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wileyonlinelibrary.com/journal/rda Reprod Dom Anim. 2018;53:588–608.
© 2018 Blackwell Verlag GmbH
1 | INTRODUCTION
Being transcriptionally inactive, proteomes are one of the promising
comprehensive tools for better understanding the molecular functions
of spermatozoa (Kwon et al., 2015; Park, Kwon, Oh, & Pang, 2012;
Pixton et al., 2004). Conventional methods for analysing semen can
only concern on different quality traits, viz. motility, morphological
characteristics, volume and concentration; however, no information
regarding the functional competence of the spermatozoa can be gen-
erated from these methodologies (Johnson, Bassham, Lipshults, &
Lamb, 1990; Petrunkina, Waberski, Günzel- Apel, & Töpfer- Petersen,
2007). Thus, these traditional methodologies may be considered as
first- line tools in the diagnosis of male fertility (Kwon, Rahman, &
Pang, 2014; Kwon, Rahman, Lee, et al., 2014).
Proteomics is one of the emerging researches in the field of post-
genomic era, which can be defined as the qualitative and quantitative
comparison of proteomes to identify the cellular mechanisms that are
involved in biological processes (Aebersold & Mann, 2003; Brewis,
1999; Tyers & Mann, 2003). Due to the cardinal role proteins for cellu-
lar function, it is therefore vital to perform the comprehensive as well
systematic identification and quantification of proteins expressed in
cells or tissues to gain new insights (Brewis, 1999). Proteomic studies
have been performed to identify various biomarkers associated with
fertility (Kwon, Rahman, & Pang, 2014; Kwon, Rahman, Lee, et al.,
2014; Kwon et al., 2015; Oliva, de Mateo, & Estanyol, 2009; Park
et al., 2012) and to understand why spermatozoa have varying levels
of fertility (Kwon, Rahman, & Pang, 2014; Kwon, Rahman, Lee, et al.,
2014; Kwon et al., 2015).
Bull spermatozoa proteomic studies identified functional at-
tributes of different sperm proteins. For instance, prostaglandin-
d- synthetase and osteopontin were reported to be the two more
abundant proteins present in seminal plasma of high fertile bulls
compared to low fertile one (Gerena et al., 1998; Henault, Killian,
Kavanaugh, & Griel, 1995). A heparin binding protein from seminal
vesicles and prostate glands so- called fertile associated antigen inter-
acts to spermatozoa membrane and thus modulates heparin–sperm
Received: 16 November 2017
|
Accepted: 10 January 2018
DOI: 10.1111/rda.13146
ORIGINAL ARTICLE
Functional proteomic analysis of crossbred (Holstein
Friesian × Sahiwal) bull spermatozoa
R Singh1 | GS Sengar1 | U Singh1 | R Deb1 | V Junghare2 | S Hazra2,3 |
S Kumar1 | S Tyagi1 | AK Das1 | TV Raja1 | A Kumar1
1Molecular Genetics Laboratory, ICAR-Central
Institute for Research on Cattle, Meerut, Uttar
Pradesh, India
2Department of Biotechnology, Indian
Institute of Technology, Roorkee, Uttarakhand,
India
3Center of Nanotechnology, Indian Institute of
Technology, Roorkee, Uttarakhand, India
Correspondence
Rajib Deb, Molecular Genetics Laboratory,
ICAR-Central Institute for Research on Cattle,
Meerut, Uttar Pradesh, India.
Email: drrajibdeb@gmail.com
Funding information
Science and Engineering Research Board,
Government of India, Grant/Award Number:
YSS/2015/001482
Contents
Male infertility is one of the prime concerns of dairy cattle production. The study was
designed to find out differentially expressed proteins in categorized crossbred
(Holstein Friesian × Sahiwal) bull semen to serve as potential biomarkers for male in-
fertility. Frozen crossbred bull semen with satisfactory phenotypic records were de-
fined as “good” and “poor” based on their fertility rates. A total of 1,547 proteins were
detected in bull spermatozoa using liquid chromatography–mass spectrometer (LC-
MS/MS) analysis. Results revealed that 558 (36.1%) and 653 (42.2%) proteins were
expressed to good and poor quality bull spermatozoa, respectively. A total of 336
proteins (21.7%) were reported to be unique for both good and poor quality bull
semen, and among the common proteins, 224 (66.7%) and 112 (33.3%) were up- and
downregulated in good and poor quality categorized bull semen, respectively. Gene
Ontology analysis of global proteomes identified different signalling pathways, and
most of them were related to cellular motility, immune systems as well as cellular me-
tabolisms. The distinctive presence of some of the proteins may provide an insight into
the molecular mechanistic role played by these proteins in crossbred bull infertility.
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SINGH et al.
interactions (McCauley, Zhang, Bellin, & Ax, 1999). Moura, Koc,
Chapman, and Killian (2006) described that accessory gland proteins
are essential for modulating sperm functions after their ejaculation
in terms of its capacitation, acrosome reaction, sperm–oocyte inter-
action as well as sperm protection.
Development of various high- throughput proteomic strategies has
made it possible to analyse the complex mixtures of different protein
in particular tissues or biological fluids (Fung, Glode, Green, & Duncan,
2004; Milardi et al., 2012) . Peddinti et al. (2008) identified differences
in the signalling pathways among high and low fertility bull sperma-
tozoa through comprehensive proteomics analysis and reported that
EGF and PDGF signalling pathways were specific to high fertility
groups.
In this study, proteins were compared between bulls with differ-
ent fertility rates and lists of peptides were catalogued through pro-
teomics analysis to identify differentially expressed proteins related
to crossbred bull fertility. In addition, to understand the molecular
attributes of the proteins detected by proteomics studies, we identi-
fied the signalling as well as metabolic pathways that the differentially
expressed proteins partake in.
2 | MATERIALS AND METHODS
All experimental procedures were approved by the Institutional
Animal Ethics Committee.
2.1 | Categorizing of crossbred bull semen
Ten frozen semen samples of crossbred bulls with superior pheno-
types like satisfactory semen volume, spermatozoal concentration,
initial progressive motility, post- thaw motility and TUNEL index were
chosen for the present study.
A total of 20 mature crossbred (Holstein Frisian × Sahiwal) bulls
maintained at Institutional Bull Rearing Unit were included in the
present experiments. Semen samples were collected from each bull
by artificial vaginal technique. Ejaculates were processed for semen
quality assessment. Instantly after collection, ejaculates were stored at
37°C in a water bath for evaluating the fresh semen quality traits, viz.
volume (ml), initial progressive motility (%) and sperm concentration
(×106/ml).
Initial progressive motility was scored at 200× magnification
with phase- contrast microscope equipped with a warm stage. Initial
progressive motility (%) was observed at four to five areas of the
slide before the recording of average values. The sperm concentra-
tion was measured with Accucell photometer (IMV Technologies,
France). The fresh semen samples were diluted with glycerol- egg
yolk- citrate- Tris extender and cryopreserved as per the standard
protocol at liquid nitrogen. After 24 hr of cryopreservation at liquid
nitrogen, the frozen samples were thawed at 37°C for 60 s and im-
mediately evaluated for post- thaw motility (%) using phase- contrast
microscope.
The amount of DNA fragmentation in frozen–thawed bull semen
samples were determined by calculating the TUNEL index percent-
age using TUNEL assay detection kit (Roche, USA) as per the man-
ufacturer’s protocol. In brief, the frozen–thawed semen samples
were washed twice in Dulbecco’s phosphate- buffered saline (DPBS)
without calcium chloride as well as magnesium chloride (Invitrogen)
followed by dilution of 10 × 106 spermatozoa/ml in DPBS for all the
samples. Sperm smears were prepared on clean glass slides, air- dried
and fixed with 2% (w/v) paraformaldehyde in DPBS for 50 min. After
washing the slides for three times with DPBS, they were permeabi-
lized with 0.1% (v/v) Triton X containing 0.1% (w/v) sodium citrate
for 2 min on ice. Fixed slides were then incubated in 50 μl of TUNEL
reaction mixture for 1 hr at 37°C in a dark humidified area. Positive
and negative controls were prepared as per the earlier described
methodologies (Takeda et al., 2015). Slides were further subjected
for washing with DPBS for three times and mounted using mounting
solutions. Positive TUNEL staining was observed under a fluorescence
microscope (Nikon ECLIPSE 80i), and the sperm TUNEL index was de-
termined by counting the positive and negative stained spermatozoa
in each of the 10 fields of vision.
Fertility phenotypes of the superior quality bull semen samples
(n = 10) have been characterized in the established field progeny test-
ing programme. According to the field testing records, three extremely
“good” with fertility rate >55% and three extremely “poor” with fertil-
ity rate <40% were subjected for further proteomics studies.
2.2 | Isolation of spermatozoa from semen samples
Total spermatozoa collected from categorized bulls were purified
through Percoll gradient centrifugation as described by Peddinti et al.
Particular Satisfactory Unsatisfactory
Semen volume (ml)** 5.56 ± 0.55 3.84 ± 0.36
Sperm concentration (million/ml) 1,087.80 ± 202.02 698.00 ± 138.53
Number of spermatozoa/Ejaculate
(million)**
6,210.40 ± 1,322.57 2,617.80 ± 577.72
Initial progressive motility (%)** 74.00 ± 2.45 52.00 ± 11.14
Post- thaw motility (%)** 54 ± 2.45 22 ± 2.00
TUNEL index (%)** 2.66 ± 0.22 5.83 ± 0.38
**p < .05.
TABLE1 Assessment of semen quality
parameters (mean ± SE)
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SINGH et al.
(2008). Percoll solution of 90% in water was developed with admixing
of 19 μM DL- Lactate, 2 μM CaCl2, 25 mM NaHCO3, 400 μM MgCl2,
3 μM KCl, 310 μM NaH2PO4, 2 mM NaCl and 10 mM HEPES. The 90%
of Percoll solution was then diluted to 45% with sperm diluent medium
containing 1 mM pyruvate, 10 mM HEPES and 0.021 mM DL- lactate in
Tyrode’s salt solution (pH 7.4). Spermatozoa were then thawed at 37°C
for 5 min followed by layered on top of the prepared Percoll gradi-
ent. Spermatozoa were subjected for centrifugation at 956 g for 5 min,
and pellets were then washed twice with phosphate- buffered solution.
After counting the total sperm using Neubauer Hemocytometer, 108
sperm cells were aliquoted and stored at −80°C till further use.
2.3 | Extraction of proteins
Extraction of total proteins was carried out from the stored sperm sam-
ples with different extraction buffers (0.1% non- idet P- 40; RIPA; urea/thi-
ourea/CHAPS and NH4HCO3) and subjected for SDS- PAGE to check the
protein profiles, and the best protein profile was observed with 50 mM
NH4HCO3 with 1% SDS. One hundred microlitres of 50 mM NH4HCO3
was added to 100 μL of samples and incubated at room temperature for
30 min. The samples were then sonicated for 10 min followed by cen-
trifugation at 600 rpm at 4°C temperature for 10 min. Further, SDS was
added to these samples from 0.1% to a final concentration of 1%. At
FIGURE1 TUNEL- positive spermatozoa obtained from crossbred bulls visualized by fluorescence microscopy. The upper panels show FITC-
stained images (a–d), and the lower panels show phase- contrast images (a′–d′). a/a′: positive control; b/b′: negative control; c/c′: satisfactory
quality bull semen; and d/d′: unsatisfactory bull semen
(a) (b) (c) (d)
(a′)(b′)(c′)(d′)
FIGURE2 Representative LC- MS- IT-
TOF total ion chromatogram (TIC) of the
identified proteins. x- axis indicates relative
intensity, while y- axis indicates mass
number/charge number
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SINGH et al.
each step, the samples were incubated at room temperature for 10 min,
followed by sonication and centrifugation. Supernatants were then col-
lected for further SDS- PAGE based quality checking. Triplicate samples
of each group (n = 3/group) were pooled and subjected for proteomic
studies through LC- MS- IT- TOF total ion chromatography.
2.4 | Proteomic data generation through LC- MS- IT-
TOF total ion chromatography
Samples were dissolved in 98% acetonitrile containing 0.1% trifluoroacetic
acid and lyophilized at −80°C under vacuum for 2 days as described by
Sharma, Agarwal, et al. (2013) and Sharma, Masaki, and Agarwal (2013).
Lyophilized samples were subjected to estimate the protein content using
standard bicinchoninic acid method. After precipitated in cold acetone, sam-
ples were centrifuged at 10,000 g for 15 min followed by poured off the
acetone and the protein pellets were allowed to dry at room temperature.
Further, protein pellets were solubilized with a solubilizing buffer (6 M urea,
100 mM Tris, pH 8.0). 100 μg of the each protein sample was reduced with
50 mM NH4HCO3 and treated with 100 mM DTT at 95°C for 1 hr followed
by alkylation with 250 mM iodoacetamide at room temperature in dark for
45 min. The sample was then digested with trypsin and incubated overnight
at 37°C. Digestion was stopped in the next day morning after addition of
10 μl of 0.1% formic acid in water to lower the pH to <6, followed by cen-
trifuged at 10,000 g to remove insoluble materials. After centrifugation, the
supernatant was collected into a separate tube and 1 μl injection volume
was used on C18 UPLC column for separation of peptides followed by
analysis on the Waters Synapt G2 Q- TOF instrument (Sandor Lifesciences
Pvt. Ltd.) for MS and MSMS. The raw data were processed by MassLynx
4.1 WATERS. The individual peptides MSMS spectra were matched to the
database sequence for protein identification on PLGS software, WATERS.
2.5 | Gene Ontology annotation
Gene Ontology (GO) resources and tools (http://www.reactome.
org) were used to identify the various molecular functions, biological
processes, protein classes and pathways represented in differentially
expressed proteins in the proteomics datasets.
2.6 | Signal pathway analysis
Signal pathway involved in the sperm fertility of bull semen was de-
termined using the Reactome pathway analysis tool (http://www.re-
actome.org). Pathway browser in reactome analysis displayed details
network of proteins and factors (Co- factors and ions) regulating the
sperm motility to its way to egg membrane. Under the Display browser
window, Bos taurus was selected as the target organism. The sperm
motility signalling pathway was retrieved under the reproduction sub-
division of the whole reactome map. We have identified the proteins in-
volved in particular pathways and corresponding functions related with
sperm fertility of bull semen. For this purpose, we mapped the network
of the target protein by submitting its Uniprot ID to the interactome
analysis tools implemented in Reactome web server.
3 | RESULTS
3.1 | Bull semen phenotypes
Based on different semen quality traits, we classified satisfactory
(n = 10) and unsatisfactory (n = 10) bull semen phenotypes (Table 1).
Both the fresh and frozen–thawed spermatozoa motility (mean ± SE)
were significantly (p < .05) higher in satisfactory group compared to
unsatisfactory one.
The sperm TUNEL index of cryopreserved crossbred bull
semen were analysed (Figure 1), and it was observed that TUNEL
index (%) was significantly (p < .05) higher in unsatisfactory bull
semen (5.83 ± 0.38) than the satisfactory (2.66 ± 0.22) groups
(Table 1).
Field testing of the superior quality bull semen samples (n = 10) provide
a range of fertility rates (%), and samples with the fertility rate more than
55% and lesser than 40% were subjected as good and poor, respectively.
FIGURE3 Distribution of proteins identified in good and low quality crossbred bull spermatozoa (a) and percentage of up-or down regulated
common proteins in good and poor quality samples (b)
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SINGH et al.
TABLE2 Upregulated proteins in good quality bull semen
Accession Description of the protein
PLG score
(Good)
Ratio of PLG score
(Good vs. Poor)
P35384 Extracellular calcium- sensing receptor
GN = CASR PE = 2 SV = 1
49.75 1.020201
F1MH24 AP2- associated protein kinase 1 OS = Bostaurus GN = AAK1 PE = 1 SV = 2 96.39 1.020201
A3KN19 Protein FAM83D OS = Bostaurus GN = FAM83D PE = 2 SV = 1 107.45 1.030455
Q08DK3 Kelch- like protein 20 OS = Bostaurus GN = KLHL20 PE = 2 SV = 3 92.05 1.040811
P31098 Osteopontin- K OS = Bostaurus PE = 2 SV = 1 1,229.79 1.040811
Q647I9 NACHT, LRR and PYD domains- containing protein 5 OS = Bostaurus GN = NLRP5
PE = 2 SV = 1
53.34 1.061837
P31096 Osteopontin OS = Bostaurus GN = SPP1 PE = 1 SV = 2 1,308.79 1.061837
O46382 Brefeldin A- inhibited guanine nucleotide- exchange protein 1 OS = Bostaurus
GN = ARFGEF1 PE = 1 SV = 1
69.77 1.072508
A6QLA0 Transcriptional repressor NF- X1 OS = Bostaurus GN = NFX1 PE = 2 SV = 1 59.05 1.072508
Q2T9R2 Protein KASH5 OS = Bostaurus GN = CCDC155 PE = 2 SV = 2 106.38 1.072508
P27922 Sodium- dependent dopamine transporter OS = Bostaurus GN = SLC6A3 PE = 2 SV = 1 147.67 1.083287
P80724 Brain acid soluble protein 1 OS = Bostaurus GN = BASP1 PE = 1 SV = 3 266.05 1.083287
A5PK21 Cytoskeleton- associated protein 2- like OS = Bostaurus GN = CKAP2L PE = 2 SV = 1 174.91 1.105171
Q2UVX4 Complement C3 OS = Bostaurus GN = C3 PE = 1 SV = 2 52.68 1.105171
Q95M18 Endoplasmin OS = Bostaurus GN = HSP90B1 PE = 2 SV = 1 183.8 1.116278
F1N5V1 Ubiquitin carboxyl- terminal hydrolase 37 OS = Bostaurus GN = USP37 PE = 3 SV = 1 88.54 1.150274
A4D7R9 Suppressor of tumorigenicity 7 protein OS = Bostaurus GN = ST7 PE = 2 SV = 1 80.52 1.173511
P80457 Xanthine dehydrogenase/oxidase OS = Bostaurus GN = XDH PE = 1 SV = 4 13.22 1.173511
O77797 A- kinase anchor protein 3 OS = Bostaurus GN = AKAP3 PE = 2 SV = 2 186.09 1.185305
Q32KN8 Tubulin alpha- 3 chain OS = Bostaurus GN = TUBA3 PE = 2 SV = 1 486.58 1.185305
A6QLV3 Leucine- rich repeat protein SHOC- 2 OS = Bostaurus GN = SHOC2 PE = 2 SV = 1 35.75 1.185305
Q5EA46 Cysteine- rich with EGF- like domain protein 1 OS = Bostaurus GN = CRELD1 PE = 2
SV = 1
133.88 1.185305
G7H7V7 Testis- and ovary- specific PAZ domain- containing protein 1 OS = Bostaurus
GN = TOPAZ1 PE = 2 SV = 1
105.11 1.185305
Q2HJ86 Tubulin alpha- 1D chain OS = Bostaurus GN = TUBA1D PE = 1 SV = 1 103.37 1.197217
Q2KJD0 Tubulin beta- 5 chain OS = Bostaurus GN = TUBB5 PE = 2 SV = 1 260.54 1.20925
Q3MHM5 Tubulin beta- 4B chain OS = Bostaurus GN = TUBB4B PE = 2 SV = 1 297.6 1.221403
Q28043 Activin receptor- type- 2A OS = Bostaurus GN = ACVR2A PE = 2 SV = 1 23.15 1.221403
Q9BE97 Aryl hydrocarbon receptor nuclear translocator OS = Bostaurus GN = ARNT PE = 2
SV = 1
97.73 1.221403
Q4JIJ3 Methionine synthase OS = Bostaurus GN = MTR PE = 2 SV = 1 88 1.233678
A6QNM8 Probable threonine–tRNA ligase 2, cytoplasmic OS = Bostaurus GN = TARSL2 PE = 2
SV = 1
130.17 1.246077
Q0VBZ5 Transcription factor jun- B OS = Bostaurus GN = JUNB PE = 2 SV = 1 19.66 1.246077
Q3ZBU7 Tubulin beta- 4A chain OS = Bostaurus GN = TUBB4A PE = 2 SV = 1 214.66 1.246077
P55252 Hyaluronan and proteoglycan link protein 1 OS = Bostaurus GN = HAPLN1 PE = 2
SV = 1
128.46 1.2586
A7E321 PNMA- like protein 1 OS = Bostaurus GN = PNMAL1 PE = 2 SV = 1 89.5 1.2586
Q28060 Desmocollin- 3 OS = Bostaurus GN = DSC3 PE = 2 SV = 1 74.34 1.2586
Q8HXQ5 Multidrug resistance- associated protein 1 OS = Bostaurus GN = ABCC1 PE = 2 SV = 1 37.21 1.2586
A7YY35 Uncharacterized protein KIAA2012 homolog OS = Bostaurus GN = KIAA2012 PE = 2
SV = 2
64.55 1.271249
(Continues)
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SINGH et al.
Accession Description of the protein
PLG score
(Good)
Ratio of PLG score
(Good vs. Poor)
Q9GLM3 X- linked retinitis pigmentosa GTPase regulator- interacting protein 1 OS = Bostaurus
GN = RPGRIP1 PE = 1 SV = 1
35.47 1.271249
F1MF21 PCNA- interacting partner OS = Bostaurus GN = PARPBP PE = 3 SV = 2 99.69 1.29693
A7MB10 Protein RRP5 homolog OS = Bostaurus GN = PDCD11 PE = 2 SV = 1 98.64 1.29693
Q6B856 Tubulin beta- 2B chain OS = Bostaurus GN = TUBB2B PE = 1 SV = 2 188.91 1.32313
P79331 A disintegrin and metalloproteinase with thrombospondin motifs 2 OS = Bostaurus
GN = ADAMTS2 PE = 1 SV = 1
100.15 1.363425
Q3SX14 Gelsolin OS = Bostaurus GN = GSN PE = 2 SV = 1 238.13 1.377128
P01001 Serine protease inhibitor Kazal- type 6 OS = Bostaurus GN = SPINK6 PE = 1 SV = 3 2,897.06 1.390968
P10894 1- phosphatidylinositol 4,5- bisphosphate phosphodiesterase beta- 1 OS = Bostaurus
GN = PLCB1 PE = 1 SV = 1
38.26 1.390968
E1BC15 RNA- binding protein 44 OS = Bostaurus GN = RBM44 PE = 3 SV = 1 133.37 1.390968
P79345 Epididymal secretory protein E1 OS = Bostaurus GN = NPC2 PE = 1 SV = 1 1,607.87 1.404948
Q5EA80 Geranylgeranyl transferase type- 2 subunit alpha OS = Bostaurus GN = RABGGTA PE = 2
SV = 1
197.46 1.404948
Q28017 Platelet- activating factor acetylhydrolase OS = Bostaurus GN = PLA2G7 PE = 2 SV = 1 375.73 1.433329
C7EXK4 Phospholipid- transporting ATPase IB OS = Bostaurus GN = ATP8A2 PE = 1 SV = 4 26.1 1.433329
Q17QZ4 Transcription factor Dp- 1 OS = Bostaurus GN = TFDP1 PE = 2 SV = 1 365.35 1.462285
Q9N1F0 Protein MRVI1 OS = Bostaurus GN = MRVI1 PE = 1 SV = 1 181.63 1.462285
Q0P5F2 Proteasome assembly chaperone 1 OS = Bostaurus GN = PSMG1 PE = 2 SV = 1 12.1 1.491825
Q27995 Nitric oxide synthase, inducible OS = Bostaurus GN = NOS2 PE = 2 SV = 3 36.78 1.521962
Q28204 Calcium- activated potassium channel subunit alpha- 1 OS = Bostaurus GN = KCNMA1
PE = 1 SV = 2
24.23 1.537258
Q0IIH7 Suppressor of tumorigenicity 14 protein homolog OS = Bostaurus GN = ST14 PE = 2
SV = 1
151.88 1.552707
A7MBB4 Mitogen- activated protein kinase kinase kinase 13 OS = Bostaurus GN = MAP3K13
PE = 2 SV = 1
25.28 1.584074
P17697 Clusterin OS = Bostaurus GN = CLU PE = 1 SV = 1 1,646.86 1.599994
A0JN52 Splicing factor 3B subunit 3 OS = Bostaurus GN = SF3B3 PE = 2 SV = 1 18.7 1.616074
Q29437 Primary amine oxidase, liver isozyme OS = Bostaurus PE = 1 SV = 1 12.98 1.616074
O18920 Angiopoietin- 1 OS = Bostaurus GN = ANGPT1 PE = 2 SV = 3 36.53 1.648721
O97583 Bifunctional heparan sulphate N- deacetylase/N- sulfotransferase 2 OS = Bostaurus
GN = NDST2 PE = 2 SV = 1
58.13 1.648721
O97831 Adhesion G protein- coupled receptor L1 OS = Bostaurus GN = ADGRL1 PE = 2 SV = 1 63.31 1.648721
A5PKL7 Leucine zipper putative tumour suppressor 2 OS = Bostaurus GN = LZTS2 PE = 2 SV = 1 63.9 1.682028
P13608 Aggrecan core protein OS = Bostaurus GN = ACAN PE = 1 SV = 3 26.66 1.682028
A6QLP2 Adenosylhomocysteinase 3 OS = Bostaurus GN = AHCYL2 PE = 1 SV = 1 226.5 1.682028
Q2HJ49 Moesin OS = Bostaurus GN = MSN PE = 2 SV = 3 120.2 1.682028
E1BB50 Cyclin- dependent kinase 12 OS = Bostaurus GN = CDK12 PE = 3 SV = 1 82.49 1.716007
E1B7X9 SWI/SNF- related matrix- associated actin- dependent regulator of chromatin subfamily A
containing DEAD/H box 1 OS = Bostaurus GN = SMARCAD1 PE = 3 SV = 2
122.36 1.750673
Q3SX46 C1GALT1- specific chaperone 1 OS = Bostaurus GN = C1GALT1C1 PE = 2 SV = 1 288.23 1.768267
Q28193 Furin OS = Bostaurus GN = FURIN PE = 1 SV = 1 60.67 1.768267
Q27966 Unconventional myosin- Ic OS = Bostaurus GN = MYO1C PE = 1 SV = 3 53.33 1.786038
A0JNJ4 Zinc finger protein 692 OS = Bostaurus GN = ZNF692 PE = 2 SV = 1 41.89 1.786038
P48820 E3 SUMO- protein ligase RanBP2 (Fragment) OS = Bostaurus GN = RANBP2 PE = 2
SV = 2
78.14 1.803988
TABLE2 (Continued)
(Continues)
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Accession Description of the protein
PLG score
(Good)
Ratio of PLG score
(Good vs. Poor)
G3MWR8 [F- actin]- methionine sulfoxide oxidase MICAL3 OS = Bostaurus GN = MICAL3 PE = 3
SV = 1
71.15 1.822119
Q08DF4 Dynamin- 1 OS = Bostaurus GN = DNM1 PE = 2 SV = 1 67.71 1.822119
Q2KJE5 Glyceraldehyde- 3- phosphate dehydrogenase, testis- specific OS = Bostaurus
GN = GAPDHS PE = 2 SV = 1
286.05 1.822119
Q58CX9 Cytosolic carboxypeptidase- like protein 5 OS = Bostaurus GN = AGBL5 PE = 2 SV = 3 49.67 1.822119
Q3ZKN0 Long- chain fatty acid transport protein 1 OS = Bostaurus GN = SLC27A1 PE = 2 SV = 1 63.77 1.840431
Q9TTA5 SWI/SNF- related matrix- associated actin- dependent regulator of chromatin subfamily
A- like protein 1 OS = Bostaurus GN = SMARCAL1 PE = 2 SV = 2
121.82 1.840431
Q29RT4 Cell division cycle- associated protein 2 OS = Bostaurus GN = CDCA2 PE = 2 SV = 1 142.85 1.858928
O02811 Phosphatidylinositol 4- kinase alpha OS = Bostaurus GN = PI4KA PE = 2 SV = 2 53.77 1.896481
Q29RL9 Transcription elongation factor A protein 1 OS = Bostaurus GN = TCEA1 PE = 2 SV = 1 107.31 1.915541
Q0VC73 Protein kintoun OS = Bostaurus GN = DNAAF2 PE = 2 SV = 2 42.6 1.934792
P79114 Unconventional myosin- X OS = Bostaurus GN = MYO10 PE = 1 SV = 1 75.2 1.954237
A6QPB3 Collagen alpha- 1(XVII) chain OS = Bostaurus GN = COL17A1 PE = 2 SV = 1 51.84 1.954237
Q17R07 ADP- ribosylation factor GTPase- activating protein 3 OS = Bostaurus GN = ARFGAP3
PE = 2 SV = 1
46.7 1.993716
Q10741 Disintegrin and metalloproteinase domain- containing protein 10 OS = Bostaurus
GN = ADAM10 PE = 1 SV = 1
167.03 2.013753
O02776 Poly(ADP- ribose) glycohydrolase OS = Bostaurus GN = PARG PE = 1 SV = 1 127.88 2.013753
E1BP74 Meiosis arrest female protein 1 OS = Bostaurus GN = MARF1 PE = 3 SV = 2 42.45 2.033991
P81019 Seminal plasma protein BSP- 30 kDa OS = Bostaurus PE = 1 SV = 2 4,392.9 2.054433
Q28205 Tubulin- specific chaperone D OS = Bostaurus GN = TBCD PE = 1 SV = 1 94.5 2.075081
Q7SIH1 Alpha- 2- macroglobulin OS = Bostaurus GN = A2M PE = 1 SV = 2 87.07 2.075081
Q29RK2 Pyruvate carboxylase, mitochondrial OS = Bostaurus GN = PC PE = 2 SV = 2 86.55 2.095936
A6QLY7 Pre- B- cell leukaemia transcription factor- interacting protein 1 OS = Bostaurus
GN = PBXIP1 PE = 2 SV = 1
71.58 2.117
Q8MJ05 Oxygen- regulated protein 1 OS = Bostaurus GN = RP1 PE = 2 SV = 1 68.45 2.138276
P20811 Calpastatin OS = Bostaurus GN = CAST PE = 1 SV = 2 110.47 2.138276
Q95116 Thrombospondin- 2 OS = Bostaurus GN = THBS2 PE = 2 SV = 2 85.03 2.159766
Q3MHE4 DNA mismatch repair protein Msh2 OS = Bostaurus GN = MSH2 PE = 2 SV = 1 62.9 2.159766
E1B7L7 Ubinuclein- 2 OS = Bostaurus GN = UBN2 PE = 3 SV = 1 28.31 2.159766
P02459 Collagen alpha- 1(II) chain OS = Bostaurus GN = COL2A1 PE = 1 SV = 4 108.21 2.181472
Q5EA28 CXXC- type zinc finger protein 1 OS = Bostaurus GN = CXXC1 PE = 2 SV = 1 86.01 2.181472
Q17QZ7 Tetratricopeptide repeat protein 27 OS = Bostaurus GN = TTC27 PE = 2 SV = 1 63.8 2.2705
A5PJS6 Ubiquitin carboxyl- terminal hydrolase 10 OS = Bostaurus GN = USP10 PE = 2 SV = 1 85.82 2.2705
E1BK52 Serine/threonine- protein kinase 10 OS = Bostaurus GN = STK10 PE = 3 SV = 3 90.16 2.2705
A1A4R8 Cell division cycle protein 23 homolog OS = Bostaurus GN = CDC23 PE = 2 SV = 1 74.91 2.2705
Q58DL5 Breast cancer anti- oestrogen resistance protein 3 OS = Bostaurus GN = BCAR3 PE = 2
SV = 2
115.53 2.2705
Q29RL1 Cilia- and flagella- associated protein 206 OS = Bostaurus GN = CFAP206 PE = 2 SV = 2 174.56 2.2705
P01030 Complement C4 (Fragments) OS = Bostaurus GN = C4 PE = 1 SV = 2 64.96 2.293319
Q24K09 DNA (cytosine- 5)- methyltransferase 1 OS = Bostaurus GN = DNMT1 PE = 2 SV = 1 50.68 2.316367
P55106 Growth/differentiation factor 6 OS = Bostaurus GN = GDF6 PE = 2 SV = 2 159 2.339647
O77788 Neurofilament medium polypeptide OS = Bostaurus GN = NEFM PE = 1 SV = 3 91.23 2.363161
TABLE2 (Continued)
(Continues)
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SINGH et al.
Accession Description of the protein
PLG score
(Good)
Ratio of PLG score
(Good vs. Poor)
Q28156 cGMP- specific 3′,5′- cyclic phosphodiesterase OS = Bostaurus GN = PDE5A PE = 1
SV = 1
185.63 2.43513
Q17QL5 Cell cycle control protein 50A OS = Bostaurus GN = TMEM30A PE = 1 SV = 1 148.09 2.459603
Q2TBV1 Replication factor C subunit 3 OS = Bostaurus GN = RFC3 PE = 2 SV = 1 139.92 2.459603
P10152 Angiogenin- 1 OS = Bostaurus GN = ANG1 PE = 1 SV = 4 1,892.54 2.459603
A7E2Y6 Maestro heat- like repeat- containing protein family member 1 OS = Bostaurus
GN = MROH1 PE = 2 SV = 1
67.26 2.484323
A1A4K3 DNA damage- binding protein 1 OS = Bostaurus GN = DDB1 PE = 1 SV = 1 64.32 2.484323
P10123 Retinaldehyde- binding protein 1 OS = Bostaurus GN = RLBP1 PE = 1 SV = 4 288.63 2.50929
Q1JQD6 TATA box- binding protein- associated factor RNA polymerase I subunit B OS = Bostaurus
GN = TAF1B PE = 2 SV = 1
37.06 2.534509
Q17R09 Pre- mRNA- splicing factor ATP- dependent RNA helicase PRP16 OS = Bostaurus
GN = DHX38 PE = 2 SV = 1
42.65 2.58571
F1N4M2 Myelin regulatory factor- like protein OS = Bostaurus GN = MYRFL PE = 2 SV = 1 24.03 2.58571
P01000 Acrosin inhibitor 1 OS = Bostaurus PE = 1 SV = 1 4,588.93 2.611696
Q1RMU5 RNA- binding protein 5 OS = Bostaurus GN = RBM5 PE = 2 SV = 1 27.94 2.611696
Q1LZ87 Zinc finger protein 397 OS = Bostaurus GN = ZNF397 PE = 2 SV = 1 64.08 2.637945
F1MCA7 Leucine- rich repeat- containing protein 7 OS = Bostaurus GN = LRRC7 PE = 3 SV = 3 29.62 2.664456
Q9GMB8 Serine–tRNA ligase, cytoplasmic OS = Bostaurus GN = SARS PE = 2 SV = 3 129.51 2.718282
P34933 Heat shock- related 70 kDa protein 2 OS = Bostaurus GN = HSPA2 PE = 2 SV = 2 134.01 2.745601
Q28141 ATP- dependent RNA helicase A OS = Bostaurus GN = DHX9 PE = 2 SV = 1 63.56 2.773195
F1MJW3 Amiloride- sensitive sodium channel subunit gamma OS = Bostaurus GN = SCNN1G
PE = 3 SV = 2
58.32 2.829217
A6QNM7 Ubiquitin carboxyl- terminal hydrolase 33 OS = Bostaurus GN = USP33 PE = 2 SV = 1 105.11 2.829217
E1BBQ2 Probable G protein- coupled receptor 158 OS = Bostaurus GN = GPR158 PE = 3 SV = 2 15.42 2.829217
Q28178 Thrombospondin- 1 OS = Bostaurus GN = THBS1 PE = 2 SV = 2 40.55 2.857651
Q3SWY8 Zinc finger with UFM1- specific peptidase domain protein OS = Bostaurus GN = ZUFSP
PE = 2 SV = 1
117.66 2.886371
A5PK39 Tripeptidyl- peptidase 2 OS = Bostaurus GN = TPP2 PE = 2 SV = 1 90.19 2.886371
A1A4J7 Protein SMG8 OS = Bostaurus GN = SMG8 PE = 2 SV = 2 103.64 2.91538
P33545 Desmocollin- 2 (Fragment) OS = Bostaurus GN = DSC2 PE = 2 SV = 1 17.47 2.91538
P02784 Seminal plasma protein PDC- 109 OS = Bostaurus PE = 1 SV = 2 17,426.57 2.974274
Q9XSC1 Secreted frizzled- related protein 5 OS = Bostaurus GN = SFRP5 PE = 2 SV = 1 64.35 2.974274
P29392 Spermadhesin- 1 OS = Bostaurus GN = SPADH1 PE = 1 SV = 1 15,502.63 3.004166
P82292 Spermadhesin Z13 OS = Bostaurus PE = 1 SV = 1 1,234.88 3.064854
Q28019 Latent- transforming growth factor beta- binding protein 2 OS = Bostaurus GN = LTBP2
PE = 1 SV = 2
20.6 3.095656
Q29RY7 Fibroleukin OS = Bostaurus GN = FGL2 PE = 2 SV = 1 97.04 3.158193
A6QLE6 Anoctamin- 4 OS = Bostaurus GN = ANO4 PE = 2 SV = 1 53.5 3.189933
P02453 Collagen alpha- 1(I) chain OS = Bostaurus GN = COL1A1 PE = 1 SV = 3 73.51 3.254374
E1B7Q7 E3 ubiquitin–protein ligase TRIP12 OS = Bostaurus GN = TRIP12 PE = 2 SV = 2 21.2 3.287081
Q58CR3 Eukaryotic translation initiation factor 2D OS = Bostaurus GN = EIF2D PE = 2 SV = 1 76.45 3.287081
Q08DZ2 Serine/threonine- protein kinase PRP4 homolog OS = Bostaurus GN = PRPF4B PE = 2
SV = 1
45.12 3.455613
E1BGH8 Protein MMS22- like OS = Bostaurus GN = MMS22L PE = 3 SV = 1 59.88 3.455613
Q0VCA1 Glutamate decarboxylase 1 OS = Bostaurus GN = GAD1 PE = 2 SV = 1 69.3 3.525421
TABLE2 (Continued)
(Continues)
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Accession Description of the protein
PLG score
(Good)
Ratio of PLG score
(Good vs. Poor)
Q08DA3 Ubiquitin carboxyl- terminal hydrolase 16 OS = Bostaurus GN = USP16 PE = 2 SV = 2 69.78 3.560852
Q2TLZ3 Macoilin OS = Bostaurus GN = TMEM57 PE = 2 SV = 1 33.79 3.560852
Q28056 Aspartyl/asparaginyl beta- hydroxylase OS = Bostaurus GN = ASPH PE = 1 SV = 1 36.97 3.59664
Q2HJE4 Ubiquitin carboxyl- terminal hydrolase 15 OS = Bostaurus GN = USP15 PE = 2 SV = 1 63.3 3.59664
Q28085 Complement factor H OS = Bostaurus GN = CFH PE = 1 SV = 3 33.44 3.669296
Q32PJ7 Tensin- 4 OS = Bostaurus GN = TNS4 PE = 2 SV = 2 200.48 3.781044
Q27991 Myosin- 10 OS = Bostaurus GN = MYH10 PE = 2 SV = 2 80.07 3.819044
Q2T9M4 Dynein regulatory complex subunit 7 OS = Bostaurus GN = DRC7 PE = 2 SV = 1 54.08 3.819044
Q2KJE0 Tax1- binding protein 1 homolog OS = Bostaurus GN = TAX1BP1 PE = 2 SV = 1 38.2 3.819044
O02740 Retinal guanylyl cyclase 2 OS = Bostaurus GN = GUCY2F PE = 2 SV = 1 70.7 4.220696
F1MUS9 PH and SEC7 domain- containing protein 1 OS = Bostaurus GN = PSD PE = 2 SV = 1 33.35 4.220696
P05059 Chromogranin- A OS = Bostaurus GN = CHGA PE = 1 SV = 1 172.99 4.526731
Q1RMS4 Leucine- rich repeat and fibronectin type- III domain- containing protein 3 OS = Bostaurus
GN = LRFN3 PE = 2 SV = 1
139.24 4.526731
P98072 Enteropeptidase OS = Bostaurus GN = TMPRSS15 PE = 1 SV = 1 117.66 4.526731
G5E5X0 LIM domain- containing protein 1 OS = Bostaurus GN = LIMD1 PE = 3 SV = 1 70.5 4.618177
Q8MI28 Limbin OS = Bostaurus GN = EVC2 PE = 2 SV = 1 63.62 4.618177
Q28198 Tissue- type plasminogen activator OS = Bostaurus GN = PLAT PE = 2 SV = 1 36.75 4.66459
A3KMV5 Ubiquitin- like modifier- activating enzyme 1 OS = Bostaurus GN = UBA1 PE = 2 SV = 1 57.88 4.758821
P79122 Pinin OS = Bostaurus GN = PNN PE = 2 SV = 3 84.88 4.806648
A7YWK3 Keratin, type II cytoskeletal 73 OS = Bostaurus GN = KRT73 PE = 2 SV = 1 100.6 4.806648
Q2T9M9 Uncharacterized protein C11orf63 homolog OS = Bostaurus PE = 2 SV = 1 39.65 4.806648
Q5E9N5 Caseinolytic peptidase B protein homolog OS = Bostaurus GN = CLPB PE = 2 SV = 1 50.37 4.854956
Q28083 Collagen alpha- 1(XI) chain (Fragment) OS = Bostaurus GN = COL11A1 PE = 1 SV = 1 58.98 4.903749
P32871 Phosphatidylinositol 4,5- bisphosphate 3- kinase catalytic subunit alpha isoform
OS = Bostaurus GN = PIK3CA PE = 1 SV = 1
67.08 4.903749
A7YY57 Rho GTPase- activating protein 29 OS = Bostaurus GN = ARHGAP29 PE = 2 SV = 1 46.71 5.002811
A2VDP1 E3 ubiquitin–protein ligase BRE1A OS = Bostaurus GN = RNF20 PE = 2 SV = 1 129.33 5.002811
A6QLU7 TRPM8 channel- associated factor 2 OS = Bostaurus GN = TCAF2 PE = 2 SV = 1 69.06 5.05309
A6QR11 Protein kinase C- binding protein NELL2 OS = Bostaurus GN = NELL2 PE = 2 SV = 1 52.59 5.05309
P80012 von Willebrand factor (Fragment) OS = Bostaurus GN = VWF PE = 1 SV = 2 56.23 5.103875
Q76LV1 Heat shock protein HSP 90 beta OS = Bostaurus GN = HSP90AB1 PE = 2 SV = 3 108.61 5.155169
Q24K22 Hepatocyte growth factor- like protein OS = Bostaurus GN = MST1 PE = 2 SV = 1 214.08 5.155169
Q9GLM4 Tensin- 1 OS = Bostaurus GN = TNS1 PE = 2 SV = 1 90.06 5.473948
Q2TBR5 Protein FAM166B OS = Bostaurus GN = FAM166B PE = 2 SV = 1 215.63 5.473948
Q29RV1 Protein disulphide- isomerase A4 OS = Bostaurus GN = PDIA4 PE = 2 SV = 1 158.05 5.528962
A7E320 E3 ubiquitin–protein ligase UHRF1 OS = Bostaurus GN = UHRF1 PE = 2 SV = 1 104.2 5.584529
Q9BE41 Myosin- 2 OS = Bostaurus GN = MYH2 PE = 2 SV = 1 96.32 5.584529
Q0III9 Alpha- actinin- 3 OS = Bostaurus GN = ACTN3 PE = 2 SV = 1 47.25 5.697343
Q8HYY4 Uveal autoantigen with coiled- coil domains and ankyrin repeats protein OS = Bostaurus
GN = UACA PE = 1 SV = 1
62.77 5.929856
Q9BE40 Myosin- 1 OS = Bostaurus GN = MYH1 PE = 2 SV = 2 89.35 6.233887
Q76LV2 Heat shock protein HSP 90- alpha OS = Bostaurus GN = HSP90AA1 PE = 1 SV = 3 80.19 6.296538
Q58D55 Beta- galactosidase OS = Bostaurus GN = GLB1 PE = 2 SV = 1 68.18 6.296538
P21671 1- phosphatidylinositol 4,5- bisphosphate phosphodiesterase delta- 4 OS = Bostaurus
GN = PLCD4 PE = 1 SV = 2
169.45 6.35982
TABLE2 (Continued)
(Continues)
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SINGH et al.
3.2 | Proteome profiles of spermatozoa from
high and low fertility bulls
A total 1,547 proteins were identified in bull spermatozoa using liquid
chromatography–mass spectrometer (LC- MS/MS) analysis (Figure 2).
Our results showed that the expression of 558 (36.1%) and 653
(42.2%) proteins was specific to good and poor quality bull sperma-
tozoa, respectively. It was observed that 336 proteins (21.7%) were
common for both good and poor quality bull semen (Figure 3). Out
of the common proteins, 224 (66.7%) and 112 (33.3%) were up- and
downregulated in good and poor quality categorized bull semen, re-
spectively (Tables 2 and 3).
Accession Description of the protein
PLG score
(Good)
Ratio of PLG score
(Good vs. Poor)
P39873 Brain ribonuclease OS = Bostaurus GN = BRN PE = 1 SV = 3 658.74 6.553505
O46385 Supervillin OS = Bostaurus GN = SVIL PE = 1 SV = 2 54.02 6.753089
Q3MHI4 Phosphorylated adapter RNA export protein OS = Bostaurus GN = PHAX PE = 2 SV = 2 293.53 6.820958
P61823 Ribonuclease pancreatic OS = Bostaurus GN = RNASE1 PE = 1 SV = 1 371.61 6.958751
A7MBJ2 Sentrin- specific protease 7 OS = Bostaurus GN = SENP7 PE = 2 SV = 1 62.76 7.170677
Q0II24 Complement C1q and tumour necrosis factor- related protein 9 OS = Bostaurus
GN = C1QTNF9 PE = 2 SV = 1
91.17 7.242743
P07224 Vitamin K- dependent protein S OS = Bostaurus GN = PROS1 PE = 1 SV = 1 57.39 7.242743
Q28044 Beta- 2 adrenergic receptor OS = Bostaurus GN = ADRB2 PE = 2 SV = 2 87.04 7.315534
A6QP16 Ubiquitin thioesterase ZRANB1 OS = Bostaurus GN = ZRANB1 PE = 1 SV = 1 140.52 7.315534
Q0IIG5 ATP- dependent 6- phosphofructokinase, muscle type OS = Bostaurus GN = PFKM
PE = 2 SV = 1
80.99 7.315534
Q3SZP7 Villin- 1 OS = Bostaurus GN = VIL1 PE = 2 SV = 3 60.37 7.538325
P12661 Retinol- binding protein 3 OS = Bostaurus GN = RBP3 PE = 1 SV = 1 60.93 7.614086
A7MBJ4 Receptor- type tyrosine–protein phosphatase F OS = Bostaurus GN = PTPRF PE = 2
SV = 1
58.77 7.845969
Q2T9U1 Signal recognition particle 54 kDa protein OS = Bostaurus GN = SRP54 PE = 2 SV = 1 60.58 8.414868
E1BB52 Cyclin- dependent kinase 13 OS = Bostaurus GN = CDK13 PE = 3 SV = 1 42.29 8.758285
Q06846 Rabphilin- 3A OS = Bostaurus GN = RPH3A PE = 1 SV = 1 93.12 8.935214
A6QP75 ALS2 C- terminal- like protein OS = Bostaurus GN = ALS2CL PE = 2 SV = 1 80.53 9.874937
A3KN33 Pikachurin OS = Bostaurus GN = EGFLAM PE = 2 SV = 1 91.96 9.974182
Q2TBI7 IQ domain- containing protein C OS = Bostaurus GN = IQCC PE = 2 SV = 1 62.75 11.35888
A2VE78 F- box/LRR- repeat protein 5 OS = Bostaurus GN = FBXL5 PE = 2 SV = 1 22.16 11.70481
P11024 NAD(P) transhydrogenase, mitochondrial OS = Bostaurus GN = NNT PE = 1 SV = 3 47.08 12.67967
P49259 Secretory phospholipase A2 receptor OS = Bostaurus GN = PLA2R1 PE = 1 SV = 1 59.75 12.93582
Q9BE39 Myosin- 7 OS = Bostaurus GN = MYH7 PE = 1 SV = 1 52.03 14.87973
E1BMP7 DNA replication ATP- dependent helicase/nuclease DNA2 OS = Bostaurus GN = DNA2
PE = 3 SV = 3
53.69 17.46153
E1BPH3 Tudor domain- containing protein 5 OS = Bostaurus GN = TDRD5 PE = 3 SV = 1 36.77 24.28843
A5PK51 Nicotinate phosphoribosyltransferase OS = Bostaurus GN = NAPRT PE = 2 SV = 2 7.99 24.53253
O18737 Calcium- binding and coiled- coil domain- containing protein 2 OS = Bostaurus
GN = CALCOCO2 PE = 2 SV = 1
8.88 24.53253
Q07175 Vitamin K- dependent gamma- carboxylase OS = Bostaurus GN = GGCX PE = 1 SV = 1 145.14 26.84286
A6QQJ8 Ribonuclease ZC3H12A OS = Bostaurus GN = ZC3H12A PE = 2 SV = 1 4.67 31.18696
Q0P5G1 Tonsoku- like protein OS = Bostaurus GN = TONSL PE = 2 SV = 1 23.25 31.50039
P35071 Cystic fibrosis transmembrane conductance regulator OS = Bostaurus GN = CFTR PE = 2
SV = 2
41.95 34.81332
Q28007 Dihydropyrimidine dehydrogenase [NADP(+)] OS = Bostaurus GN = DPYD PE = 1 SV = 1 45.47 57.97431
GN, general name; PE, protein existence; SV, sequence version; MWt, molecular weight.
TABLE2 (Continued)
598
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SINGH et al.
TABLE3 Downregulated proteins in good quality bull semen
Accession Description of the protein
PLG score
(Good)
Ratio of PLG score
(Good vs. Poor)
P56560 Amine oxidase [flavin- containing] B
GN = MAOB PE = 1 SV = 4
56.58 0.018873
Q58CU2 Band 4.1- like protein 5 OS = Bostaurus GN = EPB41L5 PE = 2 SV = 1 57.98 0.025223
O97594 Structural maintenance of chromosomes protein 3 OS = Bostaurus GN = SMC3 PE = 1 SV = 1 95.31 0.037628
Q9TU23 Centrosomal protein of 290 kDa (Fragment) OS = Bostaurus GN = CEP290 PE = 1 SV = 2 78.39 0.041172
A6QNM2 Ribosome- releasing factor 2, mitochondrial OS = Bostaurus GN = GFM2 PE = 2 SV = 1 61.53 0.048316
Q2T9X8 Spindle and centriole- associated protein 1 OS = Bostaurus GN = SPICE1 PE = 2 SV = 1 137 0.06457
Q29RP8 Histone- lysine N- methyltransferase KMT5B OS = Bostaurus GN = KMT5B PE = 2 SV = 1 48.78 0.093481
F1MF74 [F- actin]- methionine sulfoxide oxidase MICAL2 OS = Bostaurus GN = MICAL2 PE = 3 SV = 2 35.34 0.116484
Q8MIT6 Rho- associated protein kinase 1 (Fragment) OS = Bostaurus GN = ROCK1 PE = 1 SV = 1 285.61 0.133989
Q8SQB8 Integrin beta- 6 OS = Bostaurus GN = ITGB6 PE = 1 SV = 1 158.87 0.182684
P68399 Casein kinase II subunit alpha OS = Bostaurus GN = CSNK2A1 PE = 1 SV = 1 90.22 0.205975
A5D794 GTPase- activating protein and VPS9 domain- containing protein 1 OS = Bostaurus
GN = GAPVD1 PE = 2 SV = 1
27.49 0.214381
P02548 Neurofilament light polypeptide OS = Bostaurus GN = NEFL PE = 1 SV = 3 262.52 0.22313
A4IFA3 General transcription factor II- I repeat domain- containing protein 2 OS = Bostaurus
GN = GTF2IRD2 PE = 2 SV = 1
52.34 0.225373
A0JNI1 LysM and putative peptidoglycan- binding domain- containing protein 1 OS = Bostaurus
GN = LYSMD1 PE = 2 SV = 1
294.19 0.232236
A0JNH1 Centrosomal protein kizuna OS = Bostaurus GN = KIZ PE = 2 SV = 2 75.73 0.232236
A6QNX5 Keratin, type II cytoskeletal 78 OS = Bostaurus GN = KRT78 PE = 2 SV = 1 44.1 0.23457
A6QQP7 Dysferlin OS = Bostaurus GN = DYSF PE = 2 SV = 1 34.26 0.23457
Q8SPJ1 Junction plakoglobin OS = Bostaurus GN = JUP PE = 2 SV = 1 20.33 0.251579
A4IFJ5 Cytosolic phospholipase A2 OS = Bostaurus GN = PLA2G4A PE = 1 SV = 1 26.39 0.256661
Q3T0X8 Na(+)/H(+) exchange regulatory cofactor NHE- RF3 OS = Bostaurus GN = PDZK1 PE = 2 SV = 1 83.8 0.278037
Q2T9P4 Nuclear autoantigenic sperm protein OS = Bostaurus GN = NASP PE = 2 SV = 2 54.14 0.280832
E1BLP6 AT- rich interactive domain- containing protein 5B OS = Bostaurus GN = ARID5B PE = 3 SV = 1 181.8 0.29523
P00669 Seminal ribonuclease OS = Bostaurus GN = SRN PE = 1 SV = 2 14,532.17 0.313486
Q29RN6 Suppressor of cytokine signalling 5 OS = Bostaurus GN = SOCS5 PE = 2 SV = 1 248.58 0.332871
P02465 Collagen alpha- 2(I) chain OS = Bostaurus GN = COL1A2 PE = 1 SV = 2 85.71 0.339596
Q0VC16 Melanoma inhibitory activity protein 3 OS = Bostaurus GN = MIA3 PE = 2 SV = 2 63.23 0.343008
P13600 Beta- nerve growth factor OS = Bostaurus GN = NGF PE = 2 SV = 3 1,450.53 0.349938
D3K0R6 Plasma membrane calcium- transporting ATPase 4 OS = Bostaurus GN = ATP2B4 PE = 1 SV = 2 172.38 0.349938
A7MB70 Semaphorin- 3C OS = Bostaurus GN = SEMA3C PE = 2 SV = 1 111.75 0.353455
Q864U1 Breast cancer type 1 susceptibility protein homolog OS = Bostaurus GN = BRCA1 PE = 1 SV = 1 68.94 0.353455
P80747 Integrin beta- 5 OS = Bostaurus GN = ITGB5 PE = 1 SV = 2 16.22 0.357007
P20237 Gamma- aminobutyric acid receptor subunit alpha- 4 OS = Bostaurus GN = GABRA4 PE = 2
SV = 2
41.48 0.390628
P35605 Coatomer subunit beta’ OS = Bostaurus GN = COPB2 PE = 1 SV = 3 51.61 0.40657
Q2TBI4 Heat shock protein 75 kDa, mitochondrial OS = Bostaurus GN = TRAP1 PE = 2 SV = 1 79.06 0.40657
P09851 RasGTPase- activating protein 1 OS = Bostaurus GN = RASA1 PE = 1 SV = 1 51.71 0.414783
Q5W5U4 Probable ATP- dependent RNA helicase DDX4 OS = Bostaurus GN = DDX4 PE = 2 SV = 1 105.51 0.453845
P04258 Collagen alpha- 1(III) chain OS = Bostaurus GN = COL3A1 PE = 1 SV = 1 75.76 0.458406
A3KN28 Suppressor of tumorigenicity 7 protein- like OS = Bostaurus GN = ST7L PE = 2 SV = 1 93.02 0.472367
P30205 Antigen WC1.1 OS = Bostaurus PE = 2 SV = 1 45.41 0.481909
(Continues)
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SINGH et al.
Accession Description of the protein
PLG score
(Good)
Ratio of PLG score
(Good vs. Poor)
Q28106 Contactin- 1 OS = Bostaurus GN = CNTN1 PE = 2 SV = 1 88.07 0.481909
Q3SYT7 26S proteasome non- ATPase regulatory subunit 8 OS = Bostaurus GN = PSMD8 PE = 2 SV = 3 61.8 0.481909
A7MB80 General transcription factor II- I OS = Bostaurus GN = GTF2I PE = 2 SV = 1 116.08 0.481909
Q1JPD6 Speckle targeted PIP5K1A- regulated poly(A) polymerase OS = Bostaurus GN = TUT1 PE = 2
SV = 1
130.11 0.506617
P28291 C- C motif chemokine 2 OS = Bostaurus GN = CCL2 PE = 3 SV = 1 8,081.27 0.511709
Q28062 Brevican core protein OS = Bostaurus GN = BCAN PE = 1 SV = 1 51.91 0.511709
A4IFD0 Adenylate kinase isoenzyme 5 OS = Bostaurus GN = Ak5 PE = 2 SV = 1 141.03 0.516851
A7E300 Rho GTPase- activating protein 7 OS = Bostaurus GN = DLC1 PE = 2 SV = 1 47.29 0.522046
E1BEQ5 Angiomotin- like protein 1 OS = Bostaurus GN = AMOTL1 PE = 3 SV = 1 99.78 0.522046
Q2KIZ8 DNA replication licensing factor MCM6 OS = Bostaurus GN = MCM6 PE = 2 SV = 1 50.01 0.527292
P35445 Cartilage oligomeric matrix protein OS = Bostaurus GN = COMP PE = 1 SV = 2 53.5 0.532592
O97593 Structural maintenance of chromosomes protein 1A OS = Bostaurus GN = SMC1A PE = 1
SV = 1
60.12 0.537944
A5D7D1 Alpha- actinin- 4 OS = Bostaurus GN = ACTN4 PE = 2 SV = 1 45.12 0.548812
A7MB40 Protein FAM193B OS = Bostaurus GN = FAM193B PE = 2 SV = 1 177.49 0.565525
Q58CZ9 Tyrosine aminotransferase OS = Bostaurus GN = TAT PE = 2 SV = 1 72.47 0.565525
P21809 Biglycan OS = Bostaurus GN = BGN PE = 1 SV = 3 114.89 0.57695
Q1RMV0 Peroxisomal targeting signal 1 receptor OS = Bostaurus GN = PEX5 PE = 2 SV = 1 54.83 0.57695
Q0P569 Nucleobindin- 1 OS = Bostaurus GN = NUCB1 PE = 2 SV = 1 1,440.07 0.582748
Q9N2I2 Plasma serine protease inhibitor OS = Bostaurus GN = SERPINA5 PE = 1 SV = 1 4,041.07 0.588605
E1BE02 Transcription factor E2F7 OS = Bostaurus GN = E2F7 PE = 3 SV = 1 24.46 0.612626
A2VDR8 Conserved oligomeric Golgi complex subunit 7 OS = Bostaurus GN = COG7 PE = 2 SV = 1 131.73 0.618783
Q08DX0 Sorting nexin- 29 OS = Bostaurus GN = SNX29 PE = 2 SV = 1 72.71 0.625002
Q28021 Rho- associated protein kinase 2 OS = Bostaurus GN = ROCK2 PE = 1 SV = 1 84.62 0.625002
Q6TUI4 Endoribonuclease Dicer OS = Bostaurus GN = DICER1 PE = 2 SV = 3 70.92 0.637628
Q0P5K1 Vasculin OS = Bostaurus GN = GPBP1 PE = 2 SV = 1 161.89 0.650509
O97827 Adhesion G protein- coupled receptor L3 OS = Bostaurus GN = ADGRL3 PE = 2 SV = 1 28.16 0.650509
Q3ZC12 Eukaryotic translation initiation factor 3 subunit G OS = Bostaurus GN = EIF3G PE = 2 SV = 1 462.13 0.650509
F1MSG6 Rap guanine nucleotide- exchange factor 2 OS = Bostaurus GN = RAPGEF2 PE = 1 SV = 2 93.02 0.657047
P55206 C- type natriuretic peptide OS = Bostaurus GN = NPPC PE = 2 SV = 1 12,740.9 0.66365
A2VDQ5 Neurolysin, mitochondrial OS = Bostaurus GN = NLN PE = 2 SV = 1 81.99 0.66365
A7MBJ5 Cullin- associated NEDD8- dissociated protein 1 OS = Bostaurus GN = CAND1 PE = 2 SV = 1 54.44 0.66365
Q6R8F2 Cadherin- 1 OS = Bostaurus GN = CDH1 PE = 2 SV = 1 119.94 0.677057
A6QQV9 Actin filament- associated protein 1- like 1 OS = Bostaurus GN = AFAP1L1 PE = 2 SV = 1 48.65 0.677057
P26779 Prosaposin OS = Bostaurus GN = PSAP PE = 1 SV = 3 387.1 0.690734
Q9N0W2 Alpha- (1,6)- fucosyltransferase OS = Bostaurus GN = FUT8 PE = 2 SV = 1 45.68 0.697676
Q32LG3 Malate dehydrogenase, mitochondrial OS = Bostaurus GN = MDH2 PE = 1 SV = 1 60.98 0.733447
Q28146 Neurexin- 1 OS = Bostaurus GN = NRXN1 PE = 1 SV = 1 10.52 0.748264
F1MBP6 Kelch- like protein 3 OS = Bostaurus GN = KLHL3 PE = 3 SV = 3 285.72 0.786628
E1BPQ3 G protein- coupled receptor family C group 6 member A OS = Bostaurus GN = GPRC6A PE = 3
SV = 1
52.72 0.794534
Q0VCA3 LETM1 and EF- hand domain- containing protein 1, mitochondrial OS = Bostaurus GN = LETM1
PE = 2 SV = 1
57.14 0.794534
A5PKL1 Oxidation resistance protein 1 OS = Bostaurus GN = OXR1 PE = 2 SV = 2 385.64 0.802519
TABLE3 (Continued)
(Continues)
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SINGH et al.
3.3 | GO analysis and signalling pathway
Gene Ontology analysis of global proteomes identified differ-
ent signalling pathways represented between the good and poor
quality bull spermatozoa. Table 4 represents the GO analysis of
identified differential proteins to represent their different func-
tions, location as well as signalling pathways. Major significant
(p < .05) biological process of GO annotated differentially ex-
pressed proteins in good categorized bull semen is illustrated
(Table 5). Significant pathways (p < .05) are shown in Table 6.
Most of the identified signalling pathways are related to cellu-
lar motility, immune systems and cellular metabolisms. Reactome
signalling pathway of sperm motility in the bull semen depicts the
importance of Ca2+- mediated CatSper channel. It was also identi-
fied that additional proteins, viz. Izumo and CD9, can make a com-
bination to facilitate the sperm motility as well as binding with
egg membrane (Figure 4). Top 10 functions/diseases and their
respective top 10 signalling pathways for up- and downregulated
proteins in good quality bull semen are depicted in Tables 7 and
8, respectively.
Accession Description of the protein
PLG score
(Good)
Ratio of PLG score
(Good vs. Poor)
A4IF62 DNA- directed RNA polymerase III subunit RPC1 OS = Bostaurus GN = POLR3A PE = 2 SV = 1 67.2 0.802519
P15690 NADH- ubiquinone oxidoreductase 75 kDa subunit, mitochondrial OS = Bostaurus
GN = NDUFS1 PE = 1 SV = 1
125.84 0.802519
A2VE10 Protein CASC4 OS = Bostaurus GN = CASC4 PE = 2 SV = 1 58.84 0.810584
E1BBG2 MICAL- like protein 1 OS = Bostaurus GN = MICALL1 PE = 1 SV = 2 38.77 0.810584
A6QP06 Synaptotagmin- like protein 2 OS = Bostaurus GN = SYTL2 PE = 2 SV = 1 36.55 0.818731
Q9XS59 Sodium- dependent neutral amino acid transporter B(0)AT2 OS = Bostaurus GN = SLC6A15
PE = 2 SV = 1
56.99 0.818731
A4IFB6 CCR4- NOT transcription complex subunit 10 OS = Bostaurus GN = CNOT10 PE = 2 SV = 1 52.82 0.843665
A7MBI1 Zinc finger protein ZFP69 OS = Bostaurus GN = ZFP69 PE = 2 SV = 1 145.12 0.860708
P08487 1- phosphatidylinositol 4,5- bisphosphate phosphodiesterase gamma- 1 OS = Bostaurus
GN = PLCG1 PE = 1 SV = 1
72.72 0.860708
Q3B7N2 Alpha- actinin- 1 OS = Bostaurus GN = ACTN1 PE = 2 SV = 1 80.38 0.88692
Q28036 Sodium/hydrogen exchanger 1 OS = Bostaurus GN = SLC9A1 PE = 2 SV = 1 127.12 0.88692
P04557 Seminal plasma protein A3 OS = Bostaurus PE = 1 SV = 2 5,795.49 0.88692
P32592 Integrin beta- 2 OS = Bostaurus GN = ITGB2 PE = 1 SV = 1 93.44 0.88692
A4IFB1 Serrate RNA effector molecule homolog OS = Bostaurus GN = SRRT PE = 2 SV = 1 100.44 0.895834
P21214 Transforming growth factor beta- 2 OS = Bostaurus GN = TGFB2 PE = 1 SV = 3 41.22 0.904837
Q49BZ4 C- type lectin domain family 7 member A OS = Bostaurus GN = CLEC7A PE = 2 SV = 1 151.87 0.913931
P16368 Metalloproteinase inhibitor 2 OS = Bostaurus GN = TIMP2 PE = 1 SV = 2 3,606.12 0.913931
O46631 Tyrosine–protein phosphatase non- receptor- type substrate 1 OS = Bostaurus GN = SIRPA
PE = 2 SV = 1
156.39 0.923116
A3KMX0 DNA excision repair protein ERCC- 6- like 2 OS = Bostaurus GN = ERCC6L2 PE = 2 SV = 3 23.83 0.923116
Q0VCM5 Interalpha- trypsin inhibitor heavy chain H1 OS = Bostaurus GN = ITIH1 PE = 1 SV = 1 65.34 0.932394
Q0VCX5 Eukaryotic peptide chain release factor subunit 1 OS = Bostaurus GN = ETF1 PE = 2 SV = 3 51.72 0.941765
Q28046 Adseverin OS = Bostaurus GN = SCIN PE = 1 SV = 1 62.41 0.960789
Q2T9U2 Outer dense fibre protein 2 OS = Bostaurus GN = ODF2 PE = 2 SV = 1 725.72 0.960789
E1BM58 Periaxin OS = Bostaurus GN = PRX PE = 1 SV = 3 29.67 0.960789
Q08DV9 Transmembrane protein 131- like OS = Bostaurus PE = 2 SV = 2 16.81 0.970446
Q1RMR2 U1 small nuclear ribonucleoprotein 70 kDa OS = Bostaurus GN = SNRNP70 PE = 2 SV = 1 48.53 0.970446
A7XYH9 Sine oculis- binding protein homolog OS = Bostaurus GN = SOBP PE = 2 SV = 1 140.95 0.970446
Q0V898 Negative elongation factor E OS = Bostaurus GN = NELFE PE = 2 SV = 1 104.5 0.980199
P14099 cGMP- dependent 3′,5′- cyclic phosphodiesterase OS = Bostaurus GN = PDE2A PE = 1 SV = 2 138.29 0.980199
P25500 Poly(A) polymerase alpha OS = Bostaurus GN = PAPOLA PE = 1 SV = 3 30.9 0.980199
Q3ZCH0 Stress- 70 protein, mitochondrial OS = Bostaurus GN = HSPA9 PE = 2 SV = 1 60.96 0.980199
GN, general name; PE, protein existence; SV, sequence version; MWt, molecular weight.
TABLE3 (Continued)
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SINGH et al.
4 | DISCUSSION
Fertile spermatozoa can be described by its capability to fertilize
oocytes followed by zygotes formation which continue to develop
embryos (Eid, Lorton, & Parrish, 1994). According to extensive fertility
data and progeny records, it was documented that only spermatozoa
motility and morphology of high merit bulls are unable to produce suc-
cessful full- term pregnancies (Chenoweth, 2007; Parkinson, 2004),
and thus, the molecular defects play important factors which may af-
fect the ability of spermatozoa to fertilize oocytes as well as embryo
development (Dejarnette, 2005; Lewis, 2007; Peddinti et al., 2008;
Sharma, Agarwal, et al., 2013; Sharma, Masaki, and Agarwal, 2013).
Quality of semen phenotypes is habitually assessed according to
their spermatozoa characteristics such as concentration, volume, mo-
tility, as well as microscopic morphology. Results obtained from the
present study revealed that motility of the satisfactory quality groups
is significantly (p < .05) superior than unsatisfactory one. Again, sperm
DNA damage is one of the fundamental factors in successive preg-
nancy losses and in the malfunction of conception rates (Singh &
Agarwal, 2011). TUNEL assay is most commonly used to detect DNA
strand breaks which are chiefly induced by reactive oxygen species
or abortive apoptosis (Sharma, Agarwal, et al., 2013; Sharma, Masaki,
and Agarwal, 2013; Tremellen, 2008). Couples of reports suggested
that TUNEL assay may be the most suitable tool for assessing sperm
DNA strand breaks (Henkel et al., 2004; Irvine et al., 2000; Lopes,
Jurisicova, Sun, & Casper, 1998; Sharma et al., 2010; Takeda et al.,
2015). Similarly, TUNEL index was also evaluated for determining the
sperm DNA damage in quality bull semen.
In the present study, we have identified proteins that are common,
unique and differentially expressed among the spermatozoa samples
of good and poor quality crossbreed bull semen based on their con-
ception rates. Identified protein profiles of crossbred bull spermato-
zoa may play an important role to understand the bull spermatozoal
physiology which may attribute sperm functions. Most of the studies
pertaining to identification of functional proteins in semen were based
on two- dimensional electrophoresis (Hozumi et al., 2004; Kwon et al.,
2015; Martinez- Heredia, Estanyol, Ballesca, & Oliva, 2006; Pixton
et al., 2004; Tan, Fan, Luo, Zhu, & Lu, 2004). Scanty of reports are avail-
able on comprehensive non- electrophoretic proteomic study of bull
sperm proteome (Peddinti et al., 2008). Mass spectrometer has be-
come the cutting- edge instrument for LC/MDS/MS- based approaches
to characterize the spermatozoa proteome (Sharma, Agarwal, et al.,
TABLE4 Identified ontological parameters of the overall
proteomes among good and poor quality crossbred bull semen
SL. no. Ontology Good Poor
1. Molecular function
a. Binding 225 (34.3%) 255 (35%)
i. Ca ion 16 (6.5%) 19 (6.9%)
ii. Ca- dependent
phospholipid
9 (3.6%) 7 (2.7%)
iii. Chromatin binding 4 (1.6%) 3 (1.1%)
iv. Lipid 5 (2%) 9 (3.3%)
v. Nucleic acid 87 (35.1%) 88 (32.1%)
vi. Nucleotide 4 (1.6%) 8 (2.9%)
vii. Protein 123 (49.6%) 140 (51.1%)
b. Catalytic activity 226 (40.5%) 306 (42.0%)
c. Channel regulatory
activity
2 (0.3%) 1 (0.1%)
d. Receptor activity 31 (4.7%) 36 (4.9%)
e. Signal transducer
activity
9(1.4%) 13 (1.8%)
f. Structural molecular
activity
85 (13%) 51 (7.0%)
g. Translational
regulatory activity
3 (0.5%) 11 (1.5%)
h. Transporter activity 35 (5.3%) 53 (7.3%)
i. Antioxidant activity Nil 2 (0.3%)
2. Biological process
a. Biological adhesion 26 (1.9%) 24 (1.6%)
b. Biological regulation 107 (7.8%) 134 (9%)
c. Cellular component
organization or
biogenesis
111 (8.1%) 111 (7.4%)
d. Cellular process 406 (29.7%) 437 (29.3%)
e. Developmental
process
77 (5.6%) 89 (6%)
f. Growth 1 (0.1%) 3 (0.2%)
g. Immune system
process
30 (2.2%) 31 (2.1%)
h. Localization 108 (7.9%) 124 (8.3%)
i. Locomotion 7 (0.5%) 7 (0.5%)
j. Metabolic process 314 (23%) 339 (22.7%)
k. Multicellular organis-
mal process
64 (4.7%) 74 (7.7%)
l. Reproduction 11 (0.8%) 7 (0.5%)
m. Response to stimulus 103 (7.5%) 113 (7.6%0
n. Rhythmic process 2 (0.1%) Nil
3. Cellular component
a. Cell junction 7 (1.1%) 10 (1.4%0
b. Cell part 289 (45.7%) 306 (44.1%)
c. Extracellular matrix 11 (1.7%) 10 (1.4%)
(Continues)
SL. no. Ontology Good Poor
d. Extracellular region 39 (6.2%) 40 (5.8%)
e. Macromolecular
complex
86 (13.6%) 99 (14.3%)
f. Membrane 36 (5.7%) 51 (7.3%)
g. Organelle 162 (25.6%) 176 (25.4%)
h. Synapse 3 (0.5%) 2 (0.3%)
TABLE4 (Continued)
602
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SINGH et al.
Biological process Fold enrichment +/− p Values
Cellular component morphogenesis 2.59 + 8.79E- 07
Mitosis 2.28 + 5.22E- 03
Intracellular protein transport 1.66 + 2.41E- 02
Nitrogen compound metabolic process 1.49 + 7.81E- 04
Unclassified 0.85 −0.00E00
G protein- coupled receptor signalling pathway 0.37 −4.98E- 03
Sensory perception of chemical stimulus <0.2 −8.56E- 14
+, upregulated; −, downregulated.
TABLE5 GO annotated significant
biological process (p < .05) of the
differentially expressed proteins in good
quality bull semen
TABLE6 Identified reactome and panther pathways of GO annotated of significantly significant (p < .05) altered proteins between good and
poor quality spermatozoa
Pathways Fold enrichment +/− p Values
Good quality bull semen
Reactome
Formation of annular gap junctions 19.63 + 1.46E- 04
Cell- extracellular matrix interactions 18.23 + 4.13E- 06
Gap junction degradation 18.23 + 2.40E- 04
RHO GTPases activate IQGAPs 16.20 + 7.68E- 05
Gap junction trafficking 15.01 + 8.71E- 04
Interaction between L1 and Ankyrins 13.67 + 9.47E- 03
Gap junction trafficking and regulation 13.43 + 1.81E- 03
Recycling pathway of L1 11.60 + 4.71E- 03
RHO GTPases activate WASPs and WAVEs 8.10 + 1.28E- 02
Panther
Cytoskeletal regulation by Rho GTPase 5.58 + 5.87E- 07
Blood coagulation 4.98 + 7.36E- 03
Endothelin signalling pathway 3.68 + 1.27E- 02
Nicotinic acetylcholine receptor signalling pathway 3.62 + 4.45E- 03
Integrin signalling pathway 3.42 + 1.05E- 05
CCKR signalling map 2.81 + 5.18E- 03
Inflammation mediated by chemokine and cytokine signalling pathway 2.80 + 1.25E- 04
Huntington disease 2.71 + 2.85E- 02
Gonadotropin- releasing hormone receptor pathway 2.41 + 8.33E- 03
Unclassified 0.89 −0.00E00
Poor quality bull semen
Reactome
Extracellular matrix organization 3.26 + 1.08E- 05
Integrin cell surface interactions 4.02 + 4.67E- 02
Degradation of the extracellular matrix 3.95 + 7.60E- 03
Toll- like receptors Cascades 3.30 + 2.22E- 02
Hemostasis 2.05 + 1.21E- 02
Metabolism of proteins 1.72 + 5.07E- 03
Metabolism 1.55 + 2.72E- 03
Unclassified 0.79 −0.00E00
GPCR downstream signalling 0.42 −1.09E- 02
Panther
Plasminogen activating cascade 8.54 + 3.77E- 03
Blood coagulation 5.86 + 9.79E- 05
Unclassified 0.91 −0.00E00
+, upregulated; −, downregulated.
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SINGH et al.
2013; Sharma, Masaki, and Agarwal, 2013). In our present study, sper-
matozoal samples of two categorized bull semen were pooled and
subjected for proteomics analysis for three replicas from each group.
There are several studies in the proteomic literature that refer to the
profit of pooling samples where it may not be practicable to analyse
individual samples due to limitations of the sample, cost- effectiveness
or the study design (Batruch et al., 2011; Fung et al., 2004; Sharma,
Agarwal, et al., 2013; Sharma, Masaki, and Agarwal, 2013; Rolland
et al., 2013).
As per the earlier report by Peddinti et al. (2008), only proteins
identified by at least three peptides were included in the analysis
for differential expression. Peddinti et al. (2008) identified that 2,051
and 2,281 proteins were specifically expressed to high and low
fertility Holstein bull spermatozoa, respectively, and among them,
1,518 proteins were common to both. They showed that 125 pro-
teins were significantly expressed between high and low fertility bull
spermatozoa.
Biological systems exploit extremely complex, interconnected
metabolic as well as signalling pathways to its function. Consequently,
GO analysis was carried out to identify different molecular as well as
biological functions, cellular components, protein classes and signalling
pathways of our proteomic datasets. The molecular mechanisms and
signal transduction pathways mediating the processes of capacitation
as well as acrosome reaction have been partially defined (Braundmeier
& Miller, 2001). Proteomic analysis of the bull spermatozoa cytosolic
fraction showed enrichment for tyrosine kinases which are import-
ant for specific sperm proteins phosphorylation during capacitation
(Lalancette, Faure, & Leclerc, 2006).
From GO analysis of crossbred bull spermatozoa, it was observed
that catalytic activity, binding to different biological macromolecules
and structural molecular activities were comparatively superior.
Interactions with different receptor protein macromolecules were
mostly present in both spermatozoal proteomes; however, growth/
differentiation factor 6, alpha- 2- macroglobulin and complement C3/
C4- D were identified as top most cytokine receptors. Metabolism is
ranked to be the top most biological process in the identified proteome
datasets of crossbred bull spermatozoa. Ferrandi et al. (1987) reported
that spermatozoa capacitation is coupled with specific type of metab-
olism that is glycolysis or oxidative respiration. Peddinti et al. (2008)
reported that pyruvate metabolism and glycolysis were the top most
significant metabolic pathways represented in high fertility Holstein
Friesian sperm proteome, where 1-phosphatidylinositol 4,5-bisphos-
phate phosphodiesterase beta-1 (PLCB1) and NAD (P) transhydrogenic
mitochondrial protein were identified to be associated with metabolic
activities of crossbred bull spermatozoa. Mitochondrial proteins play a
critical role in variety of physiological process especially controlling the
FIGURE4 Different proteins and factors involved in sperm fertility and motility
604
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SINGH et al.
TABLE7 Top 10 functions/diseases and their respective top 10 signalling pathways for up regulated proteins in good quality bull semen
Signalling pathways
Gamma-
carboxylation of
protein precursors
Gamma- carboxylation,
transport, and
amino- terminal
cleavage of proteins
GABA
synthesis
GP1b- IX- V
activation
signalling
TP53 regulates
transcription of
several
additional cell
death genes
DNA
damage
bypass
Collagen
biosynthesis
and modifying
enzymes
Intraflagellar
transport
G beta: gamma
signalling
through PLC
beta
Post- chaperonin
tubulin folding
pathway
Physiological functions
Post- chaperonin
tubulin folding
pathway
2 (Q07175,
P07224)
Metabolism of protein 2 (Q07175, P07224)
Neuronal synthesis 1 (Q0VCA1)
Haemostasis 1 (P02453)
Gene expression
(Transcription)
1 (Q5EA80)
DNA repair 3 (A1A4K3,
Q2TBV1,
A5PJS6)
Extracellular matrix
organization
4 (P02453,
P79331,
P02459,
A6QPB3)
Organelle biogenesis
and maintenance
3 (Q3MHM5,
Q6B856,
Q3ZBU7)
Signal transduction 1 (P10894)
Metabolism of protein 1 (Q28205)
Q07175, Vitamin K- dependent gamma- carboxylase; P07224, vitamin K- dependent protein; Q0VCA1, glutamate decarboxylase; P02453, collagen alpha- 1(I) chain; Q5EA80, geranylgeranyl transferase type- 2
subunit alpha; A1A4K3, DNA damage- binding protein; Q2TBV1, replication factor C subunit 3; A5PJS6, ubiquitin carboxyl- terminal hydrolase; P79331, A disintegrin and metalloproteinase with thrombospondin
motifs 2; P02459, collagen alpha- 1(II) chain; A6QPB3, collagen alpha- 1(XVII) chain; Q3MHM5, tubulin beta- 4B chain; Q6B856, tubulin beta- 2B chain; Q3ZBU7, tubulin beta- 4A chain; P10894, 1- phosphatidylinositol
4,5- bisphosphate phosphodiesterase beta- 1; Q28205, tubulin- specific chaperone D.
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SINGH et al.
TABLE8 Top 10 functions/diseases and their respective top 10 signalling pathways for downregulated proteins in good quality bull semen
Signalling
pathways
TRKA
activation
by NGF
Regulation of cytoskeletal
remodelling and cell
spreading by IPP complex
components
Physiological
factors
Cross- linking
of collagen
fibrils
Eukaryotic
translation
termination
Acyl chain
remodelling
of CL
TP53 regulates
transcription of
cell cycle genes
Molecules
associated with
elastic fibres
Dectin- 2
family
GABA A
receptor
activation
Physiological functions
Signal
transduction
1 (P13600)
Cell- Cell
communication
1(Q3B7N2)
Muscle
contraction
1 (P55206)
Haemostasis 1 (P02465)
Metabolism of
protein
1(Q0VCX5)
Metabolism 1 (A4IFJ5)
Gene expression
(Transcription)
2 (E1BE02,
A4IFB6)
Extracellular
matrix
organization
2 (Q8SQB8,
P21214)
Immune system 1
(P08487)
Neuronal system 1 (P20237)
P13600, Beta- nerve growth factor; Q3B7N2, alpha- actinin- 1; P55206, C- type natriuretic peptide; P02465, collagen alpha- 2(I) chain; Q0VCX5, eukaryotic peptide chain release factor subunit 1; A4IFJ5, cytosolic
phospholipase A2; E1BE02, transcription factor E2F7; A4IFB6, CCR4- NOT transcription complex subunit 10; Q8SQB8, integrin beta- 6; P21214, transforming growth factor beta- 2; P08487, 1- phosphatidylinositol
4,5- bisphosphate phosphodiesterase gamma- 1; P20237, gamma- aminobutyric acid receptor subunit alpha- 4.
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SINGH et al.
oxidative energy supply and thus are central to growth, development
and differentiation. Sperm motility is greatly dependent on the ATP
generated by oxidative phosphorylation in the mitochondrial sheath
and thus results in flagellar movement (Shamsi et al., 2008). One of the
importantly identified phosphoprotein associated with different bio-
logical activities was osteopontin (OPN). OPN was identified in various
tissues and fluids including those of the male and female reproduc-
tive tracts. Erikson, Way, Chapman, and Killian (2007) demonstrated
that OPN exists at multiple molecular weight forms in the Holstein
bull reproductive tract and its presence on ejaculated sperm may sig-
nal its importance in fertilization by interacting with integrins or other
proteins on the oocyte plasma membrane. El-Haggar, Rashed, Saleh,
Taymour, and Mostafa (2013), however, reported a significant nega-
tive correlation of OPN with sperm count, sperm motility and seminal
glutathione peroxidase.
Potassium channels are essential for normal sperm physiology as
they regulate membrane potential as well as cell motility (Mannowetz,
Naidoo, Choo, Smith, & Lishko, 2013). A calcium- activated potassium
channel subunit alpha- 1 proteins were found to be involved in bio-
logical regulation of bull spermatozoa. Earlier studies revealed that an
alkalinization- sensitive sperm K+ channel, encoded by the Sol3, was
shown to be essential for male fertility in mice (Zeng, Yang, Kim, Lingle,
& Xia, 2011). Mannowetz et al. (2013) showed that Slo1 is the principal
potassium channel of human spermatozoa. Another biologically regu-
latory protein identified was nitric oxide synthetase- inducible protein.
Balercia et al. (2004) identified the role of nitric oxide concentrations
on human sperm motility. They revealed that nitric oxide concentra-
tion in human spermatozoa is negatively correlated with percentage
of total sperm motility. Beta 2 adrenergic receptor proteins were
found to be linked with biological activities of the bull spermatozoa.
Beta1- /2- subtypes of adrenergic receptor were shown to be predomi-
nantly expressed in sperm leydig and sertoli cells (Eikvar, Bjornerheim,
Attramadal, & Hansson, 1993; Hellgren, Sylven, & Magnusson, 2000;
Tolszczuk, Follea, & Pelletier, 1988; Troispoux, Reiter, Combarnous,
& Guillou, 1998). In vitro studies using selective adrenergic agonists
or antagonists indicated that beta adrenergic receptor might be in-
volved in the neuroendocrine control of testicular functions (Cooke,
Golding, Dix, & Hunter, 1982; Wanderley et al., 1989). Besides these,
DNA mismatch repairing protein, tubulin beta 4A,4B chain protein,
phospholipid- transporting protein, dynein- A, myosin- 1, tubulin-
specific chaperone D, Na- dependent dopamine transporter protein,
etc., were shown to be linked with different biological process of the
crossbred bull spermatozoa.
Importance of Ca2+- mediated CatSper channel towards sperm mo-
tility in the bull semen was depicted through reactome signalling path-
way. Slo3 and HV1 regulated channel helps to transport K+ and H+
ions, respectively. SPAM1 protein binds with hyaluronic acid which im-
proves sperm motility and velocity. Huszar, Willetts, & Corrales, (1990)
investigated that the hyaluronic acid improves retention of sperm mo-
tility and velocity in normospermic and oligospermic specimens. The
heavy and light chain of acrosin from the sperm controls the association
of Adam and B4GALT1 with zona pellucida of egg membrane. In addi-
tion with this complex formation, another two proteins Izumo and CD9
also make a combination to facilitate the sperm motility and binding
with egg membrane.
To signify the impact of upregulated and downregulated protein
controlling the sperm fertility in bull semen, we have tabulated 10 most
important signalling pathways and their corresponding physiological
functions. Different ion channel mediator proteins, neuronal system
regulatory proteins and factors were determined in this pathway anno-
tation. These proteins and factors are consecutively upregulated and
downregulated in the process of maintaining sperm fertility. Vitamin
K- dependent gamma- carboxylase helps in the Gamma- carboxylation
of precursors protein involved in the post- chaperonin tubulin folding
pathway. C- type natriuretic peptide manages the muscle contractions
which have an indirect effect on sperm motility. Collagen alpha- 2 (I)
chain helps in cross- linking the collagen fibrils during haemostasis.
DNA damage- binding protein and ubiquitin carboxyl- terminal hydro-
lase help in DNA repair. Tubulin beta- 4B chain, tubulin beta- 2B chain
and tubulin beta- 4A chain were involved in organelles biogenesis and
maintenance through intraflagellar transport. Tubilin- specific chaper-
one D helps in protein metabolism by post- chaperonin tubulin folding
pathway. Transcription factor E2F7 regulates transcription of cell cycle
genes involved in the sperm motility of bull semen. CCR4- NOT tran-
scription complex subunit 10 also works as same as E2F7.
In summary, our preliminary findings identified a list of proteins
in crossbred bull spermatozoa which may provide descriptive impli-
cations in sperm capacitation, acrosomal reaction and sperm–oocyte
communication, which may add some reference for developing protein
biomarkers. However, further studies with a large number of samples
and functional validation are necessary for providing comprehensive
conclusions.
ACKNOWLEDGEMENTS
The authors are thankful to the Director, ICAR- CIRC, and Meerut
for providing necessary facilities to carry out the present research.
Authors acknowledge the Science and Engineering Research Board,
Government of India for providing financial support under the pro-
ject YSS/2015/001482 to RS and RD (Mentor). We are also thankful
to Military Farm, Meerut, India, for providing experimental animals.
Authors also acknowledge ICAR- All India Coordinated Research
Project (AICRP) on Cattle for field progeny testing programme.
CONFLICT OF INTEREST
None of the authors have any conflict of interest to declare.
AUTHOR CONTRIBUTIONS
Rani Singh: Main author and PI. Gyanendra Singh Sengar involved in
protein extraction, proteomic data analysis, TUNEL assay and manu-
script drafting. Umesh Singh involved in sample collection. Rajib Deb:
involved in manuscript revision, implementation of the core idea of
the research. Vivek Jungare involved in identification of different
proteins and factors involved in sperm fertility and motility. Saugata
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SINGH et al.
Hazra involved in GO analysis. Sushil Kumar involved in semen sam-
ple collection. Shrikant Tyagi: involved in evaluation of semen quality.
Achintya K. Das: involved in field progeny testing. T V Raja: involved in
statistical data analysis. Ashish Kumar involved in SDS- PAGE.
ORCID
R Deb http://orcid.org/0000-0001-8670-4583
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How to cite this article: Singh R, Sengar GS, Singh U, et al.
Functional proteomic analysis of crossbred (Holstein
Friesian × Sahiwal) bull spermatozoa. Reprod Dom Anim.
2018;53:588–608. https://doi.org/10.1111/rda.13146
