The conserved fertility factor SPACA4/Bouncer has divergent modes of action in vertebrate fertilization.

Fertilization is the fundamental process that initiates the development of a new individual in all sexually reproducing species. Despite its importance, our understanding of the molecular players that govern mammalian sperm-egg interaction is incomplete, partly because many of the essential factors found in nonmammalian species do not have obvious mammalian homologs. We have recently identified the lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) protein Bouncer as an essential fertilization factor in zebrafish [S. Herberg, K. R. Gert, A. Schleiffer, A. Pauli, Science 361, 1029-1033 (2018)]. Here, we show that Bouncer's homolog in mammals, Sperm Acrosome Associated 4 (SPACA4), is also required for efficient fertilization in mice. In contrast to fish, in which Bouncer is expressed specifically in the egg, SPACA4 is expressed exclusively in the sperm. Male knockout mice are severely subfertile, and sperm lacking SPACA4 fail to fertilize wild-type eggs in vitro. Interestingly, removal of the zona pellucida rescues the fertilization defect of Spaca4-deficient sperm in vitro, indicating that SPACA4 is not required for the interaction of sperm and the oolemma but rather of sperm and the zona pellucida. Our work identifies SPACA4 as an important sperm protein necessary for zona pellucida penetration during mammalian fertilization.