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 ΔHPX (-/-) 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.

Egg activation in physiologically polyspermic newt eggs: involvement of IP3 receptor, PLCγ, and microtubules in calcium wave induction.

The egg of the polyspermic newt is activated by Ca(2+) waves induced by several sperm at fertilization. A major component of the sperm factor for egg activation is the sperm-specific citrate synthase (CS), which is introduced into the egg cytoplasm after sperm-egg fusion. We tried to clarify the mechanism for sperm-specific CS to induce [Ca(2+)]i increase in egg cytoplasm. The injection of the sperm factor into the unfertilized egg induces a [Ca(2+)]i increase that propagates over the whole egg surface as a Ca(2+) wave. The propagation of the Ca(2+) wave is inhibited by depolymerization of microtubules in the egg cytoplasm. The sperm-specific CS is highly phosphorylated and binds the component containing microtubules and the IP3 receptor. The sperm CS localized in the midpiece region was dispersed in the egg cytoplasm, but most of the CS accumulates at the sperm entry site and is distributed in association with the microtubules around the midpiece region and the nucleus. Phospholipase C (PLC) γ in egg cytoplasm also accumulates around the midpiece region in association with the sperm CS. Thus, CS at the initiation site of the Ca(2+) wave forms a complex of microtubules and endoplasmic reticulum (ER) with the IP3 receptor, in addition to PLCγ, indicating close involvement of those complexes in Ca(2+) releases from the ER by the sperm factor.

The need of MMP-2 on the sperm surface for Xenopus fertilization: its role in a fast electrical block to polyspermy.

Monospermic fertilization in the frog, Xenopus laevis, is ensured by a fast-rising, positive fertilization potential to prevent polyspermy on the fertilized egg, followed by a slow block with the formation of a fertilization envelope over the egg surface. In this paper, we found that not only the enzymatic activity of sperm matrix metalloproteinase-2 (MMP-2) was necessary for a sperm to bind and/or pass through the extracellular coat of vitelline envelope, but also the hemopexin (HPX) domain of MMP-2 on the sperm surface was involved in binding and membrane fusion between the sperm and eggs. A peptide with a partial amino acid sequence of the HPX domain caused egg activation accompanied by an increase in [Ca(2+)]i in a voltage-dependent manner, similar to that in fertilization. The membrane microdomain (MD) of unfertilized eggs bound the HPX peptide, and this was inhibited by ganglioside GM1 distributed in the MD. The treatment of sperm with GM1 or anti-MMP-2 HPX antibody allows the sperm to fertilize an egg clamped at 0 mV, which untreated sperm cannot achieve. We propose a model accounting for the mechanism of voltage-dependent fertilization based on an interaction between the positively charged HPX domain in the sperm membrane and negatively-charged GM1 in the egg plasma membrane.

Role of the PI3K-TOR-S6K pathway in the onset of cell cycle elongation during Xenopus early embryogenesis.

In the early embryogenesis of the frog, Xenopus laevis, cells proliferate by rapid and synchronous divisions, followed by cell cycle elongation and prolongation of the S phases, and then the appearance of the G2 and G1 phases after the midblastula transition (MBT). The beginning of cell cycle elongation was thought to depend on an increase in the nucleo-cytoplasmic (N/C) ratio in blastomeres and a decrease in cortical cytoplasmic factors necessary for cell cycle progression, although these factors are unknown. In the present study, we demonstrated that a regulatory subunit of PI3K (p85α) was localized in the cortical cytoplasm of the blastomere during the MBT. When the embryos were treated with a PI3K inhibitor, LY294002, or a TOR inhibitor, rapamycin, cell cycle elongation was initiated before the MBT. In addition, the inhibition of S6K expression by antisense morpholino oligo enhanced the initiation of cell cycle elongation. In contrast, the activation of PI3K-TOR by Rheb-S16H expression delayed the initiation of cell cycle elongation. These results indicate that a decrease in translational activity dependent on the PI3K-TOR-S6K pathway causes the initiation of cell cycle elongation at the onset of the MBT.