Review Article Sperm Proteomics: Road to Male Fertility...

Hindawi Publishing CorporationInternational Journal of EndocrinologyVolume 2013 Article ID 360986 11 pageshttpdxdoiorg1011552013360986

Review ArticleSperm Proteomics Road to Male Fertility and Contraception

Md Saidur Rahman June-Sub Lee Woo-Sung Kwon and Myung-Geol Pang

Department of Animal Science and Technology School of Bioresource and Bioscience Chung-Ang University4726 Seodong-daero Anseong Gyeonggi-Do 456-756 Republic of Korea

Correspondence should be addressed to Myung-Geol Pang mgpangcauackr

Received 3 June 2013 Revised 1 November 2013 Accepted 2 November 2013

Academic Editor Mario Maggi

Copyright copy 2013 Md Saidur Rahman et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Spermatozoa are highly specialized cells that can be easily obtained and purified Mature spermatozoa are transcriptionallyand translationally inactive and incapable of protein synthesis In addition spermatozoa contain relatively higher amounts ofmembrane proteins compared to other cells therefore they are very suitable for proteomic studies Recently the application ofproteomic approaches such as the two-dimensional polyacrylamide gel electrophoresis mass spectrometry and differential in-gelelectrophoresis has identified several sperm-specific proteins These findings have provided a further understanding of proteinfunctions involved in different sperm processes as well as of the differentiation of normal state from an abnormal one In additionstudies on the sperm proteome have demonstrated the importance of spermatozoal posttranslationalmodifications and their abilityto induce physiological changes responsible for fertilization Large-scale proteomic studies to identify hundreds to thousands ofsperm proteins will ultimately result in the development of novel biomarkers that may help to detect fertility the state of completecontraception and beyond Eventually these protein biomarkers will allow for a better diagnosis of sperm dysfunctions and aidin drug development This paper reviews the recent scientific publications available from the PubMed database to address spermproteomics and its potential application to characterize male fertility and contraception

1 Introduction

Spermatozoa are highly specialized cells with specificmetabolic pathways compartmentalized in different regionsUnlike other body cells spermatozoa are exceptionallydifferentiated and show large variations in their geneticcellular and chromatin structures which allow them tocontrol fertilityinfertility embryo development and hered-ity Physiologically normal spermatozoa are required forsuccessful fertilization whereas suboptimal sperm qualitywith abnormal motility morphology concentration DNAfragmentation chromatin stability and genetic compositionhas been directly linked to male factors of infertility [1ndash3]However our current understanding of spermatozoa includ-ing their different physiological and pathological aspects isstill limited and not well defined The human genome hasbeen sequenced more than 10 years ago with a large readilyaccessible database which is considered the starting pointfor the understanding of how a tissue-specific cell functionson the molecular level in health and disease conditions [4]

However without considering proteomics DNA sequencedata alone answer very few or no questions about the levelof protein expression Therefore to explore the biologicaldisplay of the human genome the physiological functions ofeach protein should be understood first [4 5]

Proteomics involves the comprehensive study of proteinswith their particular structural and functional aspects [6]Recent studies of spermatozoa from the proteomic pointof view have allowed the identification of different pro-teins in spermatozoa that are responsible for the regulationof normaldefective sperm functions Mature spermatozoaare inactive with regard to transcription translation andprotein synthesis [1] and are therefore very suitable forproteomic analysis [7] As such proteomics has the potentialto transform our understanding of how mature sperm cellswork In addition it is important to note that a spermcell contributes about half of its nuclear genetic materialto the successive diploid offspring during fertilization thusexamining the genetic composition of spermatozoa mightalso provide beneficial insights into congenital disorders

2 International Journal of Endocrinology

At present numerous proteomics techniques includingtwo-dimensional (2D) polyacrylamide gel electrophoresismass spectrometry (MS) and differential in-gel electrophore-sis are widely used to identify particular sperm-specificproteins These approaches have offered a concise but deepunderstanding of different functional aspects of sperm pro-teins for example motility capacitation acrosomal reac-tion fertilization and posttranslational modifications suchas phosphorylation glycosylation proteolytic cleavages andmutations In addition proteomic research has also provideda new horizon to study different functional states of sperma-tozoa for example normal versus malformed immature ver-sus mature capacitated versus incapacitated low versus highsperm count low versus high fertility and normalhighlyfertile versus state of complete contraception [1 8ndash10]which are all necessary and important to identify a suitablebiomarker and to select the desired sire Furthermore designand construction of agonisticantagonistic approaches usingcommercially synthesized purified chemicals with regard todifferentially expressed proteins of spermatozoa might behelpful to develop fertility enhancers new treatments forinfertility and contraception pills The present work reviewsthe latest articles published by other laboratories aswell as ourresearch team on the proteomics aspect of spermatozoa andtheir potential implications to assist fertility or to develop amale contraceptive strategy

2 Biology of the Sperm Cell

Spermatozoa are mature male gametes that are producedthrough a unique process called spermatogenesis In mam-mals spermatogenesis occurs in the male testes and epi-didymis in a stepwise fashion that is highly dependent onoptimal conditions for the process to work correctly Sper-matozoa undergo morphological biochemical and physio-logical modifications in the testes and epididymis [11] How-ever the complete differentiation process largely dependson changes at the genetic cellular and chromatin level [11ndash14] Cellular adjustment that occurs during spermiogenesisincludes nuclear condensation movement of the nucleus tothe periphery of the cell and formation of the acrosome andflagella (depicted in Figure 1) To attain successful fertilizationboth in vitro and in vivo mammalian spermatozoa mustundergo capacitation followed by the acrosome reactionwhich are important processes that finally allow the maturesperm cell to penetrate the zona pellucida and to fuse withthe oocyte plasma membrane during fertilization and earlyembryonic development [15] (see Figure 1(a)) In contrast theentire process of spermiogenesis results in significant changesin the sperm chromatin structure and DNA strand breaksto allow the accompanying change in DNA topology (seeFigure 1(b)) Both in vitro and in vivo evidence suggests thatthe DNA-binding and condensing activities of a set of basicnuclear ldquotransition proteinsrdquo is critical to the integrity of theentire chromatin remodeling process [16]

3 Background of Sperm Proteomic Research

Comprehensive identification and quantification of variousproteins expressed in cells and tissues offer important andfascinating insights into the dynamics of cellular functionsand differentiation The novel approaches of characterizingproteins by means of both qualitative and quantitative analy-ses are known as proteomics Proteins carry out a wide rangeof functions from forming structural materials to catalyzingchemical reactions thus knowing exactly which proteins arein sperm cells is a major step forward in understanding Theso-called proteome of spermatozoa contains everything thesperm needs to survive and function correctly The studyof sperm proteins was in fact initiated with the remarkablefinding by Friedrich Miescher in 1874 Miescher was ableto identify the basic component of salmon spermatozoacalled ldquoprotaminerdquo that was coupled with nuclein whichwas known as DNA afterward Oliver and Dixon [17] andBalhorn [18] noted that the protamines initially isolated byMiescher are the most abundant sperm nuclear proteins inmany animal species as well as the initial proteins identifiedin the spermatozoal nucleus However the founders of spermproteomics study were Naaby-Hansen and his colleaguesfrom Virginia and their elegant studies addressed the firsthumanrsquos sperm protein database of 1400 spots [19] With therecent development of modern proteomic approaches suchas the application of narrow-range isoelectric focusing stripsandmultiple 2D gels this numbermight bemuch higher thanthat previously reported [20ndash22]

4 Basic Techniques of Sperm ProteomicResearch

In order to understand how sperm proteins can be identifiedand characterized the fundamental process of proteomicanalysis will be addressed in brief After collection and liq-uefaction of a semen sample it can be used as it is or it can besubjected to additional processing for purification of sperma-tozoa The ejaculate comprises various cellular componentsfor example seminal plasma secretions from the accessorysex gland blood cells and epithelial cells Therefore duringsperm proteomic analysis contamination of seminal plasmaproteins might be the reason for the substantial differencesobserved between samples from the same donors [23 24]To minimize this type of contamination the direct swim-upmethod and the density gradientmethod (using Percoll) havebeen suggested previously for sperm isolation in humans [2324] However the Percoll method demonstrated much betterreproducibility than the swim-up method [23] Recentlydifferent density gradient methods have been applied toisolate spermatozoa from both human and animal subjectsFor example a discontinuous density gradient of 90minus45Percoll was used to remove extender debris seminal plasmaand dead spermatozoa in bovine semen [24 25] A 35PureSperm solution was used to separate caput and caudaepididymal sperm inmice [26] In swine a two-layer isotonicPercoll gradient (90minus40) was used to purify epididymalsperm [27] whereas 80minus55 Percoll was used to separate

International Journal of Endocrinology 3

(a) (b)

∙ DNA replication∙ Transcription∙ Repair

∙ Transcription∙ Repair

∙ Genetic recombination

Transition proteins

Protaminesbinding

Histone activation

Epigenetic modification

Differentiation

bullTranscriptionbullRepair

Spermatogonia2N

Meiosis I

Primaryspermatocyte

tetrad (2N)

Secondaryspermatocyte

Meiosis II

Spermatidmonad (N)

Spermatozoa(N)

Mitosis

Capacitation

Acrosomereaction

FertilizationMaturesperm

Epigenetic marks

coupled with histone

Nucleohistone

toroid

toroid

Matrix attachment region

Pronucleus (male)

DNA in the nucleosome

nucleoprotamine toroidProtamine-DNA

DNA sensitive

Donut-loop model(protamine-DNA within the toroid)

dyad (N)

sim(5ndash15) sperm DNA sim(85ndash95) sperm DNA in

Figure 1 Schematic diagram showing the cellular genetic and chromatin changes during spermatogenesis (a) The figure represents thecellular changes during spermatogenesis coupled with its genetic modifications Following spermatogenesis the male primordial germcells spermatogonia first differentiate to primary spermatocytes and undergo genetic recombination to produce haploid round spermatidsThe round spermatids then participate in another differentiation process to produce the mature spermatozoa For successful fertilizationmammalian spermatozoa undergo first capacitation and then the acrosome reaction which collectively allows spermatozoa to penetrate thezona pellucida and to fuse with the oocyte plasma membrane both in vivo and in vitro (b) The figure shows chromatin changes that causethe transition of the mammalian nucleohistone to nucleoprotamine [17 18] In addition the Donut-Loop model has been inserted at the endto represent the internal structure of the protamine-DNA fibers inside the toroid [17 18] The red color indicates histones and the blue colorindicates DNA

spermatozoa from the ejaculate [28] On the basis of theaforementioned studies it is important to select purificationmethods for sperm cells that are suitable for the speciesstudied

The basic techniques used in proteomics research largelyfocus on illuminating the structure and conformation purify-ing and measuring the concentration of sperm proteins Theproteomic techniques that are used for the structural analysisof sperm proteins include MS and electrophoresis [26 27]nuclear magnetic resonance spectroscopy [29 30] and X-ray crystallography [31 32] However liquid chromatography

(LC) [33 34] and the recently developed microfluidic sepa-ration technique [35 36] are widely used for the purificationof proteins from spermatozoa or other samples In additionwestern blot [37 38] and chemical microarrays [39] areused to determine the concentrationdensity of a particularprotein in sperm cells by using the corresponding antibodyof the protein of interest Furthermore various stains suchas Coomassie brilliant blue and silver stain are used toidentify the positions of individual proteins within the gelwhich appear as spots or smudges Data from the structuralanalysis can be used to identify the functions of various


sperm proteins by comparison with similar proteins in thespermwith known functions However this process generallyrequires a large protein conformation as well as availablefunctional databases sophisticated software to analyze thedata and an algorithm platform

However the most frequently used extraction and anal-ysis process of proteins from spermatozoa involves 2D gelelectrophoresis followed by excision of protein spots fromthe 2D gel and matrix-assisted laser desorptionionization-MS (MALDI-MS) or tandem MS (MSMS) identification[20 40 41] Alternatively the initial protein extract is digestedinto peptides separated through LC and analyzed byMSMS(see Figure 2) [42 43]

5 Proteomic Approach to Define MaleFertility

Recently proteomic tools have gained a unique positionbecause of their significant contribution in protein researchAs a major technique such proteomic approaches are largelyapplied to identify proteins that regulate the biologicalfunctions of a cell In the field of sperm cell biology theidentification of a wide range of proteins provides informa-tion to uncover the regulatory mechanism of male fertilityas well as infertility for which the reason is unknown orpoorly understood The current section reviews the value ofproteomics in male fertility by using the recently availablescientific information

Reports show that the male factors account for approxi-mately 50 of all infertility in human and bovine when artifi-cial insemination has been used [1] however the underlyingmolecularmechanisms are still not understood [44]Over thepast few decades the traditionally availablemethods to detectmale fertility include assessments of sperm morphology[45] motility [46] mucus penetration test (cervical) [47]membrane intactness [48] the acrosome reaction [49] ATPconcentration in semen [50] and sperm-zona binding capa-bility [51] however their clinical value is always debated [52]Therefore the identification of marker proteins of fertilitymight bemore appropriate to determine themale potentialityfor successful reproduction

In recent work from our laboratory we have identifieddifferential protein expression profiles from high- and low-fertility bulls in order to characterize high-fertility bull sper-matozoa [1] In this study the lower limit of the higher fertilitywas defined as gt70 nonreturn rate Database searchinghelped to identify proteins that are highly expressed infertile bull spermatozoa The identified proteins involvedenolase 1 ATP synthase H+ transporting mitochondrial F1complex beta subunit apoptosis-stimulating of p53 protein2 alpha-2-HS-glycoprotein and phospholipid hydroperox-ide glutathione peroxide these proteins primarily regulatemetabolism posttranslational modification and motilityof spermatozoa However 3 proteins of low-fertility bullswere highly represented that is voltage dependent anionchannel 2 (VDAC2) ropporin-1 and ubiquinol-cytochrome-c reductase complex core protein 2 These findings arein accordance with the results of Liu et al [53] who

reported that VDAC2 may induce infertility with idiopathicasthenozoospermia However several reports have shownthat ubiquinol-cytochrome-c reductase complex core protein2 is associated with oxidative stress and ROS generation inmitochondria [54 55] in spermatozoa both events play aparadoxical role in the regulation of fertility However wewere unable to represent a mechanism by which ropporin-1 correlates with low fertility Finally we demonstrated thatenolase 1 VDAC2 and ubiquinol-cytochrome-c reductasecomplex core protein 2 were significantly correlated withindividual fertility and essentially represent the true fertilitymarkers for the respective bull Therefore a similar studywill certainly be helpful to construct and design a newbiomarker to identify fertile individuals Gaviraghi et al [56]have reported that the candidate protein of a high-fertilitybull is involved in sperm-egg interactions and cell cycleregulation In their study 2D electrophoresis coupled withMALDI-MS techniques was used to identify the differentiallyexpressed proteins Notably Peddinti et al [25] employed thedifferential detergent fractionation multidimensional pro-tein identification technology and identified 125 putativebiomarkers of fertility In particular they found that high-fertility bull spermatozoa contained higher levels of proteinsthat are involved in energy metabolism cell communica-tion spermatogenesis and cell motility In addition similarmethods together with 2D electrophoresis and LC-MSMSwere considered by DrsquoAmours et al [57] to identify high-fertile Holstein bulls However these researchers were ableto identify only 2 proteins (adenylate kinase isoenzyme 1 andphosphatidylethanolamine-binding protein 1) that aremainlyexpressed in fertile bull spermatozoa and that can explain64 (119875 lt 0001) of the fertility scores of the respective bullTherefore it is important to note that a major variation inprotein identification always exists in these studies The moststraightforward explanation for this variation might be toconsider the different breeds of animals as well as the factthat in most of these studies 2D electrophoresis analysis wasconducted with pooled samples In addition contaminationof seminal plasma proteins might be another reason forthe substantial differences observed between samples fromthe same donors Therefore intensive studies on individualproteins and their contribution to reproduction should beconducted to address these open questions Furthermoreimmunolocalization and expression patterns of such proteinsshould also be considered as they can readily be used toobtain some information about their function (see Figure 3)

For successful fertilization spermatozoa should firstundergo capacitation [58ndash60] and then the acrosome reac-tion [15] which is the common sequence of biochemicaland physiological modifications facilitating the sperm cellto penetrate the zona pellucida and to fuse with the oocyteplasma membrane A capacitation-induced alteration oracrosome reaction of the sperm proteome can thereforebe a consequence of posttranslational modifications of pre-existing proteins or of the expression of new proteins orof both In any case proteomic approaches are mandatoryfor the elucidation of the molecular processes underlyingcapacitation and acrosomal reaction both of which shouldbe described in more detail For a better understanding of


SemenDensitygradient

Spermatozoa

Seminal fluid (after washing)

Extraction of proteins

Digest

pH10

pH3

Crude extracts

Digestpeptides

Excise

Digested

PeptideLC(HPLC)

Reverse phaseseperation

MSMS

Database searchProtein identification

Laser

Source Drift tubeTOF tube

Time of light

MS data

MS (MALDI-TOF)

1000

1500

2000

2500

3000

3500

4000

1D PAGE

65

Molecularweight (KDa)

200pI-3 pI-112D PAGE

7M urea 2M Thio U1 chaps 10mM DTT

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

100

90

80

70

60

50

40

30

20

10

0

2[c]A3

800 g

(KD

a)

220

116170

977659

41

30

2014

(s)

Inte

nsity

()

mzmz

Figure 2 Schematic diagram showing the extraction and analysis of proteins from sperm cells After collection and liquefaction of a wholesemen sample it can be used as it is or it can be subjected to density gradient centrifugation to separate mature and immature spermpopulations The recovered spermatozoa can be extracted and purified after which the protein concentration can be determined in order toallow for an equal protein-loading gradient on electrophoretic gelsThe further analysis of the sperm proteome is done from an aliquot of thesperm cells by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by excision of protein spots from the 2D-PAGEgels and matrix-assisted laser desorptionionization-mass spectroscopy (MALDI-MS) or tandem MS (MSMS) identification Alternativelythe initial protein extract can be digested into peptides which can be separated via liquid chromatography followed by MSMS analysis

the fertilizing capability of males attempts have been madein order to identify spermatozoa that differentially expressproteins related to such fundamentally important events InTable 1 a brief summary is given of recent animal studieswhere proteomic approaches have been utilized towardsaddressing male fertility

Since 10 years relatively few studies in the literature haveexamined the proteome of human spermatozoa in orderto define the fertile man however recently the number

of similar studies on human spermatozoa has been rapidlyincreasing Pixton et al [41] utilized a man who experiencedfailed fertilization at in vitro fertilization (patient) and differ-ent fertile controlsThey reported that although the proteomevariation among the fertile donors was very low they wereable to identify 20 proteins that were differentially expressedbetween fertile and non-fertile patients However a maindrawback of this study is that they considered only 1 patientTherefore consideration of a wider range of samples with


Acrosome

NucleusNuclear membrane

MitochondriaDense fibers

Flagellum

Fibrous sheath

Circumferential fibers

End piece

AnnulusMicrotubules

Axial filamentH

ead

Mid

pie

ceTa

il

Acrosome Acrosomal cap

Proximal centriole

CRISP2 ESP HSP70 PK-SSPACA1 IZUMO1 VDAC2

VDAC3 Arp 23 complex and so on

PLC zeta SHTAPcalicin cylicin I II ACP

arp 23 complex and so on

GAPDH PKA AVPR2GPX4 ropporin Arp

23 complex AQPs and so on

AVPR2 VDAC2VDAC3 ropporin Arp


ENO1 CRISP2 SHTAP

Figure 3 Diagram showing a mature human spermatozoon with its different parts Previously published studies were used as a referenceto summarize the list of proteins localized in different regions of the spermatozoon by immunolocalization Proteins localized in acrosomehead midpiece and tail are mainly involved in capacitation and acrosomal reaction spermatozoa-oocyte interaction (zona binding) energyproduction and metabolism and motility and metabolism respectively The localization of a single protein at multiple sites in the spermcell indicates its potential involvement in different physiological processes of the spermatozoon The listed proteins include protein kinase A(PKA) phospholipase C zeta (PLC zeta) sperm head- and tail-associated protein (SHTAP) calicin cylicins I and II actin-capping proteins(ACP) enolase 1 (ENO1) cysteine-rich secretory protein 2 (CRISP2) arginine vasopressin receptor (AVPR) voltage-dependent anion channel(VDAC) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) glutathione peroxidase 4 (GPX4) equatorial segment protein (ESP) heatshock protein 70 (HSP70) novel pyruvate kinase (PK-S) sperm acrosomemembrane-associated protein 1 (SPACA1) izumo sperm-egg fusion1 (IZUMO1) aquaporin 7 (AQPs) and the actin-related protein 23 complex (Arp23 complex)

available field data (fertilizing capability nonreturn rate inanimals) might be helpful to identify particular differences inthe protein expression that can represent the fertile individualmore accurately Eventually such studies will enable furtherelucidation of the underlining molecular mechanisms andmight shed additional light on key sperm proteins involvedin infertility

Apart from the fact that there are few other studiesthat attempted to characterize the sperm plasma membrane[71] spermatozoa-specific processes such as calcium-bindingability and capacitation [72] also provide important infor-mation on the fertility of human as well as animal speciesAlthough it was almost more than 15 years ago that tyrosinephosphorylationwas identified as an indicator of capacitationand it is known to play an essential role in fertilizationthe role of the proteins and their associated sequence ofactivation is still a matter of question Only a small numberof candidate proteins for this important event of fertilizationhave been identified to date [73] Reports demonstrated that

the phospholipid hydroperoxide glutathione peroxidase inspermatozoa of hamster is phosphorylated during tyrosinephosphorylation following capacitation [74] However itis still a long way to reveal the whole picture of humansperm physiology especially from the proteomic point ofview We summarized some proteomics studies on humanspermatozoawhere attempts have beenmade to identifymalefertility (Table 2)

6 Sperm Proteomic versus MaleContraception

The world population continues to increase more rapidlyeven after female contraception has been significantlyemphasizedThis pressure of increased population has causedvarious environmental hazards global warming hungerand diseases Consequently researchers are now looking forsuitable ways to control the birth rate of humans as well


Table 1 Summary of major proteomic studies to define the fertile male (animal models)

Sample Proteomic technique Study outcome AuthorsMouse spermatozoa LC-MSMS Identification of 52 proteins following capacitation Baker et al [61]

Boar spermatozoa LC-MS Identification of proteins from the sperm surface thatare responsible for binding of the sperm to the oocyte Belleannee et al [27]

Bull spermatozoa LC-MSMS Identification of 131 proteins to differentiate normal andpyriform sperm Shojaei Saadi et al [62]

Bull spermatozoa (Holstein) LC-MSMSIdentification of proteins of impaired sperm functiondue to elevated testicular temperature with importantimplications for fertility predictions

Newton et al [63]

Accessory sex gland fluid (Holstein) LC-MSMS Increased expression of bovine seminal plasma proteinidentified in high-fertility bulls Moura et al [64]

Accessory sex gland (dairy bull) LC-MSMS Identification of isoforms of osteopontin andphospholipase A2 (PLA2) in high-fertility bulls Moura et al [65]

Mouse spermatozoa MSMSIdentification of 299 testis-specific proteins anddetection of 155 novel proteins involved inspermiogenesis

Guo et al [66]

Mouse spermatozoa MSMS Identification of 100 proteins that are expressed onmature sperm at the site of sperm-oocyte interactions Stein et al [67]

Rat spermatozoa LC-MSMS Identification of 5123 peptides from rat spermatozoa Baker et al [68]

Mouse spermatozoa MSMSIdentification of capacitation-associated changes in thephosphorylation status (55 in vivo sites) of mousesperm proteins

Piatt et al [69]

Mouse spermatozoa PMF LC-MSMS Identification of 100 proteins of capacitation Nixon et al [70]LC-MSMS liquid chromatography-tandem mass spectrometry LC-MS liquid chromatography-mass spectrometry MSMS tandem mass spectrometryPMF peptide mass fingerprints

Table 2 Proteomic studies on human spermatozoa to access male fertility

Authors Proteomic technique Study outcome

Zhao et al [75] MALDI-TOFIdentification of 10 differentially expressed proteins inasthenozoospermic patients compared with those ofnormozoospermic donors

Martınez-Heredia et al [40] MALDI-TOF Identification of 131 proteins to provide a reference 2Dmap of mature human sperm spermatozoa

Johnston et al [76] LC-MSMS Identification of 1760 proteins that comprise humansperm

Domagala et al [77] LC-MS Identification of 35 proteins as sperm immunogenicantigens

Secciani et al [78] MSMS Identification of protein profiles of capacitated versusejaculated spermatozoa

Li et al [20] MALDI-MS Establishment of a high-resolution 2D map of humansperm proteins by identifying 16 proteins

Chan et al [79] MADLI-TOF Identification of 12 proteins to address altered proteinphosphorylation and aberrant sperm motility

Baker et al [42] LC-MSMS Identification of 1056 gene products from human spermpopulations

Lefievre et al [80] MSMS Identification of 240 S-nitrosylated proteins detected toaddress nitric oxide-induced capacitation

De Mateo et al [81] MALDI-TOF Identification of 101 spots from infertile patients

Bohring et al [22] MALDI-MS Identification of 18 proteins (sperm membraneantigens)

MALDI-TOF matrix-assisted laser desorptionionization-time of flight LC-MSMS liquid chromatography-tandem mass spectrometry LC-MS liquidchromatography-mass spectrometry MSMS tandem mass spectrometry


as of domestic animals especially pets Reports have shownthat about half of all human pregnancies are unwanted orunexpected [82] Therefore it becomes obvious to considerthe participation of men in contraceptive programs Themost available male contraception methods involve the useof condoms and vasectomy The efficacy of such methodsis quite satisfactory however their popularity is arguedAt present few hormonal methods (injected implanted ortaken orally) are also used for male contraception whichis believed to play an important role to serve this purpose[10] Although these methods have some drawbacks as theycause minor but significant effects on a number of non-reproductive parameters some of them are used with con-siderable popularity However frequently arising questionsagainst the use of such methods include their potentialefficacy for providing a state of contraception Therefore it isimportant to determine the efficacy of such methods in addi-tion to searching for new methods that could potentially beused to serve this purpose while providing higher protectionThe present section will review the application of proteomicapproaches to confirm the state of complete contraception byusing existing contraception methods as well as to establishnew methods for male contraception

In an advance headed for a long-sought newmale contra-ceptive researchers have attempted to identify key proteins inmen that could effectively suppress the production of spermwhich might become a new target for a potential male birthcontrol pill Inmale hormonal contraception the administra-tion of supraphysiologic doses of testosterone (TU) has beenshown to significantly reduce circulating gonadotropins andinduce severe oligozoospermia or azoospermia [82] How-ever supraphysiologic doses of TU are unable to suppressspermatogenesis uniformly and are associated with weightgain and suppression of high-density lipoprotein cholesterol[83 84] Exogenous progestins are also known to suppresscirculating gonadotropins and might synergistically suppressspermatogenesis when combined with exogenous TUThere-fore various combination regimens of TU plus a progestinsuch as levonorgestrel and cyproterone acetate have beenreported as potential hormonal contraceptives An elegantstudy conducted by Cui et al [10] reported that TU combinedwith levonorgestrel was able to change 31 sperm proteinscompared to only 13 proteins that changed in men givenonly testosterone In addition this research group noted thattreatment ofmenwith TU alone or TU+ levonorgestrel effec-tively suppressed spermatogenesis throughmultiple signalingpathways by altering the expression of different proteinsTherefore it is tempting to hypothesize that the identifiedproteins could serve as both targets for new male contracep-tives as well as medications for treating men that experienceinfertility However repeated trials are necessary in order torepresent a specific group of proteins to address the state ofcomplete contraception In addition Kwon et al [38] havereported that exposure of mouse spermatozoa to deamino[Cys 1 d-ArgS] vasopressin an arginine vasopressin receptor-2 agonist significantly decreased spermmotility intracellularpH and phospho-protein kinase A substrates and increasedCa2+ concentration This research helped to summarize thata higher level of arginine vasopressin a neurohypophysial

hormone has a damaging effect on overall sperm functionTherefore such studymight also be important to the progressof diagnostic tests to access male potentiality for fertilization

Nonhormonal methods including the use of compoundsthat target sperm functionality might also be attractivebecause of their theoretical promise of specificity for thereproductive tract The study of sperm proteomics continuesto identify possible targets for this approach Such non-hormonal agents will likely enter clinical trials in the nearfuture Recently we have reported that blocking VDAC2and VDAC3 proteins with 441015840-diisothiocyanostilbene-221015840-disulfonic acid significantly decreased sperm motility via-bility acrosome reaction capacitation tyrosine phosphory-lation fertilization and embryo development and proposedthat VDACs are essential for successful reproduction [37]This study indicated that the abnormal functioning ofVDACsnegatively affects sperm functions and that VDACs actas one of the key regulators for the fertilizing ability ofspermatozoa More recently Shukla et al [85] reported thatnutlin-3a inhibits the functions of ubiquinol-cytochrome-creductase core protein 2 in spermatozoa during capacitationresulting ultimately in decreased male fertility Thereforeit is suggested that similar studies should be conducted toidentify particular proteins that could play pivotal roles inmale fertilityinfertility Moreover a wide range of studiestargeting a single protein with a high number of repetitionscan improve its application in clinical conditions

7 Conclusion and Future Directions of SpermProteomic Research Targeting Male Fertilityand Contraception

The belief in the modern world is that spermatozoa providea potential source of study to investigate the numerousoutstanding findings that control reproduction in generalProteomics represents an extraordinary tool for evaluatingthe molecular mechanisms regulating sperm function forshedding light on our understanding of the male fertil-ityinfertility and for characterizing the state of completecontraception It can be considered to address spermatozoalposttranslational modifications as well as to elucidate themolecular basis of defective sperm functions with implica-tions for our ability to both diagnose and treat this conditionTherefore the identified proteins should be rigorously testedto determine whether they have any clinical value for fer-tilityinfertility evaluation Simultaneously factors that canaffect proteomics should be considered and all of these mustbe taken into account when translating discovery proteomicsinto clinical trials and practice

Several databases of sperm proteins are now acting asan important source of knowledge Catalogues of proteinsderived from proteomic approaches are valuable as they rep-resent the group of proteins that can be exposed in a partic-ular physiologicalpathological condition more specificallyhowever the functions of each protein are still unknown[40 80 86] Some proteins exhibit various functions theymay play different roles in the whole organism but mayhave a specific function in spermatozoa Therefore intensive


studies on individual proteins (ie agonistic and antagonistchemicals and knockout and knock-inmodels) and their con-tribution to reproduction should be undertaken to addressthese open questions

Comparison of data from the proteome and transcrip-tome also provides a more reliable basis to evaluate theexact mechanisms of the respective protein that controlsfertilityinfertility or contraception Taken together it mayprovide new avenues for the management of male reproduc-tion in general and novel targets for expected reproductiveoutcomes

Conflict of Interests

There is no conflict of interests to report

Acknowledgments

This study was financially supported by the CooperativeResearch Program for Agriculture Science and TechnologyDevelopment (project no PJ008415) Rural DevelopmentAdministration Republic of Korea Md Saidur Rahman wassupported through the ldquoChung-Ang University Young Scien-tist Scholarship (CAYSS)rdquo Chung-Ang University Korea

References

[1] Y J ParkW S Kwon S AOh et al ldquoFertility-related proteomicprofiling bull spermatozoa separated by percollrdquo Journal ofProteome Research vol 11 no 8 pp 4162ndash4168 2012

[2] P J Turek ldquoPractical approaches to the diagnosis and manage-ment of male infertilityrdquoNature Clinical Practice Urology vol 2no 5 pp 226ndash238 2005

[3] I D Sharlip J P JarowAM Belker et al ldquoBest practice policiesfor male infertilityrdquo Fertility and Sterility vol 77 no 5 pp 873ndash882 2002

[4] S S du Plessis A H Kashou D J Benjamin et al ldquoProteomicsa subcellular look at spermatozoardquo Reproductive Biology andEndocrinology vol 22 no 9 article 36 2011

[5] I A Brewis ldquoThe potential importance of proteomics toresearch in reproductionrdquo Human Reproduction vol 14 no 12pp 2927ndash2929 1999

[6] N L Anderson and N G Anderson ldquoProteome and pro-teomics new technologies new concepts and new wordsrdquoElectrophoresis vol 19 no 11 pp 1853ndash1861 1998

[7] T F Wu and D S Chu ldquoSperm chromatin fertile grounds forproteomic discovery of clinical toolsrdquo Molecular and CellularProteomics vol 7 no 10 pp 1876ndash1886 2008

[8] W Xu H Hu Z Wang et al ldquoProteomic characteristics ofspermatozoa in normozoospermic patients with infertilityrdquoJournal of Proteomics vol 18 no 75 pp 5426ndash5436 2012

[9] G Jagan Mohanarao and S K Atreja ldquoIdentification of capaci-tation associated tyrosine phosphoproteins in buffalo (Bubalusbubalis) and cattle spermatozoardquo Animal Reproduction Sciencevol 123 no 1-2 pp 40ndash47 2011

[10] Y Cui H Zhu Y Zhu et al ldquoProteomic analysis of testis biop-sies in men treated with injectable testosterone undecanoatealone or in combination with oral levonorgestrel as potentialmale contraceptiverdquo Journal of Proteome Research vol 7 no 9pp 3984ndash3993 2008

[11] S Kimmins and P Sassone-Corsi ldquoChromatin remodelling andepigenetic features of germ cellsrdquo Nature vol 434 no 7033 pp583ndash589 2005

[12] S Rousseaux C Caron J Govin C Lestrat A-K Faureand S Khochbin ldquoEstablishment of male-specific epigeneticinformationrdquo Gene vol 345 no 2 pp 139ndash153 2005

[13] E Seli and D Sakkas ldquoSpermatozoal nuclear determinants ofreproductive outcome implications for ARTrdquo Human Repro-duction Update vol 11 no 4 pp 337ndash349 2005

[14] A L Kierszenbaum and L L Tres ldquoThe acrosome-acroplax-ome-manchette complex and the shaping of the spermatidheadrdquo Archives of Histology and Cytology vol 67 no 4 pp 271ndash284 2004

[15] L R Fraser ldquoSperm capacitation and the acrosome reactionrdquoHuman Reproduction vol 13 no 1 pp 9ndash19 1998

[16] G Boissonneault ldquoChromatin remodeling during spermiogen-esis a possible role for the transition proteins in DNA strandbreak repairrdquo FEBS Letters vol 514 no 2-3 pp 111ndash114 2002

[17] R Oliva and G H Dixon ldquoVertebrate protamine genes and thehistone-to-protamine replacement reactionrdquo Progress in NucleicAcid Research and Molecular Biology vol 40 pp 25ndash94 1991

[18] R Balhorn ldquoThe protamine family of sperm nuclear proteinsrdquoGenome Biology vol 8 no 9 article 227 2007

[19] S Naaby-Hansen C J Flickinger and J C Herr ldquoTwo-dimensional gel electrophoretic analysis of vectorially labeledsurface proteins of human spermatozoardquo Biology of Reproduc-tion vol 56 no 3 pp 771ndash787 1997

[20] L-W Li L-Q Fan W-B Zhu et al ldquoEstablishment of a high-resolution 2-D reference map of human spermatozoal proteinsfrom 12 fertile sperm-bank donorsrdquoAsian Journal of Andrologyvol 9 no 3 pp 321ndash329 2007

[21] M R Wilkins J-C Sanchez A A Gooley et al ldquoProgresswith proteome projects why all proteins expressed by a genomeshould be identified andhow to do itrdquoBiotechnology andGeneticEngineering Reviews vol 13 pp 19ndash50 1996

[22] C Bohring E Krause BHabermann andWKrause ldquoIsolationand identification of sperm membrane antigens recognized byantisperm antibodies and their possible role in immunologicalinfertility diseaserdquoMolecular Human Reproduction vol 7 no 2pp 113ndash118 2001

[23] K L Pixton E D Deeks F M Flesch et al ldquoSperm proteomemapping of a patient who experiencd failed fertilization at IVFreveals altered expression of at least 20 proteins compared withfertile donors case reportrdquo Human Reproduction vol 19 no 6pp 1438ndash1447 2004

[24] Y J Park J Kim Y A You et al ldquoProteomic revolution toimprove tools for evaluating male fertility in animalsrdquo Journalof Proteome Research vol 12 no 11 pp 4738ndash4747 2013

[25] D Peddinti B Nanduri A Kaya J M Feugang S C Burgessand E Memili ldquoComprehensive proteomic analysis of bovinespermatozoa of varying fertility rates and identification ofbiomarkers associated with fertilityrdquo BMC Systems Biology vol22 no 2 pp 1ndash9 2008

[26] T W Ijiri T Merdiushev W Cao and G L Gerton ldquoIdentifi-cation and validation of mouse sperm proteins correlated withepididymal maturationrdquo Proteomics vol 11 no 20 pp 4047ndash4062 2011

[27] C Belleannee M Belghazi V Labas et al ldquoPurification andidentification of sperm surface proteins and changes duringepididymal maturationrdquo Proteomics vol 11 no 10 pp 1952ndash1964 2011


[28] F M Flesch B Colenbrander L M G van Golde and BM Gadella ldquoCapacitation induces tyrosine phosphorylation ofproteins in the boar sperm plasmamembranerdquo Biochemical andBiophysical Research Communications vol 262 no 3 pp 787ndash792 1999

[29] T H Hereng P H Backe J Kahmann et al ldquoStructure andfunction of the human sperm-specific isoform of protein kinaseA (PKA) catalytic subunit C1205722rdquo Journal of Structural Biologyvol 178 no 3 pp 300ndash310 2012

[30] F Odet S A Gabel JWilliams R E London E Goldberg andE M Eddy ldquoLactate dehydrogenase C and energy metabolisminmouse spermrdquoBiology of Reproduction vol 85 no 3 pp 556ndash564 2011

[31] T A Soboleva M Nekrasov A Pahwa R Williams G AHuttley and D J Tremethick ldquoA unique H2A histone variantoccupies the transcriptional start site of active genesrdquo NatureStructural and Molecular Biology vol 19 no 1 pp 25ndash31 2012

[32] J Frayne A Taylor G Cameron and A T Hadfield ldquoStructureof insoluble rat sperm glyceraldehyde-3- phosphate dehydroge-nase (GAPDH) via heterotetramer formation with escherichiacoli GAPDH reveals target for contraceptive designrdquo Journal ofBiological Chemistry vol 284 no 34 pp 22703ndash22712 2009

[33] R Kandrsquoar P Drabkova K Myslıkova et al ldquoDeterminationof retinol and 120572-tocopherol in human seminal plasma using anHPLC with UV detectionrdquo Andrologia 2013

[34] C M Zumoffen R Gil A M Caille et al ldquoA protein isolatedfrom human oviductal tissue in vitro secretion identified ashuman lactoferrin interacts with spermatozoa and oocytes andmodulates gamete interactionrdquo Human Reproduction vol 28no 5 pp 1297ndash1308 2013

[35] D Sakkas ldquoNovel technologies for selecting the best spermfor in vitro fertilization and intracytoplasmic sperm injectionrdquoFertil Steril vol 99 no 4 pp 1023ndash1029 2013

[36] Y-J Ko J-HMaeng B-C Lee S Lee S Y Hwang and Y AhnldquoSeparation of progressive motile sperm from mouse semenusing on-chip chemotaxisrdquoAnalytical Sciences vol 28 no 1 pp27ndash32 2012

[37] W S Kwon Y J Park el S A Mohamed et al ldquoVoltage-dependent anion channels are a key factor of male fertilityrdquoFertility and Sterility vol 99 no 2 pp 354ndash361 2013

[38] W S Kwon Y J Park Y H Kim et al ldquoVasopressin effectivelysuppresses male fertilityrdquo PLoS ONE vol 8 no 1 Article IDe54192 2013

[39] T-L Lee O M Rennert and W-Y Chan ldquoRevealing thetranscriptome landscape of mouse spermatogonial cells bytiling microarrayrdquo Methods in Molecular Biology vol 825 pp75ndash92 2012

[40] J Martınez-Heredia J M Estanyol J L Ballesca and ROliva ldquoProteomic identification of human sperm proteinsrdquoProteomics vol 6 no 15 pp 4356ndash4369 2006


[42] M A Baker G Reeves L Hetherington J Muller I Baur andR J Aitken ldquoIdentification of gene products present in TritonX-100 soluble and insoluble fractions of human spermatozoalysates using LC-MSMS analysisrdquo Proteomics vol 1 no 5 pp524ndash532 2007

[43] J Moriniere S Rousseaux U Steuerwald et al ldquoCooperativebinding of two acetylation marks on a histone tail by a singlebromodomainrdquo Nature vol 461 no 7264 pp 664ndash668 2009

[44] J R Kovac A W Pastuszak and D J Lamb ldquoThe useof genomics proteomics and metabolomics in identifyingbiomarkers of male infertilityrdquo Fertility and Sterility vol 99 no4 pp 998ndash1007 2013

[45] J P Bonde E Ernst T K Jensen et al ldquoRelation betweensemen quality and fertility a population-based study of 430first-pregnancy plannersrdquoThe Lancet vol 10 no 352 pp 1172ndash1177 1998

[46] P R Budworth R P Amann and P L Chapman ldquoRelationshipsbetween computerized measurements of motion of frozen-thawedbull spermatozoa and fertilityrdquo Journal of Andrology vol9 no 1 pp 41ndash54 1988

[47] R J Aitken M Sutton P Warner and D W RichardsonldquoRelationship between the movement characteristics of humanspermatozoa and their ability to penetrate cervical mucusand zona-free hamster oocytesrdquo Journal of Reproduction andFertility vol 73 no 2 pp 441ndash449 1985

[48] J Thundathil J Gil A Januskauskas et al ldquoRelationshipbetween the proportion of capacitated spermatozoa present infrozen-thawed bull semen and fertility with artificial insemina-tionrdquo International Journal of Andrology vol 22 no 6 pp 366ndash373 1999

[49] H Kjaestad E Ropstad and K A Berg ldquoEvaluation of sperma-tological parameters used to predict the fertility of frozen bullsemenrdquo Acta Veterinaria Scandinavica vol 34 no 3 pp 299ndash303 1993

[50] L Sooderquist H Rodriguez-Martinez and L Janson ldquoPost-thaw motility ATP content and cytochrome C oxidase activityof AI Bull spermatozoa in relation to fertilityrdquo ZentralblVeterinaermed A vol 38 no 3 pp 165ndash174 1991

[51] A G Braundmeier J M Demers R D Shanks and D JMiller ldquoThe relationship of porcine sperm zona-binding abilityto fertilityrdquo Journal of Animal Science vol 82 no 2 pp 452ndash4582004

[52] S E M Lewis ldquoIs sperm evaluation useful in predicting humanfertilityrdquo Reproduction vol 134 no 1 pp 31ndash40 2007

[53] B Liu P Wang Z Wang et al ldquoAnalysis and difference ofvoltage-dependent anion channel mRNA in ejaculated sper-matozoa from normozoospermic fertile donors and infertilepatients with idiopathic asthenozoospermiardquo Journal of AssistedReproduction and Genetics vol 27 no 12 pp 719ndash724 2010

[54] T Cocco M Di Paola S Papa and M Lorusso ldquoChemicalmodification of the bovine mitochondrial bc1 complex revealscritical acidic residues involved in the proton pumping activityrdquoBiochemistry vol 37 no 7 pp 2037ndash2043 1998

[55] L Aguilera-Aguirre A Bacsi A Saavedra-Molina A KuroskyS Sur and I Boldogh ldquoMitochondrial dysfunction increasesallergic airway inflammationrdquo Journal of Immunology vol 183no 8 pp 5379ndash5387 2009

[56] A Gaviraghi F Deriu A Soggiu et al ldquoProteomics to investi-gate fertility in bullsrdquoVeterinary Research Communications vol1 supplement 1 pp S33ndashS36 2010

[57] O DrsquoAmours G FrenetteM Fortier P Leclerc and R SullivanldquoProteomic comparison of detergent-extracted sperm proteinsfrom bulls with different fertility indexesrdquo Reproduction vol139 no 3 pp 545ndash556 2010

[58] C R Austin ldquoObservations on the penetration of the sperm inthemammalian eggrdquoAustralian Journal of Scientific Research Bvol 4 no 4 pp 581ndash596 1951


[59] M C Chang ldquoFertilizing capacity of spermatozoa depositedinto the fallopian tubesrdquoNature vol 168 no 4277 pp 697ndash6981951

[60] R Yanagimachi ldquoMammalian fertilizationrdquo in Physiology ofReproduction E N J Knobil Ed pp 189ndash317 Raven Press NewYork NY USA 1994

[61] MA Baker G Reeves LHetherington andR J Aitken ldquoAnal-ysis of proteomic changes associated with sperm capacitationthrough the combined use of IPG-strip prefractionation fol-lowed byRP chromatography LC-MSMS analysisrdquo Proteomicsvol 10 no 3 pp 482ndash495 2010

[62] H A Shojaei Saadi E van Riemsdijk A L Dance et alldquoProteins associated with critical sperm functions and spermhead shape are differentially expressed in morphologicallyabnormal bovine sperm induced by scrotal insulationrdquo Journalof Proteomics vol 26 no 82 pp 64ndash80 2013

[63] L D Newton J P Kastelic B Wong F Van Der Hoorn and JThundathil ldquoElevated testicular temperaturemodulates expres-sion patterns of sperm proteins in Holstein bullsrdquo MolecularReproduction and Development vol 76 no 1 pp 109ndash118 2009

[64] A A Moura D A Chapman H Koc and G J Killian ldquoAcomprehensive proteomic analysis of the accessory sex glandfluid frommature Holstein bullsrdquoAnimal Reproduction Sciencevol 98 no 3-4 pp 169ndash188 2007

[65] A A Moura H Koc D A Chapman and G J KillianldquoIdentification of proteins in the accessory sex gland fluidassociated with fertility indexes of dairy bulls a proteomicapproachrdquo Journal of Andrology vol 27 no 2 pp 201ndash211 2006

[66] X Guo J Shen Z Xia et al ldquoProteomic analysis of proteinsinvolved in spermiogenesis in mouserdquo Journal of ProteomeResearch vol 9 no 3 pp 1246ndash1256 2010

[67] K K Stein J C Go W S Lane P Primakoff and D G MylesldquoProteomic analysis of sperm regions that mediate sperm-egginteractionsrdquo Proteomics vol 6 no 12 pp 3533ndash3543 2006

[68] M A Baker L Hetherington G Reeves J Muller and R JAitken ldquoThe rat sperm proteome characterized via IPG stripprefractionation and LC-MSMS identificationrdquo Proteomicsvol 8 no 11 pp 2312ndash2321 2008

[69] MD Piatt AM SalicioniD FHunt andP EVisconti ldquoUse ofdifferential isotopic labeling and mass spectrometry to analyzecapacitation-associated changes in the phosphorylation statusof mouse sperm proteinsrdquo Journal of Proteome Research vol 8no 3 pp 1431ndash1440 2009

[70] B Nixon A Bielanowicz E AMclaughlin N Tanphaichitr MA Ensslin and R J Aitken ldquoComposition and significance ofdetergent resistant membranes in mouse spermatozoardquo Journalof Cellular Physiology vol 218 no 1 pp 122ndash134 2009

[71] J Shetty A B Diekman F C Jayes et al ldquoDifferential extrac-tion and enrichment of human sperm surface proteins in aproteome identification of immunocontraceptive candidatesrdquoElectrophoresis vol 22 no 14 pp 3053ndash3066 2001

[72] S Ficarro O Chertihin V A Westbrook et al ldquoPhospho-proteome analysis of capacitated human sperm evidence oftyrosine phosphorylation of a kinase-anchoring protein 3 andvalosin-containing proteinp97 during capacitationrdquo Journal ofBiological Chemistry vol 278 no 13 pp 11579ndash11589 2003

[73] R K Naz and P B Rajesh ldquoRole of tyrosine phosphorylationin sperm capacitationacrosome reactionrdquo Reproductive Biologyand Endocrinology vol 9 no 2 2004

[74] S K NagDas V P Winfrey and G E Olson ldquoTyrosine phos-phorylation generates multiple isoforms of the mitochondrial

capsule protein phospholipid hydroperoxide glutathione per-oxidase (PHGPx) during hamster sperm capacitationrdquo Biologyof Reproduction vol 72 no 1 pp 164ndash171 2005

[75] C Zhao R Huo F-Q Wang M Lin Z-M Zhou and J-HSha ldquoIdentification of several proteins involved in regulation ofspermmotility by proteomic analysisrdquo Fertility and Sterility vol87 no 2 pp 436ndash438 2007

[76] D S Johnston J Wooters G S Kopf Y Qiu and K P RobertsldquoAnalysis of the human sperm proteomerdquo Annals of the NewYork Academy of Sciences vol 1061 pp 190ndash202 2005

[77] A Domagała S PulidoM Kurpisz and J C Herr ldquoApplicationof proteomicmethods for identification of sperm immunogenicantigensrdquoMolecularHumanReproduction vol 13 no 7 pp 437ndash444 2007

[78] F Secciani L Bianchi L Ermini et al ldquoProtein profile of capac-itated versus ejaculated human spermrdquo Journal of ProteomeResearch vol 8 no 7 pp 3377ndash3389 2009

[79] C-C Chan H-A Shui C-H Wu et al ldquoMotility and proteinphosphorylation in healthy and asthenozoospermic spermrdquoJournal of Proteome Research vol 8 no 11 pp 5382ndash5386 2009

[80] L Lefievre Y Chen S J Conner et al ldquoHuman spermatozoacontain multiple targets for protein S-nitrosylation an alterna-tive mechanism of the modulation of sperm function by nitricoxiderdquo Proteomics vol 7 no 17 pp 3066ndash3084 2007

[81] S de Mateo J Martınez-Heredia J M Estanyol et al ldquoMarkedcorrelations in protein expression identified by proteomicanalysis of human spermatozoardquo Proteomics vol 7 no 23 pp4264ndash4277 2007

[82] S T Page J K Amory and W J Bremner ldquoAdvances in malecontraceptionrdquo Endocrine Reviews vol 29 no 4 pp 465ndash4932008

[83] B D Anawalt J K Amory K L Herbst et al ldquoIntramusculartestosterone enanthate plus very low dosage oral levonorgestrelsuppresses spermatogenesis without causing weight gain innormal young men a randomized clinical trialrdquo Journal ofAndrology vol 26 no 3 pp 405ndash413 2005

[84] B D Anawalt R A Bebb W J Bremner and A M Mat-sumoto ldquoA lower dosage levonorgestrel and testosterone com-bination effectively suppresses spermatogenesis and circulatinggonadotropin levels with fewer metabolic effects than higherdosage combinationsrdquo Journal of Andrology vol 20 no 3 pp407ndash414 1999

[85] K K Shukla W S Kwon M S Rahman et al ldquoNutlin-3adecreases male fertility via UQCRC2rdquo PLoS ONE vol 8 no 102013

[86] Y-F Zhu Y-G Cui X-J Guo et al ldquoProteomic analysis ofeffect of hyperthermia on spermatogenesis in adult male micerdquoJournal of Proteome Research vol 5 no 9 pp 2217ndash2225 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


At present numerous proteomics techniques includingtwo-dimensional (2D) polyacrylamide gel electrophoresismass spectrometry (MS) and differential in-gel electrophore-sis are widely used to identify particular sperm-specificproteins These approaches have offered a concise but deepunderstanding of different functional aspects of sperm pro-teins for example motility capacitation acrosomal reac-tion fertilization and posttranslational modifications suchas phosphorylation glycosylation proteolytic cleavages andmutations In addition proteomic research has also provideda new horizon to study different functional states of sperma-tozoa for example normal versus malformed immature ver-sus mature capacitated versus incapacitated low versus highsperm count low versus high fertility and normalhighlyfertile versus state of complete contraception [1 8ndash10]which are all necessary and important to identify a suitablebiomarker and to select the desired sire Furthermore designand construction of agonisticantagonistic approaches usingcommercially synthesized purified chemicals with regard todifferentially expressed proteins of spermatozoa might behelpful to develop fertility enhancers new treatments forinfertility and contraception pills The present work reviewsthe latest articles published by other laboratories aswell as ourresearch team on the proteomics aspect of spermatozoa andtheir potential implications to assist fertility or to develop amale contraceptive strategy

2 Biology of the Sperm Cell

Spermatozoa are mature male gametes that are producedthrough a unique process called spermatogenesis In mam-mals spermatogenesis occurs in the male testes and epi-didymis in a stepwise fashion that is highly dependent onoptimal conditions for the process to work correctly Sper-matozoa undergo morphological biochemical and physio-logical modifications in the testes and epididymis [11] How-ever the complete differentiation process largely dependson changes at the genetic cellular and chromatin level [11ndash14] Cellular adjustment that occurs during spermiogenesisincludes nuclear condensation movement of the nucleus tothe periphery of the cell and formation of the acrosome andflagella (depicted in Figure 1) To attain successful fertilizationboth in vitro and in vivo mammalian spermatozoa mustundergo capacitation followed by the acrosome reactionwhich are important processes that finally allow the maturesperm cell to penetrate the zona pellucida and to fuse withthe oocyte plasma membrane during fertilization and earlyembryonic development [15] (see Figure 1(a)) In contrast theentire process of spermiogenesis results in significant changesin the sperm chromatin structure and DNA strand breaksto allow the accompanying change in DNA topology (seeFigure 1(b)) Both in vitro and in vivo evidence suggests thatthe DNA-binding and condensing activities of a set of basicnuclear ldquotransition proteinsrdquo is critical to the integrity of theentire chromatin remodeling process [16]

3 Background of Sperm Proteomic Research

Comprehensive identification and quantification of variousproteins expressed in cells and tissues offer important andfascinating insights into the dynamics of cellular functionsand differentiation The novel approaches of characterizingproteins by means of both qualitative and quantitative analy-ses are known as proteomics Proteins carry out a wide rangeof functions from forming structural materials to catalyzingchemical reactions thus knowing exactly which proteins arein sperm cells is a major step forward in understanding Theso-called proteome of spermatozoa contains everything thesperm needs to survive and function correctly The studyof sperm proteins was in fact initiated with the remarkablefinding by Friedrich Miescher in 1874 Miescher was ableto identify the basic component of salmon spermatozoacalled ldquoprotaminerdquo that was coupled with nuclein whichwas known as DNA afterward Oliver and Dixon [17] andBalhorn [18] noted that the protamines initially isolated byMiescher are the most abundant sperm nuclear proteins inmany animal species as well as the initial proteins identifiedin the spermatozoal nucleus However the founders of spermproteomics study were Naaby-Hansen and his colleaguesfrom Virginia and their elegant studies addressed the firsthumanrsquos sperm protein database of 1400 spots [19] With therecent development of modern proteomic approaches suchas the application of narrow-range isoelectric focusing stripsandmultiple 2D gels this numbermight bemuch higher thanthat previously reported [20ndash22]

4 Basic Techniques of Sperm ProteomicResearch

In order to understand how sperm proteins can be identifiedand characterized the fundamental process of proteomicanalysis will be addressed in brief After collection and liq-uefaction of a semen sample it can be used as it is or it can besubjected to additional processing for purification of sperma-tozoa The ejaculate comprises various cellular componentsfor example seminal plasma secretions from the accessorysex gland blood cells and epithelial cells Therefore duringsperm proteomic analysis contamination of seminal plasmaproteins might be the reason for the substantial differencesobserved between samples from the same donors [23 24]To minimize this type of contamination the direct swim-upmethod and the density gradientmethod (using Percoll) havebeen suggested previously for sperm isolation in humans [2324] However the Percoll method demonstrated much betterreproducibility than the swim-up method [23] Recentlydifferent density gradient methods have been applied toisolate spermatozoa from both human and animal subjectsFor example a discontinuous density gradient of 90minus45Percoll was used to remove extender debris seminal plasmaand dead spermatozoa in bovine semen [24 25] A 35PureSperm solution was used to separate caput and caudaepididymal sperm inmice [26] In swine a two-layer isotonicPercoll gradient (90minus40) was used to purify epididymalsperm [27] whereas 80minus55 Percoll was used to separate


(a) (b)




Transition proteins

Protaminesbinding

Histone activation


Differentiation


Spermatogonia2N

Meiosis I

Primaryspermatocyte

tetrad (2N)


Meiosis II

Spermatidmonad (N)

Spermatozoa(N)

Mitosis

Capacitation

Acrosomereaction


Epigenetic marks


Nucleohistone

toroid

toroid


Pronucleus (male)



DNA sensitive


dyad (N)

















Spermatozoa



Digest

pH10

pH3

Crude extracts

Digestpeptides

Excise

Digested

PeptideLC(HPLC)


MSMS


Laser


Time of light

MS data

MS (MALDI-TOF)

1000

1500

2000

2500

3000

3500

4000

1D PAGE

65




800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

100

90

80

70

60

50

40

30

20

10

0

2[c]A3

800 g

(KD

a)

220

116170

977659

41

30

2014

(s)

Inte

nsity

()

mzmz






Acrosome



Flagellum

Fibrous sheath


End piece

AnnulusMicrotubules

Axial filamentH

ead

Mid

pie

ceTa

il


Proximal centriole









ENO1 CRISP2 SHTAP













Newton et al [63]




Guo et al [66]




Piatt et al [69]





























Acknowledgments


References































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)




Transition proteins

Protaminesbinding

Histone activation


Differentiation


Spermatogonia2N

Meiosis I

Primaryspermatocyte

tetrad (2N)


Meiosis II

Spermatidmonad (N)

Spermatozoa(N)

Mitosis

Capacitation

Acrosomereaction


Epigenetic marks


Nucleohistone

toroid

toroid


Pronucleus (male)



DNA sensitive


dyad (N)

















Spermatozoa



Digest

pH10

pH3

Crude extracts

Digestpeptides

Excise

Digested

PeptideLC(HPLC)


MSMS


Laser


Time of light

MS data

MS (MALDI-TOF)

1000

1500

2000

2500

3000

3500

4000

1D PAGE

65




800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

100

90

80

70

60

50

40

30

20

10

0

2[c]A3

800 g

(KD

a)

220

116170

977659

41

30

2014

(s)

Inte

nsity

()

mzmz






Acrosome



Flagellum

Fibrous sheath


End piece

AnnulusMicrotubules

Axial filamentH

ead

Mid

pie

ceTa

il


Proximal centriole









ENO1 CRISP2 SHTAP













Newton et al [63]




Guo et al [66]




Piatt et al [69]





























Acknowledgments


References































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























Spermatozoa



Digest

pH10

pH3

Crude extracts

Digestpeptides

Excise

Digested

PeptideLC(HPLC)


MSMS


Laser


Time of light

MS data

MS (MALDI-TOF)

1000

1500

2000

2500

3000

3500

4000

1D PAGE

65




800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

100

90

80

70

60

50

40

30

20

10

0

2[c]A3

800 g

(KD

a)

220

116170

977659

41

30

2014

(s)

Inte

nsity

()

mzmz






Acrosome



Flagellum

Fibrous sheath


End piece

AnnulusMicrotubules

Axial filamentH

ead

Mid

pie

ceTa

il


Proximal centriole









ENO1 CRISP2 SHTAP













Newton et al [63]




Guo et al [66]




Piatt et al [69]





























Acknowledgments


References































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Acrosome



Flagellum

Fibrous sheath


End piece

AnnulusMicrotubules

Axial filamentH

ead

Mid

pie

ceTa

il


Proximal centriole









ENO1 CRISP2 SHTAP













Newton et al [63]




Guo et al [66]




Piatt et al [69]





























Acknowledgments


References































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Newton et al [63]




Guo et al [66]




Piatt et al [69]





























Acknowledgments


References































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























Acknowledgments


References































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Review Article Sperm Proteomics: Road to Male Fertility...

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Transcript of Review Article Sperm Proteomics: Road to Male Fertility...