Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation

Molecular Human Reproduction vol.5 no.11 pp. 1017–1026, 1999 Regulation of human sperm capacitation by a cholesterol efflux-stimulated signal transduction pathway leading to protein kinase A-mediated up-regulation of protein tyrosine phosphorylation Joseph E.Osheroff1,3, Pablo E.Visconti1, Juan Pablo Valenzuela1, Alexander J.Travis1, Juan Alvarez2 and Gregory S. Kopf1,4 1Center for Research on Reproduction and Women’s Health, Room 1315, Biomedical Research Building II, University of Pennsylvania School of Medicine, 421 Curie Boulevard, Philadelphia, PA 19104–6142, and 2Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, Philadelphia, PA 19104–6080, USA 3Current address: Center for Reproductive Endocrinology, 95 Mount Kemble Avenue, Thebaud Building, 2nd Floor, Morristown, NJ 07922, USA 4To whom correspondence should be addressed Protein tyrosine phosphorylation is an important intracellular event accompanying the in-vitro capacitation of mouse, bovine and human spermatozoa. Here, we demonstrate that bovine serum albumin (BSA) and NaHCO3 are required for protein tyrosine phosphorylation in ejaculated human spermatozoa. The absence of protein tyrosine phosphorylation in media minus these two constituents could be recovered by addition to the media of cAMP analogues and/or phosphodiesterase inhibitors. Since BSA is postulated to modulate capacitation by removal of cholesterol from the sperm plasma membrane, we determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Incubation of spermatozoa in media containing BSA resulted in the release of significant amounts of cholesterol when compared with media devoid of BSA. Preloading BSA with cholesterol-SO4 inhibited protein tyrosine phosphorylation, as well as capacitation, and this inhibitory effect was overcome by the addition of dibutyryl cAMP plus isobutylmethylxanthine (IBMX). The functional significance of BSA-mediated cholesterol release, protein tyrosine phosphorylation and capacitation was confirmed by examining the effects of the cholesterol-binding heptasaccharides, methyl-βcyclodextrin or OH-propyl-β-cyclodextrin. Both cyclodextrins caused cholesterol efflux from the spermatozoa, increased protein tyrosine phosphorylation, and stimulated capacitation. Therefore, cholesterol release is associated with the activation of a signal transduction pathway involving protein kinase A and tyrosine kinase second messenger systems, and resulting in protein tyrosine phosphorylation and capacitation. Key words: cAMP/cholesterol/cyclodextrins/human sperm capacitation/protein tyrosine phosphorylation Introduction Although freshly ejaculated mammalian spermatozoa are motile and appear to be morphologically mature, they do not have the ability to fertilize an egg. Spermatozoa must first undergo a poorly understood maturational process during their period of residence in the female reproductive tract before they gain the ability to fertilize. This time-dependent acquisition of fertilization competence is known as capacitation (Austin, 1951, 1952; Chang, 1951). The definition of capacitation has been modified over the years to include the acquisition of the ability of acrosome-intact spermatozoa to undergo the acrosome reaction in response to the zona pellucida or to progesterone (Ward and Storey, 1984; Florman and Babcock, 1991; Kopf and Gerton, 1991; Shi and Roldan, 1995; Aitken, 1997). Capacitation has been shown to be correlated with changes in sperm plasma membrane fluidity, intracellular ion concentrations, metabolism and motility (Yanagimachi, 1994; Visconti et al., 1998). Although these changes have been known to accompany the process of capacitation, the molecular basis underlying these events is poorly understood. The changes in membrane fluidity leading to capacitation are postulated to © European Society of Human Reproduction and Embryology arise from a reduction in the cholesterol:phospholipid ratio as a consequence of the efflux of cholesterol from the plasma membrane to a protein acceptor (Langlais and Roberts, 1985; Hoshi et al., 1990; Lin and Kan, 1996; Gamzu et al., 1997; Cross, 1998); bovine serum albumin (BSA) is thought to serve as a cholesterol acceptor to mediate capacitation in vitro (Go and Wolf, 1985). This change in membrane lipid composition is thought to alter the bulk biophysical properties of the membrane by changing membrane fluidity, which may impact directly or indirectly on membrane protein function, leading to changes in ion channel and/or enzymatic activity (Kopf et al., 1999). Recent studies have established a correlation between capacitation and phosphorylation on tyrosine residues of multiple proteins in murine (Visconti et al., 1995a,b), bovine (Galantino-Homer et al., 1997), and human (Aitken et al., 1995; Carrera et al., 1996; Leclerc et al., 1996; Luconi et al., 1996; Emiliozzi and Fenichel, 1997; Brewis et al., 1998; Tomes et al., 1998) spermatozoa. In these studies, capacitation was assessed by the ability of the spermatozoa to undergo an induced acrosome reaction and, in some cases, to fertilize eggs in vitro. Studies in the mouse have demonstrated that there is 1017 J.E.Osheroff et al. an absolute requirement for serum albumin, HCO3– and Ca21 in the incubation medium for both these protein tyrosine phosphorylations and capacitation to occur (Visconti et al., 1995a). In addition, the protein tyrosine phosphorylations and capacitation are up-regulated by cAMP at the level of protein kinase A (PK-A) (Visconti et al., 1995b). Based on these and other studies (Zeng et al., 1995; Leclerc et al., 1996; de Lamirande et al., 1997; Emiliozzi and Fenichel, 1997), we have developed an hypothesis for the signalling pathways regulating mammalian sperm capacitation that includes initial changes in plasma membrane dynamics that lead to changes in ion fluxes across the membrane and stimulation of adenylyl cyclase. The resulting increase in intracellular cAMP concentrations leads to an activation of PK-A which then interacts with a sperm protein tyrosine kinase/phosphatase pathway to regulate further capacitation events. The purpose of the present study was to determine whether the signalling events involved in sperm capacitation previously described in other mammalian species are present in human spermatozoa. Specifically, we wished to demonstrate: (i) the association of sperm protein tyrosine phosphorylation with the ability of the spermatozoa to undergo an induced acrosome reaction, an index of capacitation; (ii) the requirement for NaHCO3 in regulating sperm protein tyrosine phosphorylation; (iii) the requirement for serum albumin in regulating sperm protein tyrosine phosphorylation and the possible mechanism by which this serum protein functions, i.e. through the removal of plasma membrane cholesterol; and (iv) the role of cAMP in regulating this cascade of events. Materials and methods Rea (...truncated)