Evolutionary Conservation of Mammalian Sperm Proteins Associates

Evolutionary Conservation of Mammalian Sperm Proteins Associateswith Overall, not Tyrosine, Phosphorylation in Human SpermatozoaJulia Schumacher,† Sanja Ramljak,‡ Abdul R. Asif,§ Michael Schaffrath,∥ Hans Zischler,†

and Holger Herlyn*,†

†Institute of Anthropology, University Mainz, Anselm-Franz-von-Bentzel-Weg 7, Mainz 55128, Germany‡IKFE-Institute for Clinical Research and Development, Parcusstrasse 8, Mainz 55116, Germany§Department of Clinical Chemistry, University Medical Center, Georg-August-Universitat, Robert-Koch-Strasse 40, Gottingen 37075,Germany∥Department of Obstetrics and Gynecology, University Hospital, University Mainz, Langenbeckstrasse 1, Mainz 55131, Germany

*S Supporting Information

ABSTRACT: We investigated possible associations between sequenceevolution of mammalian sperm proteins and their phosphorylation status inhumans. As a reference, spermatozoa from three normozoospermic menwere analyzed combining two-dimensional gel electrophoresis, immuno-blotting, and mass spectrometry. We identified 99 sperm proteins (thereof42 newly described) and determined the phosphorylation status for most ofthem. Sequence evolution was studied across six mammalian species usingnonsynonymous/synonymous rate ratios (dN/dS) and amino aciddistances. Site-specific purifying selection was assessed employing averageratios of evolutionary rates at phosphorylated versus nonphosphorylatedamino acids (α). According to our data, mammalian sperm proteins do notshow statistically significant sequence conservation difference, no matter ifthe human ortholog is a phosphoprotein with or without tyrosine (Y)phosphorylation. In contrast, overall phosphorylation of human sperm proteins, i.e., phosphorylation at serine (S), threonine(T), and/or Y residues, associates with above-average conservation of sequences. Complementary investigations suggest thatnumerous protein−protein interactants constrain sequence evolution of sperm phosphoproteins. Although our findings reject aspecial relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantiallycontributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as primecandidates for diagnosis and treatment of reduced male fertility.

KEYWORDS: phosphorylation, spermatozoa, primates, evolution, selection, functional constraint, 2DE, mass spectrometry, fertility,dN/dS

■ INTRODUCTION

In eukaryotes, phosphorylation at serine (S), threonine (T),and tyrosine (Y) residues regulates protein activity, bindingproperties, assembly of protein complexes, cell cycle, and signaltransduction.1−4 However, protein phosphorylation is notlimited to eukaryotes but extends to bacteria and viruses.5−7

In multicellular animals, protein phosphorylation triggersprocesses in both somatic and germ cells and is generallyconsidered to be a fundamental regulatory mechanism.8 Withrespect to male reproduction, changes in phosphorylation and,in particular, in Y phosphorylation are of utmost importance forprocesses such as sperm capacitation and hyperactivation aswell as for acrosome reaction and binding of the spermatozoonto the zona pellucida.9−15 Other studies demonstrate highrelevance of (Y) phosphorylation for the regulation of spermmotility,16−18 most probably through modulation of micro-tubule and fibrous sheath sliding properties.19,20 The outlinedinvolvement of phosphorylation in diverse sperm functions

could explain why sub- and infertility in men has beenrepeatedly linked to disturbed phosphorylation patterns,21,22

especially with respect to Y phosphorylation.23,24 The recurrentevidence for an involvement of Y phosphorylation in reducedmale fertility brings up the question of potentially higher impactof Y versus ST phosphorylation on sperm functioning. Specificways how Y and ST sites undergo phosphorylation anddifferences in incidence and binding energies within thephosphorylatated moieties25,26 would certainly endorse such ascenario.Phosphorylation plays a significant role not only in regulation

of cell processes but also in maintenance of cell structure, whichsuggests high levels of functional constraint. Accordingly,phosphoproteins, phosphorylation sites (phosphosites), andthe mechanisms of phosphorylation have been reported to be

Received: December 4, 2012Published: August 6, 2013

Article

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© 2013 American Chemical Society 5370 dx.doi.org/10.1021/pr400228c | J. Proteome Res. 2013, 12, 5370−5382

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evolutionarily conserved across eubacteria and eukar-yotes.6,27−33 On the other hand, a growing body of dataillustrates that phosphosites, phosphoproteins, as well as themechanisms of phosphorylation underwent phases of accel-erated and adaptive evolution.32,34−36 Indeed, large-scalechanges in phosphorylation may be pertinent for functionaldiversification and retention of paralogs following genomeduplication.36−38 Moreover, changes in phosphorylation aremost likely engaged in the evolution of receptor−ligandsystems, either in terms of immune evasion or in terms ofcoevolving receptor−ligand systems39−41 (see also ref 34).Considering the relevance of both phenomena in fertiliza-tion,42,43 changes in phosphorylation may represent a factoraccelerating sequence evolution of mammalian fertilizationproteins.41,44−47 However, the definite impact of phosphor-ylation on sequence evolution of mammalian sperm proteinsstill needs to be assessed. Hence, the present study aims atclarifying if overall STY phosphorylation of human spermproteins associates with increased or with lowered levels ofcross-species conservation, thereby taking sperm proteinsexhibiting no phosphorylation in humans and genome-wideaverages33,48 as references. As the regulation of diverseprocesses by Y phosphorylation may be particularly relevantfor sperm functioning,25 we additionally examine potentialassociations between this posttranslational modification andrates of sequence evolution.In order to avoid that false predictions compromise analyses

we grouped sperm proteins according to their experimentallyvalidated phosphorylation status in humans. Taking spermato-zoa of three healthy and normozoospermic men as a referenceand combining two-dimensional gel electrophoresis (2DE),immunoblotting, and mass spectrometry (MS), we determineda total of 99 human sperm proteins and clarified the majority’sphosphorylation status. On the basis of coding DNAs (cDNAs)of six mammalian species we compared levels of cross-speciesconservation between (i) sperm proteins that are either STYphosphorylated or nonphosphorylated in humans, and (ii)sperm proteins that are phosphoproteins either with or withoutY phosphorylation in human probands. Sequence evolutionacross sites was assessed using nonsynonymous to synonymoussubstitution rate ratios (dN/dS, also Ka/Ks or ω). Since dSshould reflect neutral evolution, dN/dS values larger, equating,or smaller than 1 are commonly accepted as evidence foradaptive evolution through positive selection, neutral evolution,and evolutionary conservation through negative selection,respectively. Codon-based analyses of sequence evolution arecomplemented by investigations of amino acid distances acrosssites and average ratios of evolutionary rates at phosphositesversus nonphosphorylated counterparts (α), whereby α values<1 point to purifying selection at phosphosites.33 Finally, weconsidered protein-specific levels of functional constraint asassessed by gene ontology and protein−protein interactions(PPI). The latter refers to evidence from network biologywhereupon proteins with more interaction partners, andtherefore more central positions in network topologies, showhigher essentiality and evolutionary conservation than theproteins with fewer interaction partners that are located at thenetwork periphery.49−53

■ MATERIALS AND METHODS

Study Participants, Semen Collection, and SpermPreparation

As a reference we analyzed the phosphorylation status ofhuman (Homo sapiens) sperm proteins. In detail, ejaculates ofthree healthy and normozoospermic men (Europeans; 23−28years) were included. Sampling was approved by the localethics committee. Ejaculates were collected into sterile cupsafter at least two days of sexual abstinence. Ejaculates wereliquefied at 37 °C and sperm parameters evaluated according toWorld Health Organization (WHO) guidelines.54 Spermatozoawere separated from seminal plasma by a succession of washingsteps with human tubal fluid medium (HTF) supplementedwith HEPES, gentamycin, and 3% bovine serum albumin(BSA), and centrifugation at 300g for 9 min (37 °C). Inconcordance with WHO standards54 and other studies onsperm proteins55 including those on Y phosphorylation,56

spermatozoa were capacitated by swim-up (HTF, 30 min, 37°C).Protein Extraction and Purification

Frozen sperm samples were thawed at 4 °C and solubilized atroom temperature (RT) in lysis buffer containing 7 M urea, 2M thiourea, 4% CHAPS, and 1% ampholytes (pH 3−10; 100×;Bio-Rad), DTT (w/v), protease inhibitor cocktail (Sigma-Aldrich), and phosphatase inhibitor cocktail (ThermoScientific). Cell debris was removed by centrifugation for 1min at maximum speed. Solubilized proteins were purifiedusing the ReadyPrep 2-D Cleanup Kit (Bio-Rad) andrehydrated in a buffer containing 7 M urea, 2 M thiourea, 4%CHAPS, 1.5% DTT (w/v), and 1% ampholytes (pH 3−10;100×). Protein concentration was measured using a Bradfordassay (Bio-Rad).Isoelectric Focusing, Equilibration, SDS-PAGE, andCoomassie Staining

We loaded up to 540 μg protein per strip (7 cm IPGReadyStrip, pH 5−8; Bio-Rad). Following passive rehydrationfor 2 h at RT, strips were covered with mineral oil and activelyrehydrated for 14 h at 50 V. Isoelectric focusing (IEF; 2 h at200 V, 2 h at 500 V, 6 h at 4000 V; all steps with rapid ramp)was performed at 20 °C in a Protean IEF cell (Bio-Rad).Focused strips were equilibrated in a buffer containing 6 Murea, 0.375 M Tris (1.5 M, pH 8.8), 30% glycerol, and 2% SDS(10%, w/v). For the first equilibration step (25 min), the bufferwas supplemented with 2% DTT, and for the second one (25min), with 2.5% iodoacetamide. Second-dimension electro-phoresis (2D SDS-PAGE) was carried out at 120 V in 10%polyacrylamide gels. Documentation of Coomassie (Roti-Blue,Roth) stained gels was performed with a GS-800 calibrateddensitometer (Bio-Rad). We ran and documented a minimumof three gels per donor. Another six gels (per donor) wereutilized for Western blotting.Gel Excision, In-Gel Digestion, LC−MS/MS, and ProteinIdentification

For each protein spot, gel slices were excised and dried byvacuum centrifugation. Gel pieces were soaked with digestionbuffer supplemented with trypsin (0.01 μg/μL). Afterward,samples were incubated overnight (37 °C) in the samedigestion buffer without trypsin. The next day, peptides wereextracted by sonication in solvents of increasing acetonitrile(ACN) content. Eluates were collected, completely dried byvacuum centrifugation, and frozen at −20 °C. Peptides were

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reconstituted in 0.1% formic acid for injection into a nanoflowHPLC coupled online with Q-TOF mass spectrometer. Peptidesamples (1 μL) were consecutively introduced onto two C18-reversed phase chromatography columns (first to a trappingcolumn; C18 pepMap: 300 μm × 5 mm; 5 μm particle size, andsecond eluted through the analytical column; C18 pepMap100nanoanalytical column: 75 μm × 15 cm; 3 μm particle size)using a nanoflow CapLC autosampler (Waters). Peptides wereeluted with an increasing gradient of ACN and analyzed on aQ-TOF Ultima Global mass spectrometer (Micromass)equipped with a nanoflow ESI Z-spray source in the positiveion mode.57 The data were analyzed with the MassLynx v. 4.0software and processed by using the Protein Lynx global server(version 2.2; Waters), which generates .pkl files. The followingsettings were used: 80% centroid with minimum peak of fourchannels, 10% noise reduction, and medium deisotoping with a3% threshold. The obtained peak patterns were searchedagainst the SwissProt database version 57 (525 997 sequences;185 874 894 residues; http://expasy.org/sprot) using theMASCOT (Matrix Science, http://www.matrixscience.com)search engine. The following parameters were specified: trypsinas an enzyme for digestion, up to a maximum of one missedcleavage site allowed, monoisotopic mass value, unrestrictedmolecular weight (MW), peptide tolerance ±0.5 Da, and MS/MS tolerance ±0.5 Da. Proteins were identified on the basis ofa minimum of one peptide, whose ions score exceeded thethreshold of P < 0.05. Only those spots that contained a singleprotein according to LC−MS/MS results were furtherconsidered. Moreover, we thoroughly checked if molecularweight (MW) and isoelectric point (pI) of a detected proteincorresponded with the actual position of the respective proteinspot. If expected and actual MW and pI were in conflict, aprotein was excluded. Comigrating fragments of other proteinsshould hence not have biased our results.

Immunoblotting and Image Analysis

Gels were blotted onto a polyvinylidene difluoride membrane(Hybond-P, GE Healthcare) for 75 min at 53 mA, using atransfer buffer containing 192 mM glycine, 25 mM Tris, and20% methanol. Blots were blocked for 1 h at RT in Tris-buffered saline containing Tween 20 (TBST) and 5% protease-free BSA. Three Western blots per subject were incubated (5%BSA in TBST; 18 h at 4 °C) with anti-pSTY (mousemonoclonal antibody to phosphoserine/threonine/tyrosine; 4μg/mL; Abcam) and another three with anti-pY (mousemonoclonal antibody to phosphotyrosine; 1.5 μg/mL; Abcam).Following washing, blots were incubated for another hour atRT with horseradish peroxidase-conjugated sheep polyclonalantibody (3.2 μg/mL for anti-pSTY Western blots; 2 μg/mLfor anti-pY Western blots; antibodies-online) in TBSTcontaining 1% BSA. Afterward, Western blots were docu-mented with a ChemoCam Imager (Intas).

Determining the Phosphorylation Status of Human SpermProteins

The phosphorylation status of a sperm protein was determinedby combining information from LC−MS/MS, three gel scans,three anti-pSTY Western blots, and three anti-pY Western blots(per donor, each), thus merging the data from technical andbiological replicates. We matched the data with the aid of thePDQuest (Bio-Rad) spot detection software and consideredexclusively those spots that scored a single protein according toLC−MS/MS results. As outlined above, spots containing >1protein according to LC−MS/MS were excluded, as were the

proteins whose expected and actual MW and pI were inconflict. Proteins giving inconsistent phosphorylation signalsbetween the samples of different men were not taken intoaccount either. Thus, we classified a protein as beingphosphorylated at STY only in the case of consistent signalsin anti-pSTY Western blots. In turn, a sperm protein wasconsidered as being nonphosphorylated if no signal appeared inany of the anti-pSTY and anti-pY Western blots and if the sameprotein was neither reported as being phosphorylated in vivonor in vitro according to Phospho.ELM58 (http://phospho.elm.eu.org/; evaluation date 20th June 2013) and PhosphoPep2.059 databases (http://www.phosphopep.org/; valuation date20th June 2013).Furthermore, we regarded consistent signals from anti-pSTY

and anti-pY Western blots as an evidence for phosphorylationof human sperm proteins at Y sites and possible additionalphosphorylation at S and/or T sites. We here refer to suchproteins as sperm phosphoproteins with Y phosphorylation. Incase of exclusive signals in anti-pSTY Western blots, a proteinwas regarded as being phosphorylated at S and/or T sites andbeing nonphosphorylated at Y residues. The latter proteins aredesignated as sperm phosphoproteins without Y phosphor-ylation. Using the NetPhos 2.0 program60 (http://www.cbs.dtu.dk/services/NetPhos/) we ensured for the orthologs of allsampled species that neural network predictions confirm theexperimentally validated phosphorylation patterns.Applying the above criteria, comigrating fragments of

undetected proteins cannot be excluded. However, theprocedure diminished the probability that decisions on thephosphorylation status were confounded by comigratingfragments. Moreover, although predictions support cross-species conservation, we do not postulate identical phosphor-ylation patterns in nonhuman species. Thus, whenever wename sperm proteins according to the phosphorylation statusof the human ortholog we do so for simplicity.

Generation of Alignments

Codon-alignments were generated for each sperm protein withclarified phosphorylation status in human probands. Thealignments comprised cDNAs encoding the human referenceand orthologs from Rhesus monkey (Macaca mulatta), housemouse (Mus musculus), Norway rat (Rattus norvegicus), bovine(Bos taurus), and pig (Sus scrofa) that were retrieved fromNCBI and ENSEMBL databases. In the case of missing entriesfor one or more orthologs a gene was excluded from allsubsequent analyses. Raw data sets were aligned in the codonmode using the ClustalW algorithm implemented in theGUIDANCE web-server (http://guidance.tau.ac.il/),61 whichalso pruned data sets from unreliably aligned regions byrejecting columns with confidence scores below 0.93 (defaultthreshold). The same cDNAs were used for the generation ofamino acid alignments that were pruned from unreliablyaligned regions, too (GUIDANCE; default threshold of 0.93 forthe rejection of columns). Whether taking codon or amino acidalignments, gap-positions were ignored in downstream analysesby specific settings (see below). Supporting Information Tables1 and 2 list protein names and accession numbers of cDNAorthologs used for sequence analyses.

Analyses of Sequence Evolution

Sequence evolution of sperm proteins with clarified phosphor-ylation status (see above for criteria) was studied at the codonlevel using the aforementioned cDNA alignments (sixmammalian species, pruned from gaps) and the following

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tree topology: ((bovine, pig), (mouse, rat), (Rhesus monkey,human)). Analyses were carried out employing the CODEMLprogram implemented in PAML (Phylogenetic Analysis byMaximum Likelihood) package v. 4.4.62 Codon-frequencieswere estimated from the data (F3 × 4), and sites withambiguity data were ignored (cleandata = 1). More precisely,we tested for the presence of positively selected codon sitesusing a likelihood ratio test (LRT) comparing the fit of twoversions of model M8.44 Both model versions assume a βdistribution of codon sites in the dN/dS interval (0,1).However, while the alternative version (M8) allows for anextra site class under positive selection (dN/dS ≥ 1), dN/dS ofthis extra site class is fixed at 1 in the null version (M8A). ForLRT, 2Δl was compared to critical values following a 50:50mixture of a point mass at zero and a χ-square distribution withdegrees of freedom (df) equal to the difference in the numberof free parameters between M8A and M8 (=1). To reduce thenumber of false positives we applied a 1% level of significance(critical value = 5.41). To detect local optima, we ran M8 twiceusing different initial dN/dS values. Supporting InformationTables 3 and 4 list likelihood values and results from LRT.Sequence evolution was additionally investigated across sites

using M8A estimates of dN/dS. These codon-based analyseswere complemented by investigations at the amino acid level.Thus, we inferred pairwise amino acid distances from theabove-mentioned amino acid alignments (six mammalianspecies, pruned from gaps and unreliably aligned regions)using AAML implemented in PAML package v. 4.4.62 We thencalculated protein-specific mean values from the pairwiseJones−Taylor−Thornton (JTT) distances63 reported inAAML result files.The amino acid alignments were also used to analyze levels

of purifying selection at phosphorylated S, T, and Y (pS, pT,pY) sites. Taking our own experimental data as a reference, wefocused on pS and pT sites in sperm phosphoproteins withoutY phosphorylation and on pY sites in sperm phosphoproteinswith Y phosphorylation in humans. Levels of purifying selectionwere assessed on the basis of average ratios of evolutionaryrates at phosphosites versus evolutionary rates at theirnonphosphorylated counterparts (α).33,64 Phosphosites (pS,pT, pY) and nonphosphorylated counterparts (S, T, Y) weredistinguished by neural network predictions on humanorthologs as conducted by the NetPhos 2.0 program.60

Evolutionary rates at sites of interest were defined as totalnumbers of amino acid substitutions per site per billion years.64

Numbers of amino acid substitutions at sites of interest werecalculated applying maximum parsimony [AAML (rst) files].The total time elapsed on the tree represented by our six-species sample (=94.4 million years) was obtained from theTimeTree resource (www.timetree.org65). Site-specific evolu-tionary rates as well as α values are reported for each protein inSupporting Information Table 5.

Assessing Levels of Functional Constraint

Protein-specific levels of functional constraint were assessed onthe basis of gene ontologies and numbers of protein interactionpartners. On the basis of UniProt entries and original literaturewe assigned each of the identified human sperm proteins to oneof the gene ontology (GO) classes distinguished by Martınez-Heredia et al.,66 i.e., “energy production”, “transcription,protein synthesis, transport, folding, and turnover”, “cell cycle,apoptosis, and oxidative stress”, “signal transduction”, “cytoske-leton, flagella, and cell movement”, “cell recognition”, and

“metabolism” (see Supporting Information Tables 6 and 7).Moreover, using human protein IDs as search items (seeSupporting Information Tables 8−11) we extracted numbers ofPPI partners (nPPI) per human ortholog from 17 out of 25databases available through PSICQUIC (Proteomics StandardInitiative Common QUery InterfaCe; http://www.ebi.ac.uk/Tools/webservices/psicquic/view/main.xhtml; state 5th June2013). The GeneMANIA, iRefIndex, and Interoporc databaseswere opted out in order to avoid redundant hits and a strongskew from empirical evidence toward assumed interactions.Results from BindingDB and ChEMBL were ignored as theylist interactions between proteins and drug-like moleculesinstead of protein−protein interactions. Additionally, weexcluded hits from the MPIDB and VirHostNet databanks,which contain interactions with microbial and viral proteins, aswell as all other results referring to interactions between humanproteins and proteins of other species including pathogens.Finally, we ignored search results with the attribute “predictivetext mining”, “inferred by curator”, and/or “unspecifiedmethod” (quotation marks highlight PSICQUIC terminology).Supporting Information Tables 6 and 7 contain nPPI data persampled sperm protein.Statistical Analyses

Taking our own results on human probands as a reference andapplying the above criteria, we generated two sample pairs, i.e,.(i) phosphorylated and nonphosphorylated sperm proteins and(ii) sperm phosphoproteins with and without Y phosphor-ylation. Employing χ-square tests, we tested for differencesbetween groups of proteins with respect to the incidence ofsignificant support for site-specific positive selection and thedistribution across GO classes. Using Mann−Whitney U testwe additionally analyzed if levels of dN/dS (M8A), meanamino acid distances, and nPPI differed between sample pairs.Additionally, we conducted Spearman’s rank correlationbetween dN/dS values across sites (M8A) and nPPI acrossall studied sperm proteins, thus testing for a potentialassociation between these two variables. Focusing on oursample of phosphorylated sperm proteins we finally tested if thefraction of proteins with α < 1 differed from the nullexpectation of 50%, employing a Z-test.33 We determined95% confidence intervals for median and average values on thebasis of 100 000 bootstrap replicates using self-written Perlscripts, which are available upon request. We adjusted P valuesfor multiple comparisons applying sequential Bonferronicorrection. All tests were conducted using SPSS version 20.0(IBM).

■ RESULTS

Determined Sperm Proteins Show DifferentPhosphorylation States

Mass spectrometry allowed for identification of altogether 99human sperm proteins (Table 1, Supporting InformationTables 8−11; for annotated mass spectra of the peptides withmaximum ions score, see Supporting Information Table 12)from denser and fainter spots (Figure 1; SupportingInformation Figures 1−3). Out of these 99 human spermproteins, 42 were up to now not categorized as being expressedin human sperm or testis/testes in the UniProt database (state:1st July 2013), nor were they included in the reference study ofMartınez-Heredia et al.66 Conspicuously, nine of the 99identified proteins were proteasome subunits (Table 1;Supporting Information Tables 8, 9, and 11).

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Out of the sperm proteins giving consistent signals in anti-pSTY Western blots, cDNA orthologs from human, Rhesusmonkey, house mouse, Norway rat, bovine, and pig wereT

able

1.Pho

spho

rylation

Status

of99

Hum

anSperm

ProteinsIdentified

byMassSpectrom

etrya

status

identifi

edproteins

(abbreviations)

sperm

phosphoproteins

with

outY

phosphorylation(33)

ACADS,

AK8,APE

Hn ,CAPZ

Bn ,CRISP1

,ENO1,GDI2

n ,GK2,GLU

L,GOT1n,H

SPA9n,H

SPD1,ID

H3A

n ,LA

P3,L

ZTFL

1,NME5

,NME7,ODF1,P

CYT2,PD

HB,P

HBn ,PP

P1CBn ,PR

DX4,

PSMA1n,P

SMA6n,P

SMB2,

PSMB3,

PSMC2n,P

SMD13

n ,SP

AG6,

TUBA1A

n ,TUBB2A

n ,YWHAZn

sperm

phosphoproteins

with

Yphosphorylation

(18)

ACRBP,

ACTA2n,A

NXA1n,C

APZ

A1n,D

NAJB11

n ,EC

I1n ,GST

M3,

HIBADH,H

SPA1L

,MPS

T,P

ARK7,PS

MD11

n ,PS

MD14

n ,ROPN

1,SD

HAn ,TEKT1,

TUBB2C

n ,UQCRC1

nonphosphorylatedsperm

proteins

(8)

ALD

H9A

1n,C

RISP2

,DLD

,DNAJB8,

ECHS1

n ,PR

DX2n,S

PESP

1,TEK

T2

sperm

proteins

excluded

from

analyses

(40)

AARSD

1n,A

BHD10

n ,ACTB,A

CTBL2

n ,ACTR1A

,ACTR1B

,ACTRT2,ANXA3n,A

PCS,ASR

GL1

,ATP5

B,C

LU,C

TSD

n ,EE

F1G,FGL1

n ,HPR

T1n,H

SP90AA1n,H

SP90B1n,H

SPA1A

/HSP

A1B

,HSP

A2,HSP

A5n,H

SPA8,ODF2,P

GCn ,PG

K2,PG

Pn,P

RKAR2A

,PSM

B4,RUVBL1

,RUVBL2

,SEM

G1,SE

MG2,SP

ANXB/SPA

NXB1,ST

IP1n,T

EKT4,TPI1,TSG

A10,T

UBA3C

,TUBB3n,

TUBB4A

n

aValuesin

parenthesesreferto

thenumberof

proteins

sampled.F

orprotein-specificdata

from

massspectrom

etry,and

reasonswhy

40sperm

proteins

wereexcluded

from

downstream

analyses,see

Supportin

gInform

ationTables8−

11.Proteinsthatwerenotcategorized

asbeingexpressedinhuman

spermor

testis/testesinUniProt

database

(state:1stJuly2013)andwerenotincludedinthestudyof

Martın

ez-H

erediaet

al.66arehighlighted

byn(n).

Figure 1. Two-dimensional gel (A), anti-pSTY (B), and anti-pYWestern blot (C) prepared from spermatozoa of subject 1 (Homosapiens). Only proteins with unambiguous phosphorylation status thatwere used for evolutionary analyses are highlighted. The images arerepresentative of three technical replicates per application and threenormozoospermic men (subjects 1−3). For gel and blot images withhigher resolution of subjects 1−3, see Supporting Information Figures1−3.

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available for 51. These 51 phosphorylated sperm proteins couldfurther be distinguished according to signals (presence/absence) in anti-pY Western blots. According to thiscomplementary approach, 33 human sperm proteins werecategorized as sperm phosphoproteins without Y phosphor-ylation and another 18 as sperm phosphoproteins with Yphosphorylation (Table 1; Supporting Information Tables 8and 9). The required six mammalian cDNA orthologs werefurther available for eight of the sperm proteins that gave nosignal in any of the Western blots. As the same eight proteinswere neither listed in Phospho.ELM nor in PhosphoPepdatabases, they were regarded as nonphosphorylated (Table 1,Supporting Information Table 10). These altogether 59 spermproteins with consistent phosphorylation status in our humansample (thereof eight proteasome subunits) represent the basisof downstream investigations.Neural network predictions provided supportive evidence for

our experimental data. In particular, NetPhos60 predicted pYsites for all mammalian proteins considered when the humanortholog was classified as a phosphoprotein with Y phosphor-ylation. Moreover, network-based predictions suggested thepresence of pS and/or pT sites across all investigated proteins,in case the human ortholog was categorized as a spermphosphoprotein without Y phosphorylation. Cross-speciesconservation of phosphorylation patterns is also probable,considering overall low evolutionary rates at phosphosites (seebelow). Obviously, these prediction-based data do not proveconserved phosphorylation patterns across the sampled spermproteins. However, they show that such cross-speciesconservation is probable, perhaps not in terms of singlephosphosites, but very likely in terms of entire proteins. Besidesthe possibility of variation across species, the phosphorylationstates of the sampled proteins might as well vary across theentire human population. On the other hand, the phosphor-ylation state of each protein was assessed applying strict criteria,and we expect our data to be representative for humans, at leastin quantitative terms.As mentioned above, 59 sperm proteins were used for

evolutionary analyses. The remaining 40 (thereof 9 withclarified phosphorylation state) out of 99 sperm proteins werenot used for evolutionary analyses (Table 1; SupportingInformation Table 11): due to missing database entries of atleast one cDNA ortholog we excluded seven phosphoproteinswithout Y phosphorylation (ACTBL2, PGC, PGP, PRKAR2A,RUVBL1, TEKT4, TUBA3C) and two nonphosphorylatedsperm proteins (HPRT1, SEMG1). Four sperm proteinsincluding one proteasome subunit gave consistent signals inanti-pSTY Western blots, but inconsistent signals in anti-pYWestern blots (ABHD10, ACTRT2, EEF1G, PSMB4).Discrepancies between expected and actual MW and pI ledto exclusion of six more sperm proteins. A final set of 21 othersperm proteins was not further considered in evolutionaryanalyses as their phosphorylation status could not be definitelysettled at the STY level. This was due to inconsistent signals inanti-pSTY Western blots (10 proteins), conflicts between ourown findings and database entries (4 proteins), and multipledetections from single spots (7 proteins) (Table 1; SupportingInformation Table 11). Due to the strict exclusion ofambiguous data relating to differences in phosphorylationpatterns between probands and insecure interpretations ofphosphorylation signals, false positives should not havecompromised evolutionary analyses.

No Support for an Association of TyrosinePhosphorylation with Levels of Evolutionary Conservationand Functional Constraint

On the basis of cDNAs representing a constant sample of sixmammalian species, test statistic rejected site-specific positiveselection for the vast majority of proteins showing phosphor-ylation in human probands. In detail, LRT (M8A/M8; 1% levelof significance) supported site-specific positive selection onlyfor a single sperm phosphoprotein without Y phosphorylation(CAPZB) and for none of the sperm phosphoproteins with Yphosphorylation (Table 2). Consequently, χ-square testrejected differential prevalence of site-specific positive selectionin both samples (Yates’ P > 0.05; Table 2; SupportingInformation Table 3).

Analyses of sequence evolution across sites confirmed similarlevels of evolutionary conservation in both protein samples, nomatter if the human ortholog was a sperm phosphoprotein withor without Y phosphorylation. Although the employedparameters tended to be increased in sperm phosphoproteinswith Y phosphorylation as compared to their counterpartswithout Y phosphorylation, median dN/dS values (M8A) werelow in both groups (median dN/dS = 0.097 and 0.036,respectively; Figure 2A; Supporting Information Table 3).Moreover, highly overlapping 95% confidence intervalsindicated similar levels of dN/dS in both groups (Figure 2A).Consequently, Mann−Whitney U test did not supportdifferential levels of dN/dS in sperm phosphoproteins withand without Y phosphorylation (P > 0.05; Table 3). Thepattern was reproduced at the amino acid level where weobserved higher, but not significantly increased, amino aciddistances in sperm phosphoproteins with Y phosphorylation(median = 0.080) relative to sperm phosphoproteins without Yphosphorylation (median = 0.030, P > 0.05, Mann−Whitney Utest, Table 3, Figure 2A).Prevalent evolutionary conservation of sperm phosphopro-

teins with and without Y phosphorylation in human probandswas reproduced with respect to site-specific levels of purifyingselection. Thus, fractions of proteins with α values less than 1were persistently high in both subgroups, sperm phosphopro-teins with Y phosphorylation (αY < 1 = 90.9%) and spermphosphoproteins without Y phosphorylation (αS < 1 = 69.6%;αT < 1 = 92.3%; Table 4; Supporting Information Table 5). Incase of αY (P < 0.05) and αT values (P = 0.001), support fromZ-test for a deviation from the null expectation of 50% was evensignificant. Despite overall prevailing purifying selection at pSsites, small sample size led to only tentative, but not significant,

Table 2. Incidence of Site-Specific Positive Selection inSperm Phosphoproteins with and without YPhosphorylation in Human Probandsa

condition 1/condition 2

spermphosphoproteins

with Yphosphorylation

spermphosphoproteins

without Yphosphorylation

Yates’P, χ-squaretest

number of genes withsignificant support forsite-specific positiveselection/without suchsupport

0/18 1/32 >0.05

aP, probability of α error. Support and nonsupport for site-specificpositive selection refers to a likelihood ratio test comparing the fit oftwo versions of CODEML model M8 at the 1% level of significance.See Supporting Information Table 3 for gene-specific data.

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support in case of αS values (P > 0.05; Z-test; Table 4). Thedetailed average evolutionary rates were 0.127 (pY) and 0.391(Y) in sperm phosphoproteins with Y phosphorylation, and0.285 (pS), 0.311 (S), 0.214 (pT), and 0.512 (T) in spermphosphoproteins without Y phosphorylation (Figure 3;Supporting Information Table 5). We regard these results asevidence for overall similar levels of purifying selection at

phosphosites in sperm phosphoproteins, irrespective of theirphosphorylation status at Y residues in humans.Despite some minor differences, human sperm phosphopro-

teins with and without Y phosphorylation showed similardistributions across GO classes (Yates’ P > 0.05; χ-square test;see also Figure 4 and Supporting Information Table 6).Actually, the respective P value was close to 1 indicating nearlyidentical distributions of both protein groups across GO classes.This applies especially to nearly identical proportions of spermproteins playing a role in cytoskeleton, flagella, and cellmovement (22.2% and 18.2%); energy production (22.2% and18.2%); and metabolism (5.6% and 6.1%). Finally, interactionlevels were in the same range in both groups, as exhibited bymedians of 24 and 31 PPI partners in sperm phosphoproteinswith and without Y phosphorylation, respectively (P > 0.05,Mann−Whitney U test, Table 3, Figure 2B, SupportingInformation Table 6). Thus, levels of cross-species conservationand functional constraint of sperm phosphoproteins werewidely independent of the phosphorylation status at Y residuesin human probands.

Increased Levels of Evolutionary Conservation andFunctional Constraint in Phosphorylated Relative toNonphosphorylated Sperm Proteins

Using cDNA orthologs of human, Rhesus monkey, housemouse, Norway rat, bovine, and pig, M8A estimates of dN/dSwere consistently smaller than 1 (Supporting InformationTables 3 and 4). This illustrates the predominant role ofnegative selection in our sample of mammalian sperm proteins,regardless of whether they were or were not phosphorylated inhuman probands. Irrespective of this general tendency forevolutionary conservation in both samples, levels of dN/dSestimates were significantly increased in sperm proteins without(median dN/dS = 0.156) relative to sperm proteins withphosphorylation in human probands (median dN/dS = 0.052;Figure 5A). Support from Mann−Whitney U test fordifferential levels of dN/dS even withstood correction formultiple testing (P < 0.05; Table 5). This pattern wasreproduced on the basis of average amino acid distances(JTT) generated by AAML. Thus, median amino acid distanceswere significantly higher (P < 0.01; Mann−Whitney U test) insperm proteins without phosphorylation (median = 0.113) thanin those with phosphorylation in humans (median = 0.041,Figure 5A, Table 5, Supporting Information Tables 3 and 4).This consistency of results demonstrates that differential levelsof dN/dS reflect differing nonsynonymous, but not synon-ymous, substitution rates in phosphorylated and nonphos-phorylated sperm proteins. In line with this, LRT (M8A/M8,1% level of significance) supported the occurrence of positivelyselected sites for 3 out of 8 nonphosphorylated sperm proteinsand for only 1 out of 51 phosphorylated sperm proteins(Supporting Information Tables 3 and 4). Consequently, χ-square test supported differential incidence of site-specificpositive selection across both samples with high significance(Yates’ P < 0.01, χ-square test, Table 6).We observed additional differences between phosphorylated

and nonphosphorylated sperm proteins regarding theirdistribution across GO classes (Supporting Information Tables6 and 7). Although χ-square test rejected unequal distributionof both samples across all distinguished GO classes (Yates’ P >0.05), sperm proteins playing a role in transcription, proteinsynthesis, transport, folding, and turnover (35.3% vs 12.5%);energy production (19.6% vs 12.5%); or cytoskeleton, flagella,

Figure 2. Sequence evolution (A) and protein interactions (B) ofsperm proteins that were classified as phosphoproteins with or withoutY phosphorylation. Protein-specific dN/dS values were inferred acrosssix mammalian orthologs applying model version M8A (CODEML).Protein-specific amino acid (aa) distances refer to mean pairwisedistances among the same six mammalian orthologs, using the JTTsubstitution matrix. The PSICQUIC metaserver was searched for nPPIdata. The graphic depicts medians (see numbers at the top of bars)and 95% confidence intervals of medians (whiskers) that were inferredfrom 100 000 bootstrap replicates (see Table 3). Gene/protein-specificvalues are reported in Supporting Information Tables 3 and 6. Thephosphorylation status of proteins refers to human spermatozoa.

Table 3. Sequence Evolution across Sites and Protein−Protein Interactions of Sperm Phosphoproteins with andwithout Y Phosphorylation in Human Probandsa

median valuescompared

spermphosphoproteins withY phosphorylation

sperm phosphoproteinswithout Y

phosphorylation

P (2-sided)MWU

dN/dS acrosscodon sites(M8A)

0.097 (0.043−0.147) 0.036 (0.027−0.084) >0.05

amino aciddistances(JTT)

0.080 (0.029−0.110) 0.030 (0.021−0.052) >0.05

nPPI 24 (10−43) 31 (8−55) >0.05adN/dS, ratio of nonsynonymous to synonymous substitution rates;MWU, Mann−Whitney U test; nPPI, number of protein interactionpartners; P, probability of α error. Values in parentheses refer to 95%confidence intervals of medians that were inferred from 100 000bootstrap replicates, each. See Supporting Information Tables 3 and 6for gene/protein-specific data.

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and cell movement (19.6% vs 12.5%) occurred more frequentlyin the phosphorylated relative to the nonphosphorylatedsample (Figure 6). In contrast, proteins involved in cellrecognition were clearly overrepresented in the nonphosphory-lated relative to the phosphorylated sample (37.5% vs 3.9%). Itis noteworthy that sperm proteins involved in signal trans-duction (LZTFL1, YWHAZ) were restricted only to the sampleof phosphorylated sperm proteins (3.9% vs 0%; Figure 6) and,in particular, to the sampled sperm phosphoproteins without Yphosphorylation (Figure 4).Importantly, the sampled sperm proteins also differed with

respect to their role in human PPI networks. Thus, spermproteins that were phosphorylated in human probands havesignificantly more protein−protein interaction partners (me-dian nPPI = 29) than nonphosphorylated human spermproteins (median nPPI = 3; P < 0.05, Table 5, Figure 5B,Supporting Information Tables 6 and 7). Additionally,

Spearman’s rank provided highly significant support for anegative correlation between nPPI and dN/dS (M8A) acrosssperm proteins that also withstood correction for multipletesting (rS = −0.709, P < 0.001, Supporting Information Figure4). Thus, generally more interaction partners in phosphorylatedas compared to nonphosphorylated sperm proteins may beconsidered as a factor contributing to higher levels of functionalconstraint and evolutionary conservation in the former relativeto the latter group. Eight proteasome subunits, all of themshowing phosphorylation in human probands, were particularlyconspicuous in this regard: with a median dN/dS value of 0.012(M8A) and a median amino acid distance of 0.009 (JTT), theywere all highly conserved. Moreover, with a median of 79

Table 4. Evolutionary Rates at Predicted Phosphosites and Their Nonphosphorylated Counterparts and Ratios of Both Rates inSperm Phosphoproteins with and without Y Phosphorylation in Human Probandsa

sperm phosphoproteins amino acid site av evolutionary rate proteins with α < 1/α > 1 fraction α < 1 (%) P (2-sided) Z-test

with Y phosph pY 0.127 (0.020−0.294) 10/1 90.9 <0.05*Y 0.391 (0.189−0.625)

without Y phosph pS 0.285 (0.161−0.442) 16/7 69.6 >0.05S 0.311 (0.213−0.418)

without Y phosph pT 0.214 (0.088−0.378) 24/2 92.3 0.001**T 0.512 (0.334−0.726)

aα, ratio of evolutionary rates at predicted phosphosites (pS, pT, pY) vs evolutionary rates at nonphosphorylated counterparts (S, T, Y); av, average;P, probability of α error; phosph, phosphorylation. Values in parentheses refer to 95% confidence intervals of averages that were inferred from100 000 bootstrap replicates, each. Asterisks (* and **) highlight significance at the 5% and 1% level, respectively, after correction for multipletesting. See Supporting Information Table 5 for protein-specific data.

Figure 3. Evolutionary rates at pY, pS, and pT residues and theirnonphosphorylated counterparts in sperm phosphoproteins with Yphosphorylation (Y residues) and without Y phosphorylation (S and Tresidues). The graphic shows average evolutionary rates (see numbersat the top of bars) and 95% confidence intervals (whiskers) that werecalculated from 100 000 bootstrap replicates (see Table 4). Protein-specific rates were derived across six mammalian orthologs as averagenumbers of substitutions per site per billion years. The phosphor-ylation status of proteins refers to human spermatozoa. Phosphositeswere predicted by NetPhos 2.0 program.60 For protein-specific data,see Supporting Information Table 5.

Figure 4. GO classes of human sperm phosphoproteins with andwithout phosphorylation at Y (absolute numbers and proportions).(A) Sperm phosphoproteins with Y phosphorylation and (B) spermphosphoproteins without Y phosphorylation. Assignment to GOclasses according to published data (UniProt, original literature; seeSupporting Information Table 6).

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protein interactants, all of them appeared to have a centralposition in the sperm interactome (see Supporting InformationTables 3 and 6).

In summary, our data suggest that overall stronger functionalconstraint, due to more interactants, accounts for higher levelsof cross-species conservation of phosphorylated as compared tononphosphorylated sperm proteins. By contrast, differences inthe distribution across GO classes seem to contribute less to

Figure 5. Sequence evolution (A) and protein interactions (B) ofsperm proteins that were classified as being phosphorylated andnonphosphorylated. Protein-specific dN/dS values were inferredacross six mammalian orthologs applying model version M8A(CODEML). Protein-specific amino acid (aa) distances refer tomean pairwise distances among the same six mammalian orthologs,using the JTT substitution matrix. The PSICQUIC meta-server wassearched for nPPI data. The graphic depicts medians (see numbers atthe top of bars) and 95% confidence intervals of medians (whiskers)that were derived from 100 000 bootstrap replicates (see Table 5).Gene/protein-specific values are reported in Supporting InformationTables 3, 4, 6, and 7. Asterisks (* and **) indicate significance at the5% and 1% level after correction for multiple testing (Mann−WhitneyU test). The phosphorylation state of proteins refers to humanspermatozoa.

Table 5. Sequence Evolution across Sites and Protein−Protein Interactions of Sperm Proteins That Are EitherPhosphorylated or Nonphosphorylated in HumanProbandsa

median valuescompared

phosphorylatedsperm proteins

nonphosphorylatedsperm proteins

P (2-sided)MWU

dN/dS across codonsites (M8A)

0.052 (0.034−0.088)

0.156 (0.076−0.268)

<0.05*

amino acid distances(JTT)

0.041 (0.027−0.071)

0.113 (0.090−0.183)

<0.01**

nPPI 29 (14−43) 3 (0−26) <0.05*adN/dS, ratio of nonsynonymous to synonymous substitution rates;MWU, Mann−Whitney U test; nPPI, number of protein interactionpartners; P, probability of α error. Values in parentheses refer to 95%confidence intervals of medians that were inferred from 100 000bootstrap replicates, each. Asterisks (* and **) highlight significanceat the 5% and 1% level, respectively, following correction for multipletesting. See Supporting Information Tables 3, 4, 6, and 7 for gene/protein-specific data.

Table 6. Incidence of Site-Specific Positive Selection inSperm Proteins That Are Either Phosphorylated orNonphosphorylated in Human Probandsa

condition 1/condition 2

phosphorylatedsperm proteins

nonphosphorylatedsperm proteins

Yates’P, χ-square

test

number of genes withsignificant supportfor site-specificpositive selection/without suchsupport

1/50 3/5 <0.01**

aP, probability of α error. Support and nonsupport for site-specificpositive selection refers to a likelihood ratio test comparing the fit oftwo versions of CODEML model M8 at the 1% level of significance.Asterisks (**) highlight significance at the 1% level after correction formultiple testing. See Supporting Information Tables 3 and 4 for gene-specific data.

Figure 6. GO classes of human sperm proteins as distinguished byoverall phosphorylation status in human probands (absolute numbersand proportions). (A) Phosphorylated sperm proteins: overrepresen-tation of proteins involved in cytoskeleton, flagella, and cellmovement; energy production; transcription, protein synthesis,transport, folding, and turnover. (B) Nonphosphorylated spermproteins: overrepresentation of proteins participating in cellrecognition. Assignment to GO classes according to published data(UniProt, original literature; see Supporting Information Tables 6 and7).

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the unequal evolutionary rates of sperm proteins with andwithout phosphorylation in humans. Moreover, the degree ofpurifying selection does not associate with phosphorylation ofhuman sperm proteins at Y residues. If at all, levels ofevolutionary conservation across mammals are only slightly, butnot significantly, higher in sperm phosphoproteins without Yphosphorylation than in sperm phosphoproteins with Yphosphorylation in humans.

■ DISCUSSION

Evolutionary Conservation of Mammalian Sperm ProteinsIs Associated with Overall, but not with TyrosinePhosphorylation in Humans

Forty-two out of 99 sperm proteins that we detected by massspectrometry were so far not classified as being expressed inhuman sperm, testis or testes in the UniProt database (state:first July 2013), nor were they included in the study ofMartınez-Heredia et al.66 (Table 1). Hence, the present studycontributes to the growing knowledge on the composition ofthe human sperm proteome.10,22 In addition, our data showthat overall phosphorylation, but not Y phosphorylation, affectsevolutionary rates of sperm proteins. Indeed, we found similarlevels of evolutionary conservation in sperm proteins, no matterif they were classified as phosphoproteins with or without Yphosphorylation according to evidence from human probands(Figures 2A and 3, Tables 2−5; for protein classification, seeMaterials and Methods section). The consistencies betweenboth groups of sperm proteins probably result from similarlevels of functional constraint as shown by similar distributionsacross GO classes and comparable ranges in numbers of proteininteraction partners (Figure 2B and 4; Table 3). Thus, thepresent data reject a special relevance of pY versus pST sites forsperm functioning, although pY and pST sites generally differ inrespect to incidence, mode of phosphorylation, and bindingenergies.7,26

Moreover, our data clearly answer the question of inter-relation between overall phosphorylation (at S, T, and Yresidues) of human sperm proteins and rates of sequenceevolution. Using cDNAs and amino acid sequences of sixmammalian species, we were able to reproduce an increasedevolutionary conservation of phosphorylated as compared tononphosphorylated sperm proteins in human spermatozoa(Figure 5A, Tables 5 and 6). Considering support from neuralnetwork predictions for conserved phosphorylation statesacross the sampled species (see Results section), spermphosphoproteins generally seem to evolve with lowered ratesin mammals. The respective dN/dS values were below(phosphorylated sperm proteins: median dN/dS = 0.052)and above (nonphosphorylated sperm proteins: median dN/dS= 0.156) the genome-wide average of dN/dS as derived fromanalyses of 14 963 one-to-one orthologs in the mouse-ratcomparison (=0.128).48 This suggests that nonphosphorylationof sperm proteins associates with a slight acceleration ofsequence evolution, whereas phosphorylation is linked to aboveaverage conservation. However, this conclusion has to be takenwith caution, as different species samples and tree depthshamper comparisons between the two studies. Anyhow, spermproteins showing phosphorylation in humans were moreconserved than their nonphosphorylated counterparts. Thus,the evolution of mammalian sperm proteins is in compliancewith a general tendency for increased conservation of

phosphorylation versus nonphosphorylation in bacteria andeukaryotes.6,31

Cross-species conservation of phosphorylated sperm proteinswas also apparent at the level of predicted phosphosites:whether taking sperm phosphoproteins with Y (αY < 1 =90.9%) or without Y phosphorylation in humans (αS < 1 =69.6%; αT < 1 = 92.3%; Table 4), fractions of proteins with αvalues <1 were consistently higher than genome-wide averagesinferred from a total of 4484 proteins across 44 mammalianspecies (αS < 1 = 68.1%; αT < 1 = 63.6%; αY < 1 =63.5%).33,64 Furthermore, mean evolutionary rates at phospho-sites as derived from our data (pY, 0.127; pS, 0.285; pT, 0.214;Figure 3) were decreased compared to genome-wide referencevalues (pY, 1.03; pS, 0.83; pT, 0.87).33 Again, conclusions haveto be drawn with care considering variations in sample size andtree depth between the present and the reference study.33

Nevertheless, both studies demonstrate that phosphositesevolve under strong purifying selection. This agrees withresults originating from studies with methodically differentapproaches that showed evolutionary conservation of predictedand, especially, of functional phosphosites as well as ofphosphorylation mechanisms in both bacteria and eukar-yotes6,27−29,32,67 (see also ref 68). Moreover, prevalentevolutionary conservation of phosphorylation is not in conflictwith recurrent evidence questioning raised levels of purifyingselection at phosphosites.31,35 The seeming contradiction ratherreflects that entire phosphoproteins are commonly moreconserved than single phosphosites.31 Reports on phases ofaccelerated and adaptive evolution of phosphosites, phospho-proteins, and entire phosphoproteomes32,35,36,41 are not inconflict with a general tendency for conservation in theevolution of phosphorylation either. The present study showsthat the sperm (phospho)proteome fully complies with thisuniversally valid pattern of prevailing evolutionary conservation.

Differential Functional Constraint between Sperm ProteinsThat Are Phosphorylated and Sperm Proteins That Are NotPhosphorylated in Humans

Lower evolutionary conservation of sperm proteins detected asnonphosphorylated in human probands is probably aconsequence of a high proportion of cell recognition proteins(Figure 6) that are frequently located in the sperm membraneand acrosomal matrix47,69 and are commonly known for rapidevolution.44,47,70−73 In contrast, increased evolutionary con-servation of sperm proteins showing phosphorylation in humanprobands appears to reflect their stronger involvement in highlyessential cellular processes such as transcription, proteinsynthesis, transport, folding, and turnover as well as incytoskeleton, flagella, and cell movement (Figure 6).74,75

Such increased relevance is further emphasized in this studyby the above-mentioned signal transduction proteins and theirstrict confinement to the group of phosphorylated spermproteins (Figures 4 and 6). Although differential frequenciesacross GO classes might be part of the explanation, the positionwithin the sperm interactome seems to be the deeper reasonbehind differential levels of cross-species conservation ofphosphorylated relative to nonphosphorylated sperm proteins.Indeed, Spearman’s rank correlation between numbers ofinteraction partners and dN/dS values (M8A) indicates astrong negative relationship between these two variables in oursperm protein sample (Supporting Information Figure 4).Considering this fact, significantly higher numbers ofinteractants could explain stronger conservation of sperm

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proteins with than of those without phosphorylation in humanprobands (Figure 5B, Table 5). As more PPI partners areassociated with higher centrality in PPI networks and higherprotein essentiality,49,51−53 stricter conservation of phosphory-lated sperm proteins presumably results from their utmostimportance for sperm functioning and successful fertilization.Similar findings from analyses of yeast (Saccharomyces

cerevisiae) proteins show that more PPI partners and higheressentiality in phosphorylated relative to nonphosphorylatedproteins reflect a general pattern in eukaryotes.76 Most likely,the need for compensatory exchanges in additional interactionpartners imposes higher levels of functional constraint andevolutionary conservation on phosphorylated versus non-phosphorylated sperm proteins.77 Yet another factor increasingevolutionary conservation of phosphorylated sperm proteinsmight be the higher number of interacting domains, each beingunder functional constraint, in proteins with more interactionpartners.50 It must be further considered that kinase−substraterecognition is mediated not only by phosphosites and kinasedocking motifs, but also by adaptor and scaffold proteins.26

This again imposes increased levels of functional constraint onphosphoproteins. In the present study, the determinedproteasome subunits, all of which are phosphorylated, may bethe best example for such a pattern: proteasomes accomplishhighly essential decomposition of misfolded, damaged, orunnecessary proteins and are involved in capacitation, acrosomereaction, and fertilization in various metazoan species.78

Increased numbers of protein interaction partners and highlevels of evolutionary conservation of the sampled proteasomesubunits (Supporting Information Tables 3 and 6) demonstratehow the quantity of interactants might influence proteinevolution. Hence, following duplication and functionaldiversification under relaxed functional constraint proteasomesubunits obviously evolved under increased levels of functionalconstraint.79−81 Thus, as exemplarily stated for proteasomesubunits, it seems that strong conservation of phosphorylatedsperm proteins is a consequence of manifold PPI partners andhigh centrality in intracellular PPI networks.49,50 On thecontrary, less evolutionary conservation of nonphosphorylatedsperm proteins reflects a general tendency for proteins at thenetwork periphery to be frequently surface proteins coevolvingwith their extracellular interaction partners.53,71

■ CONCLUSIONSOn the basis of experimentally obtained phosphorylation dataof human sperm proteins we show that levels of evolutionaryconservation of mammalian sperm phosphoproteins areunaffected by Y phosphorylation. Conversely, phosphorylationat S, T, and Y residues and hence overall phosphorylation inhumans associates with increased cross-species conservationacross sperm proteins and at predicted phosphosites. Thus,sperm phosphoproteins seem to be more representative of thepredominant pattern in the evolution of male reproductiveproteins, i.e., purifying selection,48,82 whereas sequenceevolution of nonphosphorylated sperm proteins is morerepresentative of the patterns usually associated withfertilization proteins, i.e., accelerated and adaptive evolu-tion.44−47,72,83 Furthermore, our data imply that more PPIpartners might be an important factor contributing to highercross-species conservation of phosphorylated sperm proteinsand predicted phosphosites. As phosphorylated sperm proteinshave already been shown to play a role in reduced male fertility(see, e.g., ref 22), evolutionarily conserved sperm phosphopro-

teins might be prime candidates for its diagnosis and treatment.In turn, nonphosphorylated sperm proteins are promisingtargets for contraceptive vaccines for men and could beconsidered as biomarkers for different grades of fertility inanimal husbandry: due to fewer interaction partners and atendency toward higher reproduction specificity, their impair-ment or blocking should have less serious adverse effects.

■ ASSOCIATED CONTENT*S Supporting Information

Additional tables and figures. This material is available free ofcharge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author

*E-mail: [email protected]. Phone: +49 (0) 6131 3923179.Fax: +49 (0) 6131 3923799.Author Contributions

The manuscript was written through contributions of allauthors. All authors approved the final version of themanuscript.Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThe authors are grateful to anonymous reviewers whosecomments and suggestions on earlier drafts greatly helped toimprove the manuscript. The study was supported by grantsfrom the German Research Council (DFG, HE 3487/2-1) andJohannes Gutenberg-University Mainz to H.H. (“Stufe 1”).

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