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 Alpha Fold. (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

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-/- testis 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).

Slc35g3 is essential for sperm head formation.

(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.

Slc35g3-/--derived spermatozoa are defective in ZP binding and oolemma fusion.

(A) The 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.

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.

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. 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.

Slc35g3 -/- testis showed impaired glycan structure

(A) Lectin blot (LB) analyses using ConA, AAL, PNA, MAL-II, and LSL-N in Slc35g3+/+ 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. mSlc35b2, mSlc35b4 and mSlc35g3 were expressed in SLC35B4 deficient cells. (D) LB analyses of LSL-N and ConA in hSLC35G3 mutant transfected SLC35B4 deficient cells, with GAPDH, were used as a loading control. FS: T179HfsTer27.