SLC35G3 is a UDP-N-acetylglucosamine transporter for sperm glycoprotein formation and underpins male fertility in mice
- Department of Experimental Genome Research, Research Institute for Microbial Diseases, The University of Osaka, Japan
- Immunology Frontier Research Center, The University of Osaka, Japan
- Department of Pathology and Immunology, Baylor College of Medicine, United States
- Center for Drug Discovery, Baylor College of Medicine, United States
- Center for Advanced Modalities and Drug Delivery System, The University of Osaka, Japan
- Center for Infectious Disease Education and Research, The University of Osaka, Japan
- The Institute of Medical Science, The University of Tokyo, Japan
Figures
Figure 1 with 2 supplements
SLC35G3 is a multi-pass transmembrane protein with unique testes-specific expression in the Golgi apparatus during early spermiogenesis.
(A) Phylogenetic tree of Slc35g3 from the TreeFam database, with dark green areas indicating the presence and light green areas indicating the absence of Slc35g3. (B) RT-PCR results across multiple tissues (upper panel) and from testes at various days postpartum (lower panel); Br: brain, Th: thymus, Lu: lung, He: heart, Li: liver, Sp: spleen, Ki: kidney, Te: testis; Epi: epididymis, Cap: caput epididymis, Cor: corpus epididymis, Cau: cauda epididymis; SV: seminal vesicle, Pr: prostate, CG: coagulating gland, Ut: uterus, Ov: ovary. β-actin (Actb) was used as the loading control. (C) scRNA-seq prediction of cells strongly expressing Slc35g3 mRNA (Mouse Cell Atlas). Dots with low transparency represent cells with predicted expression. (D) SLC35G3 structure predicted using AlphaFold. (E) From left to right: Hoechst33342 staining image, SLC35G3 immunostaining image, GM130 immunostaining image, and merged image of wild-type testicular germ cells. Scale bar: 10 µm, RS: round spermatid.
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Figure 1—source data 1
Gel for Figure 1, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/107494/elife-107494-fig1-data1-v1.pdf
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Figure 1—source data 2
Original files for gel analysis displayed in Figure 1.
- https://cdn.elifesciences.org/articles/107494/elife-107494-fig1-data2-v1.zip
Figure 1—figure supplement 1
Slc35g3 is the sole Slc35 family member that is testis-specific, with highest mRNA levels in round spermatids.
Levels of mRNA for Slc35 family genes in organs and testis ages were created using Mammalian Reproductive Genetics Database V2.
Figure 1—figure supplement 2
SLC35G3 has the potential to form a homodimer.
(A) Transfection of pCAG1.1-Slc35g3-mCherry into HEK293T cells followed by Western blot analysis using an mCherry antibody revealed a signal at the predicted size of 62.2 kDa (black arrowhead). Simultaneously, a signal was also detected at approximately twice the size (red arrowhead). (B) The predicted SLC35G3 structure using AlphaFold2 shows a potential homodimeric arrangement. (C) The predicted aligned error map from AlphaFold2 indicates that the aligned errors for both SLC35G3 proteins, denoted as A and B, are within 15 Å. The predictions for A–B and B–A interactions are also below 15 Å, suggesting a plausible structural model for SLC35G3 homodimer formation.
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Figure 1—figure supplement 2—source data 1
Gel for Figure 1—figure supplement 2, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/107494/elife-107494-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Original files for gel analysis displayed in Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/107494/elife-107494-fig1-figsupp2-data2-v1.zip
Figure 2 with 4 supplements
Slc35g3-/- induces male sterility.
(A) Depiction of Slc35g3 gene location and structure, gRNA/primer design, and the sequencing result of the mutant (deleted) allele. (B) PCR genotyping results for Slc35g3+/+, Slc35g3+/-, Slc35g3-/-, and water are presented. (C) Western blot analysis results obtained with Slc35g3+/- and Slc35g3-/- testicular germ cells (TGC) lysates and Slc35g3+/- and Slc35g3-/--derived cauda epididymal sperm lysates are shown. (D, E) Similar testis sizes (D) and weights (E) from Slc35g3+/+ and Slc35g3-/- mice (two-sided Student’s t-test; p=0.42). (F) Comparison of the number of pups per vaginal plug between Slc35g3+/+ and Slc35g3-/- mice (Wilcoxon rank-sum test; p=2.87 × 10–10). (G) Histological analysis of testis sections from Slc35g3+/+ mice (upper panels) and those from Slc35g3-/- mice (lower panels); images depict stages III (Golgi phase), VIII (acrosome phase), and XII (maturation phase).
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Figure 2—source data 1
Western blots and gel for Figure 2, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/107494/elife-107494-fig2-data1-v1.pdf
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Figure 2—source data 2
Original files for western blot and gel analysis displayed in Figure 2.
- https://cdn.elifesciences.org/articles/107494/elife-107494-fig2-data2-v1.zip
Figure 2—figure supplement 1
Differentially expressed genes (DEG) analysis using testis RNA-seq data.
(A) Genes called by RNA-seq using wild-type (WT; n=3) and knockout (KO; n=3) testis followed by DEG analysis. Blue indicates a gene that is decreased in KO (Slc35g3), and yellow indicates a gene that is increased in KO (Gm1993) compared to WT. Numbers indicate FPKM. (B) Log2 fold change, p value, gene locus, and feature of each DEG are shown.
Figure 2—figure supplement 2
SLC35G3 is localized in the Golgi apparatus, and the signal disappears in the knockout.
The top row displays immunostaining images of Slc35g3+/+ testicular cells, while the bottom row displays immunostaining images of Slc35g3-/- testicular cells. Each image shows the cell nucleus obtained by crushing the seminiferous tubule (Hoechst33342), SLC35G3, GM130, and a merged image.
Figure 2—figure supplement 3
Epididymis sections from Slc35g3+/+ and Slc35g3-/- mice Caput and cauda epididymis sections from Slc35g3+/+ and Slc35g3-/- mice are comparable.
Figure 2—figure supplement 4
No significant differences in CASA parameters of sperm from Slc35g3-/- mice compared to sperm from Slc35g3+/+ mice.
(A) Definition of parameters representing sperm motility; Velocity of average path (VAP) is indicated in green, Velocity of curved path (VCL) is indicated in blue, and Velocity of straight path (VSL) is indicated in red. Motile sperm is defined as having a VSL >0 µm/s, while progressive sperm is defined as having VSL/VAP % ≥80 and VAP ≥50 µm/s. (B) Motility of sperm from wild-type (+/+) and knockout (-/-) mice showed no significant (n.s.) differences at 10 and 120 min after suspension in the medium. (C) Percentage of progressive sperm from Slc35g3+/+ and Slc35g3-/- mice showed no significant differences at 10 and 120 min after suspension in the medium. (D) VAP of wild-type and knockout sperm showed no significant differences at 10 and 120 min after suspension in the medium. (E) VCL of wild-type and knockout sperm showed no significant differences at 10 and 120 min after suspension in the medium. (F) VSL of wild-type and knockout sperm shows no significant differences at 10 and 120 min after suspension in the medium.
Figure 3 with 1 supplement
Slc35g3 is involved in the regulation of sperm head morphology.
(A) Bright-field (BF) views of Slc35g3+/+-derived sperm (upper panels) versus Slc35g3 -/--derived sperm (lower panels); red frames are images enlarged four times. Scale bar: 50 µm for BF images, 10 µm for enlarged ones. (B) Morphological characteristics are indicated by mean ± SD of each principal component (PC) following elliptic Fourier analysis; the upper value represents SD, with zero indicating average morphology. (C, D) Plots of PC1-PC2 (C) and PC1-PC3 (D) coordinates of the elliptic Fourier analysis of sperm from Slc35g3+/+ mice (blue encircled) versus Slc35g3-/- mice (red encircled); circles represent 95% confidence ellipses. Scale bar = 10 µm.
Figure 3—figure supplement 1
Sperm from Slc35g3-/- mice exhibit morphology similar to sperm from Fam71f2-/- mice.
(A) Appearance of Fam71f2 KO sperm. Red arrowheads indicate the tips of the spermatozoa. (B) Comparison among sperm from wildtype, Fam71f2-/-, and Slc35g3-/- mice by elliptic Fourier analysis.
Figure 4 with 2 supplements
Slc35g3-/--derived spermatozoa are defective in ZP binding and oolemma fusion.
(A) The in vitro fertilization (IVF) fertilization rate of cumulus-intact oocytes using Slc35g3+/- and Slc35g3-/--derived sperm. Wilcoxon rank-sum test p=0.014. (B) Outline of the procedure of cumulus cell-free IVF. (C) Slc35g3+/--derived and Slc35g3-/--derived sperm binding to cumulus-free oocytes after insemination. Scale bar = 50 µm. (D) The number of bound sperm per egg for Slc35g3+/--derived and Slc35g3-/--derived sperm (Wilcoxon rank-sum test p=2.20 × 10–18). (E) The fertilization rate of cumulus cell-free IVF using Slc35g3+/--derived and Slc35g3-/--derived sperm. (F) The procedure of ZP-free IVF. Wilcoxon rank-sum test; p=0.0079. (G) Brightfield and Hoechst33342 staining of oocytes and Slc35g3+/--derived and Slc35g3-/--derived sperm after insemination into ZP-free oocytes; Yellow arrowheads indicate fused spermatozoa and light blue asterisks indicate metaphase II-arrested chromosomes. (H) The number of fused sperm per egg using Slc35g3+/--derived and Slc35g3-/--derived sperm (2×105 sperm/mL and 2×106 sperm/mL, respectively). Significant differences are indicated by distinct symbols. (I) The fertilization rate of ZP-free IVF using Slc35g3+/--derived and Slc35g3-/--derived sperm (2×105 sperm/mL and 2×106 sperm/mL, respectively). Significant differences are indicated by distinct symbols.
Figure 4—figure supplement 1
Oolema fusion observed under high sperm concentration conditions.
(A) Schematic diagram of fusion test using 2x106 sperm/mL. (B) Bright field and Hoechst-stained images. The yellow arrowheads show fused spermatozoa and sky-blue asterisks show metaphase II-arrested chromosomes.
Figure 4—figure supplement 2
Pups were produced from spermatozoa from Slc35g3-/-.
(A) Two-pronuclear embryos were obtained by in vitro fertilization (IVF) using 2x106 sperm. (B) Offspring obtained by embryo transfer.
Figure 5
Slc35g3-deficient mice show impaired sperm migration to the oviduct.
(A) Illustration of Tg (CAG/su9-DsRed2, Acr3-eGFP) sperm. (B) A schematic diagram of the sperm migration assay. (C) Bright field (top panel) and Dsred2 (bottom panel) images of the uteri and oviducts of females after mating with control Slc35g3+/- and Slc35g3-/- male mice. The yellow dashed line indicates the uterotubal junction (UTJ), and the yellow arrowhead represents the sperm from control Slc35g3+/- male mice that have traversed the UTJ.
Figure 6 with 2 supplements
Disruption of Slc35g3 leads to its reduced testicular expression and abnormal processing of multiple sperm proteins.
(A) Western blot analyses of SPACA1, ZPBP1, and GOPC in Slc35g3+/+ and Slc35g3-/- testes, with BASIGIN used as a loading control. (B) Western blot analysis of PNGaseF-treated or non-treated SPACA1 in Slc35g3+/+ and Slc35g3-/- testes, with BASIGIN used as a loading control. (C) Western blot analysis of PNGaseF-treated or non-treated SPACA1 in Slc35g3+/+-derived and Slc35g3-/--derived spermatozoa, with BASIGIN used as a loading control. (D) Western blot analyses of ADAM1B, ADAM3, SPACA4, LY6K, TEX101, t-ACE, LYPD4, CMTM2A, CMTM2B, IZUMO1, EQTN, and SPACA6 in Slc35g3+/- and Slc35g3-/- testes, with BASIGIN used as a loading control. All protein samples were processed under reducing and denaturing conditions unless otherwise specified. Non-reducing and non-denaturing conditions are denoted as NR. For SPACA6 detection, fractions of testis proteins from wild-type and knockout specimens, extracted using Triton X-114, were utilized (abbreviated as DET). Genes marked with blue asterisks show reduced ZP binding upon knockout, whereas ADAM3 remains unaffected. (E) Western blot analyses of ADAM1B, ADAM3, SPACA4, t-ACE, LYPD4, CMTM2A, CMTM2B, IZUMO1, EQTN, and SPACA6 in Slc35g3+/+-derived and Slc35g3-/--derived spermatozoa, BASIGIN used as a loading control. The black arrowhead indicates the predicted protein size, whereas the red arrowhead indicates an aberrantly processed protein isoform. Additionally, the light blue and purple arrowheads mark the two bands observed in the wild-type sample.
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Figure 6—source data 1
Western blots for Figure 6, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/107494/elife-107494-fig6-data1-v1.pdf
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Figure 6—source data 2
Original files for western blot analysis displayed in Figure 6.
- https://cdn.elifesciences.org/articles/107494/elife-107494-fig6-data2-v1.zip
Figure 6—figure supplement 1
ADAM3 patterns were comparable between Slc35g3+/- and Slc35g3-/- after PNGaseF treatment.
Western blot analysis of PNGaseF treated or non-treated ADAM3 from testis and sperm of Slc35g3+/- and Slc35g3-/- mice. BASIGIN was analyzed as a loading control.
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Figure 6—figure supplement 1—source data 1
Western blots for Figure 6—figure supplement 1, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/107494/elife-107494-fig6-figsupp1-data1-v1.pdf
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Figure 6—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/107494/elife-107494-fig6-figsupp1-data2-v1.zip
Figure 6—figure supplement 2
Distribution of IZUMO1 is expanded in sperm from Slc35g3-/- mice after the acrosome reaction.
Acrosome-intact sperm (upper row) and acrosome-reacted sperm (lower row) from Slc35g3+/+ mice (left column) and Slc35g3-/- mice (right column).
Figure 7 with 1 supplement
Slc35g3 -/- testis showed impaired glycan structure.
(A) Lectin blot (LB) analyses using ConA, AAL, PNA, MAL-II, and LSL-N in Slc35g3+/+ and Slc35g3-/- testes, BASIGIN as a loading control. Green circles represent mannose, red triangles fucose, yellow squares GalNAc, yellow circles galactose, purple diamonds sialic acid, and blue squares GlcNAc. (B) LB analyses using ConA, AAL, PNA, MAL-II, and LSL-N in Slc35g3+/+ and Slc35g3-/- derived spermatozoa. (C) LB analyses of LSL-N and ConA in SLC35B4-deficient HEK293T cells, with GAPDH as a loading control. Slc35b2, Slc35b4, and Slc35g3 were expressed in SLC35B4 deficient cells. (D) LB analyses of LSL-N and ConA in SLC35G3 mutant transfected SLC35B4 deficient cells, with GAPDH, were used as a loading control. FS: T179HfsTer27.
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Figure 7—source data 1
Western blots for Figure 7, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/107494/elife-107494-fig7-data1-v1.pdf
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Figure 7—source data 2
Original files for western blot analysis displayed in Figure 7.
- https://cdn.elifesciences.org/articles/107494/elife-107494-fig7-data2-v1.zip
Figure 7—figure supplement 1
Design of gRNAs and timeline of the experiment.
(A) The gRNAs were designed to remove almost the entire coding region of the exon. (B) After the introduction of pX459, selection was performed using puromycin as shown.
Author response image 1
Additional files
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Supplementary file 1
Mass spectrometry data using sperm lysates from Slc35g3+/+ and Slc35g3-/- mice.
- https://cdn.elifesciences.org/articles/107494/elife-107494-supp1-v1.xls
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Supplementary file 2
Primer and gRNA sequences used in the present studies.
- https://cdn.elifesciences.org/articles/107494/elife-107494-supp2-v1.xlsx
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Supplementary file 3
Antibodies used in the present studies.
- https://cdn.elifesciences.org/articles/107494/elife-107494-supp3-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107494/elife-107494-mdarchecklist1-v1.docx
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SLC35G3 is a UDP-N-acetylglucosamine transporter for sperm glycoprotein formation and underpins male fertility in mice
eLife 14:RP107494.
https://doi.org/10.7554/eLife.107494.3
