Fig. 3.

Fig. 3.

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.