Article

pubs.acs.org/jpr

Evolutionary Conservation of Mammalian Sperm Proteins Associates

with Overall, not Tyrosine, Phosphorylation in Human Spermatozoa
Julia Schumacher, Sanja Ramljak, Abdul R. Asif, Michael Scharath, 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
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Publication Date (Web): September 27, 2013 | doi: 10.1021/pr400228c

S Supporting Information
*

ABSTRACT: We investigated possible associations between sequence

evolution of mammalian sperm proteins and their phosphorylation status in
humans. As a reference, spermatozoa from three normozoospermic men
were analyzed combining two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry. We identied 99 sperm proteins (thereof
42 newly described) and determined the phosphorylation status for most of
them. Sequence evolution was studied across six mammalian species using
nonsynonymous/synonymous rate ratios (dN/dS) and amino acid
distances. Site-specic purifying selection was assessed employing average
ratios of evolutionary rates at phosphorylated versus nonphosphorylated
amino acids (). According to our data, mammalian sperm proteins do not
show statistically signicant sequence conservation dierence, no matter if
the 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 that
numerous proteinprotein interactants constrain sequence evolution of sperm phosphoproteins. Although our ndings reject a
special relevance of Y phosphorylation for sperm functioning, they still indicate that overall phosphorylation substantially
contributes to proper functioning of sperm proteins. Hence, phosphorylated sperm proteins might be considered as prime
candidates 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, binding
properties, assembly of protein complexes, cell cycle, and signal
transduction.14 However, protein phosphorylation is not
limited to eukaryotes but extends to bacteria and viruses.57
In multicellular animals, protein phosphorylation triggers
processes in both somatic and germ cells and is generally
considered to be a fundamental regulatory mechanism.8 With
respect to male reproduction, changes in phosphorylation and,
in particular, in Y phosphorylation are of utmost importance for
processes such as sperm capacitation and hyperactivation as
well as for acrosome reaction and binding of the spermatozoon
to the zona pellucida.915 Other studies demonstrate high
relevance of (Y) phosphorylation for the regulation of sperm
motility,1618 most probably through modulation of microtubule and brous sheath sliding properties.19,20 The outlined
involvement of phosphorylation in diverse sperm functions
2013 American Chemical Society

could explain why sub- and infertility in men has been

repeatedly linked to disturbed phosphorylation patterns,21,22
especially with respect to Y phosphorylation.23,24 The recurrent
evidence for an involvement of Y phosphorylation in reduced
male fertility brings up the question of potentially higher impact
of Y versus ST phosphorylation on sperm functioning. Specic
ways how Y and ST sites undergo phosphorylation and
dierences in incidence and binding energies within the
phosphorylatated moieties25,26 would certainly endorse such a
scenario.
Phosphorylation plays a signicant role not only in regulation
of cell processes but also in maintenance of cell structure, which
suggests high levels of functional constraint. Accordingly,
phosphoproteins, phosphorylation sites (phosphosites), and
the mechanisms of phosphorylation have been reported to be
Received: December 4, 2012
Published: August 6, 2013
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evolutionarily conserved across eubacteria and eukaryotes.6,2733 On the other hand, a growing body of data
illustrates that phosphosites, phosphoproteins, as well as the
mechanisms of phosphorylation underwent phases of accelerated and adaptive evolution.32,3436 Indeed, large-scale
changes in phosphorylation may be pertinent for functional
diversication and retention of paralogs following genome
duplication.3638 Moreover, changes in phosphorylation are
most likely engaged in the evolution of receptorligand
systems, either in terms of immune evasion or in terms of
coevolving receptorligand systems3941 (see also ref 34).
Considering the relevance of both phenomena in fertilization,42,43 changes in phosphorylation may represent a factor
accelerating sequence evolution of mammalian fertilization
proteins.41,4447 However, the denite impact of phosphorylation on sequence evolution of mammalian sperm proteins
still needs to be assessed. Hence, the present study aims at
clarifying if overall STY phosphorylation of human sperm
proteins associates with increased or with lowered levels of
cross-species conservation, thereby taking sperm proteins
exhibiting no phosphorylation in humans and genome-wide
averages33,48 as references. As the regulation of diverse
processes by Y phosphorylation may be particularly relevant
for sperm functioning,25 we additionally examine potential
associations between this posttranslational modication and
rates of sequence evolution.
In order to avoid that false predictions compromise analyses
we grouped sperm proteins according to their experimentally
validated phosphorylation status in humans. Taking spermatozoa of three healthy and normozoospermic men as a reference
and combining two-dimensional gel electrophoresis (2DE),
immunoblotting, and mass spectrometry (MS), we determined
a total of 99 human sperm proteins and claried the majoritys
phosphorylation status. On the basis of coding DNAs (cDNAs)
of six mammalian species we compared levels of cross-species
conservation between (i) sperm proteins that are either STY
phosphorylated or nonphosphorylated in humans, and (ii)
sperm proteins that are phosphoproteins either with or without
Y phosphorylation in human probands. Sequence evolution
across sites was assessed using nonsynonymous to synonymous
substitution rate ratios (dN/dS, also Ka/Ks or ). Since dS
should reect neutral evolution, dN/dS values larger, equating,
or smaller than 1 are commonly accepted as evidence for
adaptive evolution through positive selection, neutral evolution,
and evolutionary conservation through negative selection,
respectively. Codon-based analyses of sequence evolution are
complemented by investigations of amino acid distances across
sites and average ratios of evolutionary rates at phosphosites
versus nonphosphorylated counterparts (), whereby values
<1 point to purifying selection at phosphosites.33 Finally, we
considered protein-specic levels of functional constraint as
assessed by gene ontology and proteinprotein interactions
(PPI). The latter refers to evidence from network biology
whereupon proteins with more interaction partners, and
therefore more central positions in network topologies, show
higher essentiality and evolutionary conservation than the
proteins with fewer interaction partners that are located at the
network periphery.4953

Article

MATERIALS AND METHODS

Study Participants, Semen Collection, and Sperm

Preparation

As a reference we analyzed the phosphorylation status of

human (Homo sapiens) sperm proteins. In detail, ejaculates of
three healthy and normozoospermic men (Europeans; 2328
years) were included. Sampling was approved by the local
ethics committee. Ejaculates were collected into sterile cups
after at least two days of sexual abstinence. Ejaculates were
liqueed at 37 C and sperm parameters evaluated according to
World Health Organization (WHO) guidelines.54 Spermatozoa
were separated from seminal plasma by a succession of washing
steps with human tubal uid medium (HTF) supplemented
with HEPES, gentamycin, and 3% bovine serum albumin
(BSA), and centrifugation at 300g for 9 min (37 C). In
concordance with WHO standards54 and other studies on
sperm proteins55 including those on Y phosphorylation,56
spermatozoa were capacitated by swim-up (HTF, 30 min, 37
C).
Protein Extraction and Purication

Frozen sperm samples were thawed at 4 C and solubilized at

room temperature (RT) in lysis buer containing 7 M urea, 2
M thiourea, 4% CHAPS, and 1% ampholytes (pH 310; 100;
Bio-Rad), DTT (w/v), protease inhibitor cocktail (SigmaAldrich), and phosphatase inhibitor cocktail (Thermo
Scientic). Cell debris was removed by centrifugation for 1
min at maximum speed. Solubilized proteins were puried
using the ReadyPrep 2-D Cleanup Kit (Bio-Rad) and
rehydrated in a buer containing 7 M urea, 2 M thiourea, 4%
CHAPS, 1.5% DTT (w/v), and 1% ampholytes (pH 310;
100). Protein concentration was measured using a Bradford
assay (Bio-Rad).
Isoelectric Focusing, Equilibration, SDS-PAGE, and
Coomassie Staining

We loaded up to 540 g protein per strip (7 cm IPG

ReadyStrip, pH 58; Bio-Rad). Following passive rehydration
for 2 h at RT, strips were covered with mineral oil and actively
rehydrated for 14 h at 50 V. Isoelectric focusing (IEF; 2 h at
200 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 buer containing 6 M
urea, 0.375 M Tris (1.5 M, pH 8.8), 30% glycerol, and 2% SDS
(10%, w/v). For the rst equilibration step (25 min), the buer
was supplemented with 2% DTT, and for the second one (25
min), with 2.5% iodoacetamide. Second-dimension electrophoresis (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 calibrated
densitometer (Bio-Rad). We ran and documented a minimum
of three gels per donor. Another six gels (per donor) were
utilized for Western blotting.
Gel Excision, In-Gel Digestion, LCMS/MS, and Protein
Identication

For each protein spot, gel slices were excised and dried by
vacuum centrifugation. Gel pieces were soaked with digestion
buer supplemented with trypsin (0.01 g/L). Afterward,
samples were incubated overnight (37 C) in the same
digestion buer without trypsin. The next day, peptides were
extracted by sonication in solvents of increasing acetonitrile
(ACN) content. Eluates were collected, completely dried by
vacuum centrifugation, and frozen at 20 C. Peptides were
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proteins whose expected and actual MW and pI were in

conict. Proteins giving inconsistent phosphorylation signals
between the samples of dierent men were not taken into
account either. Thus, we classied a protein as being
phosphorylated at STY only in the case of consistent signals
in anti-pSTY Western blots. In turn, a sperm protein was
considered as being nonphosphorylated if no signal appeared in
any of the anti-pSTY and anti-pY Western blots and if the same
protein was neither reported as being phosphorylated in vivo
nor in vitro according to Phospho.ELM58 (http://phospho.elm.
eu.org/; evaluation date 20th June 2013) and PhosphoPep
2.059 databases (http://www.phosphopep.org/; valuation date
20th June 2013).
Furthermore, we regarded consistent signals from anti-pSTY
and anti-pY Western blots as an evidence for phosphorylation
of human sperm proteins at Y sites and possible additional
phosphorylation at S and/or T sites. We here refer to such
proteins as sperm phosphoproteins with Y phosphorylation. In
case of exclusive signals in anti-pSTY Western blots, a protein
was regarded as being phosphorylated at S and/or T sites and
being nonphosphorylated at Y residues. The latter proteins are
designated as sperm phosphoproteins without Y phosphorylation. Using the NetPhos 2.0 program60 (http://www.cbs.
dtu.dk/services/NetPhos/) we ensured for the orthologs of all
sampled species that neural network predictions conrm the
experimentally validated phosphorylation patterns.
Applying the above criteria, comigrating fragments of
undetected proteins cannot be excluded. However, the
procedure diminished the probability that decisions on the
phosphorylation status were confounded by comigrating
fragments. Moreover, although predictions support crossspecies conservation, we do not postulate identical phosphorylation patterns in nonhuman species. Thus, whenever we
name sperm proteins according to the phosphorylation status
of the human ortholog we do so for simplicity.

reconstituted in 0.1% formic acid for injection into a nanoow

HPLC coupled online with Q-TOF mass spectrometer. Peptide
samples (1 L) were consecutively introduced onto two C18reversed phase chromatography columns (rst to a trapping
column; C18 pepMap: 300 m 5 mm; 5 m particle size, and
second eluted through the analytical column; C18 pepMap100
nanoanalytical column: 75 m 15 cm; 3 m particle size)
using a nanoow CapLC autosampler (Waters). Peptides were
eluted with an increasing gradient of ACN and analyzed on a
Q-TOF Ultima Global mass spectrometer (Micromass)
equipped with a nanoow ESI Z-spray source in the positive
ion mode.57 The data were analyzed with the MassLynx v. 4.0
software and processed by using the Protein Lynx global server
(version 2.2; Waters), which generates .pkl les. The following
settings were used: 80% centroid with minimum peak of four
channels, 10% noise reduction, and medium deisotoping with a
3% threshold. The obtained peak patterns were searched
against the SwissProt database version 57 (525 997 sequences;
185 874 894 residues; http://expasy.org/sprot) using the
MASCOT (Matrix Science, http://www.matrixscience.com)
search engine. The following parameters were specied: trypsin
as an enzyme for digestion, up to a maximum of one missed
cleavage site allowed, monoisotopic mass value, unrestricted
molecular weight (MW), peptide tolerance 0.5 Da, and MS/
MS tolerance 0.5 Da. Proteins were identied on the basis of
a minimum of one peptide, whose ions score exceeded the
threshold of P < 0.05. Only those spots that contained a single
protein according to LCMS/MS results were further
considered. Moreover, we thoroughly checked if molecular
weight (MW) and isoelectric point (pI) of a detected protein
corresponded with the actual position of the respective protein
spot. If expected and actual MW and pI were in conict, a
protein was excluded. Comigrating fragments of other proteins
should hence not have biased our results.
Immunoblotting and Image Analysis

Generation of Alignments

Gels were blotted onto a polyvinylidene diuoride membrane

(Hybond-P, GE Healthcare) for 75 min at 53 mA, using a
transfer buer containing 192 mM glycine, 25 mM Tris, and
20% methanol. Blots were blocked for 1 h at RT in Trisbuered saline containing Tween 20 (TBST) and 5% proteasefree BSA. Three Western blots per subject were incubated (5%
BSA in TBST; 18 h at 4 C) with anti-pSTY (mouse
monoclonal antibody to phosphoserine/threonine/tyrosine; 4
g/mL; Abcam) and another three with anti-pY (mouse
monoclonal antibody to phosphotyrosine; 1.5 g/mL; Abcam).
Following washing, blots were incubated for another hour at
RT with horseradish peroxidase-conjugated sheep polyclonal
antibody (3.2 g/mL for anti-pSTY Western blots; 2 g/mL
for anti-pY Western blots; antibodies-online) in TBST
containing 1% BSA. Afterward, Western blots were documented with a ChemoCam Imager (Intas).

Codon-alignments were generated for each sperm protein with

claried phosphorylation status in human probands. The
alignments comprised cDNAs encoding the human reference
and orthologs from Rhesus monkey (Macaca mulatta), house
mouse (Mus musculus), Norway rat (Rattus norvegicus), bovine
(Bos taurus), and pig (Sus scrofa) that were retrieved from
NCBI and ENSEMBL databases. In the case of missing entries
for one or more orthologs a gene was excluded from all
subsequent analyses. Raw data sets were aligned in the codon
mode using the ClustalW algorithm implemented in the
GUIDANCE web-server (http://guidance.tau.ac.il/),61 which
also pruned data sets from unreliably aligned regions by
rejecting columns with condence scores below 0.93 (default
threshold). The same cDNAs were used for the generation of
amino acid alignments that were pruned from unreliably
aligned regions, too (GUIDANCE; default threshold of 0.93 for
the rejection of columns). Whether taking codon or amino acid
alignments, gap-positions were ignored in downstream analyses
by specic settings (see below). Supporting Information Tables
1 and 2 list protein names and accession numbers of cDNA
orthologs used for sequence analyses.

Determining the Phosphorylation Status of Human Sperm

Proteins

The phosphorylation status of a sperm protein was determined

by combining information from LCMS/MS, three gel scans,
three anti-pSTY Western blots, and three anti-pY Western blots
(per donor, each), thus merging the data from technical and
biological replicates. We matched the data with the aid of the
PDQuest (Bio-Rad) spot detection software and considered
exclusively those spots that scored a single protein according to
LCMS/MS results. As outlined above, spots containing >1
protein according to LCMS/MS were excluded, as were the

Analyses of Sequence Evolution

Sequence evolution of sperm proteins with claried phosphorylation status (see above for criteria) was studied at the codon
level using the aforementioned cDNA alignments (six
mammalian species, pruned from gaps) and the following
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metabolism (see Supporting Information Tables 6 and 7).

Moreover, using human protein IDs as search items (see
Supporting Information Tables 811) we extracted numbers of
PPI partners (nPPI) per human ortholog from 17 out of 25
databases available through PSICQUIC (Proteomics Standard
Initiative Common QUery InterfaCe; http://www.ebi.ac.uk/
Tools/webservices/psicquic/view/main.xhtml; state 5th June
2013). The GeneMANIA, iRefIndex, and Interoporc databases
were opted out in order to avoid redundant hits and a strong
skew from empirical evidence toward assumed interactions.
Results from BindingDB and ChEMBL were ignored as they
list interactions between proteins and drug-like molecules
instead of proteinprotein interactions. Additionally, we
excluded hits from the MPIDB and VirHostNet databanks,
which contain interactions with microbial and viral proteins, as
well as all other results referring to interactions between human
proteins and proteins of other species including pathogens.
Finally, we ignored search results with the attribute predictive
text mining, inferred by curator, and/or unspecied
method (quotation marks highlight PSICQUIC terminology).
Supporting Information Tables 6 and 7 contain nPPI data per
sampled sperm protein.

tree topology: ((bovine, pig), (mouse, rat), (Rhesus monkey,

human)). Analyses were carried out employing the CODEML
program implemented in PAML (Phylogenetic Analysis by
Maximum Likelihood) package v. 4.4.62 Codon-frequencies
were estimated from the data (F3 4), and sites with
ambiguity data were ignored (cleandata = 1). More precisely,
we tested for the presence of positively selected codon sites
using a likelihood ratio test (LRT) comparing the t of two
versions of model M8.44 Both model versions assume a
distribution of codon sites in the dN/dS interval (0,1).
However, while the alternative version (M8) allows for an
extra site class under positive selection (dN/dS 1), dN/dS of
this extra site class is xed at 1 in the null version (M8A). For
LRT, 2l was compared to critical values following a 50:50
mixture of a point mass at zero and a -square distribution with
degrees of freedom (df) equal to the dierence in the number
of free parameters between M8A and M8 (=1). To reduce the
number of false positives we applied a 1% level of signicance
(critical value = 5.41). To detect local optima, we ran M8 twice
using dierent initial dN/dS values. Supporting Information
Tables 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 analyses
were complemented by investigations at the amino acid level.
Thus, we inferred pairwise amino acid distances from the
above-mentioned amino acid alignments (six mammalian
species, pruned from gaps and unreliably aligned regions)
using AAML implemented in PAML package v. 4.4.62 We then
calculated protein-specic mean values from the pairwise
JonesTaylorThornton (JTT) distances63 reported in
AAML result les.
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, we
focused on pS and pT sites in sperm phosphoproteins without
Y phosphorylation and on pY sites in sperm phosphoproteins
with Y phosphorylation in humans. Levels of purifying selection
were assessed on the basis of average ratios of evolutionary
rates at phosphosites versus evolutionary rates at their
nonphosphorylated counterparts ().33,64 Phosphosites (pS,
pT, pY) and nonphosphorylated counterparts (S, T, Y) were
distinguished by neural network predictions on human
orthologs as conducted by the NetPhos 2.0 program.60
Evolutionary rates at sites of interest were dened as total
numbers of amino acid substitutions per site per billion years.64
Numbers of amino acid substitutions at sites of interest were
calculated applying maximum parsimony [AAML (rst) les].
The total time elapsed on the tree represented by our sixspecies sample (=94.4 million years) was obtained from the
TimeTree resource (www.timetree.org65). Site-specic evolutionary rates as well as values are reported for each protein in
Supporting Information Table 5.

Statistical Analyses

Taking our own results on human probands as a reference and

applying the above criteria, we generated two sample pairs, i.e,.
(i) phosphorylated and nonphosphorylated sperm proteins and
(ii) sperm phosphoproteins with and without Y phosphorylation. Employing -square tests, we tested for dierences
between groups of proteins with respect to the incidence of
signicant support for site-specic positive selection and the
distribution across GO classes. Using MannWhitney U test
we additionally analyzed if levels of dN/dS (M8A), mean
amino acid distances, and nPPI diered between sample pairs.
Additionally, we conducted Spearmans rank correlation
between dN/dS values across sites (M8A) and nPPI across
all studied sperm proteins, thus testing for a potential
association between these two variables. Focusing on our
sample of phosphorylated sperm proteins we nally tested if the
fraction of proteins with < 1 diered from the null
expectation of 50%, employing a Z-test.33 We determined
95% condence intervals for median and average values on the
basis of 100 000 bootstrap replicates using self-written Perl
scripts, which are available upon request. We adjusted P values
for multiple comparisons applying sequential Bonferroni
correction. All tests were conducted using SPSS version 20.0
(IBM).

RESULTS

Determined Sperm Proteins Show Dierent

Phosphorylation States

Mass spectrometry allowed for identication of altogether 99

human sperm proteins (Table 1, Supporting Information
Tables 811; for annotated mass spectra of the peptides with
maximum ions score, see Supporting Information Table 12)
from denser and fainter spots (Figure 1; Supporting
Information Figures 13). Out of these 99 human sperm
proteins, 42 were up to now not categorized as being expressed
in human sperm or testis/testes in the UniProt database (state:
1st July 2013), nor were they included in the reference study of
Mart nez-Heredia et al.66 Conspicuously, nine of the 99
identied proteins were proteasome subunits (Table 1;
Supporting Information Tables 8, 9, and 11).

Assessing Levels of Functional Constraint

Protein-specic levels of functional constraint were assessed on

the basis of gene ontologies and numbers of protein interaction
partners. On the basis of UniProt entries and original literature
we assigned each of the identied human sperm proteins to one
of the gene ontology (GO) classes distinguished by Mart nezHeredia et al.,66 i.e., energy production, transcription,
protein synthesis, transport, folding, and turnover, cell cycle,
apoptosis, and oxidative stress, signal transduction, cytoskeleton, agella, and cell movement, cell recognition, and
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identied proteins (abbreviations)

AARSD1n, ABHD10n, ACTB, ACTBL2n, ACTR1A, ACTR1B, ACTRT2, ANXA3n, APCS, ASRGL1, ATP5B, CLU, CTSDn, EEF1G, FGL1n, HPRT1n, HSP90AA1n, HSP90B1n, HSPA1A/HSPA1B,
HSPA2, HSPA5n, HSPA8, ODF2, PGCn, PGK2, PGPn, PRKAR2A, PSMB4, RUVBL1, RUVBL2, SEMG1, SEMG2, SPANXB/SPANXB1, STIP1n, TEKT4, TPI1, TSGA10, TUBA3C, TUBB3n,
TUBB4An

ALDH9A1n, CRISP2, DLD, DNAJB8, ECHS1n, PRDX2n, SPESP1, TEKT2

ACRBP, ACTA2n, ANXA1n, CAPZA1n, DNAJB11n, ECI1n, GSTM3, HIBADH, HSPA1L, MPST, PARK7, PSMD11n, PSMD14n, ROPN1, SDHAn, TEKT1, TUBB2Cn, UQCRC1

ACADS, AK8, APEH , CAPZB , CRISP1, ENO1, GDI2 , GK2, GLUL, GOT1 , HSPA9n, HSPD1, IDH3An, LAP3, LZTFL1, NME5, NME7, ODF1, PCYT2, PDHB, PHBn, PPP1CBn, PRDX4,
PSMA1n, PSMA6n, PSMB2, PSMB3, PSMC2n, PSMD13n, SPAG6, TUBA1An, TUBB2An, YWHAZn

Values in parentheses refer to the number of proteins sampled. For protein-specic data from mass spectrometry, and reasons why 40 sperm proteins were excluded from downstream analyses, see
Supporting Information Tables 811. Proteins that were not categorized as being expressed in human sperm or testis/testes in UniProt database (state: 1st July 2013) and were not included in the study of
Mart nez-Heredia et al.66 are highlighted by n (n).

sperm phosphoproteins
without Y
phosphorylation (33)
sperm phosphoproteins
with Y phosphorylation
(18)
nonphosphorylated sperm
proteins (8)
sperm proteins excluded
from analyses (40)

status

Table 1. Phosphorylation Status of 99 Human Sperm Proteins Identied by Mass Spectrometrya

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Figure 1. Two-dimensional gel (A), anti-pSTY (B), and anti-pY

Western blot (C) prepared from spermatozoa of subject 1 (Homo
sapiens). Only proteins with unambiguous phosphorylation status that
were used for evolutionary analyses are highlighted. The images are
representative of three technical replicates per application and three
normozoospermic men (subjects 13). For gel and blot images with
higher resolution of subjects 13, see Supporting Information Figures
13.

Out of the sperm proteins giving consistent signals in antipSTY Western blots, cDNA orthologs from human, Rhesus
monkey, house mouse, Norway rat, bovine, and pig were

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No Support for an Association of Tyrosine

Phosphorylation with Levels of Evolutionary Conservation
and Functional Constraint

available for 51. These 51 phosphorylated sperm proteins could

further be distinguished according to signals (presence/
absence) in anti-pY Western blots. According to this
complementary approach, 33 human sperm proteins were
categorized as sperm phosphoproteins without Y phosphorylation and another 18 as sperm phosphoproteins with Y
phosphorylation (Table 1; Supporting Information Tables 8
and 9). The required six mammalian cDNA orthologs were
further available for eight of the sperm proteins that gave no
signal in any of the Western blots. As the same eight proteins
were neither listed in Phospho.ELM nor in PhosphoPep
databases, they were regarded as nonphosphorylated (Table 1,
Supporting Information Table 10). These altogether 59 sperm
proteins with consistent phosphorylation status in our human
sample (thereof eight proteasome subunits) represent the basis
of downstream investigations.
Neural network predictions provided supportive evidence for
our experimental data. In particular, NetPhos60 predicted pY
sites for all mammalian proteins considered when the human
ortholog was classied as a phosphoprotein with Y phosphorylation. Moreover, network-based predictions suggested the
presence of pS and/or pT sites across all investigated proteins,
in case the human ortholog was categorized as a sperm
phosphoprotein without Y phosphorylation. Cross-species
conservation of phosphorylation patterns is also probable,
considering overall low evolutionary rates at phosphosites (see
below). Obviously, these prediction-based data do not prove
conserved phosphorylation patterns across the sampled sperm
proteins. However, they show that such cross-species
conservation is probable, perhaps not in terms of single
phosphosites, but very likely in terms of entire proteins. Besides
the possibility of variation across species, the phosphorylation
states of the sampled proteins might as well vary across the
entire human population. On the other hand, the phosphorylation state of each protein was assessed applying strict criteria,
and we expect our data to be representative for humans, at least
in quantitative terms.
As mentioned above, 59 sperm proteins were used for
evolutionary analyses. The remaining 40 (thereof 9 with
claried phosphorylation state) out of 99 sperm proteins were
not used for evolutionary analyses (Table 1; Supporting
Information Table 11): due to missing database entries of at
least one cDNA ortholog we excluded seven phosphoproteins
without Y phosphorylation (ACTBL2, PGC, PGP, PRKAR2A,
RUVBL1, TEKT4, TUBA3C) and two nonphosphorylated
sperm proteins (HPRT1, SEMG1). Four sperm proteins
including one proteasome subunit gave consistent signals in
anti-pSTY Western blots, but inconsistent signals in anti-pY
Western blots (ABHD10, ACTRT2, EEF1G, PSMB4).
Discrepancies between expected and actual MW and pI led
to exclusion of six more sperm proteins. A nal set of 21 other
sperm proteins was not further considered in evolutionary
analyses as their phosphorylation status could not be denitely
settled at the STY level. This was due to inconsistent signals in
anti-pSTY Western blots (10 proteins), conicts between our
own ndings and database entries (4 proteins), and multiple
detections from single spots (7 proteins) (Table 1; Supporting
Information Table 11). Due to the strict exclusion of
ambiguous data relating to dierences in phosphorylation
patterns between probands and insecure interpretations of
phosphorylation signals, false positives should not have
compromised evolutionary analyses.

On the basis of cDNAs representing a constant sample of six

mammalian species, test statistic rejected site-specic positive
selection for the vast majority of proteins showing phosphorylation in human probands. In detail, LRT (M8A/M8; 1% level
of signicance) supported site-specic positive selection only
for a single sperm phosphoprotein without Y phosphorylation
(CAPZB) and for none of the sperm phosphoproteins with Y
phosphorylation (Table 2). Consequently, -square test
rejected dierential prevalence of site-specic positive selection
in both samples (Yates P > 0.05; Table 2; Supporting
Information Table 3).
Table 2. Incidence of Site-Specic Positive Selection in
Sperm Phosphoproteins with and without Y
Phosphorylation in Human Probandsa

condition 1/condition 2
number of genes with
signicant support for
site-specic positive
selection/without such
support

sperm
phosphoproteins
with Y
phosphorylation

sperm
phosphoproteins
without Y
phosphorylation

Yates
P, square
test

0/18

1/32

>0.05

P, probability of error. Support and nonsupport for site-specic

positive selection refers to a likelihood ratio test comparing the t of
two versions of CODEML model M8 at the 1% level of signicance.
See Supporting Information Table 3 for gene-specic data.
a

Analyses of sequence evolution across sites conrmed similar

levels of evolutionary conservation in both protein samples, no
matter if the human ortholog was a sperm phosphoprotein with
or without Y phosphorylation. Although the employed
parameters tended to be increased in sperm phosphoproteins
with Y phosphorylation as compared to their counterparts
without Y phosphorylation, median dN/dS values (M8A) were
low in both groups (median dN/dS = 0.097 and 0.036,
respectively; Figure 2A; Supporting Information Table 3).
Moreover, highly overlapping 95% condence intervals
indicated similar levels of dN/dS in both groups (Figure 2A).
Consequently, MannWhitney U test did not support
dierential levels of dN/dS in sperm phosphoproteins with
and without Y phosphorylation (P > 0.05; Table 3). The
pattern was reproduced at the amino acid level where we
observed higher, but not signicantly increased, amino acid
distances in sperm phosphoproteins with Y phosphorylation
(median = 0.080) relative to sperm phosphoproteins without Y
phosphorylation (median = 0.030, P > 0.05, MannWhitney U
test, Table 3, Figure 2A).
Prevalent evolutionary conservation of sperm phosphoproteins with and without Y phosphorylation in human probands
was reproduced with respect to site-specic levels of purifying
selection. Thus, fractions of proteins with values less than 1
were persistently high in both subgroups, sperm phosphoproteins with Y phosphorylation (Y < 1 = 90.9%) and sperm
phosphoproteins without Y phosphorylation (S < 1 = 69.6%;
T < 1 = 92.3%; Table 4; Supporting Information Table 5). In
case of Y (P < 0.05) and T values (P = 0.001), support from
Z-test for a deviation from the null expectation of 50% was even
signicant. Despite overall prevailing purifying selection at pS
sites, small sample size led to only tentative, but not signicant,
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phosphosites in sperm phosphoproteins, irrespective of their

phosphorylation status at Y residues in humans.
Despite some minor dierences, human sperm phosphoproteins with and without Y phosphorylation showed similar
distributions across GO classes (Yates P > 0.05; -square test;
see also Figure 4 and Supporting Information Table 6).
Actually, the respective P value was close to 1 indicating nearly
identical distributions of both protein groups across GO classes.
This applies especially to nearly identical proportions of sperm
proteins playing a role in cytoskeleton, agella, and cell
movement (22.2% and 18.2%); energy production (22.2% and
18.2%); and metabolism (5.6% and 6.1%). Finally, interaction
levels were in the same range in both groups, as exhibited by
medians of 24 and 31 PPI partners in sperm phosphoproteins
with and without Y phosphorylation, respectively (P > 0.05,
MannWhitney U test, Table 3, Figure 2B, Supporting
Information Table 6). Thus, levels of cross-species conservation
and functional constraint of sperm phosphoproteins were
widely independent of the phosphorylation status at Y residues
in human probands.
Increased Levels of Evolutionary Conservation and
Functional Constraint in Phosphorylated Relative to
Nonphosphorylated Sperm Proteins

Using cDNA orthologs of human, Rhesus monkey, house

mouse, Norway rat, bovine, and pig, M8A estimates of dN/dS
were consistently smaller than 1 (Supporting Information
Tables 3 and 4). This illustrates the predominant role of
negative selection in our sample of mammalian sperm proteins,
regardless of whether they were or were not phosphorylated in
human probands. Irrespective of this general tendency for
evolutionary conservation in both samples, levels of dN/dS
estimates were signicantly increased in sperm proteins without
(median dN/dS = 0.156) relative to sperm proteins with
phosphorylation in human probands (median dN/dS = 0.052;
Figure 5A). Support from MannWhitney U test for
dierential levels of dN/dS even withstood correction for
multiple testing (P < 0.05; Table 5). This pattern was
reproduced on the basis of average amino acid distances
(JTT) generated by AAML. Thus, median amino acid distances
were signicantly higher (P < 0.01; MannWhitney U test) in
sperm proteins without phosphorylation (median = 0.113) than
in those with phosphorylation in humans (median = 0.041,
Figure 5A, Table 5, Supporting Information Tables 3 and 4).
This consistency of results demonstrates that dierential levels
of dN/dS reect diering nonsynonymous, but not synonymous, substitution rates in phosphorylated and nonphosphorylated sperm proteins. In line with this, LRT (M8A/M8,
1% level of signicance) supported the occurrence of positively
selected sites for 3 out of 8 nonphosphorylated sperm proteins
and for only 1 out of 51 phosphorylated sperm proteins
(Supporting Information Tables 3 and 4). Consequently, square test supported dierential incidence of site-specic
positive selection across both samples with high signicance
(Yates P < 0.01, -square test, Table 6).
We observed additional dierences between phosphorylated
and nonphosphorylated sperm proteins regarding their
distribution across GO classes (Supporting Information Tables
6 and 7). Although -square test rejected unequal distribution
of both samples across all distinguished GO classes (Yates P >
0.05), sperm proteins playing a role in transcription, protein
synthesis, transport, folding, and turnover (35.3% vs 12.5%);
energy production (19.6% vs 12.5%); or cytoskeleton, agella,

Figure 2. Sequence evolution (A) and protein interactions (B) of

sperm proteins that were classied as phosphoproteins with or without
Y phosphorylation. Protein-specic dN/dS values were inferred across
six mammalian orthologs applying model version M8A (CODEML).
Protein-specic amino acid (aa) distances refer to mean pairwise
distances among the same six mammalian orthologs, using the JTT
substitution matrix. The PSICQUIC metaserver was searched for nPPI
data. The graphic depicts medians (see numbers at the top of bars)
and 95% condence intervals of medians (whiskers) that were inferred
from 100 000 bootstrap replicates (see Table 3). Gene/protein-specic
values are reported in Supporting Information Tables 3 and 6. The
phosphorylation status of proteins refers to human spermatozoa.

Table 3. Sequence Evolution across Sites and Protein

Protein Interactions of Sperm Phosphoproteins with and
without Y Phosphorylation in Human Probandsa
median values
compared
dN/dS across
codon sites
(M8A)
amino acid
distances
(JTT)
nPPI

sperm
phosphoproteins with
Y phosphorylation

sperm phosphoproteins
without Y
phosphorylation

P (2sided)
MWU

0.097 (0.0430.147)

0.036 (0.0270.084)

>0.05

0.080 (0.0290.110)

0.030 (0.0210.052)

>0.05

24 (1043)

31 (855)

>0.05

dN/dS, ratio of nonsynonymous to synonymous substitution rates;

MWU, MannWhitney U test; nPPI, number of protein interaction
partners; P, probability of error. Values in parentheses refer to 95%
condence intervals of medians that were inferred from 100 000
bootstrap replicates, each. See Supporting Information Tables 3 and 6
for gene/protein-specic data.

support in case of S values (P > 0.05; Z-test; Table 4). The

detailed average evolutionary rates were 0.127 (pY) and 0.391
(Y) in sperm phosphoproteins with Y phosphorylation, and
0.285 (pS), 0.311 (S), 0.214 (pT), and 0.512 (T) in sperm
phosphoproteins without Y phosphorylation (Figure 3;
Supporting Information Table 5). We regard these results as
evidence for overall similar levels of purifying selection at
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Table 4. Evolutionary Rates at Predicted Phosphosites and Their Nonphosphorylated Counterparts and Ratios of Both Rates in
Sperm Phosphoproteins with and without Y Phosphorylation in Human Probandsa
sperm phosphoproteins
with Y phosph
without Y phosph
without Y phosph

amino acid site

pY
Y
pS
S
pT
T

av evolutionary rate
0.127
0.391
0.285
0.311
0.214
0.512

(0.0200.294)
(0.1890.625)
(0.1610.442)
(0.2130.418)
(0.0880.378)
(0.3340.726)

proteins with < 1/ > 1

fraction < 1 (%)

P (2-sided) Z-test

10/1

90.9

<0.05*

16/7

69.6

>0.05

24/2

92.3

0.001**

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a
, ratio of evolutionary rates at predicted phosphosites (pS, pT, pY) vs evolutionary rates at nonphosphorylated counterparts (S, T, Y); av, average;
P, probability of error; phosph, phosphorylation. Values in parentheses refer to 95% condence intervals of averages that were inferred from
100 000 bootstrap replicates, each. Asterisks (* and **) highlight signicance at the 5% and 1% level, respectively, after correction for multiple
testing. See Supporting Information Table 5 for protein-specic data.

Figure 3. Evolutionary rates at pY, pS, and pT residues and their

nonphosphorylated counterparts in sperm phosphoproteins with Y
phosphorylation (Y residues) and without Y phosphorylation (S and T
residues). The graphic shows average evolutionary rates (see numbers
at the top of bars) and 95% condence intervals (whiskers) that were
calculated from 100 000 bootstrap replicates (see Table 4). Proteinspecic rates were derived across six mammalian orthologs as average
numbers of substitutions per site per billion years. The phosphorylation status of proteins refers to human spermatozoa. Phosphosites
were predicted by NetPhos 2.0 program.60 For protein-specic data,
see Supporting Information Table 5.

Figure 4. GO classes of human sperm phosphoproteins with and

without phosphorylation at Y (absolute numbers and proportions).
(A) Sperm phosphoproteins with Y phosphorylation and (B) sperm
phosphoproteins without Y phosphorylation. Assignment to GO
classes according to published data (UniProt, original literature; see
Supporting Information Table 6).

and cell movement (19.6% vs 12.5%) occurred more frequently

in the phosphorylated relative to the nonphosphorylated
sample (Figure 6). In contrast, proteins involved in cell
recognition were clearly overrepresented in the nonphosphorylated relative to the phosphorylated sample (37.5% vs 3.9%). It
is noteworthy that sperm proteins involved in signal transduction (LZTFL1, YWHAZ) were restricted only to the sample
of phosphorylated sperm proteins (3.9% vs 0%; Figure 6) and,
in particular, to the sampled sperm phosphoproteins without Y
phosphorylation (Figure 4).
Importantly, the sampled sperm proteins also diered with
respect to their role in human PPI networks. Thus, sperm
proteins that were phosphorylated in human probands have
signicantly more proteinprotein interaction partners (median nPPI = 29) than nonphosphorylated human sperm
proteins (median nPPI = 3; P < 0.05, Table 5, Figure 5B,
Supporting Information Tables 6 and 7). Additionally,

Spearmans rank provided highly signicant support for a

negative correlation between nPPI and dN/dS (M8A) across
sperm proteins that also withstood correction for multiple
testing (rS = 0.709, P < 0.001, Supporting Information Figure
4). Thus, generally more interaction partners in phosphorylated
as compared to nonphosphorylated sperm proteins may be
considered as a factor contributing to higher levels of functional
constraint and evolutionary conservation in the former relative
to the latter group. Eight proteasome subunits, all of them
showing phosphorylation in human probands, were particularly
conspicuous in this regard: with a median dN/dS value of 0.012
(M8A) and a median amino acid distance of 0.009 (JTT), they
were all highly conserved. Moreover, with a median of 79
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Table 6. Incidence of Site-Specic Positive Selection in

Sperm Proteins That Are Either Phosphorylated or
Nonphosphorylated in Human Probandsa
condition 1/
condition 2

phosphorylated
sperm proteins

nonphosphorylated
sperm proteins

Yates
P, -square
test

number of genes with

signicant support
for site-specic
positive selection/
without such
support

1/50

3/5

<0.01**

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a
P, probability of error. Support and nonsupport for site-specic
positive selection refers to a likelihood ratio test comparing the t of
two versions of CODEML model M8 at the 1% level of signicance.
Asterisks (**) highlight signicance at the 1% level after correction for
multiple testing. See Supporting Information Tables 3 and 4 for genespecic data.

Figure 5. Sequence evolution (A) and protein interactions (B) of

sperm proteins that were classied as being phosphorylated and
nonphosphorylated. Protein-specic dN/dS values were inferred
across six mammalian orthologs applying model version M8A
(CODEML). Protein-specic amino acid (aa) distances refer to
mean pairwise distances among the same six mammalian orthologs,
using the JTT substitution matrix. The PSICQUIC meta-server was
searched for nPPI data. The graphic depicts medians (see numbers at
the top of bars) and 95% condence intervals of medians (whiskers)
that were derived from 100 000 bootstrap replicates (see Table 5).
Gene/protein-specic values are reported in Supporting Information
Tables 3, 4, 6, and 7. Asterisks (* and **) indicate signicance at the
5% and 1% level after correction for multiple testing (MannWhitney
U test). The phosphorylation state of proteins refers to human
spermatozoa.

Table 5. Sequence Evolution across Sites and Protein

Protein Interactions of Sperm Proteins That Are Either
Phosphorylated or Nonphosphorylated in Human
Probandsa
median values
compared
dN/dS across codon
sites (M8A)
amino acid distances
(JTT)
nPPI

phosphorylated
sperm proteins
0.052 (0.034
0.088)
0.041 (0.027
0.071)
29 (1443)

nonphosphorylated
sperm proteins
0.156 (0.076
0.268)
0.113 (0.090
0.183)
3 (026)

P (2sided)
MWU
<0.05*

Figure 6. GO classes of human sperm proteins as distinguished by

overall phosphorylation status in human probands (absolute numbers
and proportions). (A) Phosphorylated sperm proteins: overrepresentation of proteins involved in cytoskeleton, agella, and cell
movement; energy production; transcription, protein synthesis,
transport, folding, and turnover. (B) Nonphosphorylated sperm
proteins: overrepresentation of proteins participating in cell
recognition. Assignment to GO classes according to published data
(UniProt, original literature; see Supporting Information Tables 6 and
7).

<0.01**
<0.05*

dN/dS, ratio of nonsynonymous to synonymous substitution rates;

MWU, MannWhitney U test; nPPI, number of protein interaction
partners; P, probability of error. Values in parentheses refer to 95%
condence intervals of medians that were inferred from 100 000
bootstrap replicates, each. Asterisks (* and **) highlight signicance
at the 5% and 1% level, respectively, following correction for multiple
testing. See Supporting Information Tables 3, 4, 6, and 7 for gene/
protein-specic data.

In summary, our data suggest that overall stronger functional

constraint, due to more interactants, accounts for higher levels
of cross-species conservation of phosphorylated as compared to
nonphosphorylated sperm proteins. By contrast, dierences in
the distribution across GO classes seem to contribute less to

protein interactants, all of them appeared to have a central

position in the sperm interactome (see Supporting Information
Tables 3 and 6).
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phosphorylation versus nonphosphorylation in bacteria and

eukaryotes.6,31
Cross-species conservation of phosphorylated sperm proteins
was also apparent at the level of predicted phosphosites:
whether taking sperm phosphoproteins with Y (Y < 1 =
90.9%) or without Y phosphorylation in humans (S < 1 =
69.6%; T < 1 = 92.3%; Table 4), fractions of proteins with
values <1 were consistently higher than genome-wide averages
inferred from a total of 4484 proteins across 44 mammalian
species (S < 1 = 68.1%; T < 1 = 63.6%; Y < 1 =
63.5%).33,64 Furthermore, mean evolutionary rates at phosphosites as derived from our data (pY, 0.127; pS, 0.285; pT, 0.214;
Figure 3) were decreased compared to genome-wide reference
values (pY, 1.03; pS, 0.83; pT, 0.87).33 Again, conclusions have
to be drawn with care considering variations in sample size and
tree depth between the present and the reference study.33
Nevertheless, both studies demonstrate that phosphosites
evolve under strong purifying selection. This agrees with
results originating from studies with methodically dierent
approaches that showed evolutionary conservation of predicted
and, especially, of functional phosphosites as well as of
phosphorylation mechanisms in both bacteria and eukaryotes6,2729,32,67 (see also ref 68). Moreover, prevalent
evolutionary conservation of phosphorylation is not in conict
with recurrent evidence questioning raised levels of purifying
selection at phosphosites.31,35 The seeming contradiction rather
reects that entire phosphoproteins are commonly more
conserved than single phosphosites.31 Reports on phases of
accelerated and adaptive evolution of phosphosites, phosphoproteins, and entire phosphoproteomes32,35,36,41 are not in
conict with a general tendency for conservation in the
evolution of phosphorylation either. The present study shows
that the sperm (phospho)proteome fully complies with this
universally valid pattern of prevailing evolutionary conservation.

the unequal evolutionary rates of sperm proteins with and

without phosphorylation in humans. Moreover, the degree of
purifying selection does not associate with phosphorylation of
human sperm proteins at Y residues. If at all, levels of
evolutionary conservation across mammals are only slightly, but
not signicantly, higher in sperm phosphoproteins without Y
phosphorylation than in sperm phosphoproteins with Y
phosphorylation in humans.

DISCUSSION

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Evolutionary Conservation of Mammalian Sperm Proteins

Is Associated with Overall, but not with Tyrosine
Phosphorylation in Humans

Forty-two out of 99 sperm proteins that we detected by mass

spectrometry were so far not classied as being expressed in
human sperm, testis or testes in the UniProt database (state:
rst July 2013), nor were they included in the study of
Mart nez-Heredia et al.66 (Table 1). Hence, the present study
contributes to the growing knowledge on the composition of
the human sperm proteome.10,22 In addition, our data show
that overall phosphorylation, but not Y phosphorylation, aects
evolutionary rates of sperm proteins. Indeed, we found similar
levels of evolutionary conservation in sperm proteins, no matter
if they were classied as phosphoproteins with or without Y
phosphorylation according to evidence from human probands
(Figures 2A and 3, Tables 25; for protein classication, see
Materials and Methods section). The consistencies between
both groups of sperm proteins probably result from similar
levels of functional constraint as shown by similar distributions
across GO classes and comparable ranges in numbers of protein
interaction partners (Figure 2B and 4; Table 3). Thus, the
present data reject a special relevance of pY versus pST sites for
sperm functioning, although pY and pST sites generally dier in
respect to incidence, mode of phosphorylation, and binding
energies.7,26
Moreover, our data clearly answer the question of interrelation between overall phosphorylation (at S, T, and Y
residues) of human sperm proteins and rates of sequence
evolution. Using cDNAs and amino acid sequences of six
mammalian species, we were able to reproduce an increased
evolutionary conservation of phosphorylated as compared to
nonphosphorylated sperm proteins in human spermatozoa
(Figure 5A, Tables 5 and 6). Considering support from neural
network predictions for conserved phosphorylation states
across the sampled species (see Results section), sperm
phosphoproteins generally seem to evolve with lowered rates
in 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 from
analyses of 14 963 one-to-one orthologs in the mouse-rat
comparison (=0.128).48 This suggests that nonphosphorylation
of sperm proteins associates with a slight acceleration of
sequence evolution, whereas phosphorylation is linked to above
average conservation. However, this conclusion has to be taken
with caution, as dierent species samples and tree depths
hamper comparisons between the two studies. Anyhow, sperm
proteins showing phosphorylation in humans were more
conserved than their nonphosphorylated counterparts. Thus,
the evolution of mammalian sperm proteins is in compliance
with a general tendency for increased conservation of

Dierential Functional Constraint between Sperm Proteins

That Are Phosphorylated and Sperm Proteins That Are Not
Phosphorylated in Humans

Lower evolutionary conservation of sperm proteins detected as

nonphosphorylated in human probands is probably a
consequence of a high proportion of cell recognition proteins
(Figure 6) that are frequently located in the sperm membrane
and acrosomal matrix47,69 and are commonly known for rapid
evolution.44,47,7073 In contrast, increased evolutionary conservation of sperm proteins showing phosphorylation in human
probands appears to reect their stronger involvement in highly
essential cellular processes such as transcription, protein
synthesis, transport, folding, and turnover as well as in
cytoskeleton, agella, and cell movement (Figure 6).74,75
Such increased relevance is further emphasized in this study
by the above-mentioned signal transduction proteins and their
strict connement to the group of phosphorylated sperm
proteins (Figures 4 and 6). Although dierential frequencies
across GO classes might be part of the explanation, the position
within the sperm interactome seems to be the deeper reason
behind dierential levels of cross-species conservation of
phosphorylated relative to nonphosphorylated sperm proteins.
Indeed, Spearmans rank correlation between numbers of
interaction partners and dN/dS values (M8A) indicates a
strong negative relationship between these two variables in our
sperm protein sample (Supporting Information Figure 4).
Considering this fact, signicantly higher numbers of
interactants could explain stronger conservation of sperm
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teins might be prime candidates for its diagnosis and treatment.

In turn, nonphosphorylated sperm proteins are promising
targets for contraceptive vaccines for men and could be
considered as biomarkers for dierent grades of fertility in
animal husbandry: due to fewer interaction partners and a
tendency toward higher reproduction specicity, their impairment or blocking should have less serious adverse eects.

proteins with than of those without phosphorylation in human

probands (Figure 5B, Table 5). As more PPI partners are
associated with higher centrality in PPI networks and higher
protein essentiality,49,5153 stricter conservation of phosphorylated sperm proteins presumably results from their utmost
importance for sperm functioning and successful fertilization.
Similar ndings from analyses of yeast (Saccharomyces
cerevisiae) proteins show that more PPI partners and higher
essentiality in phosphorylated relative to nonphosphorylated
proteins reect a general pattern in eukaryotes.76 Most likely,
the need for compensatory exchanges in additional interaction
partners imposes higher levels of functional constraint and
evolutionary conservation on phosphorylated versus nonphosphorylated sperm proteins.77 Yet another factor increasing
evolutionary conservation of phosphorylated sperm proteins
might be the higher number of interacting domains, each being
under functional constraint, in proteins with more interaction
partners.50 It must be further considered that kinasesubstrate
recognition is mediated not only by phosphosites and kinase
docking motifs, but also by adaptor and scaold proteins.26
This again imposes increased levels of functional constraint on
phosphoproteins. In the present study, the determined
proteasome subunits, all of which are phosphorylated, may be
the best example for such a pattern: proteasomes accomplish
highly essential decomposition of misfolded, damaged, or
unnecessary proteins and are involved in capacitation, acrosome
reaction, and fertilization in various metazoan species.78
Increased numbers of protein interaction partners and high
levels of evolutionary conservation of the sampled proteasome
subunits (Supporting Information Tables 3 and 6) demonstrate
how the quantity of interactants might inuence protein
evolution. Hence, following duplication and functional
diversication under relaxed functional constraint proteasome
subunits obviously evolved under increased levels of functional
constraint.7981 Thus, as exemplarily stated for proteasome
subunits, it seems that strong conservation of phosphorylated
sperm proteins is a consequence of manifold PPI partners and
high centrality in intracellular PPI networks.49,50 On the
contrary, less evolutionary conservation of nonphosphorylated
sperm proteins reects a general tendency for proteins at the
network periphery to be frequently surface proteins coevolving
with their extracellular interaction partners.53,71

ASSOCIATED CONTENT

* Supporting Information
S

Additional tables and gures. This material is available free of

charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*E-mail: herlyn@uni-mainz.de. Phone: +49 (0) 6131 3923179.

Fax: +49 (0) 6131 3923799.
Author Contributions

The manuscript was written through contributions of all

authors. All authors approved the nal version of the
manuscript.
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
The authors are grateful to anonymous reviewers whose
comments and suggestions on earlier drafts greatly helped to
improve the manuscript. The study was supported by grants
from the German Research Council (DFG, HE 3487/2-1) and
Johannes Gutenberg-University Mainz to H.H. (Stufe 1).

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CONCLUSIONS
On the basis of experimentally obtained phosphorylation data
of human sperm proteins we show that levels of evolutionary
conservation of mammalian sperm phosphoproteins are
unaected by Y phosphorylation. Conversely, phosphorylation
at S, T, and Y residues and hence overall phosphorylation in
humans associates with increased cross-species conservation
across sperm proteins and at predicted phosphosites. Thus,
sperm phosphoproteins seem to be more representative of the
predominant pattern in the evolution of male reproductive
proteins, i.e., purifying selection,48,82 whereas sequence
evolution of nonphosphorylated sperm proteins is more
representative of the patterns usually associated with
fertilization proteins, i.e., accelerated and adaptive evolution.4447,72,83 Furthermore, our data imply that more PPI
partners might be an important factor contributing to higher
cross-species conservation of phosphorylated sperm proteins
and predicted phosphosites. As phosphorylated sperm proteins
have already been shown to play a role in reduced male fertility
(see, e.g., ref 22), evolutionarily conserved sperm phosphopro5380

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