Regulation of Protein Phosphorylation during Sperm Capacitation¹

^a Center for Research on Reproduction & Women's Health, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6080

	INTRODUCTION

TOP INTRODUCTION MOLECULAR BASIS OF CAPACITATION SUMMARY REFERENCES

 
After leaving the testis, mammalian spermatozoa from many species are morphologically differentiated but have acquired neither progressive motility nor the ability to fertilize a metaphase II-arrested egg. During epididymal transit, sperm acquire the ability to move progressively; however, they are still fertilization incompetent. Fertilization capacity is gained after residence in the female tract for a finite period of time. The physiological changes that confer on the sperm the ability to fertilize are collectively called "capacitation." Capacitation was first described and defined independently by Chang [1, 2] and Austin [3, 4]. The definition of this poorly understood phenomenon has been modified and narrowed over the years. Although fertilization still represents the benchmark endpoint of a capacitated sperm, the ability of the sperm to undergo a regulated acrosome reaction (e.g., in response to the zona pellucida) can be taken as an earlier, upstream endpoint of this extratesticular maturational event. It must be stressed at this point that capacitation is also correlated with changes in sperm motility patterns, designated as sperm hyperactivation, in a number of species [5, 6]. There are examples of cases in which capacitation and hyperactivation can be dissociated experimentally [7], but one cannot yet argue that hyperactivation of motility represents an event completely independent of the capacitation process [6]. Therefore, when one attempts to understand the process of capacitation at the molecular level, it is necessary to consider events occurring both in the head (i.e., acrosome reaction) and in the tail (i.e., motility changes).

The physiological site of capacitation in vivo is the oviduct or the uterus, depending on the species [5]. However, capacitation in vitro has been accomplished using cauda and/or ejaculated sperm incubated under a variety of conditions in defined media that mimic the electrolyte composition of the oviduct fluid. In most cases, these media contain energy substrates such as pyruvate, lactate, and glucose (depending on the species); a protein source that usually is serum albumin; NaHCO₃; and Ca²⁺. The action of these media components to promote capacitation at the molecular level is poorly understood and will be discussed in this review. This review is not intended to provide an exhaustive analysis of capacitation but to offer an update and to discuss future avenues of research directed toward an understanding of the molecular basis of this important biological event. The reader is referred to the excellent reviews of capacitation by Yanagimachi [5], Florman and Babcock [8], Eisenbach [9], and de Lamirande et al. [10], as well as the companion minireviews in this issue [11,12].

	MOLECULAR BASIS OF CAPACITATION

TOP INTRODUCTION MOLECULAR BASIS OF CAPACITATION SUMMARY REFERENCES

 
Although the biological phenomenon of sperm capacitation has been known for close to half a century, the molecular basis of this process is still poorly understood. However, recent work by several laboratories is beginning to lead to a unified hypothesis of how this event is controlled, and this is delineated in Figure 1. The bold labels on the figure (A1-B7) identify pathways that correspond to similarly labeled major points of discussion below.

View larger version (30K): [in this window] [in a new window]   FIG. 1. Schematic representation of the transmembrane and intracellular signaling pathways hypothesized to play a role in regulating sperm capacitation in mammals. The various pathways depicted in this figure are referred to in the text by the appropriate letter/number headings (e.g., A1, A2). Pathways A1, A2, A3, B1, and B2 are adapted from Visconti et al. [42]; pathways B3, B6, and B7 are adapted from Parrish et al. [78]; pathway B4 is based on the work of Zeng et al. [64]; pathway B5a is based on the works of de Lamirande and Gagnon [72]; and pathway B5b is based on the works of Leclerc et al. [73] and Aitken et al. [52]. The following abbreviations are used in this figure: cholesterol (Chol.), receptor (Rec.), protein tyrosine kinase (PTK), phosphotyrosine phosphatase (Ptyr-Ptase), cyclic nucleotide phosphodiesterase (PDE), and adenylyl cyclase (AC).

The observation that capacitation can occur in vitro spontaneously in a defined medium without the addition of biological fluids suggests that this process is intrinsically modulated by the sperm itself, such that these cells are preprogrammed to undergo capacitation when they are incubated in the appropriate medium. This does not rule out the influence of positive/negative regulatory factors in the female reproductive tract. In fact, it is possible that the regulation of capacitation lies more in the de-repression of inhibitory modulators of capacitation through the removal of decapacitating factors [5, 13–15] than in the stimulation of this process. Although there is no universal capacitation medium, and different media support capacitation in sperm from different species, nevertheless it appears that certain components of the media such as serum albumin, Ca²⁺, and HCO₃^- play important regulatory roles in promoting capacitation. It is unclear, however, how these compounds are coupled to membrane, transmembrane, and intracellular signaling events regulating this maturational process. This will be considered below.

A Pathways. Role of Media Constituents

A.1 Serum albumin. Serum albumin, usually BSA, present in the capacitation media for mammalian sperm (e.g., mouse, hamster, cattle, and human), is believed to function during capacitation in vitro as a sink for the removal of cholesterol from the sperm plasma membrane [11, 16–19] (pathway A1). The removal of this sterol could account for the membrane fluidity changes that have been documented to occur during capacitation [20, 21]. However, it has not been established whether or not cholesterol removal represents the only function of BSA, and little is known about the mechanism of action and the consequences of cholesterol removal. Experiments have demonstrated that other cholesterol-binding proteins, such as high-density lipoprotein or lipid transfer proteins present in follicular or oviductal fluids [10], can replace albumin in in vitro fertilization assays [22]; these results suggest that involvement in cholesterol movement represents a primary action of BSA. Recently, Cross and coworkers [11, 23–25] demonstrated that cholesterol is present in human semen and that it can account for the inhibitory effects of seminal plasma on human sperm capacitation, presumably by preventing cholesterol efflux from the sperm plasma membrane.

A.2 Calcium. The role of Ca²⁺ in initiating and/or regulating capacitation is currently controversial. Some authors have described an increase in intracellular Ca²⁺ during capacitation, while others have shown that intracellular Ca²⁺ does not change during this maturational event (Yanagimachi [5] and references therein). In mouse spermatozoa there is evidence that Ca²⁺ is required for capacitation [26, 27], although the authors of these studies did not measure intracellular Ca²⁺ concentrations. In another report, Baldi et al. [28] demonstrated that human sperm incubated under capacitation conditions displayed a rise in intracellular Ca²⁺ from 70 µM to 250 µM during the first 2 h of incubation and that this level did not significantly increase thereafter. Part of this controversy could be attributable to the well-demonstrated action of Ca²⁺ on the acrosome reaction and the inherent difficulties in differentiating these events. As discussed below, the action of Ca²⁺ at the level of effector enzymes involved in sperm signal transduction (e.g., adenylyl cyclase, cyclic nucleotide phosphodiesterase; see pathway A2) suggests that this divalent cation plays an important role in capacitation.

A.3 Bicarbonate. The requirement of HCO₃^- anion for capacitation has been well established in the mouse [7, 26, 29, 30] and in the hamster [31], although it remains to be demonstrated in other mammalian species. Little is known about the way HCO₃^- enters the sperm. Since 4,4'-diisothiocyanatostil-bene-2,2'-disulfonate (DIDS) and 4-acetomido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), well-known inhibitors of anion transporters, can block the actions of HCO₃^- on various sperm functions, some authors have suggested that sperm contain anion antiporters [32–35]. Although it has been reported that sperm contain a protein that is immunoreactive with an antibody to the AE1 class of anion transporters [34], little is known about the identification, localization, and function of this protein in these cells. The transmembrane movement of HCO₃^- anions could be responsible for the known increase in intracellular pH that is observed during capacitation [25, 36, 37]. An additional target for the action of this anion could be the regulation of sperm cAMP metabolism, since the mammalian sperm adenylyl cyclase is markedly stimulated by HCO₃^- [32, 38, 39] (pathway A3). The mechanism of action of this anion on the activity of this enzyme presently is not clear. Although Garty and Salomon [39] have partially purified this enzyme from bovine sperm and demonstrated that it is still responsive to HCO₃^-, its direct effects on a purified native or recombinant enzyme preparation remain to be demonstrated. From a physiological point of view, it is of interest that HCO₃^- concentrations are low in the epididymis and high in the seminal plasma and in the oviduct [40]. Moreover, since the presence of HCO₃^- in the extracellular milieu has also been positively correlated with the motility of pig sperm [38], changes in the concentration of HCO₃^- in the male and female reproductive tracts could play an important role in the suppression of capacitation in the epididymis and the promotion of this process in vivo in the female reproductive tract.

B Pathways. Effectors and Intracellular Second Messengers

For the purposes of this review, effectors and intracellular messengers mediating capacitation will be considered from two perspectives. Discussion of the regulatory systems that appear to be common among different species, thereby forming a unifying hypothesis of capacitation, will be considered first. Regulatory processes that may be unique to one or more species will then be discussed and will be integrated into this unifying hypothesis where appropriate.

B.1 Cyclic AMP metabolism. Although the idea that cAMP is involved in regulating mammalian sperm function has been in the literature for many years, only its role in sperm motility has been well established (for review see Eddy and O'Brien [41]). The role of cAMP in capacitation, as well as in the acrosome reaction, is still uncertain [5]. Reports from our group and others have suggested a role for cAMP during capacitation [42–44]. Recently, we have demonstrated that protein kinase A (PK-A) activity increases during mouse sperm capacitation [45]. These observed changes in PK-A activity reflect elevations of intracellular cAMP, further elucidating a role for this particular signaling pathway in mediating capacitation (pathway B1).

The mode of regulation of cAMP metabolism during capacitation is also of great interest, since it may be integrated with the aforementioned changes in Ca²⁺ and HCO₃^- movement. Ca²⁺ and HCO₃^- have a common mode of action in that both ions stimulate the activity of mammalian sperm adenylyl cyclases [32, 39, 46–49] (pathways A2 and A3). The mammalian sperm enzyme possesses unique properties, and its regulation has been the subject of multiple studies. However, the sequence and topology of this enzyme have not yet been established, and the exact mechanism by which this enzyme is stimulated by these ions is not clear.

B.2 Protein tyrosine phosphorylation. Recently our laboratory has also correlated mouse, human, and bovine sperm capacitation with an increase in protein tyrosine phosphorylation of a variety of substrates [26, 50, 51], and other laboratories have corroborated these results in these and other species [43, 52]. Protein tyrosine phosphorylation mediates a variety of cellular functions such as growth regulation, cell cycle control, cytoskeleton assembly, ionic current regulation, and receptor regulation [53, 54]. Using the mouse as an experimental paradigm, our laboratory has demonstrated that conditions conducive to capacitation of cauda epididymal sperm promote the tyrosine phosphorylation of a subset of proteins of M_r 40 000–120 000. The increase in protein tyrosine phosphorylation is dependent on the presence of BSA, Ca²⁺, and NaHCO₃ in the medium, and the concentrations of these compounds needed in order for protein tyrosine phosphorylation to occur are correlated with those needed for capacitation [26]. Specifically, the absence of any one of these media constituents prevented protein tyrosine phosphorylation and capacitation from occurring. Moreover, caput sperm, which do not possess the ability to undergo capacitation and fertilize eggs [5], do not display these changes in protein tyrosine phosphorylation when incubated under conditions normally conducive to capacitation [26], suggesting that the ability to become capacitated, as well as the ability to undergo an increase in protein tyrosine phosphorylation, is acquired during epididymal maturation.

The requirement for BSA, Ca²⁺, and NaHCO₃ in the extracellular medium in order for these protein tyrosine phosphorylations to occur represents an interesting mode of regulation of the signal transduction cascade in sperm leading to these posttranslational modifications. The role of BSA in regulating capacitation appears to rely on its ability to serve as a sink for the removal of cholesterol from the sperm plasma membrane, as described above (see section A.1 and Fig. 1, pathway A1). This interrelationship between BSA and cholesterol movement also appears to be important in the regulation of protein tyrosine phosphorylation, since it was found that preloading BSA with a cholesterol analogue in order to inhibit the ability of BSA to sequester sperm plasma membrane cholesterol inhibited protein tyrosine phosphorylation and sperm capacitation [55]. These and other experiments support the idea that cholesterol release is intimately tied to transmembrane signaling events in the sperm that result in protein tyrosine phosphorylation. This novel mode of signal transduction clearly warrants further investigation.

The requirement of extracellular Ca²⁺ and NaHCO₃ for both protein tyrosine phosphorylation and capacitation also represents a novel regulatory mechanism of cellular signaling, since these ions have been shown to be activators of the mammalian sperm adenylyl cyclase (pathways A2 and A3). It has been demonstrated that this enzyme, responsible for the synthesis of cAMP, is stimulated directly or indirectly by Ca²⁺ [46], calmodulin [47, 48], and NaHCO₃ [32, 39, 42, 49]. Since there appears to be a relationship between Ca²⁺, NaHCO₃, and increased adenylyl cyclase activity, it was of interest to determine whether the action of these ions on protein tyrosine phosphorylation and capacitation involved a cAMP-mediated pathway. As stated above, protein tyrosine phosphorylation does not occur when sperm are incubated in the absence of BSA, Ca²⁺, or NaHCO₃. However, when sperm are incubated in the absence of any of these compounds, but in the presence of cAMP agonists, the increase in protein tyrosine phosphorylation, as well as capacitation, is recovered [42]. Moreover, protein tyrosine phosphorylation is accelerated by active cAMP agonists in complete media that support capacitation. These observations lead to two main conclusions. First, the action of cAMP appears to be downstream of the actions of BSA, Ca²⁺, and NaHCO₃ and upstream of protein tyrosine phosphorylation. Second, the results also suggest that protein tyrosine phosphorylation and capacitation are regulated through a PK-A pathway (pathway B2). Consistent with this hypothesis is the observation that two inhibitors of PK-A that inhibit this enzyme by completely distinct mechanisms, Rp-cAMPs [56] and H-89 [57, 58], inhibit both protein tyrosine phosphorylation and capacitation of sperm in complete medium [42]. Moreover, we have shown that PK-A activity increases during capacitation [45].

Since the action of BSA appears to be tied to the removal of plasma membrane cholesterol, it is likely that cholesterol release is also upstream of the cAMP-induced protein tyrosine phosphorylation. Whether cholesterol removal is upstream of or parallel to the action of Ca²⁺ and/or NaHCO₃ is not presently known. It can be hypothesized that the removal of cholesterol, with a resultant change in sperm plasma membrane fluidity, could modulate Ca²⁺ and/or HCO₃^- ion fluxes leading to the activation of the adenylyl cyclase; this hypothesis remains to be tested. Taken together, these data suggest that protein tyrosine phosphorylation and capacitation appear to be under regulation of a cAMP/PK-A pathway. Up-regulation of protein tyrosine phosphorylation by PK-A during sperm capacitation is, to our knowledge, the first demonstration of a connection between these signal transduction pathways. Evidence exists for the down-regulation of receptor tyrosine kinase actions by cAMP [59, 60], but up-regulation has thus far not been demonstrated. Similar results have now been reported in other species of sperm, such as those of humans [43, 51] and cattle [50], suggesting that this unique mode of signal transduction cross talk may be universal to mammalian spermatozoa. Presently, it is not known whether the increase in protein tyrosine phosphorylation is due to the stimulation of a tyrosine kinase, an inhibition of a phosphotyrosine phosphatase, or both. Phosphotyrosine phosphatase activity in other cell types has been demonstrated to be both inhibited [61] and stimulated [62] by cAMP. The level at which cAMP/PKA functions to regulate the steady-state levels of protein tyrosine phosphorylation in sperm under conditions conducive to capacitation, as well as the nature of the regulatory enzymes in this unique signal transduction pathway, will remain the subject of intense scrutiny in our laboratory for the foreseeable future.

B.3 pH. Intracellular pH (pH_i) regulates several aspects of mammalian sperm function, although the transport mechanisms that control pH_i in these cells are not fully understood. Zeng et al. [37] have identified two acid efflux mechanisms in mouse sperm. One of the pathways shares the characteristics of the somatic cell Na⁺-dependent Cl^-/HCO₃^- exchanger. The second acid efflux pathway does not require extracellular ions. These authors described an increase in pH_i during capacitation, and these data are consistent with reports by Vredenburgh and Parrish [63] describing an increase in pH_i during capacitation of bovine sperm by heparin (pathway B3). Although the increase in pH_i accompanying heparin-induced bovine sperm capacitation is not inhibited by Rp-cAMP [36], this PK-A antagonist is able to block capacitation, suggesting that PK-A regulatory pathways function either in parallel to, or downstream of, pathways activated as a consequence of changes in pH_i. Similar conclusions follow the observation that the PK-A inhibitor, H-89, blocks capacitation-associated changes in protein tyrosine phosphorylation in bovine sperm [50], suggesting that protein tyrosine phosphorylation is also a pathway parallel to or downstream of the changes in pH_i.

B.4 Membrane potential. Capacitation is accompanied by the hyperpolarization of the sperm plasma membrane [64]. The VM calculated values changed during capacitation from -35 to -50 mV and from -30 to -60 mV in mouse and bovine sperm, respectively. Membrane hyperpolarization is due in part to an enhanced K⁺ permeability and could be related to the release of inhibitory modulation during capacitation [65] (pathway B4). Little is known about the consequences of this hyperpolarization; however, it is speculated that such membrane potential changes could recruit Ca²⁺ channels from an inactivated state to a closed, but activatable, state from which they could be subsequently opened by an agonist-induced depolarization (e.g., with the zona pellucida) [12, 65]. Presently, the role of membrane potential in regulating any of the aforementioned aspects of capacitation at the molecular level is not known and remains an important avenue for future investigation.

B.5 Free radicals. The action of free radicals in sperm function has been studied by a number of different laboratories. A majority of this work has focused on the role of free radicals in sperm lipid peroxidation and subsequent sperm viability [66–69]. However, more recent work using human sperm has focused on the role of superoxide anion generation related to capacitation and hyperactivation [70–72] (pathway B5a). Recently, Leclerc et al. [73] found that reactive oxygen species up-regulate protein tyrosine phosphorylation of several proteins. These results are in agreement with the work of Aitken et al. [52], who have described an increase in protein tyrosine phosphorylation after stimulation of a postulated endogenous NADPH-oxidase or after addition of H₂O₂ (pathway B5b). The localization of the free radical-generating system(s) in sperm is not known, nor is it known whether the action of superoxide anion is dependent or independent of cAMP.

B.6 Heparin. Studies of bovine sperm capacitation have shown that capacitation in vitro can be accomplished in media containing heparin [74, 75] or oviductal fluid, in which the active capacitating agent is thought to be a heparin-like glycosaminoglycan. It is thought that glycosaminoglycans may promote capacitation by binding to and removing seminal plasma proteins that are adsorbed to the sperm plasma membrane and are normally thought to function to inhibit capacitation [76, 77]. Interestingly, heparin also increases cAMP synthesis [78], elevates pH_i (see above), and regulates the capacitation-associated changes in protein tyrosine phosphorylation [50] (pathway B6). The mechanism by which this occurs, as well as its physiological relevance, is not clear.

B.7 Glucose. Whether glucose has inhibitory or stimulatory actions on capacitation is controversial and is apparently species dependent. In bovine sperm, glucose inhibits heparin-induced capacitation in vitro by a mechanism involving effects on cAMP metabolism and a reduction of pH_i [36, 78] (pathway B7). Glucose also has an inhibitory effect on the capacitation-associated increase in protein tyrosine phosphorylation observed when bovine sperm are incubated in the presence of heparin [50]. Paradoxically, other authors have found that glucose is beneficial for capacitation in other species [79–81]. Our laboratory has found that although glucose has an inhibitory effect on the increase in protein tyrosine phosphorylation in bull sperm [50], capacitation medium for mouse sperm, which contains glucose, has no apparent inhibitory effects on protein tyrosine phosphorylation [26]. The species-dependent differences in responses to this saccharide are not understood, nor is the mechanism of action.

	SUMMARY

TOP INTRODUCTION MOLECULAR BASIS OF CAPACITATION SUMMARY REFERENCES

 
Work from several laboratories is beginning to elucidate the molecular basis of sperm capacitation, leading to a unified model of this extratesticular maturational event. Several questions of considerable importance must now be addressed. First, how is cholesterol movement from the sperm plasma membrane regulated, and how does that movement initiate intracellular signaling? Second, what is the mechanism by which the cAMP/PK-A pathway is stimulated, and how does stimulation of this pathway lead to cross talk and up-regulation of protein tyrosine phosphorylation? Finally, what is the nature of the substrates that are phosphorylated on tyrosine residues, and how does the phosphorylation of these substrates impact on the major endpoints of capacitation, e.g., hyperactivation of motility, competence to undergo a regulated acrosome reaction, and fertilization? Such signaling processes underlying capacitation appear to be quite unique and therefore warrant continued investigation.

	ACKNOWLEDGMENTS

 
We would like to thank all of the members of our lab for their contributions to the work that has been described in this review.

	FOOTNOTES

 
¹ This work was supported by grants HD-06274, HD-34811, HD-22732, and HD-33052. P.E.V. was supported by the Rockefeller Foundation and HD-06274.

² Correspondence: Pablo E. Visconti, Center for Research on Reproduction and Women's Health, Room 314, John Morgan Building, University of Pennsylvania Medical Center, Philadelphia, PA 19104-6080. FAX: (215) 349-5118; pvisconti{at}obgyn.upenn.edu

Accepted: February 22, 1998.

Received: November 3, 1997.

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