Loss of XAP5 and XAP5L proteins results in male mice infertility.

(A) Detection of XAP5/XAP5L protein in different tissues of adult mice by Western blot analysis. (B) Expression of XAP5/XAP5L protein during postnatal testicular development. β-actin was used as control. (C) t-SNE plots displaying XAP5/XAP5L gene expression levels in individual cell types of mouse testis visualized using published single-cell data(Jung et al., 2019). Black arrow represents the developmental pseudotime corresponding to the developmental ordering of each cell progressing through spermatogenesis. Dot color intensity represents the expression level. Cells from different stages are color-coded. (D) Immunofluorescence staining of XAP5/XAP5L in adult WT testis. Representative spermatogonium (Sg), round spermatid (Rs) and elongating spermatid (Es) are indicated. Scale bars: 20 μm (white), 5 μm (red). (E) Similar development between WT and XAP5L KO mice. Scale bar: 1 cm. (F) Average litter size of pups obtained by fertility test. Each male was caged with two females for 2 months, 5 males were used for each genotype. ***P < 0.001, n.s. stands for not significant. (G) Similar appearance of WT and XAP5 cKO mice. Scale bar: 1 cm. (H) Average litter size of pups obtained by fertility test. Each male was caged with two WT females for 2 months, 4 males were used for each genotype. ***P < 0.001. Error bars depict means ± SEM. All P-values were calculated using an unpaired, two-tailed Student’s t-test.

XAP5L is required for normal sperm formation.

(A) Similar gross testes morphology, body weight and testis weight between WT and XAP5L KO mice. Scale bar: 1 mm. n = 6, n.s. stands for not significant. (B) H&E staining showing similar histological structure of the testes from WT and XAP5L KO mice. Scale bars: 100 μm. (C) Significantly reduced sperm total motility of XAP5L KO mice. n = 3, **P < 0.01. (D) H&E staining showing the morphology of sperm in WT and XAP5L KO mice. Scale bars: 10 μm. (E) Significantly increased abnormal sperm in XAP5L KO mice. n = 6, ***P < 0.001. (F) Electron microscopic analysis displaying ultrastructural defects in XAP5L KO sperm flagella, including cytoplasmic vacuoles enveloping mitochondrial sheath (as shown by red arrowheads), a complete lack of mitochondrial sheath (as shown by red dotted circle), two or more cross-sections of the same sperm flagellum enclosed within one cell membrane (as shown by black arrowheads), partially formed outer dense fibers, and the axonemal microtubules (as indicated by asterisks). Scale bars: 2 μm. Error bars depict means ± SEM. All P-values were calculated using an unpaired, two-tailed Student’s t-test.

Ablation of XAP5 in germ cells results in arrested spermatogenesis and absence of sperm.

(A) Testes sizes, body weight and testis weight from WT and XAP5 cKO mice at various ages were evaluated. Similar body weight was observed between WT and XAP5 cKO mice. No significant difference in size and weight was detected between WT and XAP5 cKO testes until P16. Scale bars: 1 mm. n = 5, ***P < 0.001, n.s. stands for not significant. (B) Histology of testes from WT and XAP5 cKO mice at various ages were evaluated by H&E staining. No pachytene spermatocytes were found, and many vacuolated tubules were observed in XAP5 cKO testis since P16. Black arrowheads indicate pachytene spermatocytes; black arrows indicate round spermatids. Vacuolated seminiferous tubules are indicated by asterisks. Scale bars: 20 μm (black), 2.5 μm (red). (C) Chromosome spreads staining showing pachytene spermatocytes from P16 WT male mouse. Yellow dotted circle indicates the XY body. Scale bars: 10 μm. (D) Immunofluorescence staining of PNA in adult testes indicating the absence of spermatids in XAP5 cKO mice. Scale bars: 20 μm. (E) H&E staining of adult epididymis displaying absence of sperm in XAP5 cKO mice. Scale bars: 50 μm. Error bars depict means ± SEM. All P-values were calculated using an unpaired, two-tailed Student’s t-test.

Aberrant ciliogenic and spermatogenic gene expression in XAP5L KO and XAP5 cKO male germ cells.

(A) Gene ontology analysis showing top ten biological process terms significantly enriched among upregulated genes in XAP5L KO sperm. (B) Real-time PCR validation of genes involved in ciliogenesis and spermatogenesis. n = 3, **P < 0.01, ***P < 0.001, n.s. stands for not significant. (C) Detection of FOXJ1 and RFX2 in mature sperm of WT and XAP5L KO mice by Western blot analysis. GAPDH was used as a control. (D) Gene ontology analysis showing top ten biological process terms significantly enriched among downregulated genes in P16 XAP5 cKO testes. (E) Real-time PCR validation of genes involved in ciliogenesis and spermatogenesis. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, n.s. stands for not significant. (F) Detection of FOXJ1 and RFX2 in P16 testes of WT and XAP5 cKO mice by Western blot analysis. GAPDH was used as a control. (G) A model on the involvement of XAP5 and XAP5L in the regulation of ciliogenesis during spermatogenesis. Error bars depict means ± SEM. All P-values were calculated using an unpaired, two-tailed Student’s t-test.

Localization of XAP5/XAP5L in testicular cells.

(A) Western blot analysis showing no localization of XAP5/XAP5L in mature sperm. Western blot analysis showing the subcellular localization of endogenous XAP5 (B) and XAP5L (C). LMNB used as a nuclear protein marker, GAPDH used as a cytoplasmic protein marker. (D) Immunofluorescence staining of XAP5/XAP5L in adult epididymis. Scale bars: 50 μm.

Generation of XAP5L KO and XAP5 cKO mice.

(A) Schematic illustration of the targeting strategy for generating a XAP5L knockout mouse line using CRISPR-Cas9 system. Upper panel showing genomic structure of the XAP5L gene. The location of two sgRNAs (sgRNA1 and sgRNA2) used and three primers (blue arrows) for genome PCR are indicated. Lower panel displaying nucleotide sequence of WT allele in the vicinity of two sgRNAs (red color) and the corresponding sequence of the XAP5L homozygous mutant (KO) allele. The deletion of XAP5L gene was verified by Sanger sequencing. (B) Genome PCR analysis in WT and XAP5L KO mice. (C) Detection of XAP5L in adult testes extracts of WT and XAP5L KO mice by Western blot analysis. β- actin was used as a control. (D) Schematic illustration of the targeting strategy for generating XAP5 cKO mice. The location of LoxP sites inserted and the primers (blue arrow) used for genome PCR are indicated. (E) Genome PCR analysis in WT and XAP5 cKO mice. (F) Detection of XAP5 in testes extracts of WT and XAP5 cKO mice by Western blot analysis. β-actin was used as a control.

XAP5L-mediated regulation of ciliary gene expression during mouse spermatogenesis.

(A) MA plot showing the numbers of differentially expressed genes in XAP5L KO sperm. Gene ontology analysis showing top ten cellular component (B) and molecular function (C) terms significantly enriched among upregulated genes in XAP5L KO sperm. (D) Venn diagram representing total genes upregulated/downregulated by at least twofold in XAP5L KO sperm and published cilia-related genes(Nemajerova et al., 2016). (E) Real-time PCR validation of genes involved in ciliogenesis and spermatogenesis. n = 3, *P < 0.05, ***P < 0.001. Error bars depict means ± SEM. All P-values were calculated using an unpaired, two-tailed Student’s t-test.

TULP2 KO male mice were infertile due to malformation of sperm.

(A) Detection of TULP2 protein in different tissues of adult mice by Western blot analysis.

(B) t-SNE plot displaying TULP2 gene expression pattern during spermatogenesis visualized using published single-cell data(Jung et al., 2019). Black arrow represents the developmental pseudotime corresponding to the developmental ordering of each cell progressing through spermatogenesis. Dot color intensity represents the expression level. Cells from different stages are color-coded. (C) Immunofluorescence staining showing TULP2 expression in the cytoplasm of round spermatids (Rs) and elongating spermatids (Es). Scale bars: 5 μm. (D) Similar development of adult WT and TULP2 KO mice. Scale bar: 1 cm. (E) Average litter size of pups obtained by fertility test. Each male was caged with two females for 2 months. ***P < 0.001, n.s. stands for not significant. (F) Similar gross testes morphology, body weight and testis weight between WT and TULP2 KO mice. Scale bar: 1 mm. n = 4, n.s. stands for not significant. (G) Significantly reduced caudal epididymal sperm counts in TULP2 KO mice compared with WT males. n = 7, ***P < 0.001. (H) Significantly reduced sperm total motility of TULP2 KO mice. n = 3, ***P < 0.001. Significantly increased abnormal sperm in TULP2 KO mice (I and J). Scale bars: 10 μm. n = 3, ***P < 0.001. (K) Electron microscopic analysis displaying ultrastructural defects in TULP2 KO sperm flagella, including two cross-sections of the same sperm flagellum enclosed within one cell membrane (as shown by red arrowheads), disorganized outer dense fibers and the axonemal microtubules (as shown by red dotted circle). Scale bars: 8 μm (black), 2 μm (red). Error bars depict means ± SEM. All P-values were calculated using an unpaired, two-tailed Student’s t-test.

XAP5-mediated regulation of ciliary gene expression during mouse spermatogenesis.

(A) Heat map showing variably expressed genes in P16 XAP5 cKO testes. Number of related genes was indicated. Gene ontology analysis showing top ten cellular component

(B) and molecular function (C) terms significantly enriched among downregulated genes in XAP5 cKO testes. (D) Venn diagram representing total genes upregulated/downregulated in XAP5 cKO testes and published cilia-related genes(Nemajerova et al., 2016). (E) Venn diagram representing total genes downregulated in XAP5 cKO testes, upregulated in XAP5L KO sperm by at least twofold and published cilia-related genes(Nemajerova et al., 2016). (F) Real-time PCR validation of genes involved in ciliogenesis and spermatogenesis. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. Error bars depict means ± SEM. All P-values were calculated using an unpaired, two-tailed Student’s t-test.

t-SNE plots displaying expression patterns of FOXJ1 and RFX families in individual cell types of mouse testes.

The black arrow represents the developmental pseudotime corresponding to the developmental ordering of each cell progressing through spermatogenesis. Dot color intensity represents the relative expression level. Cells from different stages are color- coded. The gene expression was visualized using the published single-cell data(Jung et al., 2019).