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

Abstract

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 <i>CatSperd^Null</i> 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.