nuclei were stained with Hoechst 33342 (white).
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Figure 5—figure supplement 1—source data 1
Uncropped and labeled blots for Figure 5—figure supplement 1A.
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Figure 5—figure supplement 1—source data 2
Raw unedited blots for Figure 5—figure supplement 1A.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig5-figsupp1-data2-v1.zip
Figure 5—figure supplement 2
Generation and analyses of Fbxo24−3×FLAG Tg mice.
(A) Schematic of the Fbxo24-3×FLAG construct used to generate Tg mice. (B) Immunodetection of FBXO24-3×FLAG in the testis from the Tg mice. β-Actin was used as a loading control. (C) Morphology of cauda epididymal spermatozoa. (D) Schematic for generating Fbxo24 knockout (KO) #2 mice using the CRISPR/Cas9 system. White boxes indicate untranslated regions while black boxes indicate protein coding regions. The gRNAs used are shown. Fw and Rv indicate the forward and reverse primers used for genotyping, respectively. (E) Genotyping of obtained Fbxo24 mutant mice. Fw #3-Rv #3 primers in (D) were used. N.C. indicates negative control (water). (F) Predicted FBXO24 amino acid sequences of KO #2 mutant mice. The 205 bp deletion and 1 bp insertion resulted in E32A mutation with a premature stop codon introduced 49 amino acids later. (G) Morphology of mature spermatozoa obtained from cauda epididymis. (H) The list of identified proteins by immunoprecipitation mass spectrometry (IP-MS) analysis. The top 15 identified proteins in Tg are shown. The quantitative value (normalized total spectra) was calculated using Scaffold proteome software. IgG-related proteins, which is likely from the antibody used for IP, were removed from the list.
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Figure 5—figure supplement 2—source data 1
Uncropped and labeled blots for Figure 5—figure supplement 2B.
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Figure 5—figure supplement 2—source data 2
Raw unedited blots for Figure 5—figure supplement 2B.
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Figure 5—figure supplement 2—source data 3
Uncropped and labeled gels for Figure 5—figure supplement 2E.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig5-figsupp2-data3-v1.zip
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Figure 5—figure supplement 2—source data 4
Raw unedited gels for Figure 5—figure supplement 2E.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig5-figsupp2-data4-v1.zip
Figure 6 with 1 supplement
KPNB1 and IPO5 are recruited to stress granules (SGs).
(A) Total RNA amounts in spermatozoa were measured by ultraviolet absorption. (B) Electrophoresis of RNA extracted from mature spermatozoa. (C) KPNB1 and IPO5 were localized to SGs under exposure to oxidative stress. COS7 cells were treated with water (upper row) or arsenite (lower row). Nuclei were stained with Hoechst 33342 (blue). (D) KPNB1 and IPO5 were localized to SGs under exposure to a proteasome inhibitor. COS7 cells were treated with DMSO (upper row) or MG132 (lower row). Nuclei were stained with Hoechst 33342 (blue). Error bars are mean ± standard deviation. Each dot indicates individual mouse. Statistical significance was assessed with a two-tailed Welch's t-test. ***p<0.001.
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Figure 6—source data 1
Uncropped and labeled gels for Figure 6B.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig6-data1-v1.zip
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Figure 6—source data 2
Raw unedited gels for Figure 6B.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig6-data2-v1.zip
Figure 6—figure supplement 1
Spermatogenic cells obtained from cauda epididymis.
Spermatozoa were obtained from Fbxo24 heterozygous or knockout (KO) male mice. Immature spermatogenic cells or somatic cells were rarely observed.
Additional files
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Supplementary file 1
Primers and gRNAs used in this study.
- https://cdn.elifesciences.org/articles/92794/elife-92794-supp1-v1.xlsx
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Supplementary file 2
Antibodies used in this study.
- https://cdn.elifesciences.org/articles/92794/elife-92794-supp2-v1.xlsx
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MDAR checklist
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FBXO24 deletion causes abnormal accumulation of membraneless electron-dense granules in sperm flagella and male infertility
eLife 13:RP92794.
https://doi.org/10.7554/eLife.92794.3
