Capacitation Induces Tyrosine Phosphorylation of Proteins in the Boar Sperm Plasma Membrane Frits M. Flesch,* , † Ben Colenbrander,* Lambert M. G. van Golde,† and Barend M. Gadella* , † *Department of Farm Animal Health, Graduate School of Animal Health, and †Department of Biochemistry and Cell Biology, Graduate School of Animal Health, Utrecht University, 3508 TD, Utrecht, The Netherlands Received August 2, 1999 Capacitation (activation) of mammalian spermato- zoa is accompanied by protein phosphorylation, eleva- tion of the intracellular calcium concentration and an increased plasma membrane fluidity. The subcellular localization of tyrosine phosphorylation during capac- itation have not yet been elucidated. The aim of this study was to investigate whether boar sperm capaci- tation induces tyrosine phosphorylation of plasma membrane proteins. Capacitation induced tyrosine phosphorylation of 3 proteins (27, 37, and 40 kDa), which coincided with an increase in the plasma mem- brane fluidity. The importance of the induced tyrosine phosphorylation in sperm binding to the zona pellu- cida and the induction of the acrosome reaction is discussed. © 1999 Academic Press Key Words: capacitation; tyrosine phosphorylation; membrane fluidity; plasma membrane proteins; boar sperm. Freshly ejaculated spermatozoa are not capable to fertilize the oocyte (1, 2). To acquire fertilizing ability, the spermatozoa have to undergo an activation process called capacitation. Although capacitation is of high importance, the biochemical understanding of this pro- cess is still far from complete. Especially the signifi- cance of capacitation induced changes for the primary binding of the sperm cell to the zona pellucida (the extracellular matrix of the oocyte) and for the subse- quent acrosome reaction (fusion of the apical plasma membrane with the underlying acrosomal membrane) is still not clear. Capacitation normally occurs in the female genital tract (3), but for in vitro fertilization, the sperm cells are capacitated in chemically defined me- dia (4). Although minor variations exist between these media, depending on the mammalian species, most of these media contain bicarbonate, calcium and macro- molecules (generally BSA) (5). Bicarbonate activates a sperm specific adenylate cyclase and thereby induces increased cAMP levels in the sperm cell (6). The sub- sequent activation of protein kinase A (PKA) (7) in- duces, via a yet unknown signaling mechanism, ty- rosine phosphorylation of several proteins (8, 9). Tyrosine phosphorylation can induce conformational changes in proteins thus leading either to their activa- tion or inactivation (10). Studies on capacitation and tyrosine phosphorylation have focused on the whole sperm cell in relation to (i) the binding of sperm cells to the zona pellucida and (ii) the acrosome reaction (9, 11–14). However, the sperm cell initially adheres to the zona pellucida only with its apical plasma membrane and therefore this site must contain primary zona binding molecules. This site of the sperm plasma mem- brane is also of significance for the acrosome reaction, since the acrosome reaction is a multiple fusion event of the apical plasma membrane with the outer acroso- mal membrane (1). However, sperm cells contain a considerable amount of tyrosine phosphorylated pro- teins that are not localized at the apical plasma mem- brane. For example, flagellar proteins that induce hy- permotility after being tyrosine phosphorylated (11). Taken together, it is of major interest to investigate protein phosphorylation during capacitation at the api- cal plasma membrane level rather than the entire sperm cell. This necessitates the isolation of the apical sperm plasma membrane proteins from other sperm proteins after in vitro capacitation. Our major goal was to identify capacitation induced changes in the tyrosine phosphorylation state of pro- teins in the plasma membrane of boar spermatozoa. Therefore, we incubated washed spermatozoa under different conditions to induce capacitation and isolated the plasma membrane of capacitated cells following a previously reported method for the isolation of plasma membranes from freshly ejaculated sperm cells (15). Abbreviations used: BSA, bovine serum albumin; BTS, Beltsville thawing solution; CTC, chlortetracycline; DMSO, dimethyl sulfoxide; ELLBA, enzyme-linked lectin binding assay; PBS, phosphate- buffered saline; PKA, protein kinase A; PMSF, phenylmethylsulfonyl fluoride; PNA, Arachis hypogaea (peanut) agglutinin; SDH, succi- nate dehydrogenase; TBS, Tris-buffered saline; TBSS, Tris-buffered sucrose solution; WGA, Triticum vulgare (wheat germ) agglutinin. Biochemical and Biophysical Research Communications 262, 787–792 (1999) Article ID bbrc.1999.1300, available online at http://www.idealibrary.com on 787 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.