The involvement of egg integrins in mammalian sperm-egg interactions has been controversial, with data from integrin inhibitor studies contrasting with evidence from knockouts showing that specific integrin subunits are not essential for fertility. An alpha4/alpha9 (ITGA4/ITGA9) integrin subfamily member has been implicated in fertilization but not extensively examined, so we tested the following three hypotheses: 1) an ITGA4/ITGA9 integrin participates in sperm-egg interactions, 2) short-term acute knockdown by RNA interference of integrin subunits would result in a fertilization phenotype differing from that of chronic depletion via knockout, and 3) detection of a fertilization phenotype is sensitive to in vitro fertilization (IVF) assay conditions. We show that mouse and human eggs express the alpha9 integrin subunit (ITGA9). RNA interference-mediated knockdown resulted in reduced levels of Itga9 mRNA and surface protein in mouse eggs. RNA interference attempts to knockdown ITGA9′s likely beta partner, beta1 (ITGB1), resulted in reduced Itgb1 mRNA but no reduction in ITGB1 surface protein. Therefore, studies using a function-blocking anti-ITGB1 antibody tested the hypothesis that ITGB1 participates in gamete interactions. Analyses of sperm-egg interactions with Itga9-knockdown eggs and anti-ITGB1 antibody-treated eggs in IVF assays using specific sperm:egg ratios revealed the following: 1) a reduction, but not complete loss, of sperm-egg binding and fusion was observed and 2) the reduction of sperm-egg binding and fusion was not detected in inseminations with high sperm:egg ratios. These data demonstrate that ITGA9 and ITGB1 participate in sperm-egg interactions but clearly are not the only molecules involved. This also shows that careful design of IVF parameters allows detection of deficiencies in gamete interactions.
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Vol. 80 • No. 4