J O U RN A L OF P ROTE O M IC S 7 9 ( 2 01 3 ) 1 1 4 –12 2
Available online at www.sciencedirect.com
www.elsevier.com/locate/jprot
In-depth proteomic analysis of the human sperm reveals
complex protein compositions
Gaigai Wang1 , Yueshuai Guo1 , Tao Zhou1 , Xiaodan Shi1 , Jun Yu, Ye Yang, Yibo Wu,
Jing Wang, Mingxi Liu, Xin Chen, Wenjiao Tu, Yan Zeng, Min Jiang, Suying Li, Pan Zhang,
Quan Zhou, Bo Zheng, Chunmei Yu, Zuomin Zhou, Xuejiang Guo⁎, Jiahao Sha⁎
State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing 210029, China
AR TIC LE I N FO
ABS TR ACT
Article history:
The male gamete (sperm) can fertilize an egg, and pass the male genetic information to the
Received 2 October 2012
offspring. It has long been thought that sperm had a simple protein composition. Efforts
Accepted 7 December 2012
have been made to identify the sperm proteome in different species, and only about 1000
Available online 23 December 2012
proteins were reported. However, with advanced mass spectrometry and an optimized
proteomics platform, we successfully identified 4675 human sperm proteins, of which
Keywords:
227 were testis-specific. This large number of identified proteins indicates the complex
Sperm
composition and function of human sperm. Comparison with the sperm transcriptome
Proteome
reveals little overlap, which shows the importance of future studies of sperm at the protein
Mass spectrometry
level. Interestingly, many signaling pathways, such as the IL-6, insulin and TGF-beta
Signaling pathway
receptor signaling pathways, were found to be overrepresented. In addition, we found that
Drug
500 proteins were annotated as targets of known drugs. Three of four drugs studied were
Human
found to affect sperm movement. This in-depth human sperm proteome will be a rich
resource for further studies of sperm function, and will provide candidate targets for the
development of male contraceptive drugs.
Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1.
Introduction
Approximately one in six couples experience difficulty in
conceiving a child. Male infertility accounts for about half
the cases in which assisted reproductive techniques are
recommended [1]. Over 85% of infertile male can actually
produce sperm [2]; however, for some reason, those sperm are
often unable to fertilize an egg.
As the only cell performing its function outside the male
human body, sperm is a highly specialized cell with distinct
morphological and compositional differences compared with
other somatic and germ cells [3]. It was long believed that the
function of sperm was only to deliver the paternal genomes to
the egg. However, recent studies have shown that sperm can
deliver a complex set of RNAs to the egg [4]. In addition, the
entire cell, including the midpiece and tail, enters the egg in
most species [5]. Current studies have suggested that sperm
defects can disrupt embryo development, even if the genome
carried by the cells is perfectly normal [6]. Thus, characterization of the protein composition of sperm can help better
understand sperm function.
The recently proposed Chromosome-Centric Human Proteome Project (C-HPP) aims to define the full set of proteins
encoded in each chromosome. The initial goal of the C-HPP is
to identify at least one representative protein encoded by each
of the approximately 20,300 human genes [7]. The genes and
⁎ Corresponding authors. Tel.: +86 25 86862038; fax: +86 25 86862908.
E-mail addresses: [email protected] (X. Guo), [email protected] (J. Sha).
1
These authors contributed equally to the work.
1874-3919/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jprot.2012.12.008
J O U RN A L OF P ROT EO M IC S 7 9 ( 2 01 3 ) 1 1 4 –1 22
proteins had tissue-dependent expression. According to previous analyses, the human testis, the male gonad producing
sperm, contains the largest number of tissue-specific genes
across the 31 human tissues [8]. Thus, in order to characterize
all the proteins, proteins in the testis and/or testicular cells
should be carefully studied. The in-depth proteomic analysis
of human sperm will produce data important for C-HPP.
To date, some efforts have been made to identify the
human sperm proteome, and in fact, a list of 1056 proteins has
been reported [9]. Additionally, Johnston et al. [10] claimed
identification of 1760 proteins in human sperm, but this
protein list is not available. Efforts to identify the sperm
proteome in other species, including drosophila and mammals, such as rat and mouse [11,12], have also been made, and
the numbers of proteins identified were all around or below
one thousand. In the present study, using the advanced mass
spectrometry and an optimized proteomics platform, we
successfully identified 4675 unique proteins from human
sperm, which showed the complex composition of human
sperm.
115
Mobile phase A = 95:5 H2O:ACN, 5 mM ammonium formate
buffer pH= 2.7, mobile phase B = mobile phase A + 800 mM
ammonium formate, pH= 2.7. The gradient used was 0–56% B
for 20 min, 56% to 100% B for 1 min, 100% B for 5 min, 100% to
0% B for 1 min, and 0% B for 20 min before the next run. In each
series of experiments, 100 μl fractions were collected every
2 min, and 20 fractions were obtained. The experiments were
repeated for 3 times.
2.3.
Mass spectrometric analysis and database search
This human study was ratified by the Ethics Committee of
Nanjing Medical University, and was in accordance with
National and International guidelines. Before initiating the
study, consent was obtained from all participants. The sperm
subjected to proteomics analysis were from 32 healthy male
volunteers with a mean age of 30 ± 4 years old (mean ± standard
deviation). These men had proven fertility and normal semen
quality, as assessed by World Health Organization criteria
(1999).
The semen samples were obtained by masturbation after at
least 3 days of abstinence. The samples were ejaculated into
sterile containers and allowed to liquefy for at least 30 min
before being processed by centrifugation in a 60% Percoll
gradient (GE Healthcare, Waukesha, WI, USA) to remove
seminal plasma, immature germ cells, and non-sperm cells
(mainly epithelial cells), as described by Loredana-Gandini et al.
[13,14]. The purified sperms were then washed in PBS three
times before subsequent proteomics analysis. For purity
evaluation, the sperm were resuspended in PBS and stained
with Hoechst H33342 (Sigma, St Louis, MO) for 30 s; 1000 cells
were counted by light microscopy.
For capillary reverse-phase liquid chromatography (LC) and
mass spectrometric analysis, each fraction was directly loaded
onto a μ-precolumn™ cartridge (0.3× 5 mm, 5 μm, 100 Å;
Dionex) at a flow rate of 20 μl/min. The trap column effluent
was then transferred to a reverse-phase microcapillary column
(0.075 × 150 mm, Acclaim® PepMap100 C18 column, 3 μm,
100 Å; Dionex). The reverse-phase separation of peptides was
performed using the following buffers: 2% ACN, 0.5% acetic acid
(buffer A) and 80% ACN, 0.5% acetic acid (buffer B); a 122 or
82-min ACN gradient (4% to 7% buffer B for 3 min, 7% to 33%
buffer B for 102 min or 62 min, 33% to 50% buffer B for 10 min,
50% to 100% buffer B for 3 min, 100% buffer B for 3 min, 100% to
4% buffer B for 1 min) was used. Peptide analysis was performed
using a LTQ Orbitrap Velos (ThermoFisher Scientific, San Jose,
CA) coupled directly to an LC column. An MS survey scan was
obtained for the m/z range 350–1800, and MS/MS spectra were
acquired from the survey scan for the 20 most intense ions (as
determined by Xcalibur mass spectrometer software in real
time). Dynamic mass exclusion windows of 60 s were used, and
siloxane (m/z 445.120025) was used as an internal standard.
RAW files for LC–MS/MS identification were processed by
MaxQuant (v1.2.2.5), and identified with the Andromeda
search engine according to standard workflow [15]. The peak
lists were searched against the UniProtKB human proteome
sequence database (2012/04/18), which contains 86,770 entries. Carbamidomethylation of cysteine (+57 Da) was set as a
fixed modification, and oxidized methionine (+ 16 Da) was set
as a variable modification. The initial mass tolerances for
protein identification on MS and MS/MS peaks were 20 ppm
and 0.5 Da, respectively. Two missed cleavages were permitted,
and full cleavage by trypsin was used. The false discovery rate
(FDR) of the identified peptides and proteins was estimated by
searching against the database with the reversed amino acid
sequence [15]. Only peptides that were a minimum of six amino
acids in length and had a FDR of 1% were considered for
identification.
2.2.
2.4.
2.
Materials and methods
2.1.
Sperm collection
Sample preparation for mass spectrometry
Human sperm were dissolved in 7 M urea, 2 M thiourea, 65 mM
DTT, and 1% (v/v) protease inhibitor cocktail, and the extracted
proteins from different men were mixed for subsequent
proteomic studies. Proteins of 240 μg were reduced, alkylated
and sequentially digested with modified trypsin (sequencing
grade, Promega, Madison, WI). These in-solution digests
were loaded onto a strong-cation exchange column (1 mm
ID× 10 cm, packed with Poros 10S, Dionex, Sunnyvale, CA) for
fractionation. A linear salt gradient ammonium formate in 5%
acetonitrile (ACN) was applied at a flow-rate of 50 μl/min.
Human sperm proteome annotation
For bioinformatics analysis, the international protein index
(IPI) accession number was converted to an Entrez Gene ID or
Ensembl Gene ID. All Ensembl Gene IDs were loaded onto the
Database for Annotation, Visualization and Integrated Discovery (DAVID) [16] to identify the enriched biological themes,
including Gene Ontology. A FDR of less than 0.05 was
considered statistically enriched. The Entrez Gene IDs were
loaded onto a Web-based Gene Set Analysis Toolkit (http://
bioinfo.vanderbilt.edu/webgestalt/) [17] to identify the
hyper-represented WikiPathways; a FDR of less than 0.05
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J O U RN A L OF P ROTE O M IC S 7 9 ( 2 01 3 ) 1 1 4 –12 2
was considered hyper-represented. Human cilia proteins
were from the Ciliaproteome Database V3 (http://www.
ciliaproteome.org/) [18]. The testis-specific genes used were
from those assembled by Dezső et al. based on mRNA
expression across 31 human tissues [8]. Drosophila othologs of
human genes were batch downloaded from Ensembl 65 using
BioMart (http://www.ensembl.org/biomart/martview/).
Known drug targets in the identified human sperm proteome was annotated using the DrugBank database (http://www.
drugbank.ca) [19], which is a database that contains more than
1500 non-redundant proteins or drug target sequences. In
addition, DrugBank is a richly annotated resource that combines detailed drug data with comprehensive drug target and
drug action information.
2.5.
Indirect immunofluorescence
Sperm were washed three times by centrifugation for 5 min
at 300 ×g, resuspended in PBS, and air-dried onto polyLysine-coated coverslips. The sperm cells were fixed in 4%
formaldehyde/phosphate buffered saline (PBS) for 30 min,
washed three times with PBS for 5 min, and then blocked
with horse serum (Beijing ZhongShan Biotechnology Co.,
Beijing, China) for 2 h at room temperature. Following incubation with primary antibodies overnight at 4 °C, the cells were
incubated with secondary antibody labeled with fluorescein
isothiocyanate (FITC; Beijing ZhongShan Biotechnology Co.)
at a dilution of 1:200 for 1 h at room temperature. Negative
controls were incubated without the primary antibodies, but
otherwise the same. The primary antibodies used are provided
in Supplementary Data 1.
2.6.
Evaluation of the effects of drugs on human sperm
motility in vitro
The drugs studied were disulfiram (Antabuse) (Selleck Chemicals
LLC, Houston, TX), propofol (Sigma), leflunomide (Selleck
Chemicals LLC) and sorafenib (Nexavar) (Selleck Chemicals
LLC). Sperm were incubated in a capacitating medium with
10 mg/ml BSA [20], with or without drugs at gradient concentrations. During the experiments, the drugs to be tested with
sperm were dissolved in distilled water or dimethylsulphoxide
(DMSO) (Sigma). The concentration of DMSO in the incubation
media never exceeded 1% (v/v), a condition that does not affect
sperm capacitation or acrosome reaction [21]. After incubation
for 0, 15, 30, 60 or 120 min at 37 °C in a 5% CO2 incubator, the
sperm motility was detected using Computer-Aided Sperm
Analysis (IVOS, Hamilton Thorne Biosciences, Beverly, MA).
3.
Results
3.1.
Identification of total proteins expressed in human sperm
We used a 60% percoll gradient to purify sperm from human
semen, as this gradient has been verified to be able to remove
seminal plasma, non-sperm cells and even immature germ cells
[13]. We counted 1000 purified sperm with nuclear staining, and
none were contaminated with non-sperm or immature germ
cells (see Supplementary Fig. 1). From the purified sperm, we
successfully identified 30,903 unique peptides, that corresponded
to 4675 unique proteins, using an advanced LTQ Orbitrap Velos
mass spectrometer. Since such a large amount of data can be
subject to high false positives, we controlled the FDR to 1% at
both the peptide and protein levels using a reversed sequence
database by MaxQuant [15]. Of the 4675 proteins, 4401 proteins
(94%) were identified in two independent experiments (Fig. 1A).
Their identification information is shown in Supplementary Data
2 and 3, and the detailed single peptide-based identification data
including annotated mass spectra are presented in Supplementary Data 4 and 5.
The human sperm proteome has been studied using
different approaches, including two-dimensional gel electrophoresis (2D-PAGE) separation [3] and liquid chromatography
separation [9]. Additionally, the human sperm nucleus has been
purified and profiled [22]. Comparison with these published
proteomes showed that 93% (206/221) of the proteins identified
by two independent studies were successfully identified in our
proteome (Fig. 1B), and 3777 proteins were newly identified in
our proteome. For example, we identified 21 different phosphodiesterases in human sperm, which have been refractory to
previous studies. Whereas, in all three published studies, only
Baker et al. [9] identified one phosphodiesterase. Phospholipase
C, zeta 1 (PLCZ1), phosphodiesterase 1A (PDE1A) and phospholipase C, delta 4 (PLCD4), which are well-studied phosphodiesterase that are important for sperm function [23–25], were only
identified in our proteome.
The Ciliary proteome database annotates 2688 human cilia
proteins, of which more than half (1510 proteins) were
identified in human sperm (Fig. 1C; Supplementary Data 2).
Such a large number of cilia proteins in sperm suggest that the
human sperm tail may have a similar mechanism to the cilia
in somatic cells. Thus, it may be possible to use the knowledge
of cilia structure and function to study the sperm tail.
3.2.
Immunofluorescence studies
To verify the identified human sperm proteome, 29 proteins
with commercial antibodies were randomly selected for
immunofluorescence studies. The results showed diverse
localizations in sperm (Fig. 2). Our immunolocalization
revealed proteins from different parts of sperm, including the
acrosome, equatorial region, post-equatorial region, neck,
midpiece, principal piece, and end piece, which confirmed the
validity of our proteome identification, and also showed that
our approach can indeed perform in-depth profiling of the
entire sperm proteome. In the human sperm proteome, there
are proteins identified only by one unique peptide, which is of
relatively lower confidence. Ras homolog family member A
(RHOA), inner membrane protein, mitochondrial (IMMT), and
histone deacetylase 1 (HDAC1) are such identified proteins.
Our immunofluorescence studies confirmed their expression
in human sperm, and also showed that our proteome was
reliable.
3.3.
Over-represented pathways in the human sperm
proteome
Assigning proteins to the WikiPathways revealed a considerable
number of overrepresented pathways, including those involved
J O U RN A L OF P ROT EO M IC S 7 9 ( 2 01 3 ) 1 1 4 –1 22
117
Fig. 1 – Comparison with the published proteomes or transcriptome. (A). Overlap of proteins between three replicates. (B). The
previously published sperm proteome, as determined by 2D-PAGE and LC–MS/MS, and sperm nucleus proteome were
compared with our proteome. The proteins were converted to Ensembl Genes, and the overlap among the protein lists is
shown in the Venn diagram. The indicated numbers are the numbers of Ensembl Genes. (C). The overlap between our sperm
proteome with the cilia proteins from the Ciliaproteome Database V3 (http://www.ciliaproteome.org/). (D). The overlap of genes
between our sperm proteome and the published sperm transcriptome [1].
in energy metabolism, signal transduction, cytoskeleton, and so
on (Supplementary Data 6).
the existence of so many different signaling pathways in
sperm.
3.3.1.
3.3.3.
Energy metabolism
After maturation in the epididymis, sperm acquire motility
which consumes a lot of energy. The proteins in many different
energy metabolism pathways, including glycolysis and gluconeogenesis, tricarboxylic acid cycle (TCA) cycle (Supplementary
Fig. 2A), oxidative phosphorylation and electron transport chain,
and glycogen metabolism pathways, were found to be overrepresented. High coverage of these pathways was achieved. For
example, 27 of the total 32 proteins in TCA cycle were identified
in the human sperm proteome (Supplementary Fig. 2A; Supplementary Data 6).
3.3.2.
Signal transduction
Calcium is an important messenger for signal transduction
during fertilization, and its influx can be induced by progesterone [26]. We observed over-representation of a pathway of
calcium regulation in the cardiac cell. During capacitation,
MAPK was activated. Suppression of MAPK inhibits sperm
capacitation, making them refractory to the progesteroneactivated acrosome reaction [27]. In the human sperm
proteome, several MAPKs were identified, including MAPK14,
MAPK1, MAPK3, MAPK9 and MAPK13. In addition, the MAPK
signaling pathway was found to be overrepresented (Supplementary Fig. 2B). The proteins identified in this pathway will
help elucidate the signal cascade of MAPK that is regulated
during capacitation.
We found that many other signaling pathways were overrepresented in sperm as well, such as the TNF-alpha/NF-κB,
EGFR1, insulin, TGF-beta, IL-6, IL-2, and IL-5 signaling pathways (Supplementary Data 6). It was interesting to discover
Cytoskeleton
The pathway of regulation of actin cytoskeleton was also found
to be over-represented in sperm (Supplementary Fig. 2C).
Remodeling of the actin-cytoskeleton occurs during sperm
capacitation and acrosome reaction [28]. Our previous studies
have shown that proteins in the pathway of regulation of actin
cytoskeleton, such as RhoA and RhoGDI, play important
functions in sperm capacitation [20].
3.4.
Testis-specific genes
Based on the mRNA expression results from 31 human tissues
[8], 227 proteins corresponding to 223 Entrez genes were found to
be testis-specific (Supplementary Data 2), and were significantly
enriched in sperm proteome compared with genome-wide
distribution (p value =2.2E−16, fold enrichment=2.1 by Fisher's
exact test). Gene ontology analysis annotated only 78 genes with
biological process terms, of which 41 (53%) were annotated to
sexual reproduction (Supplementary Data 7). The remaining
147 testis-specific genes (65%) had no functional annotation
(Supplementary Data 7). Therefore, it is clear that the function of
these testis-specific proteins is largely unknown, although they
are supposedly important for fertility in men and thus deserve
further investigation.
3.5.
Comparison with human sperm mRNA
Complex mRNA profiles have been identified in human sperm
[1]. The overlap between sperm mRNAs and proteins is not
well known. With our in-depth human sperm proteome, we
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J O U RN A L OF P ROTE O M IC S 7 9 ( 2 01 3 ) 1 1 4 –12 2
compared the sperm proteome profile and sperm RNA profile.
Of the 1915 genes with RNA detected in human sperm, only
553 (29%) have corresponding identified proteins (Fig. 1D). The
low percentage of identified proteins in human sperm indicates that the RNA in sperm may not only be residual RNA
for the translated sperm proteins, but may also function in
sperm capacitation or fertilization. Recently, scientists found
translation by mitochondrial ribosome during sperm capacitation [29], and after entering the oocyte, the RNA may be
translated in the zygote.
3.6.
Comparison with drosophila sperm proteome
Dorus et al. characterized drosophila sperm proteome, and
identified 381 proteins [30]. Recently, further proteomic
analysis of drosophila sperm identified 766 additional sperm
proteins, thereby expanding the sperm proteome to 1108
proteins [31]. Because analysis of spermatogenesis and the
involvement of paternal products during and after fertilization in drosophila is directly relevant to analogous processes
in mammals. With the available in-depth human sperm
proteome, it is possible to perform comparative proteomic
analyses of sperm between human and drosophila. This will
allow the establishment of parallel genetic models for the study
of spermatogenesis and sperm function, and will allow the
identification of sperm factors responsible for male infertility in
humans.
Of the 744 genes encoding drosophila sperm proteome
with orthologs in human, 441 (59%) have orthologs in our
human sperm proteome (see Supplementary Data 8), and
these genes can be studied in advance to better understand
human fertility. For example, oxen (FBgn0011227) was found
to be involved in spermatid development in drosophila [32]. It
will be interesting to study the function of its ortholog
UQCR10 in human spermatogenesis.
3.7.
Drug target annotation
DrugBank has been widely used to facilitate in silico drug
target discovery, drug design, drug docking or screening, drug
metabolism prediction and drug interaction prediction. We
Fig. 2 – Immunofluorenscence studies of the identified
human sperm proteins.The proteins from our human sperm
proteome with commercial antibodies were randomly
selected for immunofluorenscence studies. The nucleus was
stained with Hoechst (blue). Differential localization (green)
of the proteins in the sperm was observed (green).
(A) acrosome: GNA11; (B) equatorial region: DDX4;
(C) equatorial region and neck: alpha/beta SNAP;
(D) post-equatorial region: SUGT1; (E–O) neck: α Tubulin,
RBBP7, DCTN1, Flotillin 2, HSPA5, KIF3A, M6PRBP1, MAP2K1,
VAPA, Calnexin and PRPS2; (P–V) midpiece: ACY1, IMMT,
UCHL1, PRPF19, SOD2, YKT6 and VDAC1; (W–X) principal
piece: beta actin, ENO2; (Y) neck and principal piece: HDAC1;
(Z) neck, midpiece and principal piece: ENO1; (AA) end piece:
RBBP4; and (AB–AC) whole spermatozoon: β Tubulin and
RhoA. The negative control is shown in (AD). The fluorescent
images were overlaid on the DIC images.
J O U RN A L OF P ROT EO M IC S 7 9 ( 2 01 3 ) 1 1 4 –1 22
119
motility after adding drug to the sperm cell culture. Disulfiram
(DrugBank: DB00822) and propofol (DrugBank: DB00818) inhibited
sperm motility (Fig. 4A–B), but leflunomide (DrugBank: DB01097)
had no effect on sperm motility (Fig. 4C). Sperm motility was
generally unaffected with sorafenib (DrugBank: DB00398), except
after a 60 min incubation at a 100 μM concentration (Fig. 4D).
4.
Fig. 3 – Subcellular distribution of sperm proteins targeted by
known drugs in DrugBank. Proteins targeted by drugs
according to the DrugBank database were analyzed, and the
subcellular distribution of sperm proteins was annotated
using gene ontology.
annotated the human sperm proteome using DrugBank data
[19]. We found that 500 human sperm proteins could be
targeted by known drugs (Supplementary Data 9). Of these
proteins, 150 proteins were mitochondrial proteins according
to gene ontology annotation, which is significantly enriched
(Fig. 3; Supplementary Data 9 and 10). 162 proteins were
annotated as cilia proteins according to Ciliary proteome
database (Supplementary Data 9). The mitochondrial sheath
provides energy for sperm motility, and the sperm tail is
similar to the cilia. Inhibition of these proteins is expected to
suppress sperm motility and can thus be future methods for
contraception.
3.8.
Sperm motility assessment after drug treatment
Four drugs with sperm protein targets annotated by DrugBank
were used for functional assessment. Because cilia proteins may
play important roles in sperm motility, we evaluated the sperm
Discussion
It has been estimated that sperm contains only 400 to 1300+
proteins [33], and until now, the published sperm proteome
contained the number of proteins within this estimated range
[9,11,12,30,31]. Thus, sperm was assumed to be relatively “simple”
in protein composition [9]. However, using advanced mass
spectrometry and a proteomics platform, we identified 4675
proteins from human sperm, which is about 4-fold greater than
the previously estimated number. The large number of identified
sperm proteins demonstrates the unexpected complexity of the
human sperm protein composition.
In the human sperm proteome, we identified 223 testisspecific genes, which comprise 46% of the total 484 reported
testis-specific genes by Dezső et al. [8]. Sperm has been known to
use specific proteins to regulate its function. For example,
although glycolysis is highly conserved, this central metabolic
pathway is modified in germ cells using specifically-expressed
enzymes, such as testis-specific glyceraldehyde-3-phosphate
dehydrogenase (GAPDHS), lactate dehydrogenase C (LDHC) and
phosphoglycerate kinase type 2 (PGK2) [34]. We successfully
identified unique peptides of three germ cell-specific enzymes,
which are different from the somatic counterparts of the
enzymes, lactate glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), lactate dehydrogenase (LDH) and phosphoglycerate
kinase type 1 (PGK1). With the identification of 233 testis-specific
genes with unique peptides, this will help further our understanding of sperm function, and also contribute to the initial goal
of C-HPP, which is to characterize at least one representative
protein encoded by each of the approximately 20,300 human
genes [7].
Fig. 4 – The effect of drugs on sperm movement. Purified human sperm were incubated under capacitating conditions for 0, 15,
30, 60 or 120 min, and motility was measured in the presence of disulfirum (A), propofol (B), leflunomide (C), or sorafenib (D).
The standard deviation is shown as bars. Statistical differences by Student's t-test compared with control are annotated as “*”
for p < 0.05 or “**” for p < 0.01.
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With such a large-scale human sperm proteome, it is
possible to analyze functional pathways in sperm. In such
pathway analyses, we found enrichment of a series of energy
metabolic pathways including glycolysis and gluconeogenesis
pathway, which supplies the energy required for the movement and fertilization of spermatozoa [35]. In addition, we
also found hyperrepresentation of many signaling pathways.
The IL-6 signaling pathway is one such pathway. Previous
studies have shown that IL-6 can enhance the fertilizing
capacity of human sperm by increasing capacitation and the
acrosome reaction [36]; however, the detailed pathway and
mechanism by which IL-6 does this are still not known. We
identified ERK1/2 and p38, which are annotated to downstream of the IL-6 signaling pathway. ERK1/2 and p38 MAPK
are primarily localized to the tail of mature human spermatozoa, and are both involved in the acrosome reaction [37]. In
addition to the IL-6 signaling pathway, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-9, insulin, EGFR1, TGF beta, and TNF-alpha/NF-κB
signaling pathways were all hyperrepresented. Some of these
pathways have already been studied and verified to function
in sperm. For example, TNF alpha can affect human sperm
function by elevating nitric oxide production, and regulate
sperm motility [38,39]. Insulin can enhance human sperm
motility, acrosome reaction and nitric oxide production [40].
Activation of the EGFR at the end of capacitation is enhanced
[Ca2+], leaded to F-actin breakdown and facilitating the
acrosome reaction [41]. TGF beta 1 can regulate protein
changes in human sperm [42]. Thus, sperm can be regulated
by and respond to various external signals through signaling
pathways. In seminal plasma, different factors have been
detected, including IL-6, IL-8, VEGF, TNFalpha, IL-1beta and
TGFbeta1 [43]. When sperm leave the male body to meet the
egg, it goes through the cervical canal and fallopian tube. IL-6,
IL-5 and IL-2 have been found in cervical mucus [44], and
TGF-beta was also found to exist in the human fallopian tube
[45]. These factors may regulate sperm function, and help
completion of the fertilization process.
The availability of the human sperm proteome will not
only help in the study of sperm function, but will also help in
the development of contraceptive drugs. Annotation of drug
targets showed that 500 human sperm proteins are known
targets. The list of sperm proteins can be a rich resource
for the development contraceptive drugs. For example, the
identified Angiotensin-converting enzyme (ACE) can be
inhibited by 13 different drugs, including enalapril and
quinapril, according to the DrugBank database annotation.
ACE has been shown to be important for male fertility in mice.
The fertility of homozygous male mutants of ACE was greatly
reduced [46]. Although available inhibitors can only inhibit its
function in sperm weakly, if the drug can be optimized to
inhibit its GPIase function in sperm, it may serve as a
promising contraceptive drug [47].
Of the annotated drug targeted proteins, 154 are mitochondrial proteins and 162 are cilia proteins. Mitochondria provide
energy for sperm motility; cilia are structurally similar to
sperm flagella, which provide force for motility. The functional
assessment of 4 drugs targeting sperm proteins revealed that
three could influence the motility of human sperm. Disulfiram
targets the mitochondrial protein, aldehyde dehydrogenase 2
family (mitochondrial) (ALDH2), and may affect mitochondrial
function and affect sperm motility. Propofol targets fatty acid
amide hydrolase (FAAH), a cilia protein, and is also expected to
affect sperm motility. Sorafenib targets RAF1. RAF1 is located
in the acrosome and flagella of sperm, and is an important
protein in the Ras/Raf/MAPK pathway, which reportedly
regulates sperm motility [37,48]. Leflunomide targets DHODH.
DHODH is annotated as a mitochondrial protein, but its well
known function is to regulate transcriptional elongation [49].
Mature sperms are known to be transcriptionally dormant [1];
therefore, it is reasonable that DHODH did not affect sperm
motility. Thus, these drug target data can be used to evaluate
the reproductive toxicity of drugs in use, and these proteins
may be candidate targets for the development of spermicides
and treatments for male infertility.
5.
Conclusions
The results of the present study reveal that the proteomic
composition of human sperm is more complex than we
previously thought. In addition, the sperm proteome characterization in this study is currently the largest sperm proteome thus
far. Annotation of the sperm proteome revealed many signaling
pathways and targets of known drugs, and 227 of the identified
sperm proteins were testis-specific. Our human sperm proteome
will help further our understanding of sperm function, and will
provide candidate targets for male contraceptives. Additionally,
the identification of these sperm proteins, especially the
testis-specific ones, will contribute to the initial goal of C-HPP.
Acknowledgments
This study was supported by grants from the 973 program
(2011CB944304, 2009CB941703), and the Chinese Natural Science
Funds (81222006, 31000637, 31271245). And it was sponsored by
Qing Lan Project.
Appendix A. Supplementary data
Supplementary data to this article can be found online:
Supplementary Fig. 1 is purified human sperm stained by
Hoechst. Supplementary Fig. 2 is hyperrepresented pathways
in our human sperm proteome. Supplementary Data 1 is a
table that shows the primary antibodies used in immunofluorescence studies. Supplementary Data 2 is a table that shows
the proteins identified in the human sperm proteome with a
FDR of 1%. Supplementary Data 3 is all the peptides Identified
with FPR of 1%. Supplementary Data 4 is a table that shows
the single peptide-based protein identifications. Supplementary Data 5 corresponds to MS/MS spectra and fragment
assignments of the single peptide-based identifications.
Supplementary Data 6 is a table that shows the WikiPathway
annotation of the human sperm proteome. Supplementary
Data 7 contains tables that show the gene ontology annotation of testis-specific proteins in the human sperm proteome.
Supplementary Data 8 is a table of the orthologous relationship between genes from Drosophila sperm proteome and
J O U RN A L OF P ROT EO M IC S 7 9 ( 2 01 3 ) 1 1 4 –1 22
human sperm proteome. Supplementary Data 9 is a table of
proteins targeted by drugs according to DrugBank database in
the human sperm proteome. Supplementary Data 10 is a table
of gene ontology annotation of drug-targeted proteins in the
human sperm proteome.
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.jprot.2012.12.008.
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