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.
Little is yet known about the biological and biochemical properties of the disintegrin-like domains of ADAM (a disintegrin and metalloprotease) proteins. Mouse ADAM 2 (mADAM 2; fertilin beta) is a sperm surface protein involved in murine fertilization. We produced recombinant proteins containing the disintegrin-like domain of mADAM 2 in both insect cells and in bacteria. The protein produced in insect cells (baculo D+C) contained a signal sequence followed by the disintegrin-like and cysteine-rich domains; it was purified from the medium of recombinant baculovirus-infected cells. A bacterial construct containing the disintegrin-like domain was produced in Escherichia coli as a glutathione S-transferase chimera. Baculo D+C, as well as the D domain of the bacterial construct (released with thrombin), bound to the microvillar surface of murine eggs. Using concentrations in the range of 1 to 5 microM, both recombinant proteins strongly inhibited sperm-egg binding and fusion; the baculovirus-produced protein exhibited a somewhat greater extent of inhibition (approximately 75 versus approximately 55% maximal inhibition). Substitution of alanine for each of the five charged residues within the disintegrin loop of mADAM 2 revealed a critical importance for the aspartic acid at position nine. Binding of both recombinant proteins to the egg was inhibited by the function blocking anti-alpha(6) monoclonal antibody, GoH3, but not by a nonfunction-blocking anti-alpha(6) monoclonal antibody. Binding was also inhibited by a peptide analogue of, and with an antibody against, the disintegrin loop of mADAM 2.
