Role of the integrin-associated protein CD9 in binding between sperm ADAM 2 and the egg integrin alpha 6 beta 1: Implications for murine fertilization

CD9 is a tetraspan protein that associates with several pi integrins, inclu ding alpha 6 beta 1. Because alpha 6 beta 1 is present on murine eggs and i nteracts with the sperm-surface glycoprotein ADAM 2 (fertilin beta), we fir st 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 oocy tes 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 dos e-dependent manner. JF9 also disrupts binding of fluorescent beads coated w ith native fertilin or a recombinant fertilin beta disintegrin domain. (Bot h ligands bind to the egg via alpha 6 beta 1.) Immunohistochemistry showed that CD9 is undetectable in the uterine epithelium, appears basolaterally a nd as prominent apical patches on the epithelium in the region between the uterus and the oviduct, and then persists apically in the oviduct. The inte grin alpha 6A subunit is found in similar apical patches in the region betw een the uterus and oviduct, but is confined to the basal aspect of the epit helium in the uterus and oviduct. Hence, alpha 6A and CD8 both are expresse d on the apical epithelial surface at the uterine-oviduct junction. These f indings correlate with the observation that fertilin beta "knockout" sperm traverse the uterus but do not progress into the oviduct, contributing to t he infertility of fertilin beta(-/-) male mice. Our results suggest that hi gh-avidity binding between fertilin beta (ADAM 2) and alpha 6 beta 1 requir es cooperation between alpha 6 beta 1 and CD9. Such cooperation may assist sperm passage into the oviduct as well as sperm-egg interactions.