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• W2085155504 abstract "Sperm capacitation in vitro is highly correlated with an increase in protein tyrosine phosphorylation that is regulated by cAMP through a unique mode of signal transduction cross-talk. The activation of this signaling pathway, as well as capacitation, requires bovine serum albumin (BSA) in the incubation medium. BSA is hypothesized to modulate capacitation through its ability to remove cholesterol from the sperm plasma membrane. Here we demonstrate that the cholesterol-binding heptasaccharides, methyl-β-cyclodextrin and OH-propyl-β-cyclodextrin, promote the release of cholesterol from the mouse sperm plasma membrane in media devoid of BSA. Both of these β-cyclodextrins were also demonstrated to increase protein tyrosine phosphorylation in the absence of BSA in both mouse and bull sperm, and the patterns of phosphorylation were similar to those induced by media containing BSA. The potency of the different β-cyclodextrins to increase protein tyrosine phosphorylation in sperm was correlated with their cholesterol binding efficiencies, and preincubation of the β-cyclodextrins with cholesterol- SO4− to saturate their cholesterol-binding sites blocked the ability of these compounds to stimulate protein tyrosine phosphorylation. The β-cyclodextrin effect on protein tyrosine phosphorylation was both NaHCO3 and protein kinase A-dependent. The β-cyclodextrins were also able to capacitate mouse sperm in the absence of BSA, as measured by the ability of the zona pellucida to induce the acrosome reaction and by successful fertilization in vitro. In summary, β-cyclodextrins can completely replace BSA in media to support signal transduction leading to capacitation. These data further support the coupling of cholesterol efflux to the activation of membrane and transmembrane signaling events leading to the activation of a unique signaling pathway involving the cross-talk between cAMP and tyrosine kinase second messenger systems, thus defining a new mode of cellular signal transduction initiated by cholesterol release. Sperm capacitation in vitro is highly correlated with an increase in protein tyrosine phosphorylation that is regulated by cAMP through a unique mode of signal transduction cross-talk. The activation of this signaling pathway, as well as capacitation, requires bovine serum albumin (BSA) in the incubation medium. BSA is hypothesized to modulate capacitation through its ability to remove cholesterol from the sperm plasma membrane. Here we demonstrate that the cholesterol-binding heptasaccharides, methyl-β-cyclodextrin and OH-propyl-β-cyclodextrin, promote the release of cholesterol from the mouse sperm plasma membrane in media devoid of BSA. Both of these β-cyclodextrins were also demonstrated to increase protein tyrosine phosphorylation in the absence of BSA in both mouse and bull sperm, and the patterns of phosphorylation were similar to those induced by media containing BSA. The potency of the different β-cyclodextrins to increase protein tyrosine phosphorylation in sperm was correlated with their cholesterol binding efficiencies, and preincubation of the β-cyclodextrins with cholesterol- SO4− to saturate their cholesterol-binding sites blocked the ability of these compounds to stimulate protein tyrosine phosphorylation. The β-cyclodextrin effect on protein tyrosine phosphorylation was both NaHCO3 and protein kinase A-dependent. The β-cyclodextrins were also able to capacitate mouse sperm in the absence of BSA, as measured by the ability of the zona pellucida to induce the acrosome reaction and by successful fertilization in vitro. In summary, β-cyclodextrins can completely replace BSA in media to support signal transduction leading to capacitation. These data further support the coupling of cholesterol efflux to the activation of membrane and transmembrane signaling events leading to the activation of a unique signaling pathway involving the cross-talk between cAMP and tyrosine kinase second messenger systems, thus defining a new mode of cellular signal transduction initiated by cholesterol release. Immediately after ejaculation, mammalian sperm do not possess the ability to fertilize the egg. They acquire the ability to fertilize during transit through the female tract in a poorly understood process known as capacitation (1Austin C.R. Aust. J. Sci. Res. 1951; 4: 581-596Crossref PubMed Google Scholar, 2Austin C.R. Nature. 1952; 170: 326Crossref PubMed Scopus (509) Google Scholar, 3Chang M.C. Nature. 1951; 168: 697-698Crossref PubMed Scopus (973) Google Scholar, 4Chang M.C. Nature. 1955; 175: 1036-1037Crossref PubMed Scopus (51) Google Scholar). Capacitation is defined as the time-dependent acquisition of fertilization competence, and is generally assessed as the ability of the acrosome-intact sperm to undergo an acrosome reaction in response to physiological inducers such as the ZP 1The abbreviations used are: ZP, zona pellucida; BSA, bovine serum albumin; PKA, protein kinase A; M-β-CD, methyl-β-cyclodextrin; 2-OH-p-β-CD, OH-propyl-β-cyclodextrin; PVA, polyvinyl alcohol; PAGE, polyacrylamide gel electrophoresis; IBMX, isobutylmethylxanthine; Bt2cAMP, dibutyryl cAMP. or progesterone (5Visconti P.E. Galantino-Homer H. Moore G.D. Bailey J.L. Ning X.P. Fornes M. Kopf G.S. J. Androl. 1998; 19: 242-248Crossref PubMed Google Scholar, 6Visconti P.E. Kopf G.S. Biol. Reprod. 1998; 59: 1-6Crossref PubMed Scopus (400) Google Scholar), or by the ability of the sperm to fertilize eggs (7Yanagimachi R. Knobil E. Neill J.D. The Physiology of Reproduction. 3 Ed. Raven Press, Ltd., New York1994: 189-317Google Scholar). Capacitation can be mimicked in vitro by incubating epididymal or ejaculated sperm in a defined medium containing appropriate concentrations of electrolytes, metabolic energy sources, Ca2+, HCO3−, and a protein source (usually bovine serum albumin;BSA) (7Yanagimachi R. Knobil E. Neill J.D. The Physiology of Reproduction. 3 Ed. Raven Press, Ltd., New York1994: 189-317Google Scholar). Capacitation has also been correlated with changes in sperm intracellular ion concentrations, metabolism, and motility (7Yanagimachi R. Knobil E. Neill J.D. The Physiology of Reproduction. 3 Ed. Raven Press, Ltd., New York1994: 189-317Google Scholar, 8Harrison R.A.P. Reprod. Fertil. Dev. 1996; 8: 581-594Crossref PubMed Scopus (214) Google Scholar). Although these changes have been known for many years to accompany this maturational event, the molecular basis underlying these events is poorly understood. Remarkably, capacitation in vitro can occur in the absence of any specific external stimulus, suggesting that specific aspects of the capacitation process can be initiated and controlled intrinsically by the sperm itself, and that certain minimal environmental requirements must be met. This intrinsic nature of the capacitation process is of great interest from a cell regulation standpoint. It is possible that a controlling factor(s) intrinsic to the sperm plasma membrane may regulate changes in the properties of the membrane and that a de-repression of a set of preprogrammed cellular events must ultimately occur to promote the development of the capacitated state. The requirement for BSA in regulating capacitation is thought to be due to its ability to remove cholesterol from the sperm plasma membrane (9Suzuki F. Yanagimachi R. Gem. Res. 1989; 23: 335-347Crossref PubMed Scopus (54) Google Scholar, 10Langlais J. Roberts K.D. Gamete Res. 1985; 12: 183-224Crossref Scopus (333) Google Scholar, 11Go K.J. Wolf D.P. Biol. Reprod. 1985; 32: 145-153Crossref PubMed Scopus (167) Google Scholar, 12Davis B.K. Byrne R. Hangund B. Biochim. Biophys. Acta. 1979; 558: 257-266Crossref PubMed Scopus (141) Google Scholar, 13Davis B.K. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7560-7564Crossref PubMed Scopus (251) Google Scholar, 14Davis B.K. Proc. Soc. Exp. Biol. Med. 1976; 152: 257-261Crossref PubMed Scopus (47) Google Scholar). It is proposed that cholesterol efflux then leads to changes in membrane architecture and fluidity that give rise to the capacitated state. Cholesterol efflux, therefore, may represent an integral part of this intrinsic regulatory property of sperm to undergo capacitation. Previously, we and others have demonstrated that sperm capacitation in several species is correlated with an increase in tyrosine phosphorylation of a subset of proteins (15Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1129-1137Crossref PubMed Google Scholar, 16Carrera A. Moos J. Ning X.P. Gerton G.L. Tesarik J. Kopf G.S. Moss S.B. Dev. Biol. 1996; 180: 284-296Crossref PubMed Scopus (284) Google Scholar, 17Galantino-Homer H. Visconti P.E. Kopf G.S. Biol. Reprod. 1997; 56: 707-719Crossref PubMed Scopus (355) Google Scholar, 18Leclerc P. De lamirande E. Gagnon C. Biol. Reprod. 1996; 55: 684-692Crossref PubMed Scopus (356) Google Scholar). Our laboratory has determined that both protein tyrosine phosphorylation and capacitation are regulated by cAMP at the level of protein kinase A (PKA), and requires the presence of Ca2+, HCO3−, and BSA in the medium (19Visconti P.E. Moore G.D. Bailey J.L. Leclerc P. Connors S.A. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1139-1150Crossref PubMed Google Scholar). This mode of signal transduction cross-talk is, to date, unique to sperm. Although BSA is believed to be required for capacitation as a consequence of its ability to serve as a cholesterol-binding molecule, it is still not known if the only action of BSA is through the removal of cholesterol. β-Cyclodextrins are cyclic heptasaccharides consisting of β(1–4)-glucopyranose units (20Pitha J. Irie T. Sklar P.B. Nye J.S. Life Sci. 1988; 43: 493-502Crossref PubMed Scopus (228) Google Scholar). These compounds are water-soluble, are able to effectively solubilize non-polar substances, and because of these properties, have been used to deliver hydrophobic drugs (20Pitha J. Irie T. Sklar P.B. Nye J.S. Life Sci. 1988; 43: 493-502Crossref PubMed Scopus (228) Google Scholar). These compounds are also able to promote cholesterol efflux from a variety of somatic cells (21Yancey P.G. Rodrigueza W.V. Kilsdonk E.P.C. Stoudt G.W. Johnson W.J. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1996; 271: 16026-16034Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 22Kilsdonk E.P.C. Yancey P.G. Stoudt G.W. Bangerter F.W. Johnson W.J. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1995; 270: 17250-17256Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Yancey et al. (21Yancey P.G. Rodrigueza W.V. Kilsdonk E.P.C. Stoudt G.W. Johnson W.J. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1996; 271: 16026-16034Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar) demonstrated that the order of potency in accepting cholesterol is methyl-β-cyclodextrin (M-β-CD) > OH-propyl-β-cyclodextrin (2-OH-p-β-CD) > β-cyclodextrin. These studies also showed that there is an initial rapid efflux of cholesterol from the plasma membrane of mouse L-cells and human fibroblasts in response to β-cyclodextrins (21Yancey P.G. Rodrigueza W.V. Kilsdonk E.P.C. Stoudt G.W. Johnson W.J. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1996; 271: 16026-16034Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). In the present study, we tested the hypothesis that BSA is acting through the removal of cholesterol from the sperm plasma membrane by analyzing if β-cyclodextrins are able to replace BSA in the medium to up-regulate signal transduction pathways leading to protein tyrosine phosphorylation as well as capacitation. We demonstrate that both M-β-CD and 2-OH-p-β-CD promote the release of cholesterol from the mouse sperm plasma membrane in the absence of BSA. Addition of these β-cyclodextrins to the medium also increased protein tyrosine phosphorylation in the absence of BSA in mouse and bull sperm, and the patterns of phosphorylation were similar to those patterns seen in media containing BSA. These effects of β-cyclodextrins were likely due to their ability to capture cholesterol from the sperm plasma membrane. The β-cyclodextrin effect on protein tyrosine phosphorylation was dependent on the presence of NaHCO3 in the capacitation medium and the effect was cAMP-dependent. β-Cyclodextrins were also able to capacitate mouse sperm in the absence of BSA, as measured by the ability of the ZP to induce the acrosome reaction and by successful fertilization in vitro. This work is the first to demonstrate that a chemically defined non-protein constituent can substitute in capacitation media for protein cholesterol acceptors. These data further support the idea that cholesterol efflux is, in some way, coupled to the activation of membrane and transmembrane signaling events leading to the activation of a unique cross-talk between cAMP and tyrosine kinase second messenger systems, and thus defines a new mode of cellular signal transduction. 2-OH-propyl-β-cyclodextrin, methyl-β-cyclodextrin, and BSA (Fraction V, Sigma) were purchased from Sigma. Anti-phosphotyrosine antibody (clone 4G10) was from UBI, Lake Placid, NY. Solvents were from EM Science (chromatographic grade). Analtech diphasic plates (Unibond RP18/silica gel, 10 × 12 cm, 250 μm thickness) were obtained from Analtech Inc. (Newark, DE). Whatman HP-K silica gel plates (10 × 10 cm, 250 μm thickness) were purchased from Whatman Inc. (Clifton, NJ). The basic medium used for the studies with mouse sperm was a modified Krebs-Ringer bicarbonate medium (HMB-Hepes buffered), as described by Lee and Storey (23Lee M.A. Storey B.T. Biol. Reprod. 1986; 34: 349-356Crossref PubMed Scopus (135) Google Scholar). This medium was first prepared in the absence of Ca2+, BSA, and pyruvate, sterilized by passage through a 0.20-μm filter (Nalgene), and frozen at −20 °C in aliquots for single use. Working “complete” media were prepared by adding Ca2+ (1.7 mm), pyruvate (1 mm), and BSA (3 mg/ml), followed by gassing with 5% CO2 in air to pH 7.3. A modified Tyrode's Hepes-buffered medium (SpTALPH) was used for the experiments with bull sperm, as described by Parrish et al.(24Parrish J.J. Susko-Parrish J. Winer M.A. First N.L. Biol. Reprod. 1988; 38: 1171-1180Crossref PubMed Scopus (1294) Google Scholar). This medium was modified in the following manner: BSA was replaced by 1 mg/ml polyvinyl alcohol (PVA) to reduce the ability of SpTALPH to capacitate sperm (15Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1129-1137Crossref PubMed Google Scholar) and 0.4 mm EDTA was added. The medium was first prepared in the absence of Ca2+ and pyruvate and frozen at −20 °C in aliquots for single use. Working complete SpTALPH medium was prepared by adding Ca2+ (2 mm), pyruvate (1 mm), NaHCO3 (10 mm), PVA (1 mg/ml), and gentamycin (50 mg/ml). The pH of SpTALPH was adjusted to 7.4 following equilibration for 1 h at 39 °C in room air, and sterilized by passage through a 0.20-μm filter (Nalgene, Fisher, Pittsburgh, PA). Caudal epididymal mouse sperm were collected from CD1 retired breeder males by placing one minced cauda epididymis in 0.5 ml of medium HMB without BSA at 30 °C. After 5 min the sperm in suspension were washed in 10 ml of the same medium by centrifugation at 800 × g for 10 min at room temperature (24 °C). The sperm were then resuspended to a final concentration of 5–10 × 106 cells/ml and diluted 10 times in the appropriate medium depending on the experiment performed. After incubation for 1.5 h, except where indicated, the sperm were concentrated by centrifugation at 5,000 × g for 1 min (room temperature), the sperm pellet washed in 1 ml of phosphate-buffered saline, centrifuged again, and the resultant pellet resuspended in sample buffer (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) without mercaptoethanol and boiled for 5 min. After centrifugation at 5,000 × g for 3 min, the supernatant was removed, 2-mercaptoethanol was added to a final concentration of 5% (v/v), the samples boiled for 5 min, and then subjected to SDS-PAGE as described below. Ejaculated bovine sperm (Bos taurus, Holstein), collected by artificial vagina, was generously provided by the Hofmann Center at New Bolton Center, University of Pennsylvania School of Veterinary Medicine (Kennett Square, PA). The sperm were immediately assessed for motility by light microscopy and then, at 10–20 min following collection, were diluted 1:5 into SpTALPH. These samples were then subjected to two washes by centrifugation at 375 × g for 10 min each in SpTALPH to remove seminal plasma, as described previously (17Galantino-Homer H. Visconti P.E. Kopf G.S. Biol. Reprod. 1997; 56: 707-719Crossref PubMed Scopus (355) Google Scholar, 24Parrish J.J. Susko-Parrish J. Winer M.A. First N.L. Biol. Reprod. 1988; 38: 1171-1180Crossref PubMed Scopus (1294) Google Scholar). Sperm were kept at 22–24 °C in SpTALPH following the washes for transport to the laboratory (1–2 h), and then subjected to a final wash in SpTALPH. The concentration of sperm in the SpTALPH solution was determined by hemocytometer and adjusted to 5 × 107cells/ml with SpTALPH before initiating the capacitation experiments. For capacitation, 100-μl aliquots of the sperm suspension (5 × 106 cells), as well as the appropriate test reagents (1–3 μl), were incubated in capped 1.5-ml polypropylene microcentrifuge tubes at 39 °C in a Thermolyne 37900 culture as described previously (17Galantino-Homer H. Visconti P.E. Kopf G.S. Biol. Reprod. 1997; 56: 707-719Crossref PubMed Scopus (355) Google Scholar, 24Parrish J.J. Susko-Parrish J. Winer M.A. First N.L. Biol. Reprod. 1988; 38: 1171-1180Crossref PubMed Scopus (1294) Google Scholar). Additions to the sperm suspension were made immediately before initiation of the incubation period. Following an incubation period of 4 h, the sperm were concentrated by centrifugation at 10,000 × g for 3 min at room temperature, washed in 1 ml of phosphate-buffered saline containing 0.2 mmNa3VO4 at room temperature, and the sperm pellet then resuspended in sample buffer (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) without mercaptoethanol and boiled for 5 min. After centrifuging at 10,000 × gfor 3 min, the supernatant was removed, boiled in the presence of 5% 2-mercaptoethanol for 5 min, and then subjected to SDS-PAGE as described below. For all experiments, sperm from two to three different mature Holstein bulls were assayed to control for individual variation. Mouse sperm (5 × 106) were incubated in 500 μl of capacitation medium in either the absence or presence of 3 mg/ml BSA or with the appropriate β-cyclodextrins for 1.5 h. After this period, each aliquot was centrifuged for 10 min at 10,000 × g and cholesterol, desmosterol, and cholesterol-SO4− were measured in the sperm pellet and in the resultant medium supernatant as described previously (26Alvarez J.G. Storey B.T. Mol. Reprod. Dev. 1995; 42: 334-346Crossref PubMed Scopus (389) Google Scholar). Briefly, sperm pellets were extracted with 20 volumes 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 volumes 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) (5 × 5 cm, 250 μm thickness). Aliquots (4 μl) of cholesterol, desmosterol, cholesterol sulfate, and desmosterol (Sigma) 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 sulfate (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). The integrated areas obtained for the unknowns were interpolated with the standard curves obtained for cholesterol, desmosterol, and cholesterol sulfate standards, and the values expressed as nanograms/106 cells. SDS-PAGE (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) was performed in 8 or 10% gels. Electrophoretic transfer of proteins to Immobilon P (Millipore) in all experiments was carried out according to the method of Towbin et al. (27Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar) at 70 V (constant) for 2 h at 4 °C. Immunodetection was carried out at room temperature as described previously (28Kalab P. Visconti P. Leclerc P. Kopf G.S. J. Biol. Chem. 1994; 269: 3810-3817Abstract Full Text PDF PubMed Google Scholar) using a monoclonal antibody against anti-phosphotyrosine (clone 4G10; UBI) and blots developed using enhanced chemiluminescence detection with an ECL kit (Amersham Corp.), according to the manufacturer's instructions. As one end point of capacitation, we analyzed the ZP-induced acrosome reaction in mouse sperm, based on the premise that only capacitated sperm would undergo exocytosis in response to ZP. The percentage of acrosome reactions was measured using Coomassie Blue G-250 as described by Thaler and Cardullo (29Thaler C.D. Cardullo R.A. Biochemistry. 1995; 34: 7788-7795Crossref PubMed Scopus (95) Google Scholar). Briefly, sperm were incubated under the desired experimental conditions for 1.5 h, followed by the addition of 5 ZP/μl, 5 μm A23187, or buffer for an additional 30 min. An equal volume of 2 × fixative solution (7.5% formaldehyde in phosphate-buffered saline) was then added to each tube. After 10 min, the sperm were centrifuged for 2 min at 10,000 × g and resuspended in 0.1 mammonium acetate (pH 9). After centrifugation, the sperm pellet was resuspended in 20–50 μl of the same buffer, spread on to poly-l-lysine-coated slides, and air dried. The slides were then stained with 0.04% (w/v) Coomassie Blue G-250 in 3.5% (v/v) perchloric acid for 10 min, gently rinsed with deionized H2O until they appeared blue, air dried, and then mounted with Permount. To calculate the percentage of acrosome reactions, at least 200 sperm were counted per experimental condition. The data presented are the average of at least three different experiments. In vitro fertilization of metaphase II-arrested mouse eggs was performed as described previously (30Moore G.D. Kopf G.S. Schultz R.M. Dev. Biol. 1993; 159: 669-678Crossref PubMed Scopus (86) Google Scholar), with modifications as described in Visconti et al.(15Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1129-1137Crossref PubMed Google Scholar). PVA was used to maintain the isotonic conditions when sperm were cultured in media devoid of BSA prior to insemination. When β-cyclodextrins were included in the capacitation media, the medium containing the capacitated sperm were diluted 100-fold prior to insemination of the eggs. β-Cyclodextrins were also present in the insemination media in some of the experiments. All in vitrofertilizations were performed using metaphase-arrested eggs retrieved at approximately 14 h post-human chorionic gonadotropin injection. Phase-contrast optics were used to evaluate fertilization by looking for the presence of the second polar body and the formation of both the male and female pronuclei. Previously, we demonstrated in mouse sperm that both protein tyrosine phosphorylation and capacitation required the presence of BSA in the incubation medium (15Visconti P.E. Bailey J.L. Moore G.D. Pan D. Olds-Clarke P. Kopf G.S. Development. 1995; 121: 1129-1137Crossref PubMed Google Scholar). The role of BSA in regulating signal transduction leading to capacitation has been postulated to involve the removal of cholesterol from the sperm plasma membrane (9Suzuki F. Yanagimachi R. Gem. Res. 1989; 23: 335-347Crossref PubMed Scopus (54) Google Scholar, 10Langlais J. Roberts K.D. Gamete Res. 1985; 12: 183-224Crossref Scopus (333) Google Scholar, 11Go K.J. Wolf D.P. Biol. Reprod. 1985; 32: 145-153Crossref PubMed Scopus (167) Google Scholar, 12Davis B.K. Byrne R. Hangund B. Biochim. Biophys. Acta. 1979; 558: 257-266Crossref PubMed Scopus (141) Google Scholar, 13Davis B.K. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7560-7564Crossref PubMed Scopus (251) Google Scholar, 14Davis B.K. Proc. Soc. Exp. Biol. Med. 1976; 152: 257-261Crossref PubMed Scopus (47) Google Scholar). This hypothesis would predict that other cholesterol binding compounds that also remove cholesterol from the sperm plasma membrane could substitute for BSA in media to regulate signal transduction leading to capacitation. To test this model we first determined if cholesterol, cholesterol-SO4−, and desmosterol were removed from the sperm plasma membrane by 2-OH-p-β-CD and M-β-CD under our experimental conditions. This was done by directly measuring the cholesterol released into the incubation medium, as well as that remaining associated with the sperm after incubating the sperm in the absence or presence of these compounds. Cauda epididymal sperm were incubated in HMB medium in the absence or presence of 3 mg/ml BSA, 1 and 3 mm 2-OH-p-β-CD, or 1 and 3 mm M-β-CD. As shown in Table I, a majority of the measurable cholesterol, cholesterol-SO4−, and desmosterol remained associated with the sperm following a 1.5-h incubation in media devoid of BSA. This is in contrast to sperm incubated in media containing 3 mg/ml BSA for the same period of time, where a significant release of these sterols into the media was observed. 2-OH-p-β-CD, which is a very effective cholesterol binding heptasaccharide, mediated steroid release from sperm in a concentration-dependent manner in the absence of BSA (Table I). This β-cyclodextrin was more effective than BSA in mediating steroid release from the sperm, as evidenced by the significantly higher steroid concentrations observed in the media following incubation. M-β-CD, likewise, mediated steroid release from sperm in a concentration-dependent manner, but was more potent than 2-OH-p-β-CD, consistent with the fact that M-β-CD is the most potent β-cyclodextrin with respect to its affinity for cholesterol binding (21Yancey P.G. Rodrigueza W.V. Kilsdonk E.P.C. Stoudt G.W. Johnson W.J. Phillips M.C. Rothblat G.H. J. Biol. Chem. 1996; 271: 16026-16034Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar).Table IEffect of media containing BSA, 2-OH-propyl-β-cyclodextrin, or methyl-β-cyclodextrin on the release of cholesterol, desmosterol and cholesterol-SC4 from mouse spermTreatmentFractionSteroidsCDCSO4Sperm437 ± 4253 ± 515 ± 1Media11 ± 15 ± 1NDBSA (3 mg/ml)Sperm355 ± 3197 ± 79 ± 1Media98 ± 357 ± 56 ± 12-OH-p-β-CDSperm179 ± 394 ± 31 ± 0.2(1 mm)Media267 ± 8157 ± 213 ± 12-OH-p-β-CDSperm120 ± 369 ± 37 ± 1(3 mm)Media309 ± 5173 ± 28 ± 1M-β-CDSperm166 ± 7114 ± 37 ± 0.5(1 mm)Media280 ± 4137 ± 28 ± 1M-β-CDSperm3 ± 0.615 ± 33 ± 0.3(3 mm)Media410 ± 8239 ± 213 ± 1Sperm were incubated in capacitation medium in the absence or the presence of 3 mg/ml BSA, 1 or 3 mm 2-OH-p-β-CD, or 1 or 3 mm M-β-CD. After 1.5 h, the suspension was centrifuged and the different steroids in the sperm pellet and in the media supernatant were measured. Medium alone, either in the presence or absence of BSA, contains no detectable levels of these steroids (not shown). In the presence of 3 mg/ml BSA, 1 or 3 mm2-OH-p-β-CD, and 1 or 3 mm of M-β-CD, there was an increase in cholesterol and desmosterol released from the sperm which was recovered in the media. This release was significant (P < 0.01) when compared to the control (minus BSA or minus β-cyclodextrin), as assessed using the Student ttest (42Zar J.H. Biostatistical Analysis. Third Ed. Prentice Hall, Upper Saddle RiverNJ1996Google Scholar). The following abbreviations were used: C, cholesterol; D, desmosterol; CSO4; cholesterol-SO4; ND, not detectable. Values are expressed as nanograms of steroid/106 sperm,n = 6. Open table in a new tab Sperm were incubated in capacitation medium in the absence or the presence of 3 mg/ml BSA, 1 or 3 mm 2-OH-p-β-CD, or 1 or 3 mm M-β-CD. After 1.5 h, the suspension was centrifuged and the different steroids in the sperm pellet and in the media supernatant were measured. Medium alone, either in the presence or absence of BSA, contains no detectable levels of these steroids (not shown). In the presence of 3 mg/ml BSA, 1 or 3 mm2-OH-p-β-CD, and 1 or 3 mm of M-β-CD, there was an increase in cholesterol and desmosterol released from the sperm which was recovered in the media. This release was significant (P < 0.01) when compared to the control (minus BSA or minus β-cyclodextrin), as assessed using the Student ttest (42Zar J.H. Biostatistical Analysis. Third Ed. Prentice Hall, Upper Saddle RiverNJ1996Google Scholar). The following abbreviations were used: C, cholesterol; D, desmosterol; CSO4; cholesterol-SO4; ND, not detectable. Values are expressed as nanograms of steroid/106 sperm,n = 6. As mentioned above, BSA is proposed to act through its ability to serve as an ac" @default.
• W2085155504 created "2016-06-24" @default.
• W2085155504 creator A5009652519 @default.
• W2085155504 creator A5017497606 @default.
• W2085155504 creator A5021249369 @default.
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• W2085155504 date "1999-01-01" @default.
• W2085155504 modified "2023-10-05" @default.
• W2085155504 title "Cholesterol Efflux-mediated Signal Transduction in Mammalian Sperm" @default.
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