PROTEOMICS OF THE SPERMATOZOON
Oliva R1,*, Ballescá JL2
*Corresponding Author: Professor Rafael Oliva, Laboratorio de Genética Humana, Facultad de Medicina,
Universitat de Barcelona, Hospital Clinic and Institut d’Investigacions Biomèdiques August Pi I Sunyer
(DIBAPS), C/Casanova 143, 08036 Barcelona, Spain; Tel.: +34934021877; Fax:+34934035278; E-mail:
roliva@ub.edu
page: 27
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Abstract
The study of the sperm proteins is crucial for understanding
its normal function and alterations in
infertile patients. The sperm is a highly specialized
cell with a very large flagella, with little cytoplasm
and a highly condensed nucleus. The most
abundant proteins in the nucleus of mammalian
sperm are the protamines. The main functions of
the protamines are the condensation of the DNA,
possibly contributing to the generation of a more
hydrodynamic sperm head and to the protection of
the genetic message. However, in addition to protamines,
about 5.0-15.0% of the paternal genome
is also complexed with histones and histone variants.
It has also demonstrated a differential distribution
of genes in regions associated with histone
and protamine-associated regions, suggesting a potential
epigenetic relevance in embryonic development.
More recently, detailed lists of proteins have
been described corresponding to the different compartments of the sperm cell thanks to the application
of recent proteomic techniques based on mass
spectrometry (MS). Differential proteomics is also
being applied to identify the presence of protein abnormalities
found in infertile patients. The Nucleohistone-Nucleoprotamine Transition
and Organization of the DNA in the Sperm
Nucleus. Spermatogenesis involves radical changes
in chromatin structure to give rise to the mature
sperm [1,2]. The nucleosome structure present in
spermatogonia, spermatocytes and round spermatids,
is disassembled in spermiogenesis and is temporarily
replaced by transition proteins and finally
by protamines [1-4].
While most of the genome in the sperm cell
(about 85.0-95.0%) is tightly packaged by protamines
in the form of toroidal structures, it is also
important to note that about 5.0-15.0% of sperm
DNA is organized by histone proteins, many of
which are sperm-specific variants [3-5]. The distribution
of genes in genomic regions organized
by protamine and the genomic regions organized
by histones is not random. Recent studies based
on analysis of the paternal genome associated with
each of these domains using microarrays, have led
to the basic conclusion that the regions associated with the nucleohistone are associated with gene
regulatory regions [6]. In another recent study [7],
based on massive genome sequencing, it was found
that nucleosomes associated regions are significantly
enriched in genes important for development, including
imprinted genes, microRNAs, Hox genes,
promoters and transcription developmental genes
and signaling factors. It has also been shown that
histone modifications (H3K4me2, H3K27me3) are
reached at certain loci associated with developmental
genes, and promoters associated with developmental
genes are hypomethylated in the sperm, but are methylated
during maturation [7,8].
In addition to these epigenetic marks determined
by the differential distribution of genes in
the domains associated with the nucleohistone and
nucleoprotamine, other types of epigenetic information
are potentially transmitted by the sperm nucleus
to the oocyte. One of the best known is contrasted
DNA methylation. More recently, the identification
of RNAs present in the sperm and the demonstration
of oocyte transfer, opens the possibility of
their role in fertilization. Another potential source
of epigenetic information could be the presence of
other proteins in the sperm nucleus, in addition to
histones and protamines [9,10].
More recently, proteomic analysis of proteins
identified in mature sperm has provided some unexpected
results. For example, transcription factors,
DNA binding proteins and proteins involved in the
metabolism of the chromatin in cells that are transcriptionally
inactive [9,10]. The catalogs for the
proteomes of human sperm are available [9,11,12].
Most notable is the presence of proteins such as
histone acetyltransferase and deacetylase, histone
methyltransferase, DNA methyltransferase, topoisomerase,
helicase, transcription factors, zinc finguers,
homeobox proteins, cromodominio proteins,
centrosomal proteins, and telomerase in cells that
are transcriptionally inert and have at least 85.0%
of their DNA packaged by protamines [9]. A crucial
question is whether these transcription factors
and proteins newly identified in the cores, represent
remnants of the process of spermatogenesis or are
making some regions of the paternal genome and
have an epigenetic basis [9,13].
Abnormalities in the content of protamine in
subfertile patients have already been described
over 20 years ago [14]. Subsequently, other studies
confirmed the link between abnormal protamine
content and alterations in sperm parameters in infertile
patients [15,16]. One of the potential causes
of abnormal protamine ratio (P1/P2) can be found in
abnormal processing of protamine 2 and increased
protamine precursors in a subset of patients [15,17].
The results of the content of protamines and histones
have been correlated with alterations in the integrity
of DNA and the results of assisted reproduction [18].
Identification of Sperm Proteins and Their
Alterations in the Spermatozoa Through
Proteomic Techniques. Essentially, two different
alternatives can be used to study the sperm proteome
through mass spectrometry (MS) (Figure 1):
1) two dimensional (2D) separation of the proteins
followed by their identification by Matrix-assisted
laser desorption/ionization (MALDI)-MS or liquid
chro-matography-tandem MS (LC-MS/MS),
and 2) the initial digestion of proteins to generate
peptides, followed by separation and LC-MS/
MS analysis [9]. The first alternative generally involves
the separation of proteins using isoelectric
focusing and is followed by polyacrylamide gel
electrophoresis (PAGE) in the presence of sodium
dodecyl sulfate (SDS) for separating proteins in a
second dimension based on their molecular weight.
This alternative has been widely used in the past
to identify many proteins present in the sperm cell
[11,19]. Of the two alternatives, the initial generation
of peptides and analysis by LC-MS/MS is of
much higher throughput. For example, through 2D
and MALDI-TOF (time of flight) or LC-MS/ MS,
it has been possible to identify some hundreds of
proteins [11,20], whereas the generation of peptides
followed by LC-MS/MS allows the identification of
up to about 1000 different proteins [9,12].
In addition to the generation of catalogs of proteins,
proteomics has also been applied to the identification
of the presence of anomalies in infertile
patients. There are several strategies to analyze the
differential protein content in two or more different
samples. One method is 2D-DIGE (differential
in-gel electrophoresis) and is based on the differential
identification of fluorochrome-labeled proteins
extracted from the control (for example, labeled
green) and experimental cells (for example, labeled
red). This is followed by mixing of the proteins and
their separation in the same 2D system, followed by
detection that can detect increased or decreased pro-teins, observing the deviation of the fluorescence to
one of the fluorochromes [9,12]. Another alternative
is the quantification and comparison of the relative
abundance of the different proteins in separate gels.
Newer strategies are being developed based on non
radioactive isotopic labeling of the test samples and
control [9,12].
The first description of the potential of 2D proteomic
analysis in the study of defects in sperm was
performed in a patient with repeated failure of in vitro fertilization techniques [20]. The proteome of
this patient showed 20 differences compared with
controls, and identified several proteins differentials.
It was later applied to the identification of the
differential proteins in astenozoospermic patients,
oligozoospermic patients, and patients with alterations
in the content of protamines or the integrity of
DNA [19].
The application of proteomics techniques in andrology
and reproductive biology is in its infancy but the data available to date indicate their enormous
potential. It is foreseeable that in the future it will
allow the molecular dissection of the various causes
of male infertility, allowing both the identification
of the pathophysiologic mechanisms involved and
its application to the diagnosis, prognosis, and development
of new therapeutic strategies.
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