Cholesterol content regulates acrosomal exocytosis by enhancing Rab3A plasma membrane association

The acrosome is an exocytic granule that overlies the spermatozoan nucleus. In response to different stimuli, it undergoes calcium-regulated exocytosis. Freshly ejaculated mammalian sperm are not immediately capable of undergoing acrosome reaction. The acquisition of this ability is called capacitation and involves a series of still not well-characterized changes in the sperm physiology. Plasma membrane cholesterol removal is one of the sperm modifications that are associated with capacitation. However, how sterols affect acrosomal exocytosis is unknown. Here, we show that short incubations with cyclodextrin, a cholesterol removal agent, just before stimulation promote acrosomal exocytosis. Moreover, the effect was also observed in permeabilized cells stimulated with calcium, indicating that cholesterol plays a direct role in the calcium-dependent exocytosis associated with acrosome reaction. Using a photo-inhibitable calcium chelator, we show that cholesterol affects an early event of the exocytic cascade rather than the lipid bilayers mixing. Functional data indicate that one target for the cholesterol effect is Rab3A. The sterol content does not affect the Rab3A activation-deactivation cycle but regulates its membrane anchoring. Western blot analysis and immunoelectron microscopy confirmed that cholesterol efflux facilitates Rab3A association to sperm plasma membrane. Our data indicate that the cholesterol efflux occurring during capacitation optimizes the conditions for the productive assembly of the fusion machinery required for acrosome reaction.     

Differential release of soluble and matrix components: evidence for intermediate states of secretion during spontaneous acrosomal exocytosis in mouse sperm

Although its exact role in fertilization is unknown, the acrosome is a very important, exocytotic organelle overlying the anterior aspect of sperm from many species. Structurally and functionally, the acrosome can be considered to consist of soluble and particulate compartments. One component of the particulate acrosomal matrix is the zona pellucida-binding protein sp56. Our demonstration that this protein is within the acrosomal matrix and not on the sperm plasma membrane has led us to reexamine the events of acrosomal exocytosis and the role of the sperm acrosomal matrix in the fertilization process. To visualize the soluble compartment, we have utilized sperm from transgenic mice that carry soluble green fluorescent protein (GFP) in their acrosomes and, as a means to assess the exposure of acrosomal matrix components, we have tested the ability of these sperm to bind beads coated with antibodies to sp56. The loss of GFP from the acrosomes and the binding of the beads by the sperm undergoing capacitation serve as indicators of distinct stages of acrosomal exocytosis, allowing us to define intermediates of acrosomal exocytosis that occur during the course of sperm capacitation. These experiments demonstrate that the exposure and release of acrosomal proteins during spontaneous acrosomal exocytosis is not synchronous but is regulated during capacitation. Furthermore, acrosomal exocytosis under these conditions required calcium in the medium. On the basis of these findings, we propose an alternative model for acrosomal exocytosis that considers a role for these intermediates of exocytosis during capacitation and sperm-ZP interactions.     

A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis

Efficient in vitro capacitation of stallion sperm has not yet been achieved, as suggested by low sperm penetration rates reported in in vitro fertilization (IVF) studies. Our objectives were to evaluate defined incubation conditions that would support changes consistent with capacitation in stallion sperm. Protein tyrosine phosphorylation events and the ability of sperm to undergo acrosomal exocytosis under various incubation conditions were used as end points for capacitation. Sperm incubated 4-6h in modified Whitten's (MW) with the addition of 25 mM NaHCO3 and 7 mg/mL BSA (capacitating medium) yielded high rates of protein tyrosine phosphorylation. Either HCO3(-) or BSA was required to support these changes, with the combination of both providing the most intense results. When a membrane-permeable form of cAMP and a phosphodiesterase inhibitor (IBMX) were added to MW in the absence of HCO3(-) and BSA, the tyrosine phosphorylation results obtained in our capacitating conditions could not be replicated, suggesting either effects apart from cAMP were responsible for tyrosine phosphorylation, or that stallion sperm might respond differently to these reagents as compared to sperm from other mammals. Sperm incubation in capacitating conditions was also associated with high percentages (P相似文献   

Decrease in order of human sperm lipids during capacitation

Ejaculated mammalian sperm must undergo a final maturation (capacitation) before they can acrosome-react and fertilize eggs. Loss of the sperm sterols, cholesterol and desmosterol, is an obligatory step in the capacitation of human sperm. Because sterols can increase the order of membrane phospholipids, it has been suggested that the importance of sterol loss is that it decreases membrane lipid order. The present study tested the hypotheses that sterol loss decreases sperm membrane lipid order during capacitation and that lipid disorder is a sufficient stimulus for capacitation. Steady-state fluorescence anisotropy of the membrane probe, 1,6-diphenyl-1,3,5-hexatriene, decreased during capacitation, indicating a decrease in lipid order. The decrease was dependent on the loss of sperm sterols, suggesting that it reflected diminished sterol-mediated phospholipid ordering. However, the lipid-fluidizing agents, benzyl alcohol and 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl) octanoate, did not cause sperm capacitation or overcome inhibition by cholesterol. In summary, loss of sperm sterols caused a significant decline in lipid order during capacitation; however, decreased bulk lipid order was not sufficient to trigger the subsequent events that complete capacitation.     

Effect of cholesterol and other sterols on human sperm acrosomal responsiveness

Human sperm become responsive to inducers of the acrosome reaction when they are washed free of seminal plasma and incubated in an appropriate medium. Previous work has shown that cholesterol-enriched medium prevents sperm from becoming responsive to the inducer, progesterone. Sperm that were incubated 24 hr in cholesterol-enriched medium and then treated with progesterone showed no evidence of membrane fusion, indicating that cholesterol acts at a stage before the earliest morphological change. Inhibition of acrosomal responsiveness by cholesterol was reversible. Among other sterols reported in mammalian sperm, desmosterol and cholesterol sulfate also inhibited sperm from becoming responsive, but cholesterol palmitate did not. Our results support a model in which sperm unesterified cholesterol, or a molecule in equilibrium with it, suppresses acrosomal responsiveness. Cholesterol-enriched medium also prevented sperm from becoming responsive to the calcium/proton exchanging ionophore, ionomycin, suggesting that cholesterol's effect may be, at least in part, at a point in the signal transduction pathway subsequent to the rise in intracellular-free calcium. © 1996 Wiley-Liss, Inc.     

Hypercholesterolemia impaired sperm functionality in rabbits

Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a "folded head"-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events.     

Structural features of sterols required to inhibit human sperm capacitation

Ejaculated mammalian sperm must undergo a final maturation (capacitation) before they can acrosome-react and fertilize eggs. Loss of cholesterol is an essential step in the capacitation of human sperm. Experimentally maintaining a high level of cholesterol inhibits capacitation, but the mechanism is unknown. The present study investigated the structural features that are required for cholesterol's inhibitory activity. Human sperm also contain much desmosterol, which is lost from sperm during capacitation. Preventing the loss of desmosterol inhibited capacitation (as assessed by acrosomal responsiveness), with an effectiveness approximately equal to cholesterol's inhibitory activity. Other structural analogs were added to the incubation medium to replace sperm cholesterol and desmosterol. Most inhibited capacitation, including those that lacked cholesterol's 3beta-OH group (cholesteryl methyl ether and epicholesterol) and those with modified C17 groups (ergosterol and diosgenin). Two steroids did not inhibit capacitation well. Coprostanol, which has a nonplanar steroid nucleus, had low inhibitory activity that could be explained by an elevated endogenous cholesterol concentration. Epicoprostanol, which has a nonplanar ring structure and a 3alpha-OH group, promoted rather than inhibited capacitation. The inhibitory activity of the analogs was correlated with their ability to promote order of egg phosphatidylcholine as measured by fluorescence anisotropy. In summary, a planar ring structure is required for sterol inhibitory activity, but a 3beta-OH group and a saturated cholesterol-like aliphatic tail on C17 are not required. The present results support the hypothesis that sperm sterols block capacitation by increasing order of phospholipids.     

Effect of methyl-beta-cyclodextrin on the acrosomal responsiveness of human sperm

Human sperm incubated in vitro gradually become capable of acrosome-reacting in response to the agonist, progesterone (P4). Loss of unesterified cholesterol is an obligatory step in the development of acrosomal responsiveness. These experiments tested the ability of methyl-beta-cyclodextrin (MbetaCD) to accelerate sperm cholesterol loss and the development of acrosomal responsiveness. Incubating sperm 30 min in MbetaCD (2.5-10 mM) decreased sperm cholesterol by as much as 89% in a dose-dependent fashion. MbetaCD caused some sperm (maximum of 16% following treatment with 5 mM MbetaCD) to become responsive to P4, and it caused a dose-dependent increase in spontaneous acrosome reactions. The number of responsive sperm increased in the first 3 hr following their removal from MbetaCD. Continuing incubation to 24 hr increased the numbers of spontaneously reacted sperm and dead sperm, but not P4-responsive sperm. It appears, therefore, that up to 3 hr are required for the full expression of P4-responsiveness in cholesterol-depleted sperm. The observed effects of MbetaCD are due to its cholesterol-depleting properties, because including sufficient cholesterol with MbetaCD to reduce the loss of sperm cholesterol inhibited the effects of MbetaCD on cell viability, spontaneous acrosome reactions, and responsiveness to P4. MbetaCD accelerates the appearance of the functional stages that sperm normally pass through during incubation in vitro, reinforcing the view that cholesterol loss is an important determinant of the rate at which sperm become acrosomally responsive.     

Cholesterol efflux promotes acrosome reaction in goat spermatozoa

Cholesterol efflux and membrane destabilization play an important role in sperm capacitation and membrane fusion in the acrosome reaction (AR). In this study we establish the effect of cholesterol removal from spermatozoa on acrosomal responsiveness. Mature goat spermatozoa were incubated in BSA-free medium in the presence of beta-cyclodextrin (betaCD) as cholesterol acceptor. After incubation with 8 mM betaCD, 50-60% of cholesterol was released from sperm membranes with no loss in the phospholipid content, and 35% of AR was induced. However, when 30% of cholesterol was lost, this moderate cholesterol decrease was unable to initiate AR. Cholesterol desorption was very rapid, following an exponential kinetics with a half-time of around 10 min, which is in contrast with the slow sigmoidal kinetics of acrosomal responsiveness: around 2 h was required for maximal AR. Our results suggest that cholesterol efflux has a direct influence on the onset of the AR, that is, merely removing cholesterol would trigger the AR.     

Sperm of the Shrimp Sicyonia ingentis Undergo a Bi-Phasic Capacitation Accompanied by Morphological Changes

Sperm removed from seminal receptacles of female Sicyonia ingentis can be induced to undergo a bi-phasic acrosome reaction (AR), acrosomal exocytosis followed by filament formation, using egg water (EW). Sperm removed from males will not undergo any phase of the AR when incubated with EW, indicating that these sperm undergo a capacitation process after insemination. Freshly molted females (functional virgins) were placed in aquaria with males and monitored for copulation. Mated females were isolated and allowed to carry sperm for specific periods of time. At these time points, sperm were removed and assayed for the ability to undergo the AR using EW. The results indicate that sperm are competent to undergo acrosomal exocytosis after approximately 25 hr, while competency to form acrosomal filaments is not achieved until around 145 hr post-insemination. Morphological examination of sperm removed from males and sperm removed from females revealed dramatic differences. Microscopic evidence indicates that some of the morphological changes seen during capacitation are necessary for the successful completion of the AR.     

Stimulation of voltage-dependent calcium channels during capacitation and by progesterone in human sperm

To fertilize, mammalian sperm must undergo two sequential steps that require activation of calcium entry mechanisms, capacitation and acrosomal exocytosis, induced in the latter case by the egg zona pellucida glycoprotein ZP3 or by progesterone. Voltage-dependent calcium channels (VDCC) could participate in these processes. Since patch clamp recordings are extremely difficult in mature sperm, the activity of VDCC has been alternatively analyzed with optical detectors of membrane potential and intracellular calcium in sperm populations. Using this approach, we previously reported that in human sperm there is a voltage-dependent calcium influx system that strongly indicates that human sperm are endowed with functional VDCC. In this study we developed evidence indicating that calcium influx through VDCC is significantly stimulated during sperm in vitro capacitation and by progesterone action, which is present in the follicular fluid that surrounds the egg. The observed effects of capacitation and progesterone on VDCC may be physiologically significant for sperm-egg interaction.     

Signal transduction pathways that regulate sperm capacitation and the acrosome reaction

Ejaculated spermatozoa must undergo physiological priming as they traverse the female reproductive tract before they can bind to the egg’s extracellular coat, the zona pellucida (ZP), undergo the acrosome reaction, and fertilize the egg. The preparatory changes are the net result of a series of biochemical and functional modifications collectively referred to as capacitation. Accumulated evidence suggests that the event that initiates capacitation is the efflux of cholesterol from the sperm plasma membrane (PM). The efflux increases permeability and fluidity of the sperm PM and causes influx of Ca²⁺ ions that starts a signaling cascade and result in sperm capacitation. The binding of capacitated spermatozoa to ZP further elevates intrasperm Ca²⁺ and starts a new signaling cascade which open up Ca²⁺ channels in the sperm PM and outer acrosomal membrane (OAM) and cause the sperm to undergo acrosomal exocytosis. The hydrolytic action of the acrosomal enzymes released at the site of sperm-egg (zona) binding, along with the hyperactivated beat pattern of the bound spermatozoon, are important factors in directing the sperm to penetrate the ZP and fertilize the egg. The role of Ca²⁺-signaling in sperm capacitation and induction of the acrosome reaction (acrosomal exocytosis) has been of wide interest. However, the precise mechanism(s) of its action remains elusive. In this article, we intend to highlight data from this and other laboratories on Ca²⁺ signaling cascades that regulate sperm functions.     

Reorganization of lipid rafts during capacitation of human sperm

Ejaculated mammalian sperm must complete a final maturation, termed capacitation, before they can undergo acrosomal exocytosis and fertilize an egg. In human sperm, loss of sperm sterol is an obligatory, early event in capacitation. How sterol loss leads to acrosomal responsiveness is unknown. These experiments tested the hypothesis that loss of sperm sterol affects the organization of cold detergent-resistant membrane microdomains (lipid "rafts"). The GPI-linked protein CD59, the ganglioside GM1, and the protein flotillin-2 were used as markers for lipid rafts. In uncapacitated sperm, 51% of the CD59, 41% of the GM1, and 90% of the flotillin-2 were found in the raft fraction. During capacitation, sperm lost 67% of their 3beta-hydroxysterols, and the percentages of CD59 and GM1 in the raft fraction decreased to 34% and 31%, respectively. The distribution of flotillin-2 did not change. Preventing a net loss of sperm sterol prevented the loss of CD59 and GM1 from the raft fraction. Fluorescence microscopy showed CD59 and GM1 to be distributed over the entire sperm surface. Flotillin-2 was located mainly in the posterior head and midpiece. Patching using bivalent antibodies indicated that little of the GM1 and CD59 was stably associated in the same membrane rafts. Likewise, GM1 and flotillin-2 were not associated in the same membrane rafts. In summary, lipid rafts of heterogeneous composition were identified in human sperm and the two raft components, GM1 and CD59, showed a partial sterol loss-dependent shift to the nonraft domain during capacitation.     

Transitional states of acrosomal exocytosis and proteolytic processing of the acrosomal matrix in guinea pig sperm

In this study, we adapted a FluoSphere bead-binding assay to study the exposure and release of guinea pig sperm acrosomal components during the course of capacitation and acrosomal exocytosis. Prior to capacitation or the initiation of exocytosis, acrosomal proteins were not accessible to FluoSpheres coated with antibodies against two acrosomal matrix (AM) proteins, AM67 and AM50; during the course of capacitation and ionophore-induced acrosomal exocytosis, however, we detected the transient exposure of the solid-phase AM proteins on the surface of guinea pig sperm using the antibody-coated fluorescent beads. Several different transitional stages leading to complete acrosomal exocytosis were classified, and we propose these represent true, functional intermediates since some of the AM proteins are orthologues of mouse proteins that bind the zona pellucida (ZP) of unfertilized eggs. In addition, we present evidence that implicates acrosin in the proteolytic processing of AM50 during AM disassembly. Thus, we propose that the transitional states of acrosomal exocytosis involve early binding of AM proteins to the ZP (by what visually appear to be "acrosome-intact" sperm), maintenance of ZP binding that coincides with the progressive exposure of AM proteins, and gradual proteolytic disassembly of the AM to allow sperm movement through the ZP. We feel this "transitional states" model provides a more refined view of acrosomal function that supports a move away from the widely held, overly simplistic, and binary "acrosome-reaction" model, and embraces a more dynamic view of acrosomal exocytosis that involves intermediate stages of the secretory process in ZP binding and penetration.     

Influence of a capacitation period on human sperm acrosome loss.

The present study investigates whether a 5 hour capacitation period modifies the ability of human spermatozoa to undergo induced acrosomal loss. Human sperm acrosomal loss was induced by treatment with either the calcium ionophore A23187, low concentrations of the phospholipid dilauroylphosphatidylcholine (PC12), or 2 hours incubation in conditioned medium prepared from human cumulus cells (CM/CC). The use of a dual staining method (FITC-ConA and Hoechst 33258) for simultaneous assessment of acrosomal status and viability demonstrated that induction of acrosomal loss with calcium ionophore was not dependent on a capacitation period. A short (5 hour) incubation period was not sufficient to induce acrosomal loss with CM/CC above spontaneous acrosome reaction rates in medium alone. A significant capacitation-dependent increase (P < 0.05) in acrosomal loss was observed when human spermatozoa were incubated with PC12. Induction of acrosomal loss of capacitated human spermatozoa with PC12 therefore provides a simple assay for the simultaneous assessment of human sperm capacitation and the acrosome reaction in vitro.     

Guanine-nucleotide exchange factors (RAPGEF3/RAPGEF4) induce sperm membrane depolarization and acrosomal exocytosis in capacitated stallion sperm

Capacitation encompasses the molecular changes sperm undergo to fertilize an oocyte, some of which are postulated to occur via a cAMP-PRKACA (protein kinase A)-mediated pathway. Due to the recent discovery of cAMP-activated guanine nucleotide exchange factors RAPGEF3 and RAPGEF4, we sought to investigate the separate roles of PRKACA and RAPGEF3/RAPGEF4 in modulating capacitation and acrosomal exocytosis. Indirect immunofluorescence localized RAPGEF3 to the acrosome and subacrosomal ring and RAPGEF4 to the midpiece in equine sperm. Addition of the RAPGEF3/RAPGEF4-specific cAMP analogue 8-(p-chlorophenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8pCPT) to sperm incubated under both noncapacitating and capacitating conditions had no effect on protein tyrosine phosphorylation, thus supporting a PRKACA-mediated event. Conversely, activation of RAPGEF3/RAPGEF4 with 8pCPT induced acrosomal exocytosis in capacitated equine sperm at rates (34%) similar (P > 0.05) to those obtained in progesterone- and calcium ionophore-treated sperm. In the mouse, capacitation-dependent hyperpolarization of the sperm plasma membrane has been shown to recruit low voltage-activated T-type Ca(2+) channels, which later open in response to zona pellucida-induced membrane depolarization. We hypothesized that RAPGEF3 may be inducing acrosomal exocytosis via depolarization-dependent Ca(2+) influx, as RAPGEF3/RAPGEF4 have been demonstrated to play a role in the regulation of ion channels in somatic cells. We first compared the membrane potential (E(m)) of noncapacitated (-37.11 mV) and capacitated (-53.74 mV; P = 0.002) equine sperm. Interestingly, when sperm were incubated (6 h) under capacitating conditions in the presence of 8pCPT, E(m) remained depolarized (-32.06 mV). Altogether, these experiments support the hypothesis that RAPGEF3/RAPGEF4 activation regulates acrosomal exocytosis via its modulation of E(m), a novel role for RAPGEF3/RAPGEF4 in the series of events required to achieve fertilization.     

A possible mechanism of action for fertilization promoting peptide,a TRH-related tripeptide that promotes capacitation and fertilizing ability in mammalian spermatozoa

Fertilization promoting peptide (FPP), a tripeptide structurally related to thyrotrophin releasing hormone (TRH), has been shown to stimulate capacitation and fertilizing ability in both mouse and human spermatozoa, but the mechanisms of action involved in these responses are currently unknown. In the present study utilizing epididymal mouse spermatozoa, we have compared the ability of FPP, TRH, and pyroglutamylphenylalanineprolineamide (an uncharged structurally related tripeptide found in seminal plasma) to stimulate capacitation. At 50 nM, the mean concentration of FPP found in human seminal plasma, only FPP produced a significant response. This suggests that if a receptor is involved, it is one distinct from the TRH receptor. A significant response to FPP required the presence of extracellular Ca²⁺, with 90 μm Ca²⁺ being sufficient to support a stimulation of capacitation. The addition of FPP to suspensions at later stages of capacitation indicated that the nature of the response changed, such that addition of FPP to capacitated suspensions inhibited spontaneous acrosome reactions; however, FPP-treated, cells were still able to undergo acrosomal exocytosis in response to progesterone, a physiological agonist of acrosomal exocytosis. Because earlier studies had identified a similar capacitation-related change in response to adenosine, being stimulatory early in capacitation and inhibitory later in capacitation, we investigated the possibility that FPP and adenosine might be acting via the same pathway. The combination of FPP plus adenosine, whether used at low, non-stimulatory concentrations or high, maximally-stimulatory concentrations, was more effective in promoting capacitation than either compound used individually. As observed with FPP, addition of adenosine to capacitated cells inhibited spontaneous acrosome loss but did not inhibit exocytosis in response to progesterone. This suggests that the two molecules are affecting a common pathway. Since adenosine, acting via specific cell surface receptors, can stimulate fertilizing ability and adenylate cyclase activity in uncapacitated cells and then inhibit enzyme activity in capacitated cells, we propose that FPP may act by modulating the adenylate cyclase/cyclic AMP signal transduction pathway. In vivo, FPP, which would contact spermatozoa at ejaculation and probably remain bound to cells for some time, could stimulate capacitation as the spermatozoa ascend the female tract; adenosine, present in seminal plasma and the female tract, could either augment FPP's action or replace it if FPP is lost from the cell surface. We therefore suggest that FPP and adenosine, by modulating adenylate cyclase activity to promote capacitation but inhibit spontaneous acrosomal exocytosis, may provide an endogenous mechanism that helps to optimize the fertilizing potential of the few sperm cells that reach the site of fertilization in vivo. © 1996 Wiley-Liss, Inc.     

Kinetics of spontaneous and induced acrosomal loss in human sperm incubated under capacitating and noncapacitating conditions

The kinetics of spontaneous and induced acrosomal loss have been studied in human sperm incubated in capacitating and noncapacitating media. Acrosomal status was quantitated using indirect immunofluorescence with a monoclonal antibody. The response of sperm to induction by calcium ionophores was time dependent reaching a maximum after 6 hours of incubation under capacitating conditions. The inducible population slowly decreased in size through the balance of a 24-hour incubation. The time-dependent development of ionophore responsiveness by sperm exposed to capacitating conditions corroborates the idea that only capacitated cells can respond to undergo acrosomal loss in response to ionophore. In contrast, only a small, constant percentage of sperm incubated under noncapacitating conditions responded to ionophore. Substitution experiments involving the addition or deletion of human serum albumin suggest that albumin is not absolutely required for capacitation but is essential for the maintenance of motility. Polyvinyl alcohol can be substituted for serum albumin, but it does not support capacitation or motility as well as HSA. These studies may provide a basis for optimizing capacitating conditions for human sperm in vitro as well as for diagnosing fertility or fertility potential based on measurements of spontaneous and ionophore induced acrosomal loss under defined culture conditions.     

Reactive oxygen species promote tyrosine phosphorylation and capacitation in equine spermatozoa

The objective of this study was to examine the influence of reactive oxygen species (ROS) on equine sperm capacitation. Motile equine spermatozoa were separated on a discontinuous Percoll gradient, resuspended at 10 x 10(6)ml in Tyrode's medium supplemented with BSA (0.5%) and polyvinyl alcohol (0.5%) and incubated at 39 degrees C for 2h with or without the xanthine (X; 0.1mM)-xanthine oxidase (XO; 0.01 U/ml) system or NADPH (0.25 mM). The importance of hydrogen peroxide or superoxide for capacitation was determined by the addition of catalase (CAT; 150 U/ml) or superoxide dismutase (SOD; 150 U/ml), respectively. Following incubation, acrosomal exocytosis was induced by a 5 min incubation at 39 degrees C with progesterone (3.18 microM), and sperm viability and acrosomal integrity were then determined by staining with Hoechst 33258 and fluoroisothiocyanate-conjugated Pisum sativum agglutin. To examine tyrosine phosphorylation, treatments were subjected to sodium dodecyl sulfate-polyacrylaminde gel electrophoresis (SDS-PAGE) followed by Western blot analysis with the anti-phosphotyrosine antibody (alpha-PY; clone 4G10). Capacitation with the X-XO system or NADPH led to a significant (P<0.0001) increase in live acrosome-reacted spermatozoa compared to controls. The addition of CAT or SOD prevented the increase in live acrosome-reacted spermatozoa associated with X-XO treatment. Incubation with the X-XO system was also associated with a significant (P<0.005) increase in tyrosine phosphorylation when compared to controls, which could be prevented by the addition of CAT but not SOD. This study indicates that ROS can promote equine sperm capacitation and tyrosine phosphorylation, suggesting a physiological role for ROS generation by equine spermatozoa.