actin and acrosomal process

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REPRODUCTIONREVIEW

Role of actin cytoskeleton in mammalian sperm capacitationand the acrosome reaction

Haim Breitbart, Gili Cohen and Sara Rubinstein

Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel

Correspondence should be addressed to H Breitbart; Email: [email protected]

Abstract

In order to fertilize, the mammalian spermatozoa should reside in the female reproductive tract for several hours, during

which they undergo a series of biochemical modifications collectively called capacitation. Only capacitated sperm can

undergo the acrosome reaction after binding to the egg zona pellucida, a process which enables sperm to penetrate into the

egg and fertilize it. Polymerization of globular (G)-actin to filamentous (F)-actin occurs during capacitation, depending on

protein kinase A activation, protein tyrosine phosphorylation, and phospholipase D activation. F-actin formation is importantfor the translocation of phospholipase C from the cytosol to the sperm plasma membrane during capacitation. Prior to the

occurrence of the acrosome reaction, the F-actin should undergo depolymerization, a necessary process which enables the

outer acrosomal membrane and the overlying plasma membrane to come into close proximity and fuse. The binding of the

capacitated sperm to the zona pellucida induces a fast increase in sperm intracellular calcium, activation of actin severing

proteins which break down the actin fibers, and allows the acrosome reaction to take place.

Reproduction (2005)129263268

Introduction

In mammalian species, spermegg interaction and mutualactivation are mediated by the zona pellucida (ZP), theglycoprotein coat of the egg (reviewed in Wassarman1988). The spermatozoon binds to the ZP with its plasmamembrane intact, via specific receptors that are localizedover the anterior head region of the sperm. The binding ofthe sperm to the ZP stimulates it to undergo an acrosomereaction, which enables the sperm to penetrate and ferti-lize the egg (reviewed in Yanagimachi 1994).

The binding of the sperm to the egg and the occurrenceof the acrosome reaction will take place only if the spermhas previously undergone a poorly defined maturationprocess in the female reproductive tract, known as capaci-tation. We recently reviewed the known signal transduc-tion events occurring during capacitation and the

acrosome reaction (Breitbart 2003). The possible changesand regulation of the sperm actin cytoskeleton as part ofthe mechanisms of capacitation and the acrosome reac-tion are described in this review.

Actin and related proteins in sperm

In spermatogenic cells, actin filaments have beendescribed primarily in the subacrosomal space betweenthe nucleus and the developing acrosome of spermatids

of certain mammalian species (Vogl 1989). In maturespermatozoa, however, the structure and location ofactin filaments have not been made clear. In most

reports, actin seems to be present in its monomericform, although filamentous (F)-actin has been describedin mammalian species as well (Flaherty 1987, Breed &Leigh 1991, Moreno-Fierros et al. 1992, Vogl et al.1993, de las Heras et al. 1997, Howes et al. 2001). Inhuman sperm the regions reported to contain actininclude the acrosomal space, the equatorial and postacrosomal regions, and the tail (Clarke et al. 1982, Vir-tanen et al. 1984, Ochs & Wolf 1985, Fouquet & Kann1992). The presence of actin in the tail might be import-ant for the regulation of sperm motility, and its presencein the head suggests a possible involvement in the acro-some reaction. It was reported that actin polymerization

is important for initiation of sperm motility during post-testicular maturation (Lin et al. 2002). The location ofactin in the acrosomal region of several mammalianspecies including hamster, boar, human, bull, rabbit andguinea-pig (Talbot & Kleve 1978, Camatini et al. 1986,Flaherty et al. 1988, Olson & Winfrey 1991, Moreno-Fierros et al. 1992, Yagi & Paranko 1995) supports itspossible role in sperm capacitation and the acrosomereaction. Actin polymerization is necessary for spermincorporation into the egg cytoplasm (Sanchez-Gutierrezet al. 2002) and for sperm nuclei decondensation

q 2005 Society for Reproduction and Fertility DOI: 10.1530/rep.1.00269ISSN 14701626 (paper) 17417899 (online) Online version via www.reproduction-online.org


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(Kumakiri et al. 2003). The assembly of G-actin to formF-actin is controlled by several actin-binding proteins.The existence of proteins such as calicin (von Bulowet al. 1995), the capping proteins CPb3 (von Bulow et al.1997) and CPa3 (Tanaka et al. 1994), destrin, thymosinb10, testis-specific actin capping protein (Howes et al.

2001), gelsolin (de las Heras et al. 1997), scinderin (Pel-letier et al. 1999), and the actin-related proteins Arp-T1and T2 (Heid et al. 2002) in mammalian sperm suggeststhat actin polymerization and depolymerization might beinvolved in sperm function. In boar and guinea-pigsperm, actin polymerization occurs during capacitation(Castellani-Ceresa et al. 1993, Cabello-Agueros et al.2003). Recently, we showed that actin polymerizationoccurs during capacitation of bull, mouse, human, andram sperm, whereas F-actin breakdown should occur inorder to achieve the acrosome reaction (Brener et al.2003). In human sperm, actin is lost from the acrosomalregion following the acrosome reaction (AR) (Liu et al.1999) and blocking actin polymerization inhibited ZP-

induced AR (Liu et al. 1999, 2002). In addition, inhi-bition of actin breakdown blocks bovine sperm AR(Spungin et al. 1995). Moreover, inhibition of actin pol-ymerization in guinea-pig and human sperm by cytocha-losin D, blocks sperm penetration into zona-free hamstereggs (Rogers et al. 1989) as well as the in vitro fertiliza-tion ability of boar (Castellani-Ceresa et al. 1993) andmouse (Brener et al. 2003) sperm. These evidencessuggest that remodeling of actin structure plays animportant role in sperm capacitation and the AR.

Protein tyrosine phosphorylation and actin

polymerizationIt is accepted that protein kinase A (PKA)-dependent tyro-sine phosphorylation of several proteins occurs duringsperm capacitation (Visconti et al. 1995). In human andbovine sperm, reactive oxygen species (ROS) up-regulates

protein tyrosine phosphorylation (Aitken et al. 1995,Leclercet al. 1997, Rivlinet al. 2004), consistent with thesuggestion that hydrogen peroxide activates adenylylcyclase to produce cAMP which activates PKA (Aitken1997, Rivlinet al.2004).

It is unclear whether a specific ligand induces the signal

transduction cascade leading to protein tyrosine phos-phorylation in sperm capacitation. One possible ligand isthe epidermal growth factor (EGF), which interacts with itsreceptor (EGFR) identified in the bovine sperm head (Laxet al. 1994). EGF stimulates the tyrosine phosphorylationof several sperm proteins (Breitbart et al. 1995) and acti-vates phospholipase Cg (PLCg) (Spungin et al. 1995) andactin polymerization (Brener et al. 2003, Cohen et al.2004) in bovine sperm capacitation.

There is a good correlation between actin polymeriz-ation and protein tyrosine phosphorylation in bovine andram sperm capacitation (Brener et al. 2003). In bovinesperm, the two processes do not occur in the absence ofbicarbonate; both depend on PKA and tyrosine kinaseactivities, both are enhanced by EGF, hydrogen peroxide,and the tyrosine phosphatase inhibitor vanadate, and bothare blocked by glucose (Brener et al. 2003). These datasuggest that protein tyrosine phosphorylation and actinpolymerization are related processes occurring in spermcapacitation. Interestingly, we found that EGF, hydrogenperoxide, or vanadate in the absence of bovine serumalbumin (BSA) in the incubation medium, cannot, bythemselves, induce capacitation (measured by percent ofacrosome reacted cells), although they stimulate tyrosinephosphorylation and actin polymerization (Brener et al.2003). This indicates that these two processes are necess-ary but insufficient for achieving sperm capacitation. The

role of BSA is to increase cholesterol efflux from theplasma membrane, which should occur in order tocapacitate the sperm.

We show elsewhere (Cohen et al. 2004) and here(Table 1) that actin polymerization in bovine sperm is

Table 1 The effect of various inhibitors on actin polymerization of bovine sperm following incubation with various activators. The numbersrepresent relative fluorescence intensity ^ S.E.M.The fluorescence at zero time (before starting the incubation) was 24 ^ 1. Percent inhibitionwas calculated after subtracting the zero time fluorescence. The various inhibitors were used at concentrations in which they represent specificinhibition.

Activator

Inhibitor No activator(4h) % Inhibition PMA (30 min) % Inhibition dbcAMP (10 min) % Inhibition PA (3min) % Inhibition

None 100 ^ 3 102 ^ 3 92 ^ 2 112 ^ 2 BAPTA-AM 26 ^ 2 97 25 ^ 1 99 88 ^ 1 6 100 ^ 3 14Genestein 32 ^ 1 90 39 ^ 1 81 45 ^ 1 69 83 ^ 3 33PD98059 30 ^ 1 92 28 ^ 1 95 30 ^ 2 91 89 ^ 2 26Brefeldin 28 ^ 2 95 31 ^ 2 91 30 ^ 1 91 90 ^ 2 25Neomycin 24 ^ 1 100 24 ^ 2 100 31 ^ 1 90 90 ^ 2 25

Bovine sperm were incubated for the indicated times in the presence of the PKC stimulator phorbol myristyl acetate (PMA, 100 ng/ml), PKAactivator dibutyryl cAMP (dbcAMP, 1 mM) or the PLD product phosphatidic acid (PA) (3mg/ml). Before this incubation, the cells were incubatedfor 1 h with the Ca2 chelator BAPTA-AM or for 10 min with the tyrosine kinase inhibitor genestein (20mg/ml), the MAPKK inhibitor PD98059(25mM), the ARF inhibitor brefeldin (10 mM) or the PLC inhibitor neomycin (5 mM). At the end of incubation, samples were stained withFITC-phalloidin as described by us elsewhere (Cohenet al. 2004) and the fluorescence intensity in the sperm head was determined quantitat-ively using the Meta Morph Image J and Adobe Photoshop processing software.

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significantly enhanced after short incubation of the cellswith dibutyryl-cAMP (dbcAMP) or the phorbol esterphorbol myristyl acetate (PMA), which activate PKA orprotein kinase C (PKC) respectively. This F-actin for-mation is almost completely blocked by the tyrosinekinase inhibitor genestein (Table 1). This suggests that

protein tyrosine phosphorylation is involved in PKA- andPKC-induced actin polymerization, although we couldnot observe any stimulation in tyrosine phosphorylatedproteins under these conditions (Rivlin et al. 2004). It ispossible that the determination of tyrosine phosphoryl-ation using anti-phosphotyrosine is not sensitive enoughto detect very small changes in phosphorylation. Thispoint needs further clarification. The suggested activationof the tyrosine kinase is probably needed for the acti-vation of phospholipase D (PLD) (see below), since actinpolymerization induced by exogenous phosphatidic acid(PA) (the product of PLD activity) was only slightlyinhibited by genestein (Table 1).

In our recent studies, we show that in bicarbonate-deficient medium, hydrogen peroxide could induceprotein tyrosine phosphorylation and actin polymeriz-ation, two processes which are essential but not suffi-cient for capacitation (Brener et al. 2003, Rivlin et al.2004). We also show that H2O2 stimulates sperm ade-nylyl cyclase (AC) and tyrosine phosphorylation of the80 KDa protein in addition to other proteins which aretyrosine-phosphorylated under regular capacitation con-ditions as well (Rivlin et al. 2004). The H2O2-stimu-lated phosphorylation of the 80 KDa protein as wellthe phosphorylation of an 85 KDa protein are bothdependent on intracellular Ca2 concentration ([Ca2]I)

(Rivlin et al. 2004). Actin polymerization occurredduring capacitation (Table 1) or, induced by exogenousH2O2 (not shown), was almost completely blocked bychelating intracellular Ca2. F-actin formation inducedby dbcAMP was only slightly inhibited by chelatingintracellular Ca2 (Table 1), suggesting that AC acti-vation depends upon [Ca2]i.

Since tyrosine phosphorylation of the 80 KDa proteinand actin polymerization are [Ca2]i-dependent pro-cesses but not in dbcAMP-treated cells, we suggest thatH2O2 activates a Ca

2-dependent tyrosine kinase inaddition to its direct effect on sperm AC. We mustemphasize that the bicarbonate-dependent tyrosine phos-

phorylation of 8 different sperm proteins is not depen-dent on [Ca2]i (Rivlin et al. 2004). Thus, the spermsoluble AC, known to be activated by bicarbonate (Chenet al. 2000), is relatively insensitive to [Ca2]i, whereasthe tyrosine phosphorylation of the 80 KDa and 85 KDaproteins as well as actin polymerization are [Ca2]i-dependent processes. This may suggest that actin polym-erization depends on Ca2-dependent tyrosine kinase(s)which leads to the tyrosine phosphorylation of 80 KDaand 85 KDa sperm proteins.

Actin polymerization is regulated by phospholipaseD (PLD)

In a recent study, we showed that PLD is involved inbovine sperm actin polymerization during capacitation(Cohen et al. 2004). We also demonstrated that the iso-form PLD1 is localized mainly in the acrosomal region ofbovine sperm (Garbi et al. 2000), suggesting its possibleinvolvement in the acrosome reaction. The requirement ofPLD activity for F-actin formation is based on the follow-ing: first, actin polymerization is significantly inhibited bythe PLD inhibitors butan-1-ol and C2-ceramide but not bybutan-2-ol; secondly, exogenous PLD or PA stimulatesactin polymerization which is not affected by butan-1-ol,and finally, PLD activity is enhanced during capacitationprior to F-actin formation (Cohen et al.2004).

Relatively fast (within 10 20 min) PLD activation andactin polymerization are induced by activating PKA orPKC, and these activities are completely blocked bybutan-1-ol, indicating that PLD mediates these activities.

Indeed, we show that PLD is activated in sperm by acti-vation of PKA or PKC (Cohen et al. 2004). In bovinesperm, PKCa and PLD1 co-exist as a complex, whichdecomposes after PKC activation (Garbi et al. 2000). Thiscomplex is decomposed by activating PKC using its directactivator PMA or by activating the lysophosphatidic acid(LPA) receptor (Garbi et al. 2000) resulting in PLD acti-vation (Cohenet al. 2004).

Inhibition of PKA activity throughout the four hours ofsperm capacitation completely blocked actin polymeriz-ation, while inhibition of PKC revealed only partial (40%)inhibition (Cohenet al. 2004). These findings are in agree-ment with the notion regarding the obligatory role of PKA

in sperm capacitation (Visconti et al. 1995). However,we found that activation of sperm PKC induced fast(20 30min) PKA-independent actin polymerization(Cohen et al. 2004). Moreover, when PKA was blocked(using H-89 or bicarbonate-deficient medium), there wasa rapid (30 min) increase in F-actin, which was inhibitedby PKC inhibition, as well as PKCaactivation (Cohenet al.2004). The effect of H-89 or of bicarbonate-deficient med-ium on actin polymerization was blocked by inhibition ofphospholipase C (PLC) activity, suggesting that PKA inhi-bits PLC activity in bovine sperm (Cohen et al. 2004).When PKA is blocked, PLC can be activated leading toPKC and PLD activation and actin polymerization. Neo-

mycin binds to phosphtidilinositol-4,5 biphosphate (PIP2

)(the substrate of PLC and a cofactor for PLD) and inhibitsthe activity of these two enzymes as well as actin polym-erization (Table 1). However, when actin polymerizationis induced by PMA, conditions in which there is no needfor PLC activity in order to activate PKC, actin polymeriz-ation is inhibited by neomycin (Table 1) but not by thePLC-specific inhibitor U73122 (Cohen et al. 2004), indi-cating that neomycin blocks PLD activation by its bindingto PIP2. The fact that neomycin causes only a small inhi-bition in actin polymerization induced by exogenous PA

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(Table 1), supports this notion. PA is the final product of

PLD activity, therefore endogenous PLD activity is not

needed when exogenous PA is added to the cells. We also

show here that inhibition of MAP kinase-kinase (MAPKK)

or ADP-ribosylation factor (ARF) revealed high inhibition

in actin polymerization induced under capacitation or by

PMA or dbcAMP, but relatively low inhibition whenexogenous PA is added to the cells (Table 1). This indi-

cates that MAPK and ARF are involved in PLD activation.

To summarize this point, we suggest that PLD can be acti-

vated by PKC or PKA pathways via MAPK and ARF acti-

vation leading to actin polymerization. In addition, PLD

can be activated by cAMP independent of PKA, but this

activation does not lead to actin polymerization (Cohen

et al. 2004). A model describing the various pathways isshown in Fig. 1.

The crosstalk between PKA and PKC in sperm

capacitationWe previously described the gradual binding and acti-vation of PLCg to the plasma membrane of bovine spermduring capacitation (Spungin & Breitbart 1996). Since PKCactivity mediates the acrosome reaction (Breitbart et al.1992), we assume that the described activation of PLCgwould result in PKC activation prior to the acrosome reac-tion. We showed that activation of PKC during bovinesperm capacitation causes a rapid increase in actin pol-ymerization which is followed by fast depolymerization(Cohen et al. 2004), probably due to the increase in[Ca2]i (Spungin & Breitbart 1996, Brener et al. 2003).However, because high F-actin formation is needed at the

end of the capacitation time in order to capacitate thesperm (Brener et al. 2003) and this cannot be reachedwhen PKC is highly activated (Cohen et al. 2004), capaci-tation cannot be obtained. Thus, PKC activity should bekept low during sperm capacitation and this is accom-plished by activation of PKA which blocks PLC/PKC activi-ties (Cohen et al. 2004). Activation of PLC prior to theacrosome reaction would require downregulation of PKAtowards the end of the capacitation time. This possibilityis supported by others who have shown decreasingactivity of adenyl cyclase towards the end of capacitationof mouse and human sperm (Adeoya-Osiguwa & Fraser2002, Lefievreet al. 2002).

It seems that under nonphysiological conditions, acti-vation of PKA or PKC can independently cause PLD acti-vation, leading to actin polymerization (Cohen et al.2004). However, under physiological conditions, the PKApathway is obligatory for actin polymerization and capaci-tation, whereas the PKC pathway is important for the acro-some reaction (see model in Fig. 1).

In summary, although PKA or PKC can lead to actinpolymerization, a refined balance between the twopathways is required for optimal and sustained acti-vation during sperm capacitation. The activation of PKAwould cause inhibition of PLC, and prevent PKC acti-vation during capacitation. It appears that PKA acti-vation promotes capacitation whereas early activation

of PKC jeopardizes capacitation. Thus, it would benecessary for inhibition of PKA activity to occur at theend of capacitation in order to achieve PKC activationprior to the acrosome reaction.

Acknowledgements

This research was supported by the Lhel Foundation to HB.The authors declare that there is no conflict of interest thatcould prejudice the impartiality of this work.

Figure 1Remodeling of actin in sperm capacitation and the acrosomereaction (AR). G-actin is polymerized to F-actin during sperm capaci-tation and the fibers should undergo depolymerization in order toaccomplish the AR. Actin polymerization depends on PLD activation,which occurs via the HCO3

2/cAMP/PKA pathway or via the G-proteincoupled receptor (GPCR) (LPA-receptor)/PKC pathway. One of theGPCRs in sperm is LPA-receptor which can be activated by LPA,resulting in PKC activation (Garbi et al.2000) and PLD-dependent

actin polymerization (Cohen et al. 2004). MAP-kinase (MAPK), tyro-sine kinase (TK), and ADP-ribosylation factor (ARF) are involved inPLD activation, leading to phosphatidyl-choline (PC) hydrolysis toproduce phosphatidic acid (PA), which mediates polymerization ofG-actin to F-actin. The binding of capacitated sperm to the egg zonapellucida activates sperm PLC (Tomeset al.1996) to hydrolyze PIP2to diacylglycerol (DAG) and inositol triphosphate (IP3). DAG furtheractivates PKC, and IP3activates the Ca

2 channel in the outer acroso-mal membrane resulting in an increase in intracellular Ca2 concen-tration ([Ca2]i) (OTooleet al.2000). The high increase in [Ca

2]i,activates actin-severing proteins to break down F-actin to G-actinand accomplish the AR.




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Received 17 August 2004First decision 21 October 2004Revised manuscript received 9 November 2004Accepted 15 November 2004



actin and acrosomal process

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