Human gamete fusion can bypass beta1 integrin requirement.

Human Reproduction Human gamete fusion can bypass b1 integrin requirement Ya Zhong Ji 0 2 Jean Philippe Wolf 1 Pierre Jouannet 2 Morgane Bomsel 0 0 Institut Cochin de Ge ne tique Mole culaire, U 332 , 22, rue Me chain, 75014 Paris , France 1 Laboratoire de Biologie de la Reproduction, Faculte de Me decine de Bobigny , 74 rue Marcel Cachin, Bobigny 93017 2 Laboratoire de Biologie de la Reproduction, Hopital Cochin , 123, Bd Port Royal, 75014 Paris 4To whom correspondence should be addressed - Since a6b1 integrin has been shown to function as a sperm adhesion receptor in the mouse, we investigated the potential role of b1 integrin in the gamete fusion process in humans. The expression of b1 integrin was morphologically analysed by indirect immunofluorescence and confocal microscopy. A homogeneous and intense staining was detected at the plasma membrane, and in some subcortical vesicles of germinal vesicle stage oocytes (GV). b1 almost disappeared from oolemma and cytoplasm of metaphase I (MI) oocytes, but was re-expressed as asymmetrical patches at the plasma membrane of metaphase II stage oocytes (MII). A functional fusion assay based on Hoechst or calcein-AM dye transfer from one gamete to the other showed that maturing oocytes were able to fuse with an increasing number of spermatozoa (1122 from GV to MII respectively), and that fused spermatozoa co-localized with b1 integrin patches. Human gamete fusion was only partially inhibited either by RGD-containing peptide (GRGDTP), or by blocking anti-human b1 integrin monoclonal antibody (DE9), with a maximum of 50% inhibition. Despite the combined addition of GRGDTP and blocking mouse anti-human b1 integrin DE9 in the assay, a complete inhibition of fusion could not be achieved. A mouse polyclonal antibody raised against human oocyte membranes was more potent in inhibiting the fusion. Since b1 integrin expression at the plasma membrane was not correlated to oocyte fusibility, and since it was only partially inhibited by DE9 and/or RGD peptide, we suggest that human gamete fusion can bypass the b1 requirement. b1 integrin certainly participates in human gamete fusion by acting in co-operation with multiple integrin/disintegrin couples or another cofactor, not yet identified. Key words: 1 integrin/human gamete fusion The fusion mechanism during mammalian fertilization remains unclear at the molecular level. It contains two steps that can be dissociated experimentally: the binding of the spermatozoon to the oocyte plasma membrane and the actual gamete fusion, i.e. the merging of the sperm and oocyte membranes (Yanagimachi, 1994). Integrins are a family of cell surface adhesion receptors which mediate cellcell and cellextracellular matrix interactions. Integrins are heterodimeric glycoproteins composed of and subunits, and their binding to their ligand (disintegrin) on the cell partner can affect various cellular mechanisms, such as cell differentiation and gene expression (Hynes, 1992; Miyamoto et al., 1995b; Schwartz et al., 1995). The presence of integrin and disintegrin on mammalian gamete has already been assessed. In oocytes, integrin 1, 3, 5 and 6 subunits were detected in unfertilized mouse eggs by their mRNA and by subunit-specific antibodies (Tarone et al., 1993). v, 5 and 1 subunits were detected in Syrian hamster eggs using detergent extracts by enzyme-linked immunoabsorbent assay (ELISA) and 2, 5, 6 and v were shown to be expressed in human and hamster oocytes using immunobeads (Fusi et al., 1993). In the mouse, 1, 2, 5 and v integrin subunits are present in the oocyte. Oocyte maturation was associated with a redistribution of 5 and vitronectin receptor (v3), as shown by immunoprecipitation and confocal fluorescence microscopy (Evans et al., 1995a). In a recent study, integrin subunits 2, 5, 6 and 2, 4, 5 and 6 were revealed in the plasma membrane of human and hamster oocytes respectively, by indirect immunofluorescence labelling (De Nadai et al., 1995), but the 1 subunit was not found in human or in hamster oocytes, in this study. In spermatozoa, a complex of sperm surface antigens, fertilin , was described in the guinea pig spermatozoa. The subunit shares homology with certain viral fusion glycoproteins, and fertilin subunit shares high sequence similarity with a family of soluble disintegrins found in snake venom, especially for an RGD-like motif (Primakoff et al., 1987; Blobel et al., 1992; Wolfsberg et al., 1995). The role of integrins in spermegg interaction remains elusive. Penetration of human or hamster spermatozoa in zonafree golden hamster eggs was completely inhibited by RGDcontaining peptides (Bronson and Fusi, 1990). In the zonafree hamster egg penetration assay, human sperm binding to the oolemma was inhibited by echistatin, a disintegrin known to block the binding of fibronectin and vitronectin to their respective integrin receptors (51, v3). However in the same conditions, oocytesperm fusion was not inhibited (Bronson et (...truncated)