Abstract
Spermatozoa are mature male gametes that are produced in the testes of a healthy man by spermatogenesis, with further maturation of sperm taking place during their transit through the epididymis. In the human, approximately 20 to 240 million sperm are produced per day [1]. Unlike other somatic cells present in the human body, spermatozoa contain a head, neck, mid-piece and tail region. The head region contains the genetic material which is transferred to the oocyte during the fertilization process. Apart from DNA, spermatozoa also deliver additional subcellular materials such as oocyte activating factors, RNA, microRNAs and exosomal proteins that are essential for the development of the oocyte into a zygote.
25.1 Background
Spermatozoa are mature male gametes that are produced in the testes of a healthy man by spermatogenesis, with further maturation of sperm taking place during their transit through the epididymis. In the human, approximately 20 to 240 million sperm are produced per day [1]. Unlike other somatic cells present in the human body, spermatozoa contain a head, neck, mid-piece and tail region. The head region contains the genetic material which is transferred to the oocyte during the fertilization process. Apart from DNA, spermatozoa also deliver additional subcellular materials such as oocyte activating factors, RNA, microRNAs and exosomal proteins that are essential for the development of the oocyte into a zygote.
Disturbance in the molecular events related to testicular spermatogenesis or post-testicular maturation may result in infertility. In general, infertility evaluation in men is based on conventional semen analysis which is considered as the cornerstone in the assessment of male infertility. Typically, semen parameters such as sperm concentration, total motility, normal morphology and vitality are examined according to the fifth edition reference values established by the WHO laboratory manual [2]. In addition to basic semen analysis, specialized sperm function tests (SSFT) such as oxidation-reduction potential (ORP) [3] and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) or sperm chromatin structure assay (SCSA) are carried out to evaluate the seminal oxidative stress and sperm DNA fragmentation (SDF), respectively [3, 4]. High levels of oxidative stress and SDF are associated with fertilization failure and male infertility [4]. Both semen analysis and SSFT fail to identify molecular mechanisms and subcellular pathways that are dysfunctional in the spermatozoa of infertile men [5].
Recently, proteomic approaches are being used widely to understand the molecular factors that are associated with male infertility. Proteomic studies are of different types: structural proteomics, functional proteomics and expressional proteomics. In particular, global/expressional proteomic analysis of the ejaculated spermatozoa enables us to understand the patho-physiological state of spermatozoa. The mitochondrial proteome of sperm reflects the mitochondrial membrane integrity [6]. Similarly, the expression of energy metabolism-related proteins are directly associated with sperm motility. As spermatozoa are transcriptionally and translationally silent, their proteome reflects the outcome of spermatogenesis and maturation process. Furthermore, integration of proteomics with bioinformatics serves as a promising tool in the identification of potential diagnostic and therapeutic biomarkers for the management of male infertility. The availability of advanced proteomic tools such as matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has increased the knowledge and understanding of the causes of male infertility. This chapter provides a brief overview of advanced proteomic techniques and highlights the general steps involved in processing of sperm cells for proteomics and bioinformatic analysis of the proteomic data. Furthermore, proteomic-based studies on sperm and seminal plasma are discussed in detail, along with the potential role of biomarkers in the prognosis and diagnosis of male infertility.
25.2 Proteomics of Sperm Cells
Proteomics is defined as the complete protein profiling of a tissue or cell. Sperm proteins are detected using both conventional and advanced proteomic techniques. Two-dimensional (2D) gel electrophoresis is the most commonly used technique to separate sperm proteins based on their isoelectric focusing point and molecular weight. Advanced proteomic techniques include analysis of sperm proteins using MALDI-TOF and LC-MS/MS. Semen contains cellular (spermatozoa) and non-cellular (seminal plasma) components. Other than spermatozoa, semen also contains round cells including leukocytes and immature germ cells. Round cells are of two types: spermatogenic and non-spermatogenic round cells. Hence, two types of samples are being used in sperm proteomic studies: 1) processed semen sample and 2) unprocessed or neat semen sample.
Processed semen samples contain pure fraction of spermatozoa. Density gradient technique is used to separate out the seminal plasma, round cells and leukocytes from spermatozoa. Few studies indicate that the use of sperm with round cells in the protein extraction process may contaminate the sperm proteome [7, 8, 9, 10, 11, 12]. The processing of neat semen samples for proteomic studies involves separation of spermatozoa from seminal fluid by centrifugation and subsequently, washing with phosphate buffer saline (PBS) to completely remove the seminal plasma. Recent studies explained the effect on biological pathways associated with sperm function due to the presence of round cell proteins in the proteome of sperm [13, 14]. The influence of non-spermatogenic round cell proteins was found to be very negligible or insignificant as they were masked by the sperm proteome [14]. Furthermore, the presence of these round cells and leukocyte proteins did not interfere in the molecular pathways associated with sperm function [13].
Initially, the sperm pellet is mixed with lysis buffer such as radioimmunoprecipitation assay (RIPA) and left overnight for complete lysis of spermatozoa [14]. Purity and concentration of the sperm proteins are checked prior to electrophoresis (either one-dimensional or 2D gel). Proteins separated by electrophoresis are subjected to in-gel digestion using trypsin. Further, the peptides are eluted and injected into the mass spectrometry (MS) system that detects the peptides and proteins using an unbiased approach by analyzing the mass shifts without any prior information about the structure [15]. The proteins are identified based on their mass/charge ratio (m/z) and with a very low false discovery rate. Next, the proteins and peptides detected by MS are scanned completely and compared with previously annotated and sequenced proteins available in a global database. Lists of proteins are computed using softwares such as SEQUEST, Mascot and X!-Tandem that are operated using different algorithms [16]. The proteins are then categorized as differentially expressed proteins (DEPs) based on spectral counts and abundance of each protein. These DEPs are subjected to bioinformatic analysis to identify their role in different biological processes [17]. Gene ontology (GO) analysis is used to obtain the information related to localization and distribution of proteins. Furthermore, open access bioinformatics tools such as the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) are used to display the interaction between proteins [18]. Commercially available authenticated software such as Ingenuity Pathway Analysis (IPA) and Metacore™ are used to identify interactions between the functional pathways, biological processes, proteins and the transcriptional factors regulating their expression [18].
25.3 Review of Sperm Proteomic Studies
Characterization of sperm proteome and its implication in deciphering the molecular and cellular pathways in male infertility have gained increasing attention among reproductive researchers and andrologists. Although earlier proteomic studies mainly examined specific sperm surface proteins, the first comprehensive report on human sperm proteome was published in 2005 [19]. The study reported 1760 distinct proteins using 1D-SDS-PAGE coupled with LC-MS/MS analysis and indicated the presence of all 27 proteins constituting the 26S proteasome in the sperm. However, the study did not provide a complete list of proteins identified [19]. Later, Martinez-Heredia et al. characterized the sperm proteome and reported 98 distinct proteins using 2D-PAGE coupled with MALDI-TOF MS analysis [10]. Furthermore, the study revealed the functional distribution of these proteins to be energy production (23 percent), transcription, protein synthesis, transport, folding and turnover (23 percent), cell cycle, apoptosis and oxidative stress (10 percent), signal transduction (8 percent), cytoskeleton, flagella and cell movement (10 percent), cell recognition (7 percent) and metabolism (6 percent) [10]. The sperm proteomic study conducted by Baker et al. identified 1053 proteins, which included nicotinamide adenine dinucleotide phosphate oxidase (NOX), and its homolog, dual oxidase 2 (DUOX2), and various classes of receptors that are potential regulators of sperm function [20]. Gilany et al. retrieved the human sperm proteome from the literature and analyzed it by the Database for Annotation Visualization and Integrated Discovery (DAVID) software [21]. The analysis revealed a collection of 1300 proteins involved in various metabolic pathways that were primarily localized to cytoplasm [21].
Lately, in addition to whole sperm proteomics, subcellular proteomics has also gained significant attention as it provides in-depth information regarding the sperm protein content and their exact localization. Furthermore, it allows identification of less abundant proteins. Proteomic analysis of the head and flagellar regions of spermatozoa conducted by Baker et al. identified a total of 1429 proteins with 721 proteins exclusively localized in the tail and 521 proteins exclusively localized in the head fractions [22]. This was the first study that provided novel insight into the compartmentalization of proteins, particularly receptors [22]. Analysis of isolated tail fractions of spermatozoa resulted in the identification of 1049 proteins that were mainly involved in metabolism and energy production, and sperm tail structure and motility [7]. Interestingly, the analysis also revealed a high number of peroxisomal proteins in sperm, which are believed to be lacking peroxisomes [7].
Proteomic characterizations of membrane fractions of spermatozoa led to the elucidation of proteins involved in sperm-oocyte interaction [23]. de Mateo et al. isolated and analyzed human sperm nuclei by LC-MS/MS approach and reported 403 nuclear proteins [24]. The most abundant family of proteins were histones, followed by ribosome proteins, proteasome subunits, cytokeratins, tubulins, SPANX proteins, HSPs and Tektins. This was the first study to provide an in-sight on the nuclear proteins that are potentially related to sperm epigenetic functions, proper fertilization and embryo development [24]. Furthermore, de Mateo et al. were the first to report correlation between proteomics, DNA integrity and protamine content [25]. In 2014, Amaral et al. analyzed 30 different sperm proteomic studies and reported 6198 proteins, of which 30 percent are known to be expressed in the testis [8]. The proteins were cataloged based on their functional pathways including metabolism, cell cycle, apoptosis, membrane trafficking, RNA metabolism and post-translational protein modifications [8].
25.4 Sperm Proteome Profile and Male Infertility
25.4.1 Molecular Pathways and Proteins Affected in Varicocele
Varicocele is one of the most common and correctable causes of male infertility and is prevalent in 15 percent of normal men and 40 percent of infertile men of reproductive age groups [26]. Sperm proteome analysis in these subjects have facilitated in interpreting the cellular and molecular pathways implicated in the pathophysiology of varicocele. The first proteomic study that analyzed the differences in the sperm proteome of men with and without varicocele was published by Hosseinifar et al. [27]. The study revealed 15 DEPs that predominantly included heat shock proteins (HSPs), mitochondrial and cytoskeleton proteins. Sperm proteome characterization in infertile men with unilateral varicocele against healthy controls revealed 369 DEPs that were associated with major functional pathways such as metabolism, disease, immune system, gene expression, signal transduction and apoptosis [28]. Of the 369 DEPS, 29 proteins were identified to be involved in spermatogenesis and other fundamental reproductive processes such as sperm maturation, acquisition of sperm motility, hyperactivation, capacitation, acrosome reaction and fertilization. Furthermore, it was reported that unilateral varicocele mostly affected small molecule biochemistry and post-translational modification proteins [28].
Sperm proteome characterization in infertile men with bilateral varicocele against controls resulted in the identification of 73 DEPs. The majority of the DEPs were associated with metabolic processes, stress responses, oxidoreductase activity, enzyme regulation, and immune system processes [29]. Seven DEPs (Outer dense fiber protein 2 (ODF2); Tektin‑3 (TEKT3); T‑complex protein 11 homolog (TCP11); Protein‑glutamine gamma‑glutamyl transferase 4 (TGM4); Calmegin (CLGN); Mitochondrial import receptor subunit TOM22 homolog (TOM22); Apolipoprotein A‑I (APOA1)) were involved in key sperm functions such as capacitation, motility and sperm-zona binding.
For the first time, Agarwal et al. compared the sperm proteome of unilateral and bilateral varicocele in men and reported the differences in expression of proteins that were mostly involved in post-translational modification, protein folding, protein ubiquitination, free-radical scavenging, lipid and nucleic acid metabolism, small molecule biochemistry and mitochondrial dysfunction [30]. In fact, mitochondrial dysfunction is considered as one of the major mechanisms implicated in the pathophysiology of clinical varicocele and associated infertility [31, 32]. A recent study conducted by Samanta et al. examined the proteomic signatures of sperm mitochondria in varicocele subjects and reported 23 DEPs associated with mitochondrial structure (LETM1, EFHC, MIC60, PGAM5, ISOC2 and TOM22) and function (NDFSU1, UQCRC2 and COX5B), as well as core enzymes of carbohydrate and lipid metabolism [32]. Additionally, protein associated with sperm functions (ATPase1A4, HSPA2, SPA17 and APOA1) were reported to be under-expressed. Furthermore, mitochondrial electron transport chain (ETC) proteins along with testis-specific pyruvate dehydrogenase (PDH) have been suggested as biomarkers of sperm function in varicocele subjects [32].
25.4.2 Proteomic Signature of Sperm in Testicular Cancer
The incidence of testicular cancer (TC) has been increasing drastically for decades and most commonly reported in men of reproductive age group [33]. According to the American Cancer Society, the number of newly diagnosed cases of TC is estimated to be 9560 and about 410 related deaths in 2019 [33]. The most common form of TC is germ cell tumors (GCTs), which account for about 90–95 percent of all cases. The major types of GCTs are seminomas and non-seminomas. TC is associated with decline in semen quality and fertilizing potential of spermatozoa [34]. Proteomic studies have revealed that this reduction in semen quality is associated with alterations in the expression of sperm proteins [35, 36].
A few sperm proteomic studies have been conducted to identify the potential biomarkers and molecular mechanism(s) associated with the reduced fertilizing ability of TC subjects [35, 36, 37, 38, 39]. Dias et al. compared the sperm proteomic profile of men with testicular cancer seminoma against healthy fertile men [36]. Quantitative proteomic analysis revealed 393 DEPs between the groups that were associated with spermatogenic dysfunction, reduced sperm kinematics and motility, failure in capacitation and fertilization. Comparative analysis of sperm proteome of men with non-seminoma testicular cancer and healthy fertile men revealed 189 DEPs with under expression of proteins crucial for mitochondrial function, sperm motility and fertilization [35]. About 198 proteins were identified as DEPs between normozoospermic (motility>40 percent) and asthenozoospermic (motility<40 percent) in TC patients who had cryopreserved semen samples before initiation of cancer therapy [39]. The study revealed under-expression of proteins associated with the binding to zona pellucida (CCT3), mitochondrial function (ATP5A1 and UQCRC2), sperm motility (ATP1A4) and exosomal pathway in asthenozoospermic TC patients. Another recent study reported under-expression of NDUFS1 associated with mitochondrial function and overexpression of CD63 involved in sperm maturation in both normozoospermic and asthenozoospermic TC patients when compared to normozoospermic infertile men without cancer [38].
25.4.3 Cellular Changes in Sperm of Unexplained Male Infertility
Infertility of unknown origin in men with normal semen parameters and without involvement of any female infertility factor is categorized as unexplained male infertility (UMI) [40]. Although semen analysis is the cornerstone for the evaluation of male infertility, 30 percent of normozoospermic men are diagnosed with UMI [40, 41]. This clearly indicates the limitations of conventional semen analysis in predicting the male fertility potential and reproductive outcome in couples. Sperm proteomic studies in these subjects may pave the way for identifying the etiologies as well as cellular and molecular changes associated with UMI. Frapsauce et al. analyzed the proteins in normal but non-fertilizing sperm using 2D fluorescence DIGE (difference gel electrophoresis) and reported 15 DEPs [42]. Furthermore, laminin receptor (LR67) and L-xylulose reductase (P34H) proteins involved in gamete interactions were proposed as potential targets for diagnosis and prognosis of fertilization failure in IVF [42]. Several sperm proteomic studies have been conducted in normozoospermic infertile men with IVF failure and mostly, the proteins associated with sexual reproduction, metabolic process, cell growth and/or maintenance, protein metabolism and protein transport, chromosome organization, capacitation, acrosome reaction and sperm-oocyte interaction were reported to be dysregulated [43, 44, 45, 46]. A recent study compared the sperm proteome of UMI subjects against normozoospermic fertile men using LC-MS/MS and reported 162 DEPs between the groups [47]. Analysis revealed under-expression of proteins associated with reproductive system development and function, and ubiquitination pathway in UMI subjects. Furthermore, serine protease inhibitor (SERPINA5), annexin A2 (ANXA2), and sperm surface protein Sp17 (SPA17) were suggested as biomarkers for screening the fertilization potential of spermatozoa in UMI subjects [47].
25.4.4 Sperm Proteome in Oligoasthenozoospermia
Oligoasthenozoospermia (OAT) is characterized by reduced sperm concentration and motility. Very few studies have been published on the seminal plasma proteomics, and none in the sperm proteomics of OAT subjects [47, 48, 49]. Herwig et al. identified the proteins involved in the etiology of OAT due to oxidative stress [48]. Seminal plasma proteins associated with multiple biological functions such as binding activity (lactotransferrin (LTF); prolactin-induced protein (PIP); extracellular matrix protein 1 (ECM1)), transporter activity (human epididymis-specific protein 1 (HE1); prostaglandin D2 synthase (PTGDS)), immune activity (CD177), and hydrolase activity (prostate-specific antigen (PSA)) were reported to be dysregulated in OAT subjects [49].
25.4.5 Sperm Proteome in Asthenozoospermia
Asthenozoospermia, or reduced progressive motility of spermatozoa, is the most common finding in infertile men. Several sperm proteomic studies have been conducted in these subjects that have shed light on the DEPs (Table 25.1) and related pathways implicated in the pathophysiology of asthenozoospermia [50, 51, 52, 53, 54, 55, 56]. The reduced motility has been attributed to several factors including dysregulation of energy metabolism, structural defects in sperm-tail protein components and differential expression of proteins involved in sperm motility [57]. Siva et al. compared the sperm proteome of asthenozoospermic and normozoospermic subjects using 2D-PAGE MALDI MS/MS approach. The DEPs were categorized into three functional groups, namely: “energy and metabolism” (triose-phosphate isomerase (TPIS); testis-specific glycerol kinase 2 (GKP2); and succinyl-CoA:3-ketoacid co-enzyme A transferase 1 (OXCT1), mitochondrial precursor); “movement and organization” (tubulin beta 2C (TUBB2C) and tektin 1 (TEKT1)); and “protein turnover, folding and stress response” (proteasome alpha 3 subunit (PSMA3) and heat shock-related 70 kDa protein 2 (HSPA2)) [55]. Similar categorization of key DEPs was reported by other sperm proteomic studies conducted in asthenozoospermic subjects [50, 56].
| Clinical condition | Method | DEPs | Reference |
|---|---|---|---|
| Varicocele | 2D PAGE MALDI-TOF/TOF-MS | HSPA5, ATP5D, SOD1, ACPP, CLU, PARK7, KLK3, PIP, SEMG2, SEMG2pre | Hosseinifar et al. 2013 [27] |
| 1D PAGE LC-MS/MS | CABYR, AKAP, APOPA1, SEMG1, ACR, SPA17, RSPH1, RSPH9 DNAH17, DLD, GSTM3, TGM4, NPC23, ODF2GPR64, PSMA8, HIST1H2BA, PARK7 | Agarwal et al. 2015 [28] | |
| 1D PAGE LC-MS/MS | GSTM3, SPANXB1, PARK7, PSMA8, DLD, SEMG1, SEMG2 | Agarwal et al. 2015 [30] | |
| 1D PAGE LC-MS/MS | ODF2, TEKT3, TCP11, TGM4, CLGN, TOM22, APOA1 | Agarwal et al. 2016 [29] | |
| 1D PAGE LC-MS/MS | PKAR1A, AK7, CCT6B, HSPA2, ODF2 | Agarwal et al. 2016b [31] | |
| LC-MS/MS | LETM1, EFHC, MIC60, PGAM5, ISOC2, TOM22, NDFSU1, UQCRC2, COX5B, ATPase1A4, HSPA2, SPA17, APOA1 | Samanta et al. 2018 [32] | |
| 1D PAGE LC-MS/MS | HSPA2. HSP90B1, OXPHOS complex proteins | Swain et al. 2019 [58] | |
| Testicular cancer | LC-MS/MS | PSA, PAcP, ZAG, SEMG 1 and 2, AKAP4, DNAH17 | Agarwal et al. 2015 [37] |
| 1D PAGE LC-MS/MS | NDUFS1, UQCRC2, ATP1A4, ANXA2, ATP1A2, ACR | Dias et al. 2018 [35] | |
| 1D PAGE LC-MS/MS | CCT3, ATP5A1, UQCRC2, ATP1A4, MMP9 | Panner Selvam et al. 2019 [39] | |
| 1D PAGE LC-MS/MS | NDUFS1, CD63 | Panner Selvam et al. 2019 [38] | |
| 1D PAGE LC-MS/MS | HSPA2, ATP1A4, UQCRC2, ACE | Dias et al. 2019 [36] | |
| UMI | 1D PAGE LC-MS/MS | SERPINA5, ANXA2, SPA17 | Panner Selvam et al. 2019 [46] |
| MALDI-TOF/TOF | PAEP, ODFP, SEGI, PSA, and GPx4pre | Xu et al. 2012 [45] | |
| Normozoospermic men with IVF failure | 2D-DIGE MS | P34H | Frapsauce et al. 2014 [42] |
| iTRAQ LC-MS/MS | SEMG1, PIP, GAPDHS, PGK2 | Légaré et al. 2014 [44] | |
| TMT LC-MS/MS | SRPK1 | Azpiazu et al. 2014 [43] | |
| Asthenozoospermia | 2-DE MALDI-TOF MS | IDH-α, ODF, SEMG1, ARHGDIB, GOT1, PGAM2, TPI1, CA2, GS10, MSS1 | Zhao et al. 2007 [56] |
| 2D PAGE MS | ACTB, ANXA5, COX6B, H2A, PIP, PIPpre, S100A9, CLUpre, DLDpre, FHpre, HSPA2, IMPA1, MPST/ECH1pre, PSMB3, SEMG1pre, TEX12 | Martinez-Heredia et al. 2008 [50] | |
| 2D PAGE MALDI MS/MS | TPIS, PSMA3, GKP2, HSPA2, OXCT1, TUBB2C, TEKT1 | Siva et al. 2010 [55] | |
| Nano UPLC–MSE tandem mass spectrometry | GRP78, GAPDHS, HSP70-2, TUBA4A, TUBA3C, TUBA8, ODF1, AKAP3, AKAP4, ROPN1B, SPANXB, CLU, PIP, ATP5B, ALDOA, ARGDIA | Parte et al. 2012 [52] | |
| 2-DE MALDI-TOF/TOF MS | UBB2B, ODF2, AKAP4, KRT1, CLU, COX6B, GAPDS, PHGPx, HSPA2, HSPA9, VDAC2, GSTMu3, ASRGL1, SPANXB | Hashemitabar et al 2015 [59] | |
| UPLC-MS | PLXNB2, POTEKP, NIN, PHF3, DYNLL1, PROCA1, FASCIN-3; LRRC37B, PLC | Saraswat et al 2017 [53] | |
| 2-DE MALDI-TOF MS | LFT, ATP5B, DJ-1, PARK7, ODF, TEKT1, AKAP4, ELSPBP1, PDHB, NDUS1, SUCLA2, SDHA | Nowicka-Bauer et al. 2018 [51] | |
| 2D-DIGE MALDI -TOF-MS | TEX40, ATP6V0A2, SERPINB9, PSA | Sinha et al. 2019 [54] | |
| Globozoospermia | 2D DIGE MALDI-TOF/TOF MS/MS | SAMP1, ODF2, SPANXa/d, TUBA2, TPI1, PIP | Liao et al. 2009 [60] |
Parte et al. compared the expression of phosphoproteins associated with sperm motility in asthenozoospermic and normozoospermic subjects [52]. Comparative sperm proteome analysis between normozoospermic and asthenozoospermic subjects resulted in the identification of pathways associated with altered expression of proteins (Table 25.1), which included axoneme activation and focal adhesion assembly, glycolysis, gluconeogenesis, cellular response to stress and nucleosome assembly [53]. The reported DEPs were HSPs, cytoskeletal proteins, proteins involved with fibrous sheath and energy metabolism. A recent proteomic study conducted by Nowicka-Bauer et al. using 2-DE and MALDI-TOF MS, correlated the DEPs identified with the sperm mitochondrial activity [51]. The findings of this study indicated the possible role of sperm mitochondrial dysfunction and oxidative stress in the etiology of asthenozoospermia [51]. Another recent study revealed decreased expression of proteins related to calcium ion entry (TEX40) and acrosomal acidification (ATP6V0A2) in asthenozoospermic men [54]. Lower expression of TEX40 and ATP6V0A2 leads to fewer entries of calcium ion into the spermatozoa and acrosomal de-acidification, which in turn results in diminution of sperm motility in asthenozoospermic men [54].
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