Proteomic analysis of human spermatozoa proteins with oxidative stress

Male-factor infertility contributes to 50% of infertile couples worldwide, and while the causes are multifactorial, current research has focused on oxidative stress. Specifically, oxidative stress may lead to alterations in protein expression levels in spermatozoa, causing molecular and genetic defects. Research has already shown that oxidative stress is associated with a variety of male infertility diagnosis such as varicocele, idiopathic infertility, spinal cord injury, prostatitis and leukocytospermia [1‐8]. Varicocele is implicated in 35% of men with primary infertility and in 80% of infertile men with secondary infertility, and oxidative stress is one of the many causes of varicocele related infertility [9‐11]. Oxidative stress in men with varicocele is also associated both with motility and grade of varicocele.

Understanding the protein profile of spermatozoa is essential for identifying the protein alterations that occur as a result of increased production of ROS and for better diagnosis of male infertility. The development of 2-dimensional-polyacrylamide gel electrophoresis (2-DE) coupled with Western blot have aided in the identification of proteins, in relation to sperm composition and function [12, 13]. Furthermore advancements in mass spectrometry analyses of spermatozoa have broadened our knowledge and aided in the identification of pivotal sperm proteins [14, 15]. Numerous studies have reported the presence of proteins in studies involving human spermatozoa through the implementation of proteomic techniques such as 2-DE, liquid chromatography tandem mass spectrometry (LC-MS/MS), matrix assisted laser desorption and ionization- time of flight tandem mass spectrometry (MALDI-TOF-MS/MS), and 2-dimensional difference in gel electrophoresis (DIGE) [16‐21].

In this comparative study, we employed LC- MS/ MS and functional bioinformatic analyses to identify the relative abundance of proteins in spermatozoa obtained from seminal ejaculates of infertile men who were referred to the andrology clinical laboratory for advanced semen analysis (including parameters of oxidative stress such as levels of ROS in spermatozoa, concentration of antioxidant in the seminal plasma and the extent of DNA damage). We also included healthy men with high and low levels of ROS in their seminal ejaculates compared to those with low levels of ROS. Oxidative stress plays a major role in male infertility, but the crucial proteins that are differentially expressed in ROS are not known. Understanding the role of these proteins is therefore important in our understanding of the post-genomic events. It will help identify potential proteins that could serve as biomarkers to better manage and treat oxidative stress, and consequently, male infertility.

The study was approved by the Institutional Review Board of Cleveland Clinic and was conducted in accordance with national and international guidelines. A total of 52 subjects were enrolled in the study. Twenty healthy male volunteers of unproven fertility (normal semen analysis results but had not established a pregnancy) were selected on the basis of normal semen analysis. Thirty two infertile men attending our infertility clinic were referred to the andrology lab for advanced semen parameters of oxidative stress. The breakdown of the infertile subjects was as follows: primary infertility: n = 25 (25/ 32; 78.1%); Secondary infertility: n = 7 (7/25; 28%). Of the 25 men with primary infertility, 64% (16/ 25) had <2 years and 36% (9/ 25) >2 years. Clinical varicocele (grade 1-2) was present in 10 (62.5%) of the 16 men who presented with <2 years of primary infertility and in 5 (55.6%) of the 9 men with >2 years of primary infertility. In the 7 patients with secondary infertility, 6 (85.7%) had a clinical varicocele (grade 1-2).

Semen samples were collected by masturbation after 2-3 days of sexual abstinence and analyzed according to WHO 2010 criteria [22]. A complete semen analysis was performed on both donors and patients and included both macroscopic (volume, pH, color, viscosity, liquefaction time, split or complete ejaculate) and microscopic parameters (concentration, motility, morphology, round cells and peroxidase or Endtz test). All subjects were also examined for levels of reactive oxygen species (ROS) in the seminal ejaculate. The remainder of the seminal ejaculate was frozen without any cryoprotectant and stored at -55°C for proteomic analysis.

Oxidative stress plays a key role in the etiology of male infertility, possibly by altering protein expression levels in spermatozoa, causing molecular and genetic defects. Therefore, understanding the function of each protein involved and the post-translational modifications that occur during sperm-maturation is important and may lead to a useful biomarker for male infertility. In earlier studies, we demonstrated that ROS are independent of the semen parameters [6]. Therefore, in the current study, we compared the sperm proteome from a group of men with high levels of ROS (ROS+) with that from men with low or physiological levels of ROS (ROS-). The ROS grouping was done irrespective of the patient’s clinical diagnoses and semen parameters: the only criteria was the presence or absence of ROS.

We have identified 74 proteins, 15 of which had a >2-fold difference in their expression levels in the ROS+ group when compared to the ROS- group. Based on the spectral counts of the 74 proteins, we identified 47 were overexpressed and 27 that were underexpressed in the ROS+ group compared to the ROS- group. Of the 74 proteins, 17 were determined to be either in their precursor or pre-protein form. The presence of these incompletely processed proteins is indicative of post-translational processing problems as reported by some investigators [31, 32]. Accumulation of precursor forms of protein may result in deregulation of the downstream functions of the mature protein [31]. Oxidative stress may result in the accumulation of precursors or preproteins. The cellular distribution of all the proteins as ascertained by GO analysis (Figure 1A) suggested that the majority of the proteins (68 proteins) were intracellular or organellar (62 proteins) in origin, whereas a few were localized in the extracellular (13 proteins) region. The presence of extracellular proteins may be attributed to the presence of round cells in the ejaculate from both the donors and patients. We did not remove these from subsequent processing for proteomic analysis.

Similar comparative studies have been performed previously by other groups in an effort to understand the sperm proteome [31, 33, 34]. Most notable amongst these are two studies where the expression profile of proteins was compared in asthenozoospermic and normozoospermic donors [31, 33]. In these studies, 10 proteins and 17 spots were identified using 2-DE suggesting their role in motility-related male infertility. Xu et al conducted a comparative study between infertile patients and normozoospermic group and identified 24 differentially expressed proteins that could potentially serve as diagnostic markers in identifying the underlying pathology of male infertility [35]. Our findings in this study are novel, as we have compared and identified the proteins that are differentially expressed in spermatozoa obtained from seminal ejaculates with high levels of ROS (ROS+) to those normal levels of ROS (ROS-).

On examining the association of the differentially expressed proteins with the biological processes (Figure 1B), we concluded that the majority of the proteins were involved in cellular, metabolic, and regulatory processes. A smaller distribution of proteins (3-5%) was also found in biological processes such as de novo post translational (8 proteins) and protein folding (14 proteins), and these were identified as ‘enriched processes’ for our list of proteins, by the GO Term Finder.

Even though the spermatozoa become transcriptionally inactive after spermatogenesis, we found the presence of the translational proteins and this may be indicative of the presence of some leftovers from inefficient spermatogenesis. They may also have a role in the normal physiology of the spermatozoa or affect fertilization and embryo development. Further validation through the pathway and process network analysis using IPA and Metacore™, suggested that the topmost function of the differentially expressed proteins was carbohydrate metabolism and included pathways of gluconeogenesis, glycolysis, fermentation of pyruvate to lactate and sucrose degradation. There is a great controversy pertaining to the major pathway of energy metabolism during sperm motility [36‐39]. In this context, our findings showed that while oxidative phosphorylation is important for sperm function, the major pathway for energy production for sperm motility was via the glycolytic pathway. To support this view, various respiratory enzymes involved in the glycolytic pathway including phosphoglycerate kinase 2 (PGK2), glyceraldehyde-3phosphate dehydrogenase (GAPDH), fructose- biphosphate aldolase A (ALDOA), glyceraldehyde-3 phosphate dehydrogenase-S (GAPDHS), triosephosphate isomerase 1 isoform (TPI1) were identified in our study. Furthermore, GAPDH-S protein was overexpressed in the ROS+ sample. Oxidation of GAPDH-S as a result of H₂O₂ generation has been reported to decrease sperm motility and inhibit GAPDH-S activity [40].

Various isoforms of histone proteins were also identified and these included histone cluster 1, H2aa (HIST1H2AA), histone cluster1 H2ae (HISTH1AE) and histone cluster1 and H2ba (HIST1H2BA). Histones are a group of proteins that are replaced by the protamines during sperm maturation in the epididymal region [41]. Their presence in the ejaculated spermatozoa is indicative of improper packaging of sperm chromatin and subsequent DNA damage. These findings have been attributed to oxidative stress [5, 41].

We found 17 precursor proteins to be dysregulated; of these, 10 were over-expressed and 5 were underexpressed with a 2-fold difference between the ROS+ and ROS- groups. The differentially expressed proteins were mainly intracellular, cytoplasmic or organellar in distribution. This is contrary to the findings of Xu et al, who reported a higher distribution of differentially expressed proteins in the extracellular region in the infertile groups with normal semen parameters [35]. This difference may be attributed to the import/export of epididymal and seminal vesicle proteins. Furthermore, the distribution of overexpressed and underexpressed proteins in the ROS+ group revealed their involvement in various processes. We noted that many proteins showing significant changes in expression between the ROS+ and ROS- groups had roles in sexual reproduction, metabolic processes and stress response (Figures 2 and 3).

Our data suggests that semenogelin II precursor (SEMG2), which is involved in sexual reproduction, was underexpressed in the ROS+ group. Overexpression of semenogelin I and II precursor has been reported in previous comparative studies [31, 35]. Xu et al reported an increase in both isoforms of semenogelin in the spermatozoa of infertile patients compared to the normozoospermic men [35]. Semenogelin -1 and semenogelin -2 have been reported in men with varicocele only as well as in men with varicocele with a history of moderate as well as heavy smoking. Semenogelin is known to cause a decrease in sperm motility, but it was within the normal range in the ROS+ samples. Semenogelin (SEMG) is the major protein of seminal fluid proteins (20-40%) and is secreted from seminal vesicles, epididymis, and prostate [42, 43]. It is an androgen dependent protein that exists in two forms I (52 kDa) and II (71-76 kDa). SEMG helps in the formation of the semen coagulum that is degraded later by prostate-specific antigen. SEMG counteracts oxidative stress by reducing the generation of free radicals through several mechanisms: slow sperm motility of the entrapped sperm, reduced energy consumption, free radical generation and inhibition of superoxide radical generation through direct interference with sperm NADH oxidase [44].

A great number of proteins are involved in the energy production required for the spermatozoon tail movement. We identified several proteins that are involved in energy producing processes such as the carbohydrate metabolic pathways of glycolysis and gluconeogenesis. Enolase I (ENO1) was found to be significantly underexpressed while respiratory enzyme mitochondrial malate dehydrogenase precursor (MDH2) was found to be overexpressed. MDH2 is an ROS producing enzyme localized in the sperm mitochondria [45]. It catalyzes the last step of the Krebs cycle when malate is converted into oxaloacetate using NAD+/NADH as intermediates. Several of the chaperone proteins such as heat shock protein 90 kDa beta, member 1 (HSP90B1) and heat shock 70 kDa protein 5 (HSPA5) were also seen to be overexpressed in response to stress. The association of these proteins with various processes related to cellular stressors such as heat, glucose deprivation, free radical attack, and infection indicates that ROS induces a stress response in the spermatozoa [46‐48].

The GO annotations further revealed the involvement of overexpressed proteins in cellular amino acid metabolic processes. Examples of proteins that were found to be overexpressed included transglutaminases (TGM4) and glutamine synthetases. Transglutaminases are a family of calcium-dependent enzymes that catalyze the post-translational modification of the selected glutamine residues on proteins by cross-linking with the peptide-bound lysine residues or incorporating polyamines [49, 50]. Transglutaminase that is secreted from the prostate is known as TGM4 prostate. A recent study showed increased levels of transglutaminase in response to oxidative stress in the prostate [51]. Excessive production of ROS has been reported to induce cell senescence, oxidative protein modifications, and cell membrane damage, increase intracellular calcium and activate certain cytokines such as TGFb [51]. Oxidative stress impacts the aggregation, increased molecular weight, unfolding, denaturation and proteolysis of cellular proteins. These effects may result in the initiation of cellular hyperamonemia, which probably triggers the expression of glutamate synthetase to redirect the production of the released ammonium into glutamate [52].

Glutathione peroxidase 4 isoform A precursor (GPX4) and peroxiredoxin 6 (PRDX6) function to reduce oxidative stress. The former was overexpressed and the latter was underexpressed in the ROS+ group. GPX4 is a well-known antioxidant enzyme and its increased expression in sperm in the ROS+ samples is reflective of its influence on genes synthesizing this enzyme. PRDX6 also belongs to a family of antioxidant enzyme known as peroxiredoxins. They detoxify H₂O₂ similar to catalase or glutathione peroxidases (GPXs) [53, 54]. Peroxiredoxin 6 is localized in the sperm head (postacrosomal region and equatorial segment) and sperm tail [55]. Reduced levels of peroxiredoxins (PRDX1 and PRDX6) have been reported in infertile men with oxidative stress. Reduced levels of PRDX6 have also been reported in the seminal plasma from men with varicocele. Differential amounts of peroxiredoxin 6 have been reported (41% in men with varicocele and 65% in men with idiopathic infertility) [56]. Human spermatozoa are reported to have high sensitivity to ROS and may be a result of reduced recycling of peroxiredoxins after exposure to oxidative stress [55].

Optimal levels of expression and function of androgens are central to the process of spermatogenesis. To understand the dependency of the differentially expressed proteins in the ROS+ groups, a network was derived to identify proteins regulated by androgen receptors. Spermatogenesis is dependent on androgen action and androgens act by stimulating the receptors present on the Sertoli cells [57, 58]. Transcriptional network analysis (Figure 4) showed that 21 proteins were regulated by the androgen receptor. In our study, several proteins appeared to be transcriptionally activated by the androgen receptor but the effect of the androgen receptor on several other proteins is still unknown (Figure 5). Stanton et al reported that any loss to the androgen stimulus during meiosis induces changes in the proteins that are associated with various molecular processes such as apoptosis, cell signaling, oxidative stress and RNA processing [58]. These investigators further validated this observation by immunostaining for oxidized DNA adducts – they showed that spermatocytes that undergo oxidative stress induced DNA damage during androgen suppression. The completion of meiosis requires androgen action via the Sertoli cell [59]. Low levels of testicular androgens support meiosis while a high level is required for spermiogenesis.

Testicular testosterone suppression has been shown to adversely influence antioxidant activity [60]. The present study utilizing Network analysis revealed heat shock protein beta-1 (HSPB1) and clathrin heavy chain 1 (CLTC), which are known to bind and activate androgen receptor were actually underexpressed in the ROS+ group. We also observed that GAPDH levels were overexpressed, and that it was bound with the androgen receptor to impart a stimulatory effect in the ROS+ sample. The mechanism by which the androgen stimulus is transduced by the Sertoli cell to the germ cells is still unclear. In addition, it is well known that in majority of the infertility cases attributed to a male factor, spermatogenic arrest is a common feature. The process of spermatogenesis is dependent on the cAMP responsive element modulator (CREM) signaling pathway in the testis which operates in coordination with the CREM modulators (repressors and activators) [61]. CREM is essential for male fertility and the absence of CREM-dependent transcription in post-meiotic germ cells is known to result in an arrest of spermatid differentiation and apoptosis [62, 63]. CREM repressors are expressed during pre-meiotic germ cells while CREM activators are expressed in the post-meiotic germ cells. In order to understand the impact of oxidative stress on impaired spermatogenesis, we examined the CREM signaling in testis. Our findings revealed that three of the identified proteins that were differentially expressed in the ROS+ group were modulated by CREM activators. This included ODF1 and ACE1 proteins that were underexpressed as well LDHC, which was overexpressed. These findings suggest that oxidative stress may affect the switching of genes at the spermatogenic level. Comparative studies on CREM signaling in testis have been conducted on normo- and oligozoospermic men and men with round spermatids vs. round spermatid maturation arrest [64]. These findings further support the fact that a lack of a switch in the expression of CREM gene isoforms may have an adverse impact on spermatogenesis in humans.

To summarize, in this study, we have identified various proteins that are implicated in a multitude of functions associated with response and management of oxidative stress. The increased expression of histone cluster 1H2ba (HIST1H2BA), mitochondrial malate dehydrogenase precursor (MDH2), transglutaminase 4 (TGM4), glutathione peroxidase 4 isoform A precursor (GPX4), glutamine synthetase (GLUL), heat shock proteins (HSP90B1 and HSPA5) in the ROS+ sample suggests that these proteins can serve as potential biomarkers of oxidative stress. Thus alterations reported in our study in men with and without oxidative stress may help explain pathways leading to the altered semen phenotype especially in men exhibiting oxidative stress both in infertile men as well as in infertile men with varicocele. However, the interaction between the proteins and the androgen receptors is still unclear and warrants further investigation. It is important to accurately assess the protein levels and verify that the differentially expressed proteins are indeed involved in the process of oxidative stress and are possibly modified as a result of oxidative stress. In future studies, as a follow-up to the present study, quantitative estimation of the differentially (overexpressed and underexpressed) expressed proteins, utilizing western blotting and antibodies is warranted. The findings of our study provide the groundwork for further testing including the proposition that these newly identified sperm proteins play crucial roles in oxidative stress and the pathophysiology of male infertility.