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, …
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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 …
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 …
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 …
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 …
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Figure 3—source data 1
Uncropped and labeled blots for Figure 3E.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig3-data1-v1.zip
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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 …
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Figure 3—figure supplement 1—source data 1
Uncropped and labeled blots for Figure 3—figure supplement 1D and E.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig3-figsupp1-data1-v1.zip
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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 …
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 …
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) …
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Figure 5—source data 1
Uncropped and labeled blots for Figure 5B, D, E, and F.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig5-data1-v1.zip
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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 …
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Figure 5—figure supplement 1—source data 1
Uncropped and labeled blots for Figure 5—figure supplement 1A.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig5-figsupp1-data1-v1.zip
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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 …
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Figure 5—figure supplement 2—source data 1
Uncropped and labeled blots for Figure 5—figure supplement 2B.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig5-figsupp2-data1-v1.zip
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Figure 5—figure supplement 2—source data 2
Raw unedited blots for Figure 5—figure supplement 2B.
- https://cdn.elifesciences.org/articles/92794/elife-92794-fig5-figsupp2-data2-v1.zip
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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 …
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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
- https://cdn.elifesciences.org/articles/92794/elife-92794-mdarchecklist1-v1.pdf
