Diagnosis and Prognosis of Male Infertility in Mammal: The Focusing

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Diagnosis and Prognosis of Male Infertility in Mammal: The Focusing of Tyrosine Phosphorylation and Phosphotyrosine Proteins Woo-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 pregnancy in a fertile female. In more than one-third of cases, infertility arises due to the male factor. Therefore, developing strategies for the diagnosis and prognosis of male infertility is critical. Simultaneously, a satisfactory model for the cellular mechanisms that regulate normal sperm function must be established. In this regard, tyrosine phosphorylation is one of the most common mechanisms through which several signal transduction pathways are adjusted in spermatozoa. It regulates the various aspects of sperm function, for example, motility, hyperactivation, capacitation, the acrosome reaction, fertilization, and beyond. Several recent large-scale studies have identified the proteins that are phosphorylated in spermatozoa to acquire fertilization competence. However, most of these studies are basal and have not presented an overall mechanism through which tyrosine phosphorylation regulates male infertility. In this review, we focus of this mechanism, discussing most of the tyrosine-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) and Pathway Studio (v9.0). KEYWORDS: diagnosis, prognosis, male infertility, tyrosine phosphorylation, sperm protein tion,14,18,26−28 and sperm-zona pellucida-binding.17,29 It then facilitates the subsequent fertilization of an oocyte. Therefore, understanding how tyrosine phosphorylation regulates these events in spermatozoa during successful fertilization by fertile males 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 Spermatozoa are produced in the testis, and basic maturation occurs during their epididymal passage.31,32 Consequently, spermatozoa become hyperactivated, with characteristic amplified asymmetrical flagellar beating, which pushes them toward the oocyte in the female reproductive tract.33−35 The next step is capacitation, which is a comparatively more complex and less well understood maturational process. Only capacitated sperm can undergo the acrosome reaction, a process that enables a sperm to penetrate and fertilize an egg.17,18,36,37 Recent studies have demonstrated that all these aforementioned events are regulated by multiple signaling cascades and that tyrosine phosphorylation is involved in almost every step. Yanagimachi38 has reported that spermatozoa from the caput epididymis are unable to fertilize eggs because they lack protein tyrosine phosphorylation activity. This finding suggests that spermatozoa gain their tyrosine phosphorylation capability during

1. INTRODUCTION Infertility is a common problem in humans and animals worldwide that is caused by reproductive factors in male, female, or both. Male infertility is involved in approximately 40% of infertility cases.1 The WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction2 is widely used to evaluate semen samples in laboratories and hospitals. Consequently, conventional methods for the prediction of male fertility based on sperm features such as total and progressive motility,3 morphology,4,5 and DNA quality6 have been studied extensively in the males of domestic species, including bulls,7,8 pigs,9 and rams.10 However, conventional semen analysis cannot precisely diagnose fertility or infertility or determine prognosis in the case of abnormal sperm function or molecular defects in spermatozoa.11 Therefore, research related to sperm function and male fertility must establish more accurate diagnostic and prognostic methods for the management of male infertility and/or subfertility. Spermatozoa are terminally differentiated and highly specialized cells. A mature spermatozoon is the specific output of the testes that can fertilize a functionally mature oocyte in the female reproductive tract during sexual reproduction. The sperm maturation process is predominantly regulated by multiple signaling cascades associated with tyrosine phosphorylation.12−19 Several reports have postulated that, as the most common post-translational modification, tyrosine phosphorylation extensively regulates sperm motility,20−22 hyperactivation,17,23 chemotaxis,24,25 capacitation, the acrosome reac© XXXX American Chemical Society

Special Issue: Proteomics of Human Diseases: Pathogenesis, Diagnosis, Prognosis, and Treatment Received: May 28, 2014

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dx.doi.org/10.1021/pr500524p | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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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.

dependent anion channels proteins (VDACs),14 arginine vasopressin receptor 2 (AVPR2),15 and actin-related protein 2/3 complex,45 alters motility, capacitation, the acrosome reaction, and fertilization by tyrosine phosphorylation. Therefore, studies with similar designs might identify novel proteins that are tyrosine-phosphorylated or affect tyrosine phosphorylation to alter male fertility. The current review summarizes the basic mechanism and importance of tyrosine phosphorylation in the regulation of sperm function. In addition, we focus on the sperm proteome related to tyrosine phosphorylation identified and illustrated by MedScan Reader (v5.0) and Pathway Studio (v9.0), respectively, and its potential implications for the diagnosis and prognosis of male infertility.

epididymal maturation near the time of fertilization. Si and Okuno39 identified four major tyrosine-phosphorylated proteins in sperm flagella that are correlated with motility and hyperactivation. Simultaneously, Visconti and colleagues40,41 successfully evaluated the effect of protein tyrosine phosphorylation on capacitation and the acrosome reaction. In addition, Rajesh and Naz42 have reported a relationship between tyrosine phosphorylation and sperm−zona-binding. Therefore, one can, without considering additional signaling cascades, assume that manipulation of tyrosine phosphorylation might offer a straightforward approach for regulating male fertility. Recent studies have identified several proteins that are phosphorylated in spermatozoa.16,17,43 Additionally, Schumacher et al.44 identified 99 proteins in normozoospermic human spermatozoa, and the phosphorylation status for most of them has been determined. Therefore, attempts should continue to isolate additional proteins that are phosphorylated in spermatozoa from the time of their production in the testis until fertilization to understand the signaling cascades associated with tyrosine phosphorylation and male fertility. We recently reported that blocking or manipulating the function of proteins in spermatozoa, such as ubiquinol− cytochrome c reductase core protein 2 (UQCRC2),13 voltage-

2. PROTEIN PHOSPHORYLATION AND TYROSINE PHOSPHORYLATION IN SPERMATOZOA Phosphorylation is a well-studied post-translational modification of proteins in which protein kinases phosphorylate at serine, threonine, or tyrosine residues by covalently adding a phosphate group.46,47 This phenomenon has been described not only in eukaryotes but also in bacteria and viruses.36 Phosphorylation occurs through the addition of a phosphate group to a protein (or other organic molecule), can induce B



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major conformational changes, and may predispose these proteins to either activation or inactivation. Phosphorylation plays vital roles in almost every cell function, including metabolism, transcription, cell-cycle progression, differentiation, cytoskeleton arrangement, apoptosis, intercellular communication, ionic current modulation, receptor regulation, and neuronal and immunological functions, among others.17,42,46−48 Therefore, phosphoproteomics has been established as a branch of proteomics that focuses solely on the identification and characterization 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 important for the regulation of several physiological functions of spermatozoa required for fertilization, such as motility, hyperactivation, capacitation, the acrosome reaction, and sperm−egg interaction.51−55 A literature review showed that serine/threonine and tyrosine phosphorylation of proteins have been reported in spermatozoa and that the latter is critically important and might be the primary inducer of signal transduction pathways.42 Despite enormous advances in sperm phosphoproteomic research,44,56 the mechanisms of phosphorylation, especially tyrosine phosphorylation, in mammalian spermatozoa remain poorly understood. Capacitating human spermatozoa reportedly shed their sperm cytoplasm, which contains several growth factors/cytokines, including interleukin-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 epidermal growth factor (EGF),64 Ta1,65 and thymosin β4 (Tβ4),66 in addition to several unidentified factors. Several of these factors are also present in the cervical mucus of the female reproductive tract.61 In addition, bovine serum albumin (BSA), which is frequently used in medium to capacitate sperm, has growth factor-like activity.66 In fact, these molecules trigger the phosphorylation of various membrane proteins and receptors after binding to the sperm membrane.67 A model of sperm tyrosine phosphorylation, including the several maturational events induced by this process, is depicted in Figure 1.

Hyperactivation is a vigorous swimming pattern in spermatozoa34 characterized by increased amplitude and asymmetrical flagellar bending that aids the release of spermatozoa from oviductal storage to boost them through the mucus of the oviductal lumen and the matrix of the cumulus oophorus for fertilization.23,68 Hyperactivation of spermatozoa is believed to be a capacitation-associated phenomenon (described in the next subsection); however, abundant experimental data suggest that these phenomena are independent.69−71 The original concept of sperm hyperactivation was described in 1970 by the remarkable findings of Yanagimachi in golden hamster spermatozoa in an in vitro setting.68,72 Despite extensive research on sperm motility, little is known of the molecular mechanism underlying hyperactivation. A literature review showed that the acquisition of sperm motility and hyperactivation may be related to the tyrosine phosphorylation of several sperm proteins along with numerous other factors.17,55,73,74 In this section, we summarize the published studies 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 of sperm tail proteins, thus blocking subsequent hyperactivation.73 A similar finding was reported in more detail in another study.39 Four major tyrosine-phosphorylated proteins in flagellar extracts (∼90, ∼80, ∼62, and ∼48 kDa) that were correlated with sperm motility and hyperactivation have been identified.39 In addition, the incubation of the spermatozoa with either protein kinase A (PKA) or a protein tyrosine kinase inhibitor efficiently prevents both hyperactivation and the tyrosine phosphorylation of flagellar proteins, whereas protein phosphatase inhibitors reduce protein dephosphorylation and the subsequent increase in hyperactivation.39 Conversely, protamines are the major nuclear proteins in spermatozoa. The nucleus of human spermatozoa comprises two protamine types: protamine 1 and protamine 2.75 Changing the expression of sperm protamines reportedly plays a key role in the regulation of sperm motility and fertilization.75,76 Growing evidence suggests that protamines are involved in the condensation of sperm chromatin and might remodel the sperm head shape, responsible for fast swimming velocity, and therefore generate more efficient sperm.76,77 Aleem et al.78 reported that tyrosine phosphorylation occurs in rat sperm protamine during transit to the cauda epididymis and thus could regulate fertilization. These independent experiments establish 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 or pH.23,79−82 Cyclic adenosine monophosphate has the potential to stimulate sperm motility via activation of a cAMP-dependent protein kinase.55,83 Cyclic adenosine monophosphate is believed to induce protein phosphorylation in spermatozoa and subsequently enhance sperm motility. Therefore, tyrosine phosphorylation of sperm proteins may be one of the most critical regulatory mechanisms for hyperactivation in spermatozoa. In addition, Nassar et al.55 have reported that the incubation of spermatozoa with pentoxifylline significantly enhances hyperactivation, which is positively correlated with sperm tail protein phosphorylation. However, the authors did not discuss the effects of protein phosphorylation/protein tyrosine phosphorylation on hyperactivated sperm motility.

3. DIAGNOSIS AND PROGNOSIS OF MALE INFERTILITY INVOLVED IN TYROSINE PHOSPHORYLATION Phosphorylation is among the most common regulatory mechanisms for protein function, regulating cell functions by inducing conformational changes in proteins via allosteric modification. Tyrosine phosphorylation triggers several physiological events in spermatozoa, such as hyperactivation, capacitation, and the acrosome reaction, that are necessary for fertilization both in vitro and in vivo; thus, it can alter male fertility (Figure 1).17 However, the mechanism through which tyrosine phosphorylation/tyrosine-phosphorylated proteins regulate intracellular signaling in spermatozoa and the relevance of such signaling for the diagnosis and prognosis of male infertility remain poorly understood. We discuss this topic below. 3.1. Sperm Motility and Hyperactivation

The capability of a spermatozoon to move progressively toward an egg is defined as motility. Motility is a factor in successful pregnancies because only motile spermatozoa can fertilize an oocyte; poorly motile or immotile spermatozoa cannot.23,68 C



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Table 1. List of Tyrosine-Phosphorylated Proteins in Spermatozoa species

list of proteins/size in kDa

localization on spermatozoa

Boar Boar Bull Buffalo

27, 37, and 40 35 and 46 55 All tyrosine-phosphorylated protein

Bull

Enolase 1

Equatorial, acrosomal, mid and principal piece regions Flagellum

Cat Cynomolgus Monkey Hamster Human

95 and 160 All tyrosine-phosphorylated protein

acrosome reaction Flagellum

All tyrosine-phosphorylated protein 94

Acrosomal region

Human

14−200

Human Human

46 ± 3 Calcium-binding and tyrosine phosphorylation-regulated protein Protein A-kinase anchoring protein Extra-cellular signal-regulated kinases Identified 18 and 33 sperm proteins with and without Y phosphorylation, respectively. A group of tyrosine-phosphorylated protein Phospholipid hydroperoxide glutathione peroxidase

Human Human Human Human Human and bull Human, mouse, and rat Human, mouse, rat, and rabbit Mouse Mouse

Principal piece Acrosomal region

Acrosomal region, midpiece Flagellum

12 ± 2, 25 ± 7, 46 ± 3, and 95 kDa/94 ± 3

Acrosomal region

45−100 52, 75, and 95

Midpiece region

Mouse

PDH E1 β chain (36 kDa)

Acrosomal region and principal piece

Mouse Mouse Mouse

Cytochrome b-c1 complex NADH dehydrogenase (ubiquinone) Fe−S protein 6 acrosin binding protein

Mouse

Aldolase

Mouse Mouse Mouse and bull

Heat shock protein-60 Endoplasmin Voltage-dependent anion channel

Stallion

All tyrosine-phosphorylated protein

Xenopus

42

ref 138 139 74 134

Motility and protection of oxidative stress

51, 141 140 73

Acrosome reaction and zona penetration Capacitation

Principal piece

Heat shock protein-90

functions Acrosome reaction and zona binding zona binding Motility Zona binding

Capacitation and acrosome reaction Signal transduction through the sperm surface progesterone receptor Capacitation, acrosome reaction and zona binding Capacitation and acrosome reaction Calcium signal transduction

18 132

Motility Zona binding Collectively regulates male fertility

147 150 44

Capacitation Motility and viability

56 51, 143 148

Motility and oxidative stress and ROS generation Capacitation and zona binding Zona binding and gamete fusion Capacitation, acrosome reaction and zona binding Hyperactivation and capacitation

29 137 146

52 54 136

Zona binding Zona binding Ion channel activation, calcium influx, interaction with other sperm proteins Capacitation

141, 142 141 141 141, 144 141, 145 149 149 14, 51, 141 97

Egg activation

135

Acrosomal region

Acrosome reaction and zona binding

Principal piece, acrosomal region Acrosomal region Acrosomal region Acrosomal region and flagellum Equatorial region and Flagellum

Energy metabolism

fibrous sheath function improperly.84 In addition, AKAP82, its precursor pro-AKAP82, and a fibrous sheath protein of 95 kDa (FSP95) are reportedly the most prominent tyrosinephosphorylated proteins involved in the regulation of sperm motility and hyperactivation.17,85,86 Recently, we reported that the treatment of mouse spermatozoa with nutlin-3a, a small molecule antagonist of the mouse double minute 2 repressor (MDM2), downregulates UQCRC2 and the ∼100 and 60 kDa tyrosine-phosphorylated proteins, which correlates with reduced sperm motility, hyperactivation, and early embryonic development during in vitro fertilization.13 This study provided evidence that sperm motility and hyperactivation are partially regulated by tyrosine phosphorylation. However, an interaction between UQCRC2 and tyrosine phosphorylation have to be demonstrated. Moreover, another study has proposed that Deamino [Cys 1, D-ArgS] vasopressin (dDAVP), an AVPR2 agonist, significantly

A-kinase anchoring proteins (AKAPs) are tyrosine phosphorylation related proteins that form a major component of the fibrous sheath.43 One study reported that mice lacking AKAP4 and their wild-type counterparts have a similar number of viable spermatozoa; however, the motility of the AKAP4-deficient spermatozoa decreased by nearly 10% and accompanied by sluggish flagellar motion and low amplitude, and these mutants subsequently failed to progress.84 The authors suggested that the decreased motility and hyperactivation were related to a shortened, absent, or substantially reduced fibrous sheath. However, AKAP4-deficient spermatozoa may also reduce the cyclic adenosine monophosphate (cAMP)-mediated phosphorylation of flagellar proteins, which prevents the vigorous flagellar movement with a high-amplitude waveform that characterizes hyperactivated motility.72,84 Sperm motility is lost in the absence of AKAP4 because signal transduction and the activity of several glycolytic enzymes associated with the D



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Figure 2. Interactions related to tyrosine phosphorylation-regulated motility and hyperactivation that ultimately affect male infertility in mammalian spermatozoa. 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, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; PP1, protein phosphatase 1; PRKAR2B, protein kinase, cAMP-dependent, regulatory, type II, 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).

spermatozoa once inside the tract is known as capacitation.36,37 Only capacitated spermatozoa can undergo the acrosome reaction upon binding to the zona pellucida, at which point they become capable of penetrating and fertilizing an egg.13−15 Capacitation confers a series of metabolic and structural modifications to the spermatozoa that are accompanied by changes in membrane fluidity; HCO−3, Ca2+, and cAMP levels; PKA activity; and tyrosine phosphorylation of proteins.14,15,18,88−91 Studies have demonstrated that tyrosine phosphorylation is upregulated during capacitation.92−94 Other studies have consistently reported a positive correlation between capacitation and protein tyrosine phosphorylation in various mammalian species, including humans, that is correlated with fertilization and male fertility.18,40,42,95−101 The relationship between capacitation and tyrosine phosphorylation was discovered before the beginning of the 21st century; however, the basic molecular mechanism underlying tyrosine phosphorylation-mediated control of capacitation, the acrosome reaction, and male fertility is still unclear. Our review of the literature showed that four major signaling pathways modulating tyrosine phosphorylation-guided capacitation and the acrosome reaction have been reported, namely the cAMP/PKA-dependent pathway,8−12,102−104 receptor tyrosine kinase pathway,105,106 nonreceptor protein tyrosine kinase pathway,17 and G-protein coupled receptor pathway.105−107 The cAMP/PKA-dependent pathway is the most extensively studied and is more specific to sperm cells; therefore, we discuss in detail the relationship of this pathway with tyrosine phosphorylation, male fertilization,

decreases sperm motility (%), fertilization, and blastocyst formation when tyrosine phosphorylation levels of the ∼55, ∼23, ∼22, ∼21, and ∼18 kDa proteins decrease.15 Given the aforementioned studies, it is tempting to hypothesize that tyrosine phosphorylation is a mechanism by which motility and hyperactivation in spermatozoa can be controlled with subsequent fertility. However, further studies are required to determine the exact mechanism through which particular proteins regulate protein tyrosine phosphorylation as well as spermatozoa motility, hyperactivation, and subsequent fertility and early embryonic development. Therefore, understanding the molecular basis of these processes requires characterization of the phosphoproteins involved in the relevant signal transduction pathways. In addition, the large number of reported tyrosine-phosphorylated proteins should be more thoroughly characterized. We have generated a schematic representation of the various signaling pathways involved in tyrosine phosphorylation and relationship of tyrosine phosphorylation to other protein functions. These proteins exhibit significant modifications that induce sperm hyperactivation and chemotaxis via the regulation of tyrosine phosphorylation (Table 1, Figure 2). 3.2. Capacitation and Acrosome Reaction

Mature spermatozoa cannot fertilize oocytes even if they are motile and physiologically normal.87 In internally fertilizing animals, mature spermatozoa achieve fertilization competence only after they enter the female genital tract;38 the alterations of E



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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 sperm proteins at Ser and Thr residues to regulate these sperm functions. The main hypothesis has been modified according to Visconti and Kopf.114

with chemically defined media. Furthermore, low concentrations of nitric oxide (NO)-releasing compounds induce capacitation in human115 and cryopreserved bull spermatozoa.116 Interestingly, NO synthase inhibitors meaningfully decrease this process.117,118 Jagan Mohanarao and Atreja119 hypothesized that NO-triggered capacitation in buffalo spermatozoa is associated with an increase in protein tyrosine phosphorylation. The authors identified a group of proteins that undergo tyrosine phosphorylation in response to NOmediated signaling cascades during capacitation.119 Simultaneously, previous studies have shown that Ca2+ and HCO3− have a similar effect, as both ions trigger the activity of mammalian sperm cAMP and subsequently induce capacitation following increased levels of tyrosine phosphorylation.113,120,121 However, the exact mechanism through which these ions stimulate tyrosine phosphorylation remains to be elucidated. Furthermore, the addition of BSA together with Ca2+ and HCO3− to the medium reportedly stimulates protein tyrosine phosphorylation in spermatozoa before capacitation.122 Therefore, it is tempting to hypothesize that the absence of any of these components from the medium might prevent protein tyrosine phosphorylation and capacitation. BSA regulates capacitation by removing cholesterol from the sperm plasma membrane.114 Ions that have similar functions in spermatozoa remain to be elucidated. Therefore, cholesterol release as a signaling event might be highly correlated with tyrosine phosphorylation. This unique manner of signal transduction definitely deserves further investigation. Recently, several researcher have demonstrated that free radicals (especially ROS) can affect male fertility via lipid

and embryonic development (depicted hypothetically in Figure 3).17 Previous studies have reported that tyrosine phosphorylation-dependent acquisition of capacitation and the acrosome reaction in mammalian spermatozoa are achieved through the regulation of a pathway mediated by cAMP (an intracellular second messenger) in which PKA plays a fundamental role.18,40,41,97,99,108−111 Adenosine triphosphate activates membrane bound soluble adenylyl cyclase (sAC) to generate cAMP, and the cAMP in spermatozoa activates PKA, which regulates protein tyrosine phosphorylation.13−15,18,40,41 Most previous studies of cAMP have focused on its role in the regulation of sperm motility; however, its roles in capacitation and the acrosome reaction remain unknown.38,111 Alternatively, PKA might stimulate capacitation and the acrosome reaction of spermatozoa through serine/threonine phosphorylation.112 The optimal level at which cAMP/PKA regulates steady-state levels of tyrosine phosphorylation in sperm under conditions conducive to capacitation, the acrosome reaction, and fertilization will likely be a subject of intense study in the near future. Using the mouse as an experimental model, we and other researchers have demonstrated that several factors including Ca2+,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 protein functions regulate tyrosine phosphorylation-dependent capacitation and the acrosome reaction in spermatozoa.13−15 Therefore, determination of the appropriate concentrations of such factors is critical when inducing sperm capacitation in vitro F



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Figure 4. Interactions related to tyrosine phosphorylation-regulated capacitation and the acrosome reaction that ultimately affect male infertility in mammalian spermatozoa. Abbreviations: ADCY10, adenylate cyclase 10; ATP, adenosine triphosphate; AVP, arginine vasopressin; AVPR2, arginine vasopressin receptor 2; cAMP, cyclic adenosine monophosphate; CFTR, cystic fibrosis transmembrane conductance regulator (ATP-binding cassette subfamily 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-transferase mu 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, serine protease inhibitor; Kazal type-like; SRC, v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homologue (avian); TPA, tetradecanoylphorbol acetate; ZP3, zona pellucida glycoprotein 3 (sperm receptor).

peroxidation and DNA damage.123,124 However, no reports are available on the mechanism through which free radicals affect capacitation and the acrosome reaction. Leclerc et al.125 have found that ROS upregulate the tyrosine phosphorylation levels of several proteins. A similar effect of ROS has been described by Aitken et al.126 Therefore, it seems reasonable to hypothesize that ROS affect capacitation, the acrosome reaction, and subsequent fertilization. However, the effect of ROS on cAMP or other capacitation-related signaling remains to be determined. In addition, pHi is thought to play a critical role in capacitation and the acrosome reaction. Studies have suggested that changes in pHi mainly affect capacitation via the PKA-dependent pathway110 and thus modulate protein tyrosine phosphorylation.110,127 In vitro capacitation can be achieved by incubating spermatozoa in medium containing heparin or oviductal fluid.128,129 Heparin affects tyrosine phosphorylation by upregulating cAMP synthesis, thus affecting sperm capacitation.129 By contrast, glucose has the opposite effect on capacitation. In bovine sperm, glucose inhibits heparin-induced capacitation in vitro through a mechanism involving cAMP metabolism and the reduction of pHi.129 However, a similar effect 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 key molecules and receptors must still be identified to elucidate completely the molecular mechanism and signal transduction cascade involved in capacitation. A G-protein-coupled receptor pathway has not been included in this model.

Recent studies have established that manipulating the function of a sperm protein can regulate capacitation and the acrosome reaction and ultimately affect male fertility. Our previous studies have demonstrated that blocking the function of UQCRC2 with nutlin-3a, a small molecule antagonist of MDM2, lowers male fertility by reducing capacitation rate and the acrosome reaction through downregulation of tyrosine phosphorylation (100 and 60 kDa).13 In another study, we reported that blocking VDACs with the specific inhibitor 4,4′diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) could control capacitation, the acrosome reaction, and fertility by altering tyrosine phosphorylation in ICR mice.14 Interestingly, Park et al.51 reported that VDAC2 and UQCRC2 are more highly expressed in low-fertility (

Diagnosis and Prognosis of Male Infertility in Mammal: The Focusing

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