The contribution of proteomics to understanding ...

Systems Biology in Reproductive Medicine, 2012, 58: 197–210 Copyright © 2012 Informa Healthcare USA, Inc. ISSN 1939-6368 print/1939-6376 online DOI: 10.3109/19396368.2012.663233

Special Issue: SBiRM: Focus on Proteomics and Reproduction INVITED REVIEW

The contribution of proteomics to understanding epididymal maturation of mammalian spermatozoa ∗

Jean-Louis Dacheux1 , Clémence Belleannée2, Benoit Guyonnet3, Valérie Labas4, Ana-Paula Teixeira-Gomes4, Heath Ecroyd5, Xavier Druart,1 Jean-Luc Gatti6 and Françoise Dacheux1

Syst Biol Reprod Med Downloaded from informahealthcare.com by 194.167.77.1 on 07/16/12 For personal use only.

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UMR INRA-CNRS, Nouzilly, France, 2Centre de Recherche du Centre Hospitalier de Université Laval (CHUQ) and Département d’Obstétrique-Gynécologie, Faculté de médecine, Université Laval, Québec, Canada, 3Department of Cell Biology and Biochemistry Texas Tech University Health Sciences Center, Lubbock, TX, USA, 4Laboratoire de Spectrométrie de Masse pour la Protéomique, INRA, Nouzilly, France, 5School of Biological Sciences, Faculty of Sciences, Illawarra Health and Medical Research Institute, University of Wollongong, NSW, Australia, 6UMR IBSV, INRA-CNRS-Université de Nice, Sophia Antipolis Cedex, France occur at the surface of the maturing gametes; there is a progressive removal or modification of most of the testicular surface proteins of the immature spermatozoa and they gain new proteins (transiently or permanently) into their well organized membrane surface domains [Cowan and Myles 1993]. These stages have been demonstrated to be essential for the acquisition of motility and fertility by male gametes, yet, the underlying mechanisms, both at the cell surface and intracellular level, remain enigmatic. During ‘epididymal maturation’ spermatozoa are enclosed in a specific environment that is believed to play an essential role in controlling or inducing final maturation of the spermatozoa. The milieu surrounding the spermatozoa is continuously changing, not only in terms of protein content, but also in chemical composition. The epididymal milieu is remarkable due to the presence of unusually high concentrations of some of components such as carnitine, which are not found in other body fluids. Epididymal sperm maturation is the result of complex and sequential interactions in different epididymal regions and, to our knowledge, complete maturation of testicular spermatozoa has never been achieved in vitro. Research on the post-gonadal differentiation of spermatozoa has been developed over several decades with goals of: i) understanding how epididymal maturation (or lack thereof) contributes to idiopathic male infertility, ii) utilizing immature testicular and epididymal spermatozoa in vitro fertilization, and iii) applying knowledge of epididymal sperm maturation to establish and develop male contraceptives. With the development of molecular methods, numerous transcriptomic studies have been done on the epididymis, however, these global approaches, although important at the level of the epididymal tissue, cannot be used to investigate post-testicular sperm changes since: i) spermatozoa possess a very limited capacity for biosynthesis, and ii) the protein

The acquisition of the ability of the male gamete to fertilize an ovum is the result of numerous and sequential steps of differentiation of spermatozoa that occur as they transit from the testis to the end of the epididymal tubule. The post gonadal sperm modifications are mostly related to motility, egg binding, and penetration processes. As the activity of the epididymis and its luminal fluid composition are believed to be directly related to ‘sperm maturation’, a review on epididymal proteins is presented. Comparative studies have shown that the epididymal activities are species specific. Nevertheless, for all mammalian species studied, similarities exist in the sequential proteomic changes of the luminal composition of the epididymal tubule and proteins on the sperm surface. The potential roles of these modifications are discussed. Keywords epididymis, maturation, proteomic, spermatozoa

Introduction The mammalian spermatozoon is the result of an extensive cellular differentiation process, which transforms a round spermatid into a highly polarized and fully motile cell. Most of these complex biochemical, physiological, and morphological events take place in the testis during spermatogenesis. Some of these events are under the genomic regulation of the gamete, but, when the DNA begins to condense (at the elongated spermatid stage), transcription decreases and then stops. However, testicular spermatozoa released into the seminiferous tubule are not yet motile or fertile; they require discrete but essential post-gonadal differentiational steps before they are able to fertilize eggs. These final steps of differentiation occur during their transit through the long and unique epididymal tubule, which links the testis to the deferent duct. During this transit, sequential protein modifications Received 25 July 2011; accepted 23 October 2011. ∗

Address correspondence to Jean-Louis Dacheux, UMR INRA-CNRS 6176, 37380 Nouzilly, France. E-mail: [email protected]

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changes at the surface of the gamete are the result of numerous sequential interactions with components of the surrounding epididymal medium. Consequently, itemization of epididymal and sperm plasma membrane proteins and an understanding of the interactions between these components are essential in order to decipher how spermatozoa acquire motility and fertility after leaving the testes. During the last 10 years, methodological approaches to study sperm epididymal maturation have progressed from analysis of individual proteins (obtained either from sperm or epididymal secretion) to large-scale identification using proteomic approaches. With these new methods, it has been possible to determine the protein composition of the epididymal fluid for several mammalian species and to develop a comprehensive overview of the major compounds (and their modifications) that are common to each. The development of new labeling methods coupled with these proteomic approaches now allows the study of global modifications of spermatozoa during their maturation, as well as modifications that occur in various surface domains [Baker et al. 2011; Belleannee et al. 2011a].

The epididymis: a unique tubule with a complex luminal protein composition that varies along the organ When the immature spermatozoa leave the testis from the rete testis and the efferent ducts, they enter a unique convoluted tubule with a length ranging from 1-7 meters for rodents and primates to 60-70 meters for domestic animals such as porcine and equine. The diameter of this tubule varies according to species, from 100-200 micrometers proximally to several millimeters at its distal end (mainly in the caudal region). The milieu surrounding the spermatozoa is certainly one of the most complex fluids found in any exocrine gland. This specificity is maintained by active secretion and reabsorption throughout the tract and by the presence of a continuous blood barrier formed by tight junctions between the Sertoli cells at the testis level and the epithelial cells in the epididymis [Hoffer and Hinton 1984]. Thus, this barrier isolates the spermatozoa from the body fluids, from their formation to their final maturational steps. This physical separation by the blood barrier is also very important due to its role in protection from autoimmune reactions that could occur due to: i) new specific antigens appearing on the sperm during their differentiation, and ii) specific testicular and epididymal protein secretions associated with the terminal cellular differentiation of the gametes. To study the composition of the epididymal fluid it is necessary that the sampling avoid any contamination either from serum, lymph, or epididymal cellular proteins. Several studies have been done on luminal fluids released after mincing the tissue and by applying gentle pressure to the epididymal tubule [Girouard et al. 2009; Li et al. 2011; Zhang and Martin-Deleon 2003]. However, such samples may be contaminated and have not been able to identify proteins with the same sensitivity as experiments using high performance mass spectrometers

which can detect attomole concentrations of protein components. More adapted sampling methods, such as tubule micropuncture [Turner et al. 1995; Turner et al. 1999] or microperfusion techniques, have been developed and have allowed collection of almost pure epididymal luminal fluid that is more suitable for proteomic studies [Belleannee et al. 2011b; Druart et al. 1994; Fouchecourt et al. 2000; Syntin et al. 1999].

Composition of the epididymal fluid proteome The epididymal fluid provides a milieu for the gamete like blood plasma provides a milieu for cellular tissues. But, in contrast to the stable concentration and composition of blood plasma proteins, the epididymal fluid varies greatly along the duct. For example, the protein concentration ranges from 2 to 4 mg/ml in the initial segment of the epididymis, peaks to a maximum of 50-60 mg/ml in the distal caput and returns to 20-30 mg/ml in the more distal regions of the organ [Belleannee et al. 2011b; Fouchecourt et al. 2000]. These variations in protein concentration follow the changes in water content of the fluid as assessed by changes in sperm concentration between the testis and the deferent duct. From the rete testis to the deferent duct the sperm concentration increases from 108 to 109 spermatozoa/ml, with a maximum in the first part of the epididymis. The water movement occurs principally in the efferent duct where testicular fluid is reabsorbed, and continues at a low level up to the first part of the epididymis. These water changes through aquaporin water channels are driven by the transepithelial movement of Na+, Cl−, HCO−3 and result also in important modifications in the ionic composition of the lumen fluid along the epididymal tubule [Da Silva et al. 2006]. For most mammalian species, the composition of the epididymal fluid is characterized by the presence of proteins in high concentration with no more than twenty proteins representing 80 to 90% of the total luminal proteins. For example, in the bull (Fig. 1), 13 proteins make up 95% of the total protein content of the epididymal luminal fluid: lipocalin 5 (LCN5, E-RAPB) (17.3%), clusterin (CLU) (14.0%), glutathione peroxidase (GPX5) (13.6%), albumin (BSA) (13.6%), prostaglandin D2 synthase (PTGDS) (11.7%), transferrin (TF) (7.5%), Niemann-Pick disease type C2 (NPC2) (3.6%), phosphoethanolamine binding protein 4 (PEBP4) (3.3%), beta-N-acetyl-hexosaminidase (HEXB) (3.2%), glutathione S transferase (GST) (2.3%), gelsolin (Gsn) (2.2%), actin (1.4%), and beta galactosidase (GLB1) (1%) [Belleannee et al. 2011b]. In human (Fig. 2), 77% of the total luminal proteins are represented by 10 proteins: albumin (HSA) (43.8%), CLU (7.6%), NPC2 (6%), lactoferrin (LF) (5.9%), extracellular matrix protein (ECM1) (3.2%), α1-antitrypsin (A1AT) (2.7%), PTGDS (2.2%), HE3 (1.7%), TF (1.3%), and actin binding protein (ABP) (1.2%) [Dacheux et al. 2006]. Most of these epididymal proteins are characterized by numerous isoforms which result from their high degree of post-translational modifications such as glycosylation. The pI of these Systems Biology in Reproductive Medicine


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Figure 1. Identification of the major proteins from bovine epididymal fluid according to Belleannee et al. [2011b]. Proteins identified by mass spectrometry are numbered and referenced in Table 1 to the cited publication. ALB: albumin; CLU: clusterin; GLB1: beta galactosidase; GPX5: glutathione peroxidase; Gsn: gelsolin; HEXB: beta-N-acetyl-hexosaminidase; LCN5: lipocalin 5; MANB: beta mannosidase; NPC2: Niemann-Pick disease, type C2; PTGDS: prostaglandin D2 synthase; SPADH1: spermadhesin1.

isoforms can vary widely, ranging from pH 3 to 8 for the same protein (e.g., RNase10 in the boar [Castella et al. 2004]). The degree of glycosylation for one protein can also be different according to the epididymal region studied; for example, in the horse the number of isoforms of CLU and PTGDS is different between the anterior and the posterior part of the organ [Fouchecourt et al. 2000]. Moreover, the polymorphism observed for one protein may be different between species, such as the number of the isoforms for RNase 10 in the boar and the ram [Dacheux et al. 2009] (Fig. 3). The luminal protein composition is modified all along the epididymal tubule, and each epididymal region is characterized by a specific profile which differs according to species (Figs. 3, 4). For each species several major proteins can decrease (such as, PTGDS, GPX5, BSA, and TF) or increase (such as, CLU, GSN, and HEXB) in concentration according to the epididymal regions. Most of these major proteins are soluble and constitute the bulk of the epididymal proteins found by proteomics. However, among the epididymal fluid proteins, other components less soluble are GPI anchor proteins, i.e., CD52 [Kirchhoff 1996], the prion protein [Ecroyd et al. 2005; Gatti et al. 2002], SPAM1 [Chen et al. 2006], the serine protease PRSS21 [Netzel-Arnett et al. 2009], or proteins with highly hydrophobic properties [Gatti et al. 1999]. Such poorly soluble proteins can be in part explained by the Copyright © 2012 Informa Healthcare USA, Inc.

presence of particulate components, either in the form of membrane vesicles named epididymosomes (for ‘epididymal exosomes’) or of ‘micelle-like structures’ [Frenette et al. 2002; Gatti et al. 2005] in the fluid.

Origin of the luminal epididymal proteome Almost all the testicular proteins entering the epididymis are rapidly taken up in the first part of the organ. For example transferrin (the main protein secreted by the Sertoli cells) and albumin are found in the rete testis fluid but not in fluid from the proximal caput where these proteins are reabsorbed. The only exception is in the human epididymis where these proteins are found in the luminal fluid all along the organ [Dacheux et al. 2006]. After this initial region, the protein composition of the fluid is mostly linked to the secretory activity of the epithelium. The total number of proteins secreted by the epididymal epithelium throughout the epididymal tubule has been estimated in several species by in vivo/in vitro neosynthesis using radioactive labeling of proteins and proteomic analyses [Belleannee et al. 2011b; Fouchecourt et al. 2000; Syntin et al. 1999]. All these studies demonstrate that the secretory activity of the epididymis is high in the anterior part of the organ and, overall, at least several hundred proteins are secreted. For all the species studied, less than twenty proteins, secreted all along the tubule,


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Figure 2. Protein identified by mass spectrometry in the fluid of the caudal region of the human epididymis after 2D gel electrophoresis and Coomassie blue staining according to Dacheux et al. [2006]. ALDH1: aldehyde dehydrogenase; B2M: beta-2-microglobulin; CAH4: carbonic anhydraseIV; CLU: clusterin; CRISP1: cysteine-rich secretory protein 1; ECM1: extracellular matrix protein1; EDDM3A: HE3, epididymal protein 3A; G3PB: galectin 3 binding protein; GAA: alpha glucosidase; GST: glutathione S transferase; HSP90: heat shock protein 90kDa beta; IDHC: isocitrate dehydrogenase; MDH1: cytosolic malate dehydrogenase; MIF: macrophage migration inhibitory factor; NPC2: Niemann-Pick disease, type C2; PEBP4: phosphoethanolamine binding protein 4; PHHUPN: purine-nucleoside phosphorylase; PDX2: peroxiredoxin 2; PTGDS: prostaglandin D2 synthase; SOD: superoxide dismutase; SPINT3: serine peptidase inhibitor, Kunitz type 3; TF: transferrin; TPI: triosephosphate isomerase; TXN: thioredoxin.

represent more than 80% of the total epididymal secretions (Fig. 5). Each epididymal secretome is species specific. However, for all species, the major protein found is clusterin. This protein represents around 30% of the total secreted protein for all the species studied (Figs. 5, 6). Several isoforms of clusterin can be found according to the epididymal regions where it is secreted: 3 in the horse and at least 2 in the bull and the ram. Among the other common major proteins, NPC2 and lactoferrin have been identified but their percentage of the total secreted protein varies according to species, NPC2 being highest in human (8.8% of the epididymal secretome) and lactoferrin in horse (41.2 %). Other major secreted proteins are more species specific, e.g., PTGDS

(not found in porcine), GPX5 (not found in human), LCN5 (not in human and equine), or RNase10 (only major protein in porcine). This sequential secretion of most of the proteins throughout the epididymal tubule is a specific characteristic of the epididymis and almost unique in mammalian organs. This sequential secretion is responsible for the continuous change in the proteome of the epididymal fluid (Fig. 6). According to the composition of the epididymal secretome, several epididymal regions can be identified by the presence of particular proteins, e.g., PTGDS, GPX5, and RNAse10 for the proximal epididymal regions, and several glucosidases, lactoferrin, NCP2, gelsolin (GSN), and LCN5 for the middle and distal part of the organ (Fig. 6). For each Systems Biology in Reproductive Medicine


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et al. 2005; Frenette et al. 2002; Ilio and Hess 1994]. The other origin of the continuous change in the protein composition in this organ is specific protein reabsorption that occurs throughout the tubule. These two opposite effects (secretion/reabsorption) vary for almost every protein. Some are reabsorbed as soon as they are secreted, e.g., RNase10 (porcine, ovine, and murine) [Castella et al. 2004a] and some isoforms of clusterin (equine) [Fouchecourt et al. 2000]. Others accumulate during epididymal transit (e.g., lactoferrin in equine or LCN5 in bovine) or remain throughout the tubule (e.g., PTGDS in several species) [Belleannee et al. 2011b].


Control of the composition of the epididymal fluid proteins Figure 3. Distribution of the percentage of 14 major luminal proteins present in caput and caudal epididymal regions of the bull. Taken from Belleannee et al. [2011b]. BSA: albumin; CLU: clusterin; GLB1: beta galactosidase; GPX5: glutathione peroxidase; Gsn: gelsolin; GST: glutathione S transferase; HEXB: beta-N-acetyl-hexosaminidase; LCN5: lipocalin 5; NPC2: Niemann-Pick disease, type C2; PEBP4: phosphoethanolamine binding protein 4; PTGDS: prostaglandin D2 synthase; TF: transferrin.

Figure 4. Distribution of the percentage of 17 major luminal proteins present in caput and caudal epididymal fluids from human. A1AT: Alpha 1 antitrypsin; CLU: clusterin; ECM1: extracellular matrix protein1; ENOA: Enolase; G3PB: galectin 3 binding protein; HE3: EDDM3A, epididymal protein 3A; HSA: human albumin; HE1: NPC2, Niemann-Pick disease, type C2; PDGS: prostaglandin D2 synthase; PHHUPN: purine-nucleoside phosphorylase.

species, these major secreted proteins correspond to the major proteins found in the luminal fluid. These proteins are secreted by both conventional and unconventional secretion pathways. The latter is due to several of these proteins lacking signal peptides, and some arising from a specialized pathway involving large vesicular structures (blebs) released from the principal cells [Dacheux Copyright © 2012 Informa Healthcare USA, Inc.

The establishment of the regionalization of epididymal luminal secretion takes place progressively during postnatal development and before puberty, and is strongly linked to testis development. Half of the epididymal proteins are either positively or negatively controlled by androgens which are produced by the testis and transported through the testicular and epididymal fluids [Syntin et al. 1999]. However, after efferent duct ligation, the proximal caput is the most highly affected region with regard to protein secretion. For example, in the boar epididymis there is a 50% decrease in the number of proteins (including RNase10) found in this region when the efferent ducts are ligated, and this decrease is not dependent on systemic androgen. Other specific proteins of the epididymal caput, such as GPX and HEX, are positively regulated by testosterone, but with very different levels of sensitivity. In the distal caput and corpus epididymis, lactoferrin, NCP2, and Lcn5 are also stimulated by androgens, whereas clusterin is inhibited. The molecular mechanisms resulting in the establishment of regionalized gene expression in the epididymis are mostly under testicular control, in particular by androgens, but several testicular factors (‘lumicrine factors’), such as EGF, VEGFA, or FGF2 are also involved in the control of the proximal epididymis [Cotton et al. 2008; Hinton et al. 1998; Tomsig et al. 2006; Turner et al. 2007]. There are other lumicrine factors that regulate gene expression in the epididymis which remain to be defined. Moreover, these numerous (and complex) regulatory mechanisms have not yet been fully elucidated, nor their receptors and targets. For example, we are yet to identify promoters which drive region specific gene expression in the epididymis and small RNAs are increasingly suspected to be involved in gene regulation in the epididymis [Zhang et al. 2011]. Elucidating such factors is key to our future understanding of the way in which variations in epididymal fluid composition arise in this tubule.

Sperm membrane proteomic The sequential changes in the fluid are associated with the sequential modifications to the sperm membrane which


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Figure 5. Distribution of the major luminal proteins secreted by the human, boar, ram, and horse epididymis. For each protein, the amount secreted is expressed as a percentage of the total labeled protein secreted by the epididymis. A1AT: Alpha 1 antitrypsin; A2GP: alpha 2 glycoprotein; CLU: clusterin; GPX: glutathione peroxidase; Hex: beta-N-acetyl-hexosaminidase; LCN5: lipocalin 5; LF: lactoferrin; NPC2: Niemann-Pick disease, type C2; PDGS: PTGDS, prostaglandin D2 synthase; I, M: unidentified proteins.

are believed to be directly or indirectly involved in the maturation of sperm during epididymal transit. Labeling of spermatozoa with lectins, antibodies, radioactive precursors, or other chemical probes has shown that substantial changes occur in the lipid and protein composition of the sperm membrane during epididymal transit [Dacheux et al. 1984; Voglmayr et al. 1985; Yanagimachi 1994]. These changes result from various mechanisms which include redistribution (or disappearance) after proteolytic processing, the action of glycolytic enzymes, and integration of newly synthesized components. For several species, global modifications to proteins on the sperm surface can be assessed by biotin labeling of spermatozoa sampled from different epididymal regions and analyzing the labeled proteins by 1D and 2D PAGE (Figs. 6, 7). In all the species studied to date, it appears that specific testicular sperm surface proteins are removed or further processed as the gametes pass through the epididymis. The disappearance of some proteins is clearly related to a specific mechanism of proteolysis during epididymal transit. For most of these proteins, proteolysis induces either a change in their membrane domain distribution (e.g., fertilin/PH30 [Blobel 2000]) or release of the cleaved protein into the epididymal milieu (e.g., angiotensin-converting enzyme, ACE [Gatti et al. 1999]). As several surface proteins disappear or are processed, several new antigens can be identified on the sperm. Some

of these components are transitory, but several, mostly those with a low molecular weight, are still present on sperm in the distal part of the epididymis (Fig. 6). These new components could originate from epididymal secretions as antigenic similarities between sperm membrane proteins and secreted proteins have been identified in several species. However, only a few antigens have been identified and the exact degree of similarity between the sperm surface component and the epididymal protein has rarely been reported. The proteomic approach thus appears to be the only global method allowing identification of the proteins involved in these sperm surface changes. Recently, using a large scale proteomics approach (i.e., shotgun proteomics), several hundred to thousands of proteins have been characterized and identified from whole sperm [Aitken and Baker 2008; Aitken et al. 2007; Johnston et al. 2005; Martinez-Heredia et al. 2008; Oliva et al. 2009; Shetty et al. 2001; Stein et al. 2006]. Some phosphoproteins could be related to epididymal maturation of the gamete [Baker et al. 2011; Baker et al. 2005]. Such approaches identify the most abundant proteins in cells, however, because membrane proteins often represent only a small part of the entire proteome, only a few plasma membrane proteins have been identified by this method. Enrichment of the membrane sub-proteome therefore appeared to be an essential step to reduce the complexity of the samples and to optimize the effectiveness of surface Systems Biology in Reproductive Medicine

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Figure 6. Epididymal secretome and proteome from five species. For the proteome (on the left), each plate corresponds to a 1D gel electrophoresis separation of about the same quantity of epididymal proteins from each region of the epididymis. The secretome diagrams (in the middle) represent each secreted protein from the different epididymal regions and are expressed as the percentage of total secretion of the organ. The plates illustrating the secretory activities (on the right) of the different epididymal regions for the five animal species correspond to the autoradiograms of the same five 1D gel separations presented for the proteome of boar [Syntin et al. 1996], stallion [Fouchecourt et al. 2000], ram [Druart et al. 1994], human [Dacheux et al. 2006], and bull [Belleannee et al. 2011b]. A1AT: Alpha-1-antitrypsin; A2GP: alpha-2-glycoprotein; ALB: albumin; BGAL: betagalactosidase; CALM: calmodulin; CLU: clusterin; CST3: cystatin C; EDDM3A: epididymal protein 3A; GPX5: glutathione peroxidase; Gsn, Gsn 40, Gsn 80: gelsolin; GST: glutathione-S-transferase; HEXB: beta-N-acetyl-hexosaminidase; LCN5: lipocalin 5; LF: lactoferrin; MA2B2, MABA: mannosidase; NPC2: Niemann-Pick disease, type C2; PTGDS: prostaglandin D2 synthase; SPADH1: spermadhesin1; TF: transferrin; Numbers and single letters: unidentified proteins. Copyright © 2012 Informa Healthcare USA, Inc.


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Figure 7. Distribution of the biotin surface labeled proteins from caput (2), corpus (6), and cauda (9) epididymal boar spermatozoa according to Belleannée et al. [2011a]. The labeled proteins were extracted by ionic or detergent solution, separated by SDS-PAGE and revealed by Western blotting on nitrocellulose membrane using incubation with streptavidin peroxidase. Major protein variations are indicated by asterisks.

membrane protein identification. Recently, purification of sperm protein membranes by a dedicated surface labeling technique provided an opportunity to analyze a specific sub-proteome essential to the fertilizing ability of the male gamete. By this approach, developed in the boar, numerous major sperm surface proteins were identified (Fig. 8; Table 1) [Belleannee et al. 2011a]. Several of these proteins have been independently described as surface proteins (i.e., ACE, aldose reductase, arylsulfatase A, alpha-enolase, glutathione S-transferase, Huntingtin interacting protein 1, heat shock protein HSP 90-alpha, heat shock 70 kDa protein, alphamannosidase, lactadherin, and peroxiredoxin 5), but some others are new (such as the valosin-containing protein, VCP or the T-complex protein family). Moreover, some of the proteins identified in this study are cytosolic and, thus, it is probable that these proteins interact directly with surface proteins and can be consequently isolated with them. These proteins belong to networks which associate several of these surface proteins to their cytosolic partners;

one of these potential networks being related to post-transcriptional modifications and protein folding. In the boar, this network includes about half of the major proteins identified [Belleannee et al. 2011a]. Apart from their implication in binding to the egg zona pellucida and membrane, the roles of these surface sperm proteins have not been formally established. We can hypothesize that they are involved in: i) controlling modifications of surface proteins and protein folding during epididymal maturation (due to the presence of several subunits of the TCP-1 complex, HIP1 and several isoforms of heat shock proteins); ii) protein transport during the remodeling of the epididymal plasma membrane and migration of the cytoplasmic droplet (due to the presence of actin, tubulin, VCP, Hsp70, and Hsp90) [Howes et al. 2001; Suzuki-Toyota et al. 2010; Uchiyama et al. 2002]; and iii) the direct or indirect defense of male gametes against oxidative stress (CCT2, ENO1, GSTM5, HIP1, HSP90AA1, HSPA2, PRDX5, TCP1, and VCP). Systems Biology in Reproductive Medicine

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Figure 8. Two dimensional separations of purified surface proteins extracted from immature (A) and mature (B) boar spermatozoa from Belleannée et al. [2011b]. Spot and band numbers are related to the tandem mass spectrometry (MS/MS) protein analysis referred to in Table 1. The circles on the plates indicate proteins differentially expressed between mature and immature spermatozoa. Spots with a number indicate proteins not identified by MS/MS. ACE: Angiotensin-converting enzyme; ALDR2: Aldose Reductase; ARSA: Arylsulfatase A; CCT1: T-complex protein 1 subunit alpha; CCT2: T-complex protein 1 subunit beta; CCT4: T-complex protein 1 subunit delta; ENO1: Alpha enolase; GSTM5: Glutathione S-transferase Mu 5; HSPA2: Heat shock-related 70 kDa protein 2; HSPA4L: Heat shock 70 kDa protein 4L; IMPA1: Inositol monophosphatase 1; MDH1: Malate dehydrogenase; MFGE8: Lactadherin; PRDX5: Peroxiredoxin-5; VCP: Valosin-containing protein; YWHAZ: 14-3-3 protein zeta chain; ZPBP: Zona pellucida-binding protein 1.

Relationship between sperm membrane proteome and luminal epididymal proteome Most of the sperm modifications appear to be initiated in the anterior part of the epididymis and are followed by sequential surface modifications all along the epididymis. The most Copyright © 2012 Informa Healthcare USA, Inc.

significant changes to the proteins on the sperm surface are observed in this anterior region, which is the most active in terms of the number of different proteins secreted and the concentration of proteins and sperm [Syntin et al. 1996] (Fig. 9). These sequential changes in the luminal epididymal proteome are, however, crucial for sperm post-testicular

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Table 1. Identification of purified labeled surface proteins of epididymal boar spermatozoa.


Protein description

Symbol

Accession number (a)

14-3-3 protein zeta chain Aconitase Acrosin Aldose reductase Alpha enolase Alpha-mannosidase Angiotensin-converting enzyme

YWHAZ ACO1 ACR ALDR2 ENO1 MAN2C1 ACE

253706 5821963 164703 8569627 2661039 1017779 145279215

Arylsulfatase A Beta tubulin Glutathione S-transferase Mu 5 GTP-binding protein Heat shock 70 kDa protein 4L Heat shock 70k-Da protein 1 Heat shock protein HSP 90-alpha Heat shock-related 70 kDa protein 2 Hexokinase type 1 Huntingtin interacting protein 1 Inositol 1-phosphate synthase Inositol monophosphatase 1 Lactadherin L-lactate dehydrogenase A-like 6B LY6/PLAUR domain containing 4 Malate dehydrogenase Valosin-containing protein Peroxiredoxin-5 Phosphoglycerate mutase 2 T-complex protein 1 subunit alpha T-complex protein 1 subunit beta T-complex protein 1 subunit delta T-complex protein 1 subunit theta Testis-specific phosphoglycerate kinase Zona pellucida-binding protein 1

ARSA TUBB GSTM5 RAB2 HSPA4L HSPA1L HSP90AA1 HSPA2

149759319 537407 6754086 550062 4579911 162138256 60592792 41386699

HK1 HIP1 ISYNA1 IMPA1 MFGE8 LDHAL6B LYPD4 MDH1 VCP PRDX5 PGAM2 CCT1 CCT2 CCT4 CCT8 PGK2

34670 119917142 11493904 47523516 1720726 15082234 285818398 164541 55217 47523086 84000195 194033403 77735435 84000361 62896539 40748088

ZPBP

47523020

Spermatozoa surface references

[Mori et al. 1995; Suter and Habenicht 1998] [Kobayashi et al. 2002; Frenette et al. 2003] [Gitlits et al. 2000] [Okamura et al. 1995; Pereira et al. 1998; Kuno et al. 2000] [Kohn et al. 1998; Gatti, et al. 1999; Nikolaeva et al. 2006; Aleksinskaya et al. 2006] [Nikolajczyk and O’Rand 1992; Weerachatyanukul et al. 2003] [Hemachand et al. 2002]

[Miller et al. 1992; Spinaci et al. 2005] [Miller et al. 1992]

[Rao et al. 2001; Khatchadourian et al. 2007]

[Petrunkina et al. 2003] [Ploug et al. 1991; Shetty et al. 1999]

[van Gestel et al. 2007]

[Yu et al. 2006; Mori, et al. 1995]

a) Accession code for the identification in NCBI database

differentiation since maturation of sperm has never been successful by incubating immature sperm in caudal epididymal fluid in vitro. Few relationships have been observed between the major sequential modifications of the epididymal proteome and the sequential changes in the sperm membrane proteome. None of the major proteins of the epididymal fluid are common to the major proteins found on the sperm surface. Presently there is no direct evidence for a specific role of these major luminal proteins in sperm maturation, although several of them are known to act as enzymes, inhibitors, or binding proteins in cellular systems. It is probable that the major proteins surrounding the gametes during their epididymal transit are more involved in sperm preservation rather than inducing specific and localized modifications to the sperm surface. The interactions between the sperm surface and the epididymal fluid are complex and cannot be considered by only taking into account the major components. The fact that the protein concentration is particularly high in the anterior region of the epididymis most likely enhances interactions between numerous ‘minor’ proteins secreted in this region

and the sperm surface. In this respect it is important to note that only the most concentrated proteins of the epididymal fluid have been identified by tandem mass spectrometry (MS/MS), several hundred are probably present in this medium. The identification of ‘minor’ components is limited by the large range in protein concentrations which makes it difficult to identify those that are present in low concentrations. Furthermore, some of the most hydrophobic proteins may be reduced in concentration (or never present) in a soluble form in epididymal luminal fluid. The pattern of the sperm surface modifications is species specific, humans being one species in which sperm maturation is established very early in the epididymis [Dacheux et al. 2006]. However, several common sperm surface proteins can be identified, including fertilin [Primakoff et al. 1988], CRISP1 [Roberts et al. 2006], and ACE [Gatti et al. 2002]. Yet, for each of these proteins, the modifications (isoforms) present on the sperm surface are species specific. For example, ACE is released from the sperm surface in the efferent ducts in rodents, in the anterior caput in humans, and in the posterior caput or proximal corpus in several domestic animals. Systems Biology in Reproductive Medicine

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Figure 9. Outlinear representation of the epithelial secretion, the fluid volume, the spermatocrite and the sperm surface biotin labeled proteins all along the different epididymal regions (caput, corpus, and cauda) in bovine. In the posterior caput (E3-E4), the protein secretion is the most intense where the luminal fluid volume is minimal and sperm concentration is maximal. It is exactly in this region where the sperm surface is changed. In part according to Belleannee et al. [2011b].

No apparent correlation can be drawn between the sequences by which the major sperm surface proteins are modified and the changes that occur to the surrounding milieu. However, our knowledge of the protein composition of the sperm surface is still limited; only several major components have been identified by MS. For most of the minor sperm surface proteins, their presence on the sperm surface has been identified only by the use Copyright © 2012 Informa Healthcare USA, Inc.

of antibodies and there has been no global proteomic study completed to date.

Conclusions and Perspectives Formation of the male gamete is one of the most complex cellular differentiation events. In mammals, terminal modifications of spermatozoa occur outside the gonad during


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their transit through the epididymal tubule. These steps of post-testicular differentiation are not all controlled by the gamete genome, instead most are controlled by the cellular activities of the epithelial cells of the epididymis. Most of these controls are mediated by the presence of specific proteins surrounding the male gamete. An extensive knowledge of the protein composition of the gametes, and of the epididymal milieu, is key to understanding the process of post gonadal sperm maturation. With new proteomic technologies, such as cell surface capturing for the study the sperm surface glycoproteins [Wollscheid et al. 2009], and quantitative MS/MS differential approaches, it should be possible to characterize most of the proteins present in the sperm membrane and in epididymal fluid. Such techniques will even allow the identification of minor components in these samples which, until now, have been difficult to identify. Post gonadal sperm differentiation occurs in all mammals and, hence, there may be a common mechanism which controls this maturational process. However, based on our knowledge of differences in the major proteins present in the epididymal fluids from different species, it appears that each may have developed different strategies to control this final step in sperm differentiation. Thus, it appears that multi-species studies will be indispensable in furthering our understanding of the role of the epididymis in controlling the fertility of the male gamete. The identification of most of these sperm membrane and epididymal proteins is a crucial step in this process. In the meantime, we will continue to be frustrated by our lack of knowledge as to the roles of the majority of these components in the process of the post testicular differentiation of the male gamete.

Acknowledgments We thank G. Tsikis for technical assistance and M. Belghazi for her active participation in the mass spectrometry analysis. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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