Functional proteomic analysis of seminal plasma proteins in men with various semen parameters

Infertility is a major problem in 15% of couples worldwide. Male factors may play a role in half of these cases [1]. Most causes of male infertility are idiopathic. Semen analysis remains the cornerstone in the evaluation of male infertility. However, the data generated from this routine testing do not provide any insight into the underlying problems associated with developing spermatozoa. Sperm morphology plays an important role in conception, and both fertilization and pregnancy rates are affected when morphologically normal sperms are below 5%. It is also a reflection of poor testicular physiology and is an important factor in male infertility [2‐4]. However, a significant overlap of semen parameters such as sperm count, motility and morphology have been documented [5]. Idiopathic and unexplained infertility cannot be diagnosed by routine sperm function tests [6]. Similarly, oligozoospermic men may have other underlying pathologies that may contribute to infertility. Evaluation solely based on semen analysis is insufficient to determine the fertility status of the male partner.

Spermatogenesis is a complex process that involves development of the undifferentiated germ cells into a highly specialized spermatozoon capable of fertilizing an oocyte [7]. Fertilization requires physical proximity of the spermatozoa and the oocytes. Seminal plasma composed of secretions from the testis, epididymis and male accessory glands [8] provides a favorable environment and serves as a vehicle for the spermatozoa as it travels to meet the oocyte.

Seminal plasma contains unique proteins necessary for sperm function and survival [9, 10]. Seminal plasma proteins play a variety of roles—they help protect the sperm by binding to the sperm surface during ejaculation and play a key role in capacitation, acrosome reaction, and sperm-egg fusion [11, 12]. They can also modulate immune response in male and female reproductive tracts, ensuring that the most competent spermatozoa meet the oocyte during fertilization [13]. Thus, seminal plasma proteins can serve as important biomarkers for male infertility [14].

Conventional 1-Dimensional gel electrophoresis studies have provided information in relation to sperm proteins and their function in normal and abnormal spermatozoa [15, 16]. Advancements in mass- spectrometry and proteomic-based techniques have made it possible to analyze the complex protein mixtures found in tissues and body fluids. Several attempts have been made to identify these proteins using high-throughput techniques such as matrix assisted laser desorption ionization – time of flight (MALDI-TOF) mass spectrometry (MS) and liquid chromatography – tandem mass spectrometry (LC-MS/MS) and linear ion trap (LTQ-Orbitrap) mass spectrometry [17‐21].

Alterations at the molecular level in spermatozoa and the seminal plasma may contribute to male infertility. However, even after accounting for all the advances in proteomics, there has been a great lack of detailed data in the area of comparative analysis of seminal plasma proteins associated with male infertility.

The objective of the present study was 1) to compare the differential expression of proteins in the seminal plasma from subjects with normal or abnormal sperm concentration and sperm morphology utilizing proteomic tools such as LC-MS/MS and 2) utilize the functional bioinformatics analysis to identify the cellular origin and the differentially affected processes and/or pathways of these proteins to gain insights into the mechanistic roles played by these proteins in effecting the observed phenotypes. These analyses could possibly identify potential biomarkers for male infertility.

After obtaining Institutional Review Board approval, written consent was obtained from all subjects. Semen samples were obtained from 64 subjects who were healthy male volunteers of unproven fertility (n = 21) and men presenting to our infertility clinic for evaluation (n = 43). Semen samples were collected by masturbation after 2–3 days of sexual abstinence. Samples with leukocytospermia--a high concentration of white blood cells (>1 × 10⁶ WBC/mL)--were examined for the presence of granulocytes by the peroxidase or the Endtz test. The patients with a positive Endtz test were excluded from the study. Semen analysis was conducted according to WHO criteria as described below [22].

Seminal plasma is a mixture of secretions of several male accessory glands, including prostate, seminal vesicles, epididymis and Cowper’s gland. Prostate gland is a major contributor to seminal plasma. It is a very rich source of protein with concentration ranges from 35 to 55 g/l [9, 10]. It provides a safe environment for spermatozoa to carry out their physiological functions. Understanding the protein profile of human seminal plasma is important because it has a profound impact on sperm physiology and thus may affect sperm functioning [31].

In this novel study, we identified 35 proteins based on SEQUEST scoring in the seminal plasma of men with varying semen parameters and categorized them into common, differentially expressed, and low abundant proteins. The large variation in the number of proteins identified by any given technique depends mainly on the sample preparation and mass spectrometry technology available [32‐36]. Recently, the LTQ-Orbitrap mass spectrometer has become the cutting-edge instrument for LC/MDS/MS based approaches to characterize the seminal proteome. In our study, we used in-solution digestion of proteins with the online LC-MS system. Seminal plasma from different subsets was pooled to form 4 distinct study groups. There are numerous studies in the proteomic literature that refer to the benefits of pooling samples where it may not be feasible to analyze individual samples due to limitations of the sample or the study design [8, 17, 37, 38].

Of the 11 proteins found in all the samples, 9 were associated with sperm function, and the common proteins comprised the majority of the secretions of the prostate gland, the seminal vesicles and epididymis. Some of the proteins or their isoforms detected in the seminal plasma were zinc alpha-2-glycoprotein 1, clusterin, lactotransferrin, prostate specific antigen. These were similar to those reported by Utleg et al. [39].

Prostate specific antigen is a serine protease that cleaves semenogelin by hydrolysis and thus liquefies the semen coagulum and facilitates sperm motility and capacitation [40, 41]. Our study showed that prostate specific antigen isoform I preprotein was one of the common proteins, thus indicating its importance in all the 4 groups. Prolactin-induced protein (PIP) and Sg II are important common proteins that have a profound impact on sperm physiology. PIP has specificity to fibronectin and constitutes about 1% of seminal coagulum [42]. It may play a vital role in fibronectin breakdown during liquefaction. Viscous samples have been reported to show reduced amounts of PIP and PIP precursor, which may also be a contributory factor towards incomplete liquefaction [43]. Both Sg I and II are the major proteins of the coagulum. They represent 20-40% of the seminal plasma proteins. Studies have shown increased Sg concentrations in asthenozoospermic men [44, 45]. Our study showed that both prolactin and semenogelin II were in all samples but they were not differentially expressed, indicating that men with low sperm count or abnormal morphology may not be affected by these proteins—a conclusion also made by Milardi et al. [18].

Epididymal secretory protein E I precursor, albumin preprotein, lactotransferrin, extracellular matrix protein E1 precursor, prosaposin isoform a preprotein, cathepsin D preprotein were some of the commonly expressed proteins that were not differentially expressed in any of the groups, suggesting that these proteins may not play a significant role in sperm concentration or morphology.

The highest percentage of the common proteins was seen in the extracellular region (Figure 5). Human seminal plasma proteins can bind to sperm surface proteins in the ejaculate and form a protective layer around the spermatozoon [12] as well as a sperm reservoir in the oviduct [46]. It is likely that the extracellular origin of most of the common proteins may play a key role in the binding activity of the proteins. The absence of common proteins in the ribosomal and endosomal region (Figure 5) suggests a relatively low involvement in protein metabolism, as also seen by the low distribution of common proteins in the ‘protein metabolic process’ category, (Figure 6). Zinc alpha-2 glycoprotein and clusterin play a role in signal transduction while lactotransferrin is a transport and structural protein [39]. Prostate specific antigen has been shown to have enzymatic activity. Our results show a high distribution of signal transducing protein among the common proteins, suggesting the importance of clusterin, isoform 1 and zinc alpha-2 glycoprotein 1 (Figure 6). This was also confirmed from the pathway and network analysis, which also highlighted the role these proteins play in molecular transport, cell death and survival and lipid metabolism.

A clear overlap was observed for some of the differentially expressed proteins. These included the prostate specific antigen isoform 1 preprotein, zinc alpha 2 glycoprotein 1, and clusterin isoform 1. While semenogelin II precursor was seen in common proteins, the semenogelin I isoform b preprotein was found to be upregulated in the OA group. This suggests that it may contribute to the low motility seen in this group compared to other groups.

Clusterin isoform was downregulated in the ON group. This is an interesting finding, given that clusterin isoform 1 has been shown to be downregulated in prostate cancer. The proteome of the seminal fluid is largely attributed to secretions from the prostate gland, and approximately 10% is contributed by the testis and the epididymis [8].

Prostate gland is a major contributor to seminal plasma. Furthermore, one of the proteins - prostatic acid phosphatase (PAP) was significantly increased in azoospermic men compared to oligozoospermic men and asthenozoospermic men [10]. In our study, PAP levels were down regulated in the NA and ON groups. PAP levels have also been reported to correlate with sperm concentration [47‐49]. Patients with severe oligozoospermia (<1 × 10⁶) were also shown to have increased levels of seminal PAP [50]. PAP is produced in the prostate gland and is present in the seminal fluid at a concentration of 1 mg/mL. It is an important tumor marker and acts as a negative growth regulator in prostate cancer [51].

A proteomic analysis of seminal fluid, rather than blood, has been proposed as as a better starting point to identify changes that may serve as specific and sensitive markers of prostate dysfunction [17]. These authors identified more than 100 proteins such as PSA, semenogelin I and II, clusterin etc. which have been shown to affect sperm quality.

The increased expression of semenogelin I in the OA group suggests that the accessory gland secretions have a profound impact on oligozoospermic men with abnormal morphology. Wang et al. reported semenogelin I and mucin were not differentially expressed and therefore had no effect in asthenozoospermic men [21]. Our study also included the differentially expressed proteins, especially DJ-1 secreted from the testis, epididymis and prostate [39, 52]. DJ-1 has a high level of expression in the testis. We found DJ-1 to be overexpressed in the NA and ON groups. We also documented that orosomucoid 1 precursor was downregulated in the NA group whereas expression of orosomucoid 2 was comparable in all groups, though it was present in low abundance. However, Wang et al. reported the overexpression of orosomucoid 1 and orosomucoid 2 in asthenozoospermic patients [21]. These proteins have also been found in high abundance in post vasectomy patients [8].

B2MG was found to be under-expressed in OA while over-expressed in the ON samples. B2MG is present in all nucleated cells. It is one of the two polypeptide chains of the major histocompatiblity complex (MHC) class I molecule. In humans B2MG is coded by the B2M gene. It is a marker of cellular immune system. It is a naturally occurring protein and can detect certain types of tumor cells in the blood and kidney, and some inflammatory and autoimmune disorders [53, 54]. In our study as well as that reported by Fung et al. [17], many of the proteins in the seminal plasma were identified to be forerunners of prostate disease, suggesting a pathogenic role for B2MG [55, 56]. Similarly high levels of B2MG were reported in the seminal plasma of azoospermic men compared to controls [10, 57, 58]. An inverse correlation was reported between B2MG levels in seminal fluid and sperm count [10].

Similary, we reported the underexpression of galectin 3 binding protein in oligozoospermic group. Galectin-3 is a carbohydrate-binding protein whose expression level has been shown to correlate with metastatic potential in a number of different types of tumors. Galectin-3 is downregulated in prostate cancer. The altered downregulation pattern of galectin-3 observed between tumor stages suggests different roles for galectin-3 in the progression of prostate cancer [59].

TIMP 1 can inhibit tumor growth, invasion and metastasis through their matrix metalloproteinases (MMP) inhibitory activity [60, 61]. TIMPs play an inhibitory role, and any imbalance between the two may result in progression of the disease [62]. TIMP was found to be over-expressed in the ON group.

Amongst the proteins that were uniquely under-expressed in the OA group was ankyrin repeat domain 11 (ANKRD11). Ankyrins function as adaptor proteins [63, 64] and play a vital role in membrane skeleton organization, ionic transport, maintenance of cell polarity as well as cell-cell adhesion regulation. Data suggest that ANKRD11 may act as a tumor suppressor [65]. It’s presence in the semen samples has not been well documented, but it is likely that its overexpression in oligozoospermic men may play a role in apoptosis. Clusterin isoform 1 can be both pro- and antiapoptotic depending upon the isoform expressed [66, 67]. It plays a key role in signal transduction and is involved in apoptosis of spermatocytes, sperm maturation, and spermiation. Low signal transducing proteins were seen in the OA group compared with the other groups, as shown in Figure 4.

Compared with the proteins involved in stress response, as elucidated from GO annotations (Figure 4), the NA and ON groups had a higher distribution of stress proteins such as DJ-1 whereas a lower distribution of this protein was seen in OA group. DJ-1 protein is a multifunctional, highly conserved antioxidant protein and is upregulated in hyperglycemia [68, 69]. It is mainly involved in the control of oxidative stress and is downregulated in the seminal plasma of asthenozoospermic men [21]. DJ-1 is activated in stress conditions, but with higher levels of stress (as seen in OA), there is depletion of antioxidant levels and low expression of DJ-1 protein. Furthermore, the major biological function that comes into focus through IPA in the NA group is free-radical scavenging activity. This further supports the fact that DJ-1 was activated in the NA group. Significant proteins were distributed in the lysosomal and vacuolar regions in higher amounts in the OA group (Figure 3), suggesting that proteins with phagocytic activity may be activated in stress conditions. Previous studies have reported reduced levels of DJ-1 in sperm in response to toxic exposure of male rats to ornidazole and epichlorhydrin [70].

Utleg et al. categorized prostatic acid phosphatase (PAP) precursor as an enzymatic protein, and the presence of this protein in our study suggests that it plays a similar role [39]. The distribution pattern of immune system response in all the groups is shown in Figure 4. It is likely that B2MG is the major protein in immune response and that low levels of this protein are seen in stress conditions. Low distribution of B2MG in DEP compared to normal and low abundant proteins further suggests that in addition to B2MG, there are other proteins that also play a key role in immune system processes. We further validated this observation from the pathway analysis, which showed that that the common protein prosaposin isoform, a preprotein, is involved in lipid antigen presentation by CD1.

We observed overlapping of differentially expressed proteins with the low abundant proteins in our study. These included transferrin, secretory leukocyte peptidase inhibitor precursor, ubiquitin and ribosomal protein S27a precursor, protein tyrosine phosphatase, receptor type, sigma isoform 1 precursor and acidic epidiymal glycoprotein- like 1 isoform 1 precursor. Of these proteins, protein tyrosine phosphatase, receptor type, sigma isoform 1 precursor, acidic epididymal glycoprotein-like isoform 1 precursor were amongst the low abundant proteins. Protein tyrosine phosphatase, receptor type, sigma isoform 1 precursor were upregulated while acidic epididymal glycoprotein-like isoform 1 precursor was down regulated in the NA group. The acidic epididymal glycoprotein like I isoform I precursor is a member of the cysteine - rich secretory protein (CRISP) family and is encoded by the gene CRISP-I. It is expressed in the epididymis and is secreted into the epididymal lumen. It binds to the post acrosomal region of the head, where it may play a role in sperm-egg fusion. The reported low abundance of acidic epididymal glycoprotein like I isoform I precursor protein is concordant with the findings of Batruch et al. who also reported its low abundance in the seminal plasma of post vasectomy patients compared to controls [8].

Transferrin is one of the serum proteins that has been characterized in the seminal plasma, but its role in male infertility is unclear [20]. Our study showed this protein was present in low levels in the NN group but was up regulated in NA and OA groups. Prostaglandin H2 D isomerase, cathepsin B preprotein, orosomucoid 2 and the CD177 molecule were the low abundant proteins that were not differentially expressed in any of the groups. In our study, CD177 was absent in the NN group but present in all the other groups although they were not differentially expressed. Our findings are consistent with previous publications that reported low concentrations of CD177 in fertile men (control samples) compared to postvasectomy men [8] but are contrary to the findings of Wang et al. who reported upregulation of CD177 in asthenozoospermic patients [21]. Cathepsin B preprotein was present in the NN and NA groups, showing its specificity to the normal sperm count while prostaglandin H2 D isomerase was absent in the ON group. Cathepsin B preprotein found in the lysosomal region is a thiol protease believed to participate in intracellular degradation. It has also been implicated in tumor invasion and metastasis. The prostaglandin (H2) D-isomerase is expressed in the testis, epididymis and prostate and is secreted into the seminal fluid. It binds small non-substrate lipophilic molecules and may act as a scavenger for harmful hydrophobic molecules. The prostaglandin (H2) D-isomerase and cathepsin B protein are potentially involved in biological processes such as vesicle-mediated transport and defense response. The low abundant proteins showed a marked distribution in the extracellular region and may play a regulatory role. The major pathways in which the low abundant proteins were involved were prostanoid biosynthesis and eicosanoid signaling and acute phase response signaling, but the distribution of signal transduction proteins was decreased in low abundant proteins.

In the present study, we have identified proteins that are common, unique, and differentially expressed in 4 study groups. Twenty proteins were differentially expressed in the seminal plasma of men with poor sperm quality. We have highlighted the distribution pattern of these proteins in cell organelles and described their biological processes. We have also illustrated the high involvement of proteins in cellular development signal transduction. The overexpression or underexpression of these proteins in the 4 groups illustrates their role in male infertility. Our findings from the bioinformatic analysis shows that stress proteins such as DJ-1 are differentially regulated and expressed in the different study groups, suggesting that some of these proteins may serve as a potential biomarkers in identifying the mechanistic role in men with poor sperm quality.