CSIRO PUBLISHING
Reproduction, Fertility and Development, 2016, 28, 732–741 http://dx.doi.org/10.1071/RD14322
Proteome profiling of the sperm maturation milieu in the rhesus monkey (Macaca mulatta) epididymis Xin Liu A,*, Shao-Hua Jin B,*, Xue-Xia Liu A, Wen-Juan Wang C and Fu-Jun Liu A,D A
Central Laboratory, Yantai Yu Huang Ding Hospital/Qingdao University, Yantai 264000, Shandong, P.R. China. B Clinical Laboratory, Yantai Yu Huang Ding Hospital/Qingdao University, Yantai 264000, Shandong, P.R. China. C Reproduction Medical Center, Yantai Yu Huang Ding Hospital/Qingdao University, Yantai 264000, Shandong, P.R. China. D Corresponding author. Email: [email protected]
Abstract. The mammalian spermatozoon acquires its fertilising potential during transit through the epididymis, where it interacts with epididymal luminal fluid proteins (the sperm maturation milieu). In order to highlight the epididymalspecific function of the rhesus monkey (Macaca mulatta) in sperm maturation, two-dimensional gel electrophoresis of epididymal luminal fluid proteins was followed by identification by Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF/MS) or MALDI-TOF/TOF and revealed over five hundred spots, comprising 198 non-redundant proteins. Some mass spectrometric data were confirmed by western blotting identification. Some common epididymal fluid proteins were identified, such as clusterin, a-1-antitrypsin, malate dehydrogenase, L-lactate dehydrogenase B, a-1-acid glycoprotein 1 and a-mannosidase. More than 7% of all proteins were anti-oxidative, which might control oxidative stress within the male tract. When compared with bull and human epididymal luminal fluid proteins, those in the rhesus monkey had more overlap with the human, which provides evidence of a close evolutionary relationship between the rhesus monkey and man. This study provides new proteomic information on possible rhesus monkey epididymal functions and novel potential biomarkers for the noninvasive assessment of male fertility. Additional keywords: anti-oxidative, bioinformatics, epididymal fluid, evolution, mass spectrometry. Received 29 August 2014, accepted 16 September 2014, published online 30 October 2014
Introduction Spermatozoa produced by the testis are not yet motile or fertile, but these immature spermatozoa have to survive in the female tract, move to the oviduct, penetrate the egg investments and fuse with the oocyte (Yeung and Cooper 2003). In consideration of DNA condensation, RNA transcription prevention and absence of protein translation, epididymal spermatozoa only acquire the functional capacity to perform the above individual steps of fertilisation during transit through the epididymis, which secretes proteins that sequentially remove, add and modify the surface of maturing spermatozoa. Spermatozoa are consistently enclosed in a specific microenvironment and interact with epididymal contents, especially epididymal luminal fluid proteins, for their maturation and storage (Cooper and Yeung 2010). After evidence was provided to demonstrate sperm maturation in the epididymis (Bedford 1967; Orgebin-Crist 1967), some proteins began to be reported, such as rat epididymal fluid proteins B/C, D/E (Brooks and
Higgins 1980) and the antibacterial Bin1B (Li et al. 2001). More proteins should be found to reflect epididymal-specific functions in sperm maturation. The rapid development of proteomic technology opens the door to the global mapping analysis of epididymal luminal fluid proteins (Aitken et al. 2007; Dacheux et al. 2009a, 2012). In mammals, epididymal luminal fluid proteomes of the boar (Syntin et al. 1999), stallion (Fouche´court et al. 2000) and ram (Druart et al. 1994) have been reported, and more proteins were reported, such as a-1-antitrypsin, a-2-glycoprotein, albumin, b-galactosidase, calmodulin, clusterin, cystatin C, glutathione peroxidase, gelsolin, glutathione-S-transferase, b-N-acetylhexosaminidase, lipocalin 5, lactoferrin, mannosidase, prostaglandin D2 synthase and transferrin. To understand the evolutionary significance of the epididymal micro-environment, the platypus caudal epididymidal fluid proteome has also been identified (Dacheux et al. 2009b). The results showed the platypus to be an early model mammal, whose epididymal fluid proteins were few
*
These authors contributed equally to this paper.
Journal compilation Ó CSIRO 2016
www.publish.csiro.au/journals/rfd
Rhesus monkey sperm maturation milieu
and distinctive. But the platypus epididymis shows a unique feature, that of promoting sperm bundles to enhance the cells’ motility (Nixon et al. 2011). In recent years, by using twodimensional gel electrophoresis and mass spectrometry, 172 bovine epididymal luminal proteins have been identified (Belleanne´e et al. 2011), which shed light on bovine epididymal functions and specificity. The epididymal proteins in many species have been identified, but few have defined functions that could be targeted for contraception. Knowing the proteins involved in epididymal sperm maturation deserves more attention. Although there are some identical epididymal fluid proteins among species, speciesspecific proteins also apparently exist. This study aimed to produce a proteomic database of rhesus monkey epididymal luminal fluid proteins and to compare the results with the proteomic data previously reported in other mammalian species. This could be the basis of development of diagnostic markers and therapeutic targets for infertility or contraception. Materials and methods Preparation of sperm-milieu proteins Three adult male (8–10 years old) rhesus monkeys (Macaca mulatta), 9–10 kg bodyweight, from Yantai zoo’s primate colony were used for the experiment. They all had proven breeding records. Approval was obtained for the use of rhesus monkeys by the Animal Ethics Committee of Yantai Yu Huang Ding Hospital for Animal Care and Usage. Three monkeys were individually caged in a humidity- and temperature-controlled room and the light : dark cycle was set at 12 : 12 h. During the period of experiment, water and monkey chow were regularly available. After anaesthesia with ketamine hydrochloric acid (HC1) (10 mg kg1), unilateral castrations was performed on the monkeys and then three testes with their attached epididymides were removed and collected. Blood vessels and initial segments were dissected from the remaining epididymis. All three epididymides were pooled to prepare the luminal proteins (the sperm maturation milieu) as in our previous report (Li et al. 2010, 2014). Briefly, the epididymides were coarsely minced and gentle pressure applied to release luminal fluid into phosphate buffer. After centrifugation at 5700g at 48C for 10 min and microscopic examination, the resultant sperm-free supernatant was precipitated with four volumes of ice-cold acetone. Finally the precipitates were dissolved in 3 mL protein lysis buffer (7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-cholamido-propyl) dimethylammonio]-1-propanesulfonate, 65 mM dithiothreitol). The concentration of proteins was measured by the Bradford assay (Gotham et al. 1988) and then samples stored at 808C. Gel electrophoresis, protein staining and mass spectrometric analyses Two-dimensional electrophoresis gels were performed as previously reported (Go¨rg et al. 2004). The proteins were stained by Coomassie brilliant blue R-350 (Amersham Biosciences, Uppsala, Sweden), then the protein spots were excised from the gel and digested by trypsin. The resultant peptides were identified by a Voyager DE-STR biospectrometry workstation or a 4800 Matrix-Assisted Laser Desorption/Ionization Time of
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Flight/Time of Flight Mass Spectrometry (MALDI-TOF/TOF MS) (Applied Biosystems/MDS SCIEX, Framingham, MA, USA). If one spot was identified as more than one protein, the first-ranked protein was selected for each spot. Western blotting and bioinformatic analysis Western blotting was performed by following the method of Liu et al. (2014). After the incubation with primary and secondary antibody, the immunoreactive bands were visualised by a diaminobenzidine kit (Zhong-Shan Biotechnology, Beijing, China). The functional classification of the sperm-milieu proteins was performed by the online DAVID software (Version 6.7; http://david.abcc.ncifcrf.gov/ [accessed 6 June 2013]). Results Gel electrophoresis and MALDI mass spectrometry Gel electrophoretic analysis of proteins from three rhesus monkey epididymal luminal fluids, which were pooled for protein extraction and separated on two-dimensional gels, revealed 520 spots that were consistently detected in three gels (Fig. 1). From the spots analysed by MALDI-TOF/MS or MALDI-TOF/TOF, 198 epididymal fluid proteins were identified in the sperm milieu (Table 1). Thirty two percent of the total 198 proteins were represented by two or up to more than six spots, revealing the same unique proteins, presumably representing spliced variants or isoforms. Western blotting of rhesus monkey epididymal luminal fluid proteins Ten common epididymal fluid proteins (clusterin, epididymal secretory protein E1, b-hexosaminidase B, cystatin B, cathepsin B, glyceraldehyde-3-phosphate dehydrogenase, a-1-antitrypsin, 14–3-3 protein zeta/delta, epididymis-specific a-mannosidase and selenium-binding protein) and 11 antioxidant proteins (peroxiredoxin-1, superoxide dismutase [Mn], thioredoxindependent peroxide reductase, peroxiredoxin-5, glutathione S-transferase P, epididymal secretory glutathione peroxidase, peroxiredoxin-6, peroxiredoxin-4, glutathione Stransferase Mu 3, superoxide dismutase [Cu-Zn] and peroxiredoxin-6) were selected for detection by western blotting (Fig. 2). Classification of biological function of rhesus monkey epididymal luminal fluid proteins The proteomic analysis of the functions of 198 rhesus monkey epididymal luminal fluid proteins is given in Fig. 3. The majority (37%) of proteins were related to general metabolism and the next most prevalent category (13%) involved protease and protease inhibitor functions. Eight percent were structural proteins and 7% of proteins were antioxidative; the smallest group had chaperone functions. Comparison of rhesus monkey, bull and human epididymal luminal fluid proteins Each epididymal fluid proteome is species-specific. To disclose the difference in epididymal fluid proteomes between species and to minimise different proteomic results caused by different proteomic technologies, the bull (Belleanne´e et al. 2011) and
734
Reproduction, Fertility and Development
(a)
X. Liu et al.
3
pH
10
kDa 97.0 66.0
45.0
30.0
20.0
(d)
420.2
831.4
1242.6
1653.8
(e)
3822.2
100 90 80 70 60 50 40 30 20 10 0 9.0
2065.0
1680
1940.88 1996.92 2045.97 2104.95
1839.87
1622.77 1690.76 1744.88
1283.63 1365.61 1417.69 1475.71 1541.69 1519.70 1420
1940
2200
y6(⫹1)
1160
70.1097 y1(⫹1)
y15 - 17(⫹1)
b13(⫹1) y14(⫹1)
b11(⫹1) a12(⫹1)
P
212.10 294.12 300.15 b3(⫹1) 475.20 515.19 600.24 b7(⫹1) b8 - 17(⫹1) a9(⫹1) 1047.48 y8(⫹1)
MS/MS Precursor: 1954.98 YVRPGGGFVPNFQLFEK
2200
9.0E⫹3
MS/MS Precursor: 2017.97 VSFEDSVISLSGDHSIIGR
5219.2
y3(⫹1) y4(⫹1) y5(⫹1) b6(⫹1),y6 - 17(⫹1) b7 - 18(⫹1) y8(⫹1) y9(⫹1) y10(⫹1) y11(⫹1) 1239.8123 y13(⫹1) y14(⫹1) y15(⫹1) y16(⫹1) b17(⫹1) 1886.0728 1993.6578
1940 b16 ⫹ H2O(⫹1)
1680
100 90 80 70 60 50 40 30 20 10 0 900
935.23 996.58 1045.54 1095.54 1152.52 1201.59
1954.98 1982.98 2034.96 2111.06
1698.84 1754.83 1819.89 1895.91
1470.72 1536.75 1609.75
1420
1830.34 b17(⫹1)
1160
1300.69 1360.66
936.52 963.46 985.44 1050.53 1107.58 1162.58 1211.65
6.6E⫹4
(c)
100 90 80 70 60 50 40 30 20 10 0 9.0
2017.97
100 90 80 70 60 50 40 30 20 10 0 900
F
% Intensity
(b)
1316.67
14.4
433.4
857.8
1282.2
1706.6
2131.0
Mass (m/z) Fig. 1. Separation and identification of rhesus monkey epididymal luminal fluid proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and MALDI-MS spectra. (a) Reference map of epididymal luminal fluid. Example spectra of (b, c) glutathione peroxidase and (d, e) superoxide dismutase [Cu-Zn]. (b) MS map and (c) MS/MS map marked with b ions and y ions for glutathione peroxidase identification. The sequence of precursor at m/z 1954.98 was analysed by MS/MS to be YVRPGGGFVPNFQLFEK and the protein identified as glutathione peroxidase. (d ) MS map and (e) MS/MS map marked with b ions and y ions for superoxide dismutase [Cu-Zn] identification. The sequence of precursor at m/z 2017.97 was analysed by MS/MS to be VSFEDSVISLSGDHSIIGR and identified as superoxide dismutase [Cu-Zn].
our previously identified human (Li et al. 2010) epididymal luminal fluid proteins were selected, which were both performed by two-dimensional gel electrophoresis coupled with MALDI mass spectrometer identification. The results reflect the overlap of bovine and human epididymal luminal fluid proteins with
those of rhesus monkey epididymal luminal fluid (Fig. 4). A total of 39 proteins in human and bull epididymal luminal fluid were common to rhesus monkey epididymal luminal fluid, accounting for 20% of proteins in rhesus monkey epididymal luminal fluid. 125 proteins (63% of the rhesus monkey
NCBI accession number
gi|4557032 gi|75041584 gi|19923206 gi|5803181 gi|62511005 gi|5453990 gi|118090 gi|4759274 gi|18307578 gi|225939 gi|55595921 gi|119592410 gi|4507651 gi|178132 gi|4502105 gi|4507793 gi|1421609 gi|21431617 gi|1706134 gi|38540969 gi|34761571 gi|34761579 gi|50659093 gi|16507237 gi|66346698 gi|40255043 gi|31543380 gi|312176440 gi|85397577 gi|76803548 gi|4503107 gi|114326453 gi|98985828 gi|4757960 gi|56699495 gi|8659555 gi|54037162 gi|1170338 gi|119172 gi|21903479 gi|20141877 gi|119370332 gi|12643636 gi|23831360 gi|20178296 gi|2497983 gi|127234 gi|121039 gi|461466
Protein no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
L-Lactate dehydrogenase B chain 14-kDa Phosphohistidine phosphatase Glutamine synthetase Stress-induced phosphoprotein D-3-phosphoglycerate dehydrogenase Proteasome activator complex subunit 1 Peptidyl-prolyl cis-trans isomerase b precursor Thioredoxin-like protein 1 Fructose bisphosphate aldolase A Alcohol dehydrogenase [NADPþ] Cytosolic malate dehydrogenase isoform 5 Electron-transfer flavoprotein, b polypeptide Tropomyosin a-4 chain Alcohol dehydrogenase class 3 chi chain Annexin A4 Ubiquitin-conjugating enzyme E2 N Nucleoside diphosphate kinase B Protein disulfide isomerase A3 Cysteine-rich secretory protein 1 Epididymal-specific lipocalin-6 Epididymal-specific lipocalin-8 Epididymal-specific lipocalin-12 Epididymis-specific a-mannosidase 78-kDa Glucose-regulated protein a-N-Acetylglucosaminidase b-Galactosidase-1-like protein Protein DJ-1 Epididymal peptidase inhibitor Cystatin-8 Cystatin-11 Cystatin-C Disintegrin and metalloproteinase domain-containing protein 7 Disintegrin and metalloproteinase domain-containing protein 28 Cadherin-1 Wap four-disulfide core domain protein 2 Iron-responsive element-binding protein 1 Neutral a-glucosidase AB Chromobox protein homologue 5 Elongation factor 2 Vinculin Protein-glutamine gamma-glutamyltransferase 2 Complement C3 Wd repeat protein 1 Bifunctional purine biosynthesis protein Pyruvate kinase isozymes M1/M2 Mitochondrial uncoupling protein 3 Moesin Ig gamma-1 chain C region Aminoacylase-1
Protein description
72 61 137 133 95 62 125 62 104 62 62 60 62 74 135 72 76 70 69 84 92 111 159 191 63 62 88 62 61 92 90 155 104 60 84 141 89 61 94 105 77 134 120 118 146 185 67 97 146
Score
20 47 26 22 18 22 42 29 34 19 16 18 8 20 36 26 41 16 21 47 41 42 21 21 11 11 38 34 34 56 38 22 15 6 36 17 13 32 15 14 15 12 37 27 26 20 14 29 41
Coverage %
36 615 13 824 42 037 62 599 57 356 28 705 22 728 32 231 39 395 36 550 36 403 27 826 28 504 39 698 35 860 17 127 17 287 56 747 28 462 18 033 19 068 21 486 113 908 72 288 82 115 74 110 19 878 15 273 16 265 16 495 15 789 85 613 87 123 97 396 14 337 98 337 106 807 22 211 95 146 123 591 77 280 187 030 66 152 64 575 57 900 34 193 67 778 36 083 45 856
Mr
5.71 5.65 6.43 6.4 6.29 5.78 9.33 4.4 8.3 6.32 6.91 8.24 4.67 7.45 5.84 6.13 8.52 5.98 5.57 4.84 6.84 5.48 6.74 5.07 6.1 9.01 6.33 8.52 9.05 6.51 9 6.08 6.38 4.58 6.79 6.23 5.74 5.71 6.42 5.51 5.11 6.02 6.17 6.27 7.96 9.31 6.08 8.46 5.77
pI
Table 1. Proteins of the Rhesus monkey sperm maturation milieu identified by mass spectrometry
General metabolism General metabolism General metabolism Immunity stress General metabolism Protease/protease inhibitor General metabolism Antioxidant General metabolism General metabolism General metabolism General metabolism Structure General metabolism Signal transduction Protease/protease inhibitor General metabolism Chaperone reproduction transporter transporter transporter Protease/Protease inhibitor Immunity stress Protease/protease inhibitor Protease/protease inhibitor Unclassified Protease/protease inhibitor Protease/protease inhibitor Protease/protease inhibitor Protease/protease inhibitor Protease/protease inhibitor Protease/protease inhibitor Signal transduction Immunity stress transporter Protease/protease inhibitor reproduction Unclassified Immunity stress General metabolism Immunity stress Antioxidant General metabolism General metabolism General metabolism Signal transduction Immunity stress General metabolism
6A 5A 11A 12A 8A 5A 9A 4A 7A 4A 4A 4A 4A 5A 9A 5A 5A 8A 4A 4A 5A 5A 12A 11A 6A 6A 9A 3A 3A 4A 4A 11A 8A 4A 5A 11A 10A 5A 8A 12A 8A 15A 12A 11A 12A 5A 7A 6A 13A
Reproduction, Fertility and Development
(Continued )
Functional classification
Matched peptide number
Rhesus monkey sperm maturation milieu 735
NCBI accession number
gi|148840437 gi|23396498 gi|52000887 gi|113960 gi|8247940 gi|145559446 gi|29550838 gi|21903432 gi|1708967 gi|113596 gi|1708543 gi|136464 gi|7387511 gi|296439291 gi|93141249 gi|67463772 gi|38372871 gi|548453 gi|1706870 gi|20178288 gi|116242799 gi|116848 gi|119360 gi|317373553 gi|6686275 gi|119339 gi|115449 gi|134047703 gi|38258193 gi|19855073 gi|55977767 gi|112877 gi|1729976 gi|2811007 gi|61213572 gi|108860890 gi|38258929 gi|74730916 gi|130683 gi|21264428 gi|27805668 gi|51316252 gi|119627259 gi|730451 gi|125157 gi|115612 gi|74719147 gi|21361792 gi|20981682
Protein no.
50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98
Selenium-binding protein 1 Cytosolic nonspecific dipeptidase 14-3-3 Protein zeta/delta Annexin A5 Inorganic pyrophosphatase Betaine–homocysteine s-methyltransferase 1 Golgi membrane protein 1 Isocitrate dehydrogenase Malate dehydrogenase Aldose reductase Histidine triad nucleotide-binding protein 1 Transthyretin 6-Phosphogluconolactonase Nicotinate-nucleotide pyrophosphorylase Lambda-crystallin homologue Aldose 1-epimerase Aflatoxin B1 aldehyde reductase member 2 Peroxiredoxin-1 Flavin reductase Adenylate kinase isoenzyme 1 Spectrin b chain Cofilin-1 Endoplasmin a-1B-Glycoprotein Aconitate hydratase a-Enolase Carbonic anhydrase 1 Carbonic anhydrase 3 50 -Deoxyribonucleotidase Heat-shock protein b-1 Vimentin a-1-Acid glycoprotein 1 Transketolase Pyridoxal kinase Rhogef and ph domain-containing protein 2 Plexin-A4 DNA-dependent protein kinase catalytic subunit Tektin-4 Voltage-dependent anion-selective channel protein 1 Stress-70 protein Pantothenate kinase 3 L-Lactate dehydrogenase A-like 6A Spermatogenesis associated 6 60S Ribosomal protein l13A Adenylate kinase isoenzyme 4 Calpain small subunit 1 Glycoprotein-N-acetylgalactosamine 3-b-galactosyltransferase 1 Sperm-associated antigen 4-like protein Gamma-enolase
Protein description
103 126 97 76 124 70 61 79 77 82 61 112 74 61 114 144 61 98 85 102 61 105 117 67 157 73 63 110 98 114 120 78 65 84 61 66 64 74 82 76 63 65 75 66 65 61 65 67 62
Score
26 29 31 18 41 19 13 17 20 22 30 53 30 21 30 40 17 37 49 43 4 42 14 16 20 19 31 38 432 37 20 22 10 24 14 6 4 10 25 14 15 12 18 31 27 12 17 14 17
Coverage %
Table 1. (Continued)
52 280 52 845 27 728 35 914 32 639 44 942 45 306 46 630 36 403 35 830 13 793 15 877 27 530 30 796 35 396 37 742 39 564 22 096 22 105 21 621 274 439 18 491 92 411 54 239 85 372 47 139 28 852 29 553 23 368 22 768 53 619 23 497 67 835 35 080 74 845 212 318 468 788 50 617 30 754 73 635 41 068 36 484 54 014 18 509 22 073 57 948 42 176 43 053 47 239
Mr
6.13 5.66 4.73 4.94 5.54 6.41 4.91 6.53 6.91 6.51 6.43 5.52 5.7 5.81 5.81 6.18 6.7 8.27 7.13 8.73 5.39 8.22 4.76 5.58 7.36 7.01 6.59 6.86 6.18 5.98 5.06 4.93 7.58 5.75 6.51 6.42 6.75 6.01 8.62 5.87 6.13 6.51 8.6 10.51 5.38 6.41 6.17 8.61 4.91
pI
Functional classification
transporter General metabolism Immunity stress Signal transduction General metabolism General metabolism Structure General metabolism General metabolism General metabolism transporter transporter General metabolism General metabolism Structure General metabolism General metabolism Antioxidant General metabolism General metabolism Structure Structure Immunity stress Immunity stress General metabolism General metabolism General metabolism General metabolism General metabolism Immunity stress Structure Immunity stress General metabolism General metabolism Signal transduction Signal transduction General metabolism Signal transduction transporter Immunity stress General metabolism General metabolism reproduction Structure General metabolism Signal transduction Protease/protease inhibitor reproduction General metabolism
Matched peptide number 10A 11A 9A 5A 9A 6A 5A 7A 6A 6A 4A 6A 6A 5A 8A 10A 5A 6A 7A 8A 7A 6A 11A 7A 12A 8A 6A 7A 7A 7A 11A 5A 4A 6A 5A 7A 14A 4A 4A 7A 4A 4A 5A 4A 5A 4A 5A 4A 5A
736 Reproduction, Fertility and Development X. Liu et al.
99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152
gi|20178315 gi|127981 gi|134665 gi|114074 gi|2507171 gi|317373539 gi|34222621 gi|2829431 gi|139641 gi|158936749 gi|46395636 gi|6016173 gi|130770 gi|31077177 gi|23831297 gi|215274262 gi|114517 gi|74751178 gi|317373315 gi|74730987 gi|37730278 gi|5453603 gi|13543551 gi|4503909 gi|33875419 gi|68060662 gi|75070579 gi|136062 gi|111185949 gi|4503571 gi|42558919 gi|6912328 gi|226531183 gi|662841 gi|5453549 gi|5123454 gi|14585855 gi|4757900 gi|180572 gi|48257098 gi|109013331 gi|47519616 gi|4506455 gi|339647 gi|77539752 gi|4501887 gi|58042495 gi|74136167 gi|75070821 gi|48257068 gi|13937981 gi|5453541 gi|15010550 gi|4503895
Bifunctional 30 -phosphoadenosine 50 -phosphosulfate synthetase 2 Nucleoside diphosphate kinase A Superoxide dismutase [Mn] Adenine phosphoribosyltransferase Thioredoxin-dependent peroxide reductase Peroxiredoxin-5 Abhydrolase domain-containing protein 14B Calponin-1 Vitamin d-binding protein Tyrosine protein kinase Blk 30 ,50 -Bisphosphate nucleotidase 1 Glutathione transferase omega-1 Phosphatidylinositol transfer protein a isoform Casein kinase i isoform a GTP : AMP phosphotransferase Galectin-3 Adenosine triphosphate synthase subunit a Probable lipid phosphate phosphatase PPAPDC3 Prostate and testis-expressed protein 4 Prostate and testis-expressed protein 1 Glyceraldehyde-3-phosphate dehydrogenase T-Complex protein 1 subunit b isoform 1 Proteasome (prosome, macropain) subunit, a type, 1 Guanidinoacetate N-methyltransferase isoform A Distribution of catechol-O-methyltransferase protein Phosphoglycerate mutase 1 Peroxiredoxin-6 Triosephosphate isomerase Glutathione s-transferase P a-Enolase isoform 1 Retinal dehydrogenase 1 N(g),N(g)-Dimethylarginine dimethylaminohydrolase 1 isoform 1 Epididymal secretory glutathione peroxidase Heat-shock protein 27 Peroxiredoxin-4 Heat-shock 70-kDa protein 1A/1B Phosphatidylethanolamine-binding protein Calreticulin Creatine kinase-B Valosin-containing protein Glutathione S-transferase Mu 3 Tropomyosin b chain isoform 2 Reticulocalbin-1 Thyroid hormone binding protein Tubulin a-1B chain Actin, cytoplasmic 2 Proteasome subunit a-type 5 Superoxide dismutase [Cu-Zn] Peroxiredoxin-2 Heat-shock 70-kDa protein 8 Peptidylprolyl isomerase a (cyclophilin a) Anterior gradient protein 2 homologue Heat-shock protein gp96 Galactokinase 63 95 62 62 71 116 65 69 75 61 60 60 65 62 80 60 60 61 61 62 196 214 94 91 260 93 284 334 351 231 240 322 106 257 183 228 131 135 382 155 118 263 124 146 210 370 169 124 306 213 130 123 442 211
12 42 20 30 38 41 28 16 23 10 22 22 20 16 31 19 12 28 17 18 12 7 15 14 21 11 30 30 39 10 7 22 14 27 9 9 22 6 18 7 13 14 7 6 11 16 15 21 17 8 16 20 13 15
69 457 17 138 24 707 19 595 27 675 22 012 22 332 33 150 52 929 57 702 33 371 27 548 31 786 38 890 25 550 26 172 59 714 29 429 11 407 14 270 23 850.5 57 452.1 29 578.9 26 301.1 20 032.2 28 759.8 25 005.2 26 693.8 23 423 47 139.3 54 700.8 31 101.9 25 196 22 313.3 30 520.8 69 995 25 717.7 48 111.8 42 658.3 71 021.1 26 586.2 32 969.6 38 866.2 57 068.7 50 103.6 41 765.8 22 648.4 15 972.9 19 418 64 633.1 17 999.9 19 966.6 90 138.1 42 245.6
8.18 5.83 8.35 5.78 7.67 8.85 5.94 9.16 5.4 7.98 5.46 6.23 6.11 9.59 9.15 8.58 9.16 9.98 8.97 8.28 9.17 6.01 6.15 5.75 5.36 6.67 6 6.45 5.93 7.01 6.3 5.53 8.68 7.83 5.86 5.48 5.71 4.29 5.16 4.94 5.38 4.63 4.86 4.82 4.98 5.31 4.74 6.22 5.38 5.36 7.68 9.03 4.73 6.04
5A 7A 4A 4A 5A 7A 5A 6A 7A 5A 4A 5A 5A 5A 6A 4A 5A 4A 4A 5A 2B 2B 2B 2B 3B 2B 5B 4B 5B 3B 3B 4B 2B 4B 2B 4B 2B 2B 4B 3B 2B 3B 2B 2B 3B 4B 2B 2B 4B 3B 2B 2B 7B 4B
Reproduction, Fertility and Development
(Continued )
General metabolism General metabolism Antioxidant General metabolism Antioxidant Antioxidant Protease/protease inhibitor Signal transduction transporter Signal transduction General metabolism Antioxidant transporter General metabolism General metabolism transporter General metabolism General metabolism Immunity stress Immunity stress General metabolism Structure Protease/protease inhibitor General metabolism Signal transduction General metabolism Antioxidant General metabolism Antioxidant General metabolism General metabolism General metabolism Antioxidant Immunity stress Antioxidant Immunity stress transporter Signal transduction General metabolism Protease/protease inhibitor Antioxidant Structure Signal transduction transporter Structure Structure Protease/protease inhibitor Antioxidant Antioxidant Immunity stress General metabolism Signal transduction Immunity stress General metabolism
Rhesus monkey sperm maturation milieu 737
gi|22096346 gi|14250401 gi|38492599 gi|32189394 gi|55824560 gi|404390 gi|5453678 gi|2492797 gi|24430160 gi|54300702 gi|68051994 gi|109108768 gi|4557395 gi|23958133 gi|4557871 gi|544759 gi|48255905 gi|109131308 gi|18088311 gi|5729877 gi|49457530 gi|46249758 gi|4557579 gi|49456301 gi|4506179 gi|189998 gi|2352843 gi|55846712 gi|825635 gi|741376 gi|31543397 gi|157168362 gi|14149777 gi|18645167 gi|55960189 gi|109065176 gi|9506741 gi|4008131 gi|75709200 gi|55957281 gi|5542151
gi|62898045 gi|1905874 gi|1922287 gi|62087652 gi|7709904
153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193
194 195 196 197 198
Dynactin subunit 2 Actin, b b-Hexosaminidase B Adenosine triphosphate synthase subunit b Heat-shock 70-kDa protein 5 b-Trace protein, prostaglandin D synthase,PGD synthase Epididymal secretory protein E1 Albumin 26S Protease regulatory subunit 10B Ubiquitin C splice variant a-1-Antitrypsin Apolipoprotein A-I isoform 1 Carbonic anhydrase 2 Tubulin, b 2c Serotransferrin Biliverdin-ix b reductase isozyme I Transgelin Phosphoglycerate kinase 1 Nitrilase family, member 2 Heat-shock cognate 71-kDa protein isoform 1 Creatine kinase B-type Cytovillin Fatty acid-binding protein, adipocyte Methylthioadenosine phosphorylase Proteasome subunit a type-1 isoform 2 M2-Type pyruvate kinase Sorbitol dehydrogenase Clusterin Calmodulin Cathepsin B Phosphoglycerate kinase 2 Purine nucleoside phosphorylase Haloacid dehalogenase-like domain-containing protein 2 Annexin A2 SH3 Domain-binding glutamic acid-rich-like protein Cystatin-B Glycine N-methyltransferase Chaperonin Phospholipid hydroperoxide glutathione peroxidase Esterase D/formylglutathione hydrolase Chain a, macrophage migration inhibitory factor (mif) with hydroxphenylpyruvate Pyridoxine 50 -phosphate oxidase variant Carboxyl-terminal lim domain protein Enoyl-CoA hydratase Proteasome 26s ATPase subunit 5 variant Calnexin
Protein description
8 8 7 9 16 27 23 13 7 16 8 12 21 8 4 22 11 16 9 13 25 5 20 12 13 6 8 8 46 9 8 25 19 9 47 12 7 12 7 9 10 7 5 7 5 6
46 39 80 66 107
Coverage %
136 164 160 110 617 171 98 375 144 103 227 211 323 289 360 249 100 405 145 392 599 112 143 125 205 145 152 102 448 178 145 264 262 250 223 58 135 62 89 149 48
Score
29 915 36 170 31 371 38 760 31 185
44 203.9 40 978.4 58 820.7 56 524.6 66 857.5 18 670 16 559.5 67 836.5 44 145.1 17 142.3 44 558.9 30 716 29 227.9 49 808 76 999.6 21 846.3 22 596.4 44 558.1 30 560.5 70 854.2 42 648.3 69 198.6 14 709.5 31 233.8 29 536.9 57 877 38 155.8 48 598.8 17 152 17 143 44 767.3 32 097.2 28 517.9 38 551.8 9374.8 11 102 32 742 10 582 22 174 28 226 12 345
Mr
7.01 6.8 8.34 6.07 4.61
5.1 5.56 6.43 5.26 5.22 6.95 7.56 5.85 7.1 7.93 5.75 5.72 6.86 4.83 6.81 7.31 8.87 8.02 6.82 5.37 5.34 5.94 6.59 6.75 6.15 7.95 7.56 5.52 4.06 5.44 8.74 6.45 5.84 7.57 9.42 6.4 6.55 9.44 8.69 6.29 8.24
pI
Functional classification
Structure Structure Protease/protease inhibitor General metabolism Immunity stress Protease/protease inhibitor reproduction transporter Protease/protease inhibitor Protease/protease inhibitor Protease/protease inhibitor transporter General metabolism Structure transporter General metabolism transporter General metabolism General metabolism Immunity stress General metabolism Signal transduction transporter Immunity stress Protease/protease inhibitor General metabolism General metabolism transporter Signal transduction Protease/protease inhibitor General metabolism General metabolism General metabolism Signal transduction Signal transduction Protease/protease inhibitor General metabolism Chaperone Antioxidant General metabolism Immunity stress General metabolism Structure General metabolism Protease/protease inhibitor Signal transduction
Matched peptide number 2B 2B 2B 2B 6B 2B 2B 6B 2B 2B 2B 3B 4B 3B 4B 2B 2B 4B 2B 5B 6B 2B 2B 2B 2B 2B 2B 2B 4B 2B 2B 4B 3B 3B 3B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B
A
Reproduction, Fertility and Development
Proteins identified by Voyager DE-STR biospectrometry workstation (PMF) and criteria for acceptance: all identified proteins had a significance threshold of at least P # 0.05, score $ 60 and at least four masses matched. If more than one protein was identified in a spot, the single protein member with the highest score (top rank) was chosen from the multi-protein family. B Proteins identified by 4800 MALDI TOF/TOF Analyzer (MS/MS) and criteria for acceptance: individual ion scores $ 37 (significance level, P # 0.05).
NCBI accession number
Protein no.
Table 1. (Continued)
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epididymal fluid proteome) were common to human epididymal luminal fluid but only 44 proteins (22% of the rhesus proteome) were common to bull epididymal luminal fluid. Discussion Knowledge of the complex nature of sequential addition, removal and modification of epididymal fluid proteins on the surface of testicular spermatozoa during their epididymal transit has until now been sketchy. There is no proteomic study of the rhesus monkey epididymal contribution to sperm-maturation antigens and our interest was focussed on proteins of epididymal fluid because they are necessary for sperm maturation and are potential post-testicular contraceptive targets and could be used as markers for diagnosis and therapy for infertility.
739
Here a large novel database of rhesus monkey epididymal proteins was created from systematic work-up of nearly two hundred proteins, with confirmation of some by western blotting detection. Of 198 rhesus monkey epididymal fluid proteins, 32% of proteins showed isoforms. Similar results have been found in other species, such as the boar, stallion and ram (Druart et al. 1994). From a comparison of the rhesus monkey epididymal fluid proteome with that of the bull and our previously identified human epididymal fluid proteome only 39 proteins, including clusterin, were common to the three species. Most of
Bull (Belleannée et al. 2011) kDa M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 97 66 45
122
5
31
8
21 14
39
Fig. 2. Western blotting of proteins found in rhesus monkey epididymal luminal fluid. M, molecular mass marker (kDa); Lane 1, clusterin; Lane 2, epididymal secretory protein E1; Lane 3, b-hexosaminidase B; Lane 4, cystatin B; Lane 5, cathepsin B; Lane 6, glyceraldehyde-3-phosphate dehydrogenase; Lane 7, a-1-antitrypsin; Lane 8, 14-3-3 protein zeta/delta; Lane 9, epididymis-specific a-mannosidase; Lane 10, selenium-binding protein; Lane 11, peroxiredoxin-1; Lane 12, superoxide dismutase [Mn]; Lane 13, thioredoxin-dependent peroxide reductase; Lane 14, peroxiredoxin5; Lane 15, glutathione S-transferase P; Lane 16, epididymal secretory glutathione peroxidase; Lane 17, peroxiredoxin-6; Lane 18, peroxiredoxin4; Lane 19, glutathione S-transferase Mu 3; Lane 20, superoxide dismutase [Cu-Zn]; Lane 21, peroxiredoxin-6; Lane 22, negative control.
Transporter 10% Reproduction 2% Immunity stress 11%
Rhesus monkey (present findings)
86
Human (Li et al. 2010) 118
68
Fig. 4. Venn diagrams of the overlap of epididymal luminal fluid protein subsets. Distribution of the 172 proteins found in bull epididymal luminal fluid (Belleanne´e et al. 2011) or rhesus monkey epididymal luminal fluid proteins (present findings) and their overlap with those reported in human epididymal fluid (Li et al. 2010).
Antioxidant 7%
Signal transduction 10% Structure 8%
Unclassified 1%
Protease/ protease inhibitor 13% Chaperone 1%
General metabolism 37%
Fig. 3. Pie diagram of the proportion of rhesus monkey epididymal luminal fluid proteins categorised by function. Classification by function of 198 rhesus monkey epididymal luminal fluid proteins.
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these common and major proteins constitute the bulk of the epididymal proteins, as determined by proteomics. The overlap between rhesus monkey and human (63% of the rhesus monkey epididymal fluid proteome), and between rhesus monkey and bull (22%), reveals higher similarity between rhesus monkey and human. Maturation of sperm motility in human (Yeung et al. 1993) is similar to that in Macaca fascicularis (Yeung et al. 1996). Condensation of sperm chromatin in man (Golan et al. 1996) is more impressive than that in Macaca fascicularis (Golan et al. 1997), but chromatin condensation in the rhesus monkey is similar to that in the human (Sivashanmugam and Rajalakshmi 1997). Additionally, taking 97.5% genomic similarity between rhesus monkey and human into account (Gibbs et al. 2007), the rhesus monkey cannot only be used as a non-human primate model for preclinical testing of candidate vaccines (Grimaldi 2008), but also as an epididymal sperm maturation model. Biological functions of 198 rhesus monkey epididymal fluid proteins showed that the majority (37%) was related to general metabolism but 8% were structural proteins. The origin of those structural proteins is perhaps the epididymosome. Epididymal proteins are secreted by both conventional (merocrine) and unconventional (apocrine) pathways. The latter mediates the release of those proteins lacking signal peptides, which arise from a specialised pathway involving large vesicular structures (blebs) released from the principal cells. The poorly-soluble proteins can, in part, be explained by the presence of particulate components, either in the form of membrane vesicles named epididymosomes (for ‘epididymal exosomes’) or of ‘micellelike structures’ in the fluid (Frenette et al. 2002). Proteomic technology has also been used to analyse the protein composition of human epididymosomes collected during surgical vasectomy reversal and some structural proteins were found, such as tubulin, actin, tektin, clathrin and moesin (Thimon et al. 2008). We cannot rule out the possibility that debris from epididymal spermatozoa and epithelial cell contamination during fluid collection might be the origin of those structural proteins. In further studies, epididymal fluid should be collected by the cleaner luminal perfusion method (Dacheux et al. 2006) so that epithelial cell proteins do not contaminate the fluid obtained. About 19% of luminal fluid proteins were signal transduction proteins that may be important for regulating sperm functions during epididymal sperm transit and fertilisation. Almost 20% of the proteins were involved in immune defence, preventing oxidation and molecular chaperoning. The surface proteins may well participate in immunological protection of epididymal spermatozoa in the male and female tracts. In our previous proteomic study (Li et al. 2010), peroxiredoxin-6 could maintain sperm motility under the stress of hydrogen peroxide, so it could play an antioxidant role during epididymal sperm maturation. Additionally, strong antioxidants in the epididymis may prevent its developing cancer (Yeung et al. 2012). Recent work on the rhesus monkey sperm proteome has been reported and 1247 proteins were identified by mass spectrometry (Skerget et al. 2013). Thirty-two percent of sperm maturation milieu proteins in our work were found in the sperm proteome, which showed similarities and differences. Two well-studied
X. Liu et al.
rhesus monkey sperm proteins, glycosylated 58 kDa glycoprotein (MEF1; Srivastav et al. 2004) and epididymis-specific clone 42 (ESC42) (Liu et al. 2001), were not found in the above two datasets, which indicates that both the sperm proteome and the sperm maturation milieu proteome of the rhesus monkey (Macaca mulatta) needs deeper study. In conclusion, this work has greatly expanded knowledge of the rhesus monkey epididymal fluid proteome, which includes important proteins involved in sperm maturation. The roles of the new epididymal fluid proteins have to be substantiated before they can be used to develop biomarkers of normal or abnormal sperm maturation for the rhesus monkey. The study will advance our understanding of sperm maturation and also facilitate biological interpretation of epididymal function. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (no. 81300533, 81370013 and 81000277) and Shandong Provincial Natural Science Foundation, China (ZR2013HQ002). The authors thank Guo Lihai (Shanghai Asia Pacific Application Support Center, Applied Biosystems, Shanghai, China) for help identifying the twodimensional gel spots with a 4800 MALDI-TOF/TOF analyser. The authors declare no competing interests.
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Reproduction, Fertility and Development, 2016, 28, 732–741 http://dx.doi.org/10.1071/RD14322
Proteome profiling of the sperm maturation milieu in the rhesus monkey (Macaca mulatta) epididymis Xin Liu A,*, Shao-Hua Jin B,*, Xue-Xia Liu A, Wen-Juan Wang C and Fu-Jun Liu A,D A
Central Laboratory, Yantai Yu Huang Ding Hospital/Qingdao University, Yantai 264000, Shandong, P.R. China. B Clinical Laboratory, Yantai Yu Huang Ding Hospital/Qingdao University, Yantai 264000, Shandong, P.R. China. C Reproduction Medical Center, Yantai Yu Huang Ding Hospital/Qingdao University, Yantai 264000, Shandong, P.R. China. D Corresponding author. Email: [email protected]
Abstract. The mammalian spermatozoon acquires its fertilising potential during transit through the epididymis, where it interacts with epididymal luminal fluid proteins (the sperm maturation milieu). In order to highlight the epididymalspecific function of the rhesus monkey (Macaca mulatta) in sperm maturation, two-dimensional gel electrophoresis of epididymal luminal fluid proteins was followed by identification by Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry (MALDI-TOF/MS) or MALDI-TOF/TOF and revealed over five hundred spots, comprising 198 non-redundant proteins. Some mass spectrometric data were confirmed by western blotting identification. Some common epididymal fluid proteins were identified, such as clusterin, a-1-antitrypsin, malate dehydrogenase, L-lactate dehydrogenase B, a-1-acid glycoprotein 1 and a-mannosidase. More than 7% of all proteins were anti-oxidative, which might control oxidative stress within the male tract. When compared with bull and human epididymal luminal fluid proteins, those in the rhesus monkey had more overlap with the human, which provides evidence of a close evolutionary relationship between the rhesus monkey and man. This study provides new proteomic information on possible rhesus monkey epididymal functions and novel potential biomarkers for the noninvasive assessment of male fertility. Additional keywords: anti-oxidative, bioinformatics, epididymal fluid, evolution, mass spectrometry. Received 29 August 2014, accepted 16 September 2014, published online 30 October 2014
Introduction Spermatozoa produced by the testis are not yet motile or fertile, but these immature spermatozoa have to survive in the female tract, move to the oviduct, penetrate the egg investments and fuse with the oocyte (Yeung and Cooper 2003). In consideration of DNA condensation, RNA transcription prevention and absence of protein translation, epididymal spermatozoa only acquire the functional capacity to perform the above individual steps of fertilisation during transit through the epididymis, which secretes proteins that sequentially remove, add and modify the surface of maturing spermatozoa. Spermatozoa are consistently enclosed in a specific microenvironment and interact with epididymal contents, especially epididymal luminal fluid proteins, for their maturation and storage (Cooper and Yeung 2010). After evidence was provided to demonstrate sperm maturation in the epididymis (Bedford 1967; Orgebin-Crist 1967), some proteins began to be reported, such as rat epididymal fluid proteins B/C, D/E (Brooks and
Higgins 1980) and the antibacterial Bin1B (Li et al. 2001). More proteins should be found to reflect epididymal-specific functions in sperm maturation. The rapid development of proteomic technology opens the door to the global mapping analysis of epididymal luminal fluid proteins (Aitken et al. 2007; Dacheux et al. 2009a, 2012). In mammals, epididymal luminal fluid proteomes of the boar (Syntin et al. 1999), stallion (Fouche´court et al. 2000) and ram (Druart et al. 1994) have been reported, and more proteins were reported, such as a-1-antitrypsin, a-2-glycoprotein, albumin, b-galactosidase, calmodulin, clusterin, cystatin C, glutathione peroxidase, gelsolin, glutathione-S-transferase, b-N-acetylhexosaminidase, lipocalin 5, lactoferrin, mannosidase, prostaglandin D2 synthase and transferrin. To understand the evolutionary significance of the epididymal micro-environment, the platypus caudal epididymidal fluid proteome has also been identified (Dacheux et al. 2009b). The results showed the platypus to be an early model mammal, whose epididymal fluid proteins were few
*
These authors contributed equally to this paper.
Journal compilation Ó CSIRO 2016
www.publish.csiro.au/journals/rfd
Rhesus monkey sperm maturation milieu
and distinctive. But the platypus epididymis shows a unique feature, that of promoting sperm bundles to enhance the cells’ motility (Nixon et al. 2011). In recent years, by using twodimensional gel electrophoresis and mass spectrometry, 172 bovine epididymal luminal proteins have been identified (Belleanne´e et al. 2011), which shed light on bovine epididymal functions and specificity. The epididymal proteins in many species have been identified, but few have defined functions that could be targeted for contraception. Knowing the proteins involved in epididymal sperm maturation deserves more attention. Although there are some identical epididymal fluid proteins among species, speciesspecific proteins also apparently exist. This study aimed to produce a proteomic database of rhesus monkey epididymal luminal fluid proteins and to compare the results with the proteomic data previously reported in other mammalian species. This could be the basis of development of diagnostic markers and therapeutic targets for infertility or contraception. Materials and methods Preparation of sperm-milieu proteins Three adult male (8–10 years old) rhesus monkeys (Macaca mulatta), 9–10 kg bodyweight, from Yantai zoo’s primate colony were used for the experiment. They all had proven breeding records. Approval was obtained for the use of rhesus monkeys by the Animal Ethics Committee of Yantai Yu Huang Ding Hospital for Animal Care and Usage. Three monkeys were individually caged in a humidity- and temperature-controlled room and the light : dark cycle was set at 12 : 12 h. During the period of experiment, water and monkey chow were regularly available. After anaesthesia with ketamine hydrochloric acid (HC1) (10 mg kg1), unilateral castrations was performed on the monkeys and then three testes with their attached epididymides were removed and collected. Blood vessels and initial segments were dissected from the remaining epididymis. All three epididymides were pooled to prepare the luminal proteins (the sperm maturation milieu) as in our previous report (Li et al. 2010, 2014). Briefly, the epididymides were coarsely minced and gentle pressure applied to release luminal fluid into phosphate buffer. After centrifugation at 5700g at 48C for 10 min and microscopic examination, the resultant sperm-free supernatant was precipitated with four volumes of ice-cold acetone. Finally the precipitates were dissolved in 3 mL protein lysis buffer (7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-cholamido-propyl) dimethylammonio]-1-propanesulfonate, 65 mM dithiothreitol). The concentration of proteins was measured by the Bradford assay (Gotham et al. 1988) and then samples stored at 808C. Gel electrophoresis, protein staining and mass spectrometric analyses Two-dimensional electrophoresis gels were performed as previously reported (Go¨rg et al. 2004). The proteins were stained by Coomassie brilliant blue R-350 (Amersham Biosciences, Uppsala, Sweden), then the protein spots were excised from the gel and digested by trypsin. The resultant peptides were identified by a Voyager DE-STR biospectrometry workstation or a 4800 Matrix-Assisted Laser Desorption/Ionization Time of
Reproduction, Fertility and Development
733
Flight/Time of Flight Mass Spectrometry (MALDI-TOF/TOF MS) (Applied Biosystems/MDS SCIEX, Framingham, MA, USA). If one spot was identified as more than one protein, the first-ranked protein was selected for each spot. Western blotting and bioinformatic analysis Western blotting was performed by following the method of Liu et al. (2014). After the incubation with primary and secondary antibody, the immunoreactive bands were visualised by a diaminobenzidine kit (Zhong-Shan Biotechnology, Beijing, China). The functional classification of the sperm-milieu proteins was performed by the online DAVID software (Version 6.7; http://david.abcc.ncifcrf.gov/ [accessed 6 June 2013]). Results Gel electrophoresis and MALDI mass spectrometry Gel electrophoretic analysis of proteins from three rhesus monkey epididymal luminal fluids, which were pooled for protein extraction and separated on two-dimensional gels, revealed 520 spots that were consistently detected in three gels (Fig. 1). From the spots analysed by MALDI-TOF/MS or MALDI-TOF/TOF, 198 epididymal fluid proteins were identified in the sperm milieu (Table 1). Thirty two percent of the total 198 proteins were represented by two or up to more than six spots, revealing the same unique proteins, presumably representing spliced variants or isoforms. Western blotting of rhesus monkey epididymal luminal fluid proteins Ten common epididymal fluid proteins (clusterin, epididymal secretory protein E1, b-hexosaminidase B, cystatin B, cathepsin B, glyceraldehyde-3-phosphate dehydrogenase, a-1-antitrypsin, 14–3-3 protein zeta/delta, epididymis-specific a-mannosidase and selenium-binding protein) and 11 antioxidant proteins (peroxiredoxin-1, superoxide dismutase [Mn], thioredoxindependent peroxide reductase, peroxiredoxin-5, glutathione S-transferase P, epididymal secretory glutathione peroxidase, peroxiredoxin-6, peroxiredoxin-4, glutathione Stransferase Mu 3, superoxide dismutase [Cu-Zn] and peroxiredoxin-6) were selected for detection by western blotting (Fig. 2). Classification of biological function of rhesus monkey epididymal luminal fluid proteins The proteomic analysis of the functions of 198 rhesus monkey epididymal luminal fluid proteins is given in Fig. 3. The majority (37%) of proteins were related to general metabolism and the next most prevalent category (13%) involved protease and protease inhibitor functions. Eight percent were structural proteins and 7% of proteins were antioxidative; the smallest group had chaperone functions. Comparison of rhesus monkey, bull and human epididymal luminal fluid proteins Each epididymal fluid proteome is species-specific. To disclose the difference in epididymal fluid proteomes between species and to minimise different proteomic results caused by different proteomic technologies, the bull (Belleanne´e et al. 2011) and
734
Reproduction, Fertility and Development
(a)
X. Liu et al.
3
pH
10
kDa 97.0 66.0
45.0
30.0
20.0
(d)
420.2
831.4
1242.6
1653.8
(e)
3822.2
100 90 80 70 60 50 40 30 20 10 0 9.0
2065.0
1680
1940.88 1996.92 2045.97 2104.95
1839.87
1622.77 1690.76 1744.88
1283.63 1365.61 1417.69 1475.71 1541.69 1519.70 1420
1940
2200
y6(⫹1)
1160
70.1097 y1(⫹1)
y15 - 17(⫹1)
b13(⫹1) y14(⫹1)
b11(⫹1) a12(⫹1)
P
212.10 294.12 300.15 b3(⫹1) 475.20 515.19 600.24 b7(⫹1) b8 - 17(⫹1) a9(⫹1) 1047.48 y8(⫹1)
MS/MS Precursor: 1954.98 YVRPGGGFVPNFQLFEK
2200
9.0E⫹3
MS/MS Precursor: 2017.97 VSFEDSVISLSGDHSIIGR
5219.2
y3(⫹1) y4(⫹1) y5(⫹1) b6(⫹1),y6 - 17(⫹1) b7 - 18(⫹1) y8(⫹1) y9(⫹1) y10(⫹1) y11(⫹1) 1239.8123 y13(⫹1) y14(⫹1) y15(⫹1) y16(⫹1) b17(⫹1) 1886.0728 1993.6578
1940 b16 ⫹ H2O(⫹1)
1680
100 90 80 70 60 50 40 30 20 10 0 900
935.23 996.58 1045.54 1095.54 1152.52 1201.59
1954.98 1982.98 2034.96 2111.06
1698.84 1754.83 1819.89 1895.91
1470.72 1536.75 1609.75
1420
1830.34 b17(⫹1)
1160
1300.69 1360.66
936.52 963.46 985.44 1050.53 1107.58 1162.58 1211.65
6.6E⫹4
(c)
100 90 80 70 60 50 40 30 20 10 0 9.0
2017.97
100 90 80 70 60 50 40 30 20 10 0 900
F
% Intensity
(b)
1316.67
14.4
433.4
857.8
1282.2
1706.6
2131.0
Mass (m/z) Fig. 1. Separation and identification of rhesus monkey epididymal luminal fluid proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and MALDI-MS spectra. (a) Reference map of epididymal luminal fluid. Example spectra of (b, c) glutathione peroxidase and (d, e) superoxide dismutase [Cu-Zn]. (b) MS map and (c) MS/MS map marked with b ions and y ions for glutathione peroxidase identification. The sequence of precursor at m/z 1954.98 was analysed by MS/MS to be YVRPGGGFVPNFQLFEK and the protein identified as glutathione peroxidase. (d ) MS map and (e) MS/MS map marked with b ions and y ions for superoxide dismutase [Cu-Zn] identification. The sequence of precursor at m/z 2017.97 was analysed by MS/MS to be VSFEDSVISLSGDHSIIGR and identified as superoxide dismutase [Cu-Zn].
our previously identified human (Li et al. 2010) epididymal luminal fluid proteins were selected, which were both performed by two-dimensional gel electrophoresis coupled with MALDI mass spectrometer identification. The results reflect the overlap of bovine and human epididymal luminal fluid proteins with
those of rhesus monkey epididymal luminal fluid (Fig. 4). A total of 39 proteins in human and bull epididymal luminal fluid were common to rhesus monkey epididymal luminal fluid, accounting for 20% of proteins in rhesus monkey epididymal luminal fluid. 125 proteins (63% of the rhesus monkey
NCBI accession number
gi|4557032 gi|75041584 gi|19923206 gi|5803181 gi|62511005 gi|5453990 gi|118090 gi|4759274 gi|18307578 gi|225939 gi|55595921 gi|119592410 gi|4507651 gi|178132 gi|4502105 gi|4507793 gi|1421609 gi|21431617 gi|1706134 gi|38540969 gi|34761571 gi|34761579 gi|50659093 gi|16507237 gi|66346698 gi|40255043 gi|31543380 gi|312176440 gi|85397577 gi|76803548 gi|4503107 gi|114326453 gi|98985828 gi|4757960 gi|56699495 gi|8659555 gi|54037162 gi|1170338 gi|119172 gi|21903479 gi|20141877 gi|119370332 gi|12643636 gi|23831360 gi|20178296 gi|2497983 gi|127234 gi|121039 gi|461466
Protein no.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
L-Lactate dehydrogenase B chain 14-kDa Phosphohistidine phosphatase Glutamine synthetase Stress-induced phosphoprotein D-3-phosphoglycerate dehydrogenase Proteasome activator complex subunit 1 Peptidyl-prolyl cis-trans isomerase b precursor Thioredoxin-like protein 1 Fructose bisphosphate aldolase A Alcohol dehydrogenase [NADPþ] Cytosolic malate dehydrogenase isoform 5 Electron-transfer flavoprotein, b polypeptide Tropomyosin a-4 chain Alcohol dehydrogenase class 3 chi chain Annexin A4 Ubiquitin-conjugating enzyme E2 N Nucleoside diphosphate kinase B Protein disulfide isomerase A3 Cysteine-rich secretory protein 1 Epididymal-specific lipocalin-6 Epididymal-specific lipocalin-8 Epididymal-specific lipocalin-12 Epididymis-specific a-mannosidase 78-kDa Glucose-regulated protein a-N-Acetylglucosaminidase b-Galactosidase-1-like protein Protein DJ-1 Epididymal peptidase inhibitor Cystatin-8 Cystatin-11 Cystatin-C Disintegrin and metalloproteinase domain-containing protein 7 Disintegrin and metalloproteinase domain-containing protein 28 Cadherin-1 Wap four-disulfide core domain protein 2 Iron-responsive element-binding protein 1 Neutral a-glucosidase AB Chromobox protein homologue 5 Elongation factor 2 Vinculin Protein-glutamine gamma-glutamyltransferase 2 Complement C3 Wd repeat protein 1 Bifunctional purine biosynthesis protein Pyruvate kinase isozymes M1/M2 Mitochondrial uncoupling protein 3 Moesin Ig gamma-1 chain C region Aminoacylase-1
Protein description
72 61 137 133 95 62 125 62 104 62 62 60 62 74 135 72 76 70 69 84 92 111 159 191 63 62 88 62 61 92 90 155 104 60 84 141 89 61 94 105 77 134 120 118 146 185 67 97 146
Score
20 47 26 22 18 22 42 29 34 19 16 18 8 20 36 26 41 16 21 47 41 42 21 21 11 11 38 34 34 56 38 22 15 6 36 17 13 32 15 14 15 12 37 27 26 20 14 29 41
Coverage %
36 615 13 824 42 037 62 599 57 356 28 705 22 728 32 231 39 395 36 550 36 403 27 826 28 504 39 698 35 860 17 127 17 287 56 747 28 462 18 033 19 068 21 486 113 908 72 288 82 115 74 110 19 878 15 273 16 265 16 495 15 789 85 613 87 123 97 396 14 337 98 337 106 807 22 211 95 146 123 591 77 280 187 030 66 152 64 575 57 900 34 193 67 778 36 083 45 856
Mr
5.71 5.65 6.43 6.4 6.29 5.78 9.33 4.4 8.3 6.32 6.91 8.24 4.67 7.45 5.84 6.13 8.52 5.98 5.57 4.84 6.84 5.48 6.74 5.07 6.1 9.01 6.33 8.52 9.05 6.51 9 6.08 6.38 4.58 6.79 6.23 5.74 5.71 6.42 5.51 5.11 6.02 6.17 6.27 7.96 9.31 6.08 8.46 5.77
pI
Table 1. Proteins of the Rhesus monkey sperm maturation milieu identified by mass spectrometry
General metabolism General metabolism General metabolism Immunity stress General metabolism Protease/protease inhibitor General metabolism Antioxidant General metabolism General metabolism General metabolism General metabolism Structure General metabolism Signal transduction Protease/protease inhibitor General metabolism Chaperone reproduction transporter transporter transporter Protease/Protease inhibitor Immunity stress Protease/protease inhibitor Protease/protease inhibitor Unclassified Protease/protease inhibitor Protease/protease inhibitor Protease/protease inhibitor Protease/protease inhibitor Protease/protease inhibitor Protease/protease inhibitor Signal transduction Immunity stress transporter Protease/protease inhibitor reproduction Unclassified Immunity stress General metabolism Immunity stress Antioxidant General metabolism General metabolism General metabolism Signal transduction Immunity stress General metabolism
6A 5A 11A 12A 8A 5A 9A 4A 7A 4A 4A 4A 4A 5A 9A 5A 5A 8A 4A 4A 5A 5A 12A 11A 6A 6A 9A 3A 3A 4A 4A 11A 8A 4A 5A 11A 10A 5A 8A 12A 8A 15A 12A 11A 12A 5A 7A 6A 13A
Reproduction, Fertility and Development
(Continued )
Functional classification
Matched peptide number
Rhesus monkey sperm maturation milieu 735
NCBI accession number
gi|148840437 gi|23396498 gi|52000887 gi|113960 gi|8247940 gi|145559446 gi|29550838 gi|21903432 gi|1708967 gi|113596 gi|1708543 gi|136464 gi|7387511 gi|296439291 gi|93141249 gi|67463772 gi|38372871 gi|548453 gi|1706870 gi|20178288 gi|116242799 gi|116848 gi|119360 gi|317373553 gi|6686275 gi|119339 gi|115449 gi|134047703 gi|38258193 gi|19855073 gi|55977767 gi|112877 gi|1729976 gi|2811007 gi|61213572 gi|108860890 gi|38258929 gi|74730916 gi|130683 gi|21264428 gi|27805668 gi|51316252 gi|119627259 gi|730451 gi|125157 gi|115612 gi|74719147 gi|21361792 gi|20981682
Protein no.
50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98
Selenium-binding protein 1 Cytosolic nonspecific dipeptidase 14-3-3 Protein zeta/delta Annexin A5 Inorganic pyrophosphatase Betaine–homocysteine s-methyltransferase 1 Golgi membrane protein 1 Isocitrate dehydrogenase Malate dehydrogenase Aldose reductase Histidine triad nucleotide-binding protein 1 Transthyretin 6-Phosphogluconolactonase Nicotinate-nucleotide pyrophosphorylase Lambda-crystallin homologue Aldose 1-epimerase Aflatoxin B1 aldehyde reductase member 2 Peroxiredoxin-1 Flavin reductase Adenylate kinase isoenzyme 1 Spectrin b chain Cofilin-1 Endoplasmin a-1B-Glycoprotein Aconitate hydratase a-Enolase Carbonic anhydrase 1 Carbonic anhydrase 3 50 -Deoxyribonucleotidase Heat-shock protein b-1 Vimentin a-1-Acid glycoprotein 1 Transketolase Pyridoxal kinase Rhogef and ph domain-containing protein 2 Plexin-A4 DNA-dependent protein kinase catalytic subunit Tektin-4 Voltage-dependent anion-selective channel protein 1 Stress-70 protein Pantothenate kinase 3 L-Lactate dehydrogenase A-like 6A Spermatogenesis associated 6 60S Ribosomal protein l13A Adenylate kinase isoenzyme 4 Calpain small subunit 1 Glycoprotein-N-acetylgalactosamine 3-b-galactosyltransferase 1 Sperm-associated antigen 4-like protein Gamma-enolase
Protein description
103 126 97 76 124 70 61 79 77 82 61 112 74 61 114 144 61 98 85 102 61 105 117 67 157 73 63 110 98 114 120 78 65 84 61 66 64 74 82 76 63 65 75 66 65 61 65 67 62
Score
26 29 31 18 41 19 13 17 20 22 30 53 30 21 30 40 17 37 49 43 4 42 14 16 20 19 31 38 432 37 20 22 10 24 14 6 4 10 25 14 15 12 18 31 27 12 17 14 17
Coverage %
Table 1. (Continued)
52 280 52 845 27 728 35 914 32 639 44 942 45 306 46 630 36 403 35 830 13 793 15 877 27 530 30 796 35 396 37 742 39 564 22 096 22 105 21 621 274 439 18 491 92 411 54 239 85 372 47 139 28 852 29 553 23 368 22 768 53 619 23 497 67 835 35 080 74 845 212 318 468 788 50 617 30 754 73 635 41 068 36 484 54 014 18 509 22 073 57 948 42 176 43 053 47 239
Mr
6.13 5.66 4.73 4.94 5.54 6.41 4.91 6.53 6.91 6.51 6.43 5.52 5.7 5.81 5.81 6.18 6.7 8.27 7.13 8.73 5.39 8.22 4.76 5.58 7.36 7.01 6.59 6.86 6.18 5.98 5.06 4.93 7.58 5.75 6.51 6.42 6.75 6.01 8.62 5.87 6.13 6.51 8.6 10.51 5.38 6.41 6.17 8.61 4.91
pI
Functional classification
transporter General metabolism Immunity stress Signal transduction General metabolism General metabolism Structure General metabolism General metabolism General metabolism transporter transporter General metabolism General metabolism Structure General metabolism General metabolism Antioxidant General metabolism General metabolism Structure Structure Immunity stress Immunity stress General metabolism General metabolism General metabolism General metabolism General metabolism Immunity stress Structure Immunity stress General metabolism General metabolism Signal transduction Signal transduction General metabolism Signal transduction transporter Immunity stress General metabolism General metabolism reproduction Structure General metabolism Signal transduction Protease/protease inhibitor reproduction General metabolism
Matched peptide number 10A 11A 9A 5A 9A 6A 5A 7A 6A 6A 4A 6A 6A 5A 8A 10A 5A 6A 7A 8A 7A 6A 11A 7A 12A 8A 6A 7A 7A 7A 11A 5A 4A 6A 5A 7A 14A 4A 4A 7A 4A 4A 5A 4A 5A 4A 5A 4A 5A
736 Reproduction, Fertility and Development X. Liu et al.
99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152
gi|20178315 gi|127981 gi|134665 gi|114074 gi|2507171 gi|317373539 gi|34222621 gi|2829431 gi|139641 gi|158936749 gi|46395636 gi|6016173 gi|130770 gi|31077177 gi|23831297 gi|215274262 gi|114517 gi|74751178 gi|317373315 gi|74730987 gi|37730278 gi|5453603 gi|13543551 gi|4503909 gi|33875419 gi|68060662 gi|75070579 gi|136062 gi|111185949 gi|4503571 gi|42558919 gi|6912328 gi|226531183 gi|662841 gi|5453549 gi|5123454 gi|14585855 gi|4757900 gi|180572 gi|48257098 gi|109013331 gi|47519616 gi|4506455 gi|339647 gi|77539752 gi|4501887 gi|58042495 gi|74136167 gi|75070821 gi|48257068 gi|13937981 gi|5453541 gi|15010550 gi|4503895
Bifunctional 30 -phosphoadenosine 50 -phosphosulfate synthetase 2 Nucleoside diphosphate kinase A Superoxide dismutase [Mn] Adenine phosphoribosyltransferase Thioredoxin-dependent peroxide reductase Peroxiredoxin-5 Abhydrolase domain-containing protein 14B Calponin-1 Vitamin d-binding protein Tyrosine protein kinase Blk 30 ,50 -Bisphosphate nucleotidase 1 Glutathione transferase omega-1 Phosphatidylinositol transfer protein a isoform Casein kinase i isoform a GTP : AMP phosphotransferase Galectin-3 Adenosine triphosphate synthase subunit a Probable lipid phosphate phosphatase PPAPDC3 Prostate and testis-expressed protein 4 Prostate and testis-expressed protein 1 Glyceraldehyde-3-phosphate dehydrogenase T-Complex protein 1 subunit b isoform 1 Proteasome (prosome, macropain) subunit, a type, 1 Guanidinoacetate N-methyltransferase isoform A Distribution of catechol-O-methyltransferase protein Phosphoglycerate mutase 1 Peroxiredoxin-6 Triosephosphate isomerase Glutathione s-transferase P a-Enolase isoform 1 Retinal dehydrogenase 1 N(g),N(g)-Dimethylarginine dimethylaminohydrolase 1 isoform 1 Epididymal secretory glutathione peroxidase Heat-shock protein 27 Peroxiredoxin-4 Heat-shock 70-kDa protein 1A/1B Phosphatidylethanolamine-binding protein Calreticulin Creatine kinase-B Valosin-containing protein Glutathione S-transferase Mu 3 Tropomyosin b chain isoform 2 Reticulocalbin-1 Thyroid hormone binding protein Tubulin a-1B chain Actin, cytoplasmic 2 Proteasome subunit a-type 5 Superoxide dismutase [Cu-Zn] Peroxiredoxin-2 Heat-shock 70-kDa protein 8 Peptidylprolyl isomerase a (cyclophilin a) Anterior gradient protein 2 homologue Heat-shock protein gp96 Galactokinase 63 95 62 62 71 116 65 69 75 61 60 60 65 62 80 60 60 61 61 62 196 214 94 91 260 93 284 334 351 231 240 322 106 257 183 228 131 135 382 155 118 263 124 146 210 370 169 124 306 213 130 123 442 211
12 42 20 30 38 41 28 16 23 10 22 22 20 16 31 19 12 28 17 18 12 7 15 14 21 11 30 30 39 10 7 22 14 27 9 9 22 6 18 7 13 14 7 6 11 16 15 21 17 8 16 20 13 15
69 457 17 138 24 707 19 595 27 675 22 012 22 332 33 150 52 929 57 702 33 371 27 548 31 786 38 890 25 550 26 172 59 714 29 429 11 407 14 270 23 850.5 57 452.1 29 578.9 26 301.1 20 032.2 28 759.8 25 005.2 26 693.8 23 423 47 139.3 54 700.8 31 101.9 25 196 22 313.3 30 520.8 69 995 25 717.7 48 111.8 42 658.3 71 021.1 26 586.2 32 969.6 38 866.2 57 068.7 50 103.6 41 765.8 22 648.4 15 972.9 19 418 64 633.1 17 999.9 19 966.6 90 138.1 42 245.6
8.18 5.83 8.35 5.78 7.67 8.85 5.94 9.16 5.4 7.98 5.46 6.23 6.11 9.59 9.15 8.58 9.16 9.98 8.97 8.28 9.17 6.01 6.15 5.75 5.36 6.67 6 6.45 5.93 7.01 6.3 5.53 8.68 7.83 5.86 5.48 5.71 4.29 5.16 4.94 5.38 4.63 4.86 4.82 4.98 5.31 4.74 6.22 5.38 5.36 7.68 9.03 4.73 6.04
5A 7A 4A 4A 5A 7A 5A 6A 7A 5A 4A 5A 5A 5A 6A 4A 5A 4A 4A 5A 2B 2B 2B 2B 3B 2B 5B 4B 5B 3B 3B 4B 2B 4B 2B 4B 2B 2B 4B 3B 2B 3B 2B 2B 3B 4B 2B 2B 4B 3B 2B 2B 7B 4B
Reproduction, Fertility and Development
(Continued )
General metabolism General metabolism Antioxidant General metabolism Antioxidant Antioxidant Protease/protease inhibitor Signal transduction transporter Signal transduction General metabolism Antioxidant transporter General metabolism General metabolism transporter General metabolism General metabolism Immunity stress Immunity stress General metabolism Structure Protease/protease inhibitor General metabolism Signal transduction General metabolism Antioxidant General metabolism Antioxidant General metabolism General metabolism General metabolism Antioxidant Immunity stress Antioxidant Immunity stress transporter Signal transduction General metabolism Protease/protease inhibitor Antioxidant Structure Signal transduction transporter Structure Structure Protease/protease inhibitor Antioxidant Antioxidant Immunity stress General metabolism Signal transduction Immunity stress General metabolism
Rhesus monkey sperm maturation milieu 737
gi|22096346 gi|14250401 gi|38492599 gi|32189394 gi|55824560 gi|404390 gi|5453678 gi|2492797 gi|24430160 gi|54300702 gi|68051994 gi|109108768 gi|4557395 gi|23958133 gi|4557871 gi|544759 gi|48255905 gi|109131308 gi|18088311 gi|5729877 gi|49457530 gi|46249758 gi|4557579 gi|49456301 gi|4506179 gi|189998 gi|2352843 gi|55846712 gi|825635 gi|741376 gi|31543397 gi|157168362 gi|14149777 gi|18645167 gi|55960189 gi|109065176 gi|9506741 gi|4008131 gi|75709200 gi|55957281 gi|5542151
gi|62898045 gi|1905874 gi|1922287 gi|62087652 gi|7709904
153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193
194 195 196 197 198
Dynactin subunit 2 Actin, b b-Hexosaminidase B Adenosine triphosphate synthase subunit b Heat-shock 70-kDa protein 5 b-Trace protein, prostaglandin D synthase,PGD synthase Epididymal secretory protein E1 Albumin 26S Protease regulatory subunit 10B Ubiquitin C splice variant a-1-Antitrypsin Apolipoprotein A-I isoform 1 Carbonic anhydrase 2 Tubulin, b 2c Serotransferrin Biliverdin-ix b reductase isozyme I Transgelin Phosphoglycerate kinase 1 Nitrilase family, member 2 Heat-shock cognate 71-kDa protein isoform 1 Creatine kinase B-type Cytovillin Fatty acid-binding protein, adipocyte Methylthioadenosine phosphorylase Proteasome subunit a type-1 isoform 2 M2-Type pyruvate kinase Sorbitol dehydrogenase Clusterin Calmodulin Cathepsin B Phosphoglycerate kinase 2 Purine nucleoside phosphorylase Haloacid dehalogenase-like domain-containing protein 2 Annexin A2 SH3 Domain-binding glutamic acid-rich-like protein Cystatin-B Glycine N-methyltransferase Chaperonin Phospholipid hydroperoxide glutathione peroxidase Esterase D/formylglutathione hydrolase Chain a, macrophage migration inhibitory factor (mif) with hydroxphenylpyruvate Pyridoxine 50 -phosphate oxidase variant Carboxyl-terminal lim domain protein Enoyl-CoA hydratase Proteasome 26s ATPase subunit 5 variant Calnexin
Protein description
8 8 7 9 16 27 23 13 7 16 8 12 21 8 4 22 11 16 9 13 25 5 20 12 13 6 8 8 46 9 8 25 19 9 47 12 7 12 7 9 10 7 5 7 5 6
46 39 80 66 107
Coverage %
136 164 160 110 617 171 98 375 144 103 227 211 323 289 360 249 100 405 145 392 599 112 143 125 205 145 152 102 448 178 145 264 262 250 223 58 135 62 89 149 48
Score
29 915 36 170 31 371 38 760 31 185
44 203.9 40 978.4 58 820.7 56 524.6 66 857.5 18 670 16 559.5 67 836.5 44 145.1 17 142.3 44 558.9 30 716 29 227.9 49 808 76 999.6 21 846.3 22 596.4 44 558.1 30 560.5 70 854.2 42 648.3 69 198.6 14 709.5 31 233.8 29 536.9 57 877 38 155.8 48 598.8 17 152 17 143 44 767.3 32 097.2 28 517.9 38 551.8 9374.8 11 102 32 742 10 582 22 174 28 226 12 345
Mr
7.01 6.8 8.34 6.07 4.61
5.1 5.56 6.43 5.26 5.22 6.95 7.56 5.85 7.1 7.93 5.75 5.72 6.86 4.83 6.81 7.31 8.87 8.02 6.82 5.37 5.34 5.94 6.59 6.75 6.15 7.95 7.56 5.52 4.06 5.44 8.74 6.45 5.84 7.57 9.42 6.4 6.55 9.44 8.69 6.29 8.24
pI
Functional classification
Structure Structure Protease/protease inhibitor General metabolism Immunity stress Protease/protease inhibitor reproduction transporter Protease/protease inhibitor Protease/protease inhibitor Protease/protease inhibitor transporter General metabolism Structure transporter General metabolism transporter General metabolism General metabolism Immunity stress General metabolism Signal transduction transporter Immunity stress Protease/protease inhibitor General metabolism General metabolism transporter Signal transduction Protease/protease inhibitor General metabolism General metabolism General metabolism Signal transduction Signal transduction Protease/protease inhibitor General metabolism Chaperone Antioxidant General metabolism Immunity stress General metabolism Structure General metabolism Protease/protease inhibitor Signal transduction
Matched peptide number 2B 2B 2B 2B 6B 2B 2B 6B 2B 2B 2B 3B 4B 3B 4B 2B 2B 4B 2B 5B 6B 2B 2B 2B 2B 2B 2B 2B 4B 2B 2B 4B 3B 3B 3B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B 1B
A
Reproduction, Fertility and Development
Proteins identified by Voyager DE-STR biospectrometry workstation (PMF) and criteria for acceptance: all identified proteins had a significance threshold of at least P # 0.05, score $ 60 and at least four masses matched. If more than one protein was identified in a spot, the single protein member with the highest score (top rank) was chosen from the multi-protein family. B Proteins identified by 4800 MALDI TOF/TOF Analyzer (MS/MS) and criteria for acceptance: individual ion scores $ 37 (significance level, P # 0.05).
NCBI accession number
Protein no.
Table 1. (Continued)
738 X. Liu et al.
Rhesus monkey sperm maturation milieu
Reproduction, Fertility and Development
epididymal fluid proteome) were common to human epididymal luminal fluid but only 44 proteins (22% of the rhesus proteome) were common to bull epididymal luminal fluid. Discussion Knowledge of the complex nature of sequential addition, removal and modification of epididymal fluid proteins on the surface of testicular spermatozoa during their epididymal transit has until now been sketchy. There is no proteomic study of the rhesus monkey epididymal contribution to sperm-maturation antigens and our interest was focussed on proteins of epididymal fluid because they are necessary for sperm maturation and are potential post-testicular contraceptive targets and could be used as markers for diagnosis and therapy for infertility.
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Here a large novel database of rhesus monkey epididymal proteins was created from systematic work-up of nearly two hundred proteins, with confirmation of some by western blotting detection. Of 198 rhesus monkey epididymal fluid proteins, 32% of proteins showed isoforms. Similar results have been found in other species, such as the boar, stallion and ram (Druart et al. 1994). From a comparison of the rhesus monkey epididymal fluid proteome with that of the bull and our previously identified human epididymal fluid proteome only 39 proteins, including clusterin, were common to the three species. Most of
Bull (Belleannée et al. 2011) kDa M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 97 66 45
122
5
31
8
21 14
39
Fig. 2. Western blotting of proteins found in rhesus monkey epididymal luminal fluid. M, molecular mass marker (kDa); Lane 1, clusterin; Lane 2, epididymal secretory protein E1; Lane 3, b-hexosaminidase B; Lane 4, cystatin B; Lane 5, cathepsin B; Lane 6, glyceraldehyde-3-phosphate dehydrogenase; Lane 7, a-1-antitrypsin; Lane 8, 14-3-3 protein zeta/delta; Lane 9, epididymis-specific a-mannosidase; Lane 10, selenium-binding protein; Lane 11, peroxiredoxin-1; Lane 12, superoxide dismutase [Mn]; Lane 13, thioredoxin-dependent peroxide reductase; Lane 14, peroxiredoxin5; Lane 15, glutathione S-transferase P; Lane 16, epididymal secretory glutathione peroxidase; Lane 17, peroxiredoxin-6; Lane 18, peroxiredoxin4; Lane 19, glutathione S-transferase Mu 3; Lane 20, superoxide dismutase [Cu-Zn]; Lane 21, peroxiredoxin-6; Lane 22, negative control.
Transporter 10% Reproduction 2% Immunity stress 11%
Rhesus monkey (present findings)
86
Human (Li et al. 2010) 118
68
Fig. 4. Venn diagrams of the overlap of epididymal luminal fluid protein subsets. Distribution of the 172 proteins found in bull epididymal luminal fluid (Belleanne´e et al. 2011) or rhesus monkey epididymal luminal fluid proteins (present findings) and their overlap with those reported in human epididymal fluid (Li et al. 2010).
Antioxidant 7%
Signal transduction 10% Structure 8%
Unclassified 1%
Protease/ protease inhibitor 13% Chaperone 1%
General metabolism 37%
Fig. 3. Pie diagram of the proportion of rhesus monkey epididymal luminal fluid proteins categorised by function. Classification by function of 198 rhesus monkey epididymal luminal fluid proteins.
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these common and major proteins constitute the bulk of the epididymal proteins, as determined by proteomics. The overlap between rhesus monkey and human (63% of the rhesus monkey epididymal fluid proteome), and between rhesus monkey and bull (22%), reveals higher similarity between rhesus monkey and human. Maturation of sperm motility in human (Yeung et al. 1993) is similar to that in Macaca fascicularis (Yeung et al. 1996). Condensation of sperm chromatin in man (Golan et al. 1996) is more impressive than that in Macaca fascicularis (Golan et al. 1997), but chromatin condensation in the rhesus monkey is similar to that in the human (Sivashanmugam and Rajalakshmi 1997). Additionally, taking 97.5% genomic similarity between rhesus monkey and human into account (Gibbs et al. 2007), the rhesus monkey cannot only be used as a non-human primate model for preclinical testing of candidate vaccines (Grimaldi 2008), but also as an epididymal sperm maturation model. Biological functions of 198 rhesus monkey epididymal fluid proteins showed that the majority (37%) was related to general metabolism but 8% were structural proteins. The origin of those structural proteins is perhaps the epididymosome. Epididymal proteins are secreted by both conventional (merocrine) and unconventional (apocrine) pathways. The latter mediates the release of those proteins lacking signal peptides, which arise from a specialised pathway involving large vesicular structures (blebs) released from the principal cells. The poorly-soluble proteins can, in part, be explained by the presence of particulate components, either in the form of membrane vesicles named epididymosomes (for ‘epididymal exosomes’) or of ‘micellelike structures’ in the fluid (Frenette et al. 2002). Proteomic technology has also been used to analyse the protein composition of human epididymosomes collected during surgical vasectomy reversal and some structural proteins were found, such as tubulin, actin, tektin, clathrin and moesin (Thimon et al. 2008). We cannot rule out the possibility that debris from epididymal spermatozoa and epithelial cell contamination during fluid collection might be the origin of those structural proteins. In further studies, epididymal fluid should be collected by the cleaner luminal perfusion method (Dacheux et al. 2006) so that epithelial cell proteins do not contaminate the fluid obtained. About 19% of luminal fluid proteins were signal transduction proteins that may be important for regulating sperm functions during epididymal sperm transit and fertilisation. Almost 20% of the proteins were involved in immune defence, preventing oxidation and molecular chaperoning. The surface proteins may well participate in immunological protection of epididymal spermatozoa in the male and female tracts. In our previous proteomic study (Li et al. 2010), peroxiredoxin-6 could maintain sperm motility under the stress of hydrogen peroxide, so it could play an antioxidant role during epididymal sperm maturation. Additionally, strong antioxidants in the epididymis may prevent its developing cancer (Yeung et al. 2012). Recent work on the rhesus monkey sperm proteome has been reported and 1247 proteins were identified by mass spectrometry (Skerget et al. 2013). Thirty-two percent of sperm maturation milieu proteins in our work were found in the sperm proteome, which showed similarities and differences. Two well-studied
X. Liu et al.
rhesus monkey sperm proteins, glycosylated 58 kDa glycoprotein (MEF1; Srivastav et al. 2004) and epididymis-specific clone 42 (ESC42) (Liu et al. 2001), were not found in the above two datasets, which indicates that both the sperm proteome and the sperm maturation milieu proteome of the rhesus monkey (Macaca mulatta) needs deeper study. In conclusion, this work has greatly expanded knowledge of the rhesus monkey epididymal fluid proteome, which includes important proteins involved in sperm maturation. The roles of the new epididymal fluid proteins have to be substantiated before they can be used to develop biomarkers of normal or abnormal sperm maturation for the rhesus monkey. The study will advance our understanding of sperm maturation and also facilitate biological interpretation of epididymal function. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (no. 81300533, 81370013 and 81000277) and Shandong Provincial Natural Science Foundation, China (ZR2013HQ002). The authors thank Guo Lihai (Shanghai Asia Pacific Application Support Center, Applied Biosystems, Shanghai, China) for help identifying the twodimensional gel spots with a 4800 MALDI-TOF/TOF analyser. The authors declare no competing interests.
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