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Center for Reproductive Medicine, Glickman Urological and Kidney Institute, Cleveland, OH, USAFaculty of Medicine, MARA University of Technology, Sungai Buloh, Selangor, Malaysia
Oxidative stress has been established as one of the main causes of male infertility and has been implicated in many diseases associated with infertile men. It results from high concentrations of free radicals and suppressed antioxidant potential, which may alter protein expression in seminal plasma and/or spermatozoa. In recent years, proteomic analyses have been performed to characterize the protein profiles of seminal ejaculate from men with different clinical conditions, such as high oxidative stress. The aim of the present review is to summarize current findings on proteomic studies performed in men with high oxidative stress compared with those with physiological concentrations of free radicals, to better understand the aetiology of oxidative stress-induced male infertility. Each of these studies has suggested candidate biomarkers of oxidative stress, among them are DJ-1, PIP, lactotransferrin and peroxiredoxin. Changes in protein concentrations in seminal plasma samples with oxidative stress conditions were related to stress responses and to regulatory pathways, while alterations in sperm proteins were mostly associated to metabolic responses (carbohydrate metabolism) and stress responses. Future studies should include assessment of post-translational modifications in the spermatozoa as well as in seminal plasma proteomes of men diagnosed with idiopathic infertility.
Oxidative stress, which occurs due to a state of imbalance between free radicals and antioxidants, has been implicated in most cases of male infertility. Cells that are in a state of oxidative stress are more likely to have altered protein expression. The aim of this review is to better understand the causes of oxidative stress-induced male infertility. To achieve this, we assessed proteomic studies performed on the seminal plasma and spermatozoa of men with high levels of oxidative stress due to various clinical conditions and compared them with men who had physiological concentrations of free radicals. A variety of sperm and seminal plasma proteins were found to be expressed either in abundance (over-expressed) or in a lesser amount (underexpressed), while other proteins were found to be unique either to men with oxidative stress or to men with a balanced ratio of antioxidants/free radicals. Each study included in this review suggested several proteins that could possibly act as biomarkers of oxidative stress-induced male infertility, such as protein DJ-1, PIP, lactotransferrin and peroxiredoxin. Pathway analysis performed in these studies revealed that the changes in seminal plasma proteins in men with oxidative stress could be attributed to stress responses and regulatory pathways, while changes in sperm proteins were linked to stress responses and metabolic responses. Subsequent studies could look into post-translational modifications in the protein profile of men with idiopathic infertility. We hope that the information in this review will contribute to a better understanding of the main causes of idiopathic male infertility.
). Infertility affects around 15% of all couples of reproductive age, with about 50% being associated with abnormalities in the male, called male factor infertility (
). A recent study using the current duration approach to assess the prevalence of infertility estimated that 9 to 14% of American men within reproductive age (i.e. 15 to 44 years old) will probably experience difficulties to conceive (
). Male infertility could result from dysfunction at various levels along the hypothalamic-pituitary-gonadal axis: pre-testicular (damage at the hypothalamus or pituitary level), testicular (failure of the testis), post-testicular (normal testicular function but with obstruction or inflammation that leads to infertility) or a combination of these. Causes of male infertility include hypogonadotrophic hypogonadism and Kallmann syndrome, direct trauma, inflammation or infection of the testis, varicocele, cryptorchidism, Y-chromosome microdeletions, testicular cancer and chemotherapy, erectile dysfunction, infrequent or retrograde ejaculation, epididymitis, congenital bilateral absence of the vas deferens, Klinefelter’s syndrome (47,XXY), and Sertoli-cell only syndrome (
in: Parekattil S.J. Agarwal A. Male Infertility: Contemporary Clinical Approaches, Andrology, ART and Antioxidants. Springer Science+Business Media,
New York2012
) and have been associated with negative changes in sperm concentration, motility and morphology, leading to poor semen parameters and eventually to infertility (
). In fact, oxidative stress has been implicated in several male infertility-associated pathologies, including leukocytospermia and varicocele as well as idiopathic infertility (
The diagnosis of male infertility routinely begins with a basic semen analysis, which measures various semen parameters including semen volume, colour, pH, liquefaction time, viscosity, sperm count and motility, sperm morphology, concentration of round cells and polymorphonucleocytes, sperm agglutination and sperm viability (if required). Two or more of these basic semen analyses are used to identify abnormalities in: sperm concentration (oligozoospermia or azoospermia), motility (asthenozoospermia) and morphology (teratozoospermia), based on reference values established by the World Health Organization (
). In addition to the routine evaluation, several advanced tests can be performed to establish the causes(s) of infertility, among them are the assessment of ROS levels, total antioxidant capacity and sperm DNA fragmentation level, DNA compaction and apoptosis, as well as presence and localization of antisperm antibodies and genetic testing (
). However, results of these tests typically either fall within the normal range or do not help determine an exact aetiology of infertility, leading to a classification of ‘idiopathic infertility’ (
). Damaged DNA in spermatozoa is indicative of poor cellular health. Sperm DNA damage reduces semen quality and is the cause of infertility in many men (
). In assisted reproduction, spermatozoa with damaged DNA lower fertilization and pregnancy rates, impair embryo development and quality and increase the risk of spontaneous miscarriage, birth defects and childhood diseases such as cancer (
). The level of DNA damage is suggestive of clinical outcome in assisted reproduction: idiopathically infertile couples with higher levels of sperm DNA fragmentation were found to have lower live-birth rates following IVF (
Highly specialized techniques such as proteomics allow characterization of the semen profile at a molecular level, proving useful in the assessment of proteins and the understanding of biological pathways that play a key role in male infertility (
). Advances in this rapidly-evolving field have allowed researchers to better identify seminal plasma and sperm proteins and to determine how their presence or concentration may differ in fertile versus infertile patients (
). Studies looking at the sperm and seminal plasma protein profiles of men with oxidative stress-induced infertility would help in identifying alterations in the protein expression and/or translational modifications that may occur during sperm maturation and functions of proteins involved. Moreover, these studies may be extended to the characterization of other pathologies associated with male infertility at the molecular level.
Despite the established role of oxidative stress in the aetiology of male infertility, there are, as of yet, relatively few studies that have investigated the correlation between ROS-induced oxidative stress and a differential protein expression profile in the human ejaculate using proteomic analysis. Our laboratory has recently published a series of studies on patients diagnosed with primary and secondary infertility and elevated ROS concentrations using proteomic approaches (
). Using similar strategies, other laboratories have also studied the proteomic profile of infertile patients with poor semen quality who were also affected with oxidative stress (
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
Thus, in this review, we aim to summarize and compare the findings of these initial studies that have utilized proteomic analysis to look into the differential expression of proteins in the seminal ejaculate of infertile men with high oxidative stress and fertile men with physiological levels of ROS. In this review, we only included proteomic studies in which the oxidative status of infertile men was measured. Our review begins with an overview of oxidative stress and its impact on male infertility as well as the methodologies and general work flow utilized in proteomic studies, in order to provide some basis to readers less familiar with the field. In addition, this review highlights seminal plasma and spermatozoa proteins identified using proteomic analysis that are likely to play a major role in oxidative stress-induced male infertility and subsequently it lists proteins that have the potential to serve as diagnostic biomarkers of male infertility. To conclude, current limitations of these research studies as well as some perspectives in this area of research are highlighted. It is hoped that proteomic studies in men with different diagnosis of infertility will eventually lead to the discovery of biomarkers for idiopathic male infertility, which would help with the diagnosis and better management of male factor infertility.
Oxidative stress and male infertility
Oxidative stress occurs when there is an imbalance between ROS and the antioxidants that scavenge surplus free radicals (
). ROS are natural products of cellular metabolism which, in physiological amounts, are essential requirements of spermatozoa for sperm processes leading to successful fertilization, such as capacitation, hyperactivated motility and acrosomal reaction (
Reactive oxygen species in semen of infertile patients: levels of superoxide dismutase- and catalase-like activities in seminal plasma and spermatozoa.
There are two principal methods in which ROS can cause male infertility: through damage of the sperm membrane and damage of the sperm DNA. Sperm membranes have large amounts of polyunsaturated fatty acids, making them susceptible to oxidative stress. This can then affect sperm motility as well as their ability to fertilize oocytes. Furthermore, DNA fragmentation may harm the paternal genetic contribution to the developing embryo (
ROS are considered a class of free radicals because they contain oxygen molecules with one or more unpaired electrons. This makes them highly reactive and susceptible to radical formation, potentially altering cellular function and ultimately endangering cell survival (
). There are three different general forms of the ROS (Figure 1): (i) the primary form of ROS, the superoxide anion radical from which secondary ROS can be derived either directly or indirectly (
); (ii) the secondary form of ROS, hydrogen peroxide (an example of a ROS that is not a free radical), hydroxyl radical and peroxyl radical; and (iii) the tertiary form of ROS, a class of free radicals that are nitrogenous compounds: peroxynitrous acid, nitroxyl anion, peroxynitrile and nitrous oxide (
The origins of oxidative stress may vary from lifestyle choices and environmental factors (exogenous), to testicular (endogenous) sources, in addition to idiopathic causes.
Lifestyle, for example, is a major contributor to ROS production; smokers see a 107% increase in ROS concentrations in the semen, increased leukocytes and a greater likelihood of DNA fragmentation compared with non-smokers (
). Alcohol abuse induces systemic oxidative stress and reduces antioxidant defences, which is likely further exacerbated by an antioxidant deficient diet (
). Diet and exercise are also important factors in oxidative stress: improper diet and sedentary lifestyles can lead to obesity. Obesity in general increases the risk of co-morbidities, such as hypertension, dyslipidaemia, type 2 diabetes, coronary heart disease, stroke, non-alcoholic fatty liver disease, osteoarthritis, sleep apnoea and several types of cancers (
). These obesity-linked systemic inflammatory conditions upset the redox balance and contribute to seminal oxidative stress, which causes detriment to sperm function (
). The accumulation of adipose tissue, which causes obesity, may increase ROS levels through the release of pro-inflammatory cytokines, increased ROS production in leukocytes and heating of the testicles (
). Conversely, intensive exercise has been linked to increased ROS concentrations, regardless of the type of exercise, because of an increased demand for energy in the muscles (
Environmental factors also have an impact in ROS production. Phthalates are chemicals that are added to plastics to increase its flexibility (plasticizers), and are used in food packaging, medical devices and personal care products. They have been linked to increased generation of ROS and reduction in antioxidants, leading to testicular oxidative stress (
). Pesticide and heavy metal exposure are associated with diminished antioxidant levels and elevated 8-hydroxy-2′-deoxyguanosine levels in sperm DNA, indicative of increased oxidative DNA damage in spermatozoa (
). Other studies have detailed how both drug administration (such as aspirin and acetaminophen) and even assisted reproduction treatment (such as IVF and intrauterine insemination) increase oxidative stress (
). This is either through increasing enzymic activity in the case of drugs or through assisted reproduction practices such as centrifugation or removal of antioxidants from semen.
ROS can also be produced as a result of infection and inflammation. Generally, pathogens will elicit a natural immune response such as an acute inflammatory response from leukocytes and macrophages (
). Varicocele is the abnormal dilation and tortuosity of the pampiniform plexus veins within the spermatic cord and is the cause for nearly 35% of male factor infertility. Clinical varicocele can be found in 35% of men with primary infertility and 80% of men with secondary infertility. Infertile men with varicocele have high levels of seminal ROS and oxidative stress, which causes significant sperm DNA damage (
Cryptorchidism is also a common testicular cause of oxidative stress; this pathology is the result of a deficient maturation of gonocytes into type A spermatogonia, which causes hypospermatogenesis (
). If there is prolonged ischaemia followed by restoration of the blood flow (spontaneous or surgical), an influx of activated leukocytes follows into both testicles (
). However, it is implied that leukocytes contribute the most to oxidative stress because compared with spermatozoa, the rate of ROS production in leukocytes is 1000-times greater (
). This is considered an ‘extrinsic source’ of ROS as opposed to the ‘intrinsic’ sources from sperm. In cases of idiopathic causes of male infertility, patients may have normozoospermia and yet are infertile, showing high ROS production and reduced antioxidant levels when compared with fertile men (
Low levels of ROS are generated naturally during processes such as spermatogenesis. Indeed, ROS are necessary, in balance with antioxidants, for proper spermatogenic mechanisms to occur. For example, hydrogen peroxide, a secondary form of ROS, stimulates acrosomal reaction, hyperactivation, and tyrosine phosphorylation in the sperm (
). This delicate balance between hydrogen peroxide and its antioxidant, catalase, illustrates the subtle equilibrium that both facilitates proper function and prevents oxidative stress.
ROS are generally produced as a byproduct of enzymic reactions in oxidative phosphorylation, which is used to produce energy in the form of ATP. These reactions that involve the reduction of oxygen usually take place in the mitochondria (
). In the sperm cell, mitochondria are located on the midpiece. Studies show that mitochondrial DNA is more susceptible to mutations than nuclear DNA, increasing the production of ROS during this process (
). In fact, elevated ROS levels have been linked to release of cytochrome C, a protein that activates apoptotic reactions, which is increased in patients with male factor infertility (
While mutations are less likely in nuclear DNA than in mitochondrial DNA, prior studies have shown that intrinsic ROS production is highly related to DNA fragmentation (
). Free radicals can attack the purine and pyrimidine bases and the deoxyribose backbone. DNA damage may ultimately lead to poor blastocyst formation in vitro (
The significance of sperm DNA oxidation in embryo development and reproductive outcome in an oocyte donation program: a new model to study a male infertility prognostic factor.
Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection.
). However, this also makes spermatozoa vulnerable to ROS attack. Seminal fluid is an important source of antioxidants in semen, as the lack of cytoplasm and DNA compaction in spermatozoa leaves very little room for translation or for antioxidant defenses (
Reactive oxygen species in semen of infertile patients: levels of superoxide dismutase- and catalase-like activities in seminal plasma and spermatozoa.
). The main concepts of oxidative stress are summarized in Figure 2.
Figure 2Model of the build up of oxidative stress in the semen. The model highlights the imbalance caused by accumulating ROS and depleting antioxidant, which brings about a state of oxidative stress. Various lifestyle and environmental factors along with testicular and seminal sources cause the generation of ROS. Antioxidants comprise both enzymatic and non-enzymatic types.
Using the seminal ejaculate of a patient consulting for infertility, a clinical diagnosis of oxidative stress can be made using two alternative approaches: (i) measurement of ROS generated by spermatozoa; or (ii) measurement of either the amount of protein that is oxidized due to the presence of ROS (such as protein carbonyl) or the concentrations of antioxidant enzymes present (such as gluthathione peroxidase, superoxide dismutase and catalase).
Measurement of intra- and extracellular ROS generated by spermatozoa in a semen sample is performed using a luminol-mediated chemiluminescence assay. This method measures ROS concentrations in a sperm suspension (
), where horseradish peroxidase is added in order to sensitize the assay to hydrogen peroxide. A chemical called luminol is then added, which is extremely sensitive to oxidation by a number of ROS at normal pH, producing luminescence and this signal is then measured by a luminometer (
). This method is used in our clinical laboratory as part of the advanced tests for seminal ejaculates of infertile patients and we have previously published reference levels used as cut-off values to determine levels of seminal ROS in patients: physiological ROS concentrations are <20 relative light units (RLU)/s/10 million spermatozoa while pathological ROS concentrations (i.e. oxidative stress) are ⩾20 RLU/s/10 million spermatozoa (
For the measurement of ROS levels in a semen sample, products of oxidation such as protein carbonyls, which are chemically stable, are useful for detection purposes (
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
Total antioxidant capacity measures the overall antioxidant capacity present in the seminal plasma. This includes: enzymatic antioxidants such as superoxide dismutase, catalase, glutathione peroxidase; non-enzymatic antioxidants such as ascorbic acid (vitamin C) and alpha-tocotrienol (vitamin E); and molecules such as albumin, ceruloplasmin, ferritin, bilirubin, uric acid and reduced glutathione. Together, these antioxidants represent the cumulative effect of the antioxidants present in the seminal plasma (
For a long time, it was thought that sequencing of the human genome would be the ultimate strategy for unravelling the different diseases expressed by the human body. Along with advances in technology, this strategy culminated in a complete sequencing of the human genome, which was then placed on a database for public consultation (
). The one-gene-one-polypeptide theory, dominant in the past, was found to be far too simplistic to explain the relationship between the genotype and phenotype. This discrepancy in explaining the phenotype by solely examining the genotype further increased with studies that claimed the presence of around a million proteins in the human body (
May, C., Brosseron, F., Chartowski, P., Schumbrutzki, C., Schoenebeck, B., Marcus, K., 2011. Instruments and methods in proteomics. In: Hamacher, M., Eisenacher, M., Stephan, C. (Eds.), Data Mining in Proteomics. In: Methods in Molecular Biology, vol. 696, pp. 3–26.
). Protein diversity may be derived through three main processes: at the DNA level (gene polymorphisms), the pre-mRNA or mRNA level (alternative splicing) or the protein level subsequent to RNA translation (post-translational modification and specific proteolytic cleavages) (
Lights and shadows of proteomic technologies for the study of protein species including isoforms, splicing variants and protein post-translational modifications.
). Post-translational modifications may involve glycosylation, phosphorylation and ubiquitination. Translation usually occurs in the cytoplasm and involves activation, initiation, elongation and termination of the polypeptide chain. After translation, proteins undergo chemical modification, by the addition of a functional chemical group (glycosylation or phosphorylation) or other proteins (ubiquitination), or undergo structural changes (proteolytic cleavage, protein folding), which modifies the immature protein before it turns into a mature protein product. This important aspect of protein development shows how sequencing the human genome alone is not enough. Proteins play a major role in the understanding of the body and the diseases that affect it.
In the 1990s, researchers came to realize that the biochemical role of proteins needed to be explored to explain what genomics could not, which pushed researchers to work towards filling this gap in scientific knowledge (
). This endeavour was supplemented with the advancement in techniques such as 2D-differential in-gel electrophoresis (2D-DIGE) and mass spectroscopy. These technical advancements helped molecular biologists to identify protein–protein interactions and gain understanding of the cell phenotype. Proteomic approaches, such as labelling and fractioning techniques, are powerful tools for the quantitative and qualitative measurement of the total proteins in a cell, where different techniques can be used to elucidate differentially expressed proteins when comparing various tissue types or the organism in different states (
Proteomics continues to rapidly evolve and it is currently considered a promising field with many applications in the future. While basic proteomic analysis helps in the identification of proteins present in a particular tissue, quantitative proteomics deals with relative quantification of proteins present in different physiological or pathological conditions to identify differentially expressed genes in order to unravel the cellular processes and their biological significance (
). Bioinformatics helps connect these initial protein lists to its biological significance in various states of disease, which is resourceful in the discovery of biomarkers of fertility (
Specific proteins that are differentially expressed in diseased states may be used as biomarkers, which can act as a highly important non-invasive diagnostic tool (
). The development of proteomics-based therapy may also prove more effective than therapies presently used. However, leaps in the field are not made without challenges. Factors such as lifestyle and nutrition, environment, race and population differences cause diversity in protein expression. Further, factors such as ageing may affect post-translational modifications in a cell, making measurements less precise and difficult to combine (
). Advancements in technology and an increase in knowledge of post-transcriptional modification may, in the future, be able to eliminate such drawbacks.
Methods used in proteomics
The evolution of proteomics parallels the development of the techniques involved. Currently, several methods are employed to identify thousands of differentially expressed proteins.
Differences in amino acid content dictate variation in functional and chemical properties of proteins. Characteristics such as size and charge help molecular biology studies, as many separation techniques rely on these properties. For example, 2D-gel electrophoresis (2D-GE) is a method where proteins from cell lysates or fluids are run on an immobilized pH gradient that facilitates their separation in the first dimension based on their isoelectric point. Proteins resolved after the first run are then separated on the second dimension based on their apparent molecular weight, using classic polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS–PAGE;
May, C., Brosseron, F., Chartowski, P., Schumbrutzki, C., Schoenebeck, B., Marcus, K., 2011. Instruments and methods in proteomics. In: Hamacher, M., Eisenacher, M., Stephan, C. (Eds.), Data Mining in Proteomics. In: Methods in Molecular Biology, vol. 696, pp. 3–26.
). SDS is a denaturing agent (detergent) that imposes a negative charge on all proteins, overriding the original charge which no longer affects protein separation. At the end of SDS–PAGE, proteins have migrated at different distances depending on their molecular size. In order to determine the proteins that are differentially expressed, their expression is compared between treated and control conditions and a cut-off value is established to take into account the presence of artefacts.
One disadvantage of 2D-GE is its inability to resolve hydrophobic molecules and its low load–separation capacity (
). In addition, 2D-GE is insensitive to proteins present in low abundance and therefore it is not a completely reliable technique. However, it does provide a general overview of protein expression and concentrations in the cell (
) and involves labelling extracted proteins from the control and experimental samples with different dyes before allowing them to run on separate gels, which are subsequently overlapped for better comparison (
). However, this method has technical limitations, as the Cy3 and Cy5 fluorescent dyes used for protein identification and quantification in the 2D-DIGE can in fact alter the protein profile obtained, depending on the lysine content, molecular mass, abundance and acidity/hydrophobicity of the proteins present in a sample (
Mass spectrometry (MS) is an automated process that has revolutionized protein detection and identification in cells. In sperm protein analysis, protein bands generated through gel electrophoresis are excised and identified via either one of two approaches. The first method, liquid chromatography-tandem MS (LC-MS/MS), involves digestion of proteins with trypsin, followed by high-performance liquid chromatography (HPLC), to separate polypeptides depending on hydrophobicity, charge and size (
). After this procedure, repeated MS with an interface is performed to analyse and identify complex extracts by resolving the charge-to-mass ratio of molecules ionized for detection. LC-MS/MS is a highly sensitive and selective technique that allows for the recognition of primary peptide sequences from complex proteins (
The second method is called matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), and involves trypsin digestion of protein bands collected from 2D-GE but excludes HPLC. After protein processing, MS is performed to identify the molecules. One advantage of this technique is the pulsatile nature of MALDI, which allows for parallel ionization and mass analysis, resulting in the detection of greater portions of the sample. TOF is a mass analyser that separates ions formed at the same time: its role is to accelerate the different particles through a fixed distance, from a starting point to the detector. The time of flight of each ion is inversely correlated to the root square of mass-to-charge ratio (m/z). By determining particles’ TOF properties, the m/z can be established and consequently the protein can be identified (
The peptide mass-to-charge ratio that is determined through MS is then compared with set masses from previously sequenced and isolated proteins loaded in a database. Proteins are identified when a minimum of three of its peptide fragment masses match its homologous peptide masses in the protein database (
). A major advantage of MS is its ability to recognize proteins that have undergone post-translational modification. Indeed, alterations in the initial structure of the protein results in a change of its molecular mass, which is reflected by the mass of the peptide where the modification has occurred (
). Finally, proteins that are differentially expressed are selected. Spectral count is a technique by which the relative protein concentration in pre-digested proteins are analysed by MS for quantification of its expression (
In proteomics, Western immunoblotting is an important step to verify the presence and, in some cases, to quantify a protein of interest in a complex sample. In this technique, proteins are separated using 2D-GE and individual protein spots are seen along the length of the gel. A replica of the protein profile is obtained on a specific blotting support (typically nitrocellulose or PVDF, polyvinylidene difluoride membranes) using a perpendicularly-directed electric field to achieve protein transfer. Proteins on the membrane can be developed using immunodetection with specific antibodies followed by incubation with a system composed of a secondary antibody coupled to an enzyme and a substrate and a reaction detection system to visualize antigen-antibody complexes (Western immunoblotting;
). In some cases, proteins immobilized on the membrane can be subjected to interaction with other proteins, after which immunodetection of the added protein is carried out to evaluate protein–protein interaction (far-Western immunoblotting;
Bioinformatics describes the scientific field dealing with the overlap between biology, engineering and computer science. It links the computer to genetics and molecular biology through the creation of a software program. Each protein detection method has its own programs that use specific algorithms. This technology is used by a bioinformatician to derive meaning from the large amount of information collected through proteomics studies (
Proteins lists that are generated from proteomic analysis are converted to gene names for functional annotations. Annotations for undefined genes or biological functions that are not listed in a particular database can be functionally annotated using prediction tools (
). Once the annotations are obtained for the list of genes, a gene ontology (GO) analysis is conducted. GO is the study of the number of genes involved and its correlation between protein localization, structure, function and involvement in cellular biochemical pathways. In GO analysis, functions of gene products are classified using structured and controlled vocabularies, consisting of: (i) cellular components (giving functional meaning to the intracellular/extracellular localization of the gene product); (ii) biological processes (defining the molecular events taking place in the cell); and (iii) molecular functions (basic molecular activities of a gene product and its regulatory activities on the process studied (
). GO studies are complemented by a pathway analysis, which centres the evaluation on how a group of genes in a defined biological process interrelate to form a complex network, and the result of this analysis is commonly visualized as a pathway map (
). An overview of the general workflow involved in protein quantification and identification of semen samples with oxidative stress is shown in Figure 3.
Figure 3Overview of the general methods used for protein isolation and identification in seminal plasma and spermatozoa. Levels of oxidative stress (OS) in the semen sample are determined by measuring the reactive oxygen species (ROS) and/or the total antioxidant capacity (TAC). Protein separation can be performed using either 2D gel electrophoresis (2D-GE) or 2D differential in-gel electrophoresis (2D-DIGE). Next, protein identification can be done using liquid chromatography-tandem mass spectrometry (LC-MS/MS) or matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) method. Isolated proteins are identified using tandem MS data analysis programs such as MASCOT or SEQUEST. Proteins of interest can be validated using Western blotting. Based on spectral counts, protein expression is quantified. Protein lists are generated and analysed using bioinformatics. Gene ontology (GO) is then used to determine the specific gene function in cellular pathways.
Proteomic studies on seminal ejaculate with oxidative stress
Spermatogenesis is the biological process involving a series of successive divisions and cellular modifications of germ cells that results in the formation of mature and functional spermatozoa. The main function of spermatozoa is to deliver the haploid paternal genome to the female gamete. Although spermatozoa are very nearly transcriptionally inactive, sperm DNA goes through numerous modifications (such as methylation) and the majority of histones are replaced by small basic proteins, called protamines. It is believed that these changes are meant to transfer epigenetic factors to the female pronucleus in order to produce a viable embryo (
). Previous studies have also demonstrated the involvement of proteins in various molecular events, such as acrosomal reaction and penetration of the egg’s extracellular matrix, the zona pellucida (
). Sperm cell exploration has been facilitated by the ease of non-invasive collection of a sizable number of cells in each semen sample; in this regard, simple laboratory techniques such as centrifugation can be applied to separate a sufficient number of cells for analysis. However, a drawback in studying sperm proteins is the small size of the cell and the low amount of protein recovered from each cell. This can be overcome by combining semen samples from men with a similar diagnosis and then analysing the sample.
Today, thousands of proteins have been profiled in human semen, and scientists have been working on comparing protein expression in fertile and infertile men using different approaches. However, notwithstanding its relevance, until the present time, there are only a few studies that have focused on oxidative stress and its ability to alter the protein expression in the semen of patients with high ROS levels. In those studies, researchers have identified several proteins that are differentially expressed, which may play a role in the regulation and response of cells with high ROS levels.
This review includes a set of proteomic studies that assessed oxidative stress levels in their subjects as quantitative proof of oxidative stress status in seminal ejaculate. The studies selected in this review are three by our group from the Centre for Reproductive Medicine, Glickman Urological and Kidney Institute, Cleveland Clinic (
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
). Patient populations in these studies are characterized as males with asthenozoospermia, idiopathic oligoasthenoteratozoospermia (OAT) or primary or secondary infertility (a majority of which have varicocele). The donors used as controls were normozoospermic (according to
criteria) males with either proven or unproven fertility. Donors with proven fertility are men who have established a clinical pregnancy that resulted in a live birth.
Table 1Summary of proteomic studies in patients with oxidative stress.
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
TBCB, AACT, ALDR (possibly connecting idiopathic OAT and oxidative stress/inflammation)
11 fertile donors (normozoospermic)
Known cause of idiopathic OAT, positive culture for any organism
Individual samples representing pooled samples for each group
46 proteins indicative of infertility: 27 proteins common to all idiopathic OAT patients; 24 proteins over-expressed (⩾1.5-fold) in idiopathic OAT samples; 5 proteins were both common to all idiopathic OAT samples and over-expressed (⩾1.5-fold) in idiopathic OAT samples
Samples with leukocyte-spermia (Endtz positive, ⩾0.1 M/ml)
TBCB, AACT, ALDR up-regulated in idiopathic OAT samples
PIP (higher in seminal plasma of ROS+ versus ROS–)
20 healthy donors with unproven fertility (normozoospermic)
7 uniquely expressed proteins: 3 proteins expressed only in ROS– samples; 4 proteins expressed only in ROS+ samples
7 differentially expressed proteins: 4 over-expressed (>2-fold) in ROS+ samples; 3 underexpressed (<0.5-fold) in ROS– samples
AZGP1, CLU, KLK3, PIP and ACPP transcriptionally regulated by the androgen receptor
Proteomic analysis strategy included information on protein separation and sequencing, peptide identification, database search software and validation studies. P< 0.05 was used for protein identification. Motility grade a + b indicates rapid and slow progression; the cut off for normal progressive motility (grade a + b) is ⩾50% within 60 min of ejaculation (
The studies in this review pertain to men with clinical oxidative stress versus those with physiological ROS levels. To support our discussion in this review, references are occasionally made to studies performed on a similar group of subjects (i.e. infertile men with various semen parameters), although there was no assessment of oxidative stress levels in that study group (
), as the seminal constituents are mainly added to the spermatozoa at ejaculation. Semen samples used in studies from our team as well as from Herwig’s group were negative for leukocytospermia (negative Endtz test), to rule out oxidative stress originating from ROS produced by leukocytes (
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
), total antioxidant capacity and sperm DNA fragmentation were also measured as a further indication of the oxidative status of the sample. Oxidative stress is likely to result in an abundance of immature proteins in their precursor or preprotein forms, which entails a deficiency of mature functional proteins (
) that could result from high ROS levels. Protein oxidation may inhibit enzymatic and protein-binding activities as well as increase molecular weight, aggregation or proteolysis (
), which may affect the number of identified proteins.
The methodology employed in these studies played a part in the number of proteins that were identified (Table 1). Protein separation in these studies was performed by SDS-PAGE, except for the study by
, which used the 2D-DIGE method. Most of the studies ran samples in replicates. All the studies used the LC-MS/MS method (either the linear trap quadrupole Orbitrap (
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
The database used for protein identification searches was comparable and most studies employed more than one database search to reduce false positives. As is commonly used in quantitative proteomics, the relative protein abundance of the proteins expressed was measured by the intensity of the spectral count. As an overall, the cut-off values for over- and under-expressed proteins were 1.5 to 3-fold, respectively. The protein(s) identified were validated by Western immunoblotting in studies reported by
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
evaluated individual samples alongside the pooled samples. Based on the results of the study, the authors reported a lower number of unique proteins in an individual idiopathic OAT sample compared with the pooled idiopathic OAT group. All the studies had performed enrichment analysis in the GO categories of the genes expressed except for our first paper on sperm proteins (
). This was followed by pathway and network analysis and protein-protein interaction analysis to determine the processes involved when the seminal plasma or the spermatozoa is in a state of oxidative stress. In the next sections, a brief summary of reported findings of proteomics studies on seminal ejaculate with oxidative stress is presented.
Seminal plasma proteins and oxidative stress
Seminal plasma is the fluid in the semen that contains secretions from the testis, epididymis, prostate, seminal vesicles and Cowper’s glands. Seminal plasma plays an important role in providing nourishment and protection to spermatozoa and acts as a buffer as well as a medium for sperm motility. Human semen is composed of lipids, ions (such as citrate, calcium, magnesium, potassium, sodium, zinc and chloride), fructose, ascorbic acid, proteins (such as semenogelin and fibronectin), albumin and globulins, amino acids and amines, cytokines and hormones. It also contains numerous enzymatic (gluthathione peroxidase, superoxide dismutase, catalase) and non-enzymatic antioxidants (vitamins C and E, zinc) that protect spermatozoa from oxidative stress (
). The study of these proteins can provide a basis for the identification of biomarkers for the assessment of male infertility disorders. Table 2, Table 3, Table 4, Table 5 give an overview of seminal fluid proteins that have been associated with elevated levels of oxidative stress.
Table 2Seminal plasma proteins over-expressed in semen samples with oxidative stress compared to semen samples without oxidative stress.
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
Seminal plasma protein expression was analysed in patients with primary and secondary infertility who presented high levels of oxidative stress (ROS+; ⩾20 RLU/s/10 million spermatozoa) versus those with physiological levels of oxidative stress (ROS−; <20 RLU/s/10 million spermatozoa) (
). Using LC-MS/MS, the study resulted in the identification of 14 proteins: seven commonly present in both ROS+ and ROS− samples, three exclusively present in ROS− samples (fibronectin I isoform 3 preprotein (FN1), macrophage migration inhibitory factor-1 peptide (MIF) and galectin 3 binding protein (G3BP)) and four expressed solely in ROS+ samples (cystatin S precursor, albumin preprotein, lactotransferrin precursor 1 peptide and prostate-specific antigen isoform 4 preprotein) (
Semenogelin 2 precursor was found to be 2-fold up-regulated in the ROS+ group, whereas semenogelin 1 isoform a was found to be down-regulated in the ROS+ group (
). Both semenogelin 1 (50 kDa) and semenogelin 2 (63 kDa, present in lesser abundance) are secreted from the seminal vesicle and represent the most abundant components (about 20–40%) of the human semen coagulum (
). Semenogelin are responsible for the coagulation of the gel matrix that encloses the spermatozoa and it helps prevent the capacitation process (the initial part of sperm activation) by inhibiting the formation of ROS (
). A small amount of ROS, generated by the spermatozoa itself, is required to facilitate the initiation of the capacitation process and the subsequent hyperactivated motility of the spermatozoa. However, a premature onset of capacitation leads to poor fertility outcomes (
Semenogelin, the main protein of semen coagulum, inhibits human sperm capacitation by interfering with the superoxide anion generated during this process.
, the quantity of semenogelin in the seminal plasma of fertile donors (sperm motility grades A + B i.e. rapid + slow progression of 48–67%) versus asthenozoospermic patients (sperm motility 6–11%) with high levels of oxidative stress was not significantly different. The results suggested that seminal vesicle proteins such as semenogelin were less likely to be associated with the regulation of sperm motility in asthenozoospermic patients compared with proteins of the epididymis and prostate, such as DJ-1 (
studied the association of SPMI and spermatozoa in asthenozoospermic infertile patients (sperm motility <50%) by labelling washed sperm cells with anti-SPMI antibody, followed by flow cytometry analysis and Western immunoblotting. Although spermatozoa from both asthenozoospermic patients and normal subjects showed similar labelling patterns, both labelling intensity and the number of labelled spermatozoa were higher in patient samples compared with normal subjects. Further, a marked negative correlation was found between labelled sperm cells and gamete motility and viability. Based on these findings, Terai’s group postulated that the presence of membrane surface-bound SPMI on the sperm head and tail was the basis for poor motility in asthenozoospermic patients rather than the presence of semenogelin in their seminal plasma (
Prostate-specific antigen (PSA, or human kallikrein 3 (hK3)) is a serine protease that is synthesized in prostate tissue and involved in semenogelin breakdown, causing liquefaction of the semen coagulum (
). PSA isoforms were found to be differentially expressed between patients with high ROS levels and donors with physiological ROS levels: PSA isoform 1 preprotein was down-regulated in ROS+ patients, while PSA isoform 4 preprotein was unique to ROS+ samples (
). The identification of these precursor forms of incompletely modified proteins can be explained by faulty post-translational modifications, which result in a decrease in the presence of the mature form of the protein and thereby the lack of its function (
Comparative analysis of interindividual variations in the seminal plasma proteome of fertile men with identification of potential markers for azoospermia in infertile patients.
). PIP has attracted much attention when it comes to seminal fluid studies. This protein plays various roles, such as fibronectin degradation during semen liquefaction, immunoregulation, antimicrobial activity, apoptosis and tumour progression (
). The expression of PIP appears to vary between different studies. In a study on men with various semen parameters, we found that PIP was commonly expressed in groups that differed in sperm concentration and morphology parameters (
Comparative analysis of interindividual variations in the seminal plasma proteome of fertile men with identification of potential markers for azoospermia in infertile patients.
). These findings support the use of PIP as a potential biomarker for azoospermia, although additional studies using a larger number of samples are warranted (
Comparative analysis of interindividual variations in the seminal plasma proteome of fertile men with identification of potential markers for azoospermia in infertile patients.
In the human testis, DJ-1 is found in spermatids, spermatogonia, spermatocytes, Sertoli cells and Leydig cells. In Sertoli and Leydig cells, DJ-1 colocalizes with the androgen receptor, suggesting a role for this protein in the regulation of spermatogenesis via the receptor (
). We found that DJ-1 was over-expressed in patients (whose oxidative stress were not assessed) with: (i) normal sperm count but with abnormal morphology; and (ii) oligozoospermia but with normal sperm morphology (
). Therefore, when under stressful conditions, it seems that DJ-1 expression increases. As stress concentrations increase, ROS concentrations increase and antioxidant concentrations fall, leading to a decline in DJ-1 expression (
). GO analysis showed that proteins in patients with normal sperm count and abnormal morphology were involved in pathways for scavenging free radicals (
). These findings further substantiate the functions attributed to DJ-1.
Clusterin (also referred to as apolipoprotein or sulphated glycoprotein 2; 70–80 kDa) is a heterodimeric glycoprotein that is produced by the Sertoli cells (
) and secreted by the epididymis and prostate. It has a widespread distribution in human tissues and it is involved in a number of biological functions including cell-to-cell interaction, apoptosis, sperm maturation and degradation of extracellular matrix (
). Moreover, clusterin protects against harmful ROS reactions, protein precipitation and aggregation of defective spermatozoa, as well as controlling complement-induced cell lysis (
). Clusterin has been proposed as a sensitive cellular biosensor of oxidative stress, since it possesses a chaperone activity that functions to protect from the harmful effects of free radicals and oxidative stress (
Regulation of clusterin/apolipoprotein J, a functional homologue to the small heat shock proteins, by oxidative stress in ageing and age-related diseases.
reported an increased clusterin precursor expression in asthenozoospermic patients with 3.3-fold higher ROS levels when compared with fertile men with physiological ROS levels, although the clusterin concentration in the asthenozoospermic ejaculate was reduced. A GO analysis performed in our study demonstrated the transcriptional regulation of the clusterin gene by the androgen receptor as well as activation of prostate induction by the androgen receptor signalling pathway (
Prostatic acid phosphatase (PAP, or prostatic-specific acid phosphatase (PSAP)) is an enzyme produced in the prostate gland that has been extensively studied as a biomarker and negative growth regulator for prostate cancer (
). Serum concentrations of PAP are especially increased in men with metastasized prostate cancer and it served as an important tumour marker and diagnostic indicator of prostate cancer, prior to the use of PSA (
reported higher concentrations of PAP (using colourimetric assay) in seminal plasma of azoospermic patients compared with those in normozoospermic, asthenozoospermic and oligozoospermic patients, although the differences between the groups were not statistically significant. In one of our other proteomic studies (oxidative stress levels not measured), PAP concentrations were down-regulated in patients with normozoospermia and abnormal sperm morphology, as well as patients with oligozoospermic semen and normal sperm morphology (
). In our subsequent study (with oxidative stress levels measured), acid phosphatase and the prostate-specific antigen isoform I preprotein (KLK3) were both present in semen ejaculates with both increased and normal ROS levels (
). However, acid phosphatase concentration was up-regulated in seminal plasma with increased ROS levels while isoform I preprotein was down-regulated in seminal plasma with physiological ROS levels (
). The differential expression of these biomarkers of prostate cancer in infertile patients with oxidative stress may help elucidate the aetiology of prostate cancer (
). Similar to clusterin, PAP is controlled by the androgen receptor and is involved in prostate induction through the androgen receptor signalling pathway (
Identifying differentially expressed proteins between abnormal and normal semen samples is only the first step in understanding the mechanisms of male infertility at the molecular level. Protein structure, localization and involvement in biological pathways all need to be analysed to determine the dynamic cellular processes that occur. We studied the function and distribution of proteins that are commonly or differentially expressed between ROS+ and ROS− groups and found that most of the common proteins are present in the extracellular compartment (
). Proteins unique to seminal plasma with increased ROS, such as cystatin S precursor and albumin preprotein, are restricted to the extracellular matrix. Polypeptides unique to seminal ejaculates with normal ROS concentrations, such as fibronectin 1, are considered to aid in the process of endocytosis due to their presence in the vesicular lumen region. It could be proposed that the absence of certain proteins in the ROS+ group make these individuals more prone to infection and inflammatory responses (
Most of the proteins found were involved in stress and regulatory pathways. These proteins were also found to play an important role in catalytic activities. Analysis of biochemical processes revealed the involvement of proteins common to ROS+ and ROS− groups in major pathways such as regulation, response to stress, interaction with neighbouring cells and organisms. On the other hand, proteins solely expressed in oxidative stress were assumed to be involved in sperm interaction, apoptosis, necrosis and cell death because of their role in cell cycling, ageing, morphogenesis and motility. Finally, proteins restricted to semen with physiological ROS concentrations were involved in enzymic reactions such as antioxidant activities, DNA binding, serine hydrolase and serine endopeptidase activity (
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
determined the protein profile of seminal fluid in idiopathic OAT patients with high levels of oxidative stress compared with normal donors. Their proteomics analysis identified 46 proteins related to infertility. GO analysis determined that the protein processes of the 27 proteins common in all idiopathic OAT patients are focused on cellular organization and modification. Our results in samples with elevated levels of oxidative stress concur with their findings (
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
, the 24 proteins highly expressed in ROS+ idiopathic OAT patients were involved in biological processes that centre on metabolism, inflammation, immunity and stress response. Pathway analysis of the proteins identified uniquely in idiopathic OAT had an enrichment of the glycerolipid metabolism pathway only (
Proteomic analysis of seminal plasma from infertile patients with oligoasthenoteratozoospermia due to oxidative stress and comparison with fertile volunteers.
Seminal proteins are not the only proteins that are of interest as potential biomarkers of oxidative stress. Proteins expressed by the spermatozoa themselves are equally as important, as they help scientists identify their roles in the spermatozoon’s various metabolic processes, capacitation reactions and oocyte fertilization. Alterations in testicular ROS can disrupt the internal milieu of the cell, resulting in sperm dysfunction and impaired viability, motility and fertilization capacity (
). Few studies have been reported regarding protein expression in spermatozoa and how they correlate with oxidative stress. Some of the important sperm proteins in relation to oxidative stress that have been isolated from infertile patients are summarized in Table 6, Table 7, Table 8.
Table 6Sperm proteins over-expressed in semen samples with oxidative stress compared to semen samples without oxidative stress.
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