Expression, Localization, and Processing of Chicken Sperm ADAM32L2 during the Acrosome Reaction: A Possible Function in the Sperm–Egg Interaction

Abstract

Sperm–egg interactions involve a complex series of molecular events. Among these, the acrosome reaction (AR) is a prerequisite for sperm penetration, facilitating the exposure of multiple acrosomal proteins that enhance sperm binding or penetration of the outer layer of the egg; however, the specific molecules involved in this process vary across species. A disintegrin and metalloproteinase (ADAM) proteins are transmembrane glycoproteins that play a role in sperm–egg interactions, with notable differences among ADAM isoforms. In a previous characterization of the chicken sperm membrane proteome, ADAM32 metallopeptidase domain 32-like 2 (ADAM32L2), a protein structurally homologous to mammalian ADAMs, but absent in mammals, was identified. ADAM32L2 was located in the acrosomal region, underwent processing during the AR, similar to certain mammalian sperm ADAMs, and likely contributed to sperm binding to the inner perivitelline layer (IPVL) in chickens. Using various protease inhibitors, it was confirmed that sperm protease activity was involved in multiple stages of sperm interaction with the IPVL. Using a specific antibody, ADAM32L2 was predominantly expressed in the testis and localized to the sperm acrosomal region. Upon separation of the acrosome cap through an inherent AR process in chicken sperm, the 80 kDa acrosomal ADAM32L2 was processed into a 45 kDa C-terminal fragment during AR. Although zymography did not detect metalloproteinase activity in this fragment, a purified ADAM32L2 antibody inhibited sperm penetration of the IPVL, suggesting that the processed form was involved in IPVL binding. These findings elucidate the mechanism of sperm–IPVL interactions and offer new insights into the functional role of ADAM proteins in avian sperm.