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 (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 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 Fig. 1E 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 three months.

FBXO24 is essential for sperm flagellar 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 min and 120 min after incubation in capacitation medium. (E) VAP (average path velocity) was analyzed at 10 min and 120 min after incubation in capacitation medium. (F) VSL (straight line velocity) was analyzed at 10 min and 120 min after incubation in capacitation medium. (G) VCL (curvilinear velocity) was analyzed at 10 min and 120 min after incubation in capacitation medium.

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 WT females mated with WT or Fbxo24 KO males carrying RBGS transgene. Female reproductive tracts were dissected 4 h 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) 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).

Numerous membraneless electron-dense granules were found in Fbxo24 KO spermatozoa.

(A) Epididymal spermatozoa of RBGS Tg mice were stained with Hoechst 33342 (nuclei). Mitochondria were labeled with su9-DsRed2. White arrowhead indicates peeled off 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.

IPO5 and KPNB1 accumulate in Fbxo24 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) In vivo 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.

KPNB1 and IPO5 are recruited to 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 stress granules (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).