Expression profiles of FBXO24 during testicular development and spermatogenesis in mice.

(A) qPCR analysis of FBXO24 mRNA levels in multiple organs in mice. n = 3/group. Data are mean ± S.D. *, p < 0.05. (B) qPCR analysis of FBXO24 mRNA levels in developing testes at postnatal day 0 (P0), P3, P7, P14, P28, and P35. n = 3/group. Data are mean ± S.D. *, p < 0.05. (C) qPCR analysis of FBXO24 mRNA levels in isolated spermatogenic cell populations, including spermatogonia (SG), Sertoli cells (SE), spermatocytes (SC), round spermatids (RS), and elongating spermatids (ES). n = 3/group. Data are mean ± S.D. *, p < 0.05. (D) Western blot analysis of FBXO24-HA expression in the tissues of adult transgenic mice. (E-F) Co-immunostaining of FBXO24-HA (red) and (E) γH2AX (green) or (F) PNA (green) in adult Fbxo24-HA testes. Scale bar, 25 μm. DNA (blue) is stained by DAPI. Spermatogenic stages are noted.

FBXO24 deletion impairs spermatogenic defects in the late steps of spermiogenesis.

(A) qPCR analysis indicates that FBXO24 mRNA is markedly decreased in the testis of Fbxo24 knockout (KO) as compared to wild-type (WT). ***, p < 0.001. (B) The fertility tests of WT and Fbxo24 KO male mice mated with fertile female mice are shown. n = 3/group. Error bars represent mean ± S.D. ***, p < 0.001; ns: not significant. (C) Histological images of testes and epididymis of WT and Fbxo24 KO mice at 8-weeks old are shown. (D) PAS-hematoxylin staining of Fbxo24 KO testis at 8-weeks old contained less-condensed late spermatids (red arrows). Spermatogenic stages are noted. RS, round spermatids; ES, elongating spermatids; Pl, preleptotene; P, pachytene; Z, zygotene; D, diplotene. Scale bars = 25 μm. (E) The number of late spermatids is significantly reduced in Fbxo24 KO testis. Ratios of spermatids and Sertoli cells in tubule cross-sections of specific stages of seminiferous epithelial cycles and corresponding spermatid development steps are shown. Data are mean ± S.D. *, p < 0.05. (F) Litter sizes of mating tests. F1 generation of intercrosses between the indicated males and Fbxo24+/− females are shown. Each dot represents one litter.

Sperm mitochondria and flagella are defective in FBXO24-deficient mice.

(A-B) Quantification of sperm counts (A) and sperm motility (B) from WT and Fbxo24 KO epididymis are shown. n = 3/group. Error bars represent mean ± S.D. **, p < 0.01. ***, p < 0.001. (C) Sperm morphological images show the defective sperm of Fbxo24 KO mice. Red arrows indicate abnormal gaps in the mitochondrial sheath. Scale bars = 5 μm. (D) Immunofluorescence images of sperm from WT and Fbxo24 KO epididymis. PNA (acrosome, green), MitoTracker (mitochondria, red), and DAPI (nucleus, blue). White arrows indicate the weak or absent staining of MitoTracker. Scale bars = 5 μm. (E) Quantifications of the length of sperm midpiece from WT and Fbxo24 KO mice are shown. n = 100/group. Error bars represent mean ± S.D. ***, p < 0.001. (F) Scanning electronic microscopy (SEM) images indicate the mitochondria detachment from the flagellum of Fbxo24 KO sperm. Right panel insets show higher magnification of sperm midpiece. The arrowheads indicate the annulus. The dashed red line indicates a region where mitochondria are absent. Scale bars = 2 μm. (G) Transmission electronic microscopy (TEM) images indicate the mitochondria defects in three regions of Fbxo24 KO sperm midpiece in the longitudinal sections. Nu, Nucleus. The dashed red lines indicate a region where mitochondria are absent. The white arrowhead indicates sperm annulus. Scale bar = 0.2 μm. (H) TEM images indicate the ultrastructure of the midpiece, principal piece and end piece of WT and Fbxo24 KO sperm flagellum in the cross-sections. Arrows indicate the mitochondria (MI), outer dense fiber (ODF) and axoneme. Scale bar = 0.2 μm. (I-J) Western blot shows the levels of proteins of mitochondria (I) and axoneme (J) of WT and Fbxo24 KO sperm. GAPDH serves as a loading control.

Ablation of FBXO24 affects mitochondria and chromatoid body architecture in the round spermatids.

(A) TEM images show vacuolar mitochondria with disorganized cristae in the round spermatids of Fbxo24 KO testes. Right panel insets show higher magnification of mitochondria. Scale bars = 1 μm. (B) Quantification of the number of vacuolar mitochondria. ***, p < 0.001. (C) TEM images showing decondensed and enlarged chromatoid body (CB) with an irregular network in the round spermatids of Fbxo24 KO testes. Right panel insets show a higher magnification of CB. Scale bars = 1 μm. (D) Quantification of size/diameters of CB. *, p < 0.05. (E) Western blot analysis expression levels of CB components and PRMT6 in testes from WT and Fbxo24 KO mice at 8 weeks old. GAPDH serves as a loading control.

FBXO24 deficiency in mice impairs sperm histone-to-protamine exchange.

(A) SEM images show the abnormality of Fbxo24 KO sperm head. AA, anterior acrosome; EQ, equatorial segment; PAS, postacrosomal segment; VS, ventral spur; HR, hook rim. Scale bars = 2 μm. (B) TEM images show the decondensed nucleus (Nu) of Fbxo24 KO sperm. Scale bars = 1 μm. (C) Western blot analysis of the expression of histones (H2A, H2B, H3, and H4), transition proteins (TNP1), and protamines (PRM2) from WT and Fbxo24 KO sperm are shown. GAPDH serves as a loading control. (D) Quantification of protein levels. Error bars represent mean ± S.D., n = 3. *, p < 0.05. (E) Western blot analysis of the expression of indicated proteins in WT and Fbxo24 KO testis. GAPDH serves as a loading control. (F) Quantification of protein levels. Error bars represent mean ± S.D., n = 3. *, p < 0.05.

RNA-seq analyses of the round spermatids from FBXO24-deficient testes.

(A) Volcano plot of differentially expressed transcripts in the round spermatids (RS) of Fbxo24 KO vs. WT mice. Each red (up-regulation) or blue (down-regulation) dot represents a significantly changed gene. (B) GO term enrichment analysis of downregulated transcripts of Fbxo24 KO RS. (C-D) Gene expressions of mitochondrion localization (C) and chromatin organization (D) in RNA-seq analysis. (E) Venn diagrams showing the overlap between downregulated genes and abnormal alternative splicing genes in Fbxo24 KO RS. (F) Summary of differential splicing evens in Fbxo24 KO RS. The number of each category of alternative splicing is indicated.

Aberrant alternative splicing of spermiogenesis genes in the round spermatids of FBXO24-deficient mice.

(A) Co-immunoprecipitation analysis of FBXO24 and the splicing regulators (SRSF2, SRSF3, and SRSF9) in Fbxo24-HA tagged mice testis. WT testis was used as a negative control. (B) Western blotting analysis of the splicing regulators in WT and Fbxo24 KO testis. GAPDH was used as a loading control. (C) Quantification of protein levels. Error bars represent mean ± S.D., n = 3. *, p < 0.05. (D-H) Validation of abnormal alternative splicing genes related to (D) mitochondria (Mfn1), (E) flagellum (Zmynd12, Map7d1, and Bbs7), (F) chromatin (Phf7 and Kat5), (G) acrosome (Nucb2), and (H) ubiquitination (Ube2j2). The top panels represent RT-PCR analysis of indicated genes in WT and Fbxo24 RS. Gapdh serves as a loading control. The middle panels show the quantification of percent spliced in (PSI) and alternative sites in RNA-seq. Error bars represent mean ± S.D., n = 2. *, p < 0.05. The bottom panels represent the schematic diagram of alternative sites exons.

FBXO24 interacts with MIWI and mediates its K48-linked polyubiquitination.

(A) HEK293T cells transfected with empty FBXO24-mCherry or mCherry vector. Anti-mCherry beads were used for immunoprecipitation (IP), and western blots were used to detect the CUL1 (left panel) and MIWI (right panel) expression. (B) The testis lysate of Fbxo24-HA tagged mice were immunoprecipitated with anti-HA beads. Western blots were used to detect the HA, CUL1, MIWI, SKP1, and RBX1expression. (C) Schematic structures of the truncated FBXO24 protein are shown. Broken boxes show the domain of F-box and regulator of chromosome condensation 1 (RCC1). (D) HEK293T cells were transfected with indicated plasmids. Immunoprecipitation was performed using the anti-mCherry antibody. (E) Western blot analysis of HEK293T cells transfected with indicated FBXO24-mCherry and 2μg MIWI-myc plasmids. The cell lysates were immunoblotted with anti-mCherry and anti-myc antibodies. (F) FBXO24 mediated the ubiquitination of MIWI in the presence of Ub(K48) not Ub (K63). (G) HEK293T cells were transfected with indicated FBXO24-mCherry, 2μg MIWI-myc, and 2μg Ub-HA plasmids. The cell lysates were immunoblotted with the anti-mCherry, anti-myc, and anti-HA antibodies. GAPDH serves as a loading control. (H) Ubiquitination analysis of MIWI in the round spermatids of Fbxo24 KO mice. The cells were treated with MG132 (10 µM) in the ubiquitination assay.

Small RNA-seq analysis of testes from FBXO24-deficient mice.

(A) A scatter plot of differentially expressed miRNA is shown. Red and green dots represent up- and down-regulated miRNA (fold change > 2, p <0.05), respectively. (B) Genomic distribution of piRNA profile in Fbxo24 KO vs. WT testis. piRNA levels were examined in each 200 bp interval of a 2 kb region upstream and downstream of the annotated genes. (C) Scatter plots of differentially expressed piRNA mapping density (reads/kb) of the coding region (CDS), 5′ and 3′ untranslated region (UTR), and intron, as well as transposable element (TE), including retrotransposon (LTR, LINE, and SINE) and DNA transposon. The piRNA read counts were normalized with miRNA. Red and green dots represent up- and down-regulated piRNA (fold change > 2, p <0.05), respectively. (D) The size distribution of piRNAs in Fbxo24 KO vs. WT testis. (E) The top 10 up-regulated piRNAs in Fbxo24 KO testis exist in MIWI immunoprecipitates of GSM822760 data.

A schematic model shows the FBXO24-mediated post-transcriptional regulation during spermiogenesis.

Fbxo24 interacts with key splicing factors (SRSF2, SRSF3, and SRSF9) to coordinate proper alternative splicing of the target mRNA transcripts involved in spermiogenesis. FBXO24 regulates the architectures of mitochondria and chromatid body through MIWI/piRNA pathway in the round spermatids.