SPACA4 is expressed in murine sperm. (A) Protein sequence alignment of the mature domain of SPACA4/Bouncer protein family members. Shown is the mature three-finger domain of SPACA4 proteins (red box) and Bouncer proteins (orange box) lacking the N-terminal signal peptide and the carboxyl-terminal GPI-anchoring sequence. Amino acid divergence among different species is indicated based on the percent amino acid identity (blue shading); predicted cysteine bridges (gray) are shown above the alignment. Adapted from ref. 42. (B) Mammalian SPACA4 is the homolog for fish Bouncer and is expressed specifically in testis. Part of a maximum-likelihood phylogenetic tree based on Ly6/uPAR protein sequence alignments across vertebrates showing SPACA4 and Bouncer (refer to SI Appendix, Fig. S2A for the full tree; adapted from ref. 42). Branches supported by ultrafast bootstrap values (≥95%) are marked with a blue dot. Z-scores of averaged gene expression values across adult tissues are shown on the right for Danio rerio (11), Xenopus laevis (75), Mus musculus (73), and Homo sapiens (www.gtexportal.org). While Bouncer/Spaca4 mRNAs are expressed in oocytes in fish and frogs, mammalian Spaca4 mRNAs are expressed in testis. (C) Mouse Ly6/uPAR genes show diverse expression patterns across adult tissues. The heatmap is color coded based on z-scores of the normalized gene expression values (average of the square root) of RNA-Seq data from murine adult tissues (73). The clustering and dendrogram (Left) are based on expression scores. Spaca4 is highlighted in red. Numbers behind gene names indicate chromosome location in mice. (D) Mouse Spaca4 mRNA is expressed in the male germline. RT-qPCR from murine cDNA from different adult tissues reveals enrichment of Spaca4 specifically in mouse testis. Primers amplifying Hypoxanthineguanine phosphoribosyl transferase (Hprt) were used as a control. (E) Mouse SPACA4 protein localizes to the sperm head. Immunostaining of SPACA4 (red) and IZUMO1 (green) under permeabilizing conditions detects SPACA4 and IZUMO1 in the sperm head of wild-type B6D2F1 mice. In contrast to IZUMO1 that relocalizes after the acrosome reaction, SPACA4 does not change its localization. BF, brightfield image. (Scale bar, 3 µm.) (F) Murine SPACA4 localizes to the inner sperm membrane. Immunofluorescence staining of SPACA4 (red) in mouse spermatozoa under nonpermeabilizing conditions. Spermatozoa were derived from transgenic mice expressing EGFP under the control of the Acrosin promoter (65), labeling sperm with intact acrosomes (green). AR spermatozoa are highlighted by an asterisk (loss of green label). Boxed areas are shown below at higher magnification (a: AR sperm; b: spermatozoa with intact acrosome). SPACA4 is detected in AR spermatozoa (a) but not in spermatozoa with intact acrosomes (b). [Scale bar, 20 µm (Top) and 5 µm (Bottom).]

SPACA4 is required for efficient fertilization in male mice. (A and B) Overview of the C57BL/6J-Spaca4 knockout alleles generated by CRISPR-Cas9–mediated targeted mutagenesis. One allele contains a 117-nt in-frame deletion after amino acid 42. The other allele contains a 77-nt out-of-frame deletion after amino acid 47. (A) Schematic of the wild-type and knockout alleles. Yellow dashed lines indicate the site of the deletions. Predicted disulfide bridges are indicated in gray. SP, signal peptide; TM, transmembrane region. (B) Genotyping of Spaca4 knockout mice. Spaca4 PCR products are separated based on their sizes on an agarose gel. (C) Spaca4 knockout male mice are subfertile. Litter sizes of C57BL/6J-Spaca4 wild-type (+/+), heterozygous (+/−), and transheterozygous (−/−) males caged with B6129F1 wild-type females or B6129F1 wild-type males caged with transheterozygous (−/−) females. Successful mating was confirmed by plug checks. Data are means ± SD. ***P < 0.0001 (Kruskal–Wallis test with Dunn multiple-comparisons test); n.s., not significant. n = number of litters; m = number of male mice tested. (D) Sperm morphology is normal in the absence of SPACA4 protein. DIC, differential interference contrast image. The sperm heads (boxed areas) are shown below at higher magnification. Immunostaining of sperm detects SPACA4 protein (red) under permeabilizing conditions in the head of sperm from wild-type (+/+) mice but not in sperm from Spaca4 knockout (−/−) mice. DAPI (cyan) staining labels the sperm nucleus. (Scale bars, 10 µm.) (E) Sperm from Spaca4 knockout mice has a severely reduced ability to fertilize wild-type oocytes. IVF of oocytes from C57BL/6J wild-type females using sperm from C57BL/6J-Spaca4 wild-type (+/+), heterozygous (+/−), or transheterozygous (−/−) males. Plotted is the percentage of two-cell stage embryos as a measure of successful fertilization. Data are means ± SD. *P = 0.014 (Kruskal–Wallis test with Dunn multiple-comparisons test); n.s., not significant. n = total number of oocytes; m = number of males tested. (F) IZUMO1 and ADAM3 proteins are expressed and processed normally in sperm and testis from Spaca4 knockout mice. Western blot analysis showing the absence of SPACA4 protein in testes and spermatozoa of Spaca4 knockout mice (Left) and expression and processing of ADAM3 and IZUMO1 proteins similar to wild-type controls. Testes and spermatozoa of wild-type mice, as well as BASIGIN protein, which undergoes proteolytic cleavage during sperm maturation (7), are used as control. (G) IZUMO1 localization is normal in Spaca4 knockout mice. Immunostaining of sperm under permeabilizing conditions shows normal localization of IZUMO1 (green) in acrosome-intact wild-type (+/+) and SPACA4-deficient (−/−) sperm and normal relocalization of IZUMO1 in AR sperm (asterisk) in both genotypes. BF, brightfield image. (Scale bars, 10 µm.) (H) Quantification of the percentage of AR sperm 2 or 4 h after incubation of capacitated sperm. Acrosome reaction was assessed by immunostaining for IZUMO1. Data are means ± SD; n.s., not significant. n = total number of sperm; m = number of males tested.

SPACA4 enables sperm to bind to and penetrate the ZP. (A) SPACA4 is required for efficient binding of sperm to the ZP. Sperm of the indicated genotypes was incubated for 30 min with hyaluronidase-treated oocytes (with intact ZP) of matching genetic backgrounds. Plotted is the number of sperm bound to ZP-containing oocytes. Data are means ± SD. P values (**P < 0.01) are by Student’s t test. n = total number of oocytes; N = number of replicates. (B) Quantification of the percentage of AR sperm derived from wild-type (+/+) and Spaca4^{77nt-del/77nt-del} (−/−) males bound to ZP-containing wild-type oocytes. Data are means ± SD. The P value (***P < 0.001) is by Student’s t test. Numbers of total sperm bound, AR sperm, oocytes (n) and males (m) tested are indicated (SI Appendix, Fig. S6 A and B). (C) Schematic of sperm bound to the COC and experimental treatments used to remove the cumulus cells (by treatment of COCs with hyaluronidase) and the ZP (by treatment of cumulus-free oocytes with acidified Tyrode’s solution) from the COCs. Acrosome-intact sperm, blue cap; sperm undergoing the acrosome reaction, green cap, and SPACA4 (red) getting exposed; AR sperm, no cap, and SPACA4 (red) exposed. (D) SPACA4 is required for ZP penetration but not for oolemma binding and fusion. IVF performed with COCs from superovulated C57BL/6J females, cumulus cell–free oocytes (oocyte with ZP), and ZP-free oocytes with sperm from either wild-type C57BL/6J males or age-matched Spaca4^{117nt-del/77nt-del} males. Plotted is the percentage of two-cell stage embryos as a measure of successful fertilization. Data are means ± SD. P values (*P < 0.05; n.s., not significant) are by a Kruskal–Wallis test with Dunn multiple-comparisons test. n = total number of oocytes; N = number of replicates. (E) Comparison of SPACA4’s function in murine fertilization versus Bouncer’s role in fish fertilization. (Top) In the mouse, sperm-expressed SPACA4 (red) is exposed in AR sperm (green cap and no cap) and is required for efficient ZP penetration (Left) but not for sperm–egg membrane binding (Right). Acrosome-intact sperm, blue cap. (Bottom) In zebrafish, sperm can access the egg membrane via the preformed funnel, the so-called micropyle, in the chorion. Egg-expressed Bouncer (orange) is required for sperm–egg membrane binding in zebrafish (42). The tick mark (yes) or cross (no) at the top right in each box indicates whether this step of fertilization can occur in the wild-type or mutant condition.