Intracellular Ca2+ threshold reversibly switches flagellar beat off and on.

Sperm motility is essential for fertilization. The asymmetry of flagellar beat in spermatozoa is finely regulated by intracellular calcium concentration ([Ca2+]i). Recently, we demonstrated that the application of high concentrations (10-20 µM) of the Ca2+ ionophore A23187 promotes sperm immobilization after 10 min, and its removal thereafter allows motility recovery, hyperactivation, and fertilization. In addition, the same ionophore treatment overcomes infertility observed in sperm from Catsper1-/-, Slo3-/-, and Adcy10-/-, but not PMCA4-/-, which strongly suggest that regulation of [Ca2+]i is mandatory for sperm motility and hyperactivation. In this study, we found that prior to inducing sperm immobilization, high A23187 concentrations (10 µM) increase flagellar beat. While 5-10 µM A23187 substantially elevates [Ca2+]i and rapidly immobilizes sperm in a few minutes, smaller concentrations (0.5 and 1 µM) provoke smaller [Ca2+]i increases and sperm hyperactivation, confirming that [Ca2+]i increases act as a motility switch. Until now, the [Ca2+]i thresholds that switch motility on and off were not fully understood. To study the relationship between [Ca2+]i and flagellar beating, we developed an automatic tool that allows the simultaneous measurement of these two parameters. Individual spermatozoa were treated with A23187, which is then washed to evaluate [Ca2+]i and flagellar beat recovery using the implemented method. We observe that [Ca2+]i must decrease below a threshold concentration range to facilitate subsequent flagellar beat recovery and sperm motility.

Molecular changes and signaling events occurring in spermatozoa during epididymal maturation.

After leaving the testis, spermatozoa have not yet acquired the ability to move progressively and are unable to fertilize oocytes. To become fertilization competent, they must go through an epididymal maturation process in the male, and capacitation in the female tract. Epididymal maturation can be defined as those changes occurring to spermatozoa in the epididymis that render the spermatozoa the ability to capacitate in the female tract. As part of this process, sperm cells undergo a series of biochemical and physiological changes that require incorporation of new molecules derived from the epididymal epithelium, as well as post-translational modifications of endogenous proteins synthesized during spermiogenesis in the testis. This review will focus on epididymal maturation events, with emphasis in recent advances in the understanding of the molecular basis of this process.

Evidence for the involvement of proline-rich tyrosine kinase 2 in tyrosine phosphorylation downstream of protein kinase A activation during human sperm capacitation.

Sperm capacitation involves an increase in intracellular Ca(2+) concentration as well as in protein kinase A (PKA)-dependent protein tyrosine (Tyr) phosphorylation. Interestingly, in humans, a decrease in extracellular Ca(2+) concentration ([Ca(2+)]e) during capacitation induces an increase in Tyr phosphorylation indicating the complexity of Ca(2+) signaling during this process. In view of this, in the present study we further investigated the Ca(2+)-mediated signaling pathways implicated in Tyr phosphorylation during human sperm capacitation. Results revealed that sperm incubation in a medium without added Ca(2+) (⊖ Ca(2+)) increased Tyr phosphorylation but did not modify PKA-mediated phosphorylation. Moreover, inhibition of either PKA or Src family kinase signaling cascades in ⊖ Ca(2+) down-regulated both PKA substrate and Tyr phosphorylations, indicating that the [Ca(2+)]e effects on Tyr phosphorylation depend on PKA targets. Inhibition of calmodulin or Ser/Thr protein phosphatase 2B also increased Tyr phosphorylation without affecting PKA-mediated phosphorylation, supporting the potential role of these Ca(2+) downstream effectors in the increase in Tyr phosphorylation observed in ⊖ Ca(2+). Experiments aimed to identify the kinase responsible for these observations revealed the presence of proline-rich tyrosine kinase 2 (PYK2), a focal adhesion kinase (FAK) family member, in human sperm, and the use of PF431396, an FAK inhibitor, supported the involvement of PYK2 in Tyr phosphorylation downstream of PKA activation. Results also showed that PYK2 was activated in ⊖ Ca(2+) as well as during capacitation and that PF431396 affected capacitated sperm motility, acrosome reaction and ability to penetrate both mouse cumulus matrix and zona-free hamster eggs. Together, our observations support PYK2 as an intermediary component of Ca(2+) signaling between PKA-mediated and Tyr phosphorylations that is required for achieving functional human sperm capacitation.

Functional human sperm capacitation requires both bicarbonate-dependent PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases.

In all mammalian species studied so far, sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by a bicarbonate-dependent cAMP/protein kinase A (PKA) pathway. Recent studies in mice revealed, however, that a Src family kinase (SFK)-induced inactivation of serine/threonine (Ser/Thr) phosphatases is also involved in the signaling pathways leading to Tyr phosphorylation. In view of these observations and with the aim of getting a better understanding of the signaling pathways involved in human sperm capacitation, in the present work we investigated the involvement of both the cAMP/PKA and SFK/phosphatase pathways in relation to the capacitation state of the cells. For this purpose, different signaling events and sperm functional parameters were analyzed as a function of capacitation time. Results revealed a very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both phospho-PKA substrates and cAMP levels (P < 0.05). However, a complete pattern of Tyr phosphorylation was detected only after 6-h incubation at which time sperm exhibited the ability to undergo the acrosome reaction (AR) and to penetrate zona-free hamster oocytes. Sperm capacitated in the presence of the SFK inhibitor SKI606 showed a decrease in both PKA substrate and Tyr phosphorylation levels, which was overcome by exposure of sperm to the Ser/Thr phosphatase inhibitor okadaic acid (OA). However, OA was unable to induce phosphorylation when sperm were incubated under PKA-inhibitory conditions (i.e. in the absence of bicarbonate or in the presence of PKA inhibitor). Moreover, the increase in PKA activity by exposure to a cAMP analog and a phosphodiesterase inhibitor did not overcome the inhibition produced by SKI606. Whereas the presence of SKI606 during capacitation produced a negative effect (P < 0.05) on sperm motility, progesterone-induced AR and fertilizing ability, none of these inhibitions were observed when sperm were exposed to SKI606 and OA. Interestingly, different concentrations of inhibitors were required to modulate human and mouse capacitation revealing the species specificity of the molecular mechanisms underlying this process. In conclusion, our results describe for the first time the involvement of both PKA activation and Ser/Thr phosphatase down-regulation in functional human sperm capacitation and provide convincing evidence that early PKA-dependent phosphorylation is the convergent regulatory point between these two signaling pathways.

K+ and Cl- channels and transporters in sperm function.

To succeed in fertilization, spermatozoa must decode environmental cues which require a set of ion channels. Recent findings have revealed that K(+) and Cl(-) channels participate in some of the main sperm functions. This work reviews the evidence indicating the involvement of K(+) and Cl(-) channels in motility, maturation, and the acrosome reaction, and the advancement in identifying their molecular identity and modes of regulation. Improving our insight on how these channels operate will strengthen our ability to surmount some infertility problems, improve animal breeding, preserve biodiversity, and develop selective and secure male contraceptives.

Roles of the oviduct in mammalian fertilization.

The oviduct or Fallopian tube is the anatomical region where every new life begins in mammalian species. After a long journey, the spermatozoa meet the oocyte in the specific site of the oviduct named ampulla and fertilization takes place. The successful fertilization depends on several biological processes that occur in the oviduct some hours before this rendezvous and affect both gametes. Estrogen and progesterone, released from the ovary, orchestrate a series of changes by genomic and nongenomic pathways in the oviductal epithelium affecting gene expression, proteome, and secretion of its cells into the fluid bathing the oviductal lumen. In addition, new regulatory molecules are being discovered playing important roles in oviductal physiology and fertilization. The present review tries to describe these processes, building a comprehensive map of the physiology of the oviduct, to better understand the importance of this organ in reproduction. With this purpose, gamete transport, sperm and oocyte changes in the oviductal environment, and other interactions between gametes and oviduct are discussed in light of recent publications in the field.

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.

Evidence for the involvement of proline-directed serine/threonine phosphorylation in sperm capacitation.

To become fertilization competent, mammalian sperm undergo changes in the female reproductive tract termed capacitation. Capacitation correlates with an increase in tyrosine phosphorylation; however, less is known about the role of serine/threonine phosphorylation in this process. Proline-directed phosphorylation is one of the major regulatory phosphorylation events in many cellular processes such as cell proliferation and differentiation. Using mitotic phosphoprotein monoclonal-2 (MPM-2) antibody in this study, we observed that several mouse sperm proteins in the range of 70-250 kDa underwent increased serine/threonine-proline phosphorylation during capacitation. In contrast to the time course of tyrosine phosphorylation, proline-directed phosphorylation could be observed at shorter time points of sperm incubation, and it was found to be independent of NaHCO(3) and adenosine 3'5'-cyclic monophosphate (cAMP). Similar to the regulation of the increase in tyrosine phosphorylation, cholesterol acceptors such as bovine serum albumin (BSA) or 2-hydroxypropyl-beta-cyclodextrin (2-OH-propyl-beta-CD) were essential for the regulation of proline-directed phosphorylation in mouse sperm. Furthermore, it was also found to be BSA dependent in human sperm. Among proline-directed kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) is present in mammalian sperm; nevertheless, U0126 and PD098059, two inhibitors of the ERK pathway, did not block this phosphorylation in mouse sperm. In conclusion, capacitation is associated with an increase in proline-directed phosphorylation linked to cholesterol efflux in the sperm.

alpha-SNAP and NSF are required in a priming step during the human sperm acrosome reaction.

The acrosome is a membrane-limited granule that overlies the nucleus of the mature spermatozoon. In response to physiological or pharmacological stimuli it undergoes a special type of Ca2+-dependent exocytosis termed the acrosome reaction (AR), which is an absolute prerequisite for fertilization. Aided by a streptolysin-O permeabilization protocol developed in our laboratory, we have previously demonstrated requirements for Rab3A, N-ethylmaleimide-sensitive factor (NSF), several soluble NSF-attachment protein receptor (SNARE) proteins, and synaptotagmin VI in the human sperm AR. Here, we show that alpha-soluble NSF-attachment protein (alpha-SNAP), a protein essential for most fusion events through its interaction with NSF and the SNARE complex, exhibits a direct role in the AR. First, the presence of alpha-SNAP is demonstrated by the Western blot of human sperm protein extracts. Immunostaining experiments reveal an acrosomal localization for this protein. Second, the Ca2+ and Rab3A-triggered ARs are inhibited by anti-alpha-SNAP antibodies. Third, bacterially expressed alpha-SNAP abolishes exocytosis in a fashion that depends on its interaction with NSF. Fourth, we show a requirement for alpha-SNAP/NSF in a prefusion step early in the exocytotic pathway, after the tethering of the acrosome to the plasma membrane and before the efflux of intra-acrosomal Ca2+. These results suggest a key role for alpha-SNAP/NSF in the AR, and strengthen our understanding of the molecular players involved in the vesicle-to-plasma membrane fusion taking place during exocytosis.

Novel signaling pathways involved in sperm acquisition of fertilizing capacity.

Capacitation is a complex series of molecular events that occurs in sperm after epididymal maturation and confers on sperm the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of oviductal fluid. In most cases, capacitation media contain energy substrates, such as pyruvate, lactate and glucose, a cholesterol acceptor (usually serum albumin), NaHCO(3), Ca(2+), low K(+), and physiological Na(+) concentrations. The mechanism of action by which these compounds promote capacitation is poorly understood at the molecular level; however, some molecular events significant to the initiation of capacitation have been identified. For example, capacitation correlates with cholesterol efflux from the sperm plasma membrane, increased membrane fluidity, modulations in intracellular ion concentrations, hyperpolarization of the sperm plasma membrane and increased protein tyrosine phosphorylation. These molecular events are required for the subsequent induction of hyperactivation and the acrosome reaction. This review discusses the recent progress that has been made in elucidating mechanisms which regulate sperm capacitation.

Cloning and chromosomal localization of a gene encoding a novel serine/threonine kinase belonging to the subfamily of testis-specific kinases.

Using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to two highly conserved motifs within the protein kinase family of catalytic domains, we isolated a PCR fragment encoding a novel member of the testis-specific serine/threonine kinases (STK) from mouse male mixed germ cell mRNA. This PCR fragment recognized a 1020-bp transcript in male germ cells by northern blot analysis and was used to clone a full-length cDNA from a mouse mixed germ cell cDNA library. This cDNA has an open reading frame of 804 bases encoding a protein of 268 amino acids. This novel gene is almost identical to Stk22c, encoding a recently described testis-specific protein kinase, except for base-pair deletions that result in a shift in the coding region and an alteration of 22 amino acids (residues 109-131). Due to its homology with Stk22c, we have called this protein kinase gene Stk22d. Northern blot analysis revealed that this protein kinase is developmentally expressed in testicular germ cells and is not present in brain, ovary, kidney, liver, or early embryonic cells. We then cloned the human homologue of this protein kinase gene (STK22C) and found it to be expressed exclusively in the testis. Fluorescence in situ hybridization with both the human and mouse cDNA clones revealed syntenic localization on chromosomes 1p34-p35 and 4E1, respectively.

Dual regulation of the T-type Ca(2+) current by serum albumin and beta-estradiol in mammalian spermatogenic cells.

This study provides evidence for a novel mechanism of voltage-gated Ca(2+) channel regulation in mammalian spermatogenic cells by two agents that affect sperm capacitation and the acrosome reaction (AR). Patch-clamp experiments demonstrated that serum albumin induced an increase in Ca(2+) T current density in a concentration-dependent manner, and significant shifts in the voltage dependence of both steady-state activation and inactivation of the channels. These actions were not related to the ability of albumin to remove cholesterol from the membrane. In contrast, beta-estradiol significantly inhibited Ca(2+) channel activity in a concentration-dependent and essentially voltage-independent fashion. In mature sperm this dual regulation may influence capacitation and/or the AR.

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.

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 NaHCO(3) 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-SO(4) 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-beta-cyclodextrin or OH-propyl-beta-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.

Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation.

We previously demonstrated that mouse sperm capacitation is accompanied by a time-dependent increase in protein tyrosine phosphorylation that is dependent on the presence of BSA, Ca2+, and NaHCO(3), all three of which are also required for this maturational event. We also demonstrated that activation of protein kinase A (PK-A) is upstream of this capacitation-associated increase in protein tyrosine phosphorylation. BSA is hypothesized to modulate capacitation through the removal of cholesterol from the sperm plasma membrane. In this report, we demonstrate that incubation of mouse sperm medium containing BSA results in a release of cholesterol from the sperm plasma membrane to the medium; release of this sterol does not occur in medium devoid of BSA. We next determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Blocking the action of BSA by adding exogenous cholesterol-SO-(4) to the BSA-containing medium inhibits the increase in protein tyrosine phosphorylation as well as capacitation. This inhibitory effect is overcome by (1) the addition of increasing concentrations of BSA at a given concentration of cholesterol-SO-(4) and (2) the addition of dibutyryl cAMP plus IBMX. High-density lipoprotein (HDL), another cholesterol binding protein, also supports the capacitation-associated increase in protein tyrosine phosphorylation through a cAMP-dependent pathway, whereas proteins that do not interact with cholesterol have no effect. HDL also supports sperm capacitation, as assessed by fertilization in vitro. Finally, we previously demonstrated that HCO-(3) is necessary for the capacitation-associated increase in protein tyrosine phosphorylation and demonstrate here, by examining the effectiveness of HCO-(3) or BSA addition to sperm on protein tyrosine phosphorylation, that the HCO-(3) effect is downstream of the site of BSA action. Taken together, these data demonstrate that cholesterol release is associated with the activation of a transmembrane signal transduction pathway involving PK-A and protein tyrosine phosphorylation, leading to functional maturation of the sperm.

Roles of bicarbonate, cAMP, and protein tyrosine phosphorylation on capacitation and the spontaneous acrosome reaction of hamster sperm.

Capacitation is a prerequisite for successful fertilization by mammalian spermatozoa. This process is generally observed in vitro in defined NaHCO3-buffered media and has been shown to be associated with changes in cAMP metabolism and protein tyrosine phosphorylation. In this study, we observed that when NaHCO3 was replaced by 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid (HEPES), hamster sperm capacitation, measured as the ability of the sperm to undergo a spontaneous acrosome reaction, did not take place. Addition of 25 mM NaHCO3 to NaHCO3-free medium in which spermatozoa had been preincubated for 3.5 h, increased the percentage of spontaneous acrosome reactions from 0% to 80% in the following 4 h. Addition of anion transport blockers such as 4,4'-diiso thiocyano-2, 2'-stilbenedisulfonate (DIDS) or 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) to the NaHCO3-containing medium inhibited the acrosome reaction, with maximal inhibition at 600 microM, and with an EC50 of 100 microM. Increasing either extracellular or intracellular pH did not induce the acrosome reaction in NaHCO3-free medium. In contrast, addition of 500 microM dibutyryl cAMP (dbcAMP), alone or together with 100 microM 1-methyl-3-isobutylxanthine (IBMX), induced the acrosome reaction in spermatozoa incubated in NaHCO3-free medium. These compounds also partially reversed the inhibition of the acrosome reaction caused by the DIDS or SITS in complete medium. In contrast to these results, IBMX or dbcAMP did not induce acrosome reactions in cells incubated in Ca2+-free medium. When hamster sperm were incubated in the absence of NaHCO3 or in the presence of NaHCO3 and DIDS, cAMP concentrations were significantly lower than the values obtained from sperm incubated in complete medium. Protein tyrosine phosphorylation has also been shown to be highly correlated with the onset of capacitation in many species. During the first hour of capacitation, an increase in protein tyrosine phosphorylation was observed in complete medium. In the absence of NaHCO3, the increase in protein tyrosine phosphorylation was delayed for 45 min, and this delay was overcome by the addition of dbcAMP and IBMX. The induction of the acrosome reaction by calcium ionophore A23187 in NaHCO3-free medium was delayed 2 h, as compared with control medium. This delay was not observed in the presence of dbcAMP and IBMX. Taken together, these results suggest that a cAMP pathway may mediate the role of NaHCO3 in the capacitation of hamster spermatozoa and that protein tyrosine phosphorylation is necessary but not sufficient for complete capacitation.

Control of the low voltage-activated calcium channel of mouse sperm by egg ZP3 and by membrane hyperpolarization during capacitation.

Sperm adhesion to egg zonae pellucidae initiates sperm acrosome reactions, an exocytotic event that is an early step during fertilization. Previously, it was suggested that zona pellucida-evoked Ca2+ entry into sperm through low voltage-activated Ca2+ channels is an essential step in acrosome reactions, based on the inhibitory effects of Ca2+ channel antagonists. However, analysis of this channel is limited by the inability to apply electrophysiological methods directly to sperm. In this report, optical methods of determining membrane potential and internal Ca2+ levels were used to demonstrate that (i) contact with zonae pellucidae activates a transient Ca2+ response in sperm that has a time course and antagonist sensitivity anticipated of low voltage-activated Ca2+ channels; (ii) these channels are unavailable for opening in uncapacitated sperm because of voltage-dependent, steady state inactivation; (iii) membrane hyperpolarization during sperm capacitation is sufficient to recruit channels into a closed state, from which they are available for opening during fertilization; and (iv) channel conductance state may be a factor in determines the efficacy with which channel antagonists inhibit fertilization. This study provides evidence for the activation of sperm Ca2+ channels during gamete adhesion and offers a mechanism that may account for aspects of the regulation of sperm fertility during capacitation through the control of channel availability. Finally, these results suggest that channel conductance state may be a central feature in the design of channel antagonists that inhibit sperm function.

Cholesterol efflux-mediated signal transduction in mammalian sperm. beta-cyclodextrins initiate transmembrane signaling leading to an increase in protein tyrosine phosphorylation and capacitation.

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-beta-cyclodextrin and OH-propyl-beta-cyclodextrin, promote the release of cholesterol from the mouse sperm plasma membrane in media devoid of BSA. Both of these beta-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 beta-cyclodextrins to increase protein tyrosine phosphorylation in sperm was correlated with their cholesterol binding efficiencies, and preincubation of the beta-cyclodextrins with cholesterol-SO4- to saturate their cholesterol-binding sites blocked the ability of these compounds to stimulate protein tyrosine phosphorylation. The beta-cyclodextrin effect on protein tyrosine phosphorylation was both NaHCO3 and protein kinase A-dependent. The beta-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, beta-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.