Human gamete fusion can bypass beta1 integrin requirement.
Since alpha6beta1 integrin has been shown to function as a sperm adhesion receptor in the mouse, we investigated the potential role of beta1 integrin in the gamete fusion process in humans. The expression of beta1 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). Beta1 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 (11-22 from GV to MII respectively), and that fused spermatozoa co-localized with beta1 integrin patches. Human gamete fusion was only partially inhibited either by RGD-containing peptide (GRGDTP), or by blocking anti-human beta1 integrin monoclonal antibody (DE9), with a maximum of 50% inhibition. Despite the combined addition of GRGDTP and blocking mouse anti-human beta1 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 beta1 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 beta1 requirement. Beta1 integrin certainly participates in human gamete fusion by acting in co-operation with multiple integrin/disintegrin couples or another cofactor, not yet identified.
Ji
Y Z
YZ
Laboratoire de Biologie de la Reproduction, Hôpital Cochin, Paris, France.
Wolf
J P
JP
Jouannet
P
P
Bomsel
M
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D005260:Female; D006801:Humans; D019012:Integrin beta1 / Q000032:analysis / Q000502:physiology*; D008297:Male; D008565:Membrane Proteins / Q000037:antagonists & inhibitors; D008677:Metaphase; D051379:Mice; D008807:Mice, Inbred BALB C; D009842:Oligopeptides / Q000494:pharmacology; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D013084:Sperm-Ovum Interactions / Q000187:drug effects
D000911:Antibodies, Monoclonal; D019012:Integrin beta1; D008565:Membrane Proteins; D009842:Oligopeptides; C047981:arginyl-glycyl-aspartic acid