JO U R N A L OF PR O TE O MI CS 82 ( 20 1 3 ) 6 4 –80

Available online at www.sciencedirect.com

www.elsevier.com/locate/jprot

Proteins associated with critical sperm functions and sperm

head shape are differentially expressed in morphologically
abnormal bovine sperm induced by scrotal insulation

Habib A. Shojaei Saadia , Evine van Riemsdijkb , Alysha L. Dancea ,

Gayathri D. Rajamanickama , John P. Kastelica , Jacob C. Thundathila,⁎
a
Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, T2N 4N1, Canada
b
Department of Animal Sciences (Adaptation Physiology Group), Wageningen Agricultural University, Box 338,
Wageningen, 6700 AH, The Netherlands

AR TIC LE I N FO ABS TR ACT

Article history: The objective was to investigate expression patterns of proteins in pyriform sperm, a common
Received 19 October 2012 morphological abnormality in bull sperm. Ejaculates were collected from sexually mature
Accepted 27 February 2013 Holstein bulls (n = 3) twice weekly for 10 weeks (pre-thermal insult samples). Testicular
Available online 14 March 2013 temperature was elevated in all bulls by scrotal insulation for 72 consecutive hours during
week 2. Total sperm proteins were extracted from pre- and post-thermal insult sperm samples
Keywords: and subjected to two-dimensional gel electrophoresis. Among the protein spots detected, 131
Bull spots were significantly expressed (False Detection Rate <0.01) with ≥2 fold changes between
Pyriform sperm defect normal and pyriform sperm. Among them, 25 spots with ≥4 fold difference in expression
Abnormal sperm morphology patterns were identified using liquid chromatography coupled with tandem mass spectrometry
Proteomics (LC–MS/MS). Expression of several proteins involved in sperm capacitation, sperm–egg inter-
2-D gel electrophoresis action and sperm cytoskeletal structure was decreased in pyriform sperm, whereas proteins
regulating antioxidant activity, apoptosis and metabolic activity were increased. Contents of
reactive oxygen species and ubiquitinated proteins were higher in pyriform sperm. In addition to
understanding the molecular basis of functional deficiencies in sperm with specific morpholog-
ical abnormalities, comparing normal versus morphologically abnormal sperm appeared to be a
suitable experimental model for identifying important sperm functional proteins.

Biological significance
To our knowledge, this study is the first report on differential expression of proteins in pyriform
bovine sperm versus morphologically normal sperm. We report that expression of several
proteins involved in sperm capacitation, sperm–egg interaction and sperm cytoskeletal
structure was decreased in pyriform sperm, whereas proteins which regulate antioxidant
activity, apoptosis and metabolic activity were increased. Contents of reactive oxygen species
and ubiquitinated proteins were higher in pyriform sperm. In addition to understanding the
molecular basis of functional deficiencies in sperm with specific morphological abnormalities,
our results suggest that comparing normal versus morphologically abnormal sperm appeared
to be a suitable experimental model for identifying important sperm functional proteins.
© 2013 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, HMRB 400,
3330 Hospital Dr., NW, Calgary, AB, Canada T2N 4N1. Tel.: + 1 403 220 8244; fax: + 1 403 210 6693.
E-mail address: jthundat@ucalgary.ca (Jacob C. Thundathil).

1874-3919/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jprot.2013.02.027
 JO U R N A L OF P ROTE O MI CS 8 2 ( 20 1 3 ) 6 4–8 0
1. Introduction
Sperm morphology has been recognized as a key determinant of
semen quality in both humans and animals. Furthermore, it has
also been considered as an excellent predictor of the outcome of
natural mating [1,2], intrauterine [3] and artificial insemination
[4], and success of in vitro fertilization [5]. There are several
classification systems for bovine sperm abnormalities based on
origin (primary, secondary or tertiary), effect on fertility (major
vs. minor), portion of sperm affected (head, midpiece, or tail), or
whether its effects on fertility can be ameliorated by increasing
the number of sperm in the inseminate (compensable vs.
uncompensable [6]). Pyriform or “pear-shaped” sperm is the
most common uncompensable sperm defect in bulls [6],
stallions [7] and cats [8]. Pyriform sperm is considered a major
sperm defect [6] and is characterized by a normally shaped
acrosome [9–12] with severe elongation and narrowing of the
post-acrosomal region [10]. Elongated human sperm heads are
classified as either pyriform or tapered [13], depending on the
severity of the defect.
In one study [6], the incidence of pyriform sperm in bulls used
for natural service or artificial insemination was 9 and 16%,
respectively. Regarding the latter, pyriform sperm ranged from
10 to more than 50% in semen samples. In humans, the average
incidence of elongated-head sperm (pyriform) in semen was
5.9% in fertile and 22% in subfertile men [13]. Elongation of the
sperm head is generally recognized as a stress-induced sperm
aberration (due to abnormal Sertoli cell function [14]). Meiotic
non-disjunctions or spermiogenesis anomalies were recently
suggested as causes of this abnormality [13]. In humans, the
incidence of this sperm abnormality was increased in patients
with male accessory gland infection and varicocele [15,16], as
well as in persons working in plants producing reinforced plastic
[17]. Pyriform sperm have normal motility [14,18] and are capable
of passing through cervical mucus in vitro [19]. Therefore, there
should not be any reduction in the number of affected sperm
reaching the zona pellucida in vivo [9,11,12,20]. Consistent with
this observation, the majority (>90%) of morphologically abnor-
mal sperm on the zona pellucida in human were pyriform [9].
Moreover, 98% of subfertile men and 100% of fertile men had
pyriform sperm in their semen samples [21].
Thundathil et al., (1999) reported that bovine pyriform sperm
had a reduced ability to bind to and penetrate the zona pellucida,
and the resulting zygotes had a reduced ability to initiate
cleavage [18]. In men with severely elongated sperm, a low ICSI
fertilization rate was reported [22,23]. In addition, semen with a
high proportion of pyriform sperm had increased both fertiliza-
tion failure and proportion of degenerated embryos in humans
[23,24], mice [25] and cattle [11,12,18,26]. Moreover, impaired
chromatin compaction, as well as slightly increased chromo-
some aneuploidies, were reported for sperm with an elongated
head [13]. Since sperm functions are regulated by proteins
already present in sperm, these functional impairments of
pyriform sperm may be associated with expression of proteins
in affected sperm.
Elevated testicular temperature induced by impaired scrotal/
testicular thermoregulation is believed to be a common cause of
abnormal spermatogenesis and impaired sperm function in
domestic farm animals and in humans [27,28], with outcomes
65
ranging from subclinical infertility to sterility [29]. This principle
has been widely utilized in research studies to induce various
sperm abnormalities, including pyriform sperm [30–33]. Using
pyriform sperm induced by scrotal insulation of bulls as an
experimental model, the objective of this study was to investi-
gate expression of sperm proteins in pyriform sperm to elucidate
the molecular basis of impaired function and development of
this sperm defect.
2. Materials and methods
2.1. Reagents and media
Unless otherwise stated, all chemicals were purchased from
Sigma Aldrich (Oakville, ON, Canada); Percoll was from GE
Health Care Biosciences (Baie D'Urfe, QC, Canada); Tris–HCl,
bromophenol blue, glycine, methanol, and Tween-20 were from
Fisher Scientific (Edmonton, AB, Canada); 10× Tris/Glycine/SDS
Buffer, and the RC DC protein assay kit, and 2-D gel electropho-
resis apparatus were from Bio-Rad (Mississauga, ON, Canada);
and Kodak scientific imaging film was from Mandel Scientific
(Guelph, ON, Canada).
2.2. Bull selection, scrotal insulation and semen collection
Three mature (3 to 6 years) Holstein bulls producing satisfactory
semen (based on current standards for motility and morphology
[34]) and normal fertility (non-return rate ranged from 66 to 69%)
were selected for this study. Animal maintenance and handling,
as well as all experimental procedures, were performed in
accordance with the guidelines of the Canadian Council on
Animal Care and the policies of the Animal Care Committee of
the University of Calgary. Ejaculates were collected (with an
artificial vagina) twice-weekly for 2 weeks to ensure that semen
from these bulls had normal motility (≥70% progressively motile
sperm) and morphology (≥70% morphologically normal sperm).
Thereafter, the scrotum of each bull was insulated for 3 days
(start of thermal insult = Day 0) using commercial disposable
baby diapers. The diaper material was applied on the scrotum
and secured with duct tape, so that it completely insulated the
scrotum (including the scrotal neck). Scrotal surface temperature
was not recorded in this study. However, it was reported that
similar insulating procedures increased scrotal temperature by 1
to 2.5 °C [33]. Semen samples were collected twice-weekly for
8 weeks (after the start of scrotal insulation) to collect sperm
containing high proportions of specific types of morphological
abnormalities, as reported [31]. This semen collection schedule
was adopted to characterize occurrence of specific sperm
abnormalities in semen [31] and procure semen samples with a
high percentage of pyriform sperm.
2.3. Evaluation of the sperm morphology
Eosin–nigrosin stained smears were prepared from each ejacu-
late and examined under oil immersion (1000×) using a Leica DM
2500 microscope (Leica Microsystems GmbH Wetzlar, Germany).
Sperm were classified based on morphology, as described [6].
Classical pyriform sperm are distinctly pear-shaped; the acroso-
mal region is full and rounded and the postacrosomal region is
66 JO U R N A L OF PR O TE O MI CS 82 ( 20 1 3 ) 6 4 –80
narrow. However, sperm slightly pinched in the postacrosomal
region and those with a severely narrowed, paddle-shaped
sperm head were also considered pyriform sperm (Fig. 1B–D).
2.4. Sperm preparation
Pre-and post-scrotal insulation ejaculates from each of the
three bulls were cryopreserved using an egg yolk-based semen
extender. Twenty straws (0.5 ml each) of frozen semen pre-
pared from the pre- and post-insulation ejaculates from the
same bull (n = 3 bulls) were thawed in a water bath maintained
at 37 °C for 1 min and the contents of the straws were pooled in
a 15 ml centrifugation tube. Post-thaw semen from the pre-
scrotal insulation ejaculates (normal control) contained ≥70%
morphologically normal sperm with ≥40% progressively motile
sperm (Table 1), whereas post-scrotal insulation semen from
these bulls contained ≥80% pyriform sperm with ≥20% progres-
sively motile sperm (Table 2). These sperm preparations (4 ml
aliquots) were loaded onto a 45% isotonic Percoll (4 ml) and
centrifuged at 700 ×g for 30 min at 24 °C. The purpose of this
modified Percoll wash was to separate sperm from semen
extender. The supernatant was removed and the sperm pellet
was washed twice in phosphate buffered saline by centrifuga-
tion at 500 ×g for 10 min. Percoll-washed sperm preparations
were evaluated for sperm morphology and motility. Washing of
frozen-thawed sperm in 45% isotonic Percoll had only a minor
effect on post-thaw sperm characteristics (Tables 1 and 2). The
resulting sperm pellets obtained from the control and treat-
ment samples were used for protein extraction, as described
below.
2.5. Extraction of sperm total proteins
Percoll-washed sperm pellets were resuspended in a protein
extraction buffer containing 8.0 M urea, 2.0 M thiourea, 20 mM
dithiothreitol (DTT), 1.2 mM tributylphosphine (TBP), 4% w/v
CHAPS, and 1× protease inhibitor cocktail. Homogenization was
performed by intermittent vortexing of the sample in extraction
buffer for 3–5 min at a time, for 1 h. Samples were kept on ice
during this process. Subsequently, samples were centrifuged for
A B
Fig. 1 – Bovine sperm (1000 X) with normal (A) and varying degrees of pyriform morphology (B–D). Pyriform sperm is
characterized by a normal acrosomal region, but abnormal narrowing of the post acrosomal region.
10 min at 10,000 ×g, and the supernatant collected. An aliquot
of the supernatant was assayed to quantify the extracted total
sperm protein (RC DC colorimetric protein assay, Bio-Rad,
Mississauga, ON, Canada). The remaining supernatant was
aliquoted and frozen at −80 °C.
2.6. 2-D gel electrophoresis
Protein extracts prepared from normal and pyriform sperm from
each of the three bulls were subjected to 2-D gel electrophoresis
to determine differential expression of sperm proteins. At least
three replicates (using the same protein samples) were complet-
ed for each bull (to account for technical variations).
2.6.1. Rehydration of IPG strips
Immobilized pH gradient (IPG) strips (17 cm, pH 3–10 Nonlinear;
Bio-Rad) were used. Strips were passively rehydrated overnight
at room temperature with 300 μg protein in extraction buffer.
Prior to rehydration, total protein (2 μg/μl in extraction buffer),
was mixed 1:1 with extraction buffer containing 1% (v/v)
broad-range ampholytes (3–10; Bio-Rad), 0.2% (v/v) each of five
narrow-range ampholytes (Bio-Rad), 3–5, 4–6, 5–7, 7–9, 8–10,
acrylamide monomer (3% v/v), and bromophenol blue (0.002%
w/v). All components were added in one step and thoroughly
mixed by vortexing. The IPG strip was laid (gel side down) on the
extraction buffer containing protein, in a rehydration tray, and
the strip was overlaid with mineral oil to prevent evaporation.
2.6.2. Isoelectric focusing
Isoelectric focusing (IEF) was performed on a Protean IEF Cell,
(Bio-Rad) at 17 °C using a focusing tray designed for 21-cm IPG
strips. Chromatography paper (3 mm thick; Fisher Scientific,
Toronto, ON, Canada) was used to make 3-cm electrode wicks. A
larger focusing tray was used to accommodate longer electrode
wicks, which enhanced removal of salts [35]. Voltage was
ramped to 250 V for 15 min; this desalting step was repeated
once. Thereafter, voltage was linearly ramped to 4000 V over
2 h, with a maximum of 50 μA/gel. Electrode wicks were
changed as needed to remove salts and facilitate linear
ramping. The IPG strips were focused at 4000 V for a total of
C D
 JO U R N A L OF P ROTE O MI CS 8 2 ( 20 1 3 ) 6 4–8 0 67

Table 1 – Post-thaw characteristics of pre-scrotal insulation samples of bovine semen.

Bull Progressively Pyriform Other Normal
motile sperm (%) sperm (%) defects (%) sperm (%)

Pre-wash a Post-wash b Pre-wash Post-wash Pre-wash Post-wash Pre-wash Post-wash

A 45 48 2 2 5 5 93 93
B 40 44 1 0 3 2 96 98
C 42 44 0 0 5 5 95 95
a
Before percoll wash;
b
After percoll wash.

75,000 V-hours. Strips were held at 500 V until taken out of the
IEF Cell; they were immediately equilibrated and run on the
second dimension.
2.6.3. SDS-PAGE
Focused IPG strips were sequentially equilibrated in buffer
containing 6.0 M urea, 375 mM Tris, 20% glycerol, 2% sodium
dodecyl sulfate, and 130 mM DTT for 10 min, followed by
alkylation for 10 min in buffer containing 350 mM acrylamide
monomer. The second dimension was carried out on large
format polyacrylamide gels (18 × 18 × 0.1 cm) with 5% stacking
gels. The focused IPG strip was sealed to the top of the stacking
gel with 0.5% low melting agarose solution in 375 mM Tris and
0.007% bromophenol blue. Electrophoresis was carried out in a
cold room (4 °C) using pre-chilled running buffer (Tris/Glycine/
SDS Buffer for SDS-PAGE applications; BioShop, Burlington, ON,
Canada). Gels were run in a Protean II xi Cell tank (Bio-Rad) at a
constant current of 24 mA/gel for ~8 h, with a maximum limit of
300 V. To minimize technical variations, four gels (control and
treatment) were cast and run simultaneously.
2.6.4. Staining and imaging of SDS-PAGE gels
After completion of the second dimension, gels were fixed over
night in 10% methanol and 7% acetic acid. Gels were washed
with double deionized water, three or four times over 1 h to
remove fixative, followed by overnight staining with SyproRuby
(Bio-Rad). Stained gels were destained in fixative solution (10%
methanol and 7% acetic acid) for 30 min, followed by washing in
double deionised water for 1 h. Gels were imaged with ProXpress
Proteomic Imaging System (PerkinElmer, Waltham, MA, USA).
2.6.5. Protein profile analysis
To evaluate protein profiles of morphologically abnormal and
normal sperm, gel images were compared and analyzed using gel
analysis software, Delta2D 4.0 (Decodon, Greifswald, Germany).
To enable quantitative comparative spot analysis, normalization
occurred at two stages; first at 2D-PAGE by loading a consistent
amount of protein; and second at analysis using Delta2D, by
expressing normalized spot volumes as a fraction of the sum of
all spot volumes in the gel [36]. Strict criteria were used for
selecting differentially expressed (DE) protein spots: (i) significant
difference in protein expression (Wilcoxon Rank Sum Test,
P < 0.05; n = 6 gels for pyriform sperm and n = 7 gels for normal
sperm); (ii) 100% reproducibility (coefficient of variation = σ/μ < 1)
across the sample population; and (iii) False Discovery Rate
(FDR) < 0.01 [37]. In addition, to assess the most accurate MW and
pI of the interested spots, one additional gel was subjected to 2-D
gel electrophoresis using a standard protein kit (SERVA Proteome
Markers, 5 vials; SERVA Electrophoresis GmbH, Heidelberg,
Germany) to make a standard gel image for estimation of MW
and pI of spots of interest, using specialized gel analysis software
(Delta2D 4.0). The standard consisted of 8 proteins (cytochrome C,
myoglobin, ß-lactoglobulin, glucose-1-dehydrogenase, lipase,
catalase, albumin, and glucose oxidase), with a pI range from
5.5 to 9.8 and a MW range from 11.7 to 77.0 kDa.
2.7. Identification of the proteins
Isoelectric point and molecular weight of differentially ex-
pressed protein spots (with ≥4 fold change in the expression
pattern) were used in the TagIdent program of ExPASy (www.
expasy.org) for selecting candidate proteins. Potential functions
of these candidate proteins in regulation of sperm function were
evaluated using NCBI databases. Subsequently, these protein
spots were dissected from the gels, digested with trypsin, and
subjected to nanoscale liquid chromatography (LC), coupled
with tandem mass spectrometry (MS/MS), and identified by
comparing their peptide sequences to a database of candidate
proteins [38]. In that regard, probability-based MASCOT scores
were used to evaluate identifications. Only matches with
P < 0.05 for random occurrence were considered significant.
Peptide sequences were queried through the NCBI database with

Table 2 – Post-thaw characteristics of post-scrotal insulation samples of bovine semen.

Bull Progressively Pyriform Other Normal
motile sperm (%) sperm (%) defects (%) sperm (%)

Pre-wash a Post-wash b Pre-wash Post-wash Pre-wash Post-wash Pre-wash Post-wash

A 30 30 82 80 10 8 8 12
B 24 25 85 82 8 4 7 14
C 20 22 88 84 9 6 3 10
a
Before percoll wash.
b
After percoll wash.
68 JO U R N A L OF PR O TE O MI CS 82 ( 20 1 3 ) 6 4 –80
the search engine MASCOT 2.103 (Matrix Science, London, UK;
http://www.matrixscience) for Bos taurus, unless the interested
protein with appropriate MW and pI was not detected for Bos
taurus and the substantial species was selected. Moreover,
bovine trypsin and keratin identification were discarded [39].
Most of the MW and pI of the selected spots assessed by standard
gels using image analysis software (as described in Section 2.6.5)
were consistent with the MW and pI values of the identified
proteins.
2.8. Pathway analysis and network construction
The Ingenuity Pathways Analysis (IPA) (Ingenuity Systems,
www.ingenuity.com) was used for indentifying the top pathway
and network of the proteins of interest differentially expressed
in pyriform sperm. In that regard, lists of identified proteins,
containing gene identifiers and corresponding expression
values were uploaded into the IPA. Each gene identifier was
mapped to its corresponding gene object in the Ingenuity
Pathways Knowledge Base. Networks of genes were then
algorithmically generated, based on their connectivity. The
“intracellular and second messenger signals” of the “canonical
pathways” tool of IPA software were used to identify the most
significant intracellular signals among the DE proteins. Fisher's
Exact test was used to calculate a P-value determining the
probability that each canonical pathway assigned to the data
set was due to chance alone.
2.9. Immunoblotting and confirmation of the differential
expression of proteins
Based on the availability of commercial antibodies, a cohort of
DE proteins (tyrosine phosphorylated proteins, CLU, HSPD1)
identified by LC–MS/MS were selected for confirmation by
immunoblotting. Sperm protein extracts were subjected to
electrophoresis (SDS-PAGE) and transferred to nitrocellulose
membranes in transfer buffer (25 mM Tris, 0.2 M glycine, 20%
Methyl alcohol, pH 8.5) at a constant voltage (100 V). The resulting
nitrocellulose membranes were stained with 0.2% Ponceau in
acetic acid to determine the efficacy of electrotransfer and equal
loading of wells. Subsequently, non-specific binding sites were
blocked with 5% w/v skim milk in Tris-buffered saline (20 mM, pH
7.8) containing Tween-20 (0.1% v/v; TTBS) for 45 min. Membranes
were incubated (overnight at 4 °C) with a primary antibody
against candidate proteins. Membranes were washed several
times in TTBS, incubated (1 h at room temperature) with
secondary antibody conjugated to horseradish peroxidase, and
washed (three times, 10 min each). Protein bands were identified
using chemiluminescence. At least two replicates were complet-
ed for each bull for immunoblotting experiments.
2.10. Functional validation of upregulation of pathways in
pyriform sperm
2.10.1. Generation of reactive oxygen species (ROS)
Generation of ROS was measured using the intracellular indicator
2, 7-dicholorofluorescein diacetate (DCFDA; Sigma-Aldrich, Oak-
ville, ON, Canada). This compound is a cell-permeable non-
fluorescent probe that is de-esterified in the cells and cleaved into
fluorescent 2′, 7′-dichlorofluorescein (DCF) upon oxidation by
ROS [40]. Briefly, frozen-thawed sperm were Percoll-washed as
described previously and sperm (1 × 106/ml) were incubated in
PBS containing 10 μM DCFDA in the dark at 37 °C for 30 min. An
aliquot of sperm preparation from each of the groups was
examined (100×) under a fluorescent microscope (Leica RM2500,
Wetzlar, Germany) to characterize DCF fluorescence (excitation
at 485 nm and emission at 535 nm). In addition, the DCF
fluorescence was quantified using a 96-well black plate reader
and expressed as ROS content in arbitrary units of fluorescence.
2.10.2. Evaluation of protein ubiquitination
Frozen-thawed sperm were separated from the extender by
washing sperm in 45% isotonic Percoll, as described above.
Since abnormal sperm are primarily ubiquitinated on the
surface [41], we chose to extract total membrane proteins. The
sperm pellets from the normal and pyriform sperm were
suspended in a lysis buffer (1 M NaCl, 20 mM imidazole, 1 mM
EDTA, 1% Triton X-100, pH 6.0, and protease inhibitor cocktail)
for 30 min at 4 °C. That there was a minor proportion of soluble
or cytoskeletal proteins in the extract after detergent extraction
cannot be excluded. Total protein concentrations were estimat-
ed using a DC protein assay kit (Bio-Rad, Mississauga, ON,
Canada). From this, 8 μg of total protein was subjected to
electrophoresis on a 10% SDS-PAGE gel, transferred to 0.2 μm
nitrocellulose membrane and blocked with 5% non-fat dried
milk for 1 h at room temperature. Subsequently, the membrane
was incubated overnight at 4 °C with the mouse monoclonal
anti-ubiquitin antibody MK-12-3 (1:250, MBL International Corp,
Nagoya, Japan) which can detect ubiquitinated substrates in
bovine species [41] and was found to cross react with human,
mouse and rat. The membrane was washed and further
incubated with horse radish peroxidase conjugated goat
anti-mouse secondary antibody (Millipore, Billerica, MA, USA)
and immunoreactive bands were identified using chemilumi-
nescence. In addition to loading samples based on protein
concentration, the resulting nitrocellulose membrane was
silver-stained to verify equal loading of proteins for normal
and pyriform sperm [42].
3. Results
3.1. Expression pattern of sperm proteins differed in
pyriform compared to morphologically normal sperm
Based on analysis of the 2-D gels obtained from pyriform versus
normal sperm, 131 protein spots were differentially expressed
(Figs. 2 and 3). Among them, 70 spots were upregulated and 61
spots were downregulated in pyriform sperm (Fig. 3). Thirty-
eight spots had ≥4 fold change in their expression patterns;
among them, only 25 spots were selected (as described previous-
ly) for protein identification by LC–MS/MS.
A list of differentially expressed proteins and their potential
function is shown (Table 3). Identified sequences of these
proteins were provided as a supplementary file (online version
only). Based on LC–MS/MS, mitochondrial heat shock protein D1
(HSPD1; 60 kDa), Calcium-binding tyrosine phosphorylation-
regulated protein (CABYR), F-actin-capping protein subunit beta
isoform 1 (CAPZB), prohibitin (PHB), Troponin C-like protein
(CALM2), two isoforms of sperm acrosome membrane-associated
 JO U R N A L OF P ROTE O MI CS 8 2 ( 20 1 3 ) 6 4–8 0 69

Fig. 2 – Two-dimensional gel demonstrating differentially expressed sperm proteins (spot boundaries marked) between
normal and pyriform sperm. Total protein extracts prepared from normal and pyriform sperm (n = 3 bulls each) were subjected
to 2-D gel electrophoresis. The gels were fixed in acetic acid–ethanol fixative and stained with Sypro Ruby. Gel images were
analyzed using Delta 2-D gel analysis software. The differentially expressed proteins were determined by comparing their
mean normalized spot volume.

protein 3 precursor (SPACA3), and Seminal fluid protein A3-
bovine (BSP-A3) were down regulated in pyriform sperm and
reported to be involved in sperm capacitation [43–48] and sperm–
egg interactions. Spermatogenesis-associated protein 19, mito-
chondrial precursor (SPATA19), Heat shock protein beta-9
(HSPB9) and two isoforms of Dynein light chain roadblock-type
2 (DYNLRB2) were down regulated in pyriform sperm and
regulating sperm cytoskeleton and flagella organization [49–52].
Various isoforms of Clusterin (CLU), Epididymal secretory
glutathione peroxidase precursor (GPX5), peroxiredoxin 5 pro-
tein (PRDX5) as well as Histidine triad nucleotide-binding protein
2 (HINT2) were upregulated in pyriform sperm and related to
cellular stress and apoptosis. L-asparaginase (ASRGL1) and two
isoforms of ATPD5 were upregulated in pyriform sperm and
70 JO U R N A L OF PR O TE O MI CS 82 ( 20 1 3 ) 6 4 –80
Mean normalized spot volume Relative expression difference
Pyriform sperm Spot more abundant in
Normal sperm pyriform sperm
Spot more abundant in
normal sperm
Fig. 3 – Differentially expressed sperm proteins in morphologically normal vs. pyriform sperm. 131 protein spots were
differentially expressed. Among them, 70 spots were upregulated and 61 spots were downregulated in pyriform sperm.
Thirty-eight spots had ≥ 4 fold change in their expression patterns.
reported to be associated with metabolism (Table 3). Most of
these identified proteins were localized in the acrosomal region
and midpiece (Table 3).
Consistent with the results of 2-D gel electrophoresis, a
cohort of phosphotyrosine containing proteins were down-
regulated in pyriform sperm compared to morphologically
normal sperm (Fig. 4; Panel A, lane 2). Similarly, Clusterin
content was increased in pyriform sperm (Fig. 4; Panel B, lane 2)
and HSPD1 was down regulated in pyriform sperm (Fig. 4; Panel
C; Lane 2), as evidenced in 2-D gel electrophoresis.
3.2. Signaling pathways regulating cell death, cellular
growth and proliferation, and cell morphology were the top
networks modulated in pyriform sperm
According to the Ingenuity pathway analysis (IPA), cell death,
cellular growth and proliferation, and cell morphology were the
significant networks for pyriform sperm. CLU (cellular stress
and apoptosis), CABYR (sperm–egg interaction and capacita-
tion), CAPZB (sperm capacitation), CST6 (structural integrity),
DYNLRB2 (sperm flagella formation), GPX5 (cellular stress and
apoptosis), HSPD1 (sperm–egg interaction and capacitation),
PHB (Sperm–egg interaction and capacitation), PRDX5 (cellular
stress and apoptosis) and SPATA19 (flagella formation) were
the identified molecules of these networks. Moreover, protein
Mean normalized spot volume Relative expression difference
Pyriform sperm Spot more abundant in
Normal sperm pyriform sperm
Spot more abundant in
normal sperm
ubiquitination pathway was the only significant pathway
identified by ingenuity software, whereas HSPD1 and HSPB9
were the molecules reported to be involved in this pathway. In
addition, proteins associated with oxidative stress were the top
identified toxicology list detected in pyriform sperm. Functional
analysis of identified proteins using the IPA revealed significant
enrichment of genes in ‘Molecular and cellular functions’
(Table 4). This table includes genes annotated for identified
proteins according to their “molecular and cellular functions”
based on Ingenuity software database. Moreover, this molecular
and cellular functional classification was not specifically for
germ cells, but was based on available reported functions for
these proteins in all types of cells. For instance, CAPZB is
involved in Cell-to-Cell Signaling and Interaction, Cellular
Development, Cell Morphology, Cellular Function and Mainte-
nance, and Cellular Assembly and Organization.
3.3. Content of ROS and ubiquitinated proteins were higher
in sperm preparations with high proportion of pyriform sperm
3.3.1. Content of ROS in pyriform sperm
Since proteins (CLU, GPX5 and PRDX5) associated with the
regulation of oxidative stress were the top identified toxico-
logical list detected in pyriform sperm, we determined ROS
generation in pyriform sperm as a functional validation of
 JO U R N A L OF P ROTE O MI CS 8 2 ( 20 1 3 ) 6 4–8 0
pathways associated with the level of oxidative stress in
pyriform sperm. Representative microscopic fields for sperm
preparations with normal and pyriform sperm are shown (Fig.
5A and B, respectively). Compared to the normal sperm sample,
the pyriform sample had more sperm with complete or partial
DCF fluorescence (fluorescence was limited to the sperm head
and appeared as dots). Based on quantification of these fluo-
rescence data, generation of ROS was higher (p < 0.05) in sperm
preparations with pyriform sperm compared to morphological-
ly normal sperm (Fig. 5C).
3.3.2. Content of ubiquitinated proteins in pyriform sperm
Since protein ubiquitination pathway was the only significant
pathway identified by IPA analysis, we determined protein
ubiquitination in pyriform sperm. The anti-ubiquitin antibody
MK-12-3 detected several immunoreactive bands in both normal
and pyriform sperm. However, the relative intensity of the
protein bands approximately at 130, 90, 75 and 45 kDa was
higher in pyriform versus morphologically normal sperm (Fig. 6).
4. Discussion
The objective of this study was to characterize expression
patterns of sperm proteins in pyriform sperm, a common
sperm abnormality in both human and animal species, which
reduces fertility. Proteomics coupled with bioinformatic ap-
proaches were used to compare expression of sperm proteins
in pyriform sperm versus morphologically normal sperm ob-
tained from the same bulls. In this study, a cohort of structural
and functional proteins were differentially expressed be-
tween normal and pyriform sperm, including proteins asso-
ciated with sperm capacitation, sperm–egg interactions,
cytoskeletal structure and sperm morphology, sperm flagella
organization, oxidative stress, apoptosis, and metabolism.
4.1. Differential expression of sperm proteins associated
with sperm capacitation and sperm–egg interactions
Sperm functional proteins involved in regulation of sperm capac-
itation (HSPD1, CAPZB, CABYR, Troponin C-like protein) and
sperm–egg interactions (SPACA3, PHB) were expressed at low
levels in pyriform sperm compared to morphologically normal
sperm. It was reported that pyriform sperm had a moderate
ability to bind to the human zona pellucida [9] and reduced
capability to bind to and penetrate the bovine zona pellucida [18].
HSPD1 (HSP 60) is a chaperone; it has been described as a key
tyrosine-phosphorylated protein on the murine sperm surface
following capacitation [43] involved in zona pellucida binding
[53]. In bull sperm, this protein was localized to the midpiece
region. Furthermore, it was present in the oviduct epithelial cell
(OEC) plasma membrane, which can be incorporated to sperm
[54]. However, additional studies are required to document the
role of this protein in regulation of sperm function in bulls.
Actin, a major cytoskeletal protein, exits as monomeric
G-actin and as polymerized microfilaments (F-actin) in mam-
malian cells [55]. Actin polymerization and depolymerization are
associated with regulation of various cell functions. F-actin
capping proteins such as CAPZB regulate actin polymerization
by capping the fast growing ends of actin filaments [56]. CAPZ
71
can exist as heterodimers of α-(2 isoforms) and ß-subunits (3
isoforms); both subunits are required for capping actin filaments
[57]. In mammalian male germ cells, actin is believed to have a
role in determination of cell shape during spermiogenesis,
regulation of sperm motility [55] and capacitation and the
acrosome reaction [44] through its remodeling. Therefore,
lower expression of CAPZB in pyriform sperm may contribute
to an abnormal head shape and impaired ability to undergo
capacitation and acrosome reaction, affecting their ability to
bind with and penetrate the zona pellucida.
Calcium-binding tyrosine phosphorylation-regulated pro-
tein (CABYR) is a signaling protein, highly polymorphic and
localized in sperm fibrous sheath [45] and reported to be
associated with AKAPs and Ropporin [58,59]. In addition,
CABYR was reported to be involved in capacitation through
tyrosine [45], serine, or threonine [46] phosphorylation. Reduced
content of this protein in pyriform sperm may alter its ability to
undergo capacitation. Troponin C-like protein, also known as
Calmodulin [60], is present in both the cytoplasm and
perinuclear space of spermatids and sperm [61], and is relocated
to the postacrosomal region during epididymal transit [62].
Calmodulin is involved in calcium-mediated intracellular
signaling and has been implicated in sperm hyperactivation
[63], capacitation, and the acrosome reaction [47].
Two isoforms of SPACA3 (also known as sperm lysosomal-
like protein 1 (SLLP1)) were down-regulated in pyriform sperm. It
has been suggested that SPACA3 is involved in sperm–egg
interactions during fertilization [48,64,65]. It was localized on
the acrosome of human sperm, and may serve as a receptor for
egg membrane saccharide N-acetylglucosamine [48]. Very re-
cently, its interaction and binding with oolemmal SAS1B (Sperm
Acrosomal SLLP1 Binding) during fertilization was reported in
mice [65].
Prohibitin (PHB), a major protein of the mammalian sperm
mitochondria, enables targeted degradation of paternal mito-
chondria (PHB ubiquitination) after fertilization, leading to
maternal inheritance of mitochondrial DNA [66]. In a previous
study, pyriform sperm had reduced rates of cleavage [18].
Reduced expression of PHB in pyriform sperm and associated
imbalance in mitochondrial inheritance may contribute to
reduced developmental competence of zygotes resulting from
fertilization of oocytes by pyriform sperm.
Since many phosphotyrosine-containing proteins (which
have a major role in sperm capacitation [67]) were differen-
tially expressed in pyriform sperm (based on 2-D gel electro-
phoresis), we used total sperm proteins extracted from
normal and pyriform sperm in immunoblotting and con-
firmed that the content of phosphotyrosine containing pro-
teins were reduced in pyriform sperm. Collectively, reduced
expression of the above-described proteins involved in sperm
capacitation and sperm–egg interaction was consistent with
our previous observation that pyriform sperm had reduced
ability to bind to and penetrate zona pellucida, as well as a
reduced ability to initiate clevage of resulting zygotes [18].
4.2. Structural proteins associated with cytoskeleton, sperm
morphology and organization of sperm flagella
A cohort of sperm structural proteins (SPATA19, HSPB9, DYNLRB2
and PFDN5) were expressed at a low level in pyriform sperm.
 72
Table 3 – Differentially expressed proteins between morphologically normal versus pyriform bovine sperm, identified by LC MS/MS following 2-D gel electrophoresis.
BLAST

JO U R N A L OF PR O TE O MI CS 82 ( 20 1 3 ) 6 4 –80
a b
Description Symbol Sp. N. Expr. Accession E value d Score e Mass pI Matches f SC. g SL. h Function(s)
number c (kDa)

Sperm–egg interactions sperm capacitation

60 kDa heat shock protein, mitochondrial HSPD1 74 ↓ 262205483 0.0 2709 60939 5.71 81 (79) 52% Ac. & Mp. Sperm capaciation through thyrosin
phosphorilation
Calcium-binding tyrosine CABYR 75 ↓ 84000105 0.0 355 48534 4.88 9 (9) 11% Pp. Sperm capaciation through thyrosin/serin
phosphorylation-regulated protein or threonine phosphorilation
F-actin-capping protein subunit beta CAPZB 81 ↓ 4826659 0.0 303 30609 5.69 7 (7) 16% Head Sperm capaciation through thyrosin
isoform 1 [Homo sapiens] phosphorilation and acrosomal reaction
Prohibitin [Homo sapiens] PHB 82 ↓ 4505773 4e-172 460 29786 5.57 20 (19) 45% Head Fertilization through targeted degradation
of paternal mitochondria
Troponin C-like protein CALM2 93 ↓ 223036 2e-101 941 16696 4.12 36 (35) 37% Un. Sperm hyperactivation, capaciation and
acrosomal reaction
Sperm acrosome membrane-associated SPACA3 98 ↓ 157279923 4e-112 2773 18087 5.87 108 (108) 6% Ac. Sperm–egg interaction
protein 3 precursor
Sperm acrosome membrane-associated SPACA3 100 ↓ 157279923 4e-112 2773 1796 5.87 84 (84) 6% Ac. Sperm–egg interaction
protein 3 precursor
Seminal fluid protein A3 - bovine BSP-A3 105 ↓ 89765 3e-89 115 13401 5.31 2 (2) 11% Ac. & Mp. Sperm capaciation through interaction
with HDL and heparin-like glycosamino-
glycans (GAG)

Sperm cytoskeleton and flagella

Spermatogenesis-associated protein 19, SPATA19 94 ↓ 115496121 3e-94 205 17888 6.60 7 (7) 33% Mp. Participating in mitochondria assembly
mitochondrial
Dynein light chain roadblock-type 2 DYNLRB2 106 ↓ 18702323 6e-63 271 10848 6.90 8 (8) 29% Un. Probably sperm tail formation
[Homo sapiens]
Dynein light chain roadblock-type 2 DYNLRB2 107 ↓ 18702323 6e-63 213 10848 6.90 6 (6) 29% Un. Probably sperm tail formation
[Homo sapiens]

Cellular stress & Appoptosis

Clusterin preproprotein CLU 15 ↑ 27806907 0.0 5346 51081 5.73 187 (187) 30% Ac. & Pp. Sperm protection, apoptosis, sperm
membrane remodeling
 BLAST
a b
Description Symbol Sp. N. Expr. Accession E value d Score e Mass pI Matches f SC. g SL. h Function(s)
number c (kDa)

Clusterin preproprotein CLU 17 ↑ 27806907 0.0 3781 51081 5.73 143 (143) 25% Ac. & Pp. Sperm protection, apoptosis, sperm
membrane remodeling
Clusterin preproprotein CLU 20 ↑ 27806907 0.0 3090 51081 5.73 143 (143) 27% Ac. & Pp. Sperm protection, apoptosis, sperm
membrane remodeling
Epididymal secretory glutathione GPX5 44 ↑ 253314494 3e-165 704 25067 8.20 32 (32) 44% Ac. ROS scavenger
peroxidase precursor
Epididymal secretory glutathione GPX5 52 ↑ 253314494 3e-165 654 25067 8.20 32 (31) 40% Ac. ROS scavenger
peroxidase precursor [Bos taurus]
PRDX5 protein PRDX5 57 ↑ 74354725 7e-103 435 17351 5.92 17 (17) 38% pAc. & Mp. ROS scavenger
Histidine triad nucleotide-binding protein HINT2 60 ↑ 27805917 2e-78 163 17136 6.96 4 (4) 15% Un. May play a role in apoptosis.
2, mitochondrial precursor

Metabolism
L-asparaginase ASRGL1 48 ↑ 116004289 0.0 297 32030 7.00 10 (10) 6% Mp. Involving in metabolism through
glycoprotein catabolism
Chain H, Bovine F1-Atpase Inhibited By ATP5D 59 ↑ 11514063 2e-83 444 15056 4.53 15 (12) 21% Un. ATP synthase, H + transporting

JO U R N A L OF P ROTE O MI CS 8 2 ( 20 1 3 ) 6 4–8 0
Dccd (Dicyclohexylcarbodiimide)
Chain H, Bovine F1-Atpase Inhibited By ATP5D 63 ↑ 11514063 2e-83 259 15056 4.53 15 (12) 19% Un. ATP synthase, H + transporting
Dccd (Dicyclohexylcarbodiimide)

Unknown function in sperm

60S acidic ribosomal protein P2 RPLP2 62 ↑ 27807523 1e-36 146 11695 4.53 4 (4) 39% Un. Unknown
Heat shock protein beta-9 HSPB9 95 ↓ 94966950 1e-100 192 16773 8.22 6 (6) 22% Un. Unknown
Prefoldin subunit 5 PFDN5 102 ↓ 27807413 6e-93 314 17374 6.31 6 (6) 19% Un. Involved in spermatogenesis
Cystatin-M precursor CST6 109 ↓ 61097917 2e-72 118 16345 7.63 3 (3) 10% Un. May has a role in preservation of sperm
integrity
a
Spot number referred to Fig. 2.
b
The status of identified protein expression in abnormal sperm head vs. morphological normal sperm.
c
Accession code for the identification in NCBI database.
d
The Expect value (E) is a parameter that describes the number of hits one can “expect” to see by chance when searching a database of a particular size. The lower the E-value, or the closer it is to zero,
the more “significant” the match is.
e
MASCOT search score. Score is _10 Log (p), where p is the probability that the observed match is a random event. Individual ions scores > 35 indicate identity or extensive homology at the p < 0.05 level.
f
Number of matched peptides.
g
SC: Sequence coverage percentage.
h
SL: Sperm localization.

73
74 JO U R N A L OF PR O TE O MI CS 82 ( 20 1 3 ) 6 4 –80

A kDa 1 2 B kDa 1 2 expression of HSPB9 was detected in spermatogonia and early

140 50 Clusterin spermatids [70]. Although HSPB9 occurs predominantly in
100
nuclei, the purpose of nuclear localization is unknown [71].
B-Tubulin The gene encoding DYNLRB2 is expressed in spermatids and
50
spermatocytes [51]. However, the role of this protein in
35 regulation of sperm function remains unknown.
Prefoldin (PFDN), a ubiquitously expressed heterohexameric
25 kDa 1 2 co-chaperone, is necessary for proper folding of nascent proteins,
15
C 60 HSPD1 in particular, actin and tubulin [52]. Although PFDN5 is expressed
in a wide variety of tissues, a missense mutation in Pfdn5 mouse
B-Tubulin
had defects in sperm and neural cell [52], demonstrating its role
B-Tubulin
in spermatogenesis. In the present study, PFDN5 was one of the
highly down-regulated proteins in pyriform sperm. This may lead
Fig. 4 – Immunoblots confirming differential expression of to reduction in formation of microfilaments, which are necessary
sperm proteins between morphologically normal (lane 1) and for development of cytoskeletal structures such as sperm head
pyriform sperm (lane 2). A cohort of differentially expressed
shaping by capping the nuclear membrane [72] and acrosome
proteins (based on the results from 2-D gel electrophoresis)
anchorage [73,74] during spermatogenesis as well as reduction of
were selected to confirm their differential expression by
microtubules, essential for cilia formation [52]. Therefore, we
immunoblotting. Total sperm protein extracts from normal
hypothesized that deformation of the sperm head in pyriform
and pyriform sperm (used for 2-D gel analysis) were
sperm was caused, at least in part, to a defect in sperm head
electrophoresed, electrotransferred and immunoblotted with
cytoskeletal structures [75], due to a reduction in microfilament
either (A) antiphosphotyrosine antibody, (B) anti-clusterin or
formation during spermatogenesis [52].
(C) anti-heat shock protein (mitochondrial; 60 kDa). These
blots were stripped and reprobed with anti-B-Tubulin to 4.3. Proteins associated with oxidative stress, apoptosis
confirm protein loading. and metabolism

In the pyriform sperm proteins involved in regulation of

Spermatogenesis Associated Protein 19, mitochondrial (SPATA19)
was specifically expressed in haploid spermatids and may
participate in mitochondrial assembly during spermiogenesis
[68]. It has a role in the maintenance of the normal mitochondrial
sheath through a mitochondria-targeting signal and works as an
adhesive molecule between individual mitochondria, leading to
formation of a stratified mitochondrial sheath [69].
Heat shock protein beta-9 (HSPB9) is specifically expressed
in testis, notably in the germ cells [50]. In human testes,
oxidative stress, apoptosis and metabolism were highly ex-
pressed in comparison to normal sperm. Clusterin, GPX5 and
PRDX5 were identified as protective proteins against oxidative
stress. There were 3 isoforms of Clusterin and 2 isoforms of PDX5
in the present study, perhaps due to post-translational modifi-
cation of sperm proteins, e.g. glycosylation [76–78]. Higher
expression of Clusterin in pyriform sperm head defects was
consistent with previous reports of its higher expression in
morphologically abnormal sperm in bulls [79], humans [80] and

Table 4 – Significantly enriched pathways in pyriform sperm identified by Ingenuity Pathway Analysis (IPA).
1
Molecular and cellular functions p-value Molecules

Energy production 7.83E-04-1.71E-03 ATP5D, HSPD1

Nucleic acid metabolism 7.83E-04-7.67E-03 ATP5D, HSPD1
Small molecule biochemistry 7.83E-04-2.78E-02 ATP5D, PRDX5, CLU, HSPD1, SPACA3
Carbohydrate metabolism 8.55E-04-8.55E-04 SPACA3
Cell death 8.55E-04-4.58E-02 CALM1, CLU, HSPD1, PHB
Cell-to-cell signaling and interaction 8.55E-04-4.52E-02 CAPZB, CLU, HSPD1, CST6, SPACA3
Cellular compromise 8.55E-04-2.45E-02 CLU
Cellular development 8.55E-04-4.31E-02 CAPZB, CLU, PFDN5, HSPD1, CST6
Cellular growth and proliferation 8.55E-04-4.52E-02 CALM1, CLU, PFDN5, HSPD1, CST6, PHB
Cellular movement 8.55E-04-7.97E-03 CLU, CST6, PHB
Free radical scavenging 8.55E-04-2.28E-02 PRDX5, CLU
Protein degradation/synthesis 1.42E-03-1.7E-02 CLU, HSPD1
Cell morphology 1.71E-03-2.53E-02 CAPZB, HSPD1, PHB
Cell cycle 3.41E-03-1.95E-02 CLU, PHB
Cell signaling 4.27E-03-1.19E-02 HSPD1, PHB
Antigen presentation 5.97E-03-2.78E-02 CLU, HSPD1, SPACA3
Cellular function and maintenance 5.97E-03-2.53E-02 CAPZB, CLU, PFDN5, HSPD1, PHB
Molecular transport 5.97E-03-3.37E-02 ATP5D, PRDX5, CLU, HSPD1
Cellular assembly and organization 7.67E-03-2.53E-02 CAPZB, CLU, PHB
Post-translational modification & protein folding 1.19E-02-1.19E-02 HSPD1
Lipid metabolism 1.86E-02-2.78E-02 CLU

1p < 0.05 (Fischer's test; the range comprises p-values of differentially expressed proteins involved in the pathways).
 JO U R N A L OF P ROTE O MI CS 8 2 ( 20 1 3 ) 6 4–8 0 75

A B

Fig. 5 – Measurement of ROS in normal and pyriform sperm. Generation of ROS was measured using the intracellular indicator
2', 7'-dicholorofluorescein diacetate (DCFDA). Sperm (1 × 106/ml) were incubated in PBS containing 10 μM DCFDA in the dark at
37 °C for 30 min. ROS converts DCFDA to 2', 7'-dicholorofluorescein (DCF). DCFDA-loaded normal and pyriform sperm were
examined (100 ×) to characterize DCF fluorescence pattern as an indication of ROS generation. Representative microscopic
fields for sperm preparations with normal (A) and pyriform sperm (B) are provided. DCF fluorescence was measured using a
96-well black plate reader with excitation at 485 nm and emission at 535 nm and expressed as ROS content in arbitrary units of
fluorescence. The graph represents mean (± SD) of three different experiments. * P < 0.05.

rams [81]. However, none of these studies specifically evaluated
its association with a specific type of sperm morphological
abnormality. Clusterin, a highly conserved heterodimeric glyco-
protein, is an extracellular version of heat shock protein [82],
produced in both testis and epididymis. In testis, it is produced
by Sertoli cells [83], secreted into the seminiferous tubule fluid,
and integrated into membranes of elongating spermatids and
mature sperm [84]. The abundance of Clusterin on mammalian
sperm and secretion of a different form in the epididymis
suggest that it has a role in both sperm differentiation and
maturation [85]. Clusterin is secreted in response to cellular
damage and involved in sperm protection, marking dead/and
damaged sperm to be removed and resorbed in the cauda
epididymis [86], as well by apoptosis [87]. Heat stress, hypoxia
and oxidative stress resulted in apoptosis of sperm. In that
regard, it has been suggested that testicular hyperthermia, in
particular scrotal insulation, may adversely affect spermatogen-
ic cells [88]. Furthermore, stress [15], varicocele [15,89] and
hyperthermia [20,90,91] induced sperm head elongation. Sertoli
cells were highly sensitive to hyperthermia, manifested by
altered cell ultrastructure and impaired secretion [92], consistent
with increased expression of this protein in pyriform sperm.
Clusterin may affect physiological processes involving matura-
tion of pyriform sperm, impairing their fertilizing ability. For
instance, dose-related effects of Clusterin on sperm membrane
remodeling during sperm maturation have been demonstrated;
high concentrations decreased whereas low concentrations
increased delivery of Sperm Adhesion Molecule1 (SPAM1) to
human and mouse sperm membranes [93]. SPAM1is the most
widely conserved mammalian sperm membrane protein, with
multiple essential roles in fertilization, including cumulus
dispersion, zona pellucida binding, and acrosomal exocytosis
[94]. Therefore, higher expression of Clusterin in pyriform sperm
may impair fertilizing ability [32] and sperm–zona binding,
compared to normal sperm. Furthermore, Clusterin may have
additional functions in fertilization process yet to be discovered.
For example, higher expression of several isoforms of Clusterin
was recently reported in capacitated human sperm [95].
76 JO U R N A L OF PR O TE O MI CS 82 ( 20 1 3 ) 6 4 –80
A B
kDA Normal Pyriform kDA Normal Pyriform
270 270
180 180
130 130
90 90
70 70
50 50
43 43
36 36
Fig. 6 – Detection of ubiquitinated proteins in normal and
pyriform sperm. A) Total protein extract was subjected to
electrophoresis on a 10% SDS-PAGE gel, electrotransferred and
blocked with 5% non-fat dried milk. Membranes were incubated
with the mouse monoclonal anti-ubiquitin antibody MK-12-3
(1:250), washed and further incubated with Horseradish
peroxidase-conjugated goat anti-mouse antibody. The bands
were detected using chemiluminescence. B) The nitrocellulose
membrane was Silver stained to confirm equal loading of
proteins between normal and pyriform sperm.
Similar to Clusterin, higher expression of PRDX5 and two
glycosylated isoforms of GPX5 proteins [78] in pyriform sperm
may be due to oxidative stress resulting from scrotal insulation
[96]. GPX5 was an efficient epididymal luminal ROS scavenger,
bound to the transiting sperm head plasma membrane in the
epididymal duct, conferring protection from ROS-mediated
damage [97]. Moreover, GPX5 is proposed to have a role in the
epididymal maturation of sperm [98]. PRDX5 is a H2O2 scavenger
(it can reduce hydroperoxides and peroxynitrite), present in the
acrosome and postacrosomal regions and midpiece in human
sperm [99]. Based on these data, in addition to oxidative stress
as the top identified toxicological list detected in pyriform
sperm by ingenuity pathway analysis, we inferred that there
was upregulation of ROS generation in the pyriform sperm
compared to normal sperm. Therefore, we determined ROS
generation in normal versus pyriform sperm. As expected, ROS
content was significantly higher in preparations of pyriform
sperm compared to morphologically normal sperm. Microscop-
ic examination of respective sperm preparations suggested that
increased content of ROS in preparation with pyriform sperm
(compared to normal sperm) was due to the presence of a
higher proportion of ROS-positive sperm. This observation was
consistent with previous reports that abnormal sperm generate
more ROS [100–102]. Oxidative stress develops once cellular
production of ROS exceeds its natural antioxidant defenses
[103]. Upregulation of Clusterin, PRDX5 and GPX5 in pyriform
sperm may be due to a compensatory mechanism developed in
the germ cells during spermatogenesis to protect them from the
deleterious effects of ROS.
Cystatin M, also known as cystatin E (Cystatin E/M; CST6),
was recently detected in bovine cauda epididymal fluid through
albumin-depleted 2-D SDS-PAGE [77]. HINT2 is a protein with an
unknown function in sperm [95]. However, in somatic cells, it
acted as an apoptotic sensitizer in mitochondria [104].
Asparaginase-like 1 protein (ASRGL1) is associated with
sperm mitochondria [105] and involved in metabolism through
glycoprotein catabolism [106]. In addition, it was recently re-
ported that expression of ASRLG1 protein was significantly
increased in human sperm during capacitation [95]. ATP5D is a
delta subunit of the central stalk in ATP synthase located in the
inner mitochondrial membrane [107]. Very recently, it was
reported that the lack of Sertoli cell-Dicer, an RNaseIII endonu-
clease essential for the production of mature miRNAs, led to
upregulation of the ATP5D proteins in mouse testis [108].
Perhaps higher expression of the ATP5D in pyriform sperm
was due to altered Sertoli cell function.
4.4. The pyriform sperm defect occurs during
late spermiogenesis.
In bulls, the last 18 days of spermatogenesis is the period of
metamorphosis of spherical spermatids into fully formed
sperm [109]. In addition, epididymal passage takes approxi-
mately 9 days in bulls. Since the highest percentage of
pyriform sperm in bulls are obtained 15–20 days after induc-
tion of testicular heat stress [91], adverse effects on testicular
germ cells leading to pyriform sperm abnormality occur in the
late spermiogenesis stage. Moreover, altered expression of
several spermatid-specific functional and structural proteins
in pyriform sperm further supported this conclusion.
The role of ubiquitination pathway in round spermatids has
been demonstrated in several species [49]. The IPA demonstrated
that protein ubiquitination pathway was the only significant
intracellular and second messenger signaling pathway in pyri-
form sperm, emphasizing the importance of protein ubiquitin
pathway in regulation of spermatid function. Consistent with
this observation, content of ubiquitinated proteins was higher in
pyriform sperm compared to morphologically normal sperm.
Increased ubiquitin immunoreactivity has been reported in
morphologically abnormal sperm from various farm animals
[110,111]. Ubiquitin, an 8.5 kDa chaperone protein (secreted by
the chief cells of the epididymal epithelium), tags defective
proteins for degradation by endocytosis or proteolytic degrada-
tion by the multisubunit protease the 26-S proteasome, or a
combination of these mechanisms [112]. Typically such defective
sperm will be degraded in the epididymis [112]. However, due to
the presence of ubiquitin-tagged pyriform sperm in the ejaculate
of bulls following heat stress, we inferred that ubiquitin-
mediated degradation of affected sperm may not be efficient
to remove large numbers of aberrant sperm present in the
epididymal lumen.
5. Conclusions
Overall, a cohort of sperm proteins was differentially expressed
in pyriform sperm induced by scrotal insulation. Down regula-
tion of sperm proteins associated with sperm capacitation and
sperm–egg interactions in pyriform sperm provided a molecular
 JO U R N A L OF P ROTE O MI CS 8 2 ( 20 1 3 ) 6 4–8 0
basis for our previous observation that pyriform sperm had
reduced ability to bind to the zona pellucida [18]. Altered
expression of sperm cytoskeletal proteins may be associated
with abnormal head morphology in pyriform sperm and reduced
developmental competence of zygotes resulting from fertiliza-
tion of oocytes by pyriform sperm. Upregulation of proteins
associated with cellular stresses (most significant toxicological
agent based on IPA) in pyriform sperm may be a compensatory
testicular mechanism to protect affected germ cells from the
deleterious effects of ROS. High content of ubiquitinated proteins
in pyriform sperm may reflect an epididymal response to
aberrant proteins to degrade them in the epididymis. We
concluded that comparing protein profiles of normal versus a
specific type of morphological defect was apparently a suitable
research model to understand the molecular basis of impaired
sperm function and abnormal spermatogenesis.
Supplementary data to this article can be found online at
http://dx.doi.org/10.1016/j.jprot.2013.02.027.
Conflict of Interest
The authors declare no conflicts of interest.
Acknowledgments
This project received funding support from the Natural Sciences
and Engineering Research Council of Canada (NSERC-DG to JT).
Ms. Evine van Riemsdijk was supported through an internship
from the Department of Animal Sciences (Adaptation Physiol-
ogy Group), Wageningen Agricultural University, Wageningen,
The Netherlands.
REFERENCES
[1] Bonde JPE, Ernst E, Jensen TK, Hjollund NHI, Kolstad H,
Scheike T, et al. Relation between semen quality and
fertility: a population-based study of 430 first-pregnancy
planners. Lancet 1998;352:1172–7.
[2] Wiltbank JN, Parish NR. Pregnancy rate in cows and heifers
bred to bulls selected for semen quality. Theriogenology
1986;25:779–83.
[3] Karabinus DS, Gelety TJ. The impact of sperm morphology
evaluated by strict criteria on intrauterine insemination
success. Fertil Steril 1997;67:536–41.
[4] Saacke RG, Dalton JC, Nadir S, Nebel RL, Bame JH. Relationship
of seminal traits and insemination time to fertilization rate
and embryo quality. Anim Reprod Sci 2000;60–61:663–77.
[5] Coetzee K, Kruge T, Lombard C. Predictive value of normal
sperm morphology: a structured literature review. Hum
Reprod Update 1998;4:73–82.
[6] Barth AD, Oko RJ. Abnormal Morphology of Bovine Spermato-
zoa. Ames: Iowa State University Press; 1989.
[7] Leonardo FCB. Evaluation of stallion sperm morphology.
Clin Tech Equine Pract 2007;6:249–64.
[8] Zambelli D, Cunto M. Semen collection in cats: techniques
and analysis. Theriogenology 2006;66:159–65.
[9] Liu DY, Baker HWG. Morphology of spermatozoa bound to
the zona pellucida of human oocytes that failed to fertilize
in vitro. J Reprod Fertil 1992;94:71–84.
77
[10] Menkveld R, Holleboom CAG, Rhemrev JPT. Measurement
and significance of sperm morphology. Asian J Androl
2011;13:59–68.
[11] Walters AH, Eyestone WE, Saacke RG, Pearson RE,
Gwazdauskas FC. Bovine embryo development after IVF
with spermatozoa having abnormal morphology.
Theriogenology 2005;63:1925–37.
[12] Walters AH, Eyestone WE, Saacke RG, Pearson RE,
Gwazdauskas FC. Sperm morphology and preparation
method affect bovine embryonic development. J Androl
2004;25:554–63.
[13] Prisant N, Escalier D, Soufir J-C, Morillon M, Schoevaert D,
Misrahi M, et al. Ultrastructural nuclear defects and
increased chromosome aneuploidies in spermatozoa with
elongated heads. Hum Reprod 2007;22:1052–9.
[14] Menkveld R, Kruger TF. Sperm morphology and male
urogenital infections. Andrologia 1998;30:49–53.
[15] Menkveld R. Clinical significance of the low normal sperm
morphology value as proposed in the fifth edition of the WHO
Laboratory Manual for the Examination and Processing of
Human Semen. Asian J Androl 2010;12:47–58.
[16] Naftulin BN, Samuels SJ, Hellstrom WJG, Lewis EL,
Overstreet JW. Semen quality in varicocele patients is
characterized by tapered sperm cells. Fertil Steril 1991;56:
149–51.
[17] Jelnes JE. Semen quality in workers producing reinforced
plastic. Reprod Toxicol (Elmsford, NY) 1988;2:209–12.
[18] Thundathil J, Palasz AT, Mapletoft RJ, Barth AD. An
investigation of the fertilizing characteristics of
pyriform-shaped bovine spermatozoa. Anim Reprod Sci
1999;57:35–50.
[19] Jeulin C, Soumah A, Jouannet P. Morphological factors
influencing the penetration of human sperm into cervical
mucus in vitro. Int J Androl 1985;8:215–23.
[20] Kawarsky SJ, Stubbings RB, Basrur PK, King WA. Pyriform
bovine spermatozoa and fertilization potential. Can Vet J
1995;36:434–6.
[21] Rousso D, Kourtis A, Mavromatidis G, Gkoutzioulis F,
Makedos G, Panidis D. Pyriform head: a frequent but
little-studied morphological abnormality of sperm. Arch
Androl 2002;48:267–72.
[22] Osawa Y, Sueoka K, Iwata S, Shinohara M, Kobayashi N, Kuji
N, et al. Assessment of the dominant abnormal form is
useful for predicting the outcome of intracytoplasmic sperm
injection in the case of severe teratozoospermia. J Assist
Reprod Genet 1999;16:436–42.
[23] Tang SS, Gao H, Robinson WP, Ho Yuen B, Ma S. An
association between sex chromosomal aneuploidy in sperm
and an abortus with 45, X of paternal origin: possible
transmission of chromosomal abnormalities through ICSI.
Hum Reprod 2004;19:147–51.
[24] Dariš B, Goropevšek A, Hojnik N, Vlaisavljević V. Sperm
morphological abnormalities as indicators of DNA
fragmentation and fertilization in ICSI. Arch Gynecol Obstet
2010;281:363–7.
[25] Meistrich ML, Kasai K, Olds-Clarke P, Macgregor GR,
Berkowitz AD, Tung KSK. Deficiency in fertilization by
morphologically abnormal sperm produced by Azh mutant
mice. Mol Reprod Dev 1994;37:69–77.
[26] Saacke RG, Dejarnette JM, Bame JH, Karabinus DS, Whitman
SS. Can spermatozoa with abnormal heads gain access to
the ovum in artificially inseminated super- and
single-ovulating cattle? Theriogenology 1998;50:117–28.
[27] Takahashi M. Heat stress on reproductive function and
fertility in mammals. Reprod Med Biol 2012;11:37–47.
[28] Marmar JL. Varicocele and male infertility: Part II: the
pathophysiology of varicoceles in the light of current
molecular and genetic information. Hum Reprod Update
2001;7:461–72.
78 JO U R N A L OF PR O TE O MI CS 82 ( 20 1 3 ) 6 4 –80
[29] Hansen PJ. Effects of heat stress on mammalian reproduction.
Philos Trans R Soc Lond B Biol Sci 2009;364:3341–50.
[30] Brito LFC, Silva AEDF, Barbosa RT, Unanian MM, Kastelic JP.
Effects of scrotal insulation on sperm production, semen
quality, and testicular echotexture in Bos indicus and Bos
indicus × Bos taurus bulls. Anim Reprod Sci 2003;79:1–15.
[31] Barth AD, Bowman PA. The sequential appearance of sperm
abnormalities after scrotal insulation or dexamethasone
treatment in bulls. Can Vet J 1994;35:93–102.
[32] Walters AH, Saacke RG, Pearson RE, Gwazdauskas FC.
Assessment of pronuclear formation following in vitro
fertilization with bovine spermatozoa obtained after thermal
insulation of the testis. Theriogenology 2006;65:1016–28.
[33] Newton LD, Krishnakumar S, Menon AG, Kastelic JP, van der
Hoorn FA, Thundathil JC. Na +/K + ATPase regulates sperm
capacitation through a mechanism involving kinases and
redistribution of its testis-specific isoform. Mol Reprod Dev
2010;77:136–48.
[34] Barth A. Bull breeding soundness evaluation manual:
Saskatoon: Western Canadian Association of Bovine
Practitioners. WCVM; 2001.
[35] Butt RH, Coorssen JR. Postfractionation for enhanced proteomic
analyses: routine electrophoretic methods increase the
resolution of standard 2D-PAGE. J Proteome Res 2005;4:982–91.
[36] Butt RH, Lee MWY, Pirshahid SA, Backlund PS, Wood S,
Coorssen JR. An initial proteomic analysis of human
preterm labor: placental membranes. J Proteome Res 2006;5:
3161–72.
[37] Hunt SMN, Thomas MR, Sebastian LT, Pedersen SK, Harcourt
RL, Sloane AJ, et al. Optimal replication and the importance of
experimental design for gel-based quantitative proteomics. J
Proteome Res 2005;4:809–19.
[38] Kolker E, Higdon R, Hogan JM. Protein identification and
expression analysis using mass spectrometry. Trends
Microbiol 2006;14:229–35.
[39] Belleannee C, Belghazi M, Labas V, Teixeira-Gomes A-P,
Gatti JL, Dacheux J-L, et al. Purification and identification of
sperm surface proteins and changes during epididymal
maturation. Proteomics 2011;11:1952–64.
[40] Jain RK, Jain A, Kumar R, Verma V, Maikhuri JP, Sharma VL,
et al. Functional attenuation of human sperm by novel,
non-surfactant spermicides: precise targeting of membrane
physiology without affecting structure. Hum Reprod
2010;25:1165–76.
[41] Sutovsky P, Moreno R, Ramalho-Santos J, Dominko T,
Thompson WE, Schatten G. A putative, ubiquitin-dependent
mechanism for the recognition and elimination of defective
spermatozoa in the mammalian epididymis. J Cell Sci
2001;114:1665–75.
[42] Lefièvre L, Chen Y, Conner SJ, Scott JL, Publicover SJ, Ford
WCL, et al. Human spermatozoa contain multiple targets for
protein S-nitrosylation: an alternative mechanism of the
modulation of sperm function by nitric oxide? Proteomics
2007;7:3066–84.
[43] Asquith KL, Harman AJ, McLaughlin EA, Nixon B, Aitken RJ.
Localization and significance of molecular chaperones, Heat
Shock Protein 1, and Tumor Rejection Antigen gp96 in the male
reproductive tract and during capacitation and acrosome
reaction. Biol Reprod 2005;72:328–37.
[44] Breitbart H, Cohen G, Rubinstein S. Role of actin cytoskeleton in
mammalian sperm capacitation and the acrosome reaction.
Reproduction 2005;129:263–8.
[45] Naaby-Hansen S, Mandal A, Wolkowicz MJ, Sen B, Westbrook
VA, Shetty J, et al. CABYR, a novel calcium-binding tyrosine
phosphorylation-regulated fibrous sheath protein involved in
capacitation. Dev Biol 2002;242:236–54.
[46] Ficarro S, Chertihin O, Westbrook VA, White F, Jayes F, Kalab
P, et al. Phosphoproteome analysis of capacitated human
sperm. J Biol Chem 2003;278:11579–89.
[47] Bendahmane M, Lynch Ii C, Tulsiani DRP. Calmodulin signals
capacitation and triggers the agonist-induced acrosome
reaction in mouse spermatozoa. Arch Biochem Biophys
2001;390:1–8.
[48] Mandal A, Klotz KL, Shetty J, Jayes FL, Wolkowicz MJ, Bolling
LC, et al. SLLP1, a unique, intra-acrosomal, non-bacteriolytic, c
lysozyme-like protein of human spermatozoa. Biol Reprod
2003;68:1525–37.
[49] Sutovsky P, Moreno RD, Ramalho-Santos J, Dominko T, Simerly
C, Schatten G. Ubiquitinated sperm mitochondria, selective
proteolysis, and the regulation of mitochondrial inheritance in
mammalian embryos. Biol Reprod 2000;63:582–90.
[50] Kappé G, Verschuure P, Philipsen RLA, Staalduinen AA, Van
de Boogaart P, Boelens WC, et al. Characterization of two
novel human small heat shock proteins: protein
kinase-related HspB8 and testis-specific HspB9. Biochim
Biophys Acta Gene Struct Expression 2001;1520:1–6.
[51] Chalmel F, Rolland AD, Niederhauser-Wiederkehr C, Chung
SSW, Demougin P, Gattiker A, et al. The conserved
transcriptome in human and rodent male gametogenesis.
Proc Natl Acad Sci 2007;104:8346–51.
[52] Lee Y, Smith RS, Jordan W, King BL, Won J, Valpuesta JM,
et al. Prefoldin 5 is required for normal sensory and
neuronal development in a murine model. J Biol Chem
2011;286:726–36.
[53] Asquith KL, Baleato RM, McLaughlin EA, Nixon B, Aitken RJ.
Tyrosine phosphorylation activates surface chaperones
facilitating sperm–zona recognition. J Cell Sci 2004;117:
3645–57.
[54] Boilard M, Reyes-Moreno C, Lachance C, Massicotte L, Bailey
JL, Sirard M-A, et al. Localization of the Chaperone Proteins
GRP78 and HSP60 on the luminal surface of bovine oviduct
epithelial cells and their association with spermatozoa. Biol
Reprod 2004;71:1879–89.
[55] Howes EA, Hurst SM, Jones R. Actin and actin-binding
proteins in bovine spermatozoa: potential role in membrane
remodeling and intracellular signaling during epididymal
maturation and the acrosome reaction. J Androl 2001;22:
62–72.
[56] Schafer DA, Korshunova YO, Schroer TA, Cooper JA. Differential
localization and sequence analysis of capping protein
beta-subunit isoforms of vertebrates. J Cell Biol 1994;127:453–65.
[57] Dos Remedios CG, Chhabra D, Kekic M, Dedova IV,
Tsubakihara M, Berry DA, et al. Actin binding proteins:
regulation of cytoskeletal microfilaments. Physiol Rev
2003;83:433–73.
[58] Li Y-F, He W, Mandal A, Kim Y-H, Digilio L, Klotz K, et al.
CABYR binds to AKAP3 and Ropporin in the human sperm
fibrous sheath. Asian J Androl 2011;13:266–74.
[59] Fiedler SE, Bajpai M, Carr DW. Identification and
characterization of RHOA-interacting proteins in bovine
spermatozoa. Biol Reprod 2008;78:184–92.
[60] Grand RJ, Perry SV. Calmodulin-binding proteins from brain
and other tissues. Biochem J 1979;183:285–95.
[61] Kann M-L, Feinberg J, Rainteau D, Dadoune J-P, Weinman S,
Fouquet J-P. Localization of Calmodulin in perinuclear
structures of spermatids and spermatozoa: a comparison of
six mammalian species. Anat Rec 1991;230:481–8.
[62] Moriya M, Katagiri C, Yagi K. Immuno-electron microscopic
localization of calmodulin and calmodulin-binding proteins
in the mouse germ cells during spermatogenesis and
maturation. Cell Tissue Res 1993;271:441–51.
[63] Ignotz GG, Suarez SS. Calcium/Calmodulin and Calmodulin
Kinase II stimulate hyperactivation in demembranated
bovine sperm. Biol Reprod 2005;73:519–26.
[64] Herrero MB, Mandal A, Digilio LC, Coonrod SA, Maier B, Herr
JC. Mouse SLLP1, a sperm lysozyme-like protein involved in
sperm–egg binding and fertilization. Dev Biol 2005;284:
126–42.
 JO U R N A L OF P ROTE O MI CS 8 2 ( 20 1 3 ) 6 4–8 0
[65] Sachdev M, Mandal A, Mulders S, Digilio LC, Panneerdoss S,
Suryavathi V, et al. Oocyte specific oolemmal SAS1B
involved in sperm binding through intra-acrosomal SLLP1
during fertilization. Dev Biol 2012;363:40–51.
[66] Thompson WE, Ramalho-Santos J, Sutovsky P.
Ubiquitination of prohibitin in mammalian sperm
mitochondria: possible roles in the regulation of mitochondrial
inheritance and sperm quality control. Biol Reprod 2003;69:
254–60.
[67] Yanagimachi R. Fertility of mammalian spermatozoa: its
development and relativity. Zygote 1994;2:371–2.
[68] Doiguchi M, Mori T, Toshimori K, Shibata Y, Iida H.
Spergen-1 might be an adhesive molecule associated with
mitochondria in the middle piece of spermatozoa. Dev Biol
2002;252:127–37.
[69] Suzuki-Toyota F, Ito C, Toyama Y, Maekawa M, Yao R, Noda
T, et al. Factors maintaining normal sperm tail structure
during epididymal maturation studied in Gopc −/− mice. Biol
Reprod 2007;77:71–82.
[70] de Wit NJW, Verschuure P, Kappé G, King SM, de Jong WW, van
Muijen GNP, et al. Testis-specific human small heat shock
protein HSPB9 is a cancer/testis antigen, and potentially
interacts with the dynein subunit TCTEL1. Eur J Cell Biol
2004;83:337–45.
[71] Sun Y, MacRae T. Small heat shock proteins: molecular
structure and chaperone function. Cell Mol Life Sci 2005;62:
2460–76.
[72] Welch JE, O'Rand MG. Identification and distribution of actin
in spermatogenic cells and spermatozoa of the rabbit. Dev
Biol 1985;109:411–7.
[73] Oko R. Occurrence and formation of cytoskeletal proteins in
mammalian spermatozoa. Andrologia 1998;30:193–206.
[74] Tachibana M, Terada Y, Murakawa H, Murakami T, Yaegashi N,
Okamura K. Dynamic changes in the cytoskeleton during
human spermiogenesis. Fertil Steril 2005;84(Supplement 2):
1241–8.
[75] Rousseaux-Prévost R, Rousseaux J, Lesur P, Collier F,
Gauthier A, Rigot JM, et al. Abnormal expression of protein
4.1 in spermatozoa of infertile men with teratospermia.
Lancet 1994;343:764–5.
[76] Sensibar JA, Qian Y, Griswold MD, Sylvester SR, Bardin CW,
Cheng CY, et al. Localization and molecular heterogeneity of
sulfated glycoprotein-2 (clusterin) among ventral prostate,
seminal vesicle, testis, and epididymis of rats. Biol Reprod
1993;49:233–42.
[77] Moura AA, Souza CE, Stanley BA, Chapman DA, Killian GJ.
Proteomics of cauda epididymal fluid from mature Holstein
bulls. J Proteomics 2010;73:2006–20.
[78] Zhang T, Chabory E, Britan A, Grignard E, Pitiot O, Saez F, et al.
GPX5, the selenium-independent glutathione
peroxidase-encoding single copy gene is differentially
expressed in mouse epididymis. Reprod Fertil Dev 2008;20:
615–25.
[79] Ibrahim NM, Gilbert GR, Loseth KJ, Crabo BG. Correlation
between clusterin-positive spermatozoa determined by flow
cytometry in bull semen and fertility. J Androl 2000;21:887–94.
[80] Thacker S, Yadav SP, Sharma RK, Kashou A, Willard B,
Zhang D, et al. Evaluation of sperm proteins in infertile men:
a proteomic approach. Fertil Steril 2011;95:2745–8.
[81] Ibrahim NM, Romano JE, Troedsson MH, Crabo BG. Effect of
scrotal insulation on clusterin-positive cells in ram semen
and their relationship to semen quality. J Androl 2001;22:
863–77.
[82] Michel D, Chatelain G, North S, Brun G. Stress-induced
transcription of the clusterin/apoJ gene. Biochem J
1997;328(Pt 1):45–50.
[83] Griswold MD, Roberts K, Bishop P. Purification and
characterization of a sulfated glycoprotein secreted by
Sertoli cells. Biochemistry 1986;25:7265–70.
79
[84] Sylvester SR, Morales C, Oko R, Griswold MD. Localization of
sulfated glycoprotein-2 (clusterin) on spermatozoa and in
the reproductive tract of the male rat. Biol Reprod 1991;45:
195–207.
[85] Howes EA, Hurst S, Laslop A, Jones R. Cellular distribution
and molecular heterogeneity of MAC393 antigen (clusterin,
beta-chain) on the surface membrane of bull spermatozoa.
Mol Hum Reprod 1998;4:673–81.
[86] Bailey RW, Aronow B, Harmony JAK, Griswold MD. Heat
shock-initiated apoptosis is accelerated and removal of
damaged cells is delayed in the testis of Clusterin/ApoJ
knock-out mice. Biol Reprod 2002;66:1042–53.
[87] Viard I, Wehrli P, Jornot L, Bullani R, Vechietti J-L, Schifferli
JA, et al. Clusterin gene expression mediates resistance to
apoptotic cell death induced by heat shock and oxidative
stress. J Invest Dermatol 1999;112:290–6.
[88] Rahman MB, Vandaele L, Rijsselaere T, Maes D, Hoogewijs M,
Frijters A, et al. Scrotal insulation and its relationship to
abnormal morphology, chromatin protamination and nuclear
shape of spermatozoa in Holstein-Friesian and Belgian Blue
bulls. Theriogenology 2011;76:1246–57.
[89] Bülow Mv, Rackwitz H-R, Zimbelmann R, Franke WW. CP β3,
a novel isoform of an actin-binding protein, is a component
of the cytoskeletal calyx of the mammalian sperm head. Exp
Cell Res 1997;233:216–24.
[90] Dada R, Gupta NP, Kucheria K. Spermatogenic arrest in men
with testicular hyperthermia. Teratog Carcinog Mutagen
2003;23:235–43.
[91] Newton LD, Kastelic JP, Wong B, van der Hoorn F,
Thundathil J. Elevated testicular temperature modulates
expression patterns of sperm proteins in Holstein bulls. Mol
Reprod Dev 2009;76:109–18.
[92] Chen M, Cai H, Yang J-L, Lu C-L, Liu T, Yang W, et al. Effect of
heat stress on expression of junction-associated molecules
and upstream factors androgen receptor and Wilms' Tumor
1 in monkey Sertoli cells. Endocrinology 2008;149:4871–82.
[93] Griffiths GS, Galileo DS, Aravindan RG, Martin-DeLeon PA.
Clusterin facilitates exchange of Glycosyl
Phosphatidylinositol-Linked SPAM1 between reproductive
luminal fluids and mouse and human sperm membranes.
Biol Reprod 2009;81:562–70.
[94] Martin-DeLeon PA. Epididymal SPAM1 and its impact on
sperm function. Mol Cell Endocrinol 2006;250:114–21.
[95] Secciani F, Bianchi L, Ermini L, Cianti R, Armini A, La Sala GB,
et al. Protein profile of capacitated versus ejaculated human
sperm. J Proteome Res 2009;8:3377–89.
[96] Ikeda M, Kodama H, Fukuda J, Shimizu Y, Murata M,
Kumagai J, et al. Role of radical oxygen species in rat
testicular germ cell apoptosis induced by heat stress. Biol
Reprod 1999;61:393–9.
[97] Chabory E, Damon C, Lenoir A, Henry-Berger J, Vernet P,
Cadet R, et al. Mammalian glutathione peroxidases control
acquisition and maintenance of spermatozoa integrity. J
Anim Sci 2010;88:1321–31.
[98] Drevet JR. The antioxidant glutathione peroxidase family
and spermatozoa: a complex story. Mol Cell Endocrinol
2006;250:70–9.
[99] O'Flaherty C, Rico de Souza A. Hydrogen peroxide modifies
human sperm peroxiredoxins in a dose-dependent manner.
Biol Reprod 2011;84:238–47.
[100] Pasqualotto FF, Sharma RK, Nelson DR, Thomas Jr AJ,
Agarwal A. Relationship between oxidative stress, semen
characteristics, and clinical diagnosis in men undergoing
infertility investigation. Fertil Steril 2000;73:459–64.
[101] Aziz N, Saleh RA, Sharma RK, Lewis-Jones I, Esfandiari N,
Thomas Jr AJ, et al. Novel association between sperm
reactive oxygen species production, sperm morphological
defects, and the sperm deformity index. Fertil Steril 2004;81:
349–54.
80 JO U R N A L OF PR O TE O MI CS 82 ( 20 1 3 ) 6 4 –80
[102] Saleh RA, Agarwal A. Oxidative stress and male infertility:
from research bench to clinical practice. J Androl 2002;23:
737–52.
[103] Tremellen K. Oxidative stress and male infertility—a clinical
perspective. Hum Reprod Update 2008;14:243–58.
[104] Martin J, Magnino F, Schmidt K, Piguet AC, Lee JS, Semela D,
et al. Hint2, A mitochondrial apoptotic sensitizer
down-regulated in hepatocellular carcinoma. Gastroenterology
2006;130:2179–88.
[105] Bush LA, Herr JC, Wolkowicz M, Sherman NE, Shore A,
Flickinger CJ. A novel asparaginase-like protein is a sperm
autoantigen in rats. Mol Reprod Dev 2002;62:233–47.
[106] Martínez-Heredia J, Estanyol JM, Ballescà JL, Oliva R. Proteomic
identification of human sperm proteins. Proteomics 2006;6:
4356–69.
[107] Gibbons C, Montgomery MG, Leslie AGW, Walker JE. The
structure of the central stalk in bovine F1-ATPase at 2.4 A
resolution. Nature of Structural and. Mol Biol 2000;7:1055–61.
[108] Papaioannou MD, Lagarrigue M, Vejnar CE, Rolland AD,
Kühne F, Aubry F, et al. Loss of dicer in Sertoli cells has a
major impact on the testicular proteome of mice. Mol Cell
Proteomics 2011;10.
[109] Barth AD. The pathogenesis of abnormal sperm production
in bulls. Bull Management and Fertility Conference, Kansas
State University, Manhattan, Kansas; 2010.
[110] Lovercamp KW, Safranski TJ, Fischer KA, Manandhar G,
Sutovsky M, Herring W, et al. Arachidonate 15-lipoxygenase
and ubiquitin as fertility markers in boars. Theriogenology
2007;67:704–18.
[111] Sutovsky P, Turner RM, Hameed S, Sutovsky M. Differential
ubiquitination of stallion sperm proteins: possible implications
for infertility and reproductive seasonality. Biol Reprod 2003;68:
688–98.
[112] Baska KM, Manandhar G, Feng D, Agca Y, Tengowski MW,
Sutovsky M, et al. Mechanism of extracellular ubiquitination
in the mammalian epididymis. J Cell Physiol 2008;215:
684–96.