Binding between sperm and egg plasma membranes is an essential step in fertilization. Whereas fertilin, a mammalian sperm surface protein, is involved in this crucial interaction, sperm receptors on the egg plasma membrane have not been identified. Because fertilin contains a predicted integrin ligand domain, we investigated the expression and function of integrin subunits in unfertilized mouse eggs. Polymerase chain reactions detected mRNAs for alpha 5, alpha 6, alpha v, beta 1, beta 3, and beta 5. Immunofluorescence revealed alpha 6 beta 1 and alpha v beta 3 on the plasma membrane. GoH3, a function-blocking anti-alpha 6 monoclonal antibody, abolished sperm binding, but a nonfunction-blocking anti-alpha 6 monoclonal antibody, a function-blocking anti-alpha v beta 3 polyclonal antibody, and an RGD peptide had no effect. Somatic cells bound sperm avidly, but only if they expressed alpha 6 beta 1. A peptide analog of the fertilin integrin ligand domain inhibited sperm binding to eggs and alpha 6 beta 1+ cells and diminished GoH3 staining of eggs. Our results indicate a novel role for the integrin alpha 6 beta 1 as a cell-cell adhesion receptor that mediates sperm-egg binding.

Integrin receptors for extracellular matrix receptors are important effectors of cell adhesion, differentiation, and migration in cultured cells and are believed to be critical effectors of these processes during development. To determine when 131 integrins become critical during embryonic development, we generated mutant mice with a targeted disruption of the [31 integrin subunit gene. Heterozygous mutant mice were normal. Homozygous loss of ~1 integrin expression was lethal during early postimplantation development. Homozygous embryos lacking B1 integrins formed normal-looking blastocysts and initiated implantation at E4.5. However, the E4.5 Ill-null embryos in situ had collapsed blastocoeles, and whereas the trophoblast penetrated the uterine epithelium, extensive invasion of the decidua was not observed. Laminin-positive endoderm cells were detected in the inner cell mass area, but endoderm morphogenesis and migration were defective. By E5.5 ~l-null embryos had degenerated extensively. In vitro analysis showed that trophoblast function in Ill-null peri-implantation embryos was largely normal, including expression of tissue-specific markers, and outgrowth on fibronectin-and vitronectin-coated, although not on laminin-coated substrates. In contrast, the inner cell mass region of 131-null blastocyst outgrowths, and inner cell masses isolated from [31-null blastocysts, showed highly retarded growth and defective extraembryonic endoderm morphogenesis and migration. These data suggest that 131 integrins are required for normal morphogenesis of the inner cell mass and are essential mediators of growth and survival of cells of the inner cell mass. Failure of continued trophoblast development in 131-null embryos after inner cell mass failure could be attributable to either an intrinsic requirement for 131 integrins for later stages of trophoblast development, or to the lack of trophic signals from the 131-null inner cell mass.[Key Words: 131 integrins; e~V-integrins; trophoblast; extraembryonic endoderm; survival; migration] Received April 4, 1995; revised version accepted June 15, 1995.Cell--extracellular matrix (ECM) interactions play critical roles in morphogenesis and in the regulation of gene expression (Damsky and Werb 1992;Hynes 1992Hynes , 1994Adams and Watt 1993;Ashkenas et al. 1994;Cross et al. 1994). The integrin family of heterodimeric transmembrane glycoproteins constitutes the major class of receptors mediating cell-ECM interactions. These receptors link the ECM to the internal cytoskeleton and to intracellular signaling pathways. In this role, they mediate cell adhesion and migration, and transduce mechanical and informational signals from the complex extracellular environment, thereby influencing both cytoarchitecture and gene expression. Integrin heterodimeric receptors for ECM can be classified into two major families: {lJ those containing the 131 subunit, and, {2} those containing the oLV subunit. There is extensive apparent redundancy in the ligandbinding preferences of these integrins. For exampl...

No abstract

Abstract. The integrin superfamily of heterodimeric transmembrane adhesion receptors mediates many cell-cell and cell-matrix interactions whose functions are believed to be critical for normal morphogenesis and differentiation. By eliminating the fll integrin gene through homologous recombination, we have assessed the role of the fll integrin family in the F9 embryonal carcinoma model for endodermal differentiation. F9 cells were unexpectedly found to maintain three copies of the fl 1 gene and complete elimination required three sequential rounds of targeting to generate triple knockout lines (/~1 TKO).Elimination of the/~1 integrin family of adhesion receptors from F9 cells resulted in reduced adhesion to fibronectin, laminin and collagen, but strongly enhanced adhesion to vitronectin. The absence of/31 integrins did not promote significant compensatory upregulation of either t3 or/~5 subunits, both of which are known to act as vitronectin receptors when associated with otv. The loss of fll integrins severely affected morphological differentiation when the/~l-deficient cells were induced to differentiate to either parietal or visceral endoderm. Parietal endoderm derived from ill-deficient cells retained a rounded morphology and migrated poorly on both fibronectin and vitronectin. Visceral endoderm derived from/31-deficient cells were also unable to form a normal, confluent epithelial monolayer; instead, a noncontiguous layer containing clumps of disorganized cells was observed. However, loss of/~1 integrins did not interfere with induction by differentiating agents of tissue-specific gene products for either visceral or parietal endoderm. These results suggest that fll integrins mediate morphological differentiation (migration and epithelial formation) but not tissue-specific gene expression in induced F9 cells, and that these two processes are not necessarily linked in this system. TIt~ proper temporal and spatial differentiation of embryonic cells is a complex process that requires signals initiated by interactions with other cells and with extracellular matrix (ECM). ~ Cells receive information from ECM through cell surface receptors. Of particular importance are the integrins, a family of heterodimeric, transmembrane receptors that interact with both ECM ligands and intracellular components, including cytoskeleton-associated proteins (Darnsky and Werb, 1992;Hynes, 1992). Studies using function-perturbing reagents (e.g., antibodies) have indicated that integrins containing the/31 subunit, in particular, communicate signals that help to regulate the terminal

Early studies on lens induction suggested that the optic vesicle, the precursor of the retina, was the primary inducer of the lens; however, more recent experiments with amphibians establish an important role for earlier inductive interactions between anterior neural plate and adjacent presumptive lens ectoderm in lens formation. We report here experiments assessing key inductive interactions in chicken embryos to see if features of amphibian systems are conserved in birds. We first examined the issue of specification of head ectoderm for a lens fate. A large region of head ectoderm, in addition to the presumptive lens ectoderm, is specified for a lens fate before the time of neural tube closure, well before the optic vesicle first contacts the presumptive lens ectoderm. This positive lens response was observed in cultures grown in a wide range of culture media. We also tested whether the optic vesicle can induce lenses in recombinant cultures with ectoderm and find that, at least with the ectodermal tissues we examined, it generally cannot induce a lens response. Finally, we addressed how lens potential is suppressed in non-lens head ectoderm and show an inhibitory role for head mesenchyme. This mesenchyme is infiltrated by neural crest cells in most regions of the head. Taken together, these results suggest that, as in amphibians, the optic vesicle cannot be solely responsible for lens induction in chicken embryos; other tissue interactions must send early signals required for lens specification, while inhibitory interactions from mesenchyme suppress lens-forming ability outside of the lens area.