Abstract
As the mammalian spermatozoa transcends from the testis to the end of the epididymal tubule, the functionally incompetent spermatozoa acquires its fertilizing capability. Molecular changes in the spermatozoa at the posttesticular level concern qualitative and quantitative modifications of proteins along with their sugar moieties and membranous lipids mostly associated with motility, egg binding, and penetration processes. Proteomic studies have identified numerous sperm-specific proteins, and recent reports have provided a further understanding of their function with respect to male fertility. High-throughput techniques such as mass spectrometry have shown drastic potential for the identification and study of sperm proteins. In fact, compelling evidence has provided that proteins are critically important in cellular remodeling event and that aberrant expression is associated with pronounced defects in sperm function. This review highlights the posttesticular functional transformation in the epididymis and female reproductive tract with due emphasis on proteomics.
Introduction
Male infertility is a troublesome yet common problem for men all over the world. Nearly 40% to 60% of infertility cases are attributed to be associated with male factors. 1 Despite current advancements in elucidating the cause of male infertility, in most of the cases, the etiology still remains unknown. Evaluation of male fertility by conventional semen analysis as stated by the World Health Organization mainly focus on number of spermatozoa in ejaculate, their morphological abnormality, number of leukocytes, and percentage of motility. 2 Nevertheless, its predictive value to assess sperm quality and fertility outcomes is limited. 3,4 As the underlying basis for both diagnosis and understanding the cause of male infertility remains idiopathic, unraveling the molecular factors, such as the expression of essential sperm proteins that regulates fertilization, holds potential.
The mechanism of spermatogenesis in humans is a highly ordered process with coordinated cell cycle and regulatory events that marks the differentiation of a haploid round spermatid cells to the highly specialized, elongated, and terminally differentiated spermatozoon, capable of delivering the paternal genome to the oocyte. Posttesticular maturation, the spermatozoa are left with limited amount of cytosolic organelles and complete loss of transcriptional ability. During epididymal transit, the spermatozoa undergo a series of posttranslational modifications that confer it with large number of structurally and functionally competent proteins. 5
The human spermatozoon is known to be highly specialized as compared to other somatic and germinal cells based upon its compositional, morphological, and functional attributes. Owing to the limited capacity for biosynthesis in the spermatozoa, transcriptome analysis as a method of choice to investigate the posttesticular changes in the spermatozoa is restricted. In this context, new advances in proteomics open possibilities in the general understanding of the dynamics of sperm cell function in a plethora of areas. Ongoing efforts are still being made to identify sperm nuclear proteins, proteins required for sperm motility, and in the understanding of the membrane proteins required for capacitation, egg interaction, and fertilization. 6 -12 With the absence of physiologically active transcription and translation, spermatozoa could be regarded as outstanding candidates for proteomic study. This is further supported by the fact that they can be purified at high concentrations and can be pharmacologically manipulated into various functional states such as capacitated, noncapacitated, and acrosome reacted. 13,14 Moreover, understanding the differences in proteomic profile in the spermatozoa such as that of caput versus cauda epididymis and ejaculated versus capacitated, will help in unraveling the posttranslational modifications relevant to sperm maturation. 15,16 In addition, comparative proteomic analysis will not only pave the way for understanding “hidden” sperm factor responsible for infertility but also provide a molecular biomarker for diagnosis and in finding an appropriate therapeutic approach to male infertility. 17
The Proteomic Approach
Proteomics aims at providing a systematic analysis to the protein constituents of a cell and defining its level of expression with respect to a functional state in a biological system. 18,19 Proteomic analysis is essentially based on few basic steps: (1) extraction of protein or peptide from the sample, (2) reducing the complexity of the protein or peptide extract, and (3) identification of proteins or peptides through the application of mass spectrometry (MS) and bioinformatics. Initial approach in reducing the complexity of the protein or peptide extract can be achieved using 2-dimensional gel electrophoresis (2DE) or using liquid chromatography (LC). 20 -24 Several strategies have been developed to identify protein on a global scale and the most common ones are emphasized in this review. The development of MS methods and improvisation in the 2-dimensional (2D) separation of proteins has opened several possibilities in the identification and analysis of a huge number of proteins. In fact, the combinatorial effect of 2DE and MS has proved to be an important analytical tool for complete characterization of protein molecules from complex mixtures extracted from tissues, cells, or subcellular fractions. Essentially, 2 different approaches have been commonly applied to study the sperm cell proteome using MS: (1) 2D separation of proteins followed by matrix-assisted laser desorption ionization MS or LC followed by tandem MS (LC-MS/MS) and (2) initial digestion of proteins to generate peptides, followed by LC-MS/MS analysis. 14,25 The 2DE is based on the principle of separating proteins by their isoelectric focusing (IEF) in the first dimension followed by their segregation according to molecular mass. Although it has been a useful method in the past, it has several disadvantages too. Due to the low sensitivity of 2DE, detection of rare- and low-abundance proteins is difficult and further isolation of the sample into different subcellular fractions is essential to reduce the complexity of protein mixtures prior to 2DE analysis. To circumvent the above-mentioned problem, there has been the development of differential in-gel electrophoresis (DIGE) where differential labeling of proteins with fluorochromes prior to separation enables the identification of low-abundance proteins. Another important technique that yields high-throughput results is the development of MS. The MS-based proteomics has been advantageous in studying sperm cell biology for it produces proteins involved in specific biological process. 26,27 The MS is an analytical tool where the masses of molecules are converted into ions by their m/z ratio for the identification of proteins or peptides. Computer-based algorithm compares the mass spectra generated to determine whether peptides found in annotated protein databases could produce spectra that resemble those observed experimentally. 26,27 The proteins thus identified are further categorized for its distribution based on Gene Ontology (GO) annotation terms. 28 Gene Ontology is a database, which aims in providing biological information of genes or proteins involved. The ontology covers 3 domains with the objective of providing consistent vocabularies for cellular component, molecular function, and biological process of the identified gene products. Several studies have functionally categorized the human sperm proteome based on GO annotation terms using the database for annotation. There have been clear observations that since one protein may have multiple functions and can be localized in several cellular compartments, most of the proteins have been annotated to more than one classification within the GO. 29,30
Landmarks in Sperm Cell Proteomics
The study on sperm protein was first initiated by Friedrich Miescher with the discovery the salmon sperm protamine (PRM). 31 Earlier investigations to study human spermatozoa proteins have relied on 2DE, and the number of protein spots distinguished in 2D maps thus far is rather low. In this regard, Naaby-Hansen obtained an electrophoretic map of about 260 distinct acidic and neutral human spermatozoa proteins. 32 Furthermore, in search of vectorially labeled surface proteins of human spermatozoa, they acquired a composite 2DE image showing 1397 human spermatozoa proteins that belong to the membrane protein fraction and established a comprehensive human sperm database of ˜1400 spots. 33 However, the first extensive analysis of the human sperm proteome, based on LC-MS/MS approach, revealed 1760 proteins, although the individual proteins identified were not reported. 23 Another study using narrow range IEF strips and multiple 2D gels did suggest that this number might be much higher as 3872 distinct protein spots were visualized. 34 Similarly, with the use of LC-MS/MS technique, Baker and his group identified about 1056 gene products in mature human sperm. 24 However, the most extensive report up to date on the identification of mature human sperm proteins used the advanced MS and an optimized proteomics platform. 11 A total of 4675 unique proteins from human sperm were successfully identified, which shows an array of overrepresented complex pathways involved in energy metabolism, signal transduction, cytoskeleton, and so on (Figure 1 and Table 1). Whatever the case might be, based on data provided by scientific literature Amaral and her coworkers suggested that till date about 6198 different proteins have been identified, an important proportion of which (around 30%) are known to be expressed in the testis. 70 These proteins were reported to be involved in various functional pathways, such as metabolism, apoptosis, cell cycle, meiosis, and membrane trafficking, among others. Further, the knowledge on the distribution pattern of the various identified proteins could be confirmed by using the GO catalogue. Apart from the expected abundance of cytoskeletal, mitochondrial, flagellar, or membrane proteins, GO has also provided reports on the presence of a large proportion of proteins involved in transcription, protein synthesis, and turnover. 21,22
Schematic representation of factors associated with male fertility and the possible role of sperm proteome. The up and down arrows represent up- and downregulation of proteins involved in sperm function. ACR indicates acrosin; ALDOA, aldolase A; AKAP, A kinase anchoring protein; cAMP, cyclic adenosine monophosphate; COX6B, cytochrome c oxidase 6B; CREM, cAMP-response element modulator; CRISP, cystein-rich secretory protein; DLD, dihydrolipoamide dehydrogenase; ECH1, ▵3,5-▵2,4-dienoyl-CoA; ELSPBP1, epididymal sperm binding protein 1; FH, fumarate hydratase; GAPD-S, glyceraldehyde phosphate dehydrogenase-S; HSPA2, heat shock protein A2; LDH,
Protein Profile of Human Spermatozoon: Localization and Function.
Abbreviations: ACR, acrosin; AKAP, A kinase anchoring protein; ANXA5, annexin 5; ALDOA, aldolase A; ATPase, adenosine triphosphatase; BPG, 2,3-bisphosphoglycerate; CABYR, calcium-binding tyrosine phosphorylation regulated; COX, cytochrome c oxidase; CRISP, cystein-rich secretory protein; DLD, dihydrolipoamide dehydrogenase; ECH1, ▵3,5-▵2,4-dienoyl-CoA; ELSPBP1, epididymal sperm binding protein 1; ERp57, reticulum resident protein 57; FH, fumarate hydratase; GAPD-S, glyceraldehyde phosphate dehydrogenase-S; GDP, guanosine diphosphate; GTP, guanosine triphosphate; HSPA2, heat shock 70 kDa protein 2; HSP90B1, HSP 90 kDa β, member 1; Ig, immunoglobulin; LDHA,
Comparative Studies on Anomalous Behavior of Sperm Proteins
Conventionally, semen evaluation methods are focused on certain limited biophysical characteristics of the sperm cell such as motility, viscosity, pH, and so on. However, there exists difference in fertility among males where ejaculates that cannot be detected or assessed through traditional semen evaluation methods. The identification of molecular biomarkers associated with semen quality parameters and fertilizing ability through recent advancements in technology could be a unique approach in the prediction of male infertility. Studies on sperm proteomics are a promising approach that can provide the protein makeup of the sperm. This can be further corroborated to the assessment of anomalies in infertile patients by comparing the proteome of abnormal sperm samples from infertile patients with the proteome of control normozoospermic samples from fertile donors. One of the initial reports in the study of sperm defects through the use of 2D proteomics reported the proteomic mapping of a patient who experienced a failure in in vitro fertilization, where 20 different proteins were identified as compared to controls. 71
Subsequently, a differential expression profile of 17 proteins was obtained in sperm of patients diagnosed with asthenozoospermia. 54 Importantly, clusterin and semenogelin were found to be increased in these patients. 10,41 A novel study targeting the patients with oligoasthenozoospermia (OAT) was undertaken by Amaral et al 62 who detected a significantly higher incidence of heterozygosity for CAG repeats, higher mitochondrial DNA (mtDNA) content, and a lower percentage of sperm expressing polymerase γ (POLG) and TFAM in the spermatozoa of OAT group.
A comparative proteomic analysis on patients with globozoospermia revealed 35 proteins to be differentially expressed, of which 9 proteins were upregulated and 26 proteins were downregulated in round-headed spermatozoa compared with normal spermatozoa. 47 These differentially expressed proteins were suggested to be involved in a variety of cellular processes and structures, including spermatogenesis, cell skeleton, metabolism, and motility of spermatozoa.
Xu and his group focused on measuring the levels of the major proteins extracted from infertile patients whose semen parameters were normal but failed in vitro fertilization. 17 The study revealed 24 proteins whose levels are either increased or decreased in the normozoospermic infertile group. These proteins with altered levels of expression were reported to be involved in energy production, structure and movement, and cell signaling and regulation.
Application of proteomic studies as potential diagnostic markers has gained importance in recent years. Comparison of a cohort of 2045 proteins obtained in case of patients with idiopathic infertility revealed 21 proteins to be differentially expressed (>1.2-fold) in men whose sperm resulted in a clinical pregnancy via assisted reproductive technology from those who did not. 50 Such findings were known to have important implications in assessment of the pathogenic mechanisms in infertility based on sperm motility, capacitation, acrosomal reaction, and sperm–oocyte interaction. Furthermore, studies were undertaken to find correlation if any between abnormal sperm protein profiles and DNA fragmentation index and/or abnormal motility percentage in patients undergoing fertility evaluation in a clinical setting. 1,55 Preliminary investigation led to identification of 4 main protein groups having a significant correlation with abnormal DNA fragmentation and/or motility. The first group included sperm nuclear proteins such as the sperm protein associated with nucleus in the X chromosome (SPANX) isoforms and several types of histones. 1 The second group contained mitochondria-related functions and oxidative stress proteins including mitochondrial ferritin, mitochondrial single-stranded DNA-binding protein, and several isoforms of peroxiredoxins. The other 2 protein groups were related to sperm motility such as microtubule-based flagellum and spindle microtubule as well as proteins related to the ubiquitin-proteasome pathway.
In a recent novel proteomic study to detect the differential protein profile in men with and without varicocele using 2DE, 72 15 consistent differences in protein expression were identified (1, spots missing; 12, less abundant; and 2, more abundant) in the varicocele group compared with the control group. The heat shock proteins (HSPs), mitochondrial proteins, and cytoskeleton proteins are the major proteins affected by varicocele disease. Particularly, a significant upregulation of HSPs (HSP70 and HSP90) was observed in patients with varicocele, which was further validated by Western blot and immunocytochemistry. 73
Assessment of sperm proteome also provides insight to unravel-specific proteins involved in providing protection against exogenous insult. Recently, the sperm proteome from groups of men with high levels of reactive oxygen species (ROS+) was compared with that from men with low or physiological levels of ROS (ROS−) in 2 separate settings by the same group. 27,41 In the first case, 27 the authors used 2D-DIGE with differential fluorescence tagging and in gel digestion of the proteins followed by LC/MS detection. A total of 1343 protein spots were detected in ROS− samples while the ROS+ samples showed 1265 spots. Of these proteins, 31 spots were differentially expressed with 6 spots significantly decreased and 25 spots increased in the ROS− sample compared with the ROS+ sample. The authors reported overabundance of 4 antioxidant proteins in ROS− sperms those may exert essential cytoprotective effects against the build-up of ROS levels. These proteins are lactotransferrin isoform 2, lactotransferrin isoform 1 precursor, peroxiredoxin 1, and Mn-SOD mitochondrial isoform A precursor. 27 In the second study, 41 the authors subjected the samples to LC-MS/MS analysis through in-solution digestion of proteins for peptide characterization. The findings of the study revealed a total of 74 proteins in spermatozoa, of which 15 proteins with a >2-fold difference were overexpressed (ROS+ group) when compared to the ROS− group. The overexpressed proteins comprised histone cluster 1, H2ba (HIST1H2BA), mitochondrial malate dehydrogenase precursor (MDH2), HSP 90 kDa β, member 1 (HSP90B1), heat shock 70 kDa protein 5 (HSPA5), glutamine synthetase (GLUL), transglutaminase 4 (prostate; TGM4), glutathione peroxidase 4 isoform A precursor (GPX4), sperm acrosomal membrane protein 4 (SPACA4), olfactomedin 4 precursor, and chromosome 20 open reading frame 3. And proteins that were found to be underexpressed included semenogelin II precursor (SEMGIIpre), peroxiredoxin 6, clathrin heavy chain 1, eukaryotic translation elongation factor 2, and enolase 1. Furthermore, there was significant change in localization profile of the differentially expressed proteins. Although the overexpressed proteins were predominantly found in the cellular compartments of intracellular, organelle, macromolecular complex region, and mitochondria, the underexpressed proteins were dominated in the cytoplasm, extracellular, plasma membrane, protein complex, and the vesicular region. Remarkably, the endosome, lipid particle, membrane-bound organelles, and the microtubules showed the exclusive abundance of overexpressed proteins in the ROS+ group, while the proteinaceous extracellular matrix was restricted only to the underexpressed proteins.
Many of the proteins in its precursor or preprotein form have been identified in studies relating to male fertility anomalies and it may be speculated that the accumulation of these protein is an indication of generalized posttranslational problem. Assumptions are also made regarding the downregulation of some of the downstream functions related to these proteins. 41,54,74
Comparative Proteome Analysis of the Sperm During Testicular and Epididymal Transit as a Function of Its Maturation
A large body of literature report on proteomic analysis of ejaculated human sperm. But there is a paucity of reports on characterization of proteins responsible for its development in the testis and its maturation during epididymal transcend. The problem faced in these cases is the unavailability of human testicular and epididymal spermatozoa from persons with known fertility status, and biopsies cannot be taken without compromising fertility. In depth analysis of human sperm protein specific to testicular origin and epididymal origin was characterized by Li et al. 75,76 They reported that of about 319 identified human sperm-located proteins, 112 (35%) were exclusively of testicular origin, 152 (48%) were exclusively of epididymal origin, and 55 (17%) were common to both organs. 76 It was further validated that these proteins had a specific pattern of localization in sperm based on their origin. The epididymal proteins were more specifically concentrated at the principal piece, while lesser at acrosomal area, in comparison to testicular proteins. Moreover, the report suggested that of the total identified proteins, 47% were intrinsic sperm proteins expressed at the stage of spermatid, while 23% were extrinsic sperm proteins, acquired during epididymal transit, and have an epididymal origin. 75 However, a recent work by Wang et al identified about 4675 proteins in human spermatozoa, of which only 227 proteins were of testicular origin. 11 Moreover, comparison of sperm proteome with its transcriptome reveals little overlap. From pool of 1915 RNA detected in human sperm, only 553 (29%) have corresponding identified proteins. Based on the difference in percentage of identified proteins in human sperm, the authors suggested that the RNA in sperm may either be residual RNA of the translated sperm proteins or controlling elements in sperm capacitation, fertilization, or zygotic development. 51
Sperm Proteome Analysis in Different Compartments of the Spermatozoa
Sperm Surface Membrane Proteins
Sperm surface membrane envelopes highly heterogenous structures such as sperm head, mid-piece, and tail (flagellum). Its structural proteins play a key role as the spermatozoa encounter the oocyte for fertilization. The surface membrane proteins undergo complex remodeling during its epididymal transit as well as during initial gamete recognition events of fertilization (capacitation, zona binding, and acrosomal exocytosis). 77 The plasma membrane is organized into several distinctive domains, each with its own composition and function, by its complement of unique testis-specific proteins, and by the addition of secretory proteins originating in the male sex accessory glands as it traverses in the male reproductive tract. 65,78
The complexity of these sequential changes in surface proteins in relation to epididymal maturation of spermatozoa and during initial fertilization events has been demonstrated through several labeling strategies that have employed membrane impermeable tags to facilitate enrichment and identification. For instance, 125 I labeling and biotinylation of surface proteins have been employed in human spermatozoa to detect immunodominant sperm surface antigens. 33,80 -82 But the above-mentioned techniques had its own drawbacks as they were not completely “plasma-membrane proof.” 77 Apart from that, isolation of the surface membrane can be done by specific sperm disruption methods such as ultrasonication and nitrogen cavitation 83 or using lectins immobilized to beads. 84 Beyond these techniques, a more recent method of subcellular membrane fractionation is the isolation of lipid rafts (also known as microdomains or detergent resistant membranes [DRMs]). It was observed that the sperm DRM fraction after capacitation becomes highly enriched in glycosyl-phosphatidyl inositol (GPI)-anchored proteins and in proteins involved in zona binding and the acrosome reaction. 85,86
The biochemical composition of sperm plasma membrane is known to play a key role in the regulation of sperm–oocyte recognition, adhesion, and fertilization. 87 A large number of earlier studies identified several sperm membrane proteins as potential receptors for zona binding, which include tyrosine kinase receptor, 88 protein kinases, 89 integrins, 90 -93 extracellular matrix proteins such as vitronectin, fibronectin, and laminin, 91,94 PH-20, with known hyaluronidase activity, 95 proto-oncogenes such as c-myc 96 and c-ras, 97 and novel protein SPA17. 98 The presence of members of metalloproteinase-like, disintegrin-like, and cysteine-rich (MDC) family proteins on human sperm membrane like transmembrane MDC (tMDC) I (cryritestin) 35 and tMDC III (ADAM 18) 38 has been reported and its role in oolemma binding has also been reported. Similarly, sialic acid-binding glycoprotein (SABP), 25 kDa protein, was reported to play a critical role for in vitro human sperm capacitation. 39 Recently, a sperm protein called GPI-anchored membrane protein, NYDSP8, was identified and it was seen that its release during sperm–egg interaction causes calcium-dependent release of progesterone, which in turn leads to the acrosome reaction. 40
Recent evidence states that HSPs including 60, 70, and 90 migrate to sperm surface and undergo phosphorylation during capacitation since only tyrosine-phosphorylated sperm heads were able to bind to the zona pellucida (ZP). 99 A novel, testis-specific member of the HSP70 family, known as HSPA2, has been identified in mature human spermatozoa, which in coordination with HSPA5 is expressed in the sperm plasma membrane. 42 Based on the studies with low expression of HSPA2 in oligoteratozoospermic men, 43 it has been proposed that human HSPA2 may act as a marker for sperm maturity. 44 It may also play a secondary role in the remodeling of the sperm plasma membrane during spermatogenesis 45 to facilitate the formation of the ZP-binding sites. This proposal takes on added significance in light of recent work that has identified HSPA2 as a major component of spermatozoa and its differential expression of oxidatively stressed sperm compared to normal spermatozoa. 41
Most of the studies on protein composition of human sperm membrane have been mostly centered to identify different surface antigens involved in immunosubfertility and their future use as contraceptive measures. 82,100 -103 About 18 sperm membrane antigens were isolated and identified using antisperm antibodies from seminal plasma of infertile patients by Bohring and his group. 104 However, the first characteristic analysis on this aspect for humans was carried out in 2004, who compared the sperm membrane protein profiles of fertile and infertile men and reported and validated a 57-kDa fertility-associated sperm antigen. 105 In 2001, Shetty et al isolated some 8 novel sperm surface proteins and proposed their role as immune contraceptives. 82
Subsequently, research were undertaken to find the differential expression of sperm plasma membrane protein in infertile patients. In a report on globozoospermia, 2 acrosome membrane proteins like spermatozoa acrosome membrane-associated protein 1 (SPAM1) and SPANX proteins were found to be downregulated in round-headed spermatozoa. 47 Similarly in another study, annexin 5 present in human sperm plasma membrane was detected at decreased levels in the infertile patient samples. 17,48 Even in oxidative-stressed semen sample, SPACA4 was found to be overexpressed. 106 Such isolated studies have documented certain functional aspect of some sperm surface proteins; however, extensive works on identification of clinical relevant surface antigens, the molecular interactions that define domain specific functions, and the nature of the capacitation-induced changes in membrane composition and activity are warranted.
Sperm Nuclear Proteins
Human sperm chromatin modeling during spermiation is accompanied by chromatin condensation where a nucleosomal architecture shifts to a toroidal structure. 106 At the end of spermatogenesis, the nucleosomes are disassembled, and there is gradual replacement of histone proteins by transition nuclear proteins followed by PRMs, to form tight toroidal complexes organizing 85% to 95% of the human sperm DNA, while 5% to 15% of the sperm chromatin remains associated with nucleosomes. 107 By far only the histones and PRMs, as they comprise the sperm chromatin, have been validated to be a part of nongenomic paternal contribution to embryo. 108 -113 However besides these, a portion of the nuclear matrix proteins and perinuclear theca proteins are also inherited by the newly fertilized embryo and can be implicated to have certain epigenetic significance. 114 Since postfertilization, the sperm nucleus is transferred into the oocyte; thus, the study to unravel the nuclear proteins is of utmost importance.
Owing to the epigenetic significance of PRMs and histones, one of the preliminary investigation on nuclear proteins in human sperm detected PRM 1, all PRM 2 (P2-related variants), and testis-specific histone H2B. 6 The most extensive study in the field of sperm nuclear proteomics was conducted by de Mateo et al who isolated pure fractions of human sperm nuclei by cetyl trimethyl ammonium bromide treatment and generated a catalogue of about 403 sperm nuclear proteins, of which a subset of the 159 proteins have been localized in the nucleus according to UniProt and GO. 7 Histones formed the largest family of proteins detected (9.7%, 39 of 403 proteins) and also represented the largest proportion of the nuclear proteins (24.5%, 39 of 159). Various isoforms of histone proteins such as histone cluster 1 H2aa (HIST1H2AA), histone cluster 1 H2ae (HISTH1AE), and histone cluster 1 H2ba (HIST1H2BA) have been identified in the ejaculated spermatozoa having higher levels of ROS. 41 Their presence in the ejaculated spermatozoa has been attributed to oxidative stress and is indicative of improper packaging of sperm chromatin and subsequent DNA damage. Although the ribosomal proteins comprised the second largest family of proteins identified in the isolated human sperm nucleus, it was followed by proteasome subunits, SPANX family, cytokeratins, and elements of the nuclear envelope including the nuclear lamina (LAMA5, BANF1, BANF2, and SYNE1) and the nuclear pore complex and RAN cycle components (NUP37, RAN, and RCC1). The novel family of proteins identified as SPANX is usually found in the sperm head, which is consistent with their current identification in the isolated human sperm nucleus. 59 -61 Of interest, the SPANX genes have been considered as possible male infertility candidates. 61 Furthermore, Castillo et al 8 in his recent review compiled a list of 581 chromatin or nuclear proteins in the human sperm cell. Of these sperm nuclear proteins, 56% were suggested to have the potential epigenetic activity, being involved in at least one of the following functions: chromosomal organization, protein–DNA complex assembly, DNA packaging, gene expression, transcription, chromatin modification, and histone modification.
Sperm Mitochondrial Proteins
Evidence suggests that a mature spermatozoon delivers a wealth of epigenetic information to the oocyte crucial for embryonic development. Although sperm mtDNA is not complexed with PRMs, the absence of protein transcription and translation in mitochondria as per a somatic cell is still a mystery.
13
In regard to this, the addition of chloramphenicol (a specific inhibitor of mitochondrial translation) inhibited the production of 22 proteins, many of which have been shown by others to have a critical involvement in sperm capacitation, including the sperm–egg fusion receptor IZUMO.
51
Further, it was stated that a mature spermatozoa is capable of translating nuclear mRNA by mitochondrial-type ribosomes.
115
Similar studies of differential proteomics conducted by Zhao et al
116
identified 44 proteins with decreased expression in
A recent report documents some protein synthesis activity in mammalian sperm mitochondria, which mainly comprises of complexes I, II, III, IV, and V of the electron transport chain (ETC); electron carriers cytochrome c and ubiquinone; acyl-CoA synthase (ACS)’ adenosine nucleotide translocator (ANT); carnitine acyl transferase (CPT); mitochondrial carnitine/acylcarnitine carrier protein (MCAT); calcium uniporter protein (MCU); permeability transition pore (PTP); and uncoupling proteins (UCP). 117 Some previous studies reported the expression of 3 mitochondrial proteins in human sperm, cytochrome c oxidase I (COXI) and VIc (COXVIc), and DNA POLG. 62,63 In fact, comparative proteomic analysis in patients with asthenozoospermia found a significant alteration in the expression of several sperm mitochondrial proteins. 10,53,54,62,118,119 These changes in expression profile suggest participation of various mitochondrial sperm proteins in regulating essential sperm functions like motility 10,118 and capacitation. 120
Sperm Tail Proteins
Subcellular proteomics in human sperm has gained importance in recent years. After obtaining tail fractions by means of sonication followed by sucrose-gradient ultracentrifugation, and sperm tail peptides were isolated and identified by LC and tandem MS. 117 About 1049 proteins were detected, more than half of which had not been previously described in human sperm. The tail proteins were further categorized into 2 main groups according to their function: proteins related to metabolism and energy production (26%) and proteins related to sperm tail structure and motility (11%). A previous report documented the presence of 721 proteins being exclusively found in the tail of the 1429 identified sperm proteins, the rest 521 being exclusively in the head, and 159 proteins being found in both subcellular fractions. 66
Functional Role of Different Sperm Proteins
Major studies on sperm proteomics have been mostly concluded in just isolating different proteins and at most identifying them. However, unraveling the organizational complexities and physiological function is more or less not done. The latter is generally done either by comparing the differential expression pattern of the respective protein in infertile patients or based on GO.
Studies by Martinez-Heredia et al21 categorized 98 proteins on the basis of their functional role such as energy production (23%), transcription, protein synthesis, transport, folding and turnover (23%), cell cycle, apoptosis and oxidative stress (10%), signal transduction (8%), cytoskeleton, flagella and cell movement (10%), cell recognition (7%), metabolism (6%), and unknown function (11%). 21 Even in the studies of Sharma et al, 41 in case of ROS+ and ROS− spermatozoa, the functional profile of the identified proteins based on GO analysis showed a striking difference. Proteins responsible for processes such as cellular amino acid metabolic processes, cellular component biogenesis, chromosome organization, cytoskeleton organization, embryo development, gluconeogenesis, and homeostatic processes were found to be overexpressed, while the proteins involved in the processes such as carbohydrate catabolic processes, cellular component movement, glycolysis, and response to unfolded protein restricted only to the underexpressed proteins.
Sperm Proteins Involved in Initiation and Regulation of Motility
Motility is considered to be a key function of a good quality spermatozoon since the spermatozoa need to travel through the female reproductive tract to the site of fertilization but despite this accepted fact there has been a paucity of information on the molecular basis of sperm motility. On a simple note, the problem of identifying proteins involved in sperm would be by inhibiting motility but this approach is not always free of ambiguities. The inhibition of sperm motility may not necessarily be due to the effect on a single protein and may involve a cascade of proteins.
In a pilot study to characterize the proteins associated with sperm motility-related male factor infertility, 10 differentially expressed proteins were identified namely, ρ-guanosine diphosphate dissociation inhibitor 1 (ρGDI 1), outer dense fiber (ODF) protein, isocitrate dehydrogenase subunit α, phosphoglycerate mutase 2, triosephosphate isomerase, glutamate oxaloacetate transaminase 1, carbonic anhydrase II, SEMGIpre, GLUL, and 26 S protease regulatory subunit 7. 10 ρGDI 1 and ODF, identified in this study, are associated with sperm structure, and the absence of ODF protein would result in nonfunctional tails. 64 The overexpression of ODF in the study can thus be regarded as a biomarker for male infertility. Further 2 testis-specific tyrosine-phosphorylated proteins associated with the fibrous sheath structure in the tail of human sperm have been identified, A kinase anchoring protein 3 (AKAP3) family and calcium-binding tyrosine phosphorylation regulated (CABYR). 65 The AKAPs and CABYR are found to be associated with several multiprotein complexes such as the RII dimerization/docking domain of the protein kinase A regulatory subunit which in turn regulates the energy supply for flagellar movement. In fact, in the studies of Xu et al, AKAP4 a dominant protein in the ODF structures of the sperm flagellum is proved to be playing a key role in regulation of motility 67 and is seen to be downregulated in infertile patients. 17 The fibrous sheath is an intensively cross-linked, highly stabilized structure that is relatively insoluble, 121 while the underexpression of this fibrous sheath protein in infertile patients is an indirect reflection of cell integrity.
In patients with globozoospermia, low expression levels of ODF2 in round-headed spermatozoa would cause tail fragmentation leading to reduced motility 47 as ODF proteins may be necessary for the maintenance of the elastic properties of the spermatozoa tail and may provide tensile strength that is necessary to protect the spermatozoa tail against shearing forces encountered during epididymal transport and especially during ejaculation. 122,123 An increase in both semenogelin (I and II) and its precursor forms, that is, SEMGIpre and SEMGIIpre, was detected in infertile patients with normozoospermia. 17 Semenogelin has the capacity to inhibit major sperm function such as motility 124 and sperm capacitation. 52 Similarly, defects in axonemal components such as tektin and dyneins were shown to be the cause of sperm immobility. 68
Proteins Involved in Energy Metabolic Pathways
Proteins involved in various metabolic pathways such as glycolysis and gluconeogenesis, tricarboxylic acid cycle, oxidative phosphorylation, and glycogen metabolism pathways have been identified by various researchers in the human spermatozoa. In the studies of Martínez-Heredia et al, 4 proteins of the “energy production” group were differentially expressed in the asthenozoospermic group.
54
Although COX6B expression was decreased, the other 3 dihydrolipoamide dehydrogenase (DLD), fumarate hydratase (FH), ▵3,5-▵2,4-dienoyl-CoA (ECH1) existed in precursor form and were overexpressed. Their presence in their precursor form suggests a generalized posttranslation processing problem of these proteins in the patients with asthenozoospermia. The potentiality of these proteins lies in the adenosine triphosphate production in spermatozoa, and increase in precursor form attributes to decrease in mature protein, which could be related to the failure of tail movement in the spermatozoa from patients with asthenozoospermia.
10,54
Further based on the findings of Sharma et al,
41
key enzymes of carbohydrate metabolism were found to be differentially expressed in oxidatively stressed sperm. Although phosphoglycerate kinase 2 and glyceraldehyde phosphate dehydrogenase-S were underexpressed in the ROS+ group, the glyceraldehyde-3-phosphate dehydrogenase, along with fructose-biphosphate aldolase A and MDH2, was overexpressed. Additionally, 2 enzymes involved in the fermentation of pyruvate to lactate—
Sperm Proteins Associated With Protein Folding and Turnover
Protein folding is important for protein stabilization, function, and protection against degradation. In this regard, the 26S proteasome complex needs mention whose function is to degrade cytosolic and nuclear proteins initially labeled with ubiquitin molecules. 125 Low levels of proteasome might result in accumulation of ubiquitinated proteins, which has been negatively correlated with sperm motility. 126 Differential expression of the proteasome complex on asthenozoospermia samples has been reported, suggesting an importance of the proteasome complex in sperm motility. The proteasome subunit α type 3 is decreased in asthenozoospermic sample in the findings of Siva et al 53 in contrast to Martínez-Heredia et al reported that proteasome β3 subunit human is increased in asthenozoospermic samples. 54 There is a likelihood that there is the existence of connecting piece defects in the spermatozoa of patients with asthenozoospermia, which may lead to loss of centriolar proteins like proteasome and is being represented through the differential expression of the proteome analysis of these samples. 53
Sperm Proteins Involved in Capacitation and Fertilization
Capacitation is a process of activation that leads to hyperactivated motility, which facilitates the sperm–oocyte interaction, binding, and preparation for the acrosome reaction to penetrate the ZP. However, in the absence of apparent protein syntheses, most of the protein changes concomittant to capacitation are likely to be due to the posttranslational modification of existing proteins. One of the most common posttranslational modifications is phosphorylation. Sperm protein phosphorylation is an important event necessary to achieve capacitation. Hyperactivated sperm flagella showed the presence of tyrosine phosphorylated proteins, an indication of its close association with capacitation. 127 -129 In this regard, endoplasmic reticulum resident protein 57 gains importance for its presence in the acrosome and tail region and its translocation to the equatorial segment during acrosome reaction. Further findings reveal that it undergoes posttranslational modification during sperm capacitation and thus plays a critical role in gamete fusion. 69
Key cytoskeletal proteins such as actin undergoes remodeling during sperm capacitation and acrosome reaction. 130 In this context, during human sperm capacitation, a network composed of ezrin, members of Ras homolog gene family (RhoGDI 1 and Rho A), F-actin, and membrane proteins undergo modifications to have a characteristic impact on the fluidity of the sperm membrane so as to promote capacitation. 79 In an interesting study, Secciani et al 16 compared the proteomes of freshly ejaculated and capacitated sperm and observed differential expression of certain proteins in the capacitated ones. The proteins that were underexpressed included tubulin β-2C chain, ODF1 AKAP4, HSPA2, were those involved in protein metabolism, and flagellar organization; on the contrary, proteins associated with cellular stress were upregulated.
Another study, where the detergent-resistant membranes in capacitated sperm was targeted, allowed the identification of 100 proteins, many of which were found to be involved in sperm–oocyte interaction. 56 Nitric oxide acts as an inducer of capacitation and its activity has also been studied to identify 240 S-nitrosylated human sperm proteins. 57 The cystein-rich secretory protein (CRISPs) family members are associated with sperm–oolemma penetration. 58 Crisp-1 comprises of 2 functional domains amino terminal pathogenesis-related-like domain and a carboxyl terminal cysteine-rich domain with 2 potential functions of Crisp-1, 1 in sperm–egg fusion and 1 in regulation of capacitation. 12 CRISP1 is infact secreted in an androgen-dependent manner in the dorsal epididymis and then relocated to the sperm head. CRISP1, epididymal sperm-binding protein 1, and acrosin were overexpressed in the spermatozoa of high DNA fragmentation reveals of a compensatory mechanism of the possible damages and alterations suffered by viable cells from an ejaculate of lower quality, allowing sperm fertilization capacity to remain intact. 55
As mentioned earlier HSP70, a sperm surface protein, has also been shown to be important during fertilization and embryo development in humans. 131 In the studies of Lima et al, it has been demonstrated that adolescents with varicocele and unaltered semen when compared to age-matched controls presented an overexpression in the HSPA2 gene, not observed in adolescents with varicocele and altered semen. 46 Furthermore, this protein was shown to be conserved in the high sperm DNA fragmentation in comparison to the low sperm DNA fragmentation group, suggesting that HSPs may indeed play a protective responsive role in male fertility, maintaining the fertilization potential. 55 Fibronectin have been identified in extracts of ejaculate human spermatozoa, 132,133 along with the presence of vitronectin on spermatozoa. 134 These proteins are known to act as a molecular velcro, binding spermatozoa to the egg through ligation of oolemmal integrins. 135 Fertilin is another sperm surface protein, which is one of a conserved family of related cysteine-rich proteins, now designated as ADAM (a disintegrin and a metalloproteinase), and is known to play an important role in sperm–egg interaction in mammals through its interaction with oolemmal integrins. 49,36,37 Family members of SPANX-A, which are expressed in postmeiotic spermatids, may play a role in acrosome biogenesis and are downregulated in globozoospermic sperm. 47
Candidate Biomarkers and Challenges in Sperm Proteomics
In the postgenomic era, studies have mainly focused on the identification of novel protein biomarkers in complex biological systems. A biomarker is a distinctive biological or biologically derived indicator of a process, event, or condition and is considered ideal if it serves the purpose of screening, diagnosis, and monitoring disease activity. In addition to that they may hold responsible for targeted therapy or would assess therapeutic response. 136 With regard to male factor infertility, the main objective of a biomarker is to evaluate in an accurate and minimally invasive manner, a man's potential to father a child. This review summarizes important studies on sperm proteomics and thus concludes that several proteins may be selected as putative biomarkers that are indicative of a state of male infertility. With relation to oxidative stress in the spermatozoa, proteins such as MDH2, TGM4, GPX4, GLUL, HSP90B1, and HSPA5 are suggested as possible biomarkers due to their increased expression in ROS+ samples compared with those without oxidative stress. 137 In case of asthenozoospermia, most importantly DLD, FH, and ECH1 can act as putative biomarker due to the overexpression of precursor form of these proteins. 54 Similarly, in the case of globozoospermic abnormality, axonemal protein such as ODF2 and 2 acrosome membrane proteins like SPAM1 and SPANX proteins may act as candidate biomarker due to the differential expression of these proteins in the pathogenic condition. 47 Whatever the case might be, the use of semen for proteomic analysis is complicated because it contains sperm as well as seminal plasma. Another important challenge sperm proteomics faces today is spermatozoa undergoes distinct physiological changes after ejaculation, which further complicates the process. Proteomic analysis proves to be advantageous as it confirms the qualitative and quantitative measure of a protein within the spermatozoa. The challenge therefore lies in identifying ideal biomarkers by being able to distinguish relevant proteins from their chemically modified forms within the setting of such a complex milieu. Studies on proteomic profiling of spermatozoa result in the generation of huge amount of data. The information so generated mainly comprises of complex protein compositions and a large number of isolated proteins whose implications in the biological world is yet to be discovered. This limits the use of proteomic approaches to elucidate the role of biological markers. 138 Moreover, variability in proteomic profiles from patients with particular infertility forms a significant drawback in the use of this technique. The various underlying causes for this variability arises from the fact that different proteomic strategies such as the in-gel digestion or in-solution digestion of proteins, database searches, and so on are used for their analysis. 139 In proteomic analysis, different database search algorithms can result in different protein identification, including qualitative and quantitative differences. 140 Furthermore, one of the major contributor to this variability would be infertile individuals may have multiple underlying conditions. Posttranslational modifications within the spermatozoa further complicates the fact.
Epilogue
The MS technologies are constantly being updated and thus more sperm proteins will certainly be identified in the next couple of years. The complexity and diversity of sperm proteins are revealed through its posttranlational modifications and thus warrants further investigations that would better explain the functional activities of the proteins of interest. With advancing technology, it is hoped that MS techniques is developed further in reducing the complexity of dealing with the translated proteins. 139 Over the last decade, there has been great innovation in MS technology coupled with the development in bioinformatics. This forms the basis of identification of molecular signatures in relation to pathogenic spermatozoa according to proteomic profiles and thus may become standard practice in the clinical laboratory. 141 Nevertheless, it is important that these new practices should be cost-effective for its wider implications among patients from different socioeconomic classes. Simultaneously, it is equally important to follow a standardized test protocol among all clinical settings in order to reduce variability and maintain uniformity of data.
Earlier, it was critical to describe what proteins constitutes the human spermatozoa or contributes to its function. Now with the elucidation of sperm protein repositories, we nearly have this complete knowledge. So, it is time to translate sperm protein lists into functional interactomes. In this context, Amaral and her coworkers have made an attempt to determine which cellular pathways are expected to be active in sperm through the analysis of the compiled sperm proteome. 70 Furthermore, future experiments should aim in deciphering the role of some of the sperm proteins whose functions still remains unclear such as the proteins relating to RNA metabolism and protein synthesis. The ability to predict fertility using biomarkers is a promising field. Proteomic studies might help the development of new techniques in order to identify novel biomarkers for a better clinical diagnosis and treatment of male infertility.
The preceding discussion emphasizes the fact that a suite of proteins are intimately involved in modulating the production and functional activity of mammalian spermatozoa. The differential expression of proteins in patient group can be attributed not only to altered expression of respective genes but also due to altered transport into the spermatozoa from the epididymal, seminal, or prostatic fluid. No doubt, current information about fertility-related proteins forms a tool to develop diagnostic or prognostic protocols. Further efforts are warranted to unravel their physiological roles as well as establish new diagnostic methods for infertility. Preferably, epididymal proteins are targeted for contraception over testicular proteins, as the blockage of sperm maturational events will not obstruct spermatogenesis and testicular endocrine function, moreover, reversible fertility is ensured. 142,143 Fertilization events both pre and post can be affected by targeting the sperm head and the flagellar protein. This necessitates the identification of sperm proteins on different domains, which are acquired by sperm mostly as they traverse the epididymis. Proteomic technologies have matured significantly in recent years and undoubtedly have broadened our knowledge of the spermatozoa over the past decade. The key challenge is thus to move from lists of identified proteins to informed understanding of biological function. The availability of several catalogues for sperm proteins paves the way for valuable research, but it is just a mark of the beginning of a new era. Once the sperm proteome is characterized, the next important step would be the determination of function of these proteins as till date there has been several proteins whose function is still unknown. Since it is evident that the conventional semen parameters is of limited clinical utility, proteomic study of the spermatozoa may offer a great potential for the development of biomarkers for sperm function.
Footnotes
Acknowledgments
GM thanks Department of Science and Technology, Government of India for providing INSPIRE Fellowship.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.