Analytical & Bioanalytical Upadhyay et al.

, J Anal Bioanal Tech 2013, 4:4

http://dx.doi.org/10.4172/2155-9872.1000172
Techniques

Research Article Open Access

Differential Proteomic and Phospho-proteomic Analysis of Normal versus

Failed Spermiation in Adult Rats by Label-Free LC-MS/MS
Rahul D Upadhyay1, Amit Kumar Yadav2, Shobha Sonawane1, Reshma Goankar1, Debasis Dash2 and BalasinorNH1*
1
Department of Neuroendocrinology, National Institute for Research in Reproductive Health, J.M. Street, Parel, Mumbai, India
2
CSIR-Institute of Genomics and Integrative Biology, South Campus, Mathura Road, SukhdevVihar, New Delhi, India

Abstract
Spermiation is the final step of spermatogenesis and involves release of mature spermatids from the Sertoli
cells. It is a complex process involving displacement and removal of spermatid cytoplasm, formation and degradation
of tubulobulbar complexes and progressive loss of adhesive junctions, including ectoplasmic specializations and
subsequent phagocytosis of residual bodies by the Sertoli cell. Spermiation occurs in stage VIII of the rat seminiferous
epithelium cycle. In spite of the important role played by spermiation process in sperm output to the epididymis, very
little is known about this process. To enhance our knowledge of the sperm release biology, we sought to understand
the molecular events occurring at the time of spermiation. Towards this aim, we induced spermiation failure condition
by 17β estradiol treatment to adult male rats and compared with the normal spermiation. On comparing both the
groups, we identified a total of 104 differentially expressed proteins and 23 differentially expressed phosphoproteins
by LC-MS/MS analysis and quantitation. Localization and expression of some of the validated differential proteins
in the testes highlight their importance during the sperm release. The present study represents an initial step to
understand the molecular basis of the process of spermiation.

Keywords: Spermiation; Cytoskeletal; Endoplasmic reticulum;
Phosphoproteins; Proteomics
Introduction
In mammals, the male gametes, spermatozoa are formed in the
seminiferous tubule of the testis. The diploid spermatogonia undergo
several mitotic and meiotic divisions, thus forming spermatocytes and
lastly the haploid spermatid [1]. The spermatid after cyto-differentiation
is released into the lumen of the seminiferous tubule via a process
known as spermiation. In the seminiferous tubule, specific association
of different types of spermatogenic cells form stages of seminiferous
epithelium. In rats, there are 14 stages and spermiation occurs in stages
VII-VIII [2].
The process of spermiation is an elaborate process and takes almost
72 hours in the rat [3]. The process involves movement of spermatid
towards the lumen, removal of excessive spermatid cytoplasm,
dissociation of testis-specific actin based atypical adheren junction
known as Ectoplasmic specialization (ES), and formation of another
testis-specific actin-based structure known as tubulobulbar complex
(TBC). The TBCs are involved in the removal of excess of spermatid
cytoplasm, recycling of junctional molecules by endocytosis, and also
act as transient attachment devices for the mature spermatid, before it
is released into the lumen. Both ES and TBCs are actin-based structures
with cisternae of Endoplasmic reticulum (ER) [4].
Newer advances in molecular biology and genomics have improved
our knowledge of spermatogenesis by allowing the identification
of a large no. of genes essential for the development of functional
male gametes [5,6]. However, although proteomics has led to major
breakthroughs in this era, the investigation of spermatogenesis by
proteomics based strategies remains at an infant stage. Recent reports
on proteomics driven studies on testis and its germ cell types suggests
regulatory mechanism and heterogeneity underlying the function of
testicular proteins [7,8]. All these proteomics studies serve as useful
resource for comparison. Nevertheless, unprecedented efforts are still
required to improve our understanding of the highly complex and
sophisticated communication networks. Many studies in proteomics
have been done to understand spermatogenesis and its regulation
[9,10], but none of them have studied the process of sperm release, i.e.
spermiation. None of the protein identified per se could be directly
correlated to the process of spermiation. Also, various phosphorylated
proteins and phosphorylation of various adhesion molecules have been
shown to be present at the site of spermiation, but not much focus have
been given to phosphoproteome profile in sperm release [11-13].
Hence, we initiated a study to identify the molecular players of
sperm release. Towards this aim, comparative study of normal and failed
spermiation condition was performed. Our earlier study following
17β-estradiol treatment to adult male rats at a dose of 100 μg/kg for
10 days led to spermiation failure [14,15]. Therefore, this treatment
model was used for differential proteomics and phospho-proteomics by
label free nano-LC-MS/MS sequencing to elucidate the importance of
proteins involved during sperm release.
Materials and Methods
Extraction of proteins and phosphoproteins from control
and 17β-estradiol treated adult male rats (normal vs failed
spermiation)
Animal treatment: Randomly bred Holtzman strain adult male rats
*Corresponding author: Balasinor NH, Ph.D, Scientist ‘E’, Departmrnt of
Neuroendocrinology, National Institute for Research in Reproductive Health, J. M.
Street, Parel, Mumbai-400012, India, Tel: 91-22-24192025/24192140; Fax: 91-22-
24139412; E-mail: balasinorn@nirrh.res.in
Received August 13, 2013; Accepted September 12, 2013; Published September
16, 2013
Citation: Upadhyay RD, Yadav AK, Sonawane S, Goankar R, Dash D (2013)
Differential Proteomic and Phospho-proteomic Analysis of Normal versus Failed
Spermiation in Adult Rats by Label-Free LC-MS/MS. J Anal Bioanal Tech 4: 172.
doi:10.4172/2155-9872.1000172
Copyright: © 2013 Upadhyay RD, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.

J Anal Bioanal Tech

ISSN:2155-9872 JABT, an open access journal Volume 4 • Issue 4 • 1000172
Citation: Upadhyay RD, Yadav AK, Sonawane S, Goankar R, Dash D (2013) Differential Proteomic and Phospho-proteomic Analysis of Normal
versus Failed Spermiation in Adult Rats by Label-Free LC-MS/MS. J Anal Bioanal Tech 4: 172. doi:10.4172/2155-9872.1000172
(75-90 days old) maintained at a temperature of 22°C-23°C, humidity
50-55% and lighting cycle of 14 h light: 10 h dark were administered
100 μg/kg bodyweight/day of 17-β Estradiol (Sigma, USA) for 10 days.
The study was approved by institutional animal ethics committee for
the use of animals. All experiments were done at least in triplicate using
tissues from different animals.
Separation of stages VII-VIII from seminiferous tubule: 24
hrs after the administration of the last dose, testes were collected
and detunicated in 0.01 M PBS. Stages of seminiferous epithelium
cycle were separated by a transillumination method [16,17]. Briefly,
the testis was teased apart and seminiferous tubules viewed under a
stereomicroscope at 10-40 X magnification. The stages follow each other
in the seminiferous tubules in a wavelike fashion and specific pattern
can be observed for different stages by trans illumination in a freshly
isolated rat seminiferous tubule. The method is based on the fact that
condensation of the elongating/elongated spermatid nuclear chromatin
is associated with increase in light absorption. Stages IX-XI has pale
weak absorption with pale zone, whereas Stages VII to VIII, having
homogenously dark centered absorption pattern, since the spermatids
are luminally arranged in bundles. The dark zone representing Stages
VII-VIII were dissected. The separated tubules were transferred to
cryovials, snap frozen in liquid nitrogen and stored in -80°C until
protein extraction.
Protein and phosphoprotein extraction: Protein extraction from
control and treated stage VII-VIII tubules: Protein was extracted using
2D lysis buffer consisting of 50 mM Tris pH 8.5, 4% CHAPS, 9M Urea,
protease inhibitor cocktail and Phospho-Stop by incubating at 4°C
for at least 1 hr,and centrifuging at 12000 g for 10 min. The extracted
protein from both control and estradiol treated animals was estimated
by Bradford’s method, and then subjected to LC-MS/MS analysis.
Phosphoproteins enrichment by Immobilized Metal Affinity
Chromatography (IMAC): A part of proteins (300 μg) extracted were
processed for phosphoproteins enrichment by IMAC method. The
protocol involves use of Pro-Q® Diamond Phosphoproteins Enrichment
Kit (Molecular Probes, USA), which enables efficient, non-radioactive
isolation of phosphoproteins from complex cellular extracts. The
enrichment was performed as per the manufacturer’s protocol with no
notable modifications.
LC-MS/MS sequencing for differential protein and
phosphoprotein analysis
Precipitation/on-pellet digestion procedure: Proteins extracted
from control and treated samples were precipitated with Chloroform-
methanol precipitation method [18]. The mixture was incubated
overnight at -20°C, and then centrifuged at 12,000 g for 20 min at 4°C.
The supernatant was then removed and the pellet was allowed to air
dry for 3 min. The on-pellet-digestion procedure consisted of 0.05%
of cleavable detergent (ProteasMAXTM) in the solution to expedite the
digestion and leads to the complete digestion. The peptide sample was
reduced with 1 mM TCEP at 95°C for 5 min, and then alkylated with
100 mM iodoacetamide at 37°C for 30 min in the dark. A second aliquot
of trypsin was added at an enzyme: substrate ratio of 1:25 (w/w). The
mixture was incubated at 37°C overnight to achieve complete digestion.
The final volume of the digestion mixture was approximately 100 μL. A
similar protocol was followed for phosphoproteins samples.
Estimation of on-pellet-digested proteins and phosphoproteins
from control and treated animals: A modified BCA method was
devised to permit comparison of the peptide recoveries for different
sample [18]. All analyses were performed in triplicate.
J Anal Bioanal Tech
ISSN:2155-9872 JABT, an open access journal
Page 2 of 9
2 sets of samples were used for the sequencing as indicated below
Set 1: IMAC enriched phosphoproteins (3controls+3 treated)
Set 2: Non-enriched proteins (total proteins) (3controls+3 treated).
Each of these samples were run in duplicate
In-solution digestion protocol: The samples, obtained following
on-pellet digestion, were dried using a stream of nitrogen. A trypsin
solution (prepared as 40 ng/μl in 10 mM ammonium bicarbonate,
containing 10% v/v acetonitrile) was added in the ratio of 1: 30. The
tubes were centrifuged at 200 rpm and incubated at 37°C for 4 hrs.
50 μl of the 1 mM DTT solution was dispensed to completely cover
the pellet, then vortexed, mixed and incubated at 95°C for 5 min. 50
μL (100 mM) of the iodoacetamide solution was added, vortexed and
incubated at 37°C for 30 min in dark. The mixture was then dried in a
vacuum concentrator. A ratio of 1:25 of 40 ng/μl trypsin was added and
incubated at 37°C overnight. Next day, after a short spin to the tubes,
5 μl of 5% formic acid was added till the pH reached 3. The tubes were
mixed well and centrifuged at 10000 rpm for 5 min. The supernatant
was then transferred into auto-sampler vials for LC-MS/MS analysis.
Sequencing and analysis by nano LC-MS/MS
LC-MS/MS analysis of isolated peptides and phosphopeptides:
150 minute RP-LC gradient was run for optimum separation of peptides
from normal and failed spermiation samples (estradiol treated). A label
free quantification strategy was followed by adding peptide standards in
a specific range. Prepared samples were spiked with 25 fmol/μl standard
tryptic BSA digest. Samples were subjected to Nano-LC-MS/MS.
Isolated phospho-peptide from normal and failed spermiation
samples were analyzed on an Agilent 1100 nano-flow system (Agilent
Technologies, Palo Alto, CA), LC connected to a LTQ FT mass
spectrometer (Thermo Electron, San Jose, CA), equipped with a
nanoelectrospray ion source. Phosphorylation screen method was
established over Nano-LC and LTQ-Orbitrap-MS. This includes
standardization of Data Dependent Neutral Loss MS3 (DDNLMS3)
strategy, involving screening of (S,T,Y) phosphorylation. The process of
selection of phosphorylation neutral loss for MS3 was standardized for
optimum gain. As a result, following m/z were chosen for MS3 neutral
loss scanning: 24.5000, 32.6700, and 49.00.
Identification of proteins and phosphoproteins from normal and
failed spermiation: Label free peak area based quantification was done
for all the samples by using standard BSA-tryptic digest. Area of all the
samples are normalized with area of BSA found in respective sample
(values taken from separate analysis performed using carbamido-
methylation as static modification).
Target-Decoy database search strategy was customized for
maximum coverage of proteome and phospho-peptides applying a
workflow merging MASCOT and SEQUEST. A decoy database search
was included in the Proteome Discoverer 1.3 (PD) enabled. High
confident peptides (≤ 1 %FDR), with prerequisite of minimum two
peptides, leading into identification of proteins were selected and list
was generated.
Spectral counts of the identified proteins were used as semi-
quantative measure to study the differential regulation of protein in
normal and failed spermiation condition. Ratios of spectral count of
Treated/control for total proteins and Phospho-treated/Phospho-
control for phosphoproteins were calculated to study differentials.
Ratios of ≥ 2.0 were considered significantly over-expressed and ratios
Volume 4 • Issue 4 • 1000172
Citation: Upadhyay RD, Yadav AK, Sonawane S, Goankar R, Dash D (2013) Differential Proteomic and Phospho-proteomic Analysis of Normal
versus Failed Spermiation in Adult Rats by Label-Free LC-MS/MS. J Anal Bioanal Tech 4: 172. doi:10.4172/2155-9872.1000172
≤ 0.5 were considered significantly under-expressed.
Validation of differentially expressed proteins
phosphoproteins
Localization of differentially expressed proteins during normal
and failed spermiation condition in the testis: Ten micron cryo-
sections were taken on poly-L-lysine coated slides and dried at 37°C
for 60 min. The sections were post-fixed in chilled acetone kept at
-20°C for 10 min and once again dried at 37°C for 60 min. This was
followed by three washes with PBS and blocking with iTTM FX signal
enhancer (Molecular Probes) for 30 min. The blocking reagent was
drained out and sections were incubated with protein-specific antibody
(Calreticulin, T-Complex protein 1, Calnexin, all the antibodies were
polyclonal from Abcam, Cambridge, UK) prepared in PBST (0.01
M PBS containing 0.05% Tween 20) overnight at 4°C. The following
day, sections were washed in PBS and incubated with appropriate
dilutions of secondary antibodies for 60 min at room temperature.
4α-6-Diamidino-2-phenylindole (DAPI) was used as a nuclear stain.
The sections were subsequently washed and mounted in Prolong
Gold Antifade (Molecular Probes). Co-localization of above primary
antibodies with F-actin (F-actin was used as a marker for testis-specific
actin-based structures, namely TBC and ES was done using phallodin
labeled with BODIPY 558/568 (red); all the other primary antibodies
were detected using goat anti-rabbit labeled with Alexa Flour 488. For
the negative control, an equivalent concentration of rabbit IgG was used,
instead of the primary antibody. Fluorescent images were captured using
Carl Zeiss-LSM510-Meta Confocal system (Oberkochen, Germany). Z
stacks images were generated.
Western blotting of differentially expressed proteins and
phosphoproteins during normal and failed spermiation condition:
The extracted proteins were run on 12% SDS-PAGE, followed by 1 hr
transfer on nitrocellulose membrane. The membranes were blocked
and then kept overnight at 4°C in the respective primary antibodies
(Calreticulin, T-Complex protein 1, Calnexin, 14-3-3 beta). Next
day, the blots then washed in PBS with 0.1% Tween-20 buffer on a
rocking platform for 30 mins. Secondary antibody (goat anti-rabbit,
Sigma, USA) was added to the blots and incubated for an hour at
room temperature. The blots were then washed in PBS with 0.1%
Tween-20 buffer on a rocking platform for an hour, and then developed
on an X-ray Film. GAPDH antibody was used as a loading control to
determine the expression levels of proteins and phosphoproteins.
Immuno-precipitation to study phosphorylation status of 14-3-
3beta protein: Immuno-precipitation was performed, as described by
Upadhyay et al. [17], except for the primary antibody used here was
polyclonal 14-3-3 beta (Epitomics, Germany). Normal rabbit serum,
instead of primary antibody, was used as negative control. Briefly, 150-
200 µg of protein extract from stages VII–VIII micro-dissected tubules
was incubated with 7.5 μg of Pan-phospho antibody (polyclonal) for
1 h at 4°C. As much as 50 μl of washed protein G slurry pre-chilled
at 4°C, was added to the immune complex and further incubated for
1 h at 4°C on a rocking platform. The immunoprecipitated complex
was then centrifuged at 10,000 g for 30 s at 4°C and the supernatant
was discarded. Beads containing bound immuno-complex were then
Samples set considered for Search Total Rattus
analysis engines proteins proteins
PC1, PC2, PC3, C1, C2, C3, Seques 2078 427
PT1, PT2, PT3, T1, T2, T3 Mascot
Table 1: Summary of results after LC-MS run of both normal and failed spermiation sample.
J Anal Bioanal Tech
ISSN:2155-9872 JABT, an open access journal
Page 3 of 9
washed thrice with 500 μl of lysis buffer, each time centrifuging for 30
s at 10,000 g. After the last wash, the supernatant was removed and 50
and μl of 1×Laemmli buffer was added to the bead pellet. The bead pellet
was boiled to 90-100°C for 5 min and centrifuged at 10,000 g for 5 min,
and the supernatant was loaded on to 10% SDS-PAGE gel, followed by
blotting on nitrocellulose membrane, and then probing with 14-3-3
beta antibody. Normal rabbit serum and mouse IgG, instead of primary
antibody, were used as negative control.
Results
Database search for identification of proteins and
phosphoproteins
LC MS/MS coupled to Mascot and Sequest database search was
used to identify the proteins and phosphoproteins. 2078 total proteins
were identified, of which 427 were of Rattus origin (Supplementary file
1). A total of 121 phospho-peptides with 201 phosphorylation sites,
and of these 23 showed Rattus assigned phospho-peptides, as shown
in Table 1.
Differentials expression analysis of proteins and
phosphoproteins
A total of 25 proteins were found to be up-regulated and 79 proteins
to be down regulated in the differential protein analysis data. A total of
11 phospho-proteins were found to be up-regulated and 12 phospho-
proteins to be down regulated in the differential phosphoprotein
analysis data.
Detailed of all the differentials are listed in Table 2 (up-regulated
protein), Table 3 (down-regulated protein), Table 4 (up-regulated
phosphoprotein) and Table 5 (down-regulated phosphoprotein).
Expression analysis of differentially regulated proteins and
phosphoproteins
Gene ontology analysis by STRAP (Software Tool for Researching
Annotation of Proteins): Analysis of the differential proteins in
terms of cellular component revealed 22% of proteins belong to the
nucleus, 17% to the cytoplasm, 7% to the cytoskeleton and 5% each
to endoplasmic reticulum and mitochondria [19] (Figure 1a). Analysis
in-terms of biological component showed 26% of proteins involved
in cellular process, 22% in regulation (Figure 1b), while molecular
function analysis demonstrated 46% of proteins involved in binding
and 23% in catalytic activity (Figure 1c).
Interestingly, gene ontology analysis by STRAP showed a number of
proteins of endoplasmic reticulum (ER) to be differentially expressed,
using cellular component as a determinant, suggesting involvement
of ER. Since, in the testis, endoplasmic reticulum (ER) forms an
essential component of ES and TBCs [4], we studied the expression of
two ER related proteins, i.e. calreticulin and calnexin, in stages where
spermiation occurs.
Biological process analysis showed the maximum no. of differential
proteins to be involved in cellular activity and its regulation. Moreover,
molecular function analysis revealed that lot of differentials to be
Total phospho- Total phosphorylation Assigned Phospho Rattus assigned
peptides sites peptides Phospho-peptides
121 201 29 23
Volume 4 • Issue 4 • 1000172
Citation: Upadhyay RD, Yadav AK, Sonawane S, Goankar R, Dash D (2013) Differential Proteomic and Phospho-proteomic Analysis of Normal
versus Failed Spermiation in Adult Rats by Label-Free LC-MS/MS. J Anal Bioanal Tech 4: 172. doi:10.4172/2155-9872.1000172

Page 4 of 9

Protein ID Protein Name Fold Change Q5HZV9 Protein phosphatase 1 regulatory subunit 7 0.13
Q3KRD5‎ Mitochondrial import receptor subunit TOM34 3.71 P13383 Nucleolin 0.08
P34058 Heat shock protein HSP 90-beta 17.53 P11442 Clathrin heavy chain 1 0.21
Q66HD0 Endoplasmin 2.14 Q6AY30 Probable saccharopine dehydrogenase 0.10
Q7TP40 PEST proteolytic signal-containing nuclear protein 2.36 Q5XI62 Uncharacterized protein C1orf56 homolog 0.20
P34058 Heat shock protein HSP 90-beta 17.53 Q62703 Reticulocalbin-2 0.32
Q66HD0 Endoplasmin 2.14 Q6T393 Spermatogenesis-associated protein 20 0.04
O08629 Transcription intermediary factor 1-beta 2.53 P04904 Glutathione S-transferase alpha-3 0.38
P11980 Pyruvate kinase isozymes M1/M2 2.70 P04785 Protein disulfide-isomerase 0.35
Q5RK28 Normal mucosa of esophagus-specific gene 1 3.37 Q9R063 Peroxiredoxin-5, mitochondrial 0.05
protein Q91Y78 Ubiquitin carboxyl-terminal hydrolase isozyme L3 0.16
Q68FR9 Elongation factor 1-delta 4.50 Q63610 Tropomyosin alpha-3 chain 0.37
P15865 Histone H1.2 4.79 O35244 Peroxiredoxin-6 0.49
P07943 Aldose reductase 2.77 P05197 Elongation factor 2 0.18
P02770 Serum albumin 2.90 Q62764 DNA-binding protein A 0.23
Q3KRD5 Mitochondrial import receptor subunit TOM34 3.71 P11884 Aldehyde dehydrogenase, mitochondrial 0.26
Q63525 Nuclear migration protein nudC 2.08 Q08163 Adenylyl cyclase-associated protein 1 0.22
Q60587 Trifunctional enzyme subunit beta, mitochondrial 6.40 P10888 Cytochrome c oxidase subunit 4 isoform 1, mitochon- 0.47
Q498U4 SAP domain-containing ribonucleoprotein 2.15 drial
Q64060 Probable ATP-dependent RNA helicase DDX4 2.14 Q06647 ATP synthase subunit O, mitochondrial 0.39
P38983 40S ribosomal protein SA 3.56 Q9Z2L0 Voltage-dependent anion-selective channel protein1 0.14
Q6P6R2 Dihydrolipoyl dehydrogenase, mitochondrial 2.33 P38656 Lupus La protein homolog 0.11
P07483 Fatty acid-binding protein, heart 2.29 P52555 Endoplasmic reticulum resident protein 29 0.17
B0K020 CDGSH iron-sulfur domain-containing protein 1 2.20 P37996 ADP-ribosylation factor-like protein 3 0.37
Q9ER34 Aconitate hydratase, mitochondrial 3.52 P17164 Tissue alpha-L-fucosidase 0.16
P54921 Alpha-soluble NSF attachment protein 8.80 P80254 D-dopachrome decarboxylase 0.34
P18418 Calnexin 3.31 O88600 Heat shock 70 kDa protein 4 0.21
P50554 4-aminobutyrate aminotransferase, mitochondrial 0.18
Table 2: List of up-regulated proteins during normal and failed spermiation
condition. P85972 Vinculin 0.24
O08651 D-3-phosphoglycerate dehydrogenase 0.23
Protein ID Protein Name Fold change P45479 Palmitoyl-protein thioesterase 1 0.34
P15999 ATP synthase subunit alpha, mitochondrial 0.32 Q5XHZ2 Synaptonemal complex central element protein 1 0.07
P11598 Protein disulfide-isomerase A3 0.23 Q5XI60 Receptor expression-enhancing protein 6 0.28
Q6AYX5 Outer dense fiber protein 2 0.47 Q07936 Annexin A2 0.26
Q68FX6 Calcium-binding and spermatid-specific protein 1 0.44 O88767 Protein DJ-1 0.24
P02401 60S acidic ribosomal protein P2 0.14 Q6P6V0 Glucose-6-phosphate isomerase 0.26
P35565 Calreticulin 0.36 P04642 L-lactate dehydrogenase A chain 0.46
Q63429 Polyubiquitin-C 0.48 P21533 60S ribosomal protein L6 0.35
P97536 Cullin-associated NEDD8-dissociated protein 1 0.47 O35774 A-kinase anchor protein 4 0.34
P10719 ATP synthase subunit beta, mitochondrial 0.45 P11762 Galectin-1 0.13
Q6PEC1 Tubulin-specific chaperone A 0.22 P11030 Acyl-CoA-binding protein 0.46
Q9EPH8 Polyadenylate-binding protein 1 0.47 B0BN93 26S proteasome non-ATPase regulatory subunit 13 0.24
Q63081 Protein disulfide-isomerase A6 0.30 Q64560 Tripeptidyl-peptidase 2 0.16
P47245 Nardilysin 0.20 P24155 Thimet oligopeptidase 0.45
Q6P502 T-complex protein 1 subunit gamma 0.43 P14668 Annexin A5 0.28
Q6AY33 Acrosin-binding protein 0.39 P30009 Myristoylated alanine-rich C-kinase substrate 0.46
P13084 Nucleophosmin 0.22 Q7TP40 PEST proteolytic signal-containing nuclear protein 0.36
P04797 Glyceraldehyde-3-phosphate dehydrogenase 0.30 Q68FY0 Cytochrome b-c1 complex subunit 1, mitochondrial 0.16
P02793 Ferritin light chain 1 0.16 P14841 Cystatin-C 0.37
Q68FQ0 T-complex protein 1 subunit epsilon 0.20 Q9ER24 Ataxin-10 0.30
Q68FS2 COP9 signalosome complex subunit 4 0.20 Table 3: List of down-regulated proteins during normal and failed spermiation
P35571 Glycerol-3-phosphate dehydrogenase, mitochondrial 0.38 condition.
Q63945 Protein SET 0.07 involved in binding of proteins. Based on these indications, we selected
P19945 60S acidic ribosomal protein P0 0.38 14-3-3 beta from differential phosphoprotein list, which have been
P45592 Cofilin-1 0.28 shown to have a large number of binding partners, and is involved in
Q63617 Hypoxia up-regulated protein 1 0.213572 various cellular activities like protein-protein interaction, cell adhesion,
Q64428 Trifunctional enzyme subunit alpha, mitochondrial 0.30 cellular trafficking, etc. [20].
P10111 Peptidyl-prolyl cis-trans isomerase A 0.25
P16036 Phosphate carrier protein, mitochondrial 0.19 Collectively, from the differential total protein list, we selected
O35180 Endophilin-A3 0.41 two down-regulated proteins (Calreticulin and T-Complex protein
Q80U96 Exportin-1 0.19 1), and one up-regulated protein (Calnexin), for validation. From

J Anal Bioanal Tech

ISSN:2155-9872 JABT, an open access journal Volume 4 • Issue 4 • 1000172
Citation: Upadhyay RD, Yadav AK, Sonawane S, Goankar R, Dash D (2013) Differential Proteomic and Phospho-proteomic Analysis of Normal
versus Failed Spermiation in Adult Rats by Label-Free LC-MS/MS. J Anal Bioanal Tech 4: 172. doi:10.4172/2155-9872.1000172

Page 5 of 9

Protein ID Protein Name Fold Change

P35213 14-3-3 protein beta/alpha 3.24
O08629 Transcription intermediary factor 1-beta 2.77
O35180 Endophilin-A3 3.39
Q4V8E4 Coiled-coil domain-containing protein 104 13.96
P31000 Vimentin 3.36
Q62703 Reticulocalbin-2 22.00
Q4QR76 Actin-like protein 7B 3.64
Q6P6S3 B box and SPRY domain-containing protein 2.09
O35987 NSFL1 cofactor p47 3.44
Q3KR86 Mitochondrial inner membrane protein (Fragment) 3.11
P05371 Clusterin 4.59
Table 4: List of up-regulated differential Phosphoproteins during normal and failed
spermiation condition.

Protein ID Protein Name Fold change

Q05962 ADP/ATP translocase 1 0.22
Q6P4Z9 COP9 signalosome complex subunit 8 0.42
Q5U216 ATP-dependent RNA helicase DDX39A 0.42
Q07647 Solute carrier family 2, facilitated glucose trans- 0.12 Figure 2: The images represent Immunofluroscence (IF) of stage VII-VIII
porter member 3 tubules in the testis showing calnexin localization during normal and failed
P21670 Proteasome subunit alpha type-4 0.07 spermiation.
Panel a shows localization in the control group (normal spermiation) and panel
Q6AYG7 NFATC2-interacting protein 0.33 b shows localization in the treated group while subpanel c show negative
P38983 40S ribosomal protein SA 0.26 control with rabbit IGg instead of primary antibody.
Q9ESW0 DNA damage-binding protein 1 0.29 Green fluorescence=primary antibody (calnexin), red fluorescence=phalloidin
(F-actin marker), blue fluorescence=DAPI (nuclear stain), Bar=10 μm.
Q10728 Protein phosphatase 1 regulatory subunit 12A 0.19
Q5XIP1 Protein pelota homolog 0.46
Q10728 Protein phosphatase 1 regulatory subunit 12A 0.19
P21670 Proteasome subunit alpha type-4 0.07

Table 5: List of down-regulated differential Phosphoproteins during normal and

failed spermiation condition.

Figure 3: The images represent Immunofluroscence (IF) of stage VII-VIII

Figure 1: The images depict representative pie diagram of STRAP analysis
of the differentials identified during normal and failed spermiation condition by
LC-MS/MS.
The sub-panels a denotes cellular component analysis of the differentials.
The sub-panels b denotes biological process analysis of the differentials.
The sub-panels c denotes molecular function analysis of the differentials.
the differential phosphoprotein list, we selected up-regulated
phosphoprotein 14-3-3 beta, to validate LC-MS/MS results. Validation
of the above proteins was performed by immunofluroscence, western
blotting and immunoprecipitation experiments.
Localization of differential proteins during normal and failed
spermiation condition: Calnexin and calreticulinare components of
tubules in the test is showing calreticulin localization during normal and failed
spermiation.
Panel a shows localization in the control group (normal spermiation) and panel
b shows localization in the treated group while subpanel c show negative
control with rabbit IGg instead of primary antibody.
Green fluorescence=primary antibody (calreticulin), red fluorescence=phalloidin
(F-actin marker), blue fluorescence=DAPI (nuclear stain), Bar=10 μm.
the quality control system that promotes correct folding of proteins
that enter the secretory pathway and targets mis-folded proteins for
degradation. Both calnexin and calreticulin reside in the ER [21]. Hence,
their localization was studied during normal and failed spermiation in
the testis.
Calnexin was seen to be localized in the adluminal compartment
of the seminiferous epithelium of the testis during normal and failed
spermiation condition (Figure 2). The signal intensity (expression

J Anal Bioanal Tech

ISSN:2155-9872 JABT, an open access journal Volume 4 • Issue 4 • 1000172
Citation: Upadhyay RD, Yadav AK, Sonawane S, Goankar R, Dash D (2013) Differential Proteomic and Phospho-proteomic Analysis of Normal
versus Failed Spermiation in Adult Rats by Label-Free LC-MS/MS. J Anal Bioanal Tech 4: 172. doi:10.4172/2155-9872.1000172

Page 6 of 9

level) of calnexin seemed to be increased during failed spermiation, i.e.

estradiol treated group (Figure 2b).
Calreticulin was seen to be localized in the adluminal compartment
surrounding round spermatids of the seminiferous epithelium of the
testis during normal and failed spermiation condition (Figure 3). The
signal intensity (expression level) of calreticulin seemed to be decreased
during failed spermiation, i.e. estradiol treated group (Figure 3b).
The expression of F-actin (marker for TBC and ES) was localized
in the TBCs and ES in control group, i.e. normal spermiation (both
Figures 2a and 3a-red panels), whereas in failed spermiation, it was
only observed in ES as TBC were absent (both Figures 2b and 3b-red
panels).
The T-complex protein 1 (TCP-1) of the mouse has been shown
to play an important role in male germ cell development. In the yeast,
TCP-1 is involved in actin and tubulin cytoskeletal protein folding [22-
24].
Hence, testicular localization of TCP-1 was performed during
normal and failed spermiation condition (Figure 4). Localization of
T-complex protein-1 during spermiation revealed that it was mainly
localized in the seminiferous epithelium. The signal intensity was seen
to localize around all the germ cell types, both in the basal and in the
adluminal compartment during normal spermiation (Figure 4a). In the
failed spermiation condition, i.e. in the treated group the expression of Figure 5: Western Blotting and expression analysis of proteins during normal
and failed spermiation condition.
TCP-1 protein was observed only at the luminal edge of the epithelium Panel I, II, III represents western blotting of the TCP-1, calnexin and calreticulin
(Figure 4b). proteins respectively from tubules at stages VII-VIII in control group and treated
group. –ve denotes negative control without primary antibody.
Expression levels of all the three proteins during normal and Subpanel a,b,c denotes graphical representation of expression of TCP-1,
failed spermiation condition: Western Blotting of calnexin, calreticulin calnexin and calreticulin proteins respectively in control (normal spermiation)
and treated (failed spermiation) groups. Data n=3 animals, *=significant (p ≥
and TCP-1: Western blotting experiments showed significant down 0.02).
regulation of Tcp-1with p-value ≤ 0.05, and trend toward increase and
decrease for calnexin and calreticulin, respectively, as compared with

Figure 6: Expression analysis of 14-3-3 beta during normal and failed

spermiation condition.
Panel a represents western blot of the 14-3-3 beta (30 kDa) and GAPDH (37
kDa, loading control) protein from tubules at stages VII-VIII in control group and
treated group. –ve denotes negative control without primary antibody. Data of
three animals are shown (3 control and 3 treated).
Panel b denotes graphical representation of the relative expression of 14-3-3
beta w.r.t loading control GAPDH.

GAPDH expression levels (loading control). The results were seen to be

Figure 4: The images represent Immunofluroscence (IF) of stage VII-VIII
tubules in the test are showing T-complex protein-1 (TCP-1) localization during
normal and failed spermiation.
Panel a shows localization in the control group (normal spermiation) and panel
b shows localization in the treated group while subpanel c show negative
control with rabbit IGg instead of primary antibody.
green fluorescence=primary antibody (TCP-1), red fluorescence=phalloidin
(F-actin marker), blue fluorescence=DAPI (nuclear stain), Bar=10 μm.
in concordance with the LC-MS differential data (Figure 5).
Detection of phosphorylation status of 14-3-3 beta
Expression of 14-3-3 protein in testis during normal and failed
spermiation: As we observed an increase in the phosphorylation of 14-
3-3 beta in the differential phosphoprotein list, we sought to determine
the expression of 14-3-3 beta at protein level during normal and failed

J Anal Bioanal Tech

ISSN:2155-9872 JABT, an open access journal Volume 4 • Issue 4 • 1000172
Citation: Upadhyay RD, Yadav AK, Sonawane S, Goankar R, Dash D (2013) Differential Proteomic and Phospho-proteomic Analysis of Normal
versus Failed Spermiation in Adult Rats by Label-Free LC-MS/MS. J Anal Bioanal Tech 4: 172. doi:10.4172/2155-9872.1000172
Figure 7: Phosphorylation status of 14-3-3 beta protein during normal and
failed spermiation condition.
The image represents western blot of the phosphorylated 14-3-3 beta
(immunoprecipitated with Pan-phospho antibody) from tubules at stages VII-
VIII in control group and treated group. –ve denotes negative control using
rabbit serum (IP -ve control).
spermiation condition, i.e. in stages VII-VIII of the seminiferous tubule.
Western blotting showed no change in the expression levels of 14-3-3
beta (30 kDa) when compared with GAPDH (37 kDa), as a loading
control in normal and failed spermiation condition (Figures 6a and 6b).
Phosphorylation status of 14-3-3 beta by immuno-precipitation:
The phosphorylation status of 14-3-3 beta by immuno-precipitation
experiment revealed increased phosphorylation of 14-3-3 beta in
the treated group as compared to controls (Figure 7), indicating up-
regulation of phosphorylation of 14-3-3 beta, as seen in the differentials
of phosphoproteins.
Discussion
Our study suggests that there is a change in the expression of
proteins and phosphoproteins during normal and failed spermiation
condition in stages VII-VIII of rat spermatogenic cycle. A total of 427
proteins and phosphoproteins belong to rattus species (rat) of proteins
identified. Differential protein and phosphoprotein profile revealed 24
up-regulated and 80 down-regulated proteins, and 11 up-regulated and
12 down-regulated phosphoproteins.
Gene Ontology study by using STRAP software [19] revealed that
a number of proteins were related to endoplasmic reticulum (ER), as
determined using cellular component as a determinant. Since ES and
TBCs have ER as a part of their structure, we studied the expression
of two ER related proteins. Biological process analysis showed the
maximum number of differential proteins to be involved in cellular
activity and its regulation. In addition, molecular function analysis
revealed that maximum number of differentials proteins to be involved
in binding.
Amongst the other proteins in the differential list, a number
of proteins important for the process of spermatogenesis were
demonstrated, For example, Aldose reductase was seen to up-regulated
in the proteomic profile; it is known to be involved in reduction of
cytotoxic metabolites produced during spermatogenesis in addition
to its metabolic function [25]. Cofilin-1, which is down-regulated, is
known to plays a role in actin turn-over by depolymerization of actin
filament, suggesting a defect in actin remodeling during stage VII-VII
in normal versus treated animals could have led to absence of TBCs
J Anal Bioanal Tech
ISSN:2155-9872 JABT, an open access journal
Page 7 of 9
reported in our earlier study and spermiation failure [15,26]. A number
of the differentials belong to mitochondrial component of the cell, as
demonstrated by STRAP software analysis, suggesting mitochondrial
dysfunction. One such protein, phosphorylated adenine nuclease
translocase-1, which was seen to be down-regulated in the present study,
has been reported to be involved in mitochondrial DNA maintenance
[27]. MARCKS (myristoylated alanine-rich C-kinase substrate) is
known to be calcium regulated, and its phosphorylation is important
for acrosomal exocytosis [28]. Down-regulation of MARCKS in the
present study suggests the importance of calcium homeostasis during
spermatogenesis and acrosomal formation. The role of the above
mentioned proteins in the process of spermiation is unclear and further
studies in this direction needs to be done.
During spermiation, endoplasmic reticulum is known to show
dynamic changes. Flattened ER (f-ER, narrow cisternae of ER), is
associated with ES and arranged in concentric layers in the Sertoli cell
cytoplasm covering the spermiated head. The f-ER disappeared just
prior to spermiation and another ER structure, i.e. tubular ER appears
during late stage-VII surrounding both dorsal and ventral surface of
the spermatid head, which also disappeared at the time of spermiation
[29]. In addition, during spermatogenesis, ER proteins are known to be
important in regulating sperm motility and acrosomes reaction [30]. In
the present study, two ER resident proteins, calnexin and calreticulin,
are shown to be up and down regulated, respectively during failed
spermiation condition, suggesting their involvement in the dynamic
changes in ER form during spermiation, but the mechanism remains
to be elucidated.
Immuno-localization studies with calnexin showed its expression
in the adluminal compartment of the seminiferous epithelium and the
intensity of staining showed an increase in treated group. In addition,
western blotting also showed the protein to be up regulated. It has been
established that it plays a role in protein folding and its quality control.
Knockout of a testis-specific variant of calnexin, calmegin, showed
mice were infertile, highlighting the importance of calnexin in male
fertility [31]. Furthermore, localization of calmegin was reported to be
in the meiotic and post meiotic germ cell in the test is suggesting its
role in spermatid differentiation [32]. In yeast cells, calnexin have been
shown to regulate the process of apoptosis during their fission [33]. In
the present study, an increase of calnexin in spermiation failure group
may be involved in similar function of regulating apoptosis, as our
earlier study showed increase in tunnel positive cell in the treatment
group [14].
Calreticulin, a multi-functional protein resides mainly in ER is
a chaperone, and is involved inthe regulation of cell adhesion, gene
expression and intracellular Ca+2 homeostasis. It has been shown to be
present in the acrosomes of rat sperm [30,34]. A decrease of calreticulin
in the treated group could have affected the process of acrosomes
formation. Over-expression of calreticulin has shown to regulate
vinculin assembly at focal contacts and decreases phosphorylation
status of β-catenin [35]. Calreticulin affects β-catenin-associated
pathway, and hence cell adhesion [36]. Moreover, β-catenin along-with
cadherins forms an actin-based adherens junctions associated with ES
during spermatogenesis [37], suggesting the involvement of calreticulin
in spermatogenesis.
Cytoskeleton reorganization is shown to be important in
maintaining the cytoarchitecture of seminiferous epithelium [38]. Our
earlier studies demonstrated disruption in the actin and microtubular
organization and spermiation failure, following 17β-estradiol
treatment [15]. The present study shows significant down regulation
Volume 4 • Issue 4 • 1000172
Citation: Upadhyay RD, Yadav AK, Sonawane S, Goankar R, Dash D (2013) Differential Proteomic and Phospho-proteomic Analysis of Normal
versus Failed Spermiation in Adult Rats by Label-Free LC-MS/MS. J Anal Bioanal Tech 4: 172. doi:10.4172/2155-9872.1000172
of T-complex protein-1, a molecular chaperone, activated when bound
to c-AMP. Mutational studies with TCP-1 genes in the yeast have
shown disorganization of actin and microtubular network [23,24]. The
disruption in actin and microtubules observed earlier could be due to
the down regulation. Futhermore, TCP-1 protein has been shown to
be present in chromatin of highly condensed spermatid, suggesting its
involvement in sperm head formation [39]. We hypothesize that down
regulation of TCP-1 affected actin/microtubule organization, affecting
spermatid differentiation and sperm release.
Moreover, we observed phosphorylated 14-3-3 beta protein to
be over expressed in the differential phosphorylation data. Studies
in humans and rats have shown binding of 14-3-3 beta to vimentin
and tubulin in the seminiferous epithelium [17,40]. The upregulation
of phosphorylated 14-3-3 beta observed in the present study may
have affected the phosphorylation status of vimentin, leading to re-
organization of vimentin and observed apical projection retention in
stages VIII in the testis, as described earlier [17]. In addition, binding of
14-3-3 to Protein Phosphatase 1 gamma isoform have been shown to be
important during sperm function and its motility [41]. Thus, increased
phosphorylation of 14-3-3 beta may have affected vimentin and tubulin
reorganization leading to spermiation failure.
In summary, the present study demonstrates differential protein
and phosphoprotein expression during spermiation and spermiation
failed condition by LC-mass spectrometry. The differential proteins
identified suggest an effect on endoplasmic reticulum and cytoskeleton
reorganization, which forms an important component of ES and TBCs,
indicating that the function of these two structures maybe affected,
leading to spermiation failure. Some of the differentials that may serve
as potential biomarker for sperm release, however, more studies need
to be done to achieve this. Alternatively, since a number of proteins
involved in calcium homeostasis and acrosome biogenesis is affected,
suggesting that estrogen treatment could have affected the spermatid
maturation. The affected spermatids are then not released but retained
and phagocytosed by the Sertoli cell. This may prevent defective
spermatid from fertilizing the oocyte. Since, spermiation is the final
stage of spermatogenesis molecules regulating this event offer a great
potential for reversible contraceptive development without affecting
the other germ cell population.
Acknowledgements
We would like to thank Department of Science and Technology, New Delhi,
India, for partially funding the study. We would acknowledge ICMR for SRF fel-
lowship to Rahul D Upadhyay. We thank the staff of the C-CAMP facility, Mass
Spectrometry Lab, National Centre for Biological Sciences, Bangalore for the LC-
MS/MS sequencing of samples, and we are grateful to Dr. Dominik Schwudke, Mr.
Karthik Kamath for their valuable assistance in the sequencing, data generation of
proteins and phosphoproteins of our samples. The assistance of Dr. Carmen Te-
kwe is also acknowledged in analyzing the spectrometry data. We would also like
to acknowledge the efforts of Mr. Suryakant Mandavkar in the animal experiment
and Mr. Deepak Shelar for his technical assistance. Authors would like to thank Ms
Annette Fonseca for her help in correcting grammatical mistakes of the manuscript.
Conflict of Interest
Authors declare no conflict of interest.
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Rats by Label-Free LC-MS/MS. J Anal Bioanal Tech 4: 172. doi:10.4172/2155-9872.1000172
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