Role and regulation of PI3K in sperm capacitation and the acrosome reaction

Molecular and Cellular Endocrinology 314 (2010) 234–238

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Review

Role and regulation of PI3K in sperm capacitation and the acrosome reaction Haim Breitbart ∗ , Tali Rotman, Sara Rubinstein, Nir Etkovitz The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

a r t i c l e

i n f o

Article history: Received 4 March 2009 Received in revised form 14 June 2009 Accepted 16 June 2009 Keywords: Spermatozoa Capacitation Acrosome reaction PI3K PKA PKC

a b s t r a c t Mammalian spermatozoa undergo several signaling and biochemical transformations in the female genital tract, collectively called capacitation. The capacitated spermatozoon binds to the egg zona pellucida, where it undergoes the acrosome reaction (AR), a process enabling it to penetrate and fertilize the egg. Actin polymerization occurs in sperm capacitation and depolymerization prior to the AR. In this review we describe the possible role and regulation of PI3K in sperm capacitation and the acrosome reaction. We claim that PI3K is activated by protein kinase A and suppressed by protein kinase C. Only partial activation of PI3K is seen during the capacitation time, however towards the end of incubation, full activation is observed. Actin polymerization during capacitation is independent on PI3K activity, suggesting that the enzyme is not involved in sperm capacitation. However, the full activation of PI3K towards the end of the capacitation suggests that it might mediate the AR, as indeed was found. © 2009 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PI3K in sperm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of PI3K in sperm capacitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of PI3K activation in sperm capacitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PI3K in sperm acrosome reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Ejaculated mammalian spermatozoa should reside in the female genital tract for several hours before gaining the ability to fertilize the egg. During this time the sperm cells undergo some physiological and biochemical changes, collectively called capacitation, which render the spermatozoa capable of fertilization. It is still not clear what are the exact mechanisms occurring in sperm capacitation, but it seems certain to involve molecules absorbed on, or integrated into the sperm plasma membrane during epididymal maturation and on contact of spermatozoa with the seminal plasma. We have suggested elsewhere that spermine can be bound and release from sperm cells, in which at relatively high concentration (mM) it serves as decapacitation factor whereas at the ␮M range it induces the acrosome reaction (Rubinstein and Breitbart, 1991; Rubinstein et al., 1995). The removal or alteration of these molecules prepares

∗ Corresponding author. Tel.: +972 3 5318201; fax: +972 3 6356041. E-mail address: [email protected] (H. Breitbart). 0303-7207/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2009.06.009

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the sperm toward successful binding to the egg and fertilization. In 1963, Yanagimachi and Chang (1963) first reported that mammalian sperm can be capacitated in vitro, making the analysis of capacitation mechanisms considerably easier. During mammalian fertilization, the capacitated spermatozoon penetrates the cumulus oophorous of the ovum, and then binds to the zona pellucida with its plasma membrane intact. Zona binding induces the sperm cell to undergo the acrosomal reaction which involves multiple fusions between the outer acrosomal membrane and the overlying plasma membrane. This process is required for sperm penetration via the zona pellucida prior to fertilization. Capacitation medium usually composed of energy substrates, a cholesterol acceptor, NaHCO3 , Ca2+ , low K+ and isoosmotic concentrations of Na+ . The mechanisms of action which promote capacitation are poorly understood. It is accepted now that initiation of capacitation correlates with cholesterol efflux from the sperm plasma membrane which increases the membrane permeability to HCO3 − and Ca2+ (Demarco et al., 2003) leading to the activation of soluble adenylyl-cyclase (sAC) (Buck et al., 1999) to produce cAMP which activates protein kinase A (PKA) (Rev. by

H. Breitbart et al. / Molecular and Cellular Endocrinology 314 (2010) 234–238

Visconti et al., 2002). The cAMP/PKA system in sperm, can be activated by activating the G-protein-coupled-receptors (GPCRs), like the AT1-R or adenosine-R (Fraser et al., 1990), however it is not clear what is the need of this mechanism when sAC is active. During capacitation there is a significant increase in tyrosine phosphorylation of several proteins (Visconti et al., 1995a,b). The regulation of this phosphorylation involves an unusual signaling cascade mediated by PKA, and driven by the increase in sperm cAMP during capacitation (Lefievre et al., 2002; White and Aitken, 1989). The major unresolved problem in this respect is the identity of the key intermediate kinase that is activated by PKA and starts the protein tyrosine phosphorylation processes. It has been suggested that the serine–threonine kinase MAPK is involved in this process (Luconi et al., 1998), however, the tyrosine kinase is still missing. Recently, it has been suggested that this kinase is Src family (Baker et al., 2006; Leclerc and Goupil, 2002), a family of kinases which to our hypothesis mediates the activation of EGFR by PKA (Etkovitz et al., 2008). PKA is a ubiquitous multifunctional serine–threonine kinase involved in the regulation of diverse cellular events. The PKA holoenzyme consists of four subunits, two catalytic and two regulatory. The regulatory subunits form dimers via an interaction at the N-terminus and the C-terminus contain the cAMP binding sites. The binding of cAMP to the regulatory subunits triggers the dissociation and activation of the catalytic subunits. The regulatory subunits also anchor PKA to its activation location in the cell through interaction with AKAPs (Dell’Acqua and Scott, 1997; Rubin, 1994; Scott and Carr, 1992). Several AKAPs can simultaneously bind PKA and other signaling molecule such as protein phosphatase 1, calcineurin, calmodulin and protein kinase C (PKC) (Coghlan et al., 1995; Klauck et al., 1996; Nauert et al., 1997; Sarkar et al., 1984; Schillace and Scott, 1999). This suggests that AKAPs can act as scaffolding proteins that coordinate and regulate the activation of several enzymes within a specific cellular location. AKAPs contain an amphipathic helix domain and bind the type II regulatory subunit (RII) of PKA via the hydrophobic face (Carr et al., 1991). It has been suggested that spermatozoa are the only cells that contain proteins other than RII that interact with the amphipathic helix domain of AKAPs (Carr et al., 2001). We demonstrated elsewhere an interesting crosstalk between PKA and PKC in regulating actin polymerization during sperm capacitation (Cohen et al., 2004; Breitbart et al., 2005). In a recent publication we demonstrate the role of PI4K and PI3K in the regulation of actin polymerization in sperm capacitation and we show that PKA can up-regulate PI3K and PKC␣ can down-regulate its activity (Etkovitz et al., 2007). It is known that membrane phosphoinositides are substrate for PI3K which phosphorylates PIP2 on the D-3 hydroxyl and provide association sites for the interaction of several proteins. These proteins are involved in signal transduction events in the cell including sperm exocytosis (Fisher et al., 1998; Feng et al., 1998, 2005). We and others suggest that PI3K is probably not involved in sperm capacitation (Etkovitz et al., 2007; Aparicio et al., 2007; Nauc et al., 2004) but rather in the acrosome reaction (Breitbart et al., 2006; Jungnickel et al., 2007). It has been suggested that ZP3 leads to the accumulation of PIP3 , the product of PI3K activity, resulting in the activation of down-stream effectors Akt and PKC␨, two components essential for the acrosome reaction (Jungnickel et al., 2007). The activity of PI3K is probably regulated during sperm capacitation in order to get it active towards the acrosome reaction which occurs at the end of the capacitation process. In this review we would like to suggest how PI3K activity is regulated. The overall objective of our work in the last few years has been to characterize the intracellular mechanisms involved in sperm capacitation and the acrosome reaction, two processes essential for successful fertilization. In this review, we present the role and regulation of PI3K mainly by the crosstalk between PKA and PKC

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and other enzymes in the cascade leading to mammalian sperm capacitation and the acrosome reaction. 2. PI3K in sperm In mammalian cells, phosphatidylinositol (3,4,5)-trisphosphate (PIP3 ) is synthesized by PI3-kinases (PI3K), which phosphorylate phosphatidylinositol (4,5) bisphosphate (PIP2 ) at the D-3 position of the inositol ring. PI3Ks consist of three main classes identified as class I, II and III (Anderson and Jackson, 2003). Although several forms of PI3Ks exist in eukaryotes, the class IA is primarily responsible for the production of PIP3 in response to growth factors (Luo and Cantley, 2005; Cantley, 2002). The class IA PI3Ks are heterodimers, consisting of a 85 or 55 kDa regulatory subunit (PIK3R1, PIK3R2, PIK3R3) and a 110-kDa catalytic subunit (PIK3CA, PIK3CB, PIK3CD) (Anderson and Jackson, 2003; Luo and Cantley, 2005; Cantley, 2002; Wymann and Pirola, 1998). The mRNA can be alternatively spliced giving rise to three new regulatory subunits, termed p55, p45, p85+8aa (55, 45, and 100 kDa respectively) in addition to the p85 (PIK3R1) (Wymann and Pirola, 1998). PIK3R1 contains two SH2 domains, one SH3 domain, a Bcr homology domain, and prolinerich sequences (Anderson and Jackson, 2003; Wymann and Pirola, 1998). When activated, the two subunits can bind and translocate to the plasma membrane where they become catalytically active. We recently demonstrated the presence of PIK3R1 (p85␣ regulatory subunit of PI3K) in bovine sperm which is localized to the sperm mid-piece and the post-acrosomal region of the head (Etkovitz et al., 2007). The enzyme was also identified in boar, hamster, mouse and human sperm, and it was suggested that it involves in sperm capacitation and the acrosome reaction (Feng et al., 1998; Nauc et al., 2004; Aparicio et al., 2005; NagDas et al., 2002; Luconi et al., 2004). Furthermore, the PI3K regulatory subunit p85 was shown to be gradually phosphorylated during mouse sperm capacitation and this phosphorylation was inhibited in mouse lacking FGFR-1 receptor (Cotton et al., 2006). It has been suggested that sperm enkurin may tether p85 to a ZP3-activated TRPC cation channel (Sutton et al., 2004) indicating a possible involvement of PI3K in the acrosome reaction. In human sperm PI3K was also shown to participate in the regulation of sperm motility (du Plessis et al., 2004; Luconi et al., 2001, 2005; Yang et al., 2006). 3. Role of PI3K in sperm capacitation In our recent study we suggested a role for PI4K and PI3K in actin polymerization (Etkovitz et al., 2007) a process that must occur in sperm capacitation (Brener et al., 2003). Our data show clearly that PI4K but not PI3K mediates actin polymerization in sperm capacitation (Etkovitz et al., 2007). Although intracellular levels of PIP3 are enhanced during sperm capacitation indicating the activation of PI3K, we show that PI3K is not involved in actin polymerization (Etkovitz et al., 2007). However when cellular PKA activity is significantly enhanced by adding dibutyryl cAMP (dbcAMP) to the cells, PI3K-dependent actin polymerization is observed (Etkovitz et al., 2007). Moreover, the cells show relatively small phosphorylation/activation of PI3K (on Tyr467 at the p85 regulatory subunit) at the beginning of the capacitation and this activation is significantly enhanced when PKA is activated by the addition of dbcAMP to the incubation medium (Etkovitz et al., 2007). Opposite results are obtained when PKC is activated in which fast actin polymerization was observed (Cohen et al., 2004), while under these conditions PI3K activation is inhibited and this inhibition could be eliminated by blocking PKC activity (Etkovitz et al., 2007). Moreover, we show elsewhere that activation of sperm PKA inhibits PKC␣ activation probably due to direct inhibition of PLC by PKA (Cohen et al., 2004). The involvement of the isoform PKC␣ in the inhibition

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H. Breitbart et al. / Molecular and Cellular Endocrinology 314 (2010) 234–238

Fig. 1. PKC␣ inhibition enhanced PI3K activation during sperm capacitation (unpublished data). Bovine sperm were incubated for 4 h in mTALP capacitation medium, in the presence or absence of 15 nM RO32-4320. Cells proteins were extracted at the indicated times using lysis buffer as previously described (Etkovitz et al., 2007) and analyzed by western blotting using anti-p(Tyr467)-PI3K specific antibody. Tubulin antibody was used to equalize the loading quantities. The results represent three independent experiments.

Table 1 The induction of mouse sperm acrosome reaction by A23187, dbcAMP or ZP is mediated by PI3K (unpubplished data). Mouse sperm were incubated in HMB for 1.5 h. At the end of this incubation 10 nM wortmannin (WT) was added as indicated for 10 min. After this incubation, 10 ␮M A23187, 1 mM dbcAMP or 7.5 ZP/␮l ZP were added to cells for additional 20 min of incubation. Acrosome reacted cells were identified by PNA-FITC staining. The percentage of acrosome reacted cells at the end of the 1.5 h incubation (26%) was subtracted to obtain the induced percentage (AR (%)). I (%) represents percentage inhibition of the acrosome reaction. The data represent the mean ± SD of duplicates from at least five experiments. Treatment

CONT. 10 nM WT

of PI3K activation is further supported by showing that the inhibition of PKC␣ by its specific inhibitor RO32-4320 caused faster and higher activation of PI3K compared to the control cells (Fig. 1). In addition, we showed here for the first time that PKC␣ is highly phosphorylated/activated at the beginning of the capacitation (Fig. 2) conditions in which PI3K is not activated (see Fig. 1). Altogether, these data in bovine sperm suggest that the cellular ratio between PKA and PKC␣ activities regulates PI3K activation state. At relatively high PKA activity and low PKC␣, PI3K is activated, however when PKC␣ activity is relatively high, PI3K activation is suppressed. In boar sperm, it has been suggested that PI3K does not regulate the capacitation process (Aparicio et al., 2007). In hamster sperm, 100 nM wortmannin, a known inhibitor of PI3K, inhibit hyperactivated motility, a phenomenon occurring in sperm capacitation (NagDas et al., 2002), however, this concentration of wortmannin is relatively high and it might inhibit PI4K as well. We found that 10 nM wortmannin completely blocked PI3K in bovine sperm (Etkovitz et al., 2007) thus, the conclusions regarding the association of PI3K in the development of hyperactivated motility should be reconsidered. Indeed it was shown that inhibition of PI3K in human sperm using LY294002, triggers an increase in intracellular levels of cAMP and tyrosine phosphorylation of AKAP3 as well as sperm motility (Luconi et al., 2004). These authors suggest that PI3K negatively regulates sperm motility by interfering with AKAP3-PKA binding. In boar sperm, PI3K significantly decreases straight-line velocity, circular and average, but does not affect progressive motility (Aparicio et al., 2005). These data together suggest that PI3K activation may suppress premature development of hyperactivated motility before the sperm arrive to the oviduct thus might interfere with the fertilization process. It is well established that PKA-dependent protein tyrosine phosphorylation occur in sperm capacitation (Visconti et al., 1995a,b) thus, PI3K might down-regulate this process. Therefore, the data presented by Luconi and Baldi (2003) may suggest that PI3K is not involved in sperm capacitation or even interfere with sperm capacitation. Moreover, inhibition of PI3K in human sperm did not affect protein tyrosine phosphorylation (Nauc et al., 2004). This conclusion supports our data in bovine sperm in which we suggest that PI3K is not involved in sperm actin polymerization a process occurring in capacitation

*

10 ␮M A23187

1 mM dbcAMP

7.5 ZP/␮l ZP

AR (%)

I (%)

AR (%)

I (%)

AR (%)

I (%)

27.9 ± 2.7 12.6 ± 6.3*

– 55

22.2 ± 4.7 10.2 ± 4.8*

– 54

14.1 ± 2.9 1.0 ± 1.7*

– 93

Significant difference from the corresponding control, P < 0.05.

(Nauc et al., 2004). Moreover, the fact that activation of PKC suppresses PI3K activation (Etkovitz et al., 2007) and the fact that PI3K is activated towards the end of the capacitation process (Fig. 1), support our notion regarding the possible negative role of PI3K in sperm capacitation. 4. Regulation of PI3K activation in sperm capacitation As suggested above it is not clear if PI3K is involved in sperm capacitation, however an increase in PIP3 production occurs in sperm capacitation (Etkovitz et al., 2007) indicating that the enzyme is at least partially active during this period of time. Since there are evidences indicating that PI3K mediates the acrosome reaction (Table 1), the enzyme is highly phosphorylated/activated towards the end of the capacitation prior to the initiation of the acrosome reaction (see Fig. 1) therefore, suggesting that this activation is part of the capacitation process. In mouse sperm PI3K is also activated when incubated under in vitro capacitation conditions (Cotton et al., 2006). We described above that PI3K is activated by PKA and its activity is suppressed by PKC probably by the isoform PKC␣. Recently it has been shown in other cell types that PKA can directly phosphorylate PI3K␣ (p85␣) regulatory subunit on Ser83, leading to its activation (Cosentino et al., 2007; De Gregorio et al., 2007). It was also shown that PKC␣ can inhibit PI3K directly or indirectly (Sipeki et al., 2006; Guan et al., 2007). We hypothesized that the ratio between PKA and PKC␣ activities dictates PI3K activation. It is accepted that PKA activity is enhanced during capacitation due to the activation of the bicarbonate and calcium-dependent-sAC which converts ATP to cAMP, the activator of PKA. We show elsewhere that activation of sperm PKA, lead to the inactivation of PKC␣ probably via direct inactivation of phospholipase C by PKA (Cohen et al., 2004). Moreover, we showed here that PKC␣ is already phosphorylated/activated at the beginning of the capacitation process whereas towards the end of the capacitation it undergoes significant dephosphorylation and degradation (Fig. 2). Altogether the data support our hypothesis in which activation of PKA and suppression of PKC␣ enables PI3K activation. 5. PI3K in sperm acrosome reaction

Fig. 2. PKC␣ is degraded and dephosphorylated during sperm capacitation (unpublished data). Bovine sperm were incubated for 4 h in mTALP capacitation medium. Cell proteins were extracted by lysis buffer at the zero time and after 4 h of incubation and analyzed by western blotting as previously described (Etkovitz et al., 2007) using anti-PKC␣ and p(Ser657)-PKC␣ antibodies. ␤-Actin antibody was used to equalize the loading quantities. The results represent seven independent experiments.

The acrosome reaction in capacitated sperm is initiated after its binding to the egg zona pellucida. The zona pellucida composed of three glycoproteins in which the glycoprotein ZP3 triggers the acrosome reaction (Bleil and Wassarman, 1983). It has been suggested that ZP3 mediates the hydrolysis of PIP2 by activating phospholipase C (Roldan et al., 1994; Fukami et al., 2003), resulting in an increase in intracellular calcium ions concentration due to the opening of TRPC channels (O’Toole et al., 2000; Jungnickel et al., 2001) and probably via calcium release from the acrosome

H. Breitbart et al. / Molecular and Cellular Endocrinology 314 (2010) 234–238 Table 2 The induction of bovine sperm acrosome reaction by A23187, dbcAMP or EGF is mediated by PI3K (unpublished data). Bovine sperm were incubated in mTALP for 4 h. At the end of this incubation 10 nM wortmannin (WT) was added as indicated for 10 min. After this incubation, 10 ␮M A23187, 1 mM dbcAMP or 1 ng/ml EGF were added to cells for additional 20 min of incubation. Acrosome reacted cells were identified by PSA-FITC staining. The percentage of acrosome reacted cells at the end of the 4 h incubation (18%) was subtracted to obtain the induced percentage (AR (%)). I (%) represents percentage inhibition of the Acrosome reaction. The data represent the mean ± SD of duplicates from at least five experiments. Treatment

CONT. 10 nM WT *

10 ␮M A23187

1 mM dbcAMP

1 ng/ml EGF

AR (%)

I (%)

AR (%)

I (%)

AR (%)

I (%)

67.0 ± 3.5 19.5 ± 7.0*

– 71

56.2 ± 3.8 25.5 ± 5.6*

– 55

49.7 ± 7.4 15.0 ± 1.4*

– 70

Significant difference from the corresponding control, P < 0.05.

(Walensky and Snyder, 1995; Dragileva et al., 1999; De Blas et al., 2002). It has been suggested that the class 1 PI3K mediates mouse sperm acrosome reaction (AR) induced by ZP3 (Jungnickel et al., 2007). Moreover, this group shows that D3-phosphoinositides inhibitors drive the AR suggesting that spontaneous AR in capacitated sperm is suppressed by phosphoinositides phosphatase activity (Jungnickel et al., 2007). This group also suggested that the produced PIP3 can lead to the activation of PDK1 which activates Akt and PKC␨ resulting in the occurrence of the AR. Other studies show that PI3K inhibitors reduce the rate of the AR (Fisher et al., 1998; Feng et al., 1998, 2005). Fisher et al. (1998) show that 10 nM wortmannin, a specific inhibitor of PI3K, inhibits human sperm AR induced by Mannose-BSA but not by the calcium ionophore A23187 or by progesterone. Interestingly, we show that 10 nM wortmannin inhibits significantly the AR induced by the calcium ionophore A23187 in bovine (Breitbart et al., 2005 and Table 2) and mouse sperm (Table 1). Another known PI3K inhibitor LY294002 did not affect human sperm AR induced by progesterone or by isolated zona pellucida (du Plessis et al., 2004). However, Jungnickel (Jungnickel et al., 2007) show that LY294002 blocked the AR induced by ZP3, and moreover, they show that WT inhibits the fertilization rate by 75% when capacitated sperm were treated for 15 min with 100 nM WT and then added to the oocyte. This result was confirmed by our finding that 10 nM WT inhibited ZP induced AR (Table 1) and reduced the fertilization rate by 30% (not shown). A model describing the regulation of PI3K activity in sperm capacitation and in the acrosome reaction is presented in Fig. 3.

Fig. 3. A model describing the regulation of PI3K in sperm capacitation and the acrosome reaction: capacitation: efflux of cholesterol from the sperm plasma membrane enhances bicarbonate permeability into the cell resulting in the activation of the soluble adenylyl-cyclase (sAC) which leads to cAMP production and PKA activation. PKA can activate PI3K but in order to do so PKC␣ should be suppressed because it blocks PI3K activation. Acrosome reaction: the activated PI3K phosphorylates PIP2 to get PIP3 which can activate PDK1 leading to the activation of Akt and PKC␨ resulting in the occurrence of the acrosome reaction, as suggested previously by the Florman’s group (Jungnickel et al., 2007).

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