Proteomic Revolution to Improve Tools for Evaluating Male Fertility in

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Proteomic Revolution to Improve Tools for Evaluating Male Fertility in Animals Yoo-Jin Park, Jin Kim, Young-Ah You, and Myung-Geol Pang* Department of Animal Science and Technology, Chung-Ang University, Anseong, Gyeonggi-do 456-756, Korea ABSTRACT: Artificial insemination has been used as a common breeding technique for the rapid dissemination of important genes to improve livestock quality. However, infertility or subfertility in the male leads to the disintegration of the breeding system and large economic losses. Therefore, the development of an accurate diagnostic protocol for male fertility is of critical importance. To this end, many basic laboratory assays have been developed on the basis of semen analysis. Although these assays may provide a preliminary estimate of male fertility, their accuracies are often unacceptably low. Therefore, it is vital to develop new semen analyses that are simple to use and accurate. Proteomic approaches will shed light on understanding sperm physiology and help in developing new diagnostic tools for male fertility. The aim of this study was to review the retrospective semen analyses and prospective proteomic studies of male fertility determination and usefulness of proteomic approaches in diagnosing male fertility potential in animal industry. KEYWORDS: infertility, subfertility, semen analysis, sperm physiology, protein profiling, spermatozoa, biomarker

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INTRODUCTION Artificial insemination (AI) is a common breeding technique for the rapid dissemination of key genes to improve livestock quality. However, only 50% of such inseminations result in successful full-term pregnancies;1 the failure of this breeding system leads to large economic losses. Because half of the failures can be attributed to decreased male fertility or infertility, the prediction and diagnosis of male fertility is of economic importance in livestock breeding. Many laboratory assays have been established for evaluating male fertility. Because fertilization and pregnancy are exceptionally complex processes that involve many sequential events, quality parameters based solely on sperm analysis usually exhibit relatively poor correlations with male fertility.2−5 To establish new diagnostic tools, Corner and Barratt6 addressed the importance of proteomic studies to evaluate the male fertility in a clinical environment. Broad and dynamic comparative proteomic and genomic studies are a prerequisite to identify novel biomarkers for male fertility; these will be subsequently applicable for use male fertility determination. Recently, the comparative and comprehensive proteomic studies have amplified potential ability for the exploration of male fertility markers7−9 and boosted the development of molecular diagnosis tools to determine the male fertility or sperm dysfunction.8,10 Moreover, to understand the functionally important protein markers and complexities in male fertility, comparative proteomic studies relevant to the sperm maturation have been suggested.11 Therefore, to examine the clinical value of retrospective semen analysis and prospective proteomic studies using spermatozoa for male fertility potential determination, this study reviewed in the following sections. First, we summarize retrospective studies on traditional semen analysis. Second, we outline the proteomic profiling of © XXXX American Chemical Society

seminal plasma and spermatozoa and schematic representations of differential signaling pathways in different stages (i.e., maturation, capacitation, the acrosome reaction, and fertilization) associated with male fertility. Finally, we address prospective proteomic strategies in terms of the technological advances that are required to improve male fertility potential.

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RETROSPECTIVE ANALYSIS FOR THE DETERMINATION OF SPERM FERTILITY Several laboratory assays have been used for the identification of fertile males as valuable stock in animal husbandry. These tests include assessments of sperm viability,12 motility,13,14 morphology,13,15 penetration of cervical mucus or sperm migration tests,16−18 membrane intactness,19 the acrosome reaction,15,20 ATP concentration in semen,21 and sperm penetration assay (SPA) using homologous eggs.22,23 In frozen−thawed bovine semen, the percentages of motile (r = 0.52 to 0.59) and viable spermatozoa (r = 0.39 to 0.54) were significantly correlated with in vivo fertility (nonreturn rate: NRR).12 NRR was also significantly and positively correlated with the percentage of capacitated spermatozoa.14 However, there are contradictory reports that observed that the percentage of motile spermatozoa,24,25 sperm zona-binding ability,24 acrosome integrity,25 and in vitro fertilization (IVF) rate26 were not significantly correlated with NRR. In boars, the sperm chromatin structure assay27 and sperm morphology28 were correlated with either farrowing rate or litter size. Recently, our laboratory reported that the percentage Special Issue: Agricultural and Environmental Proteomics Received: June 29, 2013

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dx.doi.org/10.1021/pr400639x | J. Proteome Res. XXXX, XXX, XXX−XXX

Journal of Proteome Research

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Table 1. List of Differentially Expressed Proteins in Mature Spermatozoa Compared with Immature Spermatozoa function

regulation

species

acrosin aldose reductase

protein name

ACR ALDR

symbol

sperm−egg interaction energy metabolism

angiotensin-converting enzyme

ACE

unknown

A-kinase anchor protein 3 A-kinase anchor protein 4 arginine vasopressin receptor 2 β-subunit F1-ATPase enolase 1, non-neuronal

AKAP3 AKAP4 AVPR2 ATP5B ENO1

signal transduction signal transduction sperm−egg interaction energy metabolism energy metabolism

glutathione S-transferase Mu 5 heat-shock protein 2

GSTM5 HSPA 2

anti-oxidant molecular chaperone

heat-shock protein 5 heat-shock protein 9 lactate dehydrogenase

HSPA 5 HSPA 9 LDH

energy metabolism

peroxiredoxin 4 pyruvate dehydrogenase phosphatidyl-ethanolamine binding protein sperm equatorial segment protein 1 triosephosphate isomerase 1

PRX4 PDHB PEBP SPESP1 TPI1

antioxidant energy metabolism sperm−egg interaction sperm−egg interaction energy metabolism

up up down down down up up up up up down down down down down down down down up up up down up down

swine61 bovine62 swine57 swine57 rat63 mice58 mice58 mice64 rat59 rat56,59 mice58 rat57 rat57 mice58 mice58 mice58 rat59 mice65 rat56 mice58 rat59 mice58 mice58 rat56

prerequisite to identify fertility-related markers for using these tools. Mature spermatozoa are highly specialized, multicompartmental cells that have the absence of transcription, translation, and protein synthesis. Furthermore, large numbers of spermatozoa can be reliably purified under a variety of validated pharmacological manipulations (i.e., epididymal maturation, capacitation, the acrosome reaction, and fertilization); therefore, spermatozoa are widely used as an acceptable model for proteomic analysis.8,11 Until recently, proteomic studies using spermatozoa have been promoted to understand the molecular mechanisms of male fertility based on the advantage of spermatozoa.1,9,36−38 Recent proteomic applications using 2-D gel electrophoresis (2-DE) 8,36 and mass spectrometry (MS)39,40 have been employed to identify the proteins present in spermatozoa. In addition, comparative and comprehensive proteomic analyses have led to the development of new molecular detection tools to diagnose male fertility potential or sperm dysfunction.8−10 To understand the roles of functionally important protein markers and the complexities of male fertility, Aitken and Baker11 suggested conducting comparative proteomic studies on post-translational modifications that are relevant to sperm maturation (immature versus mature spermatozoa) or capacitation (before- versus after-capacitated spermatozoa).

of noncapacitated spermatozoa showed positive correlations with either farrowing rate (r = 0.447) or litter size (r = 0.445), while the percentage of acrosome-reacted spermatozoa was negatively correlated with farrowing rate (r = −0.612).29 In contrast, Gadea et al.30 suggested that these sperm parameters possess some limitations with respect to interpretations of boar fertility. Although these tests may provide preliminary information on the quantitative aspects of male fertility, few individual tests showed significant correlations with in vivo fertility.3−5 Therefore, more accurate prediction tools for male fertility are necessary. In particular, xenogeneic SPA using zona-free hamster oocytes have been used as accurate general diagnostic tests for sperm fertility by using human,31 bovine,32 or mouse33 spermatozoa. While SPA has received widespread attention as a test for sperm fertility, the indefinite range of penetration levels that constitute normal fertility, interassay variability, and lack of quality control are inherent problems of this bioassay system.34,35 Recently, to cope with the weak point of the xenogeneic SPA system, our laboratory optimized the assay method to increase its sensitivity and establish the cutoff value using the sperm penetration index.34,35 As a result, the accuracy of determining male fertility was improved to ∼96% in porcine34 and bovine.35 However, this system is complicated, time-consuming, and expensive.34,35 Therefore, it is necessary to develop new diagnosis tools that are also simple, highly accurate, and cost-effective.

Protein Profiling of Seminal Plasma

To transport sperm into the female reproductive tract and acquire sperm fertilizing ability, spermatozoa travel through the epididymal tubule. During the transit, spermatozoa undergo gradual surface modifications, such as cytoplasmic droplets deletion, lipid reorganization, and protein transformation in plasma membrane in the seminal plasma secreted from multiple glands of the male reproductive tract.41−43 Seminal plasma contains various proteins that related to sperm motility,44 DNA integrity,45 capacitation,46 and interaction with oocyte.47

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PROTEOMIC APPROACHES TO ANALYZE SPERMATOZOA As indicated by Corner and Barratt,6 the establishment of new diagnosis tools requires the development of genomic and proteomic studies in a clinical environment. Therefore, broad and dynamic comparative proteomic and genomic studies are a B



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To understand the effect of seminal plasma on sperm fertility or sperm function, we have conducted comprehensive proteomic studies of seminal plasma proteins in bovine,48−50 equine,51 and porcine.52 Brandon et al.51 identified four proteins that showed differential expression between normal and subfertility using 2-DE in equine. Among those proteins, only one seminal plasma protein, SP-1 (72 kDa, pI 5.6), was positively correlated with fertility, while three proteins, SP-2 (75 kDa, pI 6.0), SP-3 (18 kDa, pI 6.5), and SP-4 (16 kDa, pI 6.5), were negatively correlated with fertility. Moreover, Killian et al.49 identified four seminal plasma proteins that exhibited difference in expression between normal and subfertile Holstein bulls using 2-DE. Among these proteins, two proteins (26 kDa, pI 6.2 and 55 kDa, pI 4.5) were more highly expressed in normal spermatozoa, while the other two proteins (16 kDa, pI 4.1 and pI 6.7) were more highly represented in the spermatozoa of subfertile bulls. These findings may provide information on the effect of seminal plasma proteins in sperm fertility or sperm function.

Figure 1. Schematic representation of interactions among 18 proteins related to sperm maturation. These proteins are differentially expressed in spermatozoa during maturation.

Protein Profiling of Spermatozoa during Maturation

Sperm cells are produced in the testis and released into the highly differentiated and compartmentalized epididymis. As the released spermatozoa arrive at the caput and transit to the corpus and cauda epididymis, they undergo the biological and functional modification necessary to acquire the ability to fertilize.53 Therefore, to understand the mechanism involved in male fertility, it is also important to study the maturational modifications of sperm in epididymis. To promote the acquisition of sperm functionality during epididymal maturation, the secretion of specific proteins is one of the most remarkable mechanisms observed in the epididymis.54 The proteins secreted in the epididymis are related to motility changes, and remodeling of the sperm surface and the acrosomal region can penetrate the oocyte.53,55 Recently, comparative 2-DE and MS studies using immature and mature spermatozoa were used to identify proteins during maturation.56−58 A total of 17 and 60 proteins that showed differential expression between caput and cauda spermatozoa were identified by 2-D fluorescence difference gel electrophoresis and MS in mice58 and by 2-DE and MALDI-TOF in rats,59 respectively. In boars, 32 proteins exhibited significant differences between immature and mature spermatozoa after 2DE gel separation of ionic and detergent membrane extracts.57 These reports suggested that because the function of each region in the epididymis is clearly defined, most of the proteins in the spermatozoa from caput and cauda epididymis have been identified to be consistent with their putative functions in the epididymis.56−58 We summarized the 18 proteins that are differentially expressed in the spermatozoa during maturation, categorizing them according to putative function and species (Table 1). In this Table, because of the limited information available on livestock, we included information from rodents as well. In addition, we illustrated a schematic representation of different signaling pathways involving these proteins, which show significant modification during epididymal maturation (Figure 1). Interestingly, three proteins, namely, aldose reductase (ALDR), enolase 1 (ENO1), and triosephosphate isomerase 1 (TPI1), which are associated with energy metabolism, displayed different regulation mechanisms during epididymal maturation among the studied species. On the basis of these findings, we hypothesize that different mechanisms of energy

production may be present and that this may be associated with inherent fertility differences among different species. Jones60 suggested that variations in protein composition in spermatozoa exist among different species. Therefore, to understand the distinct male fertility and to identify fertility markers of spermatozoa during the maturation stage in specific animals, comprehensive protein profiling among multiple species is necessary. Proteomic Profiling of Fertile and Subfertile Spermatozoa

Comparative studies between fertile and subfertile spermatozoa have been initiated to investigate male fertility at the protein level.1,9,36−38 Although many studies have been conducted on proteome profiling in both fertile and infertile spermatozoa in humans,37,64−68 there have been few studies in animals. Among the studies that have been conducted in animals, most have suggested bovine spermatozoa to be an ideal model for male fertility studies1,9,69 because it has several advantages, such as adequate sample size, availability of good breeding records, and abundant fertility data. Peddinti et al.1 identified 125 proteins that showed differential expression between normal and subfertile bull spermatozoa using differential detergent fractionation 2-D liquid chromatography. Among those proteins, 74 proteins were more highly represented in normal spermatozoa, whereas 51 proteins were more abundant in subfertile bull spermatozoa. Moreover, D’Amours et al.69 identified eight proteins that showed differential expression between Triton X-100 extracted proteins from normal and subfertile bull spermatozoa using 2-D difference gel electrophoresis technique. Two proteins were abundantly expressed in the normal sperm, while six proteins were more highly expressed in subfertile bull spermatozoa. In our previous study, we identified eight proteins that exhibited at least a three-fold difference in expression between normal and subfertile bull spermatozoa using 2-DE. Among these proteins, five were more highly expressed in normal spermatozoa, while the other three were more highly represented in the spermatozoa of subfertile bulls.9 In this study, ENO1 was significantly and positively correlated with NRR, while voltage-dependent anion channel 2 (VDAC2) and ubiquinol-cytochrome-c reductase complex core protein 2 (UQCRC2) showed negative correlations with NRR.9 C



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Table 2. List of Differentially Expressed Proteins in Normal Spermatozoa Compared to Sub-Fertile Spermatozoa protein name

symbol

regulation

enolase 1 voltage-dependent anion channel 2 ATP synthase, H+ transporting, mitochondrial F1 complex, β subunit ropporin-1 apoptosis-stimulating protein 2 of p53 ubiquinol-cytochrome-c reductase complex core protein 2 phospholipid hydroperoxide glutathione peroxidase, itochondrial α-2-HS-glycoprotein T-complex protein 1 subunit 3 T-complex protein 1 subunit θ epididymal sperm-binding protein E12 proteasome subunit α type-6 adenylate kinase isoenzyme 1 phosphatidylethanolamine-binding protein 1 binder of sperm 1 cytochrome c oxidase subunit III pyruvate kinase casein kinase A-kinase anchor protein 4 aldose reductase annexin A2

Eno1 VDAC2 ATP5B RON1 ASPP2 UQCRC2 GPx4 AHSG

up down up down up down up up

energy metabolism ion transport oxidative stress cell signaling oxidative stress oxidative stress oxidative stress immune system

function

references bovine9 bovine9 bovine9 bovine9 bovine9 bovine9 bovine9 bovine9

CCT5 CCT8 ELSPBP1 PSMA6 AK1 PEBP1 BSP1 Cox 3 PKM2 CK2 AKAP4 ALDR2 ANXA 2

down down down down up up down up up up up down

structure structure energy metabolism unknown energy metabolism sperm−egg interaction cell interaction oxidative stress energy metabolism cell interaction structure energy metabolism cell interaction

bovine69 bovine69 bovine69 bovine69 bovine69 bovine69 bovine69 bovine1 bovine1 bovine1 bovine1 bovine1 bovine1

Figure 2. Schematic representation of interactions among 21 proteins related to sperm fertility. These 21 proteins are differentially expressed between normal and subfertile animals. D



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Table 3. List of Differentially Expressed Proteins in Capacitated Spermatozoa Compared to Non-Capacitated Spermatozoa proteins

symbol

function

regulation

ATP synthase, H+ transporting, mitochondrial F1 complex, β subunit pyruvate dehydrogenase e1 component subunit β glutathione S-transferase F-actin capping protein subunit β2 glycerol-3-phosphate dehydrogenase 2 voltage-dependent anion-selective channel protein 2 membrane metallo-endopeptidase-like 1 A-kinase anchor protein 4 acrosin alkaline phosphatase 5′-nucleotidase sorbitol dehydrogenase cytochrome c oxidase polypeptide VIC succinyl-CoA ligase [ADP-forming] β-chain pyruvate dehydrogenase e1 component subunit α phosphoglycerate mutase 2 triosephosphate isomerase glycerol phosphate dehydrogenase 2, mitochondrial fructose-bisphosphate aldolase glycerol-3-phosphate dehydrogenase 2 A-kinase anchor protein 4 ubiquinol-cytochrome-c reductase complex core protein 1 glutathione S-transferase Mu 5

ATP5B PDB1 GSTs F_actin_cap_B GPD2 VDAC2 MMEL1 AKAP 4 ACR AP 5′-NT SORD COX6C SUCLA2 PDHE1-A type I PGAM2 TPI GPD2 FBA GPD2 AKAP 4 UQCRC1 GSTM5

oxidative stress energy metabolism oxidative stress ion transport energy metabolism ion transport sperm−zona interaction structure sperm−egg interaction structure sperm−egg interaction energy metabolism energy metabolism energy metabolism energy metabolism energy metabolism energy metabolism energy metabolism energy metabolism energy metabolism structure antioxidant antioxidant

up up up up up up up up up down down up up up up up up down down up up up up

references bovine87

swine82

rodents85

hamsters88

Figure 3. Schematic representation of the interactions among 23 proteins that undergo modification during sperm capacitation in animals.

We summarized and categorized the 21 proteins as putative biomarkers of male fertility in animals on the basis of protein function (Table 2). In addition, we illustrated the relevant signaling and metabolic pathways by using these putative biomarkers to facilitate understanding of the mechanisms

behind male fertility (Figure 2). Interestingly, all of the proteins involved in oxidative stress response, such as ATP synthase, H+ transport proteins, mitochondrial F1 complex, β subunit (ATP5B), apoptosis-stimulating protein 2 of p53 (ASPP2), phospholipid hydroperoxide glutathione peroxidase, mitochonE



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drial protein (GPx4), and cytochrome c oxidase subunit III (Cox3) were abundantly expressed in normal bull spermatozoa. The sole exception was UQCRC2 (Table 2). These findings suggest that spermatozoa possess lower resistance to oxidative stress, which may cause oxidative damage during capacitation or fertilization; this results in subfertility. Three of the four proteins related to energy metabolism, namely, ENO1, adenylate kinase isoenzyme 1, and pyruvate kinase (PKM2), were more highly expressed in normal spermatozoa than in subfertile ones. Energy metabolism is associated with ATP as an energy source during capacitation, fertilization, and sperm decondensation in oocytes after fertilization.70,71 On the basis of these findings, we hypothesize that limitations in the amounts of proteins involved in energy metabolism may contribute to an aberrant energy supply, which results in subfertility or infertility. These novel proteins that exhibit differences between normal and subfertile spermatozoa may be used as biomarkers for male fertility in animals. Because these studies were performed only in bovines, Table 2 presents data obtained from bulls.

roles in assisting spermatozoa of hyperactivated motility, penetration ability, and decondensation in oocytes.70,71 Therefore, the upregulation of these proteins may play a role in regulating the provision of energy sources in spermatozoa during capacitation for the purpose of maintaining their motility or penetration ability. These novel proteins may provide information on the dynamic mechanisms during capacitation associated with male fertility.

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CONCERN OF SPERM PURIFICATION FOR THE PROTEOMIC STUDY The ejaculate contains various cellular components, such as seminal plasma, secretion from accessory sex gland, blood cells, and epithelial cells. Therefore, the contamination of seminal plasma proteins during sperm proteomic analysis might be the cause of the substantial variation observed between samples from the same donors.67 To reduce this contamination, Pixton et al.67 evaluated the direct swim-up method and the density gradient method (using Percoll) in humans, with respect to sperm isolation. In that study, the Percoll method displayed much better reproducibility than the swim-up method.67 Furthermore, two seminal plasma proteins, prostate-specific antigen and prostatic acid phosphatase, were identified in the swim-up method, whereas these proteins were removed when the Percoll method was used.67 To this end, different density gradient methods have been used to isolate spermatozoa in both human and animal subjects. For example, a discontinuous density gradient of 90−45% Percoll was used to remove extender debris, seminal plasma, and dead spermatozoa in bovine semen.1,9 A 35% PureSperm solution was used to isolate the caput and cauda epididymal sperm in mice.58 In swine, a two-layer isotonic Percoll gradient (90−40%) was used to purify epididymal sperm,57 and 80−55% Percoll was used to isolate spermatozoa from ejaculate.82 Interestingly, different density gradient methods have been used in different species. On the basis of the aforementioned studies, we concluded that it is important to select purification methods for sperm cells that are appropriate for the species being studied to improve the reproducibility of sperm proteomic analysis.

Protein Profiling of Spermatozoa during Capacitation

Spermatozoa must undergo a multifactorial physiological and biochemical change in the female tract that termed as “capacitation”.72,73 Capacitation, as a post-translation modification, is associated with tyrosine phosphorylation59,74 that is related to the activation of tyrosine kinase by cyclic AMP,75−77 increase in membrane fluidity,75,76 calcium influx,78 and hyperactivated motility. These procedures are able to facilitate sperm to penetrate the oocyte. Therefore, to understand differences in male fertility, it is necessary to perform comparative analyses of spermatozoa between the pre- and post-capacitation stages. Of particular importance in capacitation is tyrosine phosphorylation; tyrosine phosphorylated proteins are expressed in the flagella and related to the acquisition of hyperactive motility79,80 to facilitate the penetration of spermatozoa in the cumulus cell or zona-pellucida of oocytes. In addition, the proportions of tyrosine-phosphorylated proteins in the acrosomal region were observed to increase during capacitation; this modification is involved in sperm− zona interaction and the acrosome reaction in human81 and porcine spermatozoa.82 To understand the mechanisms underlying sperm fertility involved in capacitation, comparative proteomic studies between pre- and postcapacitated spermatozoa have been reported in humans,83 mice,84,85 hamsters,86 boars,82 and bovines.87 On the basis of these reports, we categorized 23 proteins that show significant modification during sperm capacitation according to species and putative protein functions (Table 3, Figure 3). Interestingly, all of the proteins involved in oxidative stress response, such as ATP5B, GST, UQCRC1, and GSTM5, were upregulated during capacitation, regardless of species (Table 3). Although oxidative stress is associated with abnormal function and cell death, a moderate level of ROS is an important mechanism that induces capacitation.88 These findings support the hypothesis that the upregulation of these proteins during capacitation may lead to amelioration of oxidative stress and cell damage. Furthermore, 9 of the 11 proteins associated with energy metabolism are highly upregulated during capacitation (Table 3). Processes associated with energy metabolism play important

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FUTURE REQUIREMENTS It is absolutely imperative that proteomic approaches will shed light on our understanding of the male fertility, which will help in developing new tools to evaluate it. Therefore, a more comprehensive and sensitive approach is required to fully understand the complexities of male fertility.35 Furthermore, the identified proteins should be rigorously tested to determine whether the protein markers have any clinical value for fertility evaluation.6,9 To cope with the drawbacks of current proteomic studies and to identify genuine fertility-related biomarkers, we suggest that as a first step comprehensive and comparative studies between spermatozoa from normal and subfertile animals be conducted. Second, the identified proteins should be individually tested through proteomic studies in at least 20 individual semen samples to address whether the proteins are correlated with fertility.9 In addition, variations in protein composition among different species47 should be considered. To understand the mechanisms of male fertility and to identify the true biomarkers of male fertility, we suggest four strategies in a proteomic approach, building upon the findings of Corner and Barratt6 (Figure 4). First, representative samples F



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Figure 4. Strategies employed by proteomic approaches to identify the true biomarkers of male fertility. Three proteomic approaches are outlined. Samples of proteins from representative samples (normal and subfertile spermatozoa) are combined for each group to minimize individual differences in male fertility. Then, comprehensive and comparative proteomic study is conducted. The validity of proteomic result is confirmed through individual tests, and new tools are developed using accepted markers to apply in animal industry.

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(i.e., normal and subfertile) with normal semen parameters, such as sperm count, % motility, intact, and viable and morphologically normal spermatozoa, assessed by laboratory tests are need to minimize the variation of male fertility.9,49 As indicated, appropriate purification of spermatozoa is important to reduce experimental error and improve the reproducibility of proteomic analysis. Second, a comprehensive proteomic study is now necessary to identify the quantities of novel proteins present at different sperm maturation stages (i.e., epididymal maturation, ejaculation, capacitation, the acrosome reaction, and fertilization), fertility condition levels (normal and subnormal), and species levels. Third, the validity of these results will have to be confirmed through rigorous individual tests by using an abundant number of samples. Finally, protein function tests (i.e., agonist and antagonist chemicals, knockout and knock-in models) will be required to understand the mechanisms by which proteins are associated with male fertility.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was carried out with the support of grants from the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ008415), Rural Development Administration, Republic of Korea.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +82-31-670-4841. Fax: +8231-675-9001. G



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