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From the * Avicore Biotechnology Institute,
Optifarm Solution Inc, Geumjeong-Dong, Gyeonggi-Do, Korea; the
Department of Agricultural Biotechnology,
Seoul National University, Seoul, Korea; the
Department of Molecular Science and
Technology, Ajou University, Suwon, Korea; and the
Department of Microbiology, College of Natural
Sciences, Changwon National University, Changwon, Korea.
| Correspondence to: Dr Jae Yong Han, Professor, Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea (e-mail: jaehan{at}snu.ac.kr). |
| Received for publication December 17, 2008; accepted for publication April 23, 2009. |
Although chicken spermatogonial stem cells (SCs) are important in
spermatogenesis and transgenic research, little is known about these cells.
Recently, our group constructed an in vitro culture system to establish
germline stem cells (GSCs). To examine the mechanism of chicken spermatogonial
SC development, we constructed a proteome map of GSCs from 4-week-old chicken
testes. Soluble extracts of the GSCs were fractionated by 2-dimensional gel
electrophoresis (pH 4–7). Several protein spots, including those that
displayed significantly high levels, were identified by matrix-assisted laser
desorption/ionization-time of flight mass spectrometry and liquid
chromatography–tandem mass spectrometry. Of the 82/250 GSC spots
examined, 56 yielded mass spectra that matched avian proteins found in the
on-line databases. All of the identified proteins were classified into
functional groups. This type of proteome map is an important resource for
research on spermatogenesis and transgenesis.
Key words: Spermatogonial stem cell, 2-dimensional gel electrophoresis, mass spectrometry
Germline stem cells (GSCs), which include spermatogonial cells, have received a lot of attention recently, and most research on GSCs has focused on stem cells (de Rooij and Grootegoed, 1998; Nagano et al, 1998; Ehmcke et al, 2006a,b), transplantation for chimera production (Brinster and Nagano, 1998; Ogawa, 2001; McLean, 2005), male fertility (Orwig and Schlatt, 2005), and transgenesis (Olive and Cuzin, 2005; Hill and Dobrinski, 2006). To understand the molecular mechanisms of spermatogenesis, genomic studies have advanced rapidly (Eddy, 1998b). However, the molecular and physiological mechanisms of spermatogenesis remain largely unknown.
Many proteomic studies related to spermatogenesis have been conducted on the mammalian testis (Huang et al, 2005; Peddinti et al, 2008), sperm (Zhao et al, 2007; Atiken and Baker, 2008; Baker et al, 2008), and GSCs (Kurosaki et al, 2007), whereas others constructed proteome maps of the rat spermatogonia (Guillaume et al, 2001; Com et al, 2003), thereby identifying specific proteins to that cell type (Com et al, 2006).
In chickens, the testicular stroma and Sertoli cells develop from primordial cells that migrate from the extraembryonic germinal crescent and infiltrate the somatic tissue of the gonadal ridge. The gonadal ridge tissue is primed to influence the development of the primordial germ cells (PGCs) into either oogonia or spermatogonia as a result of earlier events dictated by sex-determining genes, especially the production of androgens (Thurston, 2000). These migration and development processes are different in mammals and birds.
The chicken is a useful model system for research in developmental biology and transgenesis. Since the publication of the chicken genome sequence, genetic analyses and proteomic studies in chickens have accelerated. Previously, we established chicken embryonic germ cell lines (Park and Han, 2000; Park et al, 2003), isolated chicken spermatogonial cells, produced testis-mediated germline chimeras (Lee et al, 2006), and developed an in vitro culture system for GSC-like cells (Jung et al, 2007). We have also constructed proteome maps from cultured gonadal primordial germ cells (Han et al, 2005), analyzed the embryonic gonad expressed sequence tags (ESTs; Shin et al, 2006), and constructed a germ cell EST database for the chicken (Kim et al, 2006). These advances, which improve the retrieval of chicken spermatogonial cells proteins from reference databases, facilitate proteomic studies. In this study, we capitalize on these technical advances to construct proteome maps of GSCs, which can be used to investigate avian spermatogenesis and germ cell biology.
Materials and Methods
Animals and Experimental Management
All of the procedures for animal management, reproduction, and surgery were
performed in accordance with the standard operation protocols of Seoul
National University, Seoul, Korea. Appropriate management procedures for the
experimental samples and quality control of the laboratory facilities and
equipment were also conducted. All of the chickens were maintained at the
University Animal Farm, College of Agriculture and Life Sciences, Seoul
National University.
Isolation and In Vitro Culture of Chicken Spermatogonial Cells
Spermatogonial cells were isolated from 4-week-old White Leghorn chickens,
as described previously (Jung et al,
2007). The decapsulated testes were resuspended in
phosphate-buffered saline (PBS) and approximately 1 x 106
dissociated cells were placed in a 100-mm culture dish with modified DMEM
(Gibco Invitrogen, Grand Island, New York) that contained 10% (vol/vol) fetal
bovine serum (Hyclone, Logan, Utah), 2% (vol/vol) chicken serum (Gibco
Invitrogen), 1x antibiotic-antimycotics (Gibco Invitrogen), 10 mM
nonessential amino acids (Gibco Invitrogen), 10 mM HEPES buffer (Gibco
Invitrogen), and 0.55 mM β-mercaptoethanol (Gibco Invitrogen).
Subsequently, 10 ng/mL leukemia inhibitory factor, 10 ng/mL basic fibroblast
growth factor, and 100 ng/mL insulin-like growth factor 1 were added to the
base medium according to the experimental design. The seeded cells were
cultured in an incubator at 37°C in an atmosphere of 5% CO2
with 60%–70% relative humidity. Growth factors were purchased from Sigma
(St. Louis, Missouri). After 15 days in culture, the GSC colonies were
agitated and harvested by gentle pipetting without trypsin-EDTA treatment to
compare results with those obtained by our previous method
(Han et al, 2005).
Two-Dimensional Gel Electrophoresis
Sample preparation and 2-dimensional gel electrophoresis (2-DE) analysis
were separated as described previously
(Han et al, 2005). For
estimation of GSCs purity, lectin–fluorescein isothiocyanate (FITC)
staining was employed (Jung et al,
2007). FITC-conjugated Solanum tuberosum agglutinin (STA)
was purchased from Sigma. The harvested GSCs were washed 3 times with PBS,
counted, and subsequently pelleted by centrifugation at 200 x g
for 5 minutes. Pellets that contained approximately 5 x 106
cells were frozen at –80°C until required.
The frozen cell pellets were solubilized in lysis buffer that contained a protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany). The lysates were homogenized and centrifuged at 12 000 x g for 15 minutes. Endonuclease (700 U/mL; Sigma) was added to the mixture, which was subjected to the Bradford protein assay (Bradford, 1976) and then stored at –70°C.
For 2-DE analysis, an immobilized pH 4–7 gradient (IPG) strip (Amersham Bioscience, Little Chalfont, United Kingdom) was rehydrated in swelling buffer that contained 7 M urea, 2 M thiourea, 0.4% dithiothreitol, and 4% CHAPS. The lysates (200 µg) were cup loaded into the rehydrated IPG strip with the use of a Multiphore-II apparatus (Amersham Bioscience) for a total of 57 kVh. The second dimension run was carried out on 8%–16% linear gradient sodium dodecyl sulfate (SDS)–polyacrylamide gels at 40 mA per gel for 6 hours with the use of a Hoefer Dalt gel tank (Amersham Bioscience). The gels were stained with colloidal Coomassie blue G-250 (Sigma) for 5 hours (Neuhoff et al, 1988), destained in 1% acetic acid for 12 hours, and then imaged using the GS-710 imaging calibrated densitometer (Bio-Rad, Hercules, California). Protein spot detection and image comparisons were carried out using the ImageQuant TL software (Amersham Bioscience). The Mr and isoelectric point (pI) values were estimated with external SDS polyacrylamide gel electrophoresis standards and a reference map provided by the manufacturers.
Mass Spectrometry
Protein spots were digested in-gel with sequencing-grade modified trypsin
(Promega, Madison, Wisconsin). The samples were prepared on a stainless steel
matrix-assisted laser desorption/ionization (MALDI) target according to the
dried droplet method with 
-cyano-4-hydroxycinnamic acid (CHCA) as
matrix.
MALDI time of flight (TOF) analysis was performed in reflector mode with the Voyager-DE STR MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, California) over the range of 1000–3500 thomsons. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed by reversed-phase capillary high-performance liquid chromatography directly coupled to a Finnigan LCQ ion trap mass spectrometer. A 0.075x20-mm trapping column and a 0.075x130-mm resolving column were packed in-house with Vydac 218MS low trifluoroacetic acid C18 beads (5 µm; Vydac, Hesperia, California) and placed in-line. Peptides were bound and preconcentrated in the trapping column with 5% (vol/vol) acetonitrile in 0.1% (vol/vol) formic acid. The eluting gradient was 5%–80% (vol/vol) acetonitrile in 0.1% (vol/vol) formic acid for 50 min at a flow rate of 0.15 µL/min. The eluent from the capillary column was sprayed directly into the ion trap mass spectrometer.
Database Searching and Gene Ontology Analysis
Proteins were identified on the basis of their peptide mass fingerprints by
manually searching with the MASCOT (Matrix Science, London, United Kingdom)
and MS-Fit (The Regents of the University of California, Oakland, California)
programs. The initial search parameters allowed a single missed trypsin
cleavage, propionicamide modification of cysteine residues, variable oxidation
of methionine, and a mass-to-charge ratio (m/z) error of
±50 ppm.
The LC-MS/MS data were collected in centroid mode using the "triple play" mode, which involved a full mass scan over the mass range of 395–2000 daltons (m/z), determination of the charge states of an ion on zoom scan, and acquisition of an MS/MS spectrum of the ion on full MS/MS scan, the collision energy of which was preset at 55%. The individual spectra from MS/MS were processed using the TurboSEQUEST software (Thermo Quest, San Jose, California). The generated peak list files were used to query either the MSDB database or the National Center for Biotechnology Information (NCBI) with the MASCOT program (http://www.matrixscience.com). Modifications of methionine and cysteine, peptide mass tolerance at 2 daltons, MS/MS ion mass tolerance at 0.8 daltons, allowance of missed cleavage at 2, and charge states (+1, +2, and +3) were taken into account. Only significant hits as defined by MASCOT probability analysis were considered initially.
Gene ontology (GO) annotations of identified proteins were conducted on the basis of sequence similarity with the Tentative Consensus sequences of the Gallus gallus Gene Index (GgGI, Release 11.0) at The Institute for Genomic Research (TIGR). The identified protein matched with the NCBI site to make nucleic acid sequence file and sequence similarity searches against the tentative consensus sequences of GgGI using BLASTN, and an E value below 1.0 x 10–10 for GO identification. The GO section is divided into 3 categories: molecular function, biological process, and cellular components. A chi-square goodness of fit test was used to test the significance of which GO terms were enriched in our set of identified proteins or the TIGR data, but relatively depleted in the other. The GO proportions in the total TIGR GgGI was used as the expected proportion for the null hypothesis. The test of significance was conducted at the second level from the GO root. Similar to Kim et al (2006), a particular GO term can be viewed as a function that amps gene G in go (G) = 0 or 1, according to the corresponding GO terms. Only the list of genes annotated with GO terms was counted for the test. A false discovery rate (FDR) correction (Benjamini and Hochberg, 1995) was used with a default cutoff of .05.
Results
Sample Preparation
The GSC cells were collected by gentle pipetting, and the levels of feeder
contaminants were <1%, as assessed by STA-FITC staining (data not shown).
After sample preparation, 200 µg of GSC proteins were used in the 2-DE
analysis (duplicate).
2-DE Analysis
The 2-DE protein pattern obtained from a colloidal Coomassie-stained gel
for a soluble protein extract of chicken GSCs is shown in
Figure 1. More than 250 spots
were counted per gel after background subtraction within the windows of pI
4–7 and molecular mass of about 10–200 kd by ImageQuant TL
software.
|
Protein Identification
Identification of the major spots was attempted by MALDI-MS. When peptide
mass fingerprinting gave no or ambiguous results, sequencing by ion trap mass
spectrometry was performed (Table
1). Thirty-four spots were identified by LC-MS/MS
(Table 2). Because the full
genomic sequence of the chicken is available, 10 hypothetical or unnamed
protein products were identified, whereas some other proteins remained
unmatched. Six of the spots corresponded to human and mouse proteins.
|
|
We analyzed 81 of the 250 GSC spots detected by peptide mass fingerprinting, sequencing ion trap mass spectrometry, or both. In total, 62 polypeptides were successfully identified, which corresponds to 24.8% of the proteins on the whole pH 4–7 2-DE gel. The 19 unknown proteins (23.45%) were related to chicken or mammalian species or to ESTs that have not yet been characterized in the chicken.
The 62 identified proteins corresponded to 48 different polypeptides. Indeed, 6 of the identified proteins were represented by at least 2 distinct spots on the gel (eg, vimentin spots 12–14). As demonstrated in our previous study (Han et al, 2005), several polypeptides showed different Mr and pI values for the identified protein.
GO Analysis
Figure 2 shows the
distribution of GO terms, second-level GO terms according to the GO
consortium, of the identified protein spots. The identified polypeptides were
classified according to their known or postulated functions into the following
categories: biological process, cellular component, and molecular function
(Figure 2). The most abundant
class of proteins was that related to physiological and cellular processes in
biological process (Figure 2A),
binding and catalytic activity in molecular function
(Figure 2B), and cell and
organelle part in cellular component
(Figure 2C). Because the GO
terms are not mutually exclusive, a gene could belong to several different GO
terms. The cell and organelle part, which included polypeptides of the
cytoskeleton, such as the architectural proteins - and β-actin,
vimentin, tropomyosin, and tubulin (Table
1). The next most frequent groups of identified proteins were
those implicated in metabolism and protein synthesis and processing. The
latter group included polypeptides that are involved in protein folding and
heat shock-related proteins. The functional groups that contained the lowest
numbers of polypeptides were binding and transport and nucleotide-related
proteins. EST clones that matched the polypeptides were not identified;
instead, the number of hypothetical or unnamed protein products was relatively
high. The remaining group contained proteins that could not be classified in
the functional analysis, and included the actin-capping protein
β-subunit, β-galactoside–binding lectin, and a protein that is
similar to the Abelson helper integration site. Unknown proteins were
relatively common.
|
A chi-square goodness of fit test was used to test the significant GO terms compared with the expected GO proportions (Figure 2). The GO proportions in the total TIGR GgGI were used as the expected proportion for the null hypothesis. Most of the second term did not show significant enrichiment or depletion with respect to the expected GO proportions. Development of the GO term in the biological process showed enrichment in our dataset (Figure 2A). Significant enrichment of the development term in our dataset was also consistent with biological expectations. The obsolete molecular function term was also enriched in our dataset (Figure 2B). The obsolete term in GO describes a concept that would be better represented in another way than with GO terms.
Discussion
The primary goal of this work was to generate the first proteome map and characterize the major proteins of chicken GSCs from 4-week-old chicken testes. Our starting material was not ex vivo spermatogonial cells from the mouse or rat (Com et al, 2003), but GSCs. In mammalian species, successful culturing of spermatogonia has been reported only for the mouse (Kanatsu-Shinohana et al, 2003), with limited success achieved in culturing bovine spermatogonia (Izadyar et al, 2003). Recently, in vitro sperm formation was performed successfully with cultured spermatogonial cells as the starting material (Hong et al, 2004). Spermatogonial cells are thought to represent an extremely small proportion of testicular cells. The derivation of a mass culture system to amplify GSCs is indispensable for proteomic studies. The proteins identified in the pH 4–7 range were classified according to their putative functions in an attempt to improve our understanding of the biology of spermatogenesis.
In terms of sample preparation, the impurity of the feeder cells was lower than in our previous 2-DE study (Han et al, 2005), mainly because we used a different sample preparation method. By changing to gentle pipetting methods instead of trypsin-EDTA treatment, the contaminant of feeder stroma cells dropped by a factor of 10 to 1%. We also compare the 2-DE map of remaining stroma cells after collection of the germ cells by 2 methods (data not shown). Several vimentin and tropomyosin proteins were identified with approximate pI 4.5–5.0 following the use of Tris-EDTA after pipetting, which removed the gonadal primordial germ cells (unpublished data). Compared with our previous results (Han et al, 2005), some spots (1, 2, 5, 8, 25, 55, and 59) were highly expressed and could be used as standard spots for comparative studies of the chicken germ cell proteome. The genomic sequence of the chicken is now available, and several groups have attempted to identify the chicken proteome (Agudo et al, 2005; McCarthy et al, 2006) and spermatogonial proteins (Guillaume et al, 2001; Com et al, 2003). The emerging data should enhance the identification of proteins in this type of study.
We have identified the major proteins produced by GSCs and shown that most
of these proteins have known functions in the chicken or mammals. The
developmental changes in -smooth muscle actin and vimentin
(Devkota et al, 2006) and the
distribution patterns of structural proteins (actin, desmin, vimentin, and
tubulin) in the testis during postnatal development were investigated with the
use of immunohistochemical methods (Steger
and Wrobel, 1994). Vimentin filaments play an important role in
the adaptation of Sertoli cells to the varying configurations of neighboring
cells during spermatogenesis as well as under pathological conditions
(Aumüller et al,
1988).
The pyridoxal kinase genes are detectable in all human tissues and are particularly abundant in the testes (Hanna et al, 1997). Fang et al (2004) have shown by cDNA microarray that the pyridoxal kinase PKH-T gene is highly expressed in the adult human testis and spermatozoa, and they have suggested that this protein plays an important role in spermatogenesis and is related to male infertility. The heat shock proteins are highly conserved molecular chaperone proteins that are grouped into different families according to molecular size. The role of HSP70 in spermatogenesis has been studied, and the Hsc70 and Grp78 genes are either constitutively expressed or their expression is induced by heat shock and other stresses (Eddy, 1998a). In vivo studies of the effects of the histone-deacetylase inhibitor trichostatin-A on murine spermatogenesis suggest that the regulation of histone deacetylase activity/concentration plays a major role in modulating histone hyperacetylation and probably histone replacement during spermiogenesis (Hazzouri et al, 2000). Insulin receptor substrate-1 (IRS-1) acts as a docking protein and mediates multiple interactions among other proteins, resulting in the transduction of metabolic and mitogenic signals. IRS-1 plays a crucial role in mediating the metabolic actions of insulin through the activation of the PI3 kinase-dependent pathways, whereas other proteins, such as insulin and certain cytokines that act via IRS-1, IRS-2, and the glucose transporter GLUT-3 and associated signal transduction pathways, have effects on different cell types of the human testis (Kokk et al, 2005). PLXnb2 is a family of plexins, which are receptors for semaphorins and large transmembrane proteins. In the testis, Plxna1, Plxnb1, and Plxnc1 are expressed in the developing sex chords. During development, plexins are also expressed in specific and distinct patterns in nonneuronal tissues (Perala et al, 2005).
Forkhead box (FOX) proteins have been shown to play important roles in
regulating the expression of genes involved in cell growth, proliferation,
differentiation, longevity, and transformation
(Katoh and Katoh, 2004).
Prohibitin, which is a 30-kd intracellular protein, has been shown to be a
negative growth regulatory protein that blocks DNA synthesis in normal
fibroblasts and HeLa cells (Nuell et al,
1991). In the rat, prohibitin appears to function as a mitotic or
meiotic inhibitory molecule that acts on spermatogonia or spermatocytes,
respectively, to prevent the processes of mitotic and meiotic division. In
other cells, prohibitin has been shown to be a potent antiproliferative factor
(Choongkittaworn et al, 1993).
Prohibitin is considered to be an inhibitor of proliferation that maintains
the GSC state. In an analysis of the human Werner helicase-interacting protein
1 (WRNIP1/Whip), which has been implicated in eukaryotic postreplication
repair, Tsurimoto et al (2005)
have found that human WRNIP1 (hWRNIP1) forms homo-oligomeric complexes that
physically interact with human pol 
(hpol ) and stimulate DNA
synthesis activity, mainly by increasing the frequency of initiation. Although
the exact function of this protein remains to be clarified, it might have a
similar function to that described above for GSCs.
Other groups of categorized proteins are important in GSC maintenance. The actin-capping protein (CapZ) is one of the actin regulatory proteins and is the most evolutionarily conserved of all the barbed end capping proteins, being expressed in virtually all mammalian cell types (Witke et al, 2001). Actin-capping proteins participate in the rearrangement of filamentous actin in the eukaryotic cell, and testis-specific actin-capping protein is expressed postmeiotically in round spermatids, which colocalize with filamentous actin at the base of the differentiating acrosome (Hurst et al, 1998). The β-galactoside–binding lectins (galectin) are regulated by galectin-1 during the spermatogenic cycle (Dettin et al, 2003). Ahi-1/AHI-1 (Abelson helper integration site-1) is a recently identified and widely expressed gene that encodes a family of proteins with, thus far, undetermined function. However, the findings that these proteins contain multiple SH3 binding sites, an SH3 domain, and multiple WD40 repeat domains, all of which are known to be important mediators of protein-protein interactions, suggest that Ahi-1/AHI-1 proteins are involved in cell signaling activities (Jiang et al, 2004). Thioredoxins function as general protein disulfide reductases. Mammalian male germ cells are equipped with a set of 3 testis-specific thioredoxins (Sptrx-1, -2, and -3) that are expressed either in different structures within the sperm cell or at different stages of sperm development (Jimenez et al, 2005). Three of the spots were identified as keratins, which might have been introduced during the handling of the 2-DE gels. However, a recent study identified 2 groups of keratins (keratins 5 and 9) that were expressed during mammalian spermatogenesis (Kierszenbaum, 2002).
Compared with our previous study (Han et al, 2005), the number of unidentified proteins in this study was lower (30% vs 23.5%). However, the 10 spots that were identified as hypothetical or unnamed protein products hinder domain searches and GO analyses. Recently, our group published the testis-specific tentative consensus (TCs) by in situ hybridization (Rengaraj et al, 2008). Therefore, our next study will focus on identifying the hypothetical and unnamed protein products, as well as the other functions and roles of the known proteins in GSCs. Obviously, the unknown proteins are the most interesting group, in that novel genes discovered by this proteomic approach could be germ cell specific and play major roles in spermatogenesis. Our next study will provide additional information concerning the GSC proteome by employing broad- and narrow-range 2-DE to improve the detection of low-abundance proteins, and by conducting a comparative study of gPGCs. Comparisons of germ cell development (e.g., gPGCs vs GSCs) represent excellent tools for analyses of the processes of germ cell and stem cell development.
Conclusions
We report the proteomic analysis of cultured GSCs from 4-week-old chicken testes. With the use of 2-DE, MALDI-TOF, and LC-MS/MS techniques, we have identified 82 major GSC proteins. Although the functions of most of these proteins have been characterized previously, we discovered 10 hypothetical/unnamed protein products. Thus, we have created a proteome map of cultured GSCs from 4-week-old chicken testes that will serve as a reference map for future investigations. Proteome maps of this type are important resources for studies of spermatogenesis and germ cell biology.
Footnotes
Supported by a grant from BioGreen 21 Program (20070401034010), Rural Development Administration, Republic of Korea, by a graduate fellowship from the Brain Korea 21 project, and by WCU program (R31-2008-000-10056-0) of the Ministry of Education, Science, and Technology, Korea.
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