Cross-species insemination reveals mouse sperm ability to enter and cross the fish micropyle

Extracellular matrices surrounding eggs in fish (chorion) and mammals (zona pellucida [ZP]) regulate gamete recognition before fertilization, though their mechanisms differ. Mouse sperm bind and cross the ZP at any site, while fish sperm cross the chorion through a funnel-shaped opening, the micropyle. To explore these divergent processes, we established cross-species insemination assays, mixing zebrafish eggs with mouse sperm. While mouse sperm could not bind to the chorion, a subpopulation successfully located and crossed the fish micropyle. Confocal and electron microscopy revealed that sperm entered the micropyle and accumulated in the zebrafish inter-chorion space. However, transgenic mouse sperm with mCherry-labeled acrosomes failed to undergo acrosome exocytosis efficiently in the micropyle, with both acrosome-intact and reacted sperm found in the inter-chorion space. Sperm entry and crossing were dependent on hyperactive motility, as sperm from CatSperd Null mice, which fail to undergo hyperactivation, did not interact with or cross the micropyle. These findings suggest a conserved mechanism for sperm entry into the micropyle, providing a novel platform to investigate cross-species gamete interactions and uncover novel steps in fertilization.