5826

DOI 10.1002/pmic.200500830

Proteomics 2006, 6, 58265833

RESEARCH ARTICLE

Characterization of bovine seminal plasma by

proteomics
Van C. Kelly1, Sulee Kuy1, David J. Palmer1, Zhenzhong Xu2,
Stephen R. Davis3 and Garth J. Cooper1
1

School of Biological Sciences, University of Auckland, Auckland, New Zealand

Livestock Improvement, Newstead, Hamilton, New Zealand
3
ViaLactia Biosciences (NZ) Ltd., Newmarket, Auckland, New Zealand
2

Previous investigations of bovine seminal plasma (BSP) have revealed the identities of the three
major proteins, BSP-PDC109, BSP-A3 and BSP-30 kDa, which together constitute about half of
the total protein, as well as about 30 of the minor proteins. Analyses of BSP by 2-DE have revealed
about 250 protein spots, suggesting that much of the BSP proteome remains undescribed. In this
study, BSP has been analyzed by 2-D LC-based and SDS-PAGE-based proteomic methods.
Ninety-nine proteins were identified, including 49 minor proteins that have not previously been
described in seminal plasma of any species.

Received: November 17, 2005

Revised: May 30, 2006
Accepted: July 10, 2006

Keywords:
Bovine / Proteome analysis / Seminal plasma

Introduction

Seminal plasma is a composite fluid formed from the secretions of multiple glands of the male reproductive tract. Seminal plasma functions to transport sperm into the female
reproductive tract as well as directly affecting sperm function
such as the acquisition of fertilizing capacity (see ref [1] for a
review). Previous studies have elucidated the major protein
components of bovine seminal plasma (BSP) as BSP-PDC109
(or BSP-A1/A2), BSP-A3 and BSP-30 kDa [2, 3], which together comprise 5065% of the total protein [4, 5]. These proteins bind to the sperm surface at ejaculation via interactions
with choline phospholipids in the sperm membrane and
appear to participate in the sperm membrane lipid modifications that occur during capacitation (see ref [6] for a review).
Correspondence: Dr. Garth Cooper, School of Biological
Sciences, University of Auckland, Private Bag 92019, Auckland,
New Zealand
E-mail: g.cooper@auckland.ac.nz
Fax: 164-9-3737-045
Abbreviations: BSP, bovine seminal plasma; BtGI, Bos taurus
Gene Index; GpBt, database of ab initio Gnomon protein predictions from Bos taurus genome sequence; NCBInr, National
Center for Biotechnology Information non-redundant protein
sequence database

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

About 30 of the minor proteins of BSP have been identified

in studies utilizing biochemical purification, enzyme assay,
immunoassay or proteomics, or through investigations of factors involved in male fertility or sperm preservation [734]. Proteome mapping studies of Bos taurus taurus and B. taurus indicus
seminal plasma [33, 34] by 2-DE with analysis of selected protein
spots lead to the identification of 12 and 11 distinct proteins,
respectively. However, in each study, about 250 protein spots
were visualized, suggesting that there remains a considerable
amount of the BSP proteome to identify. We aimed to obtain a
comprehensive inventory of the protein components of BSP
using shotgun proteomics methods. In this study, a 2-D LC/
MS/MS proteomics approach as well as an SDS-PAGE-based
method have been used to characterize the BSP proteome.

Materials and methods

2.1 Materials
A sample of B. taurus taurus semen was collected from a
single bull at an artificial insemination facility using an artificial vagina and immediately centrifuged (10006g, 15 min)
to remove sperm. The seminal plasma was stored at 2207C.
Protein was determined by the bicinchoninic acid method
[35] using BSA as a standard.
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Animal Proteomics

5827

2.2 Proteomics
Seminal plasma was analyzed by 2-D LC-based and SDSPAGE-based proteomic approaches as described previously
[36] but with two exceptions. First, in the 2-D LC, 22 fractions were collected during strong cation exchange chromatography of seminal plasma tryptic peptides and each
fraction was analyzed twice by RP-LC/MS/MS. The data
from these analyses were combined for the sequence database searches. Secondly, different sequence databases were
searched in this study. To identify proteins, MASCOT was
used to search filtered MS/MS data against Swiss-Prot
(mammals-only; release 48.2, 11 October 2005, Swiss Institute of Bioinformatics, Geneva, Switzerland), the National
Center for Biotechnology Information non-redundant protein sequence database (NCBInr; mammals-only, release
date 11th October 2005, National Library of Medicine,
Bethesda, MD, USA), the B. taurus Gene Index (BtGI;
release 11, The Institute for Genomic Research, Rockville,
MD, USA), and ab initio Gnomon protein predictions
from B. taurus genome sequence (GpBt) downloaded from
ftp://ftp.ncbi.nlm.nih.gov/genomes/Bos_taurus/protein/(ver
sion 1/build 1, release date 29th March 2005). To test the
validation criteria, MASCOT was used to search filtered
MS/MS data against Swiss-Prot (mammals-only; release
48.2, 11th October 2005) and NCBInr (bovine entries, release
date 11 October 2005) where the amino acid sequence of
each entry was reversed.

Results

BSP was analyzed using 2-D LC and gel-based proteomic

approaches. Ninety-six proteins were identified using the
2-D LC proteomic approach (Table 1, Supplementary Material). Fifty-five proteins were identified using an SDS-PAGEbased proteomic approach (Fig. 1, Table 1, Supplementary
Material), including 3 proteins that were not identified as
valid protein matches using the 2-D LC method. These proteins were identified through four, three and two nonredundant peptides matching adenylate kinase isoenzyme 1.
Predicted: similar to ectonucleotide pyrophosphatase/phosphodiesterase 3 and predicted: similar to tubulin alpha-1,
respectively.
Multiple sequence databases were utilized as none of the
databases are comprehensive with regard to bovine proteins.
Priority was given to reporting entries in Swiss-Prot, followed by NCBInr, BtGI, and finally GpBt, unless a significantly better match was obtained in a database of lower
priority. Three of the identified proteins are sequences from
BtGI. Two of these, quiescin Q6 and N-acetyl-beta-glucosaminidase, were superior matches to corresponding predicted
protein sequences in NCBInr, as the matched peptides were
distributed over multiple partial sequences in NCBInr. The
other BtGI entry, dipeptidyl-peptidase II, was not represented in Swiss-Prot or NCBInr as a bovine sequence. Two of
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. SDS-PAGE analysis of bovine

seminal plasma. Bovine seminal plasma
(20 mg of protein) was separated by SDSPAGE and stained with CBB. The positions of
molecular size markers are indicated on the
right of the figure.

the identified proteins matched entries in GpBT. One of

these, seminal secretoglobin, was not represented in any
other database. The other GpBT entry, tissue alpha-L-fucosidase, was superior to two predicted partial sequences in
NCBInr.
The protein names in Table 1 are given according to
the nomenclature of the database from which the sequence
was sourced, except for the two entries from GpBT, which
did not have protein-descriptive annotations. These were
named according to the results of BLAST sequence similarity searches: seminal secretoglobin matched various
mammaglobin and lipophilin sequences, which are secretoglobin family members, with about 50% sequence identity; tissue alpha-L-fucosidase matched human tissue fucosidase alpha-L-1 (Swiss-Prot P04066) with 89% sequence
identity.
Two of the identified proteins (predicted: similar to
serotransferrin and predicted: similar to seminal plasma
protein A3) appear from their names to be duplicate
entries of serotransferrin and BSP-A3. However, in addition to being distinguished by unique matching peptides,
BLAST sequence similarity searches revealed that they are
distinct proteins. The protein named similar to serotransferrin is more similar to inhibitor of carbonic anhydrase (81% sequence identity to pig inhibitor of carbonic
anhydrase, Swiss-Prot Q29545) than to serotransferrin
(66% sequence identity to horse serotransferrin, Swiss-Prot
P27425). Predicted: similar to seminal plasma protein A3
has 71% sequence identity to BSP-A3.
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Van C. Kelly et al.

Proteomics 2006, 6, 58265833

Table 1. Proteins identified in bovine seminal plasma by 2D-LC/MS/MS and/or by SDS-PAGE followed by RP-LC/MS/MS

Protein namea)

Accession no.b)

2-D LCc)

SDS-PAGEd)

14-3-3 protein zeta/delta

5-nucleotidase
Acidic seminal fluid protein
Acrosin inhibitor I
Actin, cytoplasmic 1 or 2
Adenylate kinase isoenzyme 1
Angiogenin-1
Angiogenin-2
Angiotensin-converting enzyme
Beta-2-microglobulin
Beta-mannosidase
Beta-nerve growth factor
Cathepsin B
Cathepsin L
Clusterin
Complement H factor 1
C-type natriuretic peptide precursor
Cystatin E/M
Dipeptidyl peptidase 4
Epididymal secretory protein E1
Galactosidase, beta 1
Glutathione peroxidase type 5
Heat shock 90kD protein 1, alpha
Hypothetical protein LOC512373
Immunoglobulin gamma-1 chain C region
Immunoglobulin gamma-2 chain C region
Immunoglobulin lambda light chain
Lactotransferrin
Long palate, lung and nasal epithelium carcinoma
associated protein 1
Metalloproteinase inhibitor 2
Monocyte chemotactic protein 1A
Osteopontin
Peptidyl-prolyl cis-trans isomerase B
Peroxiredoxin 5, mitochondrial
Platelet-activating factor acetylhydrolase
Predicted: hypothetical protein XP_581816
Predicted: similar to alpha-mannosidase 2C1
Predicted: similar to beta-defensin 1
Predicted: similar to beta-microseminoprotein
Predicted: similar to cauxin
Predicted: similar to chaperonin containing TCP1,
subunit 2, isoform 1, 3 or 6
Predicted: similar to chaperonin containing TCP1,
theta subunit
Predicted: similar to complement C3, isoform 1 or 2
Predicted: similar to complement factor B
Predicted: similar to complement factor H isoform 2
Predicted: similar to cysteine-rich secretory protein 1
Predicted: similar to deoxyribonuclease gammah)
Predicted: similar to ecto-ADP-ribosyltransferase 5
Predicted: similar to ectonucleotide pyrophosphatase/
phosphodiesterase 3
Predicted: similar to epididymal-specific lipocalin 8
Predicted: similar to epididymal sperm binding
protein 1

P63103
Q05927
P29392
P01000
P60712, P63258
P00570
P10152
P80929
449408
P01888
Q29444
P13600
P07688
P25975
P17697
76677897
P55206
61097917
P81425
P79345
61554628
70778757
60592792
66792842
440
89611
15088675
P24627
Q8SPF8

6 (12)
6 (9)
9 (357)
3 (52)
7 (13)

28 (28)

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3 (7)
4 (13)
15 (40)
5 (14)
9 (14)
12 (45)f)
3 (8)
2 (3)
14 (163)
31 (62)
5 (113)
7 (34)
6 (9)
9 (71)
6 (8)
2 (6)
9 (16)
5 (6)
6 (8)
3 (4)
3 (12)
21 (43)
6 (15)

P16368
P28291
P31096
P80311
Q9BGI1
Q28017
76635171
76662175
76663957
76656898
76625606
76618459, 76618463,
76618469
75812922

9 (82)
2 (18)
5 (62)
9 (27)
4 (6)
13 (58)
5 (20)
9 (20)
4 (21)
4 (28)
9 (35)
5 (8)

76622034, 76622036
76650936
76673990
76650495
76648815
76635826
76673669

4 (7)
7 (13)
6 (16)
3 (4)
13 (31)
7 (17)

76631205
76641922

6 (68)
10 (31)

3 (5)

12 (15)

Previously
describede)

[23]
[22]
[12]

45 (42)
24 (22)

85, 100115 (150)

102 (101)
15 (25)

3045, 6070 (51)

131, 169 (140)
16 (13)

18 (17)
22 (24)
85 (85)

[17, 18]
[34]
H [64]
[16]

[8]
[9]

[34]
[31]
[24]

H [65]
H [65]
85 (78)

[20]

23 (24)
14 (11)

[10]
[21]
[25]

22 (23)
55 (50)
70 (27)
120 (116)

[27]
[32]g)

65 (69)

H [66]
S [67]

55 (60)
H [68]
98 (83)

37 (35)
47 (33)
45, 117 (100)

[11]
H [69]

20 (58)
30 (34)

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Animal Proteomics

Proteomics 2006, 6, 58265833

5829

Table 1. Continued

Protein namea)

Accession no.b)

2-D LCc)

SDS-PAGEd)

Previously
describede)

Predicted: similar to epididymis-specific alphamannosidase

Predicted: similar to fructose-1,6-bisphosphatase
Predicted: similar to galectin-3 binding protein
Predicted: similar to gelsolin
Predicted: similar to glucose-6-phosphate isomerase,
isoform 2
Predicted: similar to heat shock 70kDa protein 1-like
Predicted: similar to lactate dehydrogenase C
Predicted: similar to mesothelin isoform 1
Predicted: similar to metalloproteinase inhibitor 1
Predicted: similar to nephronectin, isoform b
Predicted: similar to nucleobindin 1, isoform 1, 2 or 3

76620326

8 (12)

117 (132)

[32]g)

76660763
76645574
76625353
76640979

5 (11)
7 (22)
7 (16)
5 (12)

85 (62)
45 (86)
55 (63)

Predicted: similar to nucleobindin 2,

isoform 1, 3, 6, 7, 8 or 9
Predicted: similar to phosphoglycerate kinase,
testis-specific
Predicted: similar to phosphoglycerate mutase 2
Predicted: similar to phosphatidylethanolamine-binding
protein 4, isoform 2
Predicted: similar to seminal plasma protein A3
Predicted: similar to serine protease inhibitor
Kazal-type 6
Predicted: similar to serotransferrin
Predicted: similar to T-complex protein-1, alpha subunit
Predicted: similar to T-complex protein-1, epsilon subunit,
isoform 1, 2 or 3
Predicted: similar to T-complex protein-1, eta subunit
Predicted: similar to testis expressed sequence 101
Predicted: similar to TIP120 protein, isoform 1, 2 or 3
Predicted: similar to tubulin alpha-1
Predicted: similar to vascular non-inflammatory
molecule 2, isoform 1
Predicted: similar to WAP four-disulfide core domain
protein 2 (Epididymal secretory protein E4)
Predicted: similar to WAP four-disulfide core domain
protein 8
Proactivator polypeptide
Prostaglandin-H2 D-isomerase
Ribonuclease 4
Seminal plasma protein A3 (BSP-A3)
Seminal plasma protein BSP-30 kDa (BSP-30 kDa)
Seminal plasma protein PDC-109 (BSP-PDC109)
Seminal ribonuclease
Seminal secretoglobin
Serine or cysteine proteinase inhibitor clade A member 5
(Alpha-1-antitrypsin)
Serine protease inhibitor clade E member 2 (serpin E2)
Serotransferrin
Serum albumin
Similar to GB|AAA51828.1 (human) N-acetyl-betaglucosaminidase
Similar to UP|DPP2_MOUSE dipeptidyl-peptidase II

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

76650931
8 (14)
76657182
7 (12)
76652313
9 (24)
76675546
3 (4)
76658274
4 (8)
76641927, 76641929, 14 (35)
76641931
76635537, 76635535, 4 (12)
76635543, 76635545,
76635547, 76635549
76650497
11 (30)

[26]
H [63]
[14]

H [70]
46 (44)
H [71]
55 (55)

46 (52)

61861624
76624606

7 (21)
3 (6)

61872568
76623551

6 (31)
4 (17)

76608261
76626355
76646678, 76646674,
76646676
76628903
76687188
76661744, 76661742,
76661740
76610661
76626079

9 (16)
7 (10)
4 (10)

8 (19)

16 (52)
70 (58)

76687485

4 (221)

20 (13)

76644489

3 (11)

P26779
O02853
P15467
P04557
P81019
P02784
P00669
hmm49876
28603766

13 (27)
2 (11)
5 (21)i)
6 (80)
9 (139)
6 (246)
5 (44)
5 (18)
18 (57)

46405157
Q29443
P02769
TC290778

13 (51)
20 (46)
31 (121)
9 (20)

TC260357

4 (12)

H [72]

28 (29)

6 (10)
3 (5)
4 (5)

H [63]
[7]

27 (21)
15 (14)
16 (16)
30 (21)
15 (15)
17 (16)

[73]
[74]
[75]
[15]

5070, 85, 98 (45)

[34]

47 (44)
67 (78)
65 (69)
61 (64)

H [58]
[28]
[34]
[30]
H [76]

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Van C. Kelly et al.

Proteomics 2006, 6, 58265833

Table 1. Continued

Protein namea)

Accession no.b)

2-D LCc)

SDS-PAGEd)

Previously
describede)

Similar to UPIO00391_HUMAN quiescin Q6

(sulfhydryl oxidase)
Spermadhesin 2
Superoxide dismutase [Cu-Zn]
T-complex protein 1, gamma subunit
Tissue alpha-L-fucosidase
Tissue factor pathway inhibitor 2
Tubulin, beta-2

TC262457

14 (33)

63 (83)

[29]

67763575
P00442
62988282
hmm60426
73586850
75773583

8 (152)
3 (8)
4 (5)
2 (6)
12 (70)
4 (9)

16 (15)

[19, 77]
[13]

61 (36)
37 (27)
50 (50)

[32]
H [59]

a) Protein names are given according to the nomenclature of the database from which the sequence was sourced, except for those in italics that have been given by the authors. Multiple protein isoform names are given where it was not possible to distinguish a single
protein matching the MS/MS data.
b) Numeric-only accession numbers are from NCBInr, accession numbers beginning with TC are from the B. taurus Gene Index, accession numbers beginning with hmm are ab initio Gnomon protein predictions from B. taurus genome sequence and the others are
from Swiss-Prot. Multiple accession numbers are given for entries with multiple protein names.
c) Proteins that were identified by 2D-LC/MS/MS (2-D LC) are indicated by entries in this column. The number of non-redundant tryptic or
semi-tryptic peptides matching the identified protein is listed. The number in parentheses is the total number of MS/MS spectra corresponding to the identified protein.
d) Proteins that were identified by SDS-PAGE followed by RP-LC/MS/MS are indicated by entries in this column. The number, numbers or
number range for each entry is the apparent size(s) (Mr61023) of the identified protein. The number in parentheses is the calculated
molecular mass (kDa) of the unmodified protein precursor. Underlined numbers are calculated molecular masses for the corresponding
human proteins for entries with partial bovine sequences.
e) References are provided for proteins previously reported in bovine seminal plasma (BSP), that have been identified using proteomic
methods, immunochemical detection or activity assays, and are listed in the text. References for proteins that have not been reported in
BSP, but in seminal plasma from other species are annotated: H = human, S = sheep.
f) Two peptides matching this protein encompassed sequence conflicts in Swiss-Prot: residue 118 = F; residue 161 = K.
g) Acid and neutral alpha-mannosidase have been described in BSP, ref [32].
h) This sequence is 84% identical to the N-terminal sequence of a deoxyribonuclease I-like protein reported in BSP, ref [11].
i) Two peptides matching this protein encompassed sequence conflicts in Swiss-Prot: residue 112 = E; residue 119 = K.

The validation criteria used for protein identification

were tested by searching the MS data against reversedsequence databases using MASCOT. None of the reversed
sequences yielded a valid protein match, although there
was one protein match with a single valid peptide scoring
55. The validation criteria, which include the requirement
for at least two non-redundant peptides per protein, appear
sufficiently stringent to yield only true-positive protein
matches.
Information on protein molecular size provided by
the SDS-PAGE-based proteomic approach (Table 1) indicated that most of the proteins identified corresponded to
the size of the intact protein, allowing for known or
predicted post-translational processing. However, some of
the proteins appeared to be significantly larger or smaller
than their expected sizes. Predicted: similar to ectonucleotide pyrophosphatase/phosphodiesterase 3 was present as two distinct bands; presumably, the lower band is
a result of limited proteolysis. Predicted: hypothetical
protein XP_581816 appeared to be significantly larger
than the calculated size, even allowing for glycosylation
at the six predicted N-linked glycosylation sites, suggest 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

ing that either the sequence prediction is incorrect or

that this protein runs anomalously in SDS-PAGE. Alpha1-antitrypsin (serine or cysteine proteinase inhibitor clade
E member 2) was detected in multiple regions of the gel
corresponding to the calculated mass of 45 kDa, as well
as higher apparent masses, suggesting the presence of
SDS-resistant aggregates containing this protein. Predicted: similar to epididymal-specific lipocalin 8 appeared
to be significantly smaller than the calculated size, suggesting that either a proteolytic fragment of this protein
has been observed or the sequence prediction is incorrect.

Discussion

Ninety-nine proteins were identified in BSP using a combination of 2-D LC and gel-based proteomic methods. Sixtyfour of these proteins are minor components of BSP that
have not previously been reported in this fluid, although 15
of these have been reported in seminal plasma from other
species.
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Proteomics 2006, 6, 58265833

Forty-six of the proteins identified in this study matched

hypothetical protein sequences predicted from the B. taurus
genome. Many of these predicted sequences are supported
by mRNA or EST sequences as indicated by NCBInr
sequence annotations. The protein analysis reported here
provides further support that these predicted sequences
encode authentic proteins. The apparent and calculated sizes
of the hypothetical proteins detected by gel-based proteomics
were generally in good agreement, suggesting that most of
the sequence predictions are accurate.
Protein sequences predicted from the bovine genome are
rapidly making protein sequence databases comprehensive
sources of bovine protein sequence data. During the course
of this study, 14 proteins that we initially identified using
EST-derived sequences in BtGI were replaced by updated or
new entries corresponding to predicted proteins in NCBInr.
None of the protein identifications relied on matches to
orthologous proteins in non-bovine species, which is in contrast to some of our earlier bovine proteomics work (unpublished results) carried out before the availability of the bovine
genome sequence. Only a small number of the proteins we
identified relied on sequences in BtGI or to predicted
sequences not represented in NCBInr.
Most of the proteins identified in earlier proteomic
analyses of BSP [33, 34] were unambiguously identified in
this study, with the exception of 6 proteins. Mortarino et al.
[34] identified 12 distinct proteins by Edman sequencing
and Assumpco et al. [33] identified 11 distinct proteins by
MALDI-TOF MS, 9 and 7 of which, respectively, clearly correspond to proteins identified in this study. Spermadhesin
Z13 was reported in both earlier proteomic analyses but is
not reported here since peptides matching this protein were
a subset of those matching spermadhesin 2. Creatine
kinase, semenogelin II and spermidine synthase, identified
as matches to non-bovine sequences in ref. [33] were not
identified in this study. The reason for this discrepancy is
not clear, although it could be due to the different analytical
techniques used in the two studies or it may reflect differences in the B. taurus taurus (this study) and B. taurus indicus (Assumpco et al. [33]) cattle investigated. The protein
annotated as unknown but with similarity to human LERK1 (ephrin A1) [34] was not identified in this study as a valid
protein match, but we did observe a single valid peptide
matching predicted: similar to ephrin A1 isoform a
(giu76612106) in both the 2-D LC and gel-derived datasets.
The protein annotated as unknown but with similarity to rat
epididymal retinoic-acid-binding protein [34] appears very
similar to predicted: similar to epididymal-specific lipocalin
8 identified in this study. The N-terminal sequence of spot
11 reported by Mortarino et al. [34] has 22 of 26 residues
identical to predicted: similar to epididymal-specific lipocalin 8 and both of these lipocalins were observed as approximately 20-kDa proteins.
Some of the proteins not previously reported in BSP have
been described in epididymal fluid, which contributes to
seminal plasma, in sperm or in small membranous vesicles
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Animal Proteomics

5831

present in seminal plasma. Epididymal fluid from various

mammals is known to contain many of the proteins that we
have identified in BSP, including previously known seminal
plasma proteins (alpha-1-antitrypsin, clusterin, lactotransferrin, glutathione peroxidase, predicted: similar to
alpha-mannosidase 2C1, predicted: similar to epididymisspecific alpha-mannosidase, prostaglandin D2 synthase and
superoxide dismutase) as well as some of the newly identified seminal plasma proteins (cathepsin L, predicted: similar
to cysteine-rich secretory protein-1, predicted: similar to epididymal sperm binding protein 1, and predicted: similar to
WAP four-disulfide core domain protein 2) [3743]. Detection of epididymis-derived proteins in seminal fluid is interesting because observation of these proteins in epididymal
fluid is not necessarily indicative of their presence in seminal
plasma. As well as being a site for protein secretion, the epididymis is a site of protein reabsorption and the composition
of epididymal fluid changes as sperm mature during transit
through this tissue ([42] and see refs [41, 44] for reviews). The
reabsorption is specific and some proteins secreted in the
proximal epididymis are not detected in more distal epididymal fluid, for example the proteins annotated as train A and
train F/G in the boar epididymis [37]. The presence of epididymis-derived proteins in seminal plasma suggests roles for
these proteins in addition to their possible roles in sperm
maturation in the epididymis.
Newly identified proteins in BSP that have previously
been reported in bovine sperm or in other mammalian
sperm include actin, adenylate kinase, gelsolin, lactate dehydrogenase, phosphoglycerate mutase, tubulin, and heat
shock 70- and 90-kDa proteins [14, 4550]. Damaged sperm
could be the source of these proteins in the seminal plasma
sample. However, lactate dehydrogenase and glucose phosphate isomerase are BSP proteins that are also present in
sperm and these enzymes have been found to be released
from sperm in the absence of any apparent cellular damage
[14].
Small membranous vesicles known as prostasomes or
exosome-like vesicles are present in seminal plasma and
could be the source of some of the proteins identified in this
fluid. Such vesicles would have been present in seminal
plasma in this study and in the earlier proteomic studies of
BSP [33, 34] as the samples were not ultracentrifuged. Some
of the potential sperm-derived proteins identified in this
study (actin, tubulin and heat shock 70- and 90-kDa proteins)
have also been reported in human prostasomes [51]. Additional proteins newly identified in BSP in this study (143-3
protein zeta/delta, predicted: similar to phosphoglycerate
kinase, predicted: similar to phosphatidylethanolaminebinding protein and dipeptidyl peptidase IV) have been
reported in human prostasomes [51] and/or in exosome-like
vesicles of ram epididymal fluid [52]. Epididymal fluid
appears to be the major source of vesicles in ram, bovine and
human seminal plasma [52]. The quantitative significance of
vesicle-derived proteins in BSP is unclear; in the ram, they
represent a small fraction of epididymal fluid protein [52].
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5832

Van C. Kelly et al.

Considerable effort has been spent identifying predictors of bull fertility based on the relative abundance of
fertility-associated proteins. Thirteen previously known BSP
proteins (acidic seminal fluid protein, BSP-A3, BSPPDC109, BSP-30 kDa, clusterin, deoxyribonuclease gamma,
glutathione peroxidase, metalloproteinase inhibitor 2,
osteopontin, platelet-activating factor acetylhydrolase, prostaglandin-H2 D-isomerase, spermadhesin Z13 and superoxide dismutase) have been positively or negatively correlated with high fertility in studies of bovine bulls [5, 7, 10,
11, 24, 25, 5355].
Some of the newly identified BSP proteins, which are
minor components, could also be associated with bovine
bull fertility based on studies of orthologous proteins in
other mammals or similarity with other BSP proteins.
The BSP fertility-associated protein prostaglandin-H2 Disomerase is a lipocalin family member and the activity of
this protein in the male reproductive system, thought to
be in sperm maturation and/or storage, appears to reside
in its ability to bind lipophilic molecules rather than in its
enzyme activity [56]. By similarity, as has been suggested
for the lipocalin identified in BSP by Mortarino et al. [34],
a predicted protein similar to epididymal-specific lipocalin
8 could also traffic lipophilic molecules important for
sperm maturation and/or storage. The levels of testis-specific phosphoglycerate kinase, adenylate kinase, and serpin
E2 have been correlated with male fertility in studies of
human, porcine or mouse semen [5760]. The level of
tissue factor pathway inhibitor 2 in human seminal
plasma has been associated with sperm motility [59].
Finally, proactivator polypeptide may play a role in fertility. Synthetic peptides derived from rat and rooster
proactivator polypeptide have been shown to increase fertility when added to thawed bull sperm [61] and to thawed
or fresh rooster sperm [62]. It is not clear whether the
molecular form of proactivator polypeptide in BSP is appropriate for this activity, as we identified proactivator
polypeptide only using the 2-D LC method which does
not provide information on protein size. Proactivator
polypeptide has been identified in human seminal plasma
as the intact protein [63].
It is clear that proteins associated with fertility include
major and minor BSP components. Fertility studies in nonbovine animals suggest that low abundance proteins newly
identified in this study could include useful markers of male
fertility. A recent study of fertility-associated proteins in
bovine accessory sex gland fluid [55] has elaborated the value
of multivariate analysis of major and minor proteins in fertility prediction. In that study, Moura et al. [55] visualized
proteins by CBB staining after separations using 2-D gels,
which may not have revealed some of the low abundance
proteins detected in this study. Potentially, techniques with
more sensitive protein detection, such as 2-D gels with fluorescent protein stains or LC/MS methods, will broaden the
number of markers included in the assay and grant more
robust predictions of male fertility.
2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Proteomics 2006, 6, 58265833

References

[1] De Jonge, C., Hum. Reprod. Update 2005, 11, 205214.

[2] Desnoyers, L., Therien, I., Manjunath, P., Mol. Reprod. Dev.
1994, 37, 425435.
[3] Manjunath, P., Sairam, M. R., Biochem. J. 1987, 241, 685
692.
[4] Manjunath, P., Baillargeon, L., Marcel, Y. L., Seidah, N. G. et
al., in Chretien, M., McKerns, K. W. (Eds.), Molecular biology
of brain and endocrine peptidergic systems, Plenum Press,
New York 1988, pp. 259273.
[5] Nauc, V., Manjunath, P., Biol. Reprod. 2000, 63, 10581066.
[6] Manjunath, P., Therien, I., J. Reprod. Immunol. 2002, 53,
109119.
[7] Gerena, R. L., Irikura, D., Urade, Y., Eguchi, N. et al., Biol.
Reprod. 1998, 58, 826833.
[8] Jobim, M. I., Oberst, E. R., Salbego, C. G., Souza, D. O. et al.,
Theriogenology 2004, 61, 255266.
[9] Hosang, K., Scheit, K. H., DNA Cell Biol. 1994, 13, 409417.
[10] McCauley, T. C., Zhang, H. M., Bellin, M. E., Ax, R. L., Mol.
Reprod. Dev. 2001, 58, 336341.
[11] McCauley, T. C., Zhang, H., Bellin, M. E., Ax, R. L., Mol.
Reprod. Dev. 1999, 54, 145153.
[12] Meloun, B., Cechova, D., Jonakova, V., Hoppe Seylers Z.
Physiol. Chem. 1983, 364, 16651670.
[13] Mennella, M. R., Jones, R., Biochem. J. 1980, 191, 289297.
[14] Osinowo, O. A., J. Reprod. Fertil. 1981, 62, 549554.
[15] Suzuki, H., Parente, A., Farina, B., Greco, L. et al., Biol. Chem.
Hoppe Seyler 1987, 368, 13051312.
[16] Steele, J. G., Dalton, B. A., Hoffman, H., Underwood, P. A. et
al., Mol. Immunol. 1985, 22, 10611072.
[17] Sharma, M., Singh, U. S., J. Biochem. (Tokyo) 1988, 104, 57
61.
[18] Sharma, M., Singh, U. S., Andrologia 1991, 23, 315319.
[19] Tedeschi, G., Oungre, E., Mortarino, M., Negri, A. et al., Eur.
J. Biochem. 2000, 267, 61756179.
[20] Veselsky, L., Arch. Androl. 1981, 7, 17.
[21] Wempe, F., Henschen, A., Scheit, K. H., DNA Cell Biol. 1991,
10, 671679.
[22] Einspanier, R., Einspanier, A., Wempe, F., Scheit, K. H., Biochem. Biophys. Res. Commun. 1991, 179, 10061010.
[23] Fini, C., Talamo, F., Cherri, S., Coli, M., et al. Biochem. J.
2003, 372, 443451.
[24] Bilodeau, J. F., Chatterjee, S., Sirard, M. A., Gagnon, C., Mol.
Reprod. Dev. 2000, 55, 282288.
[25] Cancel, A. M., Chapman, D. A., Killian, G. J., Biol. Reprod.
1997, 57, 12931301.
[26] Dhanotiya, R. S., Arch. Exp. Veterinarmed. 1985, 39, 869
873.
[27] Hough, S. R., Parks, J. E., J. Androl. 1997, 18, 540548.
[28] Gilmont, R. R., Senger, P. L., Sylvester, S. R., Griswold, M. D.,
Biol. Reprod. 1990, 43, 151157.
[29] Hoober, K. L., Glynn, N. M., Burnside, J., Coppock, D. L. et al.,
J. Biol. Chem. 1999, 274, 3175931762.
[30] Khar, A., Anand, S. R., Biochim. Biophys. Acta 1977, 483,
152159.

www.proteomics-journal.com

Proteomics 2006, 6, 58265833

Animal Proteomics

5833

[31] Jauhiainen, A., VanhaPerttula, T., Int. J. Biochem. 1986, 18,

719724.

[54] Roncoletta, M., Morani Eda, S., Esper, C. R., Barnabe, V. H. et

al., Anim. Reprod. Sci. 2006, 91, 7787.

[32] Jauhiainen, A., Vanha-Perttula, T., Int. J. Androl. 1987, 10,

489494.

[55] Moura, A. A., Koc, H., Chapman, D. A., Killian, G. J., J.

Androl. 2006, 27, 201211.

[33] Assumpcao, T. I., Fontes, W., Sousa, M. V., Ricart, C. A., Protein Pept. Lett. 2005, 12, 813817.

[56] Fouchecourt, S., Charpigny, G., Reinaud, P., Dumont, P. et al.,

Biol. Reprod. 2002, 66, 458467.

[34] Mortarino, M., Tedeschi, G., Negri, A., Ceciliani, F. et al.,

Electrophoresis 1998, 19, 797801.

[57] Dolcetta, G., Busatta, E., Sommacampagna, P., Dusi, M. et

al., Andrologia 1986, 18, 184189.

[35] Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K. et al.,

Anal. Biochem. 1985, 150, 7685.

[58] Murer, V., Spetz, J. F., Hengst, U., Altrogge, L. M. et al., Proc.
Natl. Acad. Sci. USA 2001, 98, 30293033.

[36] Palmer, D. J., Kelly, V. C., Smit, A.-M., Kuy, S. et al., Proteomics 2006, 6, 22082216.

[59] Lee, J. N., Lian, J. D., Lee, J. H., Chard, T., Fertil Steril 1983,
39, 704706.

[37] Syntin, P., Dacheux, F., Druart, X., Gatti, J. L. et al., Biol.
Reprod. 1996, 55, 956974.

[60] Chen, K., Knorr, C., Moser, G., Gatphayak, K. et al., Mamm.
Genome 2004, 15, 9961006.

[38] Roberts, K. P., Ensrud, K. M., Wooters, J. L., Nolan, M. A. et

al., Mol. Cell. Endocrinol. 2006, 250, 122127.
[39] Okamura, N., Tamba, M., Liao, H. J., Onoe, S. et al., Mol.
Reprod. Dev. 1995, 42, 141148.
[40] Xu, W. D., Miao, S. Y., Zhang, M. L., Wang, L. F. et al., Arch.
Androl. 1997, 38, 16.
[41] Dacheux, J. L., Gatti, J. L., Dacheux, F., Microsc. Res. Tech.
2003, 61, 717.
[42] Dacheux, J. L., Belghazi, M., Lanson, Y., Dacheux, F., Mol.
Cell. Endocrinol. 2006, 250, 3642.
[43] Saalmann, A., Munz, S., Ellerbrock, K., Ivell, R. et al., Mol.
Reprod. Dev. 2001, 58, 88100.
[44] Gatti, J. L., Castella, S., Dacheux, F., Ecroyd, H. et al., Anim.
Reprod. Sci. 2004, 8283, 321339.
[45] Kamaruddin, M., Kroetsch, T., Basrur, P. K., Hansen, P. J. et
al., Andrologia 2004, 36, 327334.
[46] Cao, W. L., Wang, Y. X., Xiang, Z. Q., Li, Z., Asian J. Androl.
2003, 5, 4346.

[61] Amann, R. P., Seidel, G. E., Jr., Brink, Z. A., J. Androl. 1999,
20, 4246.
[62] Hammerstedt, R. H., Cramer, P. G., Barbato, G. F., Amann, R.
P. et al., J. Androl. 2001, 22, 361375.
[63] Fung, K. Y. C., Glode, L. M., Green, S., Duncan, M. W., Prostate 2004, 61, 171181.
[64] Ben Ayed, S., Gonzales, J., Foglietti, M. J., Percheron, F. et
al., Andrologia 1989, 21, 432436.
[65] Sedor, J., Callahan, H. J., Perussia, B., Lattime, E. C, et al., J.
Androl. 1993, 14, 187193.
[66] Akiyama, K., Yoshioka, Y., Schmid, K., Offner, G. D. et al.,
Biochim. Biophys. Acta 1985, 829, 288294.
[67] Ecroyd, H., Belghazi, M., Dacheux, J. L., Miyazaki, M. et al.,
Biol. Reprod. 2006, 74, 439447.
[68] Rahimi, A., Sepehri, H., Pakravesh, J., Bahar, K., Am. J.
Reprod. Immunol. 1999, 41, 330336.
[69] Haugen, H. F., Skrede, S., Clin. Chem. 1977, 23, 15311537.
[70] Li, H., Chen, Q., Li, S., Yao, W. et al., Ann. Occup. Hyg. 2001,
45, 505511.

[47] de las Heras, M. A., Valcarcel, A., Perez, L. J., Moses, D. F.,
Tissue Cell 1997, 29, 4753.

[71] Baumgart, E., Lenk, S. V., Loening, S. A., Jung, K., Hum.
Reprod. 2002, 17, 29192923.

[48] Naaby-Hansen, S., Flickinger, C. J., Herr, J. C., Biol. Reprod.

1997, 56, 771787.

[72] Schirren, C., Laudahn, G., Heinze, I., Hartmann, E. et al.,

Andrologia 1979, 11, 393401.

[49] Minelli, A., Moroni, M., Castellini, C., Lattaioli, P. et al., J.

Androl. 1999, 20, 259266.

[73] Seidah, N. G., Manjunath, P., Rochemont, J., Sairam, M. R. et

al., Biochem. J. 1987, 243, 195203.

[50] Fundele, R., Winking, H., Illmensee, K., Jagerbauer, E. M.,

Dev Biol 1987, 124, 562566.

[74] Calvete, J. J., Mann, K., Sanz, L., Raida, M. et al., FEBS Lett.
1996, 399, 147152.

[51] Utleg, A. G., Yi, E. C., Xie, T., Shannon, P. et al., Prostate 2003,
56, 150161.

[75] Esch, F. S., Ling, N. C., Bohlen, P., Ying, S. Y. et al., Biochem.
Biophys. Res. Commun. 1983, 113, 861867.

[52] Gatti, J. L., Metayer, S., Belghazi, M., Dacheux, F. et al., Biol.
Reprod. 2005, 72, 14521465.

[76] Maes, M. B., Lambeir, A. M., Gilany, K., Senten, K. et al.,

Biochem. J. 2005, 386, 315324.

[53] Ibrahim, N. M., Gilbert, G. R., Loseth, K. J., Crabo, B. G., J.

Androl. 2000, 21, 887894.

[77] Haase, B., Schlotterer, C., Hundrieser, M. E., Kuiper, H. et al.,

Gene 2005, 352, 2029.

2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

www.proteomics-journal.com