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Page 1 ofReproduction 28 Advance Publication first posted on 19 December 2013 as Manuscript REP-13-0313

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Identification of differentially expressed proteins between fresh

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and frozen-Thawed boar spermatozoa by iTRAQ-coupled 2D

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LC-MS/MS

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Xiaoli Chen1,2┼, Huabin Zhu1┼, Chuanhuo Hu2, Haisheng Hao1, Junfang Zhang1,

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Kunpeng Li1,3, Xueming Zhao1, Tong Qin1, Kan Zhao4, Huishan Zhu4 and Dong Wang1,*

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1 The Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture

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of China, Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing 100193,

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China

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2 Animal Science and Technology College, Guangxi University, Nanning 530004, China

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3 Jilin Agriculture University, Changchun, 130118, China

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4 Beijing Protein Innovation Co., Ltd., Beijing 101318, China

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* Correspondence: Dr. Dong Wang, The Key Laboratory for Farm Animal Genetic Resources and Utilization of

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Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agriculture Sciences, Beijing

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100193, China

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E-mail: [email protected]

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Tel: +86-10-62815892, +86-10-13810509281

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┼These authors contributed equally to this work.

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Abstract

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Cryodamage is a major problem in semen cryopreservation, causing changes in protein levels that

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influence the function and motility of the spermatozoa. In our study, protein samples prepared from fresh

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and frozen-thawed boar spermatozoa were compared using the iTRAQ labeling technique coupled with

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2D-LC/MS-MS analysis. A total of 41 differentially expressed proteins were identified and quantified,

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including 35 proteins that were increased and 6 proteins that were decreased in frozen-thawed

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spermatozoa by at least a mean of 1.79-fold (P < 0.05). By classifying into 10 distinct categories using

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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.

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to sperm premature capacitation, adhesions, energy supply and sperm-oocyte binding and fusion. The

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expressions of four of these proteins, SOD1, TPI1, ODF2 and AKAP3, were verified by Western blot. We

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propose that the alteration of these identified proteins affected the cryopreserved semen quality and

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ultimately lowered the fertilizing capacity. This is the first study to compare protein levels between fresh

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and frozen-thawed spermatozoa using iTRAQ technology. Our preliminary results provide an overview of

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the molecular mechanisms of cryodamage in frozen-thawed spermatozoa and theoretical guidance to

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improve the cryopreservation of boar semen.

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Introduction

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For breeding of livestock, freezing semen has become an indispensable technique in the cattle

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industry (Pons-Rejraji et al. 2009, Lemma A 2011). However, farrowing rates decrease by 20–30%

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and litter size drops by two to three piglets when using cryopreserved boar semen. Because of the

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sublethal damage, approximately 40–50% of the sperm do not survive cryopreservation, and even

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when using similar numbers of motile sperm, fertility is still lower after thawing compared with

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fresh semen (Watson 2000). As a result, cryopreserved semen is not routinely used in the pig

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industry (Bailey et al. 2008). To improve the cryopreservation technology of boar semen, many

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studies have focused on understanding the mechanism underlying the cryodamage. It has been

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shown that the most evident damage are to the plasma membrane, acrosome, mitochondrial

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sheath (mid-piece) and axonema after freezing and thawing (Cerolini et al. 2001), and studies on

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the proteins of cryopreserved sperm have revealed profound implications on fertility and embryo

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development (Oliva et al. 2009). Cryopreservation changes the functional state of many proteins,

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such as enzymes related to sperm metabolism (Huang et al. 1999), proteins related to capacitation

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and acrosome reaction (Tabuchi et al. 2008), proteins related to membrane and structure 2

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(Desrosiers et al. 2006), and proteins related to apoptosis (Jeong et al. 2009). These variations all

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influence the structural integrity, biological processes and function, ultimately reducing the

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fertilization capacity of the sperm.

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To explore the mechanism of cryodamage, the two-dimensional gel electrophoresis (2-DE) was

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used to detect the changes of protein in sperm, and many differentially expressed proteins were

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found in human sperm (Cao WL 2003), sea bass sperm (Zilli et al. 2005) and sheep sperm (Li HY

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2011) after cryopreservation. However, they did not identify those differentially expressed proteins

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in their studies. The proteome profiles of ‘good’ and ‘poor’ boar sperm after freezing were also

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compared using LC-MS/MS analysis, which indicated that boar spermatozoa contain large amount

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of proteins whose susceptibility to cryopreservation and implications for sperm function are still to

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be characterised (Feugang JM 2011). In current proteomics research, the quantification of proteins

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has developed into a combination of iTRAQ (isobaric tags for relative and absolute quantification)

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and LC-MS/MS (Niu et al. 2009). The multiplexing capability allows different protein samples to be

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simultaneously quantified with a control standard sample in the same run (Tannu & Hemby 2006).

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In our present study, the iTRAQ-coupled 2-D (two-dimensional) LC-MS/MS approach was applied

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for the first time to study the changes in proteins of fresh and frozen-thawed sperm on a global

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scale in order to understand the primary mechanism and process of cryodamage to the

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spermatozoa. Overall, we aimed to provide a foundation for optimization and improvement of

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cryopreservation technology in this study.

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Materials and methods

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Collection and Pre-treatment of Semen

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In the present experiment, animal care and samples collection procedures were approved and

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conducted under established standard of the Institute of Animal Science, Chinese Academy of 3

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Agricultural Sciences, Beijing, China. Semen samples were randomly obtained from six healthy and

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normally collected semen of Yorkshire stud boars (2–3 years old, the farrowing rate is 80-90% and

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litter size is 11-13) from the Beijing HotBoar swine AI Service Centre. The sperm-rich fraction of

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each ejaculate was manually collected using the gloved-hand method. After collection, the semen

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samples were immediately filtered through a 100-μm semen filter paper to remove gelatinous

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material and diluted at the ratio of 1:1 (v:v) at room temperature. The diluted semen samples were

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transported to our laboratory at 25–30°C within less than 1 h for quality assessment and

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subsequent analysis.

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Sperm quality analyses were performed by microscopy to ensure the quality of the ejaculates

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(motility >80%, deformation ratio