HUMAN SEMINAL PLASMA PROTEOME STUDY: A SEARCH
FOR MALE INFERTILITY BIOMARKERS
Davalieva K1,*, Kiprijanovska S1, Noveski P1, Plaseski T2, Kocevska B2,
Plaseska-Karanfilska D1
*Corresponding Author: Dr. Katarina Davalieva, Research Centre for Genetic Engineering and
Biotechnology “Georgi D. Efremov,” Macedonian Academy of Sciences and Arts, Krste Misirkov 2, 1000
Skopje, Republic of Macedonia; Tel.: +389-2-3235-410; Fax: +389-2-3115-434; E-mail: katarina@manu.edu.mk
page: 35
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INTRODUCTION
The proteomes most likely to contain clinically
useful disease biomarkers are those of human body
fluids. Proteomics has raised great expectations for
the discovery of biomarkers for improved diagnosis
or stratification of a wide range of diseases
[1]. Blood plasma and other body fluids, which also
include seminal fluids, are expected to be excellent
sources of protein biomarkers because they circulate
through, or come in contact with a variety of tissues,
and during this contact they are likely to pick up proteins
secreted or shed by tissues, a hypothesis that has
been tested and confirmed [2].
In addition to the general physiological importance
of knowing the composition of seminal fluid,
medical interest centers on two main areas: infertility
and prostate cancer. Male infertility is a widespread
medical condition with large societal and
emotional costs. Between one in six and one in 10
couples seek medical help for the problem of sub
fertility. In 20.0-25.0% of cases the infertility problem
is due to the male partner, in 30.0-40.0% the
problem is predominantly female; in approximately
30.0% of cases, abnormalities are found in both
partners, and in 15.0% no specific factor is identified
[3]. Since seminal fluid has an important role in
spermatozoa survival and overall fertilization success,
its impairment can be directly connected to
infertility. Seminal plasma contains many distinct protein
components that are important for the functioning
and survival of spermatozoa. It is a mixture of secretions
from several male accessory glands, including
prostate, seminal vesicles, epididymis, and
Cowper’s gland. The average protein concentration
of human seminal plasma ranges from 35 to 55 g/L
making it a rich, as well as an easily accessible
source for protein identification. Seminal plasma
has the feature common to many other body fluids,
namely it is characterized by a high dynamic range
of protein abundance, making low-abundance components
difficult to analyze.
Characterization of Human Seminal Plasma
Protein and Peptide Constituents. Research of
seminal plasma proteome began using two-dimensional
polyacrylamide gel electrophoresis (2-D
PAGE) and the electrophoretic transfer of proteins
to nitrocellulose sheets followed by subsequent detection
with immunological procedures in 1981 [4].
By this approach, a few seminal plasma proteins
such as prostatic acid phosphatase (PAP), some glycoproteins
and creatinin kinases, were identified.
Two-dimensional (2-D) gels coupled with mass
spectrometry (MS) were applied to study the role
of seminal plasma proteins in impaired spermatogenesis
in 2001, and about 750 spots were detected
in the 2-D maps of seminal plasma from a fertile
men [5]. However, only two seminal plasma constituents,
namely, PAP and prostatic specific antigen
(PSA) were identified by matrix-assisted laser desorption/
ionization time of flight (MALDI-TOF) in
this study, as the two major spot clusters in the 2-D
map of the seminal plasma proteome. By using 2-D
PAGE with MALDI and liquid chromatographyelectrospray
ionization (LC-ESI), Fung and colleagues
[6] identified 100 different protein constituents
in seminal plasma. This study established that
there were multiple post-translational variants of the
majority of the proteins. Overall, hormones, growth
factors and bio-active peptides were detected and
identified. Pilch and Mann [7] successfully catalogued
932 proteins in seminal plasma using Fourier
transform MS and two consecutive stages of MS
fragmentation. This study identified proteins known
to be characteristic for each of the organs contributing
to the formation of seminal plasma: prostate,
seminal vesicles, epididymis, and the bulbourethral
gland. The large proportions of the identified proteins
were extracellular, secreted by the male accessory
glands as well as extracellular matrix proteins
that are required for the classical functions of seminal
fluid. The second class of the identified proteins
was found to originate from prostasomes, while the
third class of proteins was the result of an epithelial
shredding. The overall numbers and proportions of
proteins in the identified proteome indicated that the
predominant functions are in clot formation and liquefaction,
in metabolic support and protection for
the spermatozoa and in immunological reactions.
The identification of seminal plasma protein and
peptide constituents by using combined 2-D PAGE/
MS/MS and 2D nano high performance liquid chromatography
(HPLC)/MS/MS is currently in progress
at our institution.
Identification of some well defined subgroup of
human seminal plasma proteins such as heparin-binding
proteins that are included in the fertilization process
were also carried out by the use of affinity chromatography,
2D electrophoresis and MALDI-TOF
MS [8]. Difference in the expression of sperm membrane
proteins between fertile and infertile males [9],
as well as proteins associated with human fertilization
capability [10], have also been investigated.
Comparative Analysis of Seminal Plasma
Proteome With Identification of Potential
Infertility Biomarkers. The research in the field of
comparative proteomic analysis of male infertilityassociated
seminal plasma proteins has been carried
out for almost a decade. However, extensive comparative
analysis of seminal plasma proteome in
order to establish a potential link between seminal
plasma proteins and male infertility is still lacking.
By investigating the differential expression of
proteins between two or more biologically different
states of reduced male fertility or infertility, potential
diagnostic or prognostic markers may be identified.
The gold standard techniques for this is still
2-D PAGE/2-D DIGE coupled to MS because this
approach is amenable to the separation and visualization
of a wide range of proteins, together with
their post translational variants.
Identification of 61 differentially expressed proteins
based on 2-D PAGE and tandem MS analysis
of seminal plasma of azoospermic and vasectomized
patients has been reported by Sarita-Garibaldi and
coworkers [11]. Two- dimensional DIGE combined
with MS was also carried out to find seminal plasma proteins associated specifically with azoospermia
and revealed a total of four potential markers for non
obstructive azoospermia [12]. The identified potential
biomarkers were stabilin 2 (STAB2), 135 kd
centrosomal protein (CP135), guanine nucleotide,
releasing protein (GNRP), and prolactin-inducible
protein (PIP). In a recent study, we utilized the 2-D
DIGE/MS approach to detect differential protein
expression of seminal plasma proteins between
four distinct groups of men with normozoospermia,
oligozoospermia, asthenozoospermia and azoospermia
[13]. The results from our study demonstrated
that normozoospermic, oligozoospermic and asthenozoospermic
groups have similar seminal plasma
protein profiles, resulting in no statistically significant
differences in protein expression. However, in
the azoospermic group there were a total of eight
proteins with a statistically significant increase of
expression in comparison with the rest of the studied
groups. From the eight differentially expressed
proteins, seven were successfully identified by MS
as fibronectin (FINC), prostatic acid phosphatase
(PAP), proteasome subunit α type-3 (PSA3), β-2-
microglobulin (B2MG), galectin-3 binding protein
(LG3BP), prolactin inducible protein (PIP) and cytosolic
non specific dipeptidase (CNDP2). All of
the reported proteins have already been reported
as constituents of seminal plasma. Most of these
proteins (FINC, PAP, B2MG, LG3BP, PIP) are localized
in the extracellular region and have the molecular
function of protein binding. From all the differentially
expressed spots found in this study, PAP
was found to be exclusively increased in azoospermic
patients compared with the rest of the studied
groups. Prostatic acid phosphatase is the most abundant
phosphatase in human prostate tissue and has
been studied extensively, primarily due to its clinical
relevance as a biomarker of prostate carcinoma
[14]. Several other studies have implicated PAP in
male infertility before, with enzyme levels inversely
correlated with sperm concentration [15-17].
Another study, based on the comparative analysis
of the levels of PIP in fertile and infertile men,
has proposed that PIP could be an immunoglobulin
G-binding protein [18]. Prolactin inducible protein
was shown to exist in several isoforms in seminal
plasma by Western blot. The pattern of PIP isoform
variability in seminal plasma from fertile and infertile
men is quite complex but one multimeric form of PIP was confirmed to be absent from the seminal
plasma of fertile men.
The levels of fibronectin (FINC fragments in
seminal plasma samples were found higher in seminal
plasma with abnormal semen characteristics
than in the normozoospermic group [19]. The results
suggest that seminal plasma FINC fragments
may contribute to fertilization and the analysis of
FINC fragmentation may have a diagnostic value in
andrological investigations.
Proteomic analysis of seminal plasma from asthenozoospermia
patients revealed a rich source of
biomarker candidates for male infertility, proposing
that functional abnormalities of the epididymis and
prostate can contribute to asthenozoospermia [20].
Among the identified proteins, DJ-1, a protein that
has been shown to be involved in the control of
oxidative stress, was down regulated in asthenozoospermia
patients and was proposed as a candidate
biomarker for this condition.
In a conclusion, the research in the field of seminal
plasma proteome and the search for biomarkers
of male infertility is still ongoing. An in-depth
understanding of the seminal plasma proteome
would contribute greatly to the elucidation of the
roles of seminal plasma proteins in the regulation
of motility and to the establishment of biomarkers
for male infertility. There have been different sets of
proteins proposed as biomarkers in different conditions
of reduced fertility and/or infertility. However,
only small set of proteins such as PAP, PIP, FINC
are found differentially expressed in male infertility
by independent studies. One of the reasons for this
very low percentage of overlap between independent
studies is that different proteomics techniques
and their combinations were used. Therefore, the
data from these studies is quite heterogeneous.
However, the appearance of high-throughput MSbased
techniques allows more detailed investigation
of the proteomes of interest, among which is human
seminal plasma proteome, and holds promise on
more reproducible results in the future.
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