Sperm MMP-2 is indispensable for fast electrical block to polyspermy at fertilization in Xenopus tropicalis

Sperm matrix metalloproteinase-2 (MMP-2) is necessary for frog fertilization. Monospermy is ensured by a fast, electrical block to polyspermy mediated by a positive fertilization potential. To determine the role of the MMP-2 hemopexin domain (HPX) in a fast block to polyspermy during fertilization of the frog, Xenopus tropicalis, we prepared mutant frogs deficient in mmp2 gene using the transcription activator-like effector nuclease method. mmp2 Delta HPX (-/-) sperm without MMP-2 protein were able to fertilize wild-type (WT; +/+) eggs. However, polyspermy occurred in some eggs. The mutant sperm generated a normal fertilization potential amounting to 10 mV, and were able to fertilize eggs at 10 mV, at which WT sperm never fertilized. Sensitivity during voltage-dependent fertilization decreased in mutant sperm. This study demonstrates for the first time that the genetic alteration of the MMP-2 molecule in sperm causes polyspermy during fertilization of a monospermic species. Our findings provide reliable evidence that sperm MMP-2 is indispensable for the fast, electrical block to polyspermy during Xenopus fertilization.