Post-Translational Modifications in Sperm Proteome

Biochimica et Biophysica Acta 1860 (2016) 1450–1465

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Biochimica et Biophysica Acta

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Review Post-Translational Modiﬁcations in sperm Proteome: The Chemistry of Proteome diversiﬁcations in the Pathophysiology of male factor infertility

Luna Samanta a,b, Nirlipta Swain b,AhmetAyaza, Vijay Venugopal a, Ashok Agarwal a,⁎ a American Center for Reproductive Medicine, Department of Urology, Cleveland Clinic, Cleveland, OH 44195, USA b Redox Biology Laboratory, Department of Zsoology, School of Life Sciences, Ravenshaw University, Cuttack – 753003, Odisha, India article info abstract

Article history: Background: The spermatozoa undergo a series of changes in the epididymis to mature after their release from Received 22 October 2015 the testis and subsequently in the female reproductive tract after ejaculation to get capacitated and achieve fer- Received in revised form 26 March 2016 tilization potential. Despite having a silenced protein synthesis machinery, the dynamic change in protein profile Accepted 4 April 2016 of the spermatozoa is attributed either to acquisition of new proteins via vescicular transport or to several post- Available online 6 April 2016 translational modifications (PTMs) occurring on the already expressed protein complement. Scope of review: In this review emphasis is given on the PTMs already reported on the human sperm proteins Keywords: under normal and pathologic conditions with particular reference to sperm function such as motility and fertil- Spermatozoa Proteome ization. An attempt has been made to summarize different protocols and methods used for analysis of PTMs on Post-translational modifications sperm proteins and the newer trends those were emerging. Infertility Major conclusions: Deciphering the differential occurrence of PTM on protein at ultrastructural level would give Male factor us a better insight of structure-function relationship of the particular protein. Protein with multiple PTMs could be used to generate the complex interaction network involved in a physiological function of a sperm. It can be speculated that crosstalk between different PTMs occurring either on same/ other proteins actually regulate the protein stability and activity both in physiological and pathological states. General significance: The analytical prospective of various PTMs reported in human spermatozoa and their rele- vance to sperm function particularly in various pathophysiological states, would pave way for development of biomarkers for diagnosis, prognosis and therapeutic intervention of male infertility. © 2016 Elsevier B.V. All rights reserved.

1. Introduction of defective post-translationally modified proteins and their substrates can lead to various human diseases and ailments. (See Table 1.) A proteome is the entire set of proteins that is or can be expressed by Mass spectrometry is one of the fundamental tools used to identify a cell, tissue, or organism at a given time. A cell proteome is much larger and analyze PTMs and quantify their changes. Modern proteomic and more complex than its respective encoding genome. Various factors approaches have made large-scale screening of complex mixtures of determine the structure and function of a cell proteome, including post- proteins possible, allowing us to precisely detect any chemical modifica- translational covalent modification of proteins. From a genetic point of tions. There are different branches of modification-specificproteomics view, any differences between the primary polypeptide sequence gen- based on the pattern of protein alteration–phosphoproteomics erated from translation and the final structure of a fully functional pro- for phosphorylation studies, diglycine remnant proteomics for tein are considered post-translational modification (PTM). These PTMs ubiquitination, degradomics for protein fragmentation, and redox pro- are chemical alterations in the protein structure typically catalyzed by teomics for oxidative damage to proteins [1–3]. exceedingly substrate-specific enzymes that regulate protein stability, Sperm essentially do not synthesize proteins. Thus, the dynamic na- distribution and function. Any failure to control such complex molecular ture of sperm proteome depends either on acquisition of new proteins processes could be detrimental or fatal to a cell because large amounts by vescicular transport or PTMs of already manufactured proteins. Therefore, PTMs on existing proteins play a pivotal role in regulating sperm function including maturation and acquisition of fertilizing potential. Of the 300 covalent modifications reported, phosphorylation, ⁎ Corresponding author at: Lerner College of Medicine, Andrology Center, American nitrosylation, glycosylation, methylation, lipid modification, Center for Reproductive Medicine, Cleveland Clinic, Mail Code X-11, 10681 Carnegie Avenue, Cleveland, OH, 44195, United States. ubiquitination, and acetylation are the most common [4]. Even under E-mail address: [email protected] (A. Agarwal). oxidative stress conditions, reactive oxygen species (ROS)-induced

Table 1 A list of post-translational modiﬁcations of human sperm proteins and their role in sperm function.

Sl. No. PTM Modiﬁed Protein in Human sperm Related function References

1 Phosphorylation Histone Sperm chromatin condensation [9,10] P1, P2 [13,19] Histone variants (TH2B, H2X, H1t) [20,21] [10,22], Bad, GSK-3β, HSP27, JNK/SAPK, mTOR, p38MAPK, p53 Sperm motility [30] AKAPs, F895 Capacitation [28,35,36] VCP, NSF, HSPA2 Zona binding [38] IZUMO1 Sperm-egg fusion [49] 2 Acetylation Histone Sperm chromatin condensation and epigenetic code [54,57,58,59] P1,P2 [19] 11 Tubulin proteins, ODF2, and Tektin proteins (TEKT3 and TEKT4) Sperm motility [58] 9 proteins of NADH dehydrogenase complex and 10 proteins of ATP synthase complex AKAP, CABYR, proteasome Capacitation [52,58] ACRBP, ACRV1, VDAC3 Acrosomal reaction [58] PDIA3/ERp57, ACE, ZPBP [52] RPL10A and RPL3 Ribosomal cytoplasmic protein [52] 3 Glycosylation SAGA-1 Sperm egg adhesion [74] GLIPR1L1, Acrosin, ADAM2 [64] IZUMO [75] GPX4 Antioxidant protection [64] 4 Nitrosylation HSPs, Tetkin, Tubulin, Semenoglein Sperm motility [82] Rys-R calcium channel Mobilization of Ca+2 stores in sperm [83,84] 5 Methylation Histone (H3) Epigenetic code [59] 6 Ubiquitination Histone (H2A, H2B) Histone replacement [179]. Prohibitin Marking defective spermatozoa for degradation [121] 7 SUMOylation Histone (H4) Sex chromosome silencing [127] SCP1, SCP2 Synaptonemal complex scaffold maintenance [122] RanGap1 Silencing gene translation [130,183] DRP1 Mitochondrial functioning [130] Topoisomerase IIα Chromatin condensation [183] Tubulins, ODF3 or SHIPPO, AKAP3 and AKAP4, Sperm motility [123] HSP70, HSP60, HSPgp96, GST, TCP1 complex proteins Stress related protein [123] RNE, SPAN-X, ubiquitin, VCP, Sperm maturation [123] Semenoglein Capacitation [123]

protein modifications such as glutathionylation, nitro- tyrosine and special reference to their role in capacitation, motility, hyperactivation, dityrosine formation have been reported [5]. acrosome reaction, sperm-zona binding and egg activation [9]. The primary focus of this review is to provide an overview of the var- During epididymal transit, histone phosphorylation is a primary PTM ious PTMs reported in human spermatozoa and to discuss their rele- that regulates the chromatin remodeling process. Phosphorylation- vance to sperm function, particularly in various pathophysiological dephosphorylation of the sperm histone occurs during mammalian sper- states. In addition, we will also discuss the potential of using proteomic miogenesis and is most likely involved in the proper condensation of analysis to develop biomarkers for the diagnosis, prognosis and thera- spermatid chromatin [10,11]. Phosphorylation of specific histone variants peutic management of male infertility. may protect the chromatin from proteolytic degradation during the nucleohistone-to-nucleoprotamine transition and may later help in rapid activation of specific genes post fertilization [12,13].Furthermore, 2. Post-translational modifications (PTMs) in sperm phosphorylated protamine 1 (P1) and protamine 2 (P2) comprise a significant portion of the human sperm nuclei although they are present in 2.1. Phosphorylation

Phosphorylation alters the structural conformation of a protein by introducing a covalently bound phosphate group to an amino acid (aa), especially tyrosine (tyr), serine (ser), and threonine (thr) of the polypeptide chain (Fig. 1). Because a charged and hydrophobic element gets added at the R group of the modified aa, nearby aa are affected as well, and it is these interactions that switch on or off the activity of a protein. In fact, the process of protein phosphorylation is regulated by the collaborative actions of protein kinases (PK) and protein phosphatases (PP). The former catalyzes the addition of the phosphate group while the latter facilitates its removal. Phosphorylation is one of the major PTM controlling sperm function, especially epididymal maturation, motility, capacitation and acrosomal reaction. In fact, levels of tyr phosphorylation increase in specificcom- partments of human spermatozoon following capacitation and zona binding [6]. The sperm head is predominantly phosphorylated during early capacitation [7] followed by the principal piece at the later part of capacitation and then the neck piece once the sperm binds to the Fig. 1. Phosphorylation on protein. Tyrosine, Serine and Threonine residues on proteins zona pellucida [8]. Kwon et al., have documented tyrosine phosphoryla- equally are susceptible to phosphorylation, however for convenience only Serine is tion and phosphotyrosine proteins in mammalian spermatozoa with shown here. 1452 L. Samanta et al. / Biochimica et Biophysica Acta 1860 (2016) 1450–1465 smaller amounts than their non-phosphorylated forms [14].Infact,prot- are also identified in the flagella of human spermatozoa, which means amines are immediately phosphorylated post synthesis in spermatids, but they may play a similar role. Other important proteins undergoing tyr most of the phosphate groups are removed post DNA binding [11,15], phosphorylation during capacitation in human sperm are valosin- which helps them to bind along the DNA groves [16]. However, as the containing proteins (VCP), a homolog of the SNARE-interacting protein spermatid differentiate into spermatozoon at a later stage of chromatin NSF, and two members of the AKAP family: AKAP 3 and AKAP 4 [39]. condensation, complete dephosphorylation of protamines occurs [11, Furthermore, in human spermatozoa, tyr-phosphorylation also occurs 15]. This leads to complete neutralization of the phosphate-ribose back- in the head as a subsurface event during early capacitation stages and bone by increasing the protamine’s DNA binding constant [17]. The un- is correlated with a sperm’s ability to undergo the acrosomal reaction bound cysteinyl residues of dephosphorylated protamines now become [7].Tyr phosphorylation starts at the tip of the sperm tail and then pro- cross-linked either by S-cysteinylation or Zn2+-coordination, which ceeds through the neck [8]. In fact, attainment of functional competence generate a supercondensed, crystal-like sperm nucleus [18].P2issug- during capacitation is marked by high levels of tyr phosphorylation in gested to play a role in this cycle of protamine phosphorylation- pre-existing sperm proteins [38], which makes tyr phosphorylation a dephosphorylation [19].AstudybyCastilloetal.confirmed the presence hallmark of capacitation. The Ca2+ ion acts as a powerful, potential reg- of phosphorylation sites in both P1 and P2 in mature human sperm, ulator of tyr phosphorylation in sperm. Extracellular Ca2+ is reported to namely P1S8p, P1S10p, P2S14p [20]. And according to the authors, the ob- decrease intracellular availability of ATP in sperm, suppressing tyr ki- served phosphorylated protamine in motile human sperm is a character- nase activity and thus inhibiting the tyr phosphorylation cascade during istic feature of the species and accounts for the heterogeneity of capacitation [40]. chromatin condensation, which is absent in other mammalian sperm. On binding to the zona pellucida (ZP), the capacitated sperm Multiple phosphorylation sites have also been reported in non- undergoes the acrosomal reaction (AR) by the exocytotic release of en- canonical histone variants, namely TH2B [21,22],H2AX[11],H1t[23]. zymes. This reaction enables the sperm to penetrate the ZP and subse- One especially significant change mediated by protein phosphoryla- quentlyfusewiththeeggoolema. tion is the sequential development of motility, which begins during ep- Two sperm receptors, a Gi-coupled receptor (Gi protein) and a tyr ki- ididymal transit and culminates post capacitation in the female nase (TK) receptor, bind to the ZP and are regarded as initiators of the reproductive tract. Sperm tail movement is regulated by precise phos- phosphorylation cascade [41,42].Gi protein activates membrane- phorylation patterns in various parts of the flagellum and axonema, bound enzymes such as phospholipase C (PLC) and adenylate cyclase which is coordinated by the action of PK and PP [24,25]. Two signaling (AC) and increases generation of the second messengers such as pathways in particular are involved in the above process- the cyclic cAMP, inositol trisphosphate (IP3) and diacylglycerol (DAG). On other AMP-dependent protein kinase A (cAMP–PKA) [26,27] and Ca 2+/ other hand, TK is autophosphorylating and activates coupled PLC, gen- calmodulin-dependent protein kinases (CaM kinases) [28,29]. The for- erating IP3 and DAG. Increased production of second messengers leads mer is controlled by the bicarbonate (HCO3-) and calcium ion (Ca2+) to the activation of protein kinases such as cAMP-dependent kinase levels in the luminal epididymal fluid and female reproductive tract. In- (PKA) and Ca2+ and phospholipid-dependent kinase (PKC). In human sulin like growth factor 1 receptor (IGF1R) and Insulin Receptor (INSR) sperm, PKA [43] and PKC [44] phosphorylate a series of proteins, espe- were found to be the most enriched tyrosine phosphorylation kinases in cially Ca2+ -channels, in the outer acrosomal membrane (OAM) and human sperm that interacted with the up-regulated phosphorylation the plasma membrane (PM), respectively. Because the spermatozoa substrate sites during sperm capacitation andwere also responsible for needs the Ca2+ influx to proceed further, both PKA and PKC target the hyperactivated motility [30]. Another study found that phosphorylated Ca2+ -channels [45]. A sustained enhancement of cytosolic Ca2+ in levels of several proteins (Bad, GSK-3β, HSP27, JNK/SAPK, mTOR, p38 the sperm cell results in activation of tyr phosphatases (PTPs) that de- MAPK, and p53) were significantly correlated with motility parameters phosphorylates tyr on actin. This leads to dispersion of the F-actin barri- [31]. er intervening between the OAM and the overlying PM as well as Capacitation involves a fast- and a slow- process for maintenance of subsequent fusion of the two membranes and acrosomal release [32]. sperm motility. The fast process involves activation of vigorous and Further, intracellular Ca2+ activates RAS-associated protein RAB3A asymmetric movement of the flagellum whereas the slow process in- and causes dephosphorylation of N-ethylmaleimide-sensitive factor volves alterations in flagellar movements from a symmetrical to a (NSF) by PTPs [46]. Once dephosphorylated, NSF together with synapto- hyper-activated state [32]. Both the events are regulated by a HCO3-/ some associated protein (α-SNAP) changes the assembly of NSF attach- SACY/cAMP/PKA pathway and involve entry of HCO3- and Ca2+ ions ment protein receptors (SNAREs) from cis to trans conformation, into the cell through a Na+/HCO3-cotransporter (NBC) and a sperm- causing the acrosomal membranes to fuse with sperm surface mem- specificCa2+ channel (CatSper), respectively [33]. These ions coordi- branes and the acrosomal contents to be released. nately stimulate a unique type of adenylyl cyclase (SACY) that activates Phosphorylation is also known to play a regulatory role in activat- PKA by generating cAMP and leads to phosphorylation of ser/thr resi- ing proteins needed for sperm-ZP binding. Tyr phosphorylation in dues and subsequently tyr residues [26,33]. Human sperm capacitation human sperm is strongly correlated with sperm-ZP binding capacity also requires down-regulation of ser/thr phosphatase for a functional but not with the ZP-induced AR [47]. Strong species-specific PKA-dependent phosphorylation [34,35]. differences exist in the molecular mechanisms that drive sperm– The slow process is triggered by the removal of cholesterol from the egg recognition. For example, in mice, the appearance of tyr sperm membrane and an increase in membrane fluidity [32].ProteinA- phosphorylated chaperone proteins on the sperm surface during kinase anchoring proteins (AKAPs) localized on the fibrous sheath of capacitation, facilitates sperm–ZP recognition [48]. One the other human sperm–namely AKAP82, its precursor pro-AKAP82, and FSP95– hand, in human spermatozoa, no such surface expression of are the most prominent tyr phosphorylated proteins during capacita- phosphotyrosinated chaperone proteins was initially detected [49]. tion [29,36,37]. AKAP interacts with additional signaling molecules But the testis specific chaperone HSPA2 is later reported to be tyr such as SRC in the sperm flagellum, and the latter is a key intermediate phosphorylated during capacitation and help in zona binding via for- kinase in promoting tyr phosphorylation [38]. Additionally, the physical mation of protein complexes [50]. Another interesting protein interaction of PKA and SRC in capacitated (but not uncapacitated) sper- whose phosphorylation status regulates its role in sperm- oocyte fu- matozoa supports their role in the capacitation signaling cascade, espe- sion is the IZUMO1. IZUMO 1 undergoes both N-terminal glycosyla- cially in coordinating the spatial and temporal activity of signaling tion in extracellular domain as well as tyr phosphorylation in C- pathways downstream of PKA. terminal tail [51]. The phosphorylation status of IZUMO1 varies dur- During capacitation, phosphorylation of Y416 residue activates SRC ing sperm epididymal migration in rats [52]. Moreover, in post-AR in human sperm. Other SRC family kinases such as LYN, YES, and FYN mouse sperm, IZUMO 1 covers the entire sperm head and interacts L. Samanta et al. / Biochimica et Biophysica Acta 1860 (2016) 1450–1465 1453 with its counterpart, JUNO, in the oocyte [53]. In humans, phosphor- shown that they contain acetylated H3 as well [54,61].Becausetheacet- ylation of IZUMO 1 may help regulate fertility status [51]. ylated histones have no reported functional significance, they may represent a sperm-specific epigenetic code. Post fertilization, these 2.2. Lysine Acetylation acetylated histones may act as activation markers associated with specific regions of the sperm genome and influence the embryonic gene Protein acetylation refers to the catalytic transfer of an acetyl moiety [13,54,61]. from acetyl CoA to a free amino group of the target protein. Lysine acet- Aberrant hyperacetylation of human sperm is reported to be associ- ylation, in which the ε-amino group of a lysine (lys) residue on a poly- ated with male infertility caused by spermatogenic impairment. A re- peptide accepts the acetyl moiety, is a reversible dynamic PTM cent work reported an association between acetylation-associated catalyzed by lys-acetyl transferases (KATs) (Fig. 2). Likewise, removal enzyme activity and sperm DNA fragmentation index (DFI) in ejaculat- of the acetyl group is catalyzed by deacetylases (KDACs). Considering ed human spermatozoa [59]. Although no direct association between the diverse lys-deacetylases that have been identified in the human DNA damage and histone acetylation was reported, a positive relation- sperm proteome [54], lys-acetylation most likely plays an important ship between sperm DFI and histone acetyltransferases (HAT) activity role in sperm function. Proteasomal degradation of core histones affect- was found. If histones are hyperacetylated, the histone-bound DNA ing chromatin organization and axonemal microtubule construction af- may be less tightly compacted and therefore more susceptible to DNA fecting sperm motility are regulated by reversible lys-acetylation. damage. Acetylation weakens the DNA-histone interaction by neutraliz- During post-meiotic maturation in male germ cells, the sperm chro- ing the positive charge of the lys residue. Thus, a positive correlation be- matin undergoes extensive compaction with sequential replacement of tween sperm DFI and HAT activity could be an indicator of severe DNA histones with transition proteins and then protamines. Global histone damage in spermatozoa. Elevated sperm DNA damage is associated hyperacetylation prior to histone replacement causes localized relaxa- with a late paternal effect during activation of the male genome, tion of the chromatin, creating open chromatin domains. A study of disturbing normal embryonic development [61]. mouse spermatids suggested that the N-terminal tail of histone H4 The sperm tail is composed of the axoneme (consists of 9 + 2 becomes highly acetylated during histone replacement [55]. Because arrayed microtubules), outer dense fibers (ODF), and the mitochondrial acetylation adds negative charges to nucleosomes, it is possible and fibrous sheath (FS). Eleven microtubule proteins (TUBA1A, TUBA1C, that acetylation of H4 loosens chromatin fibers to enhance histone re- TUBA3C, TUBA4A, TUBA8, TUBB, TUBB2B, TUBB3, TUBB4, TUBB4B, and placement. In addition to histone H4, other core histones are also TUBB6) were reported to have lys-acetylation sites [60].Thereversible 40 hyperacetylated at specific stages of spermatogenesis, and the pattern tubulin acetylation at K site is reported to be specific for regulating mi- of histone acetylation is similar between the mouse and humans [56]. crotubule stability in the flagella of spermatozoa [63]. In fact, the differ- Massive histone hyperacetylation facilitates recruitment of ential expression of TUBA3C and TUBA4A was crucial for the regulation proteasomes specific for histone degradation [57]. Qian et al. showed of α-tubulin acetylation in sperm, ultimately governing its motility [64]. that most proteasomes in mammalian testes (spermatoproteasomes) Other proteins such as ODF2 and Tektin 3 (TEKT3) and Tektin 4 are activated by PA200, a 200 kD nuclear proteasome activator (TEKT4), which are components of the sperm tail related to sperm mo- protein and catalyze the polyubiquitin-independent degradation of tility, were also reported to be acetylated [60]. Moreover, treating sper- histones. Acetylation on histones is required for their binding to matozoa with lys deacetylase TSA and NAM as well as anti-acetyl-lys the bromodomain-like regions in PA200 and its yeast ortholog, antibody significantly reduced motility [60]. Nine acetylated proteins Blm10facilitating the degradation of the core histones by of complex I (NADH dehydrogenase complex) and ten acetylated pro- spermatoproteosomes. [58]. teins of proton-transporting ATP synthase complex of mitochondrial Using an advanced proteomic approach, Yu et al. identified 4 poten- electron transport chain were identified in ejaculated human spermato- tial sites of acetylation in histones of mature human sperm: H4K8ac, zoa [60]. Because both the complexes are essential for the generation of H4K12ac, H4K16ac and H3K9ac [54]. Moreover, both protamine types ATP, which is needed for sperm movement, lys-acetylation seems to be P1 and P2 in mature human sperm have been reported to have acetyla- one of the regulatory factors for sperm motility. tion sites [20]. Annotation of the lys-acetylproteome of capacitated human sperma- However, site-specific lys-acetylation of histones observed in matozoa revealed that acetyated proteins are also associated with several ture ejaculated sperm [54,59–61] suggests that it plays a role regulating other sperm functions including energy metabolism, capacitation, acro- embryonic development. Earlier studies showed that human sperm some reaction, sperm–egg recognition and fusion. A considerable num- contain acetylated H4 [62]. Newer studies using proteomics have ber of proteins that have lys-acetylation sites were found to be involved in glycolysis and the TCA cycle [54,60]. Capacitation-associated proteins, namely AKAP, CABYR and proteasomes as well as AR-associated proteins, namely acrosin binding protein (ACRBP), acrosomal vesicle protein 1 (ACRV1) and voltage-dependent anion channel 3 (VDAC3), all have lys acetylation sites [54,60]. Additionally when anti-acetyl lys antibody was used in a mouse IVF inhibition assay, fertilization was significantly inhibited [60]. PDIA3/ERp57, angiotensin I converting enzyme (ACE) and zona pellucida binding protein (ZPBP) also are acetylated [60]. Apart from these, ribosomal cytoplasmic proteins (60S) RPL10A and RPL3, which have been isolated from human sperm nuclei [65], are also present in the acetylproteome [54], suggesting that lys acetylation acts as a regulator of several physiological functions of human spermatozoa.

2.3. Glycosylation

Glycosylation is one of the most diverse forms of structural PTM. In this process, a sugar moiety is attached by glycosidic bonds to an Fig. 2. Lysine Acetylation on protein. KATs: Lysine acetyl transferases; KDACs: Lysine amino acid of the protein sequence. There are two types of linkages; deacetylase. 1) N-glycosylation characterized by the addition of an oligosaccharide 1454 L. Samanta et al. / Biochimica et Biophysica Acta 1860 (2016) 1450–1465 group to Asparagine (asp) residue via N-Acetylglucosamine linker; and sperm. N-linked glycoproteins such as CD52 [76], GLIPR1L1 [66], and 2) O-glycosylation characterized by the addition of a monosaccharide IZUMO [77] have been reported to play an important role in sperm- group to ser and thr groups via N-Acetylgalactosamine linker (Fig. 3). egg adhesion. The glycosylated proteins on the sperm surface recognize In the context of sperm, carbohydrate-rich molecules on the surface ZP proteins. The sialyl-Lewis(x) sequence is the major carbohydrate li- form a thick layer of glycocalyx dominated by the N-linked glycopro- gand responsible for sperm–oocyte binding [78]. Proteins such as teins [66]. acrosin [79] and ADAM2 [80], which mediate sperm-oolemal fusion in Glycosylation of sperm proteins may occur either during testicular humans, also contain N-glycosylation sites [66]. The mitochondrial anti- synthesis or epididymal maturation. However, most studies suggest that oxidant enzyme Glutathione peroxidase 4 (GPX4) expressed in human during epididymal transit, sperm proteins undergo glycosylation or de- sperm [81] is also reported to be a N-linked glycan [66]. glycosylation to become functional. Epididymal luminal fluid is reported to contain two sets of glycan-modifying enzymes: (1) glycosyltransferases 2.4. S-Nitrosylation (GalTases), a class of synthetic enzymes that add sugar residues from a donor sugar (nucleotide sugar) to the acceptor site(s) on existing S-Nitrosylation, a selective, temporal and spatially regulated PTM, molecules and (2) glycohydrolases or glycosidases, a class of hydrolytic involves the covalent incorporation of a nitric oxide (NO) moiety into enzymes that cleave sugar residues from existing glycoconjugates [67]. thiol (-SH) groups, which are frequently found on cysteine residues of In humans, both GalTases [68] and a variety of glycosidases (β-D- the protein (Fig. 4). In sperm, S-nitrosylation mainly occurs in the mannosidase, α-L-fucosidase and N-acetyl-β-D-glucosaminidase) [69] female reproductive tract where the sperm is exposed to higher concen- have been found in seminal plasma. These enzymes add or modify the trations of NO produced from oviductal cells. Although both endothelial glycan linkages of the already existing proteins in sperm. and neuronal forms of nitric oxide synthase (endothelial, eNOS and According to Arenas et al, terminal sequences of oligosaccharide neuronal, nNOS) are detected in human sperm [82,83], S-nitrosylation side-chains in spermatocytes and, principally, spermatids are: fucose, is more likely to be caused by NO produced from cumulus cells rather mannose, Neu5Ac2, 3Gal1, 3GalNAc, Gal1, 3GalNAc, Gal1, 4GlcNAc, than being an autocrine effect of endogenous sperm NO generation. Neu5AcGalNAc and GalNAc (in O-glycosylated proteins); mannose (in During capacitation in human sperm, TUBB2C, TPIS, HSPA2 and N-glycosylated proteins); and GlcNAc (in both protein types) [70].Asi- PSMA3 undergo S-nitrosylation [84]. alic acid residue is added to galactose and GalNAc residues. S-nitrosylation modulates sperm motility. Human sperm proteins Although glycosylation of sperm proteins occurs mostly during epi- related to motility such as heat shock-related proteins (HSPs), didymal maturation, there are some testicular glycosylated proteins tektin, tubulin and semenogelin 1, are S-nitrosylated [84]. The S- that undergo deglycosylation in the epididymis in order to become nytrosoproteome in human sperm seems to regulate the mobilization functional. For example, in mice, basigin (bsg)–a transmembrane of calcium stores in the sperm neck/midpiece (probably by activating glycoprotein–is localized on the surface of the sperm tail in the epidid- RyRs calcium channels) and enhance the flagellar beating [85,86]. ymis and transported to head post capacitation [71]. Testicular bsg is a RyRs are intracellular Ca2+ channels containing a large number of sulf- glycosylated protein that undergoes molecular processing and deglyco- hydryl groups and are thus subjected to S-nitrosylation. sylation during its transit in the epididymis. Spam1 is another such protein whose activity is regulated by deglycosylation during epididymal 2.5. Methylation maturation [72]. Furthermore, a large number of glycoproteins are also incorporated into the sperm membrane form epididymal secretions Methylation is the transfer of methyl groups to amino acids in pro- [73]. One such important epididymal glycoprotein inserted on human teins; the process involves methylating enzymes or methyl transferases sperm surface is the sperm agglutination antigen-1 (SAGA-1), a (Fig. 5). Typically in sperm, methylation is associated with the retained human male reproductive tract glycoform of CD52 [74]. It has a histone proteins playing a role in epigenetic signaling. Moreover, sever- glycosylphosphatidylinositol-anchor (GPI-anchor) attached at the C- al proteins related to sperm motility also become methylated. terminus and N-linked glycans that are bound to the single potential Methylation of histones is catalyzed by the enzyme histone methyl- glycosylation site at position 3 [75].Inonestudy,CD52glycoformswere transferase (HMTases), which usually targets a lys or arginine (arg) present on both the sperm head and tail, yet only the N-glycan of the residue. Several HMTases such as histone H3 lys 9-selective methyl- head CD52 had zona or oolemma receptors [76]. Under capacitating transferases (Suv39h HMTases) have testis specific expression [87], conditions, the core peptide opens, particularly the GPI anchor [76],thus which suggests that methylation is one of the major PTM of histone dur- elucidating its potentiality in mediating sperm binding to oolemma. ing spermatogenesis. High levels of H3K4me1, H3K9me1, H3K9me2, and Because N-linked glycoproteins are considered pivotal for sperm -- H3K27me3 have been detected in ejaculated human sperm [61].Infact, egg interactions and fertility, they have been extensively studied in some reports have suggested that six lys-methylations on histones 3 and 4 are central to epigenetic mechanisms that activate specific paternal genes in the early embryo [88,89]. It was further elucidated that levels and distribution of post-translationally modified histones and protamines in human sperm are related. A strong correlation was observed between the levels of P2 and H3K9me2 [61]. Protein O-carboxylmethylation occurring in the tail region is another type of protein methylation that occurs in mature spermatozoa. Sev- eral earlier reports explored the protein O-carboxylmethyl transferase (PCM) activity of differentiating male germ cells in the testis and of spermatozoa and found that protein O-carboxylmethylation plays a possible role in the regulation of tail movement [90].Thiswasalso demonstrated in several earlier studies of different mammalian models like the rat, rabbit, bull and hamster [91–94]. PCM catalyzes the methylesterification by S-adenosylmethionine of dicarboxylic amino acid residues in proteins. PCM appears to be the only type of protein methyltransferase that is present in human ejaculated mature spermatozoa [95,96]. Furthermore, methyl acceptor proteins (MAP) have also Fig. 3. N-linked glycosylation on arginine residues on proteins. been detected in human sperm [97], making the PCM-MAP system L. Samanta et al. / Biochimica et Biophysica Acta 1860 (2016) 1450–1465 1455

Fig. 4. The process of S-nitrosylation on proteins one of the most crucial factors involved in spermatozoal motility in by the proteasome (Fig. 6). Ub is a small protein linked to other proteins humans. Another study found PCM activities to be correlated with the via an isopeptide bond between the C-terminal glycine of Ub and the motility index in human spermatozoa [95]. amino group on the side chain of a lys residue on the target protein. The protein ubiquitin proteasome pathway (UPP) is an ATP- 2.6. Ubiquitination dependent mechanism involving a three-step enzymatic process. Con- jugation of Ub to its target protein requires the hierarchal transfer of Ubiquitination involves the modiﬁcation of target proteins through Ub from an E1 ubiquitin-activating enzyme (ATP-dependent) to an E2 the covalent attachment of ubiquitin (Ub), leading to its degradation ubiquitin conjugating enzyme and ﬁnally to the target protein

Fig. 5. Methylation of argine residues on proteins by protein arginine methyltransferases (PRMTs). Adomet: S-adenosyl-L-methionine (methyl group donor), AdoHcy: S-adenosyl homocystein; MMA(ω): ω-NG-monomethylarginine; ADMA: ω-NG,NG-dimethylarginine; SDMA: ω-NG,N’G-dimethylarginine. 1456 L. Samanta et al. / Biochimica et Biophysica Acta 1860 (2016) 1450–1465

degradation sites for nucleoproteins generated during the condensation of chromatin. While ubiquitination typically occurs intra-cellularly, studies have shown that the UPS is also functional extracellularly, acting on spermatozoa during their epididymal transit [109,110].Specifically, the defective and abnormal sperms are ubiquitinated, which serves as a way to maintain ubiquitin-dependent sperm quality control [110,111].Ubis secreted by the principal cells of the epididymal epithelium into the epididymal lumen [112] and becomes a part of the seminal plasma [113]. The exact mechanism by which ubiquitination machinery recognize defective spermatozoa at molecular level has yet to be deciphered. How- ever, it has been proposed that the ubiquitin-dependent sperm quality control residing in the epididymal epithelium can detect defective spermatozoa, especially those with apoptotic or necrotic DNA [110].In- creased sperm ubiquitination was found to be inversely associated with sperm count, motility and % normal morphology and directly correlated with the percentage of spermatozoa with abnormal chromatin [111,114,115]. All these results support the use of Ub-crossreactive Fig. 6. Schematic representation of Ubiquitination of proteins. Conjugation of Ub to its sperm surface proteins as a biomarker of human semen quality. Fur- target protein requires the hierarchal transfer of Ub from an E1 ubiquitin-activating thermore, spermatozoa that undergo proper ubiquitination may have fi enzyme (ATP-dependent) to an E2 ubiquitin conjugating enzyme and nally to the a higher chance for fertilization because they are made redundant by target protein facilitated by an E3 ubiquitin ligase. the UPP in the epididymis compared to hypo-ubiquitinated spermatozoa [116]. Additionally, studies on the sperm proteasome have indicated that facilitated by an E3 ubiquitin ligase [98,99]. The protein substrate, post fertilization is somehow controlled by a gamete-associated extracellular mono-ubiquitination, can either be released for its specific purpose or UPP, as proteasome-mediated proteolytic events participate in mam- undergo polyubiquitination to add on a series of ubiquitin molecules malian sperm-zona pellucida penetration [117–119]. Thus, it is possible linked to the first ubiquitin. The usual consequence of such to detect various proteasomal subunits present on the plasma mem- ubiquitinated tagged proteins is degradation by the 26S proteasome, brane surface of human sperm [120]. Finally, Ub may also tag mamma- completing the UPP. In contrast, sometimes the deubiquitination en- lian sperm mitochondrial proteins for destruction in the zygote, zymes cleave the ubiquitination substrates from the polyubiquitin contributing to the generally exclusive maternal inheritance of the mi- chain or depolymerize polyubiquitin so that the substrate proteins are tochondrial genome [121,122]. The mitochondrial protein ‘prohibitin’ released and become functional again. is one of the ubiquitinated substrates that allows the sperm mitochon- The ubiquitination-proteasome system (UPS) mainly and largely dria to be recognized by an egg's ubiquitin-proteasome dependent pro- mediates protein turnover in spermatozoa preventing unnecessary ac- teolytic machinery after fertilization and most likely facilitates the cumulation of defective or unwanted proteins. During spermatogenesis, marking of defective spermatozoa in the epididymis for degradation the UPS serves to fulfil two major functions; one is modifying histones [123]. as they are replaced with protamines, and the second is maintaining sperm quality by proteolytic elimination of abnormal spermatozoa. 2.7. SUMOylation The mechanism and functional significance of histone ubiquitination in human spermatids have not been explored much but nevertheless, SUMOylation is a PTM in which Small Ubiquitin-like Modifier it is well typified in rat and mouse models. As the progenitor cells un- (SUMO) proteins are covalently attached to and detached from other dergo spermatocytogenesis, H2A becomes ubiquitinated, which is be- proteins in cells to modify their function. SUMO proteins are similar to lieved to be important for the silencing of sex chromosomes in the XY Ub, and SUMOylation is directed by an enzymatic cascade analogous body [100]. The ubiquitinated H2A (ubH2A) is localized mainly in the to that involved in ubiquitination. SUMO is also linked to proteins via XY bodies of primary pachytene spermatocytes [101], which is a struc- an isopeptide bond between the C-terminal glycine (gly) of Ub and ture of the pseudoautosomal region with gene transcription silenced. the amino group on the side chain of a lys residue on the target protein. This phenomena is known as meiotic sex chromosome inactivation However, in contrast to ubiquitination, which is largely but not always (MSCI) [102] and is the mechanism to prevent unpaired region of X associated with proteolytic pathways, SUMOylation does not target pro- and Y chromosomes from initiating cell death. MSCI is postulated to teins to degradation. All three major isoforms of mammalian SUMO be important for the completion of the meiotic process because disrup- proteins–viz, SUMO1, SUMO2 and SUMO3 (with 95% similarity be- tion of MSCI leads to the arrest of spermatocytes at the pachytene stage tween SUMO2 and 3, these mostly referred to as SUMO2/3)–have of meiotic prophase [103]. been detected in human sperm [124,125], which suggests that they Besides meiosis, spermatid histones H2A and H2B are also play a role in modulating sperm function. ubiquitinated as they are transiently replaced by transitional proteins One of the major functional attributes of SUMOylation in sperm is and then permanently by protamines [101,104]. Proteolytic degrada- regulating the non-homologous chromosomal synapsis and XY body tion of histones is facilitated by global ubiquitination, which occurs dur- formation. It also plays a role in meiotic sex chromosome inactivation. ing the postmeiotic stages of spermatogenesis(i.e., spermiogenesis) and In rodents as well as human spermatids, variability in the timing and marks the massive chromatin remodeling events [105]. The different concentration of SUMO-1 to the sex body was predictive of the possible testis specific ubiquitin E3 ligases involved specifically in the UPS- involvement of SUMOylation in the MSCI and XY body formation [126, aided metamorphic change are UBR2 and RNF8 [106,105,107]. 127]. Although premeiotic-to-early meiotic cells demonstrated a low- Haraguchi et al. [108] reported the presence of polyubiquitinated pro- to-undetectable level of SUMO-1, it could be observed on gonosomal teins in nuclei (nuclear dense area) of ejaculated human spermatozoa chromatin at the zygotene stage [126,128,129]. At this stage, the chro- and proteasome in the nuclear vacuoles and clear spots. Based on mosome homologues pair and the sex chromatin condensation is initi- their results, they hypothesized that the UPS is more actively operation- ated. And finally, as the human spermatocytes progress toward the al in the nuclear vacuoles of human sperm, which can be considered the endofprophaseofthefirst meiotic division, SUMO-1 can no longer be L. Samanta et al. / Biochimica et Biophysica Acta 1860 (2016) 1450–1465 1457 detectable within the sex body and pericentromeric heterochromatin its involvement in the formation/repair of DNA breakage [132].Both and instead becomes localized exclusively to the centromeres [129]. SUMO1 expression and co-localization with the three proteins were sig- It has been further proposed that SUMO-1 is not involved in faculta- nificantly higher in morphologically abnormal sperm [132],furthersug- tive heterochromatinization of human pachytene spermatocytes as ob- gesting that sumoylation is a marker of defective sperm. served in mouse spermatocytesbecause it does not accumulate on the Using proteomic data, several SUMOylation targets directly involved human XY body., It is possible that a balance is necessary between his- in the regulation of sperm motility and other functions have been iden- tone sumoylation and ubiquitination—one that is maintained in a highly tified [125]. These include proteins associated with motility namely tu- specialized manner–in order to facilitate SUMO-1-associated silencing bulins, outer dense fiber protein 3 (ODF3 or SHIPPO), and A-kinase of the sex chromosomes [127]. While SUMOylation is usually associated anchor protein 3 and 4 (AKAP3 and 4) as well as several glycolytic with transcriptional repression, and ubiquitination is generally correlat- and mitochondria enzymes related to pathways of energy metabolism ed with increased transcriptional activity, SUMO may act as blockers for including ATP synthase. Several stress-related proteins were additional- ubiquitin attachment sites [130].Consistentwiththispostulate,SUMO- ly identified as SUMO targets, including two members of the heat shock 1 was found to be associated with phosphorylated H2AX, ubiquitinated protein 70 family (HSP70 proteins 2 and 4), HSP60, HSPgp96, glutathi- H2A, and/or other post-translationally modified histones [127].More- one S-transferase and several proteins of the chaperonin-containing over, histone H4 is reported to be sumoylated in human sperm, and TCP1 complex. Moreover, several proteins associated with the redun- methylated H3 and P1 have been found in SUMO-1 positive areas dant nuclear envelope (RNE) are identified as possible targets of [129].Suchfindings suggest that SUMO-1 may be involved in multiple SUMO. RNE is reported to play a role in sperm maturation and fertiliza- cross-talk interactions involving various post-translationally modified tion events; 90% of the RNE proteins are SUMOylated. These cluster of histones in the XY region, thereby, contributing to MSCI. proteins mainly include SPAN-X (human sperm protein associated Additionally, SUMO-1 may function in the organization of with the nucleus mapped to the X chromosome), ubiquitin, VCP pericentromeric and centromeric heterochromatin domains in both (Valosin-containing protein), and semenogelins (involved in regulation round and elongating spermatids [124,126]. During spermiogenesis, of capacitation) [125]. SUMO-1 is localized in chromocenters of certain round spermatids and in the perinuclear ring and centrosomes of elongating spermatids. However, the role of SUMO-1 in microtubular nucleation and nuclear 3. Proteomics analysis of Post-translational Modifications in sperm reshaping as was seen in rodent models could not be validated in human spermatocytes. In another study, SUMO-2/3 were also observed Approximately 6198 different proteins have been identified in in human pachytene spermatocytes [124]. It was further seen that in the human spermatozoa to date [134], and these account for about 78% of testis of fertile adult men, synaptonemal complex protein SCP1, SCP2 the sperm cell proteome. Subcellular proteomic analyses of human were SUMOylated by SUMO-1, suggesting that it helps maintain the spermatozoa revealed that of 1429 proteins, 721 proteins are exclusive- SC scaffold [124]. All such studies confirm the regulatory action of ly found in the tail and 521 exclusively in the head, which provided SUMO proteins in spermatogenesis. novel insights into the compartmentalization of the human sperm pro- In human ejaculated sperm, several SUMO-1 and SUMO-2/3ylated teome [135]. A comparative account of the sperm proteome with its re- proteins are present [125,131].While only SUMO-1 is reported to be lo- spective metabolome suggested that these proteins are involved in calized in the head region [131], both SUMO1 and SUMO2/3 can be seen various functional pathways such as metabolism, apoptosis, cell cycle, in the neck area of sperm and found at detectable levels along the whole meiosis, motility and membrane trafficking [136]. length of the sperm flagella [125]. SUMOylation of human spermatozoa While the importance of PTM has been well established, the ability has been mostly regarded as an indicator of poor semen quality. The to directly determine the sites of PTM within sperm proteins in percentage of SUMOylated spermatozoa is inversely correlated with human sperm and to quantitate the extent of PTMs at these sites is a total and progressive motility [131]. Furthermore, SUMOylation is also recent development that has relied almost exclusively on advances associated with poor sperm morphology [125] and motility. Nonmotile, in the field of proteomics. In comparison to traditional protein malformed, microcephalic (small head) and acephalic (no head) sperm microsequencing tools or antibody based assays, MS is more efficient have significantly higher levels of sumoylated proteins relative to their and accurate. Although highly sensitive, the prerequisite of antibody normal counterparts. based assays is the prior knowledge of the type and the site of modifica- SUMO pathways are also involved in responses to DNA damage. A tion, which becomes its major limiting factor. positive correlation is observed between SUMO1-ylation and sperm The major hurdle faced in the detection of PTM in proteins is that the DNA fragmentation (SDF) [132]. When SDF was induced by stressful proteins are lost when they are digested into peptides during isolation. conditions (freezing and thawing and oxidative stress), SUMO1- For this reason, gel-based proteomics using 2DE gel electrophoresis ylation increased. Based on these findings, SUMOylation may be useful (DIGE) analysis may be more effective due to its high resolving power as an additional marker of defective spermatozoa during microscopic and ability to visualize PTMs directly, especially phosphorylation and sperm analysis. glycosylation of a given protein [137].Quantification and identification In order to understand the functional basis of SUMOylation in sperm, of proteins can be done with a combined DIGE approach using a the roles of different target proteins of SUMO need to be characterized. mixed internal standard followed by in-gel digestion and MS/MS of RanGap-1 (Ran GTPase-activating protein 1), one of the main targets of only the chemically modified proteins [138]. For further confirmation SUMO in somatic cells, is reported to be localized in the post-acrosomal of the nature of PTM, DIGE can be coupled with western blot and region of the head as well as in the neck area [132,133]. SUMOylation of other such techniques. RanGap-1 may play a role in silencing gene translation [133].DRP1 Nevertheless, gel-based proteomics has its own weaknesses that can (Dynamin-related protein 1), whose SUMOylation in somatic cells pro- be overcome using gel free proteomic approaches such as liquid chro- vokes alterations of mitochondrial functions, is seen to co-localize with matography tandem mass spectrometry (LC-MS/MS). In these tech- SUMO1 in the mid-piece of sperm as expected [132].Withpossible niques, instead of focusing on entire protein complement, only a anomalies in mitochondrial functioning due to SUMO-modified DRP1, subset of peptides are targeted. The prefractionation of pepetides can this protein may represent the link between SUMO and poor motility. be performed on an IPG strip followed by enrichment of targeted pep- Topoisomerase II alpha, which is necessary for chromatin condensation, tides for a particular PTM and then LC-MS/MS [52]. Enrichment of mod- is co-localized with SUMO-1 in the sperm nucleus [133]. An augmenta- ified peptides is necessary to avoid ion suppression effects during tion in co-localization and co-immunoprecipitation of SUMO1- simultaneous examination of proteolytic digests with/without PTMs in Topoisomerase Iiα was observed under stressful conditions, suggesting MS [23]. Because ionization of modified peptides is generally less 1458 L. Samanta et al. / Biochimica et Biophysica Acta 1860 (2016) 1450–1465 efficient than ionization of their unmodified counterparts, their chance important for fertility or sperm function. Additionally, the annotation of detection is compromised. of metabolic pathways showed significant enrichment of lys acetylated In spermatozoa where protein synthesis machinery is silent, proteins (125 proteins) in sperm energy metabolism and carbohydrate metabolic labeling strategies (e.g., SILAC) commonly used for metabolism [60]. relative quantitation in other cell types has limited effectiveness. None- In another study by Yu et al. [54], 973 lys-acetylated sites were iden- theless, alternative label-free strategies have been used in sperm tified that matched with 456 human sperm proteins, including 671 phosphoproteomic studies [139]. In an earlier study, a technique for novel Kac sites and 205 novel lys acetylated proteins [54].Using phosphoprotein microsequencing utilizing MS involved alignment of a Motif-X (http://motif-x.med.harvard.edu), the authors found that protein spot on a gel known to be phosphorylated with a corresponding these proteins exhibited 3 conserved motifs, i.e., XXXKYXXX, western blot; it is then cored, in-gel digested and identified using tan- XXXKFXXX, and XXXKHXXX where X denotes any amino acid. The dem MS (MS/MS). Naaby-Hansen et al. [140] used the above technique protein-protein interaction of the identified acetylated proteins was in- and identified five groups of surface proteins phosphorylated in tyro- vestigated using consensusPathDB (http://consensuspathdb.org/), sine residues. In an attempt to identify proteins that are phosphorylated PINA- Protein Interaction Network Analysis (http://cbg.garvan.unsw. during capacitation, DIGE coupled to anti-phosphotyrosine immuno- edu.au/pina/) ,HiPPIE- Human Integrated Protein-Protein Interaction blots was outperformed followed by MS/MS along with immobilized reference (http://cbdm.mdc-berlin. de/tools/hippie/ information.php) metal ion affinity chromatography (IMAC) for unbiased phosphopep- and dbPTM (http://dbptm.mbc.nctu.edu.tw/). In the annotated activi- tide enrichment [39]. This study identified 5 tyr, 56 ser and 2 thr phos- ties, catalytic activity ranked first followed by oxidoreductase activity. phorylation sites in AKAPs and VCP. A similar type of study was KEGG analysis revealed almost all key enzymes of glycolysis and TCA undertaken on rat epididymal spermatozoa where IMAC was done cycle are lys-acetylated. One hundred and ninety-one proteins were lo- using TiO2 to enrich phosphopeptidome and to study the protein phos- cated in sperm mitochondria, 108 in the membranes, 79 in the acro- phorylation changes associated with mammalian sperm maturation somes, 13 in the nuclei, 12 in the flagellum and 5 in the microtubules. during epididymal transit [141,142] and capacitation [141]. Recently, The identified lys acetylated proteins in the different proteomic studies Wang [30] employed label-free quantitative phosphoproteomics to in- were found to be essential for sperm motility and fertilization. A prote- vestigate the overall phosphorylation events and identified functional omic study of PTMs in histones revealed that levels of H3ac are lower kinases during human sperm capacitation. A total of 3,303 phosphory- than levels of H4ac, with both being present at detectable levels in the lated sites, corresponding to 986 phosphorylated proteins, were tail [61]. Acetylation in P1 of human sperm was reported for first time recognized using IMAC-TiO2 phosphopeptide continuous enrichment by Castillo et al. using MS [20]. methods by LC-MS/MS, of which the phosphorylation levels of 231 The most extensive study of the glycoproteome of human sperm sites (including 25 tyrosine sites) were increased significantly. was performed by Wang et al. [66]. Enrichment and deglycosylation of Data were analyzed by comparing the isolated sites with the N-Linked glycosylated peptides were performed using the methods of PhosphoSitePlus® database (www.phosphosite.org) [143]. Further- Zielinska et al. [146]. Eluted peptides were then analyzed using MS/ more, another proteomic analysis identified 12 protein spots as having MS coupled with the glyco-FASP. To identify the reactive domains and differential phosphorylation including gamma-tubulin complex associ- involved pathways, DAVID (http://david.abcc.ncifcrf.gov/home.jsp) ated protein 2 (GCP2) in sperm between healthy and aberrantly motile was used; and WebLogo 3 (weblogo.threeplusone.com/)wasemployed groups A similar type of comparative study was undertaken using Nano to create relative frequency plots of the aligned N-glycosylated peptide UPLC-MS, which identified 66 phosphoproteins that were differentially sequences. In this study, the authors reported 554 N-glycosylated sites regulated [64] In order to characterize the identified phosphoproteins and 297 N-glycosylated proteins corresponding to 295 Ensembl genes, involvement in relevant signal transduction pathways, a well- which were recognized to be a part of the human sperm N- illustrated schematic representation was drawn by Park et al. [145] glycoproteome. The majority of the latter (91.2%) were present in the and his group using MedScan Reader (v5.0) and Pathway Studio “membrane”, “extracellular region”, and “lysosome” and functionally (v9.0). The above representation helps to elucidate the molecular role responsible for cell recognition and fertilization. Moreover, analyzing of different phosphoproteins and their relation to other proteins as well. the INTERPRO and PROSITE domain compositions of human sperm gly- The lys acetylproteome of capacitated human sperm was deter- coproteins using DAVID, the authors identified four classes of lectins, in- mined through immunopurification enrichment using an anti-acetyl cluding L-type lectins, mannose 6- phosphate receptors with P-type lys antibody and high-throughput LC-MS/MS. Sun et al. [60] performed CRD (carbohydrate recognition domain), C-type lectin-like proteins, sequence motif analysis using iceLogo and characterized 1206 lys acet- and I-type lectins. These lectin-linked glycoproteins in sperm are poten- ylated sites corresponding to 576 lys acetylated proteins in human ca- tial candidates for egg binding. pacitated sperm. GO annotations using ToppGene database (https:// The S-nitrosoproteome was critically analysed by Lefievre et al. toppgene.cchmc.org/) and enrichment analysis of metabolic pathways [84]. The S-nitrosylated proteins in human spermatozoa were based on KEGG using WebGestalt were performed to interpret the bio- detected using the biotin switch assay and identified using MS/MS. logical significance for a specific protein list. Furthermore, the protein- Cellular distribution and functional analysis were assessed using protein interaction network was analyzed using the database STRING- Gene Ontology (GO) database (www.geneontology.org/). After Search Tool for the Retrieval of Interacting Genes/Proteins (string-db. exposing spermatozoa from normozoospermic men to NO donor, S- org/). Mapping of the location of lys acetylated sites to the functional nitrosoglutathione the authors detected 240 S-nitrosylated proteins. domains in each protein was done using the SMART (Simple Modular Of the 240 proteins 52 (22%) including tubulin, glutathione-S-trans- Architecture Research Tool_ database (smart.embl-heidelberg.de/). Bio- ferase and heat shock proteins but also novel targets including A- informatic analysis showed that these proteins were associated with kinase anchoring protein (AKAP) types 3 and 4, voltage-dependent sperm function, including motility, capacitation, acrosome reaction anion-selective channel protein 3 and semenogelin 1 and 2 were al- and sperm–egg interaction. The 576 identified acetylated human ready established as targets for S-nitrosylation in other cell types. sperm proteins were mapped to 533 Entrez genes. Annotation of cellu- The authors also documented S-nitrosylation on 3 sperm-specific lar components by ToppGene showed the localization of acetylated AKAPs, heat shock proteins particularly HspA2, RyRs (intacellular gene products in mitochondria (153 genes), microtubules (33 genes), calcium channels) and sperm specific glycolytic enzymes GAPHD flagellum (21 genes), nucleoplasm (25 genes), nucleosomes (9 genes), and LDH which warrents further study because of their known sig- cytosol (13 genes) and plasma membranes (8 genes) and the zona pel- nificance in sperm biology. Furthermore, out of all the proteins al- lucida receptor complex. The network of sperm-associated biological ready known to be necessary for sperm-egg interactions only 3 processes indicated 30 lys acetylated proteins with known functions were detected by the authors to be S-nitrosylated. Thus, it could be L. Samanta et al. / Biochimica et Biophysica Acta 1860 (2016) 1450–1465 1459 inferred that S-nitrosylation was not necessary for fertilization Current approaches for the evaluation of sperm abnormalities are focus- per se. ing on different proteomic studies. As the underlying causes of male infer- The only proteomics study on sperm methylation was undertaken by tility remains largely unknown, molecular factors such as the absence or Krejci et al. [61]. The study investigated the variability and distribution abnormal expression of some essential sperm proteins could provide an- pattern of histone PTMs in human sperm. LC-MS/MS and MALDI-TOF swers. Moreover, anomalies in the modifications of normally expressed MS were performed to measure levels of H3K4me1, H3K9me1/me2, proteins can also result in malfunctioning of various pathways relevant H3K9ac, H3K27me3, H3K36me3, H3K79me1, and H4ac in spermatozoa to important sperm functions. When the PTM patterns in spermatozoa from 17 donors. Histones H3 and H4 were isolated and characterized by of normozoospermic and fertile men are studied in comparison to MALDI-TOF MS according to the procedures by Cincarova et al. [147] pathophysiologically compromised sperm protein from infertile men, and Legartova et al. [148] with some minor modifications. The mass spec- structural anomalies in expressed proteins could be revealed. Such differ- trometric RAW data fileswereanalyzedusingtheProteomeDiscoverer ential alteration patterns in PTM could be used as an effective diagnostic software (1.4, Thermo Fisher Scientific) with in-house Mascot (2.4.1, strategy to better understand male factor infertility. Matrixscience). Mascot MS/MS ion searches were performed against the Azoospermia is defined as the complete absence of spermatozoa in UniProtKB protein database for Homo sapiens taxonomy downloaded on the ejaculate, invariably resulting in infertility; however, its presence 2014-01-01 or against cRAP contaminant database (downloaded from does not necessarily imply sterility [151]. In fact, azoospermia has http://www.thegpm.org/crap/) and an in-house histone database of the been recognized as one of the most intriguing topics in male infertility. group in parallel. Peptide identifications were manually verified, and Azoospermia is divided into two major categories: obstructive azoo- quantitative data evaluation for selected peptides was performed using spermia (OA) in which there is genital tract outflow obstruction Skyline quantification software (Skyline 2.6.0.7176). LC-MS/MS analysis blocking passage of sperm, and non-obstructive azoospermia (NOA) in indicated that levels of H3K4me1,H3K9me1, H3K9me2, and H3K27me3 which the testicle fails to produce mature sperm in the ejaculate were quite high in human spermatozoa, and these observations are con- [152]. Two PTM seemed to be associated with azoospermia; sistent with an MS analysis of post-translationally modified histones in ubiquitination and acetylation. Impaired production of different en- mouse sperm [13].H3K27me3 was also detected in the mid-piece and re- zymes belonging to UPS, namely UBE2B protein (an ubiquitin- mainder of tail in human sperm. conjugating enzyme), can cause defects in spermatozoa maturation The sperm ubiquitinome has not been comprehensively identified and may result in NOA [153,154]. Moreover, novel mutations in genes by any one group. Studies on the sperm ubiquitinome would certainly coding for testis-specific ubiquitin proteases such as USP26 [155] and shed light on how protein turn over in spermatozoa is regulated at USP9Y [156] were observed in azoospermic patients, suggesting that various stages such as maturation, capacitation and fertilization. protein ubiquitination plays a role in azoospermia. On the other hand, Vigodner et al. [125], carried out a proteomic characterization of in the OA cases, spermatids exhibited hyperacetylation in histone H4 different SUMOylated proteins in human ejaculated sperm. Nanospray [157]. Moreover, in oligozoospermic patients having ≤15 X106 sperm/ LC-MS/MS was performed, and raw data files were created with Prote- mL of seminal ejaculate [158], UPS might play some regulatory role. Al- ome Discoverer 1.2, merged and searched against the human NCBI data- teration in the production of ubiquitin-conjugating enzyme UBE2B base (May 27, 2011) using the in-house Mascot Protein Search engine. [154,159] and protease USP26 [160] in oligozoospermic semen samples Mass spectrometric analysis specifically identified 55 SUMO targets in suggesting its possible involvement. sperm, including major flagella proteins, proteins involved in sperm Asthenozoospermia (AZS) is a common cause of male infertility and maturation and differentiation, heat shock proteins and important gly- is characterized by sperm motility of less than 40% [158]. Several studies colytic and mitochondrial enzymes. The functional distribution of the have looked at the potential involvement of altered protein phosphory- proteins demonstrated that: 18% of the total identified proteins were lation in the pathogenesis of AZS and reported a direct link between the implicated in stress response and protein folding, 22% were cytoskeletal former and aberrant sperm motility. Proteomic analysis identified 12 and flagella proteins, 22% were proteins previously implicated in sperm protein spots showing differential phosphorylation patterns in AZS maturation, differentiation, acrosome reaction and cell-cell recognition, samples, which included the gamma-tubulin complex associated pro- 22% were proteins involved in transcription, RNA-binding, translation, tein 2 (GCP2) [144]. Similarly, Parte et al. [64] identified 66 differentially histones and 16% were glycolytic and mitochondrial enzymes. More- expressed phosphoproteins in AZS sperm that were mostly composed over, the identified targets included proteins with specificfunctionsin of the HSPs, cytoskeletal proteins, proteins associated with the fibrous germ cells and sperm, including heat shock-related 70-kDa protein 2, sheath, and those associated with energy metabolism. This further outer dense fiber protein 3, β-tubulin, A-kinase anchor protein 3 and suggests that altered phosphorylation can be linked to reduced motility 4, l-lactate dehydrogenase C, sperm protein associated with the nucleus as observed in AZS spermatozoa. Furthermore, a deficiency in tyr on the X chromosome B/F, valosin-containing protein, ras-related phosphorylation of tail proteins, especially those related to protein Rab-2A, proacrosin binding protein sp32 precursor, acrosomal hyperactivated motility, was also reported to be associated to AZS vesicle protein, seminogelins, histone H4, and ubiquitin. [161]. Hypophosphorylation of proteins during capacitation in these During the last two decades, hundreds to thousands of sperm pro- groups was thought to be caused by decreased membrane fluidity [162]. teins have been identified for a variety of purposes including to develop In two separate proteomic studies, several proteins containing S- novel biomarkers for diagnosis of fertility status and complete contra- nitrosylation sites were found to be differentially expressed in AZS ception [149]. However, it is apparent from the discussion vide supra sperm [163,164]. Similarly, testis-specificacetylableα-tubulin isoform that the sperm proteome is dynamic and proper physiological function- TUBA3C and TUBA8 were decreased, and TUBA4A was increased in ing of the sperm proteins requires PTM (Fig. 7). Thus, it is obvious that AZS as compared with normal spermatozoa [165]. A variation of ubiqui- abnormal PTM on spermatozoa proteins will lead to dysfunction tin protease USP26 was also reported to be directly associated with resulting in infertility. In fact, a few studies in last decade have shown human sperm motility [160]. Moreover, some AZS patients have a se- that there is a significant difference in the PTM of many proteins in vere structural anomaly in their sperm known as dysplasia of the fibrous the spermatozoa of men having various pathophysiological states. sheath (DFS). A study found that sperm with DFS were accompanied by increased ubiquitination of the sperm surface as well as the sperm mito- 4. Comparative profiling of PTMs in different pathophysiological chondrial proteins [166]. In AZS patients only, the percentage of conditions of male infertility SUMO1-positive spermatozoa was found to be inversely correlated with total and progressive motility [131]. Additionally, an association Various structural and biochemical changes that render spermato- between AZS and anomalies in the seminal glycome has also been zoa functionally active (capacitated) can be attributed to PTMs [150]. documented [167,168]. 1460 L. Samanta et al. / Biochimica et Biophysica Acta 1860 (2016) 1450–1465

Fig. 7. Schematic representation of role of post-tranlational modiﬁcation on proteins in physiological function of spermatozoa.

Teratozoospermia (TZS) is a condition in which fewer than 4% of varicocele group [175]. Furthermore, enzymes linked to protein glyco- sperm have normal morphology [169]. About six sets of sperm proteins sylation, namely acid β-glucuronidase, α-mannosidase, α-glucosidase, were found to have differential tyr phosphorylation in TZS samples α-galactosidase, β-galactosidase and β-N-acetylglucosaminidase, when compared with normospermic ones [170]. Furthermore, sperma- showed over-expression and hyperactivity in the seminal plasma of in- tozoa from TZS males had a compromised ability to undergo tyrosine fertile men with VC [176]. As these extracellular glycosidases are impli- phosphorylation following capacitation [170]. The TZS index was cated to cause modification of sperm plasma membrane glycoproteins found to be significantly and negatively correlated with phosphorylated during sperm maturational process, it could be postulated that such levels of protein Stat3 [31], suggesting that diminished protein tyrosine over-expression of acid glycosidase in the seminal plasma would mask phosphorylation is one of the factors responsible for compromised the carbohydrate-containing molecules present in ejaculated spermato- sperm function in TZS males. In addition, ubiquitination, which has al- zoa of men with VC, altering their fertilization potential. ready been shown to be correlated with abnormal sperm count, could Proteomic technology is mostly centered on the enrichment of be anticipated to play a regulatory role in the TZS group. When the phosphopeptides and glycopepetides in sperm because these PTMs RNA transcript profiling of normozoospermic men was compared with are the dominant ones in sperm [177]. Moreover, proteins with only that of TZS individuals, transcripts encoding components of UPP were one type of functional modification are usually considered in sperm pro- underrepresented in the latter group [171]. Likewise, the ejaculated teomic studies, and proteins with multiple modifications are largely ig- sperm of the TZS group showed increased binding of anti-ubiquitin an- nored. But it can be speculated that the cross-talk between different tibodies to sperm surface, reflecting the occurrence of enhanced sperm PTMs occurring either in the same or other proteins actually regulate surface ubiquitination in nonsystematic cases of TZS [118]. protein stability and activity at the physiological level. Of course, the Varicocele (VC) is a condition in which there is abnormal dilations major challenge with this approach is that searching for additional and tortuosity of the pampiniform venous plexus within the spermatic modifications greatly increases the search time and reduces the speci- cord; it affects roughly 40% of infertile males. A comparative study of ficity of identification. The objective of future PTM studies needs to be tyr-phosphorylation status in the spermatozoa of VC patients revealed centered not only on detecting the differential modification of proteins a reduced level of phosphorylation [172, 173]. An alteration in plasma occurring at ultrastructural level but also on categorizing specific pro- membrane dynamics was postulated to be responsible for the impaired teins undergoing multiple PTMs. ability of the VC spermatozoa to phosphorylate proteins in tyr residues, resulting in sperm dysfunction in the VC patients [173]. In one study by 5. Multiple PTMs and signal crosstalk in spermatozoa our group, proteomic analysis showed that 122 differentially expressed proteins were associated with acetylation in spermatozoa in VC patients Recently, a study identified roughly 500 proteins with multiple types along with alterations in ubiquitination pathway and protein folding of modifications— mainly phosphorylation, acetylation and [174]. Ubiquitination median, as a marker for functionality of the ubiquitination—in ejaculated spermatozoa of humans [4].Proteins ubiquitin-proteasome system was positively correlated with motility with multiple PTMs were mapped to BioGRID to generate a complex in- and negatively correlated with morphology of spermatozoa in the teraction network. Some of the functionally important proteins L. Samanta et al. / Biochimica et Biophysica Acta 1860 (2016) 1450–1465 1461 identified so far with multiple PTM include AKAPs, Pyruvate dehydroge- Furthermore, it was proposed that four novel PTMs—three on H2B and nase E1 component subunit alpha (PDHA2) [4] and histones [61]. one on H3 (H2BT9p, H2BK117me3, H2BK121me3, and H3R83me1)—are Brunner et al. performed an extensive multiple PTM analysis of murine testis specific PTM of histones occurring only in spermatozoa. Even spermatozoa proteins using a novel proteomic approach [13]. They used ubiquitination sites were also observed in histone H2A of mouse sper- a peptide-based bottom-up collision induced dissociation/electron matids [101]. Similar to mouse sperm, H3 and H4 are the most modified transfer dissociation MS/MS strategy as well as intact protein top- histones in human sperm. Extensive proteomic analysis of mature down electron transfer tandem MS to more accurately determine and human sperm have identified five potential sites of methylations in confirm multiple PTM sites on the same protein/ peptide sequence. H3: H3K4,H3K9,H3K27,H3K36,H3K79 [61] and a single site of acetyla- Sperm chromatin packaging is an extremely important maturational tion i.e. H3K9ac [54]. Further, 3 potential sites of acetylation in H4 of ma- process that is controlled by signal crosstalk between multiple PTM. His- ture human sperm (H4K8,H4K12,andH4K16) were observed [54]. tone replacement during protamine toroid formation is a sequential Generally, the methylated histones H3 are linked with developmental event. Histones are replaced by protamines as well as by selected genes and may play a role in defining the so-called epigenetic histone testis-specific histone variants; about 5% - 15% of histones are retained code while hyperacetylation of H4 directs the histone-protamine transi- [178]. The functional significance proposed for this intricate physiolog- tion in haploid spermatids [181]. Moreover, an earlier study found that ical phenomenon is generation of a more hydrodynamic head for better during male pro-nuclei formation post-fertilization in humans, motility, protection of paternal chromosomes from exogenous insult, decondensation of spermatozoa nuclei involves H3 phosphorylation and epigenetic control of early embryonic development [179]. There [182]. Ubiquitination of H2A and H2B in human sperm may also occur are three major structural protein components involved in sperm chro- as a silencing and activating histone modification, respectively [183]. matin organizations: (i) five types of histones (H1, H2A, H2B, H3 and An interesting regulation was observed between two different PTM in H4), (ii) two types of protamine (P1 and P2) and (iii) the major testis the same histone: Metzler-Guillemain et al. found that there was com- specific histone variant TH2B All three categories have been reported petition between sumoylation and methylation at the H4K20 site in to have multiple PTMs (phosphorylation, acetylation, methylation and the human spermatid [129]. Further, it was observed that in human ubiquitination) those may act as regulatory factors in many chromatin pachytene nuclei, the P1 chromodomain specifically binds to H3- template-mediated events in sperm. An extensive proteomic study on K9me3, with the later preventing the sumoylation of the former—this mouse sperm chromatin performed by Brunner et al. identified 26 his- is an example of interregulation of PTM between two different proteins tone PTMs and 11 protamine PTMs [13], whereas Pentakota et al. iden- in human sperm. tified 10 TH2B PTMs [180]. During stage-specific structural reorganization of sperm chromatin, Protamines that are unique to mature spermatozoa have not been novel histone variants unique to male germ cells are incorporated in well studied as far as PTMs are concerned, especially in human sperm, mammalian sperm [21]. This group of histone variants include testicular possibly because the functional aspect associated with PTM in prot- specific histone variants such as TH2A, TH2B, and TH3, and linker his- amines is mainly protecting the paternal genome rather than influenc- tone variants H1t and HILS1 [180]. Although they are less stable than ing embryonic development. Nevertheless, Brunner et al. performed a standard canonical histones, histone variants such as TH2B and H2AX proteoform profiling of mature mouse sperm using bottom-up MS/MS have been detected in mature human sperm and may be associated and found that 53% of P1 and only 16% of P2 had PTM [13].There with certain sperm functions. TH2B is one of the most abundant histone were 7 multiple PTM sites in P1, namely, 3 phosphorylation sites variants in human sperm [184]; it is associated with telomere-binding (P1S8p, P1S42p, P1T44p), 3 acetylation sites (P1-A1ac, P1S42ac, P1T44ac, complex [185]. TH2B shares roughly 85% homology with the canonical P1K49ac) and 1 methylation site (P1K49me). Similarly, there were 4 histone H2B. In human TH2B, the N-terminal segment has two potential multiple PTM sites in P2, namely, 3 acetylation sites (P2S55ac, P2S57ac, phosphorylation sites (S5 and S6), while four additional sites are located P2K64ac) and 1 phosphorylation site (P2S55p). Here, it is noteworthy in the central and C-terminal regions [22]. Although no extensive study to mention that both in P1 and P2, a single AA can possess 2 different has been undertaken targeting the PTM on TH2B of human sperm, infer- types of PTM depending on the need (P1S42p and P1S42ac, P1K49ac ences can be drawn from similar studies on rodent sperm, as it shares a and P1K49me, P2S55ac and P2S55p). Moreover, using a top-down MS ap- strong (95%) homology with TH2B from rats and mice [22].Pentakota proach, the authors found that the co-occurrence of acetylation and et al. did an extensive proteomic analysis of PTM in TH2B of mouse phosphorylation in the same protamine peptide is not possible whereas sperm isolated at various stages of spermatogenesis and identified mul- the simultaneous presence of acetylation and methylation is. Castillo tiple PTM occurring in the form of acetylation, methylation, and phos- et al. found that there were 6 potential sites for phosphorylation and phorylation [180]. TH2B from spermatocytes had six acetylation, three acetylation in P1 in human sperm (P1S8,P1S10,P1S12,P1S21,P1S28, monomethylation, and one phosphorylation site whereas TH2B from P1S31) along with a mono- and di-phosphorylated site at the N- round spermatids had four acetylation and two monomethylation terminal end [20]. In the hP2 family, phosphorylation, acetylation and sites. In similar kind of study on rat sperm, Lu et al. [21] found that methylation have been reported. It has been reported that all possible rTH2B was unique with the addition of 3 potential phosphorylation combinations of PTM, even acetylation along with phosphorylation in sites and repositioning of two (S11 and T22). Further, a novel the same peptide, are possible in human sperm in contast to mouse phosphoswitch site was reported at T116pandK117m. sperm. It could be inferred that the occurrence of multiple PTMs in prot- Analysis of different regulatory roles multiple PTM play in chromatin amines is therefore not random but rather has some functional signifi- remodeling shows just how intricately such PTM occur, modulate and cance, although the presence of a protamine code in the form of PTM control sperm physiology. The signal crosstalk between multiple PTM [13] is doubtful [20]. can be summarized in the following manner: 1) Although the same pro- The other important component of sperm chromatin is the canonical tein contained different PTM sites, only a few combinations can occur si- histones, where PTM regulates all its functions beginning from mediat- multaneously in a peptide—for example, in protamines, acetylation and ing proper replacement with protamines during chromatin structural phosphorylation cannot co-occur in the same peptide [13]. 2) There can transitions to transmission of a sperm-derived epigenetic histone be competition between two PTMs for the same site on a protein as was code. In mouse sperm, Brunner et al. detected 26 PTMs on the four his- observed between sumoylation and methylation at H4K20 [129].3)The tone types H1, H2B, H3 and H4, of which about 60% of total H3 and H4 occurrence of PTM in a protein can be suppressed by another PTM in a and b10% of total H1 and H2B had PTM [13]. H3 had multiple sites for different protein as was observed when methylation of H3 prevented lys/arg methylations, H2B had sites for both lys methylations and thr the sumoylation of P1 [129]. 4) A phosphoswitch was reported where phosphorylations, while H4 had the maximum variation in multiple phosphorylation of a site adjacent to (or nearby) a methyl (acetyl) site PTM with sites for lys/arg methylations and lys/ser acetylation. prevented the binding of a factor to the latter in human sperm H1.4 1462 L. Samanta et al. / Biochimica et Biophysica Acta 1860 (2016) 1450–1465 when simultaneous phosphorylation at S23 of H1.4 blocked the binding [6] D. Sakkas, G. Leppens-Luisier, H. 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