Diagnosis and Prognosis of Male Infertility in Mammal: The Focusing of Tyrosine Phosphorylation and Phosphotyrosine Proteins

Diagnosis and Prognosis of Male Infertility in Mammal: The Focusingof Tyrosine Phosphorylation and Phosphotyrosine ProteinsWoo-Sung Kwon, Md Saidur Rahman, and Myung-Geol Pang*

Department of Animal Science & Technology, Chung-Ang University, Anseong, Gyeonggi-do 456-756, Republic of Korea

ABSTRACT: Male infertility refers to the inability of a man to achieve a pregnancyin a fertile female. In more than one-third of cases, infertility arises due to the malefactor. Therefore, developing strategies for the diagnosis and prognosis of maleinfertility is critical. Simultaneously, a satisfactory model for the cellular mechanismsthat regulate normal sperm function must be established. In this regard, tyrosinephosphorylation is one of the most common mechanisms through which severalsignal transduction pathways are adjusted in spermatozoa. It regulates the variousaspects of sperm function, for example, motility, hyperactivation, capacitation, theacrosome reaction, fertilization, and beyond. Several recent large-scale studies haveidentified the proteins that are phosphorylated in spermatozoa to acquirefertilization competence. However, most of these studies are basal and have notpresented an overall mechanism through which tyrosine phosphorylation regulatesmale infertility. In this review, we focus of this mechanism, discussing most of thetyrosine-phosphorylated proteins in spermatozoa that have been identified to date.We categorized tyrosine-phosphorylated proteins in spermatozoa that regulate male infertility using MedScan Reader (v5.0) andPathway Studio (v9.0).

KEYWORDS: diagnosis, prognosis, male infertility, tyrosine phosphorylation, sperm protein

1. INTRODUCTION

Infertility is a common problem in humans and animalsworldwide that is caused by reproductive factors in male,female, or both. Male infertility is involved in approximately40% of infertility cases.1 The WHO Laboratory Manual for theExamination of Human Semen and Sperm-Cervical MucusInteraction2 is widely used to evaluate semen samples inlaboratories and hospitals. Consequently, conventional meth-ods for the prediction of male fertility based on sperm featuressuch as total and progressive motility,3 morphology,4,5 andDNA quality6 have been studied extensively in the males ofdomestic species, including bulls,7,8 pigs,9 and rams.10 However,conventional semen analysis cannot precisely diagnose fertilityor infertility or determine prognosis in the case of abnormalsperm function or molecular defects in spermatozoa.11

Therefore, research related to sperm function and male fertilitymust establish more accurate diagnostic and prognosticmethods for the management of male infertility and/orsubfertility.Spermatozoa are terminally differentiated and highly

specialized cells. A mature spermatozoon is the specific outputof the testes that can fertilize a functionally mature oocyte inthe female reproductive tract during sexual reproduction. Thesperm maturation process is predominantly regulated bymultiple signaling cascades associated with tyrosine phosphor-ylation.12−19 Several reports have postulated that, as the mostcommon post-translational modification, tyrosine phosphor-ylation extensively regulates sperm motility,20−22 hyperactiva-tion,17,23 chemotaxis,24,25 capacitation, the acrosome reac-

tion,14,18,26−28 and sperm-zona pellucida-binding.17,29 It thenfacilitates the subsequent fertilization of an oocyte. Therefore,understanding how tyrosine phosphorylation regulates theseevents in spermatozoa during successful fertilization by fertilemales is of paramount importance.Previous in vitro and in vivo studies have shown that

mammalian spermatozoa must undergo a series of complex,stage-specific events for successful fertilization.30 Spermatozoaare produced in the testis, and basic maturation occurs duringtheir epididymal passage.31,32 Consequently, spermatozoabecome hyperactivated, with characteristic amplified asym-metrical flagellar beating, which pushes them toward the oocytein the female reproductive tract.33−35 The next step iscapacitation, which is a comparatively more complex and lesswell understood maturational process. Only capacitated spermcan undergo the acrosome reaction, a process that enables asperm to penetrate and fertilize an egg.17,18,36,37 Recent studieshave demonstrated that all these aforementioned events areregulated by multiple signaling cascades and that tyrosinephosphorylation is involved in almost every step. Yanagima-chi38 has reported that spermatozoa from the caput epididymisare unable to fertilize eggs because they lack protein tyrosinephosphorylation activity. This finding suggests that spermato-zoa gain their tyrosine phosphorylation capability during

Special Issue: Proteomics of Human Diseases: Pathogenesis,Diagnosis, Prognosis, and Treatment

Received: May 28, 2014Published: September 16, 2014

Reviews

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epididymal maturation near the time of fertilization. Si andOkuno39 identified four major tyrosine-phosphorylated pro-teins in sperm flagella that are correlated with motility andhyperactivation. Simultaneously, Visconti and colleagues40,41

successfully evaluated the effect of protein tyrosine phosphor-ylation on capacitation and the acrosome reaction. In addition,Rajesh and Naz42 have reported a relationship between tyrosinephosphorylation and sperm−zona-binding. Therefore, one can,without considering additional signaling cascades, assume thatmanipulation of tyrosine phosphorylation might offer astraightforward approach for regulating male fertility.Recent studies have identified several proteins that are

phosphorylated in spermatozoa.16,17,43 Additionally, Schumach-er et al.44 identified 99 proteins in normozoospermic humanspermatozoa, and the phosphorylation status for most of themhas been determined. Therefore, attempts should continue toisolate additional proteins that are phosphorylated inspermatozoa from the time of their production in the testisuntil fertilization to understand the signaling cascadesassociated with tyrosine phosphorylation and male fertility.We recently reported that blocking or manipulating thefunction of proteins in spermatozoa, such as ubiquinol−cytochrome c reductase core protein 2 (UQCRC2),13 voltage-

dependent anion channels proteins (VDACs),14 argininevasopressin receptor 2 (AVPR2),15 and actin-related protein2/3 complex,45 alters motility, capacitation, the acrosomereaction, and fertilization by tyrosine phosphorylation. There-fore, studies with similar designs might identify novel proteinsthat are tyrosine-phosphorylated or affect tyrosine phosphor-ylation to alter male fertility. The current review summarizesthe basic mechanism and importance of tyrosine phosphor-ylation in the regulation of sperm function. In addition, wefocus on the sperm proteome related to tyrosine phosphor-ylation identified and illustrated by MedScan Reader (v5.0) andPathway Studio (v9.0), respectively, and its potentialimplications for the diagnosis and prognosis of male infertility.

2. PROTEIN PHOSPHORYLATION AND TYROSINEPHOSPHORYLATION IN SPERMATOZOA

Phosphorylation is a well-studied post-translational modifica-tion of proteins in which protein kinases phosphorylate atserine, threonine, or tyrosine residues by covalently adding aphosphate group.46,47 This phenomenon has been describednot only in eukaryotes but also in bacteria and viruses.36

Phosphorylation occurs through the addition of a phosphategroup to a protein (or other organic molecule), can induce

Figure 1. Tyrosine phosphorylation and related events in mammalian spermatozoa (from production to sperm−zona interaction). Abbreviations:BSA, bovine serum albumin; GF, growth factor; IL, interleukin; P4, progesterone; PAF, platelet aggregation factor; TP, tyrosine phosphorylation;ZP, zona proteins.

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major conformational changes, and may predispose theseproteins to either activation or inactivation. Phosphorylationplays vital roles in almost every cell function, includingmetabolism, transcription, cell-cycle progression, differentia-tion, cytoskeleton arrangement, apoptosis, intercellular com-munication, ionic current modulation, receptor regulation, andneuronal and immunological functions, among others.17,42,46−48

Therefore, phosphoproteomics has been established as a branchof proteomics that focuses solely on the identification andcharacterization of proteins that can be phosphorylated(Figures 2, 3, and 5).Mature spermatozoa are widely known to be transcriptionally

and translationally silent27,49 or barely capable of translation.50

Therefore, post-translational modification is potentially im-portant for the regulation of several physiological functions ofspermatozoa required for fertilization, such as motility,hyperactivation, capacitation, the acrosome reaction, andsperm−egg interaction.51−55 A literature review showed thatserine/threonine and tyrosine phosphorylation of proteins havebeen reported in spermatozoa and that the latter is criticallyimportant and might be the primary inducer of signaltransduction pathways.42 Despite enormous advances insperm phosphoproteomic research,44,56 the mechanisms ofphosphorylation, especially tyrosine phosphorylation, inmammalian spermatozoa remain poorly understood. Capacitat-ing human spermatozoa reportedly shed their sperm cytoplasm,which contains several growth factors/cytokines, includinginterleukin-2 (IL-2),57,58 interleukin-6 (IL-6),59 interleukin-8(IL-8),60 colony stimulating factor-1 (CSF-1),61 interferon-g(IFN-g),62 tumor necrosis factor-a (TNF-a),63 epidermalgrowth factor (EGF),64 Ta1,65 and thymosin β4 (Tβ4),66 inaddition to several unidentified factors. Several of these factorsare also present in the cervical mucus of the femalereproductive tract.61 In addition, bovine serum albumin(BSA), which is frequently used in medium to capacitatesperm, has growth factor-like activity.66 In fact, these moleculestrigger the phosphorylation of various membrane proteins andreceptors after binding to the sperm membrane.67 A model ofsperm tyrosine phosphorylation, including the several matura-tional events induced by this process, is depicted in Figure 1.

3. DIAGNOSIS AND PROGNOSIS OF MALEINFERTILITY INVOLVED IN TYROSINEPHOSPHORYLATION

Phosphorylation is among the most common regulatorymechanisms for protein function, regulating cell functions byinducing conformational changes in proteins via allostericmodification. Tyrosine phosphorylation triggers several phys-iological events in spermatozoa, such as hyperactivation,capacitation, and the acrosome reaction, that are necessaryfor fertilization both in vitro and in vivo; thus, it can alter malefertility (Figure 1).17 However, the mechanism through whichtyrosine phosphorylation/tyrosine-phosphorylated proteinsregulate intracellular signaling in spermatozoa and the relevanceof such signaling for the diagnosis and prognosis of maleinfertility remain poorly understood. We discuss this topicbelow.

3.1. Sperm Motility and Hyperactivation

The capability of a spermatozoon to move progressively towardan egg is defined as motility. Motility is a factor in successfulpregnancies because only motile spermatozoa can fertilize anoocyte; poorly motile or immotile spermatozoa cannot.23,68

Hyperactivation is a vigorous swimming pattern in spermato-zoa34 characterized by increased amplitude and asymmetricalflagellar bending that aids the release of spermatozoa fromoviductal storage to boost them through the mucus of theoviductal lumen and the matrix of the cumulus oophorus forfertilization.23,68 Hyperactivation of spermatozoa is believed tobe a capacitation-associated phenomenon (described in thenext subsection); however, abundant experimental data suggestthat these phenomena are independent.69−71 The originalconcept of sperm hyperactivation was described in 1970 by theremarkable findings of Yanagimachi in golden hamsterspermatozoa in an in vitro setting.68,72 Despite extensiveresearch on sperm motility, little is known of the molecularmechanism underlying hyperactivation. A literature reviewshowed that the acquisition of sperm motility and hyper-activation may be related to the tyrosine phosphorylation ofseveral sperm proteins along with numerous other fac-tors.17,55,73,74 In this section, we summarize the publishedstudies on the possible correlations among sperm motility,hyperactivation, tyrosine phosphorylation, and male fertility.In 1999, Mahony and Gwathmey showed that treating

spermatozoa with genistein, a tyrosine kinase inhibitor,significantly decreased tyrosine phosphorylation levels ofsperm tail proteins, thus blocking subsequent hyperactivation.73

A similar finding was reported in more detail in anotherstudy.39 Four major tyrosine-phosphorylated proteins inflagellar extracts (∼90, ∼80, ∼62, and ∼48 kDa) that werecorrelated with sperm motility and hyperactivation have beenidentified.39 In addition, the incubation of the spermatozoa witheither protein kinase A (PKA) or a protein tyrosine kinaseinhibitor efficiently prevents both hyperactivation and thetyrosine phosphorylation of flagellar proteins, whereas proteinphosphatase inhibitors reduce protein dephosphorylation andthe subsequent increase in hyperactivation.39 Conversely,protamines are the major nuclear proteins in spermatozoa.The nucleus of human spermatozoa comprises two protaminetypes: protamine 1 and protamine 2.75 Changing the expressionof sperm protamines reportedly plays a key role in theregulation of sperm motility and fertilization.75,76 Growingevidence suggests that protamines are involved in thecondensation of sperm chromatin and might remodel thesperm head shape, responsible for fast swimming velocity, andtherefore generate more efficient sperm.76,77 Aleem et al.78

reported that tyrosine phosphorylation occurs in rat spermprotamine during transit to the cauda epididymis and thuscould regulate fertilization. These independent experimentsestablish a strong correlation among motility, hyperactivation,tyrosine phosphorylation, and fertilization.Most studies on sperm motility have focused on the effects of

cyclic adenosine monophosphate (cAMP) and calcium orpH.23,79−82 Cyclic adenosine monophosphate has the potentialto stimulate sperm motility via activation of a cAMP-dependentprotein kinase.55,83 Cyclic adenosine monophosphate isbelieved to induce protein phosphorylation in spermatozoaand subsequently enhance sperm motility. Therefore, tyrosinephosphorylation of sperm proteins may be one of the mostcritical regulatory mechanisms for hyperactivation in sperma-tozoa. In addition, Nassar et al.55 have reported that theincubation of spermatozoa with pentoxifylline significantlyenhances hyperactivation, which is positively correlated withsperm tail protein phosphorylation. However, the authors didnot discuss the effects of protein phosphorylation/proteintyrosine phosphorylation on hyperactivated sperm motility.



A-kinase anchoring proteins (AKAPs) are tyrosine phosphor-ylation related proteins that form a major component of thefibrous sheath.43 One study reported that mice lacking AKAP4and their wild-type counterparts have a similar number of viablespermatozoa; however, the motility of the AKAP4-deficientspermatozoa decreased by nearly 10% and accompanied bysluggish flagellar motion and low amplitude, and these mutantssubsequently failed to progress.84 The authors suggested thatthe decreased motility and hyperactivation were related to ashortened, absent, or substantially reduced fibrous sheath.However, AKAP4-deficient spermatozoa may also reduce thecyclic adenosine monophosphate (cAMP)-mediated phosphor-ylation of flagellar proteins, which prevents the vigorousflagellar movement with a high-amplitude waveform thatcharacterizes hyperactivated motility.72,84 Sperm motility islost in the absence of AKAP4 because signal transduction andthe activity of several glycolytic enzymes associated with the

fibrous sheath function improperly.84 In addition, AKAP82, itsprecursor pro-AKAP82, and a fibrous sheath protein of 95 kDa(FSP95) are reportedly the most prominent tyrosine-phosphorylated proteins involved in the regulation of spermmotility and hyperactivation.17,85,86

Recently, we reported that the treatment of mousespermatozoa with nutlin-3a, a small molecule antagonist ofthe mouse double minute 2 repressor (MDM2), downregulatesUQCRC2 and the ∼100 and 60 kDa tyrosine-phosphorylatedproteins, which correlates with reduced sperm motility,hyperactivation, and early embryonic development during invitro fertilization.13 This study provided evidence that spermmotility and hyperactivation are partially regulated by tyrosinephosphorylation. However, an interaction between UQCRC2and tyrosine phosphorylation have to be demonstrated.Moreover, another study has proposed that Deamino [Cys 1,D-ArgS] vasopressin (dDAVP), an AVPR2 agonist, significantly

Table 1. List of Tyrosine-Phosphorylated Proteins in Spermatozoa

species list of proteins/size in kDa localization on spermatozoa functions ref

Boar 27, 37, and 40 Acrosome reaction and zona binding 138Boar 35 and 46 zona binding 139Bull 55 Motility 74Buffalo All tyrosine-phosphorylated protein Equatorial, acrosomal, mid

and principal piece regionsZona binding 134

Bull Enolase 1 Flagellum Motility and protection of oxidative stress 51,141

Cat 95 and 160 acrosome reaction Acrosome reaction and zona penetration 140CynomolgusMonkey

All tyrosine-phosphorylated protein Flagellum Capacitation 73

Hamster All tyrosine-phosphorylated protein Capacitation and acrosome reaction 18Human 94 Acrosomal region Signal transduction through the sperm

surface progesterone receptor132

Human 14−200 Capacitation, acrosome reaction and zonabinding

29

Human 46 ± 3 Capacitation and acrosome reaction 137Human Calcium-binding and tyrosine phosphorylation-regulated

proteinPrincipal piece Calcium signal transduction 146

Human Protein A-kinase anchoring protein Principal piece Motility 147Human Extra-cellular signal-regulated kinases Acrosomal region Zona binding 150Human Identified 18 and 33 sperm proteins with and without Y

phosphorylation, respectively.Collectively regulates male fertility 44

Human A group of tyrosine-phosphorylated protein Capacitation 56Human and bull Phospholipid hydroperoxide glutathione peroxidase Acrosomal region, midpiece Motility and viability 51,

143Human, mouse,and rat

Heat shock protein-90 Flagellum Motility and oxidative stress and ROSgeneration

148

Human, mouse,rat, and rabbit

12 ± 2, 25 ± 7, 46 ± 3, and 95 kDa/94 ± 3 Acrosomal region Capacitation and zona binding 52

Mouse 45−100 Midpiece region Zona binding and gamete fusion 54Mouse 52, 75, and 95 Capacitation, acrosome reaction and zona

binding136

Mouse PDH E1 β chain (36 kDa) Acrosomal region andprincipal piece

Hyperactivation and capacitation 141,142

Mouse Cytochrome b-c1 complex 141Mouse NADH dehydrogenase (ubiquinone) Fe−S protein 6 141Mouse acrosin binding protein Acrosomal region Acrosome reaction and zona binding 141,

144Mouse Aldolase Principal piece, acrosomal

regionEnergy metabolism 141,

145Mouse Heat shock protein-60 Acrosomal region Zona binding 149Mouse Endoplasmin Acrosomal region Zona binding 149Mouse and bull Voltage-dependent anion channel Acrosomal region and

flagellumIon channel activation, calcium influx,interaction with other sperm proteins

14, 51,141

Stallion All tyrosine-phosphorylated protein Equatorial region andFlagellum

Capacitation 97

Xenopus 42 Egg activation 135



decreases sperm motility (%), fertilization, and blastocystformation when tyrosine phosphorylation levels of the ∼55,∼23, ∼22, ∼21, and ∼18 kDa proteins decrease.15 Given theaforementioned studies, it is tempting to hypothesize thattyrosine phosphorylation is a mechanism by which motility andhyperactivation in spermatozoa can be controlled withsubsequent fertility. However, further studies are required todetermine the exact mechanism through which particularproteins regulate protein tyrosine phosphorylation as well asspermatozoa motility, hyperactivation, and subsequent fertilityand early embryonic development. Therefore, understandingthe molecular basis of these processes requires characterizationof the phosphoproteins involved in the relevant signaltransduction pathways. In addition, the large number ofreported tyrosine-phosphorylated proteins should be morethoroughly characterized. We have generated a schematicrepresentation of the various signaling pathways involved intyrosine phosphorylation and relationship of tyrosine phos-phorylation to other protein functions. These proteins exhibitsignificant modifications that induce sperm hyperactivation andchemotaxis via the regulation of tyrosine phosphorylation(Table 1, Figure 2).

3.2. Capacitation and Acrosome Reaction

Mature spermatozoa cannot fertilize oocytes even if they aremotile and physiologically normal.87 In internally fertilizinganimals, mature spermatozoa achieve fertilization competenceonly after they enter the female genital tract;38 the alterations of

spermatozoa once inside the tract is known as capacitation.36,37

Only capacitated spermatozoa can undergo the acrosomereaction upon binding to the zona pellucida, at which pointthey become capable of penetrating and fertilizing an egg.13−15

Capacitation confers a series of metabolic and structuralmodifications to the spermatozoa that are accompanied bychanges in membrane fluidity; HCO−3, Ca2+, and cAMP levels;PKA activity; and tyrosine phosphorylation of pro-teins.14,15,18,88−91 Studies have demonstrated that tyrosinephosphorylation is upregulated during capacitation.92−94

Other studies have consistently reported a positive correlationbetween capacitation and protein tyrosine phosphorylation invarious mammalian species, including humans, that is correlatedwith fertilization and male fertility.18,40,42,95−101 The relation-ship between capacitation and tyrosine phosphorylation wasdiscovered before the beginning of the 21st century; however,the basic molecular mechanism underlying tyrosine phosphor-ylation-mediated control of capacitation, the acrosome reaction,and male fertility is still unclear. Our review of the literatureshowed that four major signaling pathways modulating tyrosinephosphorylation-guided capacitation and the acrosome reactionhave been reported, namely the cAMP/PKA-dependentpathway,8−12,102−104 receptor tyrosine kinase pathway,105,106

nonreceptor protein tyrosine kinase pathway,17 and G-proteincoupled receptor pathway.105−107 The cAMP/PKA-dependentpathway is the most extensively studied and is more specific tosperm cells; therefore, we discuss in detail the relationship ofthis pathway with tyrosine phosphorylation, male fertilization,

Figure 2. Interactions related to tyrosine phosphorylation-regulated motility and hyperactivation that ultimately affect male infertility in mammalianspermatozoa. Abbreviations: ADCY10, adenylate cyclase 10; AKAP, A-kinase anchor proteins; AKAP3, A-kinase (PRKA) anchor protein 3; AKAP4,A-kinase (PRKA) anchor protein 4; ATP, adenosine triphosphate; AVP, arginine vasopressin; AVPR2, arginine vasopressin receptor 2; CABYR,calcium binding tyrosine-(Y)-phosphorylation regulated; cAMP, cyclic adenosine monophosphate; CATSPER2c cation channel, sperm associated 2;CD55, CD55 molecule; decay accelerating factor for complement; CNR1, cannabinoid receptor 1; CRISP1, cysteine-rich secretory protein 1; IP3,inositol trisphosphate; ODF2, outer dense fiber of sperm tails 2; PI3K, phosphatidylinositol 3-kinase; PKA, cAMP-dependent protein kinase, proteinkinase A; PKC, protein kinase C; PLC, phospholipase C; PP1, protein phosphatase 1; PRKAR2B, protein kinase, cAMP-dependent, regulatory, typeII, beta; PRKCA, protein kinase C, alpha; ROPN1, rhophilin associated tail protein 1; ROPN1L, rhophilin associated tail protein 1-like; ROS,reactive oxygen species; SLC9A10, solute carrier family 9, member 10; SRC, v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homologue(avian); ZP3, zona pellucida glycoprotein 3 (sperm receptor).



and embryonic development (depicted hypothetically in Figure3).17

Previous studies have reported that tyrosine phosphoryla-tion-dependent acquisition of capacitation and the acrosomereaction in mammalian spermatozoa are achieved through theregulation of a pathway mediated by cAMP (an intracellularsecond messenger) in which PKA plays a fundamentalrole.18,40,41,97,99,108−111 Adenosine triphosphate activates mem-brane bound soluble adenylyl cyclase (sAC) to generate cAMP,and the cAMP in spermatozoa activates PKA, which regulatesprotein tyrosine phosphorylation.13−15,18,40,41 Most previousstudies of cAMP have focused on its role in the regulation ofsperm motility; however, its roles in capacitation and theacrosome reaction remain unknown.38,111 Alternatively, PKAmight stimulate capacitation and the acrosome reaction ofspermatozoa through serine/threonine phosphorylation.112 Theoptimal level at which cAMP/PKA regulates steady-state levelsof tyrosine phosphorylation in sperm under conditionsconducive to capacitation, the acrosome reaction, andfertilization will likely be a subject of intense study in thenear future.Using the mouse as an experimental model, we and other

researchers have demonstrated that several factors includingCa2+,113,114 HCO3

−,115,116 BSA,117 heparin,118,119 glucose,reactive oxygen species (ROS),120,121 intracellular pH(pHi),

110,114 and upregulation or blockage of de novo proteinfunctions regulate tyrosine phosphorylation-dependent capaci-tation and the acrosome reaction in spermatozoa.13−15

Therefore, determination of the appropriate concentrations ofsuch factors is critical when inducing sperm capacitation in vitro

with chemically defined media. Furthermore, low concen-trations of nitric oxide (NO)-releasing compounds inducecapacitation in human115 and cryopreserved bull spermato-zoa.116 Interestingly, NO synthase inhibitors meaningfullydecrease this process.117,118 Jagan Mohanarao and Atreja119

hypothesized that NO-triggered capacitation in buffalospermatozoa is associated with an increase in protein tyrosinephosphorylation. The authors identified a group of proteinsthat undergo tyrosine phosphorylation in response to NO-mediated signaling cascades during capacitation.119 Simulta-neously, previous studies have shown that Ca2+ and HCO3

−

have a similar effect, as both ions trigger the activity ofmammalian sperm cAMP and subsequently induce capacitationfollowing increased levels of tyrosine phosphorylation.113,120,121

However, the exact mechanism through which these ionsstimulate tyrosine phosphorylation remains to be elucidated.Furthermore, the addition of BSA together with Ca2+ andHCO3

− to the medium reportedly stimulates protein tyrosinephosphorylation in spermatozoa before capacitation.122 There-fore, it is tempting to hypothesize that the absence of any ofthese components from the medium might prevent proteintyrosine phosphorylation and capacitation. BSA regulatescapacitation by removing cholesterol from the sperm plasmamembrane.114 Ions that have similar functions in spermatozoaremain to be elucidated. Therefore, cholesterol release as asignaling event might be highly correlated with tyrosinephosphorylation. This unique manner of signal transductiondefinitely deserves further investigation.Recently, several researcher have demonstrated that free

radicals (especially ROS) can affect male fertility via lipid

Figure 3. Possible mechanism underlying hyperpolarization, capacitation, and the acrosome reaction in mammalian spermatozoa. Protein kinase-A(PKA)-stimulated tyrosine phosphorylation induces capacitation and the acrosome reaction. PKA might also regulate the phosphorylation of spermproteins at Ser and Thr residues to regulate these sperm functions. The main hypothesis has been modified according to Visconti and Kopf.114



peroxidation and DNA damage.123,124 However, no reports areavailable on the mechanism through which free radicals affectcapacitation and the acrosome reaction. Leclerc et al.125 havefound that ROS upregulate the tyrosine phosphorylation levelsof several proteins. A similar effect of ROS has been describedby Aitken et al.126 Therefore, it seems reasonable tohypothesize that ROS affect capacitation, the acrosomereaction, and subsequent fertilization. However, the effect ofROS on cAMP or other capacitation-related signaling remainsto be determined. In addition, pHi is thought to play a criticalrole in capacitation and the acrosome reaction. Studies havesuggested that changes in pHi mainly affect capacitation via thePKA-dependent pathway110 and thus modulate protein tyrosinephosphorylation.110,127

In vitro capacitation can be achieved by incubatingspermatozoa in medium containing heparin or oviductalfluid.128,129 Heparin affects tyrosine phosphorylation byupregulating cAMP synthesis, thus affecting sperm capacita-tion.129 By contrast, glucose has the opposite effect oncapacitation. In bovine sperm, glucose inhibits heparin-inducedcapacitation in vitro through a mechanism involving cAMPmetabolism and the reduction of pHi.

129 However, a similareffect has not been demonstrated in an in vivo trial.Furthermore, these cascades may not be mutually exclusive,and may include cross-talk among several molecules. Many keymolecules and receptors must still be identified to elucidatecompletely the molecular mechanism and signal transductioncascade involved in capacitation. A G-protein-coupled receptorpathway has not been included in this model.

Recent studies have established that manipulating thefunction of a sperm protein can regulate capacitation and theacrosome reaction and ultimately affect male fertility. Ourprevious studies have demonstrated that blocking the functionof UQCRC2 with nutlin-3a, a small molecule antagonist ofMDM2, lowers male fertility by reducing capacitation rate andthe acrosome reaction through downregulation of tyrosinephosphorylation (100 and 60 kDa).13 In another study, wereported that blocking VDACs with the specific inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) could con-trol capacitation, the acrosome reaction, and fertility by alteringtyrosine phosphorylation in ICR mice.14 Interestingly, Park etal.51 reported that VDAC2 and UQCRC2 are more highlyexpressed in low-fertility (<70% Nonreturn rate) bulls. Becausehigher expression levels of VDAC2 and UQCRC2 were foundin the lower-fertility genotype, the blocking of these proteinsshould improve fertility. Possible explanations for thediscrepancies in results are that a particular protein couldaffect capacitation, the acrosome reaction, and fertilizationwhen expressed at a certain level. Alternatively, increasedexpression levels of VDAC2 and UQCRC2 in less fertile males(bulls) could be a compensation for lowered fertility. Similarly,another study has proposed that exposure of mice spermatozoato the specific AVPR2 agonist dDAVP decreases the PKAsubstrate level (23 kDa) and tyrosine phosphorylation level (30kDa) dose dependently, subsequently affecting capacitation, theacrosome reaction, and embryo development.15 Therefore,further studies should attempt to evaluate the effect of severalsperm proteins on fertilization and their association withprotein tyrosine phosphorylation. The application of Pathway

Figure 4. Interactions related to tyrosine phosphorylation-regulated capacitation and the acrosome reaction that ultimately affect male infertility inmammalian spermatozoa. Abbreviations: ADCY10, adenylate cyclase 10; ATP, adenosine triphosphate; AVP, arginine vasopressin; AVPR2, argininevasopressin receptor 2; cAMP, cyclic adenosine monophosphate; CFTR, cystic fibrosis transmembrane conductance regulator (ATP-binding cassettesubfamily C, member 7); CYCS, cytochrome c, somatic; DMBT1, deleted in malignant brain tumors 1; EGF, epidermal growth factor; EGFR,epidermal growth factor receptor; GPD2, glycerol-3-phosphate dehydrogenase 2 (mitochondrial); GSN, Gelsolin; GSTM3, glutathione S-transferasemu 3; HDL, high-density lipoprotein; IP3, inositol trisphosphate; IZUMO1, izumo sperm-egg fusion 1; LY6G6C, lymphocyte antigen 6 complex,locus G6C; MAPK3, mitogen-activated protein kinase 3; MIF, macrophage migration inhibitory factor (glycosylation-inhibiting factor); PDHA1,pyruvate dehydrogenase (lipoamide) alpha 1; PKA, cAMP-dependent protein kinase, protein kinase A; PKC, protein kinase C; Spinkl, serineprotease inhibitor; Kazal type-like; SRC, v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homologue (avian); TPA, tetradecanoylphorbolacetate; ZP3, zona pellucida glycoprotein 3 (sperm receptor).



Studio helped us analyze the proteins implicated in tyrosinephosphorylation-mediated regulation of capacitation, theacrosome reaction, and male fertility (Table 1, Figure 4).

3.3. Sperm-Zona Binding and Postfertilization EggActivation

Tyrosine phosphorylation has been recognized as an importantphenomenon correlated with various maturational processes inspermatozoa (described earlier). Several studies have alsoreported a direct association among tyrosine phosphorylation,sperm−zona-binding capability, and postfertilization eggactivation. It has been reported that zonadhesin, a spermmembrane protein, binds postacrosomally to the zonapellucida, acting as a primary gamete-recognition compo-nent.130 An elegant study conducted by Herlyn and Zischler131

reported that, together with several conformational changes,phosphorylation motifs occur in zonadhesin (D domains) andare primarily responsible for its function in various animalspecies. Accordingly, tyrosine phosphorylation is the mostcommon post-translational modification/phosphorylation inmammalian spermatozoa.17,42 Therefore, it could be hypothe-sized that tyrosine phosphorylation has a critical role in thespecies-specific binding of zonadhesin to the zona pellucida.Naz et al.52 have reported that the incubation of humanspermatozoa with an antiphosphotyrosine antibody significantlyinhibits sperm binding and penetration of zona-free hamster

eggs. In another study, Naz and Ahmad53 demonstrated thattyrosine phosphorylation occurs in three human sperm proteins(95, 51, and 14−18 kDa) capable of binding zona pellucidaproteins and stimulating sperm-zona binding. Therefore, itseems reasonable to hypothesize that tyrosine phosphorylationof sperm proteins plays a dynamic role in sperm−zonainteraction and binding.An excellent study by Urner et al.54 showed that the level of

tyrosine phosphorylation increases during zona binding andgamete fusion in mice spermatozoa. Furthermore, the authorsshowed that, following binding to the oocyte zona pellucida andoolemma, tyrosine phosphorylation in the sperm flagellum isdelayed in the absence of glucose (described earlier).Therefore, this study provides a direct link between proteintyrosine phosphorylation and gamete interaction. In addition,Nassar et al.55 have reported that exposure of humanspermatozoa to pentoxifylline increases protein phosphoryla-tion levels in sperm tail and the capability of sperm to bind tothe zona pellucida. However, the molecular mechanismunderlying protein phosphorylation/tyrosine phosphorylation-stimulated binding remains to be elucidated and might be anexcellent target for research. Progesterone is well documentedto trigger the rapid influx of Ca2+ and acrosomal exocytosis inthe spermatozoa of various animal species. Tesarik et al.132 havereported a selective increase in the tyrosine phosphorylationlevels of a 94 kDa phosphoprotein in the presence of

Figure 5. Interactions related to tyrosine phosphorylation-regulated sperm−zona interaction and egg activation that ultimately affect male fertility inmammalian spermatozoa. Abbreviations: ADCY10, adenylate cyclase 10; AGT, angiotensinogen (serpin peptidase inhibitor, clade A, member 8);ALB, albumin; Amp1, amplitude of circadian rhythm 1; ATP, adenosine triphosphate; AVP, arginine vasopressin; AVPR2, arginine vasopressinreceptor 2; cAMP, cyclic adenosine monophosphate; CCNB2, cyclin B2; CRISP1, cysteine-rich secretory protein 1; CTSB, cathepsin B; EGF,epidermal growth factor; FYN, FYN oncogene related to SRC, FGR, YES; GSTM3, glutathione S-transferase mu 3; IL6, interleukin 6 (interferon,beta 2); IP3, inositol trisphosphate; MAPK1, mitogen-activated protein kinase 1; PECAM1, platelet/endothelial cell adhesion molecule; PI3K,phosphatidylinositol 3-kinase; PKA, cAMP-dependent protein kinase, protein kinase A; PLC, phospholipase C; PP1, protein phosphatase 1; ROS,reactive oxygen species; SHC1, SHC (Src homology 2 domain containing) transforming protein 1; SRC, v-src sarcoma (Schmidt-Ruppin A-2) viraloncogene homologue (avian); TPA, tetradecanoylphorbol acetate; VDAC, voltage-dependent anion channel; ZP3, zona pellucida glycoprotein 3(sperm receptor).



progesterone. Because progesterone induces spermatozoabinding to and penetration of the oocyte plasma membrane,133

it is reasonable to hypothesize that tyrosine phosphorylation isthe signal that regulates progesterone-mediated spermactivation and fertilization. However, the molecular basis ofthis relationship remains to be determined. A similar affiliationbetween tyrosine phosphorylation and zona binding wasreported in a recent study by Kadirvel et al.134 These authorsshowed a strong correlation between the protein tyrosinephosphorylation pattern and zona-binding capability in vitro incapacitated (heparin induced) and frozen−thawed (cryocapa-citated) buffalo spermatozoa. In addition, they observed adifference in zona-binding capability between frozen−thawedand in vitro capacitated spermatozoa; however, an explanationwas not provided. By contrast, studies in Xenopus laevis (clawedfrog) eggs by Sato and colleagues135 have demonstrated proteintyrosine phosphorylation and dephosphorylation pathways insperm-induced egg activation. However, similar studies inmammalian spermatozoa have not yet been reported. Wegenerated an illustration of the sperm protein signalingpathways to facilitate understanding of the mechanismsunderlying tyrosine phosphorylation, egg activation, andsperm−zona binding (Table 1, Figure 5).

4. SUMMARY AND FUTURE PROSPECTSConventional semen analysis has several drawbacks in thediagnosis and prognosis of male infertility, subfertility, or both.Therefore, the development of new approaches is extraordi-narily important. In this regard, sperm functions and themechanism of male fertility must be fully understood. Thetyrosine phosphorylation of sperm proteins is a centralregulator of many key activities in spermatozoa, all of whichare required for fertilization. Despite previous research efforts,several questions remain to be addressed for a more completeview of tyrosine phosphorylation-regulated sperm functions andtheir roles in male fertility. Our literature review yielded manyreports that tyrosine phosphorylation in spermatozoa isregulated by several factors; however, the underlyingmechanism is less clearly defined. Moreover, future studiesshould focus on the detection and isolation of other factors thatmay affect tyrosine phosphorylation as well as male (in)fertility.Manipulation of sperm protein function using special agonistsor antagonists may regulate tyrosine phosphorylation inspermatozoa.5−7 Although recently applied proteomic ap-proaches have identified several sperm-specific proteins, theirfunctions in tyrosine phosphorylation and their association withfertility are unclear. Therefore, future studies should attempt toprovide a robust understanding of tyrosine phosphorylationand male infertility. Such findings will assist in the developmentof more accurate tools for the diagnosis and prognosis of maleinfertility.

■ AUTHOR INFORMATIONCorresponding Author

*Telephone: +82.31.670.4841. Fax: +82.31.675.9001. E-mail:[email protected]

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis study was supported by the Cooperative ResearchProgram for Agricultural Science and Technology Develop-

ment (Project No. PJ008415) of the Rural DevelopmentAdministration, Republic of Korea.

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Diagnosis and Prognosis of Male Infertility in Mammal: The Focusing of Tyrosine Phosphorylation and Phosphotyrosine Proteins