Molecular Human Reproduction, Vol. 5, No. 11, 1017-1026,
November 1999
© 1999 European Society of Human Reproduction and Embryology
Regulation of sperm function |
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
1 Center 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 2 Department of Obstetrics and Gynecology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, Philadelphia, PA 19104–6080, USA
Abstract
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-ß-cyclodextrin or OH-propyl-ß-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.
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 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 an absolute requirement for serum albumin, HCO3– and Ca2+ 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
Reagents
BSA (fraction V, # A-4503), dibutyryl-cAMP, 3-isobutyl-1-methylxanthine (IBMX), A23187, 2-OH-propyl-ß-cyclodextrin, methyl- ß-cyclodextrin and cholesterol-3-sulphate were purchased from Sigma Chemical Company (St Louis, MO, USA). Sp-cAMPS and Rp-cAMPS were obtained from Research Biochemicals International (RBI; Nantick, MA, USA). Progesterone was purchased from Cal-Biochem (La Jolla, CA, USA). Anti-phosphotyrosine antibody (clone 4G10) was obtained from Upstate Biochemical Technology Inc (UBI, Lake Placid, NY, USA) and goat anti-mouse immunoglobulin G (IgG) conjugated to horseradish peroxidase (Pierce; Rockford, IL, USA) and an enhanced chemiluminescence kit (ECL; Amersham Life Science Inc, Oakville, ON, USA) were used for immunodetection of phosphotyrosine containing proteins. Concanavalin A conjugated to fluorescein isothiocyanate (FITC–ConA) was purchased from Vector Laboratories (Burlingame, CA, USA).
Culture media
The basic medium used for all experiments was modified human tubal fluid (mHTF; Irvine Scientific, Santa Ana, CA, USA), or self-prepared according to the manufacturer's instructions; mHTF contained 21 mmol/l HEPES. `Complete' media were prepared by the addition to mHTF of BSA and NaHCO3 to final concentrations of 5 mg/ml and 10 mmol/l respectively. `Incomplete' media were either mHTF as purchased or modified so as to include 10 mmol/l NaHCO3, depending on the control desired. mHTF devoid of NaHCO3 was prepared according to the recipe of the commercial medium but without the addition of NaHCO3. For the cyclodextrin experiments, BSA was omitted from the media except where explicitly stated. The pH of all media was adjusted to 7.40–7.45 before use.
Sperm preparation and incubation
Freshly ejaculated spermatozoa from healthy volunteers were obtained by masturbation after 2 days of abstinence. After liquefaction at room temperature, the spermatozoa were analysed for viability and motility by light microscopy using World Health Organization (WHO) criteria. The semen was then split into 1 ml aliquots in 15 ml plastic conical tubes, diluted with equal volumes of mHTF and then washed by centrifugation at 350 g for 8 min. The supernatant was discarded and the remaining soft sperm pellet was carefully overlaid with 500 µl of mHTF. The tubes were then incubated at a 45° angle at 37°C in an atmosphere of 5% CO2 in air for 1 h to allow swim-up of the most viable spermatozoa. Following swim-up, the spermatozoa were again assessed for motility by light microscopy. The sperm concentration was determined with a haemocytometer and then adjusted to 20x106/ml for use in the experiments. For capacitation, 50 µl aliquots of the sperm suspension were added to 450 µl of various test media in 1.5 ml polypropylene microcentrifuge tubes for a final sperm concentration of 2x106/ml. The tubes were then incubated at 37°C in a humidified incubator that was flushed continually with 5% CO2 in air. For those experiments where we analysed the effects of NaHCO3, sperm selection by swim-up was performed as described above by incubating sealed tubes containing the spermatozoa in a water bath at 37°C. Subsequent incubation of spermatozoa prepared in the NaHCO3-free media was then perfomed by incubating the washed spermatozoa in 1.5 ml polypropylene microcentrifuge tubes at the same concentration above in a water bath at 37°C. In this case the NaHCO3-free media was buffered to pH 7.4–7.45 with 21 mmol/l HEPES.
After incubation for various time periods between 0 h and overnight (20 h), the spermatozoa were concentrated by centrifugation at 20 000 g for 2 min at room temperature and then washed once in 0.5 ml phosphate-buffered saline (PBS) at room temperature. The sperm pellet was then resuspended in sample buffer (Laemmli, 1970) without mercaptoethanol and boiled for 5 min. After centrifugation at 20 000 g for 2 min, the supernatant was removed, 2-mercaptoethanol added to a final concentration of 5%, the sample boiled for 3 min, and then subjected to sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) as described below.
Steroid measurements
Human spermatozoa (5x106) were incubated in 500 µl of capacitation medium in either the absence or presence of 3 mg/ml of BSA or with the appropriate ß-cyclodextrins for 1.5 h. After this period, each aliquot was centrifuged for 10 min at 10 000 g and cholesterol, demosterol and cholesterol-SO4 were measured in the sperm pellet and in the resultant medium supernatant as previously described (Alvarez and Storey, 1995; Visconti et al., 1999a). Briefly, sperm pellets were extracted with 20 vol of chloroform–methanol (1:1, v/v), vortexed for 10 s, centrifuged at 800 g for 3 min and the supernatant evaporated to dryness under N2. The resultant supernatants following the initial centrifugation were then extracted with 6 vol of chloroform–methanol (2:1, v/v), vortexed for 10 s, centrifuged at 800 g for 10 s, and the lower organic phase aspirated and evaporated to dryness. Both the sperm pellet and medium supernatant extracts were dissolved in 20 µl of chloroform–methanol (1:1 v/v), and 4 µl aliquots applied to silver nitrate-impregnated Whatman HP-K silica gel microplates (Whatman Inc, Clifton, NJ, USA) (5x5 cm, 250 µm thickness). Aliquots (4 µl) of cholesterol, desmosterol, cholesterol sulphate, and desmosterol (Sigma Chemical Co) at a concentration of 0.1 mg/ml, were applied on separate lanes as reference standards. The plates were pre-developed in chloroform–methanol (1:1, v/v) to 1 cm from the lower edge of the plate. This pre-development step was used to minimize eddy diffusion which results in band broadening and lower resolution. Following pre-development, the plates were thoroughly dried and then developed in chloroform–acetone (95:5, v/v) in the same dimension. Following development, the plates were thoroughly dried, dipped in a 10% solution of copper sulphate (in 8% phosphoric acid), and placed on a CAMAG Plate Heater III at 185°C for 5 min. The resulting bands were scanned at 400 nm in the reflectance mode using a Shimadzu CS-9000 spectrodensitometer (Shimadzu Scientific Instruments, Columbia, MD, USA). The integrated areas obtained for the unknowns were interpolated with the standard curves obtained for cholesterol, desmosterol and cholesterol sulphate standards, and the values expressed as ng/106 cells.
Induction of the acrosome reaction and evaluation of acrosomal status
Since fertilization of human eggs could not be performed as an assay to assess capacitation, the ability of the spermatozoa to respond to progesterone to undergo an acrosome reaction was utilized as an assay for capacitation (Florman and Babcock, 1991; Visconti et al., 1998). It has been demonstrated that the progesterone-induced acrosome reaction occurs only in capacitated spermatozoa (Shi and Roldan, 1995). We also examined the effects of different treatments on the acrosome reaction induced by the calcium ionophore A23187. Spermatozoa were incubated under various conditions at a concentration of 2x106/ml. At the conclusion of the appropriate incubation period, spermatozoa were stimulated with either progesterone (3.2 µmol/l) for 20 min or A23187 (10 µmol/l) for 1 h. The FITC–ConA staining procedure used to assess acrosomal status was modified from Nishikimi et al. (Nishikimi et al., 1997). Following the incubation, the cells in each tube were fixed with 1 ml 4% formaldehyde for 1 h, followed by centrifugation at 400 g for 2 min. The remaining pellet was then washed with PBS, re-centrifuged and incubated in 10 mg/ml FITC–ConA in PBS for 30 min. The stained spermatozoa were centrifuged at 400 g and washed once again in PBS. After centrifugation, the supernatant was discarded and the spermatozoa were resuspended in the small amount of PBS remaining in the tube (~20 µl). The suspension (10 µl) was then allowed to dry on each well of a 2-well coated slide (Cel-Line Associates, NJ, USA), which was then mounted with Vectashield mounting medium. Fluorescence was observed using a Zeiss Photomicroscope III equipped with epifluorescence at x400, and the acrosome reaction was evaluated on a total of 200 spermatozoa per slide. Spermatozoa demonstrating fluorescence over the sperm head were considered to be acrosome-reacted. Assessment of acrosome reactions were made by an observer blinded to the different incubation conditions.
SDS–PAGE and immunoblotting
SDS–PAGE was performed using 10% gels according to the method of Laemmli (Laemmli, 1970). Electrophoretic transfer of proteins to Immobilon P (Amersham Life Science, Arlington Heights, IL, USA) in all experiments was carried out according to the method of Towbin et al. (Towbin et al., 1979), at 70 V for 2 h at 4°C. Immunodetection of proteins transferred to Immobilon P was performed at room temperature as described previously (Kalab et al., 1994) using a primary monoclonal antibody against phosphotyrosine (clone 4G10; UBI), a secondary goat anti-mouse horseradish peroxidase-conjugated IgG (Pierce, Rockford, IL, USA) and ECL using an Amersham ECL kit according to the manufacturer's instructions.
Results
Time-dependent changes in protein tyrosine phosphorylation under conditions conducive to capacitation
When freshly ejaculated human spermatozoa were incubated for various periods of time in complete media, a time dependent increase in protein tyrosine phosphorylation of a subset of proteins of Mr = 43 000–200 000 was routinely observed over the 18 h period of incubation (Figure 1). It should be noted that the most prominent tyrosine phosphorylated proteins at Mr = 95 000 and Mr = 83 000 are the human homologues of the mouse proAKAP82 and AKAP82 respectively, as previously demonstrated by our group (Carrera et al., 1996). Brewis et al. (1998) have also noted a capacitation-dependent increase in the tyrosine phosphorylation of Mr = 95 000 and Mr = 80 000 proteins in human spermatozoa (Brewis et al., 1998), but the amino acid sequence identity of these proteins was not determined.
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Requirement of serum albumin for protein tyrosine phosphorylation and capacitation
The presence of serum albumin in the culture medium has previously been demonstrated to be required for sperm capacitation in a variety of mammals (Go and Wolf, 1985
; Langlais and Roberts, 1985). This requirement is thought to be due to the ability of BSA to bind cholesterol, resulting in the efflux of this sterol from the sperm plasma membrane. Previously, it has been shown that the protein tyrosine phosphorylations that accompany mouse sperm capacitation require the presence of BSA in the media (Visconti et al., 1995a). When human spermatozoa were incubated overnight in media without BSA, protein tyrosine phosphorylation was minimal (Figure 2). Incubation of spermatozoa in media containing increasing concentrations of BSA resulted in an increase in protein tyrosine phosphorylation, with maximum phosphorylation occurring at 3 mg/ml BSA (Figure 2). This requirement for BSA correlated with the capacitation state of the spermatozoa as demonstrated by the percentage of spermatozoa able to undergo an induced acrosome reaction in response to progesterone or A23187 after incubation in media with and without BSA (Figure 3). Moreover, these aforementioned effects of BSA on protein tyrosine phosphorylation and capacitation were associated with the ability of BSA to promote the release of cholesterol and desmosterol from the spermatozoa into the medium (Table I). Taken together, these data suggest that BSA-mediated cholesterol release from human spermatozoa is correlated with protein tyrosine phosphorylation and capacitation.
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Requirement of NaHCO3 for protein tyrosine phosphorylation
The presence of NaHCO3 in the medium has been demonstrated to be required for capacitation (Lee, 1986; Boatman, 1991; Shi, 1995; Visconti, 1995; Visconti et al., 1999b
), as well for the protein tyrosine phosphorylations that accompany this extratesticular maturational event (Visconti et al., 1995a). In order to determine whether extracellular NaHCO3 was required for the protein tyrosine phosphorylations in human spermatozoa, spermatozoa were incubated for various periods of time in media containing BSA, but devoid of NaHCO3. As shown in Figure 4, when compared with spermatozoa incubated in complete media containing 10 mmol/l NaHCO3, spermatozoa incubated in the absence of NaHCO3 did not display appreciable tyrosine phosphorylation. When various concentrations of NaHCO3 were added back to media either devoid of BSA or containing BSA, there was a concentration-dependent effect of NaHCO3 on protein tyrosine phosphorylation (Figure 5). In media containing 25 mmol/l NaHCO3, but devoid of BSA, the degree of protein tyrosine phosphorylation was similar to that of spermatozoa incubated with BSA at lower concentrations of NaHCO3, suggesting that the site of action of NaHCO3 is downstream of that of BSA.
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Roles of cAMP and PK-A on protein tyrosine phosphorylation
Previous studies have demonstrated a key role for cAMP and PK-A in the signalling pathways leading to sperm protein tyrosine phosphorylation (Visconti et al., 1995b
; Leclerc et al., 1996; Galantino-Homer et al., 1997) and capacitation (Visconti et al., 1995b, 1997). It has been postulated that the production of cAMP is an event downstream from the site of BSA and NaHCO3 action on spermatozoa (Visconti et al., 1995a, b). To investigate this hypothesis, spermatozoa were incubated in media devoid of BSA and with various concentrations of NaHCO3, but containing both dibutyryl-cAMP and the phosphodiesterase inhibitor IBMX. As shown in Figure 5
, the addition of db-cAMP plus IBMX increased protein tyrosine phosphorylation over the respective controls at all NaHCO3 concentrations tested. These spermatozoa demonstrated a degree of protein tyrosine phosphorylation similar to that of spermatozoa incubated with BSA. IBMX alone also appeared to increase protein tyrosine phosphorylation significantly over the respective controls, whereas this was not as obvious when similar experiments were carried out with dbcAMP alone (Figure 5). These data suggest that, similar to mouse and bovine spermatozoa, protein tyrosine phosphorylation in human spermatozoa appears to be up-regulated by cAMP. Studies in both mouse and bovine spermatozoa have demonstrated that this cAMP effect on protein tyrosine phosphorylation occurs at the level of PK-A (Visconti et al., 1995b; Galantino-Homer et al., 1997). To demonstrate whether a similar relationship exists in human spermatozoa, cells were incubated in media containing either Sp-cAMPS, a membrane permeable cAMP analogue that activates PK-A or its inactive stereoisomer, Rp-cAMPS, which can function as an antagonist of PK-A. When spermatozoa were incubated in the absence of BSA, Sp-cAMPS, but not Rp-cAMPS, was able to support protein tyrosine phosphorylation to levels similar to that seen with BSA addition alone (Figure 6). Moreover, the BSA-supported protein tyrosine phosphorylation was inhibited by the addition of Rp-cAMPS to the medium. These data support the idea that protein tyrosine phosphorylation in human spermatozoa is downstream of a cAMP pathway.
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Effects of cholesterol-3-sulphate on the BSA-induced changes in sperm protein tyrosine phosphorylation and capacitation
As stated previously, it has been postulated that BSA may function to support capacitation in vitro by acting as an acceptor for cholesterol, thus enhancing cholesterol efflux from the sperm plasma membrane (Go and Wolf, 1985
; Langlais and Roberts, 1985; de Lamirande et al., 1997; Cross, 1998). Cholesterol efflux from the spermatozoa of a variety of mammalian species under conditions conducive to capacitation has been demonstrated (de Lamirande et al., 1997) (and references therein). The resultant release of this sterol has then been postulated to entrain a series of poorly understood events leading to the capacitated state. We have postulated that the BSA requirement for protein tyrosine phosphorylation observed in mouse spermatozoa (Visconti et al., 1995a, b) and human spermatozoa is somehow related to cholesterol efflux and that the efflux of this sterol from spermatozoa changes membrane dynamics such that a signal transduction pathway is initiated leading to the elevation of cAMP. If this hypothesis were correct, one would predict that blocking the ability of BSA to serve as an acceptor for sperm membrane cholesterol by saturating these sites on BSA would result in an inhibition of protein tyrosine phosphorylation and capacitation. To test this hypothesis, BSA was pre-loaded with increasing concentrations of cholesterol (in the form of cholesterol-3-sulphate) prior to incubation with human spermatozoa. Cholesterol-3-sulphate was used in these experiments due to its solubility characteristics. As shown in Figure 7, pre-loading the BSA with cholesterol-3-sulphate followed by incubation with spermatozoa resulted in an inhibition of protein tyrosine phosphorylation in a concentration-dependent manner at cholesterol-3-sulphate concentrations of 
5 µmol/l. Concentrations of 20 µmol/l cholesterol-3-sulphate resulted in complete inhibition of protein tyrosine phosphorylation, when compared to spermatozoa incubated in media devoid of BSA (Figure 7). In addition, spermatozoa incubated in complete media containing BSA pre-loaded with 5 µmol/l cholesterol-3-sulphate were less capable of undergoing an induced acrosome reaction than spermatozoa incubated in complete media without cholesterol-3-sulphate (Table I), suggesting that capacitation was inhibited under these conditions. Moreover, these inhibitory effects of cholesterol-3-sulphate pre-loading of BSA on protein tyrosine phosphorylation could be rescued by the addition of db-cAMP and IBMX to these media (Figure 8), again suggesting that the action of cAMP was downstream of the BSA effect.
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ß-Cyclodextrins as substitutes for serum albumin in supporting sperm cholesterol efflux, protein tyrosine phosphorylation and capacitation
ß-Cyclodextrins are cyclic heptasaccharides that have the ability to efficiently bind cholesterol and induce cholesterol efflux from cells (Pitha et al., 1988
; Kilsdonk et al., 1995; Yancey et al., 1996). If cholesterol efflux is, indeed, a trigger for changes in cAMP, PK-A and protein tyrosine phosphorylation leading to capacitation, it would be predicted that cyclodextrins should be able to substitute for BSA in the induction of these events. This, in fact, has recently been demonstrated in mouse spermatozoa (Choi and Toyoda, 1998; Visconti et al., 1999a). Incubation of spermatozoa in media devoid of BSA but containing various concentrations of either methyl-ß-cyclodextrin or 2-OH-propyl-ß-cyclodextrin promoted the release of both cholesterol and desmosterol from spermatozoa (Table I). These ß-cyclodextrins were, in fact, more potent than BSA in promoting the release of these steroids. In addition, incubation of spermatozoa in media without BSA, but containing increasing concentrations of either methyl-ß-cyclodextrin or 2-OH-propyl-ß-cyclodextrin, resulted in concentration dependent increases in protein tyrosine phosphorylation, approximating those obtained when media were supplemented with BSA (Figure 9). Spermatozoa incubated with methyl-ß-cyclodextrin and Rp-cAMPS showed a significant reduction in protein tyrosine phosphorylation when compared with spermatozoa incubated in media containing this ß-cyclodextrin alone (Figure 6), demonstrating that the ß-cyclodextrin effect to increase protein tyrosine phosphorylation was likely through cAMP and PK-A, similar to that seen with BSA. Similarly, spermatozoa incubated in the absence of BSA but in the presence of either ß-cyclodextrin were able to undergo the acrosome reaction when stimulated with progesterone or A23187 (Figure 3), suggesting that these ß-cyclodextrins support capacitation in the absence of a protein supplement such as BSA. It is interesting to note that the percentage of spontaneous acrosome reactions increases under these conditions. This observation is consistent with observations made in mouse spermatozoa (Visconti et al., 1999a). The percentage of spermatozoa that were acrosome-reacted after incubation with ß-cyclodextrins and subsequent stimulation with either progesterone or A23187 was similar to that after incubation with BSA, and greater than that observed after incubation in control media (Figure 3).
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Discussion
The data presented in this report support the hypothesis that signal transduction pathways that are activated during capacitation of human spermatozoa are regulated by mechanisms similar to those previously identified in other species (Visconti et al., 1995a, b, 1997, 1999a; Carrera et al., 1996; Galantino-Homer et al., 1997). This work also extends the observations made by others studying human sperm capacitation (Aitken et al., 1995; Luconi et al., 1996; Leclerc et al., 1996, 1997). Specifically, we have shown that the protein tyrosine phosphorylation of several human sperm proteins is related to the capacitation state of these cells and that the signalling pathway regulating these processes involves the movement of cholesterol, presumably from the plasma membrane, with the subsequent activation of an intracellular signalling pathway leading to the activation of PK-A and the up-regulation of protein tyrosine phosphorylation. This protein tyrosine phosphorylation occurs in a time-dependent fashion and is initiated by the presence of factors (BSA; ß-cyclodextrins) extrinsic to the spermatozoa that can function as acceptors for cholesterol. Both BSA and the ß-cyclodextrins used in this study (methyl-ß-cyclodextrins ; 2-OH-propyl-ß-cyclodextrins) were shown to cause an efflux of cholesterol from human spermatozoa as shown by mass measurements, and these results are consistent with observations using mouse spermatozoa (Visconti et al., 1999a). It should be noted that while this paper was being written, the addition of methyl-ß-cyclodextrin to human spermatozoa was also shown to cause a release of cholesterol and desmosterol from these cells (Cross, 1999). Preloading the BSA in the capacitation media with an excess of cholesterol-3-sulphate blocks both the activation of this unique signal transduction pathway and capacitation, as assessed by the ability of the spermatozoa to undergo acrosome reactions in response to either progesterone or A23187. This most likely occurs by saturating the cholesterol acceptor sites on BSA, thereby blocking the ability of BSA to initiate the movement of cholesterol out of the sperm plasma membrane. cAMP is integral to the functioning of this pathway and it functions downstream of the cholesterol movement, at the level of PK-A. cAMP analogues can both initiate protein tyrosine phosphorylation in the absence of BSA and overcome the inhibitory effect of cholesterol-3-sulphate in media containing BSA. Moreover, Rp-cAMPS, an inhibitor of PK-A, decreases the degree of tyrosine phosphorylation initiated by either BSA or by the ß-cyclodextrins.
The observations made in this report, as well as recent work by our laboratory using mouse spermatozoa (Visconti et al., 1999a), represents one of the first examples of a transmembrane signal transduction pathway regulated by cholesterol. Although it has recently been demonstrated that signal transduction via CD59 and CD48 in Jurkat T cells leading to an increase in intracellular calcium is regulated, in some manner, by cellular cholesterol (Stulnig et al., 1997), this effect appears to be independent of effects on membrane dynamics, unlike the case that we report here. The mechanism by which changes in cholesterol content of the sperm membrane regulate transmembrane signalling events leading to capacitation is not known, although it is clear from numerous studies that this sterol alters the bulk biophysical properties of biological membranes. For example, the presence of membrane cholesterol can increase the orientation order of the membrane lipid hydrocarbon chains, thus reducing the ability of membrane proteins to undergo conformational changes that may control their functions due to the less fluid nature of the membrane. Thus, high concentrations of cholesterol in the membrane might inhibit membrane protein function. Such an indirect effect of this sterol on membrane protein function might stabilize those membrane and transmembrane events that constitute part of the `intrinsic' regulation of capacitation. Cholesterol has also been demonstrated to have direct effects by binding to and regulating membrane protein function. Such direct effects of this sterol might manifest itself as a positive or negative modulatory effect on the membrane protein in question. Direct and indirect modulatory effects of cholesterol on membrane-associated ion transporters such as the 
-aminobutyric acid (GABA) transporter and Na+, K+-ATPase have been documented (Vemuri and Philipson, 1989; Shouffani and Kanner, 1990). In this regard, it is interesting to note that losses of plasma membrane cholesterol (Visconti et al., 1999a) and changes in membrane potential (Zeng et al., 1995) accompany capacitation in the mouse. Moreover, the loss of cholesterol from the human sperm plasma membrane during capacitation has been postulated to be coupled, in some fashion, to the increase in intracellular pH that accompanies this maturational process (Cross and Razy-Faulkner, 1997). These observations support the idea that cholesterol itself and/or changes in its concentrations in the plasma membrane could modulate transmembrane ionic movements that ultimately regulate membrane potential and intracellular pH. This hypothesis remains to be tested experimentally.
Our laboratory has demonstrated that the release of cholesterol is, in some manner, tied to changes in protein tyrosine phosphorylation (this report) (Visconti et al., 1999a). Normally, cellular signalling involving the activation of tyrosine kinases and protein tyrosine phosphorylation is mediated through the activation of plasma membrane receptors. Such receptors could either possess intrinsic tyrosine kinase activity or could associate with tyrosine kinases. Spermatozoa represent a unique case in which the increase in protein tyrosine phosphorylation is regulated through a cAMP and PK-A pathway, and this modulation may be due to either a direct or indirect effect on protein tyrosine kinase and/or phosphotyrosine phosphatase activities. Presently, we do not know how cholesterol removal regulates such a pathway, but it is tempting to speculate that changes in membrane dynamics upon cholesterol removal as described above could result in a change in permeability of the spermatozoa to HCO3– and/or Ca2+, both of which are capable of stimulating the sperm adenylyl cyclase. We and others have demonstrated that these ions are important regulators of this unique signal transduction pathway and capacitation in human (this report) (Carrera et al., 1996; Luconi et al., 1996), as well as in mouse (Visconti et al., 1995a) spermatozoa. Recent work in the mouse by our group is consistent with this possibility (Visconti et al., 1999a).
Whether cholesterol loss leading to the activation of these aforementioned pathways and capacitation occurs in vivo in human spermatozoa has not been determined. If such processes did occur during capacitation in vivo, the identity of the molecule(s) mediating this cholesterol loss would be of considerable interest. In the present work we utilized two different cholesterol acceptors (i.e. BSA, ß-cyclodextrins) to activate signal transduction cascades in human spermatozoa leading to the ability to undergo an induced acrosome reaction, an index of capacitation. Independently, Cross (1999) has recently demonstrated that addition of methyl-ß-cyclodextrin to human spermatozoa increases both the percentage of spontaneous acrosome reactions as well as acrosome reactions induced by progesterone. The ability of a non-protein media supplement (ß-cyclodextrins) to bring about functional capacitation in vitro as assessed by competence to undergo a zona pellucida-induced acrosome reaction, fertilization, and generation of viable offspring has recently been demonstrated in the mouse (Choi and Toyoda, 1998; Visconti et al., 1999a). In addition to the fact that the data presented in this paper highlight a new and important role for cholesterol in initiating transmembrane signal transduction, the data also suggest that completely defined media devoid of protein could be utilized in the various assisted reproductive technologies to obtain successful fertilization in vitro. This is currently being assessed.
Acknowledgments
This work was supported by grants to GSK (NIH HD 06274 and HD 22732). PEV was supported by the Rockefeller Foundation and by NIH HD06274. JPV was supported by a training grant from the Fogarty International Center. AJT was supported by 5T32GM-7170 and HD-33052. JGA was supported by NID (IH HD-36146) and a grant from the Cystic Fibrosis Foundation.
Notes
3 Current address: Center for Reproductive Endocrinology, 95 Mount Kemble Avenue, Thebaud Building, 2nd Floor, Morristown, NJ 07922, USA 
4 To whom correspondence should be addressed
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Submitted on April 26, 1999; accepted on August 4, 1999.
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