Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin α6β1: Implications for murine fertilization

Abstract

CD9 is a tetraspan protein that associates with several β1 integrins, including α6β1. Because α6β1 is present on murine eggs and interacts with the sperm-surface glycoprotein ADAM 2 (fertilin β), we first asked whether CD9 is present on murine eggs and whether it functions in sperm–egg binding and fusion. CD9 is present on the plasma membrane of oocytes in the ovary as well as on eggs isolated from the oviduct. The anti-CD9 mAb, JF9, potently inhibits sperm–egg binding and fusion in vitro in a dose-dependent manner. JF9 also disrupts binding of fluorescent beads coated with native fertilin or a recombinant fertilin β disintegrin domain. (Both ligands bind to the egg via α6β1.) Immunohistochemistry showed that CD9 is undetectable in the uterine epithelium, appears basolaterally and as prominent apical patches on the epithelium in the region between the uterus and the oviduct, and then persists apically in the oviduct. The integrin α6A subunit is found in similar apical patches in the region between the uterus and oviduct, but is confined to the basal aspect of the epithelium in the uterus and oviduct. Hence, α6A and CD9 both are expressed on the apical epithelial surface at the uterine–oviduct junction. These findings correlate with the observation that fertilin β “knockout” sperm traverse the uterus but do not progress into the oviduct, contributing to the infertility of fertilin β^−/− male mice. Our results suggest that high-avidity binding between fertilin β (ADAM 2) and α6β1 requires cooperation between α6β1 and CD9. Such cooperation may assist sperm passage into the oviduct as well as sperm–egg interactions.