Molecular Human Reproduction vol.2 no.5 pp. 341-347, 1996 P-selectin is expressed on the oolemma of human and hamster oocytes following sperm adhesion and is also detected on the equatorial region of acrosome-reacted human spermatozoa F.M.Fusi 1 , M.Montesano 1 , N.Bernocchi 1 , C.Panzeri 2 , F.Ferrara 3 , A.Villa 2 and R.A.Bronson 4 - 5 department of Obstetrics and Gynecology, department of Pharmacology, and department of Laboratory Medicine, Istituto Scientifico San Raffaele, Milano, Italy, department of Obstetrics and Gynecology, State University of New York at Stony Brook, Stony Brook, NY 11794-8091, USA ^ o whom correspondence should be addressed Selectins are a family of adhesive molecules, involved in the interactions between leukocytes and endothelium and in platelet adhesion. P-selectin, one of the members of this family, is stored in a-granules and dense granules of platelets as well as in Weibel-Palade bodies of endothelial cells, and it is rapidly redistributed to the cell surface after activation, rt recognizes carbohydrate structures as ligands, in particular sialyi-Lewis*, which is part of the CD15 antigen. In this work we studied P-selectin expression on gametes. While zona- free human and hamster oocytes did not react with a monoclonal antibody directed against P-selectin, oocytes from both species displayed a reactivity with this antibody following their contact with human spermatozoa, as demonstrated both by covasphere binding and indirect immunofluorescence. Artificial activation of zona-intact human oocytes by means of the calcium ionophore A23187 induced the expression on the oolemma of a moiety reacting with anti-P-selectin antibody as well. P-selectin also appeared to be expressed on the sperm surface following the acrosome reaction, as demonstrated by a flow cytometric study of reactivity of spermatozoa with the anti-P-selectin antibody, using the expression of CD46 as a marker of the acrosome reaction. The localization of the P-selectin moiety on the equatorial region of the plasma membrane of acrosome reacted spermatozoa was confirmed by transmission electron microscopy using immunogold labelling. We suggest that P-selectin might be involved in gamete interactions. Key words: acrosome reaction/gamete interaction/oolemma/P-selectin/spermatozoa Introduction The selectin family of molecules is known to support the adhesion of leukocytes to the blood vessel wall and adhesive interactions between leukocytes and platelets (Rosen and Bertozzi, 1994). The three known members, called E-, P- and L- selectin, are structurally similar transmembrane glycoproteins, each containing an amino-terminal lectin-like domain, an epidermal growth factor (EGF) domain, and a variable number of complement regulatory-like repeats. While L-selectin is present on the surface of most lymphocytes, E-selectin is transcribed and expressed in a transiently up-regulated way (maximal at 2-6 h) after stimulation by endotoxins and cytokines. P-selectin (CD62) is stored in a-granules and dense granules of platelets as well as in Weibel-Palade bodies of endothelial cells, and it is rapidly (within minutes) redistributed to the cell surface after activation (Rosen and Bertozzi, 1994). Selectins recognize carbohydrate structures as ligands, in particular sialyl-Lewis x (sLe*), which is part of the CD 15 antigen (Polley et al, 1991). Cell activation leading to P- selectin expression can also be obtained through the interaction of some integrins with their ligands (Larsen et al, 1989). Evidence has accumulated that sperm—oolemmaJ interactions leading to fertilization may involve the recognition of the adhesion molecules vitronectin or fibronectin by their integrin receptors, a ^ and a$\, via the Arg-Gly-Asp (RGD) sequence. We have previously demonstrated that RGD-con- © European Society for Human Reproduction and Embryology taining peptides are able to interfere with the adherence of human and hamster spermatozoa to the oolemma of zona-free hamster oocytes (Bronson and Fusi, 1990). In addition, the expression of several integrin chains, such as o^, 05, o^, Pi, and P 3 has been described on the oolemma of human, hamster and mouse oocytes by our laboratory and others (Fusi et al, 1993; Tarone et al, 1993; Almeida et al, 1995). Several integrin chains have also been detected on human spermatozoa (Schaller et al, 1993; Fusi et al, 19%). In this work we have shown the presence of a surface moiety reacting with an anti-P-selectin monoclonal antibody on the oolemma of human and hamster oocytes after binding of human spermatozoa to the oolemma, but not on the oolemma of oocytes that had not been inseminated. This moiety also appears on the surface of human oocytes following their activation by the calcium ionophore A23187. Human spermatozoa also display P-selectin, in a functional state-dependent manner, in that acro- some-reacted but not capacitated, acrosome-intact spermatozoa reacted with the anti-P-selectin antibody, as demonstrated by both indirect immunofluorescence and immunogold labelling using transmission electron microscopy. Materials and methods Sperm preparation Spermatozoa for the experiments were obtained by a three-step Percoll centrifugation from five healthy donors whose specimens 341 Downloaded from https://academic.oup.com/molehr/article-abstract/2/5/341/985621 by guest on 24 May 2020