Systems Biology in Reproductive Medicine, 2013, Early Online: 1–7 Copyright © 2013 Informa Healthcare USA, Inc. ISSN 1939-6368 print/1939-6376 online DOI: 10.3109/19396368.2013.822612
RESEARCH COMMUNICATION
Biochemical characterization of stallion prostasomes and comparison to their human counterparts Göran K. Ronquist1, Bo Ek2, Gunnar Ronquist1, Jane Morrell3, Lena Carlsson1 and Anders Larsson1∗
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Department of Medical Sciences, Clinical Chemistry, University Hospital, Uppsala, Sweden, 2Department of Analytical Chemistry, Science for Life Laboratory, Uppsala University, Uppsala, Sweden, 3Department of Clinical Sciences, Division of Reproduction, Swedish University of Agricultural Sciences, Uppsala, Sweden
1982; Minelli et al. 1998]. Hence, most prostasomal investigations have been carried out on human specimens. The prostasomal membrane is rich in saturated fatty acids with sphingomyelin being the predominant phospholipid. The richness in saturated membrane fatty acid tails in the internal space of the membrane bilayer conveys a sterically accepting environment for cholesterol to be embedded and the prostasomal membrane displays an unusually high cholesterol/phospholipid ratio [Arvidson et al. 1989]. The membrane composition of prostasomes shows similarities with membrane domains (lipid rafts) occurring instantaneously in the plasma membrane of cells. Lipid rafts attract a broad range of membrane proteins, among which receptor complex-forming proteins and GPI-anchors are included [Nixon and Aitken 2009; Bonnon et al. 2010]. On investigation of the protein composition of human prostasomes, more than 400 prostasomal proteins were identified [Utleg et al. 2003; Poliakov et al. 2009]. Somewhat unexpectedly, chromosomal DNA was also identified in human prostasomes [Ronquist et al. 2009]. Human prostasomes have the ability to interact and under certain conditions even fuse with sperm cells enabling a transfer of molecules in favor of fertilization [Ronquist et al. 1990; Carlini et al. 1997; Arienti et al. 1997]. The prostasomes promote and prolong the motility of human sperm cells [Stegmayr and Ronquist 1982; Fabiani et al. 1995; Park et al. 2011] and their interaction also results in immunosuppressive and complement inhibitory activities [Kelly et al. 1991; Skibinski et al. 1992; Rooney et al. 1993]. Additionally, prostasomes have been assigned antioxidant [Saez et al. 1998] and antibacterial capacities [Carlsson et al. 2000], and they may play a role in the capacitation and acrosome reactions [Palmerini et al. 2003; Siciliano et al. 2008; Arienti et al. 2004; Cross and Mahasreshti 1997]. A number of features of equine pregnancy are unique compared to other mammals [Bauersachs and Wolf 2012].
Release of nanometer-sized prostasomes into human and equine semen suggests essential functions in their relationships with sperm cells and the fertilization process. The two types of prostasomes displayed ultrastructural similarities, albeit the human prostasomes were somewhat larger than the stallion prostasomes. A high ratio of saturated fatty acids was characteristic for the two prostasome types. Electrophoretic separation systems revealed an equine prostasomal pattern different from that of human. The 21 distinctive low molecular weight protein spots in the 2D-gel (with no counterparts in human prostasomes) were identified via peptide mass fingerprinting, several of which may be different isoforms. Out of the three high molecular weight bands characteristic for human prostasomes (CD10, CD13, and CD26), CD10 and CD13 were retrieved in equine prostasomes. We present some new proteins of horse prostasomes not found in their human counterparts. Further studies are warranted to reveal the function of these proteins. Keywords lipid raft, microvesicles, prostasomes, reproduction, stallion Abbreviations MALDI-MS: matrix-assisted laser desorption/ ionization-mass spectrometry; NCBInr: National Center for Biotechnology Information, non-redundant; TEM: transmission electron microscopy; QELS: quasi-elastic light scattering; PBS: phosphate buffered saline; MS: mass spectrometry.
Introduction Human prostasomes are microvesicles (40-490 nm, mean 150 nm) secreted by acinar cells of the prostate gland into the prostatic fluid and therewith into seminal fluid [Ronquist et al. 1978a; 1978b; Ronquist and Brody 1985]. There are few reports on prostasomes of other species besides homo sapiens [Siciliano et al. 2008; Breitbart and Rubinstein Received 17 October 2012; revised 02 May 2013; accepted 03 May 2013.
∗ Address correspondence to Anders Larsson, Department of Medical Sciences, Clinical Chemistry, Akademiska sjukhuset, entrance 61, 3rd floor, SE-751 85 Uppsala, Sweden. E-mail: [email protected]
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2 G. K. Ronquist et al.
Figure 1. Comparative electron microscopic pictures of human (A) and horse (B) prostasomes. Both human and horse prostasomes displayed prostasomes of various sizes. The horse prostasomes were somewhat smaller than the human prostasomes. Note the difference in magnification. Both prostasome types typically demonstrated electron dense and electron lucid vesicles generally surrounded by a classical bilayered membrane (arrows).
The bi-lobated horse prostate gland produces most of the seminal fluid [Hejmej et al. 2006] contrary to the consolidated human prostate gland that contributes only approximately one third of the volume of the seminal fluid [Eliasson and Lindholmer 1972]. Motility characteristics of stallion sperm cells are believed to be predictive of the fertility capacity at collection [Heckenbichler et al. 2011; Jasko 1992]. Mammalian sperm cells obtained from the testes are immotile and acquire their motility during and after ejaculation with the contribution of the accessory sex gland secretion [Töpfer-Petersen et al. 2005]. Recent work on human prostasomes revealed that sperm cells are not obliged to manufacture or maintain all the important signaling proteins that are motility promoting and essential for the fertilizing ability of sperm cells. Instead, they acquire some from prostasomes on their way to their target, the ovum [Park et al. 2011]. It is conceivable that similar interactive mechanisms exist between prostasomes and sperm cells of the horse provided they share some common characteristics. The present study compared the biochemical characteristics of human and stallion prostasomes.
Results Electron microscopic findings Purified prostasomes were analyzed by transmission electron microscopy. Both human and stallion prostasomes exhibited nanometer-sized, round structures surrounded by a bilayered membrane. They contained varying amounts of electron-dense material. Human prostasomes were somewhat larger than stallion prostasomes (Fig. 1A and B). Fatty acid composition Gas chromatography analysis of the prostasomal membranes of both human and horse displayed on the whole a similarity in regard to fatty acid composition with a high ratio of saturated fatty acids and, hence, the similar ratios indicated a relationship between the two membrane structures
Table 1. Membrane fatty acid composition of human and stallion prostasomes. Fatty acid C 14:0 C 16:0 C 18:0 C 20:0 C 22:0 C 24:0 Total saturated lipids C 16:1 n-7 C 18:1 n-9 C 18:1 n-7 C 20:1 n-9 Total mono-unsaturated lipids C 18:2 n-6 C 20:2 n-6 Total bi-unsaturated lipids C 18:3 n-3 C 18:3 n-6 C 20:3 n-6 Total tri-unsaturated lipids C 18:4 n-3 C 20:4 n-6 C 20:5 n-3 EPA C 22:5 n-3 DPA C 22:6 n-3 DHA Total polyunsaturated lipids Total
Human prostasomes (%)
Stallion prostasomes (%)
1.9 30.4 44.9 3.8 4.4 3.2 88.6 n.d. 7.2 n.d. n.d. 7.2
3.1 33.4 48.6 2.3 2.1 2.0 91.5 n.d. 3.0 n.d. n.d. 3.0
2.4 n.d. 2.4 n.d. n.d. n.d. 0 n.d. n.d. 0.7 n.d. 1.6 2.3
3.6 n.d. 3.6 n.d. n.d. n.d. 0 n.d. n.d. 0.5 n.d. 0.8 1.3
100
100
EPA: Eicosapentaenoic acid; DPA: Docosapentaenoic acid; DHA: Docosahexaenoic acid; n.d.: not detected
(Table 1). The production of ‘liposomes’ harboring therapeutic drugs has been a goal of many clinical researchers. To date, the lipid composition of liposomes has been empirically determined and to some extent based mostly on physiochemical knowledge acquired on lipid organization of artificial membranes. The comparison of lipid composition of prostasomes (with known fusiogenic properties) from Systems Biology in Reproductive Medicine
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Biochemical characterization of stallion prostasomes and comparison to their human counterparts
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Figure 2. SDS-PAGE of human prostasomes, horse prostasomes from five individual horses, and pooled horse prostasomes. Lane 1 was loaded with pooled human prostasomes. Lanes 2, 3, 4, 5, and 6 were loaded with prostasomes from individual horses; A, L, NW, O, and B. Lanes 7 and 8 were loaded with two different pools of horse prostasomes. The three bands around 98 kDa (see arrows), containing the characteristic proteins (CD10, CD13, and CD 26) for human prostasomes, appeared in all lanes. Proteins in the gel were disclosed by Coomassie staining. Lanes (1-8) in the right insert show the pattern of proteins > 98 kDa using the more sensitive silver staining.
two species offers a good opportunity of a ‘therapeutic vesicle’ whose lipid composition and organization could be a starting point of such a process.
1D SDS-PAGE and immunoblot analysis Purified prostasomes were separated by 1D SDS-PAGE for identification of protein band pattern. By comparison, prostasomes from human (Fig. 2, lane 1) and stallion (Fig. 2, lanes 2-8) demonstrated bands with similar molecular weights in the high molecular weight range including the three characteristic proteins; CD10, CD13, and CD26 previously found typical for human prostasomes [Ronquist et al. 2011]. Distinguishing features were present and were featured prominently in the low molecular weight area. Several prominent bands characteristic of the stallion prostasomes were not present in the human prostasomes. In order to test the reproducibility of the banding pattern, prostasomes from individual stallions were analyzed. As shown in Figure 2, lanes 2-6, there was a satisfactory conformity between the banding pattern from 2 pooled samples of prostasomes derived from a number of other stallions (Fig. 2, lanes 2-6 and 7-8). Hence, the results were generally consistent among individuals. After separation by 1D SDS-PAGE the separated protein was electrotransfered onto nitrocellulose in preparation for immunoblotting. Immunoblotting with a polyclonal chicken-antibody directed against human prostasomes revealed, as expected, a broad variety of human prostasomal proteins (results not shown). Moreover, some of the horse prostasomal antigens were also identified by the same antibody (results not shown). This indicated a certain degree of similarity between the human and stallion prostasome antigenic structures. Still, it should be noted that due to a lack of anti-equine antibodies, reverse western blot confirmation is prevented in most cases. Copyright © 2013 Informa Healthcare USA, Inc.
Figure 3. A comparative survey of 2D-separated proteins from human and horse prostasomes. The separations were performed in two dimensions, first a horizontal isoelectric separation followed by a vertical molecular weight separation. Prostasomal proteins from horse (bottom) displayed several marked proteins in the lower molecular weight bands (analyzed by Matrix-assisted laser desorption/ionization (MALDI)-Mass spectrometry (MS)), not seen in the corresponding 2D of human prostasomal proteins (top).
2D-electrophoretic separation and identification by mass spectrometry Purified prostasomes of both species were first separated by isoelectric focusing and then separated by SDS-PAGE to achieve a 2D-separation. The 2D-electrophoretic separation of proteins from purified human prostasomes (Fig. 3) compared with the corresponding 2D-separation of horse prostasomal proteins. Figure 3 displayed strong differences in the
4 G. K. Ronquist et al. Table 2. Identified proteins in stallion prostasomes (Equus caballus). Spot number
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Protein
Entry
Seminal plasma protein-1 Seminal plasma protein HSP-1 Seminal plasma protein-1 Seminal plasma protein-1 Seminal plasma protein-1 Seminal plasma protein-1 Impossible to identify Caveolin-1 Seminal plasma protein-1 Similar to mitochondrial import inner membrane translocase subunit Tim23 Similar to growth hormone releasing factor Similar to mitochondrial ribosomal protein S12 Similar to inositol polyphosphate phosphatase-like 1 Similar to type I keratin KA17 isoform 3 Similar to Keratin 7 Similar to seminal plasma protein 2 TP53-regulated inhibitor of apoptosis 1 Similar to glutamate receptor ionotropic kainate 4 isoform 1 Similar to seminal plasma protein 2 Similar to seminal plasma protein 2 Similar to seminal plasma protein 2
Q70GG6 P81121 Q70GG6 Q70GG6 Q70GG6 Q70GG6 Q2QLB0 Q70GG6 F2Q0S0 Q9XS89 F7BBW5 Q6P549 Q6IFU8 F7DZA2 Q70GG4 Q9D8Z2 F6SKB7 Q70GG4 Q70GG4 Q70GG4
Table 3. Compilation of exosome-related proteins identified in both horse and human prostasomes by either mass spectrometry or immunoblotting. 14-3-3 proteins 14-3-3 protein beta 14-3-3 protein epsilon 14-3-3 protein gamma 14-3-3 protein zeta/delta Annexins Annexin A1 Annexin A11 Annexin A2 Annexin A3 CD-antigens CD9 antigen (Member of the tetraspanin family) CD10 antigen (Neprilysin) CD13 antigen (Aminopeptidase N) CD26 antigen (Dipeptidyl peptidase 4)
Clathrin/Clusterin Clathrin heavy chain 1 Clusterin Ezrin/Moesin/Radixin Ezrin Moesin Radixin Vesicle-associated membrane proteins Vesicle-associated membrane protein 3
levels of the low molecular weight proteins similar to that observed by 1D SDS-PAGE (Fig. 2). Three bands from the 1D-separated and 21 spots from the 2D-separated stallion prostasomes were cut out and digested with trypsin prior to Matrix-assisted laser desorption/ionization-Mass spectrometry (MALDI-MS) analysis. The MALDI-MS analysis of the three bands, in the 1D-separation of horse proteins, with a similar molecular weight to those repeatedly occurring in the 1D-gel of human prostasomes (CD10, CD13, and CD26) showed that CD10 and CD13 but not CD26 could be identified. Analysis of the 21 distinctive low-molecular weight proteins (Fig. 3, bottom
panel) in the 2D-gel of horse proteins via peptide mass fingerprinting using the National Center for Biotechnology Information non-redundant (NCBInr) database are listed in Table 2. It may be mentioned in this context that only 9 spots were definitely new proteins while the 12 others might represent different isoforms/degradation products. Eighteen proteins were detected in both prostasome types subdivided into 14-3-3 protein, annexins, CD-antigens, Cathrin/Clusterin, Ezrin/Moesin/Radixin, and Vesicleassociated membrane proteins (Table 3).
Discussion Prostasomes were previously reported to be present in stallion seminal plasma [Minelli et al. 1998; Arienti et al. 1998; Aalberts et al. 2012]. Here we provide evidence of similarities but also dissimilarities between human and stallion prostasomes. The investigation by transmission electron microscopy (TEM) displayed nanometer-sized prostasomes for both species, with a somewhat smaller average size for the prostasomes from horse. This was contradictory to the report of Arienti et al. [1998] who found the average diameter of horse prostasomes being somewhat larger than that of human prostasomes when using the quasi-elastic light scattering (QELS)-technique. The TEM-pictures of prostasomes from stallions showed the contribution of an amorphous substance that apparently eluded the chromatography step in the prostasome purification. This was in agreement with a previous investigation [Minelli et al. 1998]. The origin of this amorphous substance is not known. It is possible that the amorphous substance of stallion is aggregated plasma that created high molecular complexes that were found together with prostasomes in the void volume during gel chromatography. Gas chromatography analyses revealed a similar composition of fatty acids in both human and horse prostasomes with a high degree of saturated fatty acids consistent with previous findings [Arienti et al. 2001]. The separation of prostasomal proteins registered in the 1D-patterns from 5 different horses was consistent and this pattern also corresponded with poolings of prostasomes from other horses. Moreover, human prostasomal 1D-patterns from different preparations over time were found to have a high similarity between themselves [Ronquist et al. 2011] although being different from those of horses. Nevertheless, similarities in the banding patterns existed as well. The three bands characteristic for human prostasomes identified as CD10 (neprilysin), CD13 (aminopeptidase N), and CD26 (dipeptidyl peptidase 4) had analogous bands in stallion prostasomes, of which CD10 and CD13 could be identified by mass-spectrometry. It should be kept in mind, however, that the molecular weight determined from 2D-SDS gels do not always agree with mass spectrometry results. Additional marked, low-molecular weight bands were typical of horse prostasomes. These low-molecular weight proteins had a charge range between at least pH 3 and 12 when separated by 2D-gel electrophoresis. Identification of these proteins revealed several isoforms. Among Systems Biology in Reproductive Medicine
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Biochemical characterization of stallion prostasomes and comparison to their human counterparts the unique proteins, seminal plasma proteins 1 and 2, involved in the capacitation reaction in the bovine species [Manjunath et al. 2009] and the seminal plasma protein, horse seminal protein-1, are of known reproductive function [Calvete et al. 1995]. This characteristic pattern, of prostasome proteins can be used to differentiate prostasomes from epididymosomes that have a different protein pattern [Thimon et al. 2008]. This also tallies with our previous investigation showing no difference in the total amount of prostasomes between normospermic and azoospermic individuals ruling out any significant contribution of epididymosomes in our prostasome preparation [Carlsson et al. 2006]. Surprisingly, two of the low-molecular weight proteins were mitochondrial. This could be the formation of prostasomes in the late endosomes or early stages of lysosomes [Sahlen et al. 2002], organelles where the milieu of cellular proteins are degraded. The formation of prostasomes inside late endosomes takes place by an invagination of specific pockets of the membrane, called lipid rafts. They contain a high proportion of saturated fatty acids and are found as part of the process by which caveola incorporate caveolin [Williams and Lisanti 2004]. This protien was also identified in horse prostasomes. Several other proteins were also identified in horse prostasomes. For example, the growth hormone releasing factor, a protein that elevates the level of circulating growth hormone and insulin-like growth factor, was identified. At present the exact role of this hormone in a prostasomal context is unclear. Inositol polyphosphate phosphatase-like 1 protein that regulates phosphoinositide 3-pathways related to numerous cell activities [Raaijmakers et al. 2007], the keratins, fibrous structural proteins with different functions [Kirfel et al. 2003], and the TP53-regulated inhibitor of apoptosis 1 important in controlling the cell survival [Park and Nakamura 2005] were also identified. As stated by Mellor [2006] the protein “Similar to glutamate receptor ionotropic kainate 4 isoform 1” is a kainate receptor subtype forming gated ion-channels. Like TP53, these proteins have not been observed in human prostasomes [Utleg et al. 2003; Poliakov et al. 2009]. A recent report [Park et al. 2011] described the role of Ca2+ -channel protein transfer from prostasomes to sperms as an important factor in promoting sperm motility. They also highlighted the problem of degradation of proteins in sperms between the time transcription ceases, before spermiogenesis, and the time when proteins are most needed in the female reproductive tract. They therefore proposed that a fusion with prostasomes containing newly synthesized proteins provides a protective function. If correct, this would suggest a vital role that could be attributed to prostasomes that should be observed in other mammalian species. Prostasomes have been identified in other species including ram [Breitbart and Rubinstein 1982], dog [Frenette et al. 1985], stallion [Minelli et al. 1998; Arienti et al. 1998], and boar [Siciliano et al. 2008] but the search is far from exhaustive. However, as yet, no in-depth comparisons have been made between prostasomes in different species. Our ambition to highlight equine prostasomes in a reproductive context should be considered in the light of the importance and great commercial interest of achieving optimal progenies. Copyright © 2013 Informa Healthcare USA, Inc.
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The prostasome is likely to add another factor for consideration. Modern equine breeding management and technologies have positively influenced the conception rates, healthy pregnancies, and successful foaling. An increased knowledge of horse prostasomes and comparison with their human counterparts may be a further step forward towards a more optimized reproduction in horses.
Materials and Methods This study was approved by the human and animal Ethics Committees of the University of Uppsala (ethical number: Ups 01-367) and the Swedish University of Agricultural Sciences (SLU, ethical number: C 345/9), Uppsala.
Purification of prostasomes from human and horses Human seminal plasma was received from the Reproduction Unit at Uppsala University Hospital. Seminal plasma from horses was obtained from the Division of Reproduction, SLU. All preparatory procedures, unless otherwise stated, were performed at 4°C. The seminal plasma was first centrifuged at 10,000 x g for 30 min to pellet and remove possible cell debris. The supernatant was then ultracentrifuged at 100,000 x g for 2 h to pellet prostasomes and the pellet was resuspended in 30 mmol/L of Tris-HCl buffer, containing 130 mmol/L NaCl, pH 7.6 (isotonic Tris-HCl buffer). The prostasome pellet was then loaded on a Superdex column (Superdex, GE Healthcare, Uppsala, Sweden) equilibrated with the isotonic Tris-HCl buffer, to separate the prostasomes from an amorphous substance [Stegmayr and Ronquist 1982]. Fractions were collected every 20 min at a flow rate of 4 mL/h and the prostasome-containing fractions were identified by elevated absorbances at both 260 nm and 280 nm. The column-purified prostasomes were ultracentrifuged at 100,000 x g for 2 h to pellet the prostasomes. The pellet was subsequently resuspended in phosphate buffered saline, PBS (150 mmol/L NaCl, 10 mmol/L Na2HPO4, 2 mmol/L NaH2PO4), pH 7.4, and the concentration on a protein basis was adjusted to 2 mg/mL (ESL protein kit, Roche Diagnostics, Mannheim, Germany).
Electron microscopy Purified prostasomes (2 mL, 4 mg) from both sources were pelleted by ultracentrifugation and the pellet was fixed with 2.5% glutaraldehyde in PBS before embedding in Epon according to conventional techniques [Sahlen et al. 2002]. The plastic block was cut in 2 µm sections on an LKB microtome and subsequently trimmed to be cut into 40-60 nm sections. The sections were placed on slot grids with 0.5% Formvar film, contrasted with lead citrate and uranyl acetate and examined in a Zeiss Supra 35-VP (Zeiss Supra 35-VP, Carl Zeiss SMT) field emission scanning electron microscope, equipped with a STEM detector for transmission electron microscopy.
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6 G. K. Ronquist et al. Fatty acid analyses Prostasomal samples from human and horse were analyzed by gas chromatography for fatty acid composition of the prostasomal membranes (Triomega analys).
human and equine whole prostasome lysates (cf.: Table 3) was carried out as previously described [Ronquist et al. 2013].
SDS-PAGE (1D-electrophoresis) In all experiments, a final amount of 5.5 µL of purified prostasomes from either human or stallion (2 mg/mL, corresponding to 11 µg prostasomal protein) was separated on a 4-12% NuPAGE-gel at 200 V for 35 min. Protein bands were visualized by Coomassie staining or silver staining according to the manufacturer’s protocol. All SDS-PAGE materials were from InVitrogen (Paisley, UK). Immunoblotting was performed as previously described [Ronquist et al. 2012].
Declaration of interest: Biochemical analyses and data collection were funded by: The Swedish Medical Research Council 1D 2004/C1779, Liońs Cancer Fund, Uppsala, Sweden, and Stiftelsen Familjeplaneringsfonden, Uppsala, Sweden. JM was funded by the Swedish Foundation for Equine Research, Stockholm, project number H1047052. None of the authors have any conflicts of interest to declare.
2D-electrophoresis Prostasomes purified from seminal plasma from humans and horses (2 mg) were each pelleted and resuspended in 5 mL rehydration buffer (urea, 7 mol/L; thiourea, 2 mol/L; chaps, 4% (w/v); ASB-14, 2% (w/v) (all from SigmaAldrich, St. Louis, MO, USA) and dithiotreitol, (DTT) 5% (w/v) (Amersham Bioscience, Uppsala, Sweden). For the first-dimension electrophoresis, the isoelectric focusing (IEF), using immobilized pH-gradient, was carried out by loading 240 µL of rehydrated prostasomes (approximately 0.1 mg protein) onto the 11 cm IEF-strip (IEF pH 3-10, BioRad) with passive in-gel rehydration (21Ί C), overnight. Focusing was performed to a total of 20 kVh using an IPG-phor unit (21°C). The focused strips were equilibrated for 10 min, each in BioRad ReadyPrep 2-D starter kit equilibration buffer I and II (BioRad). The second dimension was carried out in a 4-12% SDS gel (Tris-HCl, 1 mm thick, 11 cm IPG well), and protein visualization was by Coomassie staining (InVitrogen). Mass spectrometry Preparative gels were loaded with 11 µg protein for 1D-separation and approximately 400 µg protein for 2D-separation. Three bands of the 1D-separated stallion proteins were cut out and 21 spots of the 2D-separated stallion proteins were punched out. In-gel digestion was carried out with trypsin [Havlis et al. 2003]. A preliminary MALDI-analysis was performed by removing 0.6 µL digest and spotting this on the MALDI target plate (ground steel 24 x 16 spots) and mixing with 0.6 µL 1:1 (v/v) saturated matrix solution of α-cyano-4-hydroxycinnamic acid in 30% acetonitrile/0.1% trifluoroacetic acid. Mass spectrometry (MS) -analysis was performed in a Bruker Ultraflex II (Bruker Corporation, Bremen, Germany). Spectra were analyzed by using the program, mMass [Strohalm et al. 2010] and from there, manual PMF searches in Mascot were initiated. Searches were usually done at the 60 ppm error level using “Other mammalia” and NCBInr (20110429 or close to this date). If judged necessary, i.e., no clear hit was obtained, a more thorough extraction of the digest was performed, using StageTips [Rappsilber et al. 2007] and subjected to a new MALDI-MS analysis. Comparative mass spectrometry of
Author contributions: Conceived and designed the experiments: GR, AL, JM; Performed the experiments: GKR, BE; Analyzed the data: GKR, BE, LC; Contributed reagents/ materials/ analysis tools: GKR, BE, GR, JM, LC, AL; Wrote the manuscript: GKR, BE, GR, AL.
References Aalberts, M., Sostaric, E., Wubbolts, R., Wauben, M.W., Nolte-’t Hoen, E.N., Gadella, B.M., et al. (2012) Spermatozoa recruit prostasomes in response to capacitation induction. Biochim Biophys Acta S1570–9639(12)00181–1. doi: 10.1016/j.bbapap.2012.08.008. Arienti, G., Carlini, E., De Cosmo, A.M., Di, P.P. and Palmerini, C.A. (1998) Prostasome-like particles in stallion semen. Biol Reprod 59:309–313. Arienti, G., Carlini, E., Saccardi, C. and Palmerini, C.A. (2004) Role of human prostasomes in the activation of spermatozoa. J Cell Mol Med 8:77–84. Arienti, G., Polci, A., Carlini, E. and Palmerini, C.A. (1997) Transfer of CD26/dipeptidyl peptidase IV (E.C. 3.5.4.4) from prostasomes to sperm. FEBS Lett 410:343–346. Arienti, G., Polci, A., De, C.A., Saccardi, C., Carlini, E. and Palmerini, C.A. (2001) Lipid fatty acid and protein pattern of equine prostasome-like vesicles. Comp Biochem Physiol B Biochem Mol Biol 128:661–666. Arvidson, G., Ronquist, G., Wikander, G. and Ojteg, A.C. (1989) Human prostasome membranes exhibit very high cholesterol/ phospholipid ratios yielding high molecular ordering. Biochim Biophys Acta 984:167–173. Bauersachs, S. and Wolf, E. (2012) Transcriptome analyses of bovine, porcine and equine endometrium during the pre-implantation phase. Anim Reprod Sci 134:84–94. Bonnon, C., Wendeler, M.W., Paccaud, J.P. and Hauri, H.P. (2010) Selective export of human GPI-anchored proteins from the endoplasmic reticulum. J Cell Sci 123:1705–1715. Breitbart, H. and Rubinstein, S. (1982) Characterization of Mg2+- and Ca2+-ATPase activity in membrane vesicles from ejaculated ram seminal plasma. Arch Androl 9:147–157. Calvete, J.J., Mann, K., Schafer, W., Sanz, L., Reinert, M., Nessau, S., et al. (1995) Amino acid sequence of HSP-1, a major protein of stallion seminal plasma: effect of glycosylation on its heparin- and gelatin-binding capabilities. Biochem J 310 (Pt 2):615–622. Carlini, E., Palmerini, C.A., Cosmi, E.V. and Arienti, G. (1997) Fusion of sperm with prostasomes: effects on membrane fluidity. Arch Biochem Biophys 343:6–12. Carlsson, L., Pahlson, C., Bergquist, M., Ronquist, G. and Stridsberg, M. (2000) Antibacterial activity of human prostasomes. Prostate 44:279–286. Carlsson, L., Ronquist, G., Eliasson, R., Egberg, N. and Larsson, A. (2006) Flow cytometric technique for determination of prostasomal quantity, size and expression of CD10, CD13, CD26 and CD59 in human seminal plasma. Int J Androl 29:331–338. Systems Biology in Reproductive Medicine
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Biochemical characterization of stallion prostasomes and comparison to their human counterparts Cross, N.L. and Mahasreshti, P. (1997) Prostasome fraction of human seminal plasma prevents sperm from becoming acrosomally responsive to the agonist progesterone. Arch Androl 39:39–44. Eliasson, R. and Lindholmer, C. (1972) Distribution and properties of spermatozoa in different fractions of split ejaculates. Fertil Steril 23:252–256. Fabiani, R., Johansson, L., Lundkvist, O. and Ronquist, G. (1995) Prolongation and improvement of prostasome promotive effect on sperm forward motility. Eur J Obstet Gynecol Reprod Biol 58:191–198. Frenette, G., Dube, J.Y., Marcotte, J.R. and Tremblay, R.R. (1985) Arginine esterase from isolated dog prostate secretory granules is fully active enzymatically. Can J Physiol Pharmacol 63:1603–1607. Havlis, J., Thomas, H., Sebela, M. and Shevchenko, A. (2003) Fastresponse proteomics by accelerated in-gel digestion of proteins. Anal Chem 75:1300–1306. Heckenbichler, S., Deichsel, K., Peters, P. and Aurich, C. (2011) Quality and fertility of cooled-shipped stallion semen at the time of insemination. Theriogenology 75:849–856. Hejmej, A., Wiszniewska, B., Kosiniak-Kamysz, K., Sadowska, J. and Bilinska, B. (2006) The presence of androgen receptors in the epididymis and prostate of the stallion and cryptorchid horse–a preliminary study. Vet J 171:373–379. Jasko, D.J. (1992) Evaluation of stallion semen. Vet Clin North Am Equine Pract 8:129–148. Kelly, R.W., Holland, P., Skibinski, G., Harrison, C., McMillan, L., Hargreave, T. et al. (1991) Extracellular organelles (prostasomes) are immunosuppressive components of human semen. Clin Exp Immunol 86:550–556. Kirfel, J., Magin, T.M. and Reichelt, J. (2003) Keratins: a structural scaffold with emerging functions. Cell Mol Life Sci 60:56–71. Manjunath, P., Lefebvre, J., Jois, P.S., Fan, J. and Wright, M.W. (2009) New nomenclature for mammalian BSP genes. Biol Reprod 80:394–397. Mellor, J.R. (2006) Synaptic plasticity of kainate receptors. Biochem Soc Trans 34:949–951. Minelli, A., Moroni, M., Martinez, E., Mezzasoma, I. and Ronquist, G. (1998) Occurrence of prostasome-like membrane vesicles in equine seminal plasma. J Reprod Fertil 114:237–243. Nixon, B. and Aitken, R.J. (2009) The biological significance of detergent-resistant membranes in spermatozoa. J Reprod Immunol 83:8–13. Palmerini, C.A., Saccardi, C., Carlini, E., Fabiani, R. and Arienti, G. (2003) Fusion of prostasomes to human spermatozoa stimulates the acrosome reaction. Fertil Steril 80:1181–1184. Park, K.H., Kim, B.J., Kang, J., Nam, T.S., Lim, J.M., Kim, H.T., et al. (2011) Ca2+ signaling tools acquired from prostasomes are required for progesterone-induced sperm motility. Sci Signal 4:ra31. Park, W.R. and Nakamura, Y. (2005) p53CSV, a novel p53–inducible gene involved in the p53-dependent cell-survival pathway. Cancer Res 65:1197–1206. Poliakov, A., Spilman, M., Dokland, T., Amling, C.L. and Mobley, J.A. (2009) Structural heterogeneity and protein composition of exosome-like vesicles (prostasomes) in human semen. Prostate 69:159–167. Raaijmakers, J.H., Deneubourg, L., Rehmann, H., de Koning, J., Zhang, Z., Krugmann, S., et al. (2007) The PI3K effector Arap3 interacts with the PI(3,4,5)P3 phosphatase SHIP2 in a SAM domain-dependent manner. Cell Signal 19:1249–1257. Rappsilber, J., Mann, M. and Ishihama, Y. (2007) Protocol for micropurification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906.
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Ronquist, G. and Brody, I. (1985) The prostasome: its secretion and function in man. Biochim Biophys Acta 822:203–218. Ronquist, G., Brody, I., Gottfries, A. and Stegmayr, B. (1978a) An Mg2+ and Ca2+-stimulated adenosine triphosphatase in human prostatic fluid: part I. Andrologia 10:261–272. Ronquist, G., Brody, I., Gottfries, A. and Stegmayr, B. (1978b) An Mg2+ and Ca2+-stimulated adenosine triphosphatase in human prostatic fluid: part II. Andrologia 10:427–433. Ronquist, K.G., Ek, B., Stavreus-Evers, A., Larsson, A. and Ronquist, G. (2013) Human prostasomes express glycolytic enzymes with capacity for ATP production. Am J Physiol Endocrinol Metab 304:E576–582. Ronquist, K.G., Larsson, A., Ronquist, G., Isaksson, A., Hreinsson, J., Carlsson, L., et al. (2011) Prostasomal DNA characterization and transfer into human sperm. Mol Reprod Dev 78:467–476. Ronquist, K.G., Larsson, A., Stavreus-Evers, A. and Ronquist, G. (2012) Prostasomes are heterogeneous regarding size and appearance but affiliated to one DNA-containing exosome family. Prostate 72:1736–745. Ronquist, G., Nilsson, B.O. and Hjerten, S. (1990) Interaction between prostasomes and spermatozoa from human semen. Arch Androl 24:147–157. Ronquist, K.G., Ronquist, G., Carlsson, L. and Larsson, A. (2009) Human prostasomes contain chromosomal DNA. Prostate 69:737–743. Rooney, I.A., Atkinson, J.P., Krul, E.S., Schonfeld, G., Polakoski, K., Saffitz, J.E., et al. (1993) Physiologic relevance of the membrane attack complex inhibitory protein CD59 in human seminal plasma: CD59 is present on extracellular organelles (prostasomes), binds cell membranes, and inhibits complement-mediated lysis. J Exp Med 177:1409–1420. Saez, F., Motta, C., Boucher, D. and Grizard, G. (1998) Antioxidant capacity of prostasomes in human semen. Mol Hum Reprod 4:667–672. Sahlen, G.E., Egevad, L., Ahlander, A., Norlen, B.J., Ronquist, G. and Nilsson, B.O. (2002) Ultrastructure of the secretion of prostasomes from benign and malignant epithelial cells in the prostate. Prostate 53:192–199. Siciliano, L., Marciano, V. and Carpino, A. (2008) Prostasome-like vesicles stimulate acrosome reaction of pig spermatozoa. Reprod Biol Endocrinol 6:5. Skibinski, G., Kelly, R.W., Harkiss, D. and James, K. (1992) Immunosuppression by human seminal plasma–extracellular organelles (prostasomes) modulate activity of phagocytic cells. Am J Reprod Immunol 28:97–103. Stegmayr, B. and Ronquist, G. (1982) Promotive effect on human sperm progressive motility by prostasomes. Urol Res 10:253–257. Strohalm, M., Kavan, D., Novak, P., Volny, M. and Havlicek, V. (2010) mMass 3: a cross-platform software environment for precise analysis of mass spectrometric data. Anal Chem 82:4648–4651. Thimon, V., Frenette, G., Saez, F., Thabet, M. and Sullivan, R. (2008) Protein composition of human epididymosomes collected during surgical vasectomy reversal: a proteomic and genomic approach. Hum Reprod 23:1698–1707. Töpfer-Petersen, E., Ekhlasi-Hundrieser, M., Kirchhoff, C., Leeb, T. and Sieme, H. (2005) The role of stallion seminal proteins in fertilisation. Anim Reprod Sci 89:159–170. Utleg, A.G., Yi, E.C., Xie, T., Shannon, P., White, J.T., Goodlett, D.R., et al. (2003) Proteomic analysis of human prostasomes. Prostate 56:150–161. Williams, T.M., and Lisanti, M.P. (2004) The caveolin proteins. Genome Biol 5:214.
RESEARCH COMMUNICATION
Biochemical characterization of stallion prostasomes and comparison to their human counterparts Göran K. Ronquist1, Bo Ek2, Gunnar Ronquist1, Jane Morrell3, Lena Carlsson1 and Anders Larsson1∗
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1
Department of Medical Sciences, Clinical Chemistry, University Hospital, Uppsala, Sweden, 2Department of Analytical Chemistry, Science for Life Laboratory, Uppsala University, Uppsala, Sweden, 3Department of Clinical Sciences, Division of Reproduction, Swedish University of Agricultural Sciences, Uppsala, Sweden
1982; Minelli et al. 1998]. Hence, most prostasomal investigations have been carried out on human specimens. The prostasomal membrane is rich in saturated fatty acids with sphingomyelin being the predominant phospholipid. The richness in saturated membrane fatty acid tails in the internal space of the membrane bilayer conveys a sterically accepting environment for cholesterol to be embedded and the prostasomal membrane displays an unusually high cholesterol/phospholipid ratio [Arvidson et al. 1989]. The membrane composition of prostasomes shows similarities with membrane domains (lipid rafts) occurring instantaneously in the plasma membrane of cells. Lipid rafts attract a broad range of membrane proteins, among which receptor complex-forming proteins and GPI-anchors are included [Nixon and Aitken 2009; Bonnon et al. 2010]. On investigation of the protein composition of human prostasomes, more than 400 prostasomal proteins were identified [Utleg et al. 2003; Poliakov et al. 2009]. Somewhat unexpectedly, chromosomal DNA was also identified in human prostasomes [Ronquist et al. 2009]. Human prostasomes have the ability to interact and under certain conditions even fuse with sperm cells enabling a transfer of molecules in favor of fertilization [Ronquist et al. 1990; Carlini et al. 1997; Arienti et al. 1997]. The prostasomes promote and prolong the motility of human sperm cells [Stegmayr and Ronquist 1982; Fabiani et al. 1995; Park et al. 2011] and their interaction also results in immunosuppressive and complement inhibitory activities [Kelly et al. 1991; Skibinski et al. 1992; Rooney et al. 1993]. Additionally, prostasomes have been assigned antioxidant [Saez et al. 1998] and antibacterial capacities [Carlsson et al. 2000], and they may play a role in the capacitation and acrosome reactions [Palmerini et al. 2003; Siciliano et al. 2008; Arienti et al. 2004; Cross and Mahasreshti 1997]. A number of features of equine pregnancy are unique compared to other mammals [Bauersachs and Wolf 2012].
Release of nanometer-sized prostasomes into human and equine semen suggests essential functions in their relationships with sperm cells and the fertilization process. The two types of prostasomes displayed ultrastructural similarities, albeit the human prostasomes were somewhat larger than the stallion prostasomes. A high ratio of saturated fatty acids was characteristic for the two prostasome types. Electrophoretic separation systems revealed an equine prostasomal pattern different from that of human. The 21 distinctive low molecular weight protein spots in the 2D-gel (with no counterparts in human prostasomes) were identified via peptide mass fingerprinting, several of which may be different isoforms. Out of the three high molecular weight bands characteristic for human prostasomes (CD10, CD13, and CD26), CD10 and CD13 were retrieved in equine prostasomes. We present some new proteins of horse prostasomes not found in their human counterparts. Further studies are warranted to reveal the function of these proteins. Keywords lipid raft, microvesicles, prostasomes, reproduction, stallion Abbreviations MALDI-MS: matrix-assisted laser desorption/ ionization-mass spectrometry; NCBInr: National Center for Biotechnology Information, non-redundant; TEM: transmission electron microscopy; QELS: quasi-elastic light scattering; PBS: phosphate buffered saline; MS: mass spectrometry.
Introduction Human prostasomes are microvesicles (40-490 nm, mean 150 nm) secreted by acinar cells of the prostate gland into the prostatic fluid and therewith into seminal fluid [Ronquist et al. 1978a; 1978b; Ronquist and Brody 1985]. There are few reports on prostasomes of other species besides homo sapiens [Siciliano et al. 2008; Breitbart and Rubinstein Received 17 October 2012; revised 02 May 2013; accepted 03 May 2013.
∗ Address correspondence to Anders Larsson, Department of Medical Sciences, Clinical Chemistry, Akademiska sjukhuset, entrance 61, 3rd floor, SE-751 85 Uppsala, Sweden. E-mail: [email protected]
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2 G. K. Ronquist et al.
Figure 1. Comparative electron microscopic pictures of human (A) and horse (B) prostasomes. Both human and horse prostasomes displayed prostasomes of various sizes. The horse prostasomes were somewhat smaller than the human prostasomes. Note the difference in magnification. Both prostasome types typically demonstrated electron dense and electron lucid vesicles generally surrounded by a classical bilayered membrane (arrows).
The bi-lobated horse prostate gland produces most of the seminal fluid [Hejmej et al. 2006] contrary to the consolidated human prostate gland that contributes only approximately one third of the volume of the seminal fluid [Eliasson and Lindholmer 1972]. Motility characteristics of stallion sperm cells are believed to be predictive of the fertility capacity at collection [Heckenbichler et al. 2011; Jasko 1992]. Mammalian sperm cells obtained from the testes are immotile and acquire their motility during and after ejaculation with the contribution of the accessory sex gland secretion [Töpfer-Petersen et al. 2005]. Recent work on human prostasomes revealed that sperm cells are not obliged to manufacture or maintain all the important signaling proteins that are motility promoting and essential for the fertilizing ability of sperm cells. Instead, they acquire some from prostasomes on their way to their target, the ovum [Park et al. 2011]. It is conceivable that similar interactive mechanisms exist between prostasomes and sperm cells of the horse provided they share some common characteristics. The present study compared the biochemical characteristics of human and stallion prostasomes.
Results Electron microscopic findings Purified prostasomes were analyzed by transmission electron microscopy. Both human and stallion prostasomes exhibited nanometer-sized, round structures surrounded by a bilayered membrane. They contained varying amounts of electron-dense material. Human prostasomes were somewhat larger than stallion prostasomes (Fig. 1A and B). Fatty acid composition Gas chromatography analysis of the prostasomal membranes of both human and horse displayed on the whole a similarity in regard to fatty acid composition with a high ratio of saturated fatty acids and, hence, the similar ratios indicated a relationship between the two membrane structures
Table 1. Membrane fatty acid composition of human and stallion prostasomes. Fatty acid C 14:0 C 16:0 C 18:0 C 20:0 C 22:0 C 24:0 Total saturated lipids C 16:1 n-7 C 18:1 n-9 C 18:1 n-7 C 20:1 n-9 Total mono-unsaturated lipids C 18:2 n-6 C 20:2 n-6 Total bi-unsaturated lipids C 18:3 n-3 C 18:3 n-6 C 20:3 n-6 Total tri-unsaturated lipids C 18:4 n-3 C 20:4 n-6 C 20:5 n-3 EPA C 22:5 n-3 DPA C 22:6 n-3 DHA Total polyunsaturated lipids Total
Human prostasomes (%)
Stallion prostasomes (%)
1.9 30.4 44.9 3.8 4.4 3.2 88.6 n.d. 7.2 n.d. n.d. 7.2
3.1 33.4 48.6 2.3 2.1 2.0 91.5 n.d. 3.0 n.d. n.d. 3.0
2.4 n.d. 2.4 n.d. n.d. n.d. 0 n.d. n.d. 0.7 n.d. 1.6 2.3
3.6 n.d. 3.6 n.d. n.d. n.d. 0 n.d. n.d. 0.5 n.d. 0.8 1.3
100
100
EPA: Eicosapentaenoic acid; DPA: Docosapentaenoic acid; DHA: Docosahexaenoic acid; n.d.: not detected
(Table 1). The production of ‘liposomes’ harboring therapeutic drugs has been a goal of many clinical researchers. To date, the lipid composition of liposomes has been empirically determined and to some extent based mostly on physiochemical knowledge acquired on lipid organization of artificial membranes. The comparison of lipid composition of prostasomes (with known fusiogenic properties) from Systems Biology in Reproductive Medicine
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Biochemical characterization of stallion prostasomes and comparison to their human counterparts
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Figure 2. SDS-PAGE of human prostasomes, horse prostasomes from five individual horses, and pooled horse prostasomes. Lane 1 was loaded with pooled human prostasomes. Lanes 2, 3, 4, 5, and 6 were loaded with prostasomes from individual horses; A, L, NW, O, and B. Lanes 7 and 8 were loaded with two different pools of horse prostasomes. The three bands around 98 kDa (see arrows), containing the characteristic proteins (CD10, CD13, and CD 26) for human prostasomes, appeared in all lanes. Proteins in the gel were disclosed by Coomassie staining. Lanes (1-8) in the right insert show the pattern of proteins > 98 kDa using the more sensitive silver staining.
two species offers a good opportunity of a ‘therapeutic vesicle’ whose lipid composition and organization could be a starting point of such a process.
1D SDS-PAGE and immunoblot analysis Purified prostasomes were separated by 1D SDS-PAGE for identification of protein band pattern. By comparison, prostasomes from human (Fig. 2, lane 1) and stallion (Fig. 2, lanes 2-8) demonstrated bands with similar molecular weights in the high molecular weight range including the three characteristic proteins; CD10, CD13, and CD26 previously found typical for human prostasomes [Ronquist et al. 2011]. Distinguishing features were present and were featured prominently in the low molecular weight area. Several prominent bands characteristic of the stallion prostasomes were not present in the human prostasomes. In order to test the reproducibility of the banding pattern, prostasomes from individual stallions were analyzed. As shown in Figure 2, lanes 2-6, there was a satisfactory conformity between the banding pattern from 2 pooled samples of prostasomes derived from a number of other stallions (Fig. 2, lanes 2-6 and 7-8). Hence, the results were generally consistent among individuals. After separation by 1D SDS-PAGE the separated protein was electrotransfered onto nitrocellulose in preparation for immunoblotting. Immunoblotting with a polyclonal chicken-antibody directed against human prostasomes revealed, as expected, a broad variety of human prostasomal proteins (results not shown). Moreover, some of the horse prostasomal antigens were also identified by the same antibody (results not shown). This indicated a certain degree of similarity between the human and stallion prostasome antigenic structures. Still, it should be noted that due to a lack of anti-equine antibodies, reverse western blot confirmation is prevented in most cases. Copyright © 2013 Informa Healthcare USA, Inc.
Figure 3. A comparative survey of 2D-separated proteins from human and horse prostasomes. The separations were performed in two dimensions, first a horizontal isoelectric separation followed by a vertical molecular weight separation. Prostasomal proteins from horse (bottom) displayed several marked proteins in the lower molecular weight bands (analyzed by Matrix-assisted laser desorption/ionization (MALDI)-Mass spectrometry (MS)), not seen in the corresponding 2D of human prostasomal proteins (top).
2D-electrophoretic separation and identification by mass spectrometry Purified prostasomes of both species were first separated by isoelectric focusing and then separated by SDS-PAGE to achieve a 2D-separation. The 2D-electrophoretic separation of proteins from purified human prostasomes (Fig. 3) compared with the corresponding 2D-separation of horse prostasomal proteins. Figure 3 displayed strong differences in the
4 G. K. Ronquist et al. Table 2. Identified proteins in stallion prostasomes (Equus caballus). Spot number
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Protein
Entry
Seminal plasma protein-1 Seminal plasma protein HSP-1 Seminal plasma protein-1 Seminal plasma protein-1 Seminal plasma protein-1 Seminal plasma protein-1 Impossible to identify Caveolin-1 Seminal plasma protein-1 Similar to mitochondrial import inner membrane translocase subunit Tim23 Similar to growth hormone releasing factor Similar to mitochondrial ribosomal protein S12 Similar to inositol polyphosphate phosphatase-like 1 Similar to type I keratin KA17 isoform 3 Similar to Keratin 7 Similar to seminal plasma protein 2 TP53-regulated inhibitor of apoptosis 1 Similar to glutamate receptor ionotropic kainate 4 isoform 1 Similar to seminal plasma protein 2 Similar to seminal plasma protein 2 Similar to seminal plasma protein 2
Q70GG6 P81121 Q70GG6 Q70GG6 Q70GG6 Q70GG6 Q2QLB0 Q70GG6 F2Q0S0 Q9XS89 F7BBW5 Q6P549 Q6IFU8 F7DZA2 Q70GG4 Q9D8Z2 F6SKB7 Q70GG4 Q70GG4 Q70GG4
Table 3. Compilation of exosome-related proteins identified in both horse and human prostasomes by either mass spectrometry or immunoblotting. 14-3-3 proteins 14-3-3 protein beta 14-3-3 protein epsilon 14-3-3 protein gamma 14-3-3 protein zeta/delta Annexins Annexin A1 Annexin A11 Annexin A2 Annexin A3 CD-antigens CD9 antigen (Member of the tetraspanin family) CD10 antigen (Neprilysin) CD13 antigen (Aminopeptidase N) CD26 antigen (Dipeptidyl peptidase 4)
Clathrin/Clusterin Clathrin heavy chain 1 Clusterin Ezrin/Moesin/Radixin Ezrin Moesin Radixin Vesicle-associated membrane proteins Vesicle-associated membrane protein 3
levels of the low molecular weight proteins similar to that observed by 1D SDS-PAGE (Fig. 2). Three bands from the 1D-separated and 21 spots from the 2D-separated stallion prostasomes were cut out and digested with trypsin prior to Matrix-assisted laser desorption/ionization-Mass spectrometry (MALDI-MS) analysis. The MALDI-MS analysis of the three bands, in the 1D-separation of horse proteins, with a similar molecular weight to those repeatedly occurring in the 1D-gel of human prostasomes (CD10, CD13, and CD26) showed that CD10 and CD13 but not CD26 could be identified. Analysis of the 21 distinctive low-molecular weight proteins (Fig. 3, bottom
panel) in the 2D-gel of horse proteins via peptide mass fingerprinting using the National Center for Biotechnology Information non-redundant (NCBInr) database are listed in Table 2. It may be mentioned in this context that only 9 spots were definitely new proteins while the 12 others might represent different isoforms/degradation products. Eighteen proteins were detected in both prostasome types subdivided into 14-3-3 protein, annexins, CD-antigens, Cathrin/Clusterin, Ezrin/Moesin/Radixin, and Vesicleassociated membrane proteins (Table 3).
Discussion Prostasomes were previously reported to be present in stallion seminal plasma [Minelli et al. 1998; Arienti et al. 1998; Aalberts et al. 2012]. Here we provide evidence of similarities but also dissimilarities between human and stallion prostasomes. The investigation by transmission electron microscopy (TEM) displayed nanometer-sized prostasomes for both species, with a somewhat smaller average size for the prostasomes from horse. This was contradictory to the report of Arienti et al. [1998] who found the average diameter of horse prostasomes being somewhat larger than that of human prostasomes when using the quasi-elastic light scattering (QELS)-technique. The TEM-pictures of prostasomes from stallions showed the contribution of an amorphous substance that apparently eluded the chromatography step in the prostasome purification. This was in agreement with a previous investigation [Minelli et al. 1998]. The origin of this amorphous substance is not known. It is possible that the amorphous substance of stallion is aggregated plasma that created high molecular complexes that were found together with prostasomes in the void volume during gel chromatography. Gas chromatography analyses revealed a similar composition of fatty acids in both human and horse prostasomes with a high degree of saturated fatty acids consistent with previous findings [Arienti et al. 2001]. The separation of prostasomal proteins registered in the 1D-patterns from 5 different horses was consistent and this pattern also corresponded with poolings of prostasomes from other horses. Moreover, human prostasomal 1D-patterns from different preparations over time were found to have a high similarity between themselves [Ronquist et al. 2011] although being different from those of horses. Nevertheless, similarities in the banding patterns existed as well. The three bands characteristic for human prostasomes identified as CD10 (neprilysin), CD13 (aminopeptidase N), and CD26 (dipeptidyl peptidase 4) had analogous bands in stallion prostasomes, of which CD10 and CD13 could be identified by mass-spectrometry. It should be kept in mind, however, that the molecular weight determined from 2D-SDS gels do not always agree with mass spectrometry results. Additional marked, low-molecular weight bands were typical of horse prostasomes. These low-molecular weight proteins had a charge range between at least pH 3 and 12 when separated by 2D-gel electrophoresis. Identification of these proteins revealed several isoforms. Among Systems Biology in Reproductive Medicine
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Biochemical characterization of stallion prostasomes and comparison to their human counterparts the unique proteins, seminal plasma proteins 1 and 2, involved in the capacitation reaction in the bovine species [Manjunath et al. 2009] and the seminal plasma protein, horse seminal protein-1, are of known reproductive function [Calvete et al. 1995]. This characteristic pattern, of prostasome proteins can be used to differentiate prostasomes from epididymosomes that have a different protein pattern [Thimon et al. 2008]. This also tallies with our previous investigation showing no difference in the total amount of prostasomes between normospermic and azoospermic individuals ruling out any significant contribution of epididymosomes in our prostasome preparation [Carlsson et al. 2006]. Surprisingly, two of the low-molecular weight proteins were mitochondrial. This could be the formation of prostasomes in the late endosomes or early stages of lysosomes [Sahlen et al. 2002], organelles where the milieu of cellular proteins are degraded. The formation of prostasomes inside late endosomes takes place by an invagination of specific pockets of the membrane, called lipid rafts. They contain a high proportion of saturated fatty acids and are found as part of the process by which caveola incorporate caveolin [Williams and Lisanti 2004]. This protien was also identified in horse prostasomes. Several other proteins were also identified in horse prostasomes. For example, the growth hormone releasing factor, a protein that elevates the level of circulating growth hormone and insulin-like growth factor, was identified. At present the exact role of this hormone in a prostasomal context is unclear. Inositol polyphosphate phosphatase-like 1 protein that regulates phosphoinositide 3-pathways related to numerous cell activities [Raaijmakers et al. 2007], the keratins, fibrous structural proteins with different functions [Kirfel et al. 2003], and the TP53-regulated inhibitor of apoptosis 1 important in controlling the cell survival [Park and Nakamura 2005] were also identified. As stated by Mellor [2006] the protein “Similar to glutamate receptor ionotropic kainate 4 isoform 1” is a kainate receptor subtype forming gated ion-channels. Like TP53, these proteins have not been observed in human prostasomes [Utleg et al. 2003; Poliakov et al. 2009]. A recent report [Park et al. 2011] described the role of Ca2+ -channel protein transfer from prostasomes to sperms as an important factor in promoting sperm motility. They also highlighted the problem of degradation of proteins in sperms between the time transcription ceases, before spermiogenesis, and the time when proteins are most needed in the female reproductive tract. They therefore proposed that a fusion with prostasomes containing newly synthesized proteins provides a protective function. If correct, this would suggest a vital role that could be attributed to prostasomes that should be observed in other mammalian species. Prostasomes have been identified in other species including ram [Breitbart and Rubinstein 1982], dog [Frenette et al. 1985], stallion [Minelli et al. 1998; Arienti et al. 1998], and boar [Siciliano et al. 2008] but the search is far from exhaustive. However, as yet, no in-depth comparisons have been made between prostasomes in different species. Our ambition to highlight equine prostasomes in a reproductive context should be considered in the light of the importance and great commercial interest of achieving optimal progenies. Copyright © 2013 Informa Healthcare USA, Inc.
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The prostasome is likely to add another factor for consideration. Modern equine breeding management and technologies have positively influenced the conception rates, healthy pregnancies, and successful foaling. An increased knowledge of horse prostasomes and comparison with their human counterparts may be a further step forward towards a more optimized reproduction in horses.
Materials and Methods This study was approved by the human and animal Ethics Committees of the University of Uppsala (ethical number: Ups 01-367) and the Swedish University of Agricultural Sciences (SLU, ethical number: C 345/9), Uppsala.
Purification of prostasomes from human and horses Human seminal plasma was received from the Reproduction Unit at Uppsala University Hospital. Seminal plasma from horses was obtained from the Division of Reproduction, SLU. All preparatory procedures, unless otherwise stated, were performed at 4°C. The seminal plasma was first centrifuged at 10,000 x g for 30 min to pellet and remove possible cell debris. The supernatant was then ultracentrifuged at 100,000 x g for 2 h to pellet prostasomes and the pellet was resuspended in 30 mmol/L of Tris-HCl buffer, containing 130 mmol/L NaCl, pH 7.6 (isotonic Tris-HCl buffer). The prostasome pellet was then loaded on a Superdex column (Superdex, GE Healthcare, Uppsala, Sweden) equilibrated with the isotonic Tris-HCl buffer, to separate the prostasomes from an amorphous substance [Stegmayr and Ronquist 1982]. Fractions were collected every 20 min at a flow rate of 4 mL/h and the prostasome-containing fractions were identified by elevated absorbances at both 260 nm and 280 nm. The column-purified prostasomes were ultracentrifuged at 100,000 x g for 2 h to pellet the prostasomes. The pellet was subsequently resuspended in phosphate buffered saline, PBS (150 mmol/L NaCl, 10 mmol/L Na2HPO4, 2 mmol/L NaH2PO4), pH 7.4, and the concentration on a protein basis was adjusted to 2 mg/mL (ESL protein kit, Roche Diagnostics, Mannheim, Germany).
Electron microscopy Purified prostasomes (2 mL, 4 mg) from both sources were pelleted by ultracentrifugation and the pellet was fixed with 2.5% glutaraldehyde in PBS before embedding in Epon according to conventional techniques [Sahlen et al. 2002]. The plastic block was cut in 2 µm sections on an LKB microtome and subsequently trimmed to be cut into 40-60 nm sections. The sections were placed on slot grids with 0.5% Formvar film, contrasted with lead citrate and uranyl acetate and examined in a Zeiss Supra 35-VP (Zeiss Supra 35-VP, Carl Zeiss SMT) field emission scanning electron microscope, equipped with a STEM detector for transmission electron microscopy.
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6 G. K. Ronquist et al. Fatty acid analyses Prostasomal samples from human and horse were analyzed by gas chromatography for fatty acid composition of the prostasomal membranes (Triomega analys).
human and equine whole prostasome lysates (cf.: Table 3) was carried out as previously described [Ronquist et al. 2013].
SDS-PAGE (1D-electrophoresis) In all experiments, a final amount of 5.5 µL of purified prostasomes from either human or stallion (2 mg/mL, corresponding to 11 µg prostasomal protein) was separated on a 4-12% NuPAGE-gel at 200 V for 35 min. Protein bands were visualized by Coomassie staining or silver staining according to the manufacturer’s protocol. All SDS-PAGE materials were from InVitrogen (Paisley, UK). Immunoblotting was performed as previously described [Ronquist et al. 2012].
Declaration of interest: Biochemical analyses and data collection were funded by: The Swedish Medical Research Council 1D 2004/C1779, Liońs Cancer Fund, Uppsala, Sweden, and Stiftelsen Familjeplaneringsfonden, Uppsala, Sweden. JM was funded by the Swedish Foundation for Equine Research, Stockholm, project number H1047052. None of the authors have any conflicts of interest to declare.
2D-electrophoresis Prostasomes purified from seminal plasma from humans and horses (2 mg) were each pelleted and resuspended in 5 mL rehydration buffer (urea, 7 mol/L; thiourea, 2 mol/L; chaps, 4% (w/v); ASB-14, 2% (w/v) (all from SigmaAldrich, St. Louis, MO, USA) and dithiotreitol, (DTT) 5% (w/v) (Amersham Bioscience, Uppsala, Sweden). For the first-dimension electrophoresis, the isoelectric focusing (IEF), using immobilized pH-gradient, was carried out by loading 240 µL of rehydrated prostasomes (approximately 0.1 mg protein) onto the 11 cm IEF-strip (IEF pH 3-10, BioRad) with passive in-gel rehydration (21Ί C), overnight. Focusing was performed to a total of 20 kVh using an IPG-phor unit (21°C). The focused strips were equilibrated for 10 min, each in BioRad ReadyPrep 2-D starter kit equilibration buffer I and II (BioRad). The second dimension was carried out in a 4-12% SDS gel (Tris-HCl, 1 mm thick, 11 cm IPG well), and protein visualization was by Coomassie staining (InVitrogen). Mass spectrometry Preparative gels were loaded with 11 µg protein for 1D-separation and approximately 400 µg protein for 2D-separation. Three bands of the 1D-separated stallion proteins were cut out and 21 spots of the 2D-separated stallion proteins were punched out. In-gel digestion was carried out with trypsin [Havlis et al. 2003]. A preliminary MALDI-analysis was performed by removing 0.6 µL digest and spotting this on the MALDI target plate (ground steel 24 x 16 spots) and mixing with 0.6 µL 1:1 (v/v) saturated matrix solution of α-cyano-4-hydroxycinnamic acid in 30% acetonitrile/0.1% trifluoroacetic acid. Mass spectrometry (MS) -analysis was performed in a Bruker Ultraflex II (Bruker Corporation, Bremen, Germany). Spectra were analyzed by using the program, mMass [Strohalm et al. 2010] and from there, manual PMF searches in Mascot were initiated. Searches were usually done at the 60 ppm error level using “Other mammalia” and NCBInr (20110429 or close to this date). If judged necessary, i.e., no clear hit was obtained, a more thorough extraction of the digest was performed, using StageTips [Rappsilber et al. 2007] and subjected to a new MALDI-MS analysis. Comparative mass spectrometry of
Author contributions: Conceived and designed the experiments: GR, AL, JM; Performed the experiments: GKR, BE; Analyzed the data: GKR, BE, LC; Contributed reagents/ materials/ analysis tools: GKR, BE, GR, JM, LC, AL; Wrote the manuscript: GKR, BE, GR, AL.
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