Page 1 ofReproduction 28 Advance Publication first posted on 19 December 2013 as Manuscript REP-13-0313
1
Identification of differentially expressed proteins between fresh
2
and frozen-Thawed boar spermatozoa by iTRAQ-coupled 2D
3
LC-MS/MS
4 5
Xiaoli Chen1,2┼, Huabin Zhu1┼, Chuanhuo Hu2, Haisheng Hao1, Junfang Zhang1,
6
Kunpeng Li1,3, Xueming Zhao1, Tong Qin1, Kan Zhao4, Huishan Zhu4 and Dong Wang1,*
7 8
1 The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture
9
of China, Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193,
10
China
11
2 Animal Science and Technology College, Guangxi University, Nanning 530004, China
12
3 Jilin Agriculture University, Changchun, 130118, China
13
4 Beijing Protein Innovation Co., Ltd., Beijing 101318, China
14 15
* Correspondence: Dr. Dong Wang, The Key Laboratory for Farm Animal Genetic Resources and Utilization of
16
Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing
17
100193, China
18
E-mail:
[email protected]
19
Tel: +86-10-62815892, +86-10-13810509281
20
┼These authors contributed equally to this work.
21 22
Abstract
23
Cryodamage is a major problem in semen cryopreservation, causing changes in protein levels that
24
influence the function and motility of the spermatozoa. In our study, protein samples prepared from fresh
25
and frozen-thawed boar spermatozoa were compared using the iTRAQ labeling technique coupled with
26
2D-LC/MS-MS analysis. A total of 41 differentially expressed proteins were identified and quantified,
27
including 35 proteins that were increased and 6 proteins that were decreased in frozen-thawed
28
spermatozoa by at least a mean of 1.79-fold (P < 0.05). By classifying into 10 distinct categories using
29
bioinformatic analysis, most of the 41 differentially expressed proteins were found to be closely relevant 1
Copyright © 2013 by the Society for Reproduction and Fertility.
Page 2 of 28
30
to sperm premature capacitation, adhesions, energy supply and sperm-oocyte binding and fusion. The
31
expressions of four of these proteins, SOD1, TPI1, ODF2 and AKAP3, were verified by Western blot. We
32
propose that the alteration of these identified proteins affected the cryopreserved semen quality and
33
ultimately lowered the fertilizing capacity. This is the first study to compare protein levels between fresh
34
and frozen-thawed spermatozoa using iTRAQ technology. Our preliminary results provide an overview of
35
the molecular mechanisms of cryodamage in frozen-thawed spermatozoa and theoretical guidance to
36
improve the cryopreservation of boar semen.
37 38
Introduction
39
For breeding of livestock, freezing semen has become an indispensable technique in the cattle
40
industry (Pons-Rejraji et al. 2009, Lemma A 2011). However, farrowing rates decrease by 20–30%
41
and litter size drops by two to three piglets when using cryopreserved boar semen. Because of the
42
sublethal damage, approximately 40–50% of the sperm do not survive cryopreservation, and even
43
when using similar numbers of motile sperm, fertility is still lower after thawing compared with
44
fresh semen (Watson 2000). As a result, cryopreserved semen is not routinely used in the pig
45
industry (Bailey et al. 2008). To improve the cryopreservation technology of boar semen, many
46
studies have focused on understanding the mechanism underlying the cryodamage. It has been
47
shown that the most evident damage are to the plasma membrane, acrosome, mitochondrial
48
sheath (mid-piece) and axonema after freezing and thawing (Cerolini et al. 2001), and studies on
49
the proteins of cryopreserved sperm have revealed profound implications on fertility and embryo
50
development (Oliva et al. 2009). Cryopreservation changes the functional state of many proteins,
51
such as enzymes related to sperm metabolism (Huang et al. 1999), proteins related to capacitation
52
and acrosome reaction (Tabuchi et al. 2008), proteins related to membrane and structure 2
Page 3 of 28
53
(Desrosiers et al. 2006), and proteins related to apoptosis (Jeong et al. 2009). These variations all
54
influence the structural integrity, biological processes and function, ultimately reducing the
55
fertilization capacity of the sperm.
56
To explore the mechanism of cryodamage, the two-dimensional gel electrophoresis (2-DE) was
57
used to detect the changes of protein in sperm, and many differentially expressed proteins were
58
found in human sperm (Cao WL 2003), sea bass sperm (Zilli et al. 2005) and sheep sperm (Li HY
59
2011) after cryopreservation. However, they did not identify those differentially expressed proteins
60
in their studies. The proteome profiles of ‘good’ and ‘poor’ boar sperm after freezing were also
61
compared using LC-MS/MS analysis, which indicated that boar spermatozoa contain large amount
62
of proteins whose susceptibility to cryopreservation and implications for sperm function are still to
63
be characterised (Feugang JM 2011). In current proteomics research, the quantification of proteins
64
has developed into a combination of iTRAQ (isobaric tags for relative and absolute quantification)
65
and LC-MS/MS (Niu et al. 2009). The multiplexing capability allows different protein samples to be
66
simultaneously quantified with a control standard sample in the same run (Tannu & Hemby 2006).
67
In our present study, the iTRAQ-coupled 2-D (two-dimensional) LC-MS/MS approach was applied
68
for the first time to study the changes in proteins of fresh and frozen-thawed sperm on a global
69
scale in order to understand the primary mechanism and process of cryodamage to the
70
spermatozoa. Overall, we aimed to provide a foundation for optimization and improvement of
71
cryopreservation technology in this study.
72
Materials and methods
73
Collection and Pre-treatment of Semen
74
In the present experiment, animal care and samples collection procedures were approved and
75
conducted under established standard of the Institute of Animal Science, Chinese Academy of 3
Page 4 of 28
76
Agricultural Sciences, Beijing, China. Semen samples were randomly obtained from six healthy and
77
normally collected semen of Yorkshire stud boars (2–3 years old, the farrowing rate is 80-90% and
78
litter size is 11-13) from the Beijing HotBoar swine AI Service Centre. The sperm-rich fraction of
79
each ejaculate was manually collected using the gloved-hand method. After collection, the semen
80
samples were immediately filtered through a 100-μm semen filter paper to remove gelatinous
81
material and diluted at the ratio of 1:1 (v:v) at room temperature. The diluted semen samples were
82
transported to our laboratory at 25–30°C within less than 1 h for quality assessment and
83
subsequent analysis.
84
Sperm quality analyses were performed by microscopy to ensure the quality of the ejaculates
85
(motility >80%, deformation ratio