- Research article
- Open Access
Identification of testis-relevant genes using in silico analysis from testis ESTs and cDNA microarray in the black tiger shrimp (Penaeus monodon)
- Thidathip Wongsurawat†1,
- Rungnapa Leelatanawit†1, 2,
- Natechanok Thamniemdee1,
- Umaporn Uawisetwathana1,
- Nitsara Karoonuthaisiri1Email author,
- Piamsak Menasveta3, 4 and
- Sirawut Klinbunga1, 3
© Wongsurawat et al; licensee BioMed Central Ltd. 2010
Received: 26 January 2010
Accepted: 9 August 2010
Published: 9 August 2010
Poor reproductive maturation of the black tiger shrimp (Penaeus monodon) in captivity is one of the serious threats to sustainability of the shrimp farming industry. Understanding molecular mechanisms governing reproductive maturation processes requires the fundamental knowledge of integrated expression profiles in gonads of this economically important species. In P. monodon, a non-model species for which the genome sequence is not available, expressed sequence tag (EST) and cDNA microarray analyses can help reveal important transcripts relevant to reproduction and facilitate functional characterization of transcripts with important roles in male reproductive development and maturation.
In this study, a conventional testis EST library was exploited to reveal novel transcripts. A total of 4,803 ESTs were unidirectionally sequenced and analyzed in silico using a customizable data analysis package, ESTplus. After sequence assembly, 2,702 unique sequences comprised of 424 contigs and 2,278 singletons were identified; of these, 1,133 sequences are homologous to genes with known functions. The sequences were further characterized according to gene ontology categories (41% biological process, 24% molecular function, 35% cellular component). Through comparison with EST libraries of other tissues of P. monodon, 1,579 transcripts found only in the testis cDNA library were identified. A total of 621 ESTs have not been identified in penaeid shrimp. Furthermore, cDNA microarray analysis revealed several ESTs homologous to testis-relevant genes were more preferentially expressed in testis than in ovary. Representatives of these transcripts, homologs of saposin (PmSap) and Dmc1 (PmDmc1), were further characterized by RACE-PCR. The more abundant expression levels in testis than ovary of PmSap and PmDmc1 were verified by quantitative real-time PCR in juveniles and wild broodstock of P. monodon.
Without a genome sequence, a combination of EST analysis and high-throughput cDNA microarray technology can be a useful integrated tool as an initial step towards the identification of transcripts with important biological functions. Identification and expression analysis of saposin and Dmc1 homologs demonstrate the power of these methods for characterizing functionally important genes in P. monodon.
The black tiger shrimp (Penaeus monodon) is an aquatic animal of central importance as it brings an annual income of over one billion USD in Thailand . However, domestication of the black tiger shrimp (Penaeus monodon) is impeded by poor reproductive maturation of both male and female brooders in captivity. Ovarian development of penaeid shrimp is induced by a unilateral eyestalk ablation technique; however, the technique does not have the same effect in male reproductive maturation . No molecular markers pinpointing the maturation stage of testis or sperm quality in penaeid shrimp are currently known. Domesticated male P. monodon yields lower fertilization rates of zygotes and lower survival rates of offspring than wild male P. monodon . However, the role of genes implicated in the regulation of spermatogenesis and their patterns of expression in penaeid shrimp are still poorly understood.
To gain insight on the molecular mechanisms governing the male reproduction process of this important species, EST libraries from P. monodon broodstock testis were previously constructed for gene discovery [4, 5]. A testis-specific transcript, PMTST1 (P. monodon testis-specific transcript 1), was identified, and expression levels of 51 additional putative testis-specific genes in cultured and wild P. monodon were examined by reverse-transcription (RT)-PCR, semiquantitative RT-PCR, and real-time RT-PCR . Nevertheless, more exhaustive gene discovery is needed to unravel testis-relevant genes and their possible functions.
In this study, a total of 4,803 ESTs from the testis library were sequenced and analyzed in silico using a customizable data analysis package, ESTplus . Many transcripts known to be relevant to testicular development in other organisms were identified. A total of 1,076 of these testis EST sequences were included in the construction of a new microarray. Gene expression profiles of testis were simultaneously compared to those of ovary in both juveniles and broodstock. Among transcripts with differential expression levels, saposin and Dmc1 homologs were further examined by quantitative real-time PCR. Furthermore, full-length sequences of saposin and Dmc1 cDNAs were obtained by RACE-PCR.
Results and Discussion
Characteristics and functional annotation of testis ESTs of P. monodon
Distribution of species that the testis ESTs were matched to known transcripts in the database using BlastX
The 2,702 unique sequences were searched using BlastN against the P. monodon EST data (PmDB: http://pmonodon.biotec.or.th) excluding previously identified testis EST libraries (Figure 2B). A total of 1,579 sequences did not match ESTs from other tissues of P. monodon making them potential testis-specific transcripts or rare transcripts in other shrimp tissues. Examples of genes in this group relevant to testicular development are phospholipase A2, mago nashi proliferation-associated protein, actin-depolymerizing factor and profilin. Phospholipase A2 is required for the acrosome reaction (AR), a special exocytotic process promoted by signal transduction pathways, and capacitation, a process for maturation of spermatozoa [8, 9]. Protein mago nashi originally identified in Drosophila is essential for germplasm assembly . Actin polymerization is important for a wide range of cellular functions and properties, including cell division, cell motility, cell polarity and cell-cell contacts. Profilins are widely expressed small actin-binding proteins which are functionally involved in the regulation of actin dynamics [11, 12]. On the other hand, cofilin/actin-depolymerizing protein is an important factor in spermatogenesis which disassembles actin filaments when unphosphorylated .
To reveal novel transcripts which are not found in other Penaeidae ESTs, the testis ESTs from this study were also compared to Penaeid ESTs (207,852 sequences, using a keyword of "Penaeidae") from the NCBI database (Figure 2C). A total of 2,081 (77%) sequences were homologous to those from the NCBI's penaeid shrimp database. 621 newly identified transcripts (23%) were found, 475 of which (18%) have not been reported in the PmDB database.
Most of the obtained EST sequences ranged from 201-1,100 bp in length (Figure 3A). Apart from a large number of singletons (2,278 unique sequences, 84% of discovered sequences), most of the contigs (187 contigs, 7%) were assembled from two ESTs and only 100 contigs (4%) were deduced from greater than five ESTs. Only three contigs; cytochrome C oxidase subunit 1 (COI), elongation factor 1-alpha (EF-1α), and cell division cycle 2 (cdc2) were assembled from over 50 sequences (Figure 3B). EF-1α functions in protein synthesis by promoting the binding of aminoacyl tRNA to the 80 S ribosome, and it is one of the most abundant soluble proteins in eukaryotes . Mitochondrial COI has been found to be more highly expressed in well differentiated cells with high activity than in moderately and poorly differentiated cells [15, 16]. Cdc2 is the key regulator of the eukaryotic cell cycle, and its activity is controlled by interaction with other proteins such as cyclin A and cyclin B1 . Mitotic cyclins and cdc2 are involved in capacitation and/or acrosome reaction of sperm . During the meiotic cell cycle, the G2/M phase transition is controlled by the maturation promoting factor (MPF), a complex of cdc2 (cdk1) and cyclin B1 . High redundancies of these transcripts in the ESTs are consistent with their essential cellular functions during spermatogenesis and testicular development.
In the biological process category, ESTs involved in the cellular process were predominant (30% of examined ESTs), followed by those involved in the metabolic process (25% of examined ESTs). In the cellular component category, EST functionally involved in the cell (39% of examined ESTs) predominated followed by those functionally displayed in the organelles (30% of examined ESTs) and macromolecular complex (21% of examined ESTs). In the molecular function category, ESTs displaying binding function (43% of examined ESTs) predominated followed by those displaying catalytic activity (28% of examined ESTs).
The reproduction group, including mago-nashi proliferation-associated protein, ubiquitin-conjugating enzyme E2, and small ubiquitin-related modifier precursor (SUMO), contributes ~2% of the total number of contigs. This discovery rate is comparable to that identified from the conventional ovarian cDNA library of P. monodon .
The transcripts found in the reproduction group exhibit relevant functions to male reproduction. For instance, SUMO plays an important role in diverse reproductive functions such as spermatogenesis and modulation of steroid receptor activity . In the sumoylation pathway, SUMO is transferred to lysine residues of the protein substrates through the thioester cascade of ubiquitin activating enzyme E1 and ubiquitin conjugating enzyme E2 (UBE2), and SUMO ligase E3 . In the kuruma shrimp Marsupenaeus japonicus, UBE2 was expressed at a higher level in testis than in ovary. The expression at stage I (GSI = 0.33 ± 0.004, N = 5) was significantly lower than that of stage II (GSI = 0.45 ± 0.12, N = 5) but comparable to that of stage III (GSI = 0.57 ± 0.006, N = 5) of testis . The analysis of baseline information acquired by this study addresses the paucity of data and provides a better understanding of reproductive maturation in male P. monodon.
Differential Gene Expression Patterns by cDNA Microarrays
Of 5,568 transcripts on the microarray, 2,133 transcripts were present in at least 8 of the 9 microarrays and had relative expression levels greater than the median value ± 1SD in at least one microarray (Figure 5A). Within this subset, 244 transcripts with higher abundance (3 fold) in ovary than testis and 239 transcripts with higher abundance (3 fold) in testis than ovary were identified (Figure 5B). From these differentially expressed genes, there were transcripts whose putative functions are possibly involved in testicular development (Figure 5C).
When examining the library sources of transcripts more abundantly expressed in ovaries, we found 51% were from the ovary, 22% from testis, 9% from hemocyte and heart each, 8% from gill, 1% from lymphoid organ and <1% from the remaining tissues (Figure 5D). These transcripts included peritrophin (also called cortical rod protein, CRP), thrombospondin (TSP) and nuclear autoantigenic sperm protein (NASP). In the kuruma shrimp, CRP and TSP are first scattered throughout the cytoplasm of oocytes and are subsequently localized in cortical rods during the cortical rod formation . NASP is a histone-binding protein. Overexpression of NASP in mice testis affects progression of cell proliferation through the cell cycle [26, 27]. Expression analysis of these genes [20, 28] and dolichyl diphosphooligocharide protein glycotransferase, asparagenyl tRNA synthetase and 3-oxoacid CoA transferase by semiquantitative RT-PCR (S. Klinbunga, unpublished data) revealed greater expression levels in ovary than testis of wild P. monodon broodstock. This validated the accuracy of the microarray analysis for gene expression analysis in P. monodon.
Transcripts with higher expression in testis were found mainly from the testis EST library (65%), 8% from heart, 7% from hemocyte, 5% from gill, 3% from ovary, 2% from hepatopancreas, 1% from intestine, and 1.5% from the rest (Figure 5E). Examples of these transcripts include argonaute 1, dynactin 5, and orphan 2. Argonaute proteins are key mediators for RNA silencing mechanisms . Cytoplasmic dynactin forms a complex with dynein and plays a functional role in spermatid growth in Drosophila .
Based on the distribution of library sources where the differentially expressed transcripts came from, it is evident that we can obtain additional information by including cDNA probes from various EST libraries beyond testis and ovary libraries. This more comprehensive coverage of microarray probes demonstrates the usefulness of the present microarray version in gene expression analysis of P. monodon.
Within the 1,076 cDNA spots from testis ESTs that were fabricated onto the present version of cDNA microarrays, there were 169 transcripts with greater abundance in testis than ovary. Of these transcripts saposin-related protein and Dmc1 were consistently more abundant in testis than in ovary for both juvenile and broodstock comparisons. Moreover, these genes have previously been reported to be relevant to male reproduction in other organisms.
In vertebrates, saposins are a group of four small glycoproteins (A, B, C, and D), derived from a common precursor protein called prosaposin, which is reported to activate glycosphingolipid hydrolysis . Inactivation of prosaposin gene resulted in accumulation of lactosylceramide, glucosylceramid, digalactosyl ceramide, sulfactide, ceramide, and globotriaosylceramide in lysosomes. A prosaposin -/- mouse model demonstrated that the mice die at day 35-40 after birth due to neurological defects . The disruption of the prosaposin gene resulted in a reduction in size and weight: 30% of the testis, 37% of the epididymis, 75% of the seminal vesicles, and 60% of the prostate glands. Moreover, the smaller testes from the mutant mice were associated with reduced spermiogenesis, especially in the late spermatids .
Dmc1 (a RAC A-like recombinase) is known to be a specific factor for meiotic recombination and has been identified as a gene product specifically expressed during the early meiotic prophase . Recently, the full-length Dmc1 cDNA was cloned from the testis of the Japanese eel (Anguilla japonica). Dmc1 mRNA was abundantly expressed in the testis and ovary with lower expressed in the brain. In situ hybridization revealed that Dmc1 of A. japonica was localized only in the primary spermatocytes implying its important role during the initial stages of spermatogenesis .
Isolation and characterization of the full-length cDNA of saposin (PmSap) and Dmc1 (PmDmc1) in P. monodon
Based on their differential expression levels seen in the microarray analysis and their possible involvement in testicular development from previous studies in other organisms, saposin-related protein (PmSap) and Dmc1 (PmDmc1) were further characterized by RACE-PCR to obtain their full-length cDNA sequences.
Most saposins in vertebrate and invertebrate contain a signal peptide. Typically, vertebrate prosaposins contain two conserved SapA domains and four conserved SapB domains in which six equally placed cysteines are found in each SapB domain. In contrast, the numbers of saposin domains in invertebrates is less conserved. The absence of some of SapA domain produced variants of saposin-related proteins in various taxa (Figure 6B). Moreover, there are variable numbers of SapB domains in invertebrates, from 6 domains present in the pea aphid, Acyrthosiphon pisum (Homoptera), to up to 9 domains in the black legged tick Ixodes scapularis (Acari) and Nasonia vitripennis (Hymenoptera) (Figure 6B). Additional SapB domains in invertebrate saposins may be explained by several rounds of tandem internal gene duplication as previously proposed for the creation of the four domain saposins in vertebrate .
In P. monodon, the deduced PmSap protein contained a signal peptide with a cleavage site between Ala21 and Glu22, two SapA and seven SapB domains (SapA: positions 25-58 and 823-856 with E-value = 2.74e-12 and 1.56e-07, respectively; SapB: positions 68-144, 178-251, 272-346, 437-512, 531-606, 646-721, and 738-813 with E-value = 1.32e-22, 5.32e-09, 7.28e-16, 4.34e-23, 4.61e-27, 2.63e-22, and 5.83e-15, respectively). Interestingly, six fixed positions of cysteine were found in each SapB domain of PmSap but conserved prolines in identical positions as previously reported in vertebrate saposins  were not found (data not shown). Five predicted glycosylation sites were found in SapB domains 1 (NET, positions 87-89), 3 (NTT, positions 291-293), 5 (NRT and NLS, positions 550-552 and 593-595, respectively) and 6 (NAT, positions 665-667). These predicted SapB domains significantly matched saposin-related protein A (E-value = 8e-24; Uniprot No. Q9Y125), BmP109 (E-value = 7.4e-20; Uniprot No. O15997), Saposin C (E-value = 6.4e-14; Uniprot No. P220097) and BmP109 (E-value = 3.4e-7; Uniprot No. O15997), respectively.
Expression of PmSap and PmDmc1 in testis of juveniles and domesticated and wild broodstock of P. monodon
Tissue expression analysis is important for providing basic information needed to prioritize further analysis of functional genes. Based on the fact that a particular gene may be expressed in several tissues and possess different functions, rapid detection of PmSap and PmDmc1 expression profiles in gonads of P. monodon by cDNA microarray was further confirmed by quantitative real-time PCR.
Molecular mechanisms and expression patterns of genes controlling different steps of sperm maturation and testicular development should be examined for a better understanding of P. monodon reproductive maturation in captivity. The limited number of known genes expressed in testis of this economically important species was partially resolved by EST analysis in the present study. Typically, molecular studies of the hormonal and environmental effectors involving shrimp reproduction have been limited to one or a few genes. Here, we illustrate that cDNA microarrays are relatively simple, reliable and cost-effective for examining integrated interactions among genes in gonads of P. monodon. This method can help accelerate studies on reproductive development and maturation of P. monodon.
The testis cDNA library of P. monodon was previously constructed as described in . The lambda library was converted to the pBluescript library by in vivo excision. Colony PCR was performed in a 25 μl reaction mixture containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, Enhancer solution, 200 mM of each dNTP, 2 mM MgCl2, 0.2 μM each of M13-F (5'-ACG TTG TAA AAC GAC GGC CAG-3') and M13-R (5'-ACA GGA AAC AGC TAT GAC CAT G-3'), and 1.25 unit of i-Taq™DNA Polymerase (iNtRON Biotechnology). PCR was carried out in a thermocycler consisting of predenaturation at 94°C for 5 min followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 1 min and extension at 72°C for 3 min. The final extension was carried out at the same temperature for 7 min. The colony PCR products were size-fractionated on 1.5% agarose gel and visualized after ethidium bromide staining. Clones with >400 bp inserts were selected for further sequencing. Plasmid was extracted using 96-well plate format (EZ-10 96-well spin column, Bio Basic). The concentrations of plasmids were measured using a NanoDrop 8000. Nucleotide sequence of each clone was obtained using an automated DNA sequencer. The 4,803 sequences of the testis ESTs from this study were deposited in NCBI dbEST http://www.ncbi.nlm.nih.gov/dbEST/ with accession numbers of GW992816-GW996323, HO000142-HO000184, GE613296 - GE614547.
In silicoanalysis of ESTs
Nucleotide sequences were analyzed using ESTplus, an integrative system for comprehensive and customized EST analysis and proteomic data matching , as summarized in Figure 1. Briefly, the sequencing data were analyzed by SeqClean (removal of the polyA/polyT tail, low-quality ends, short sequences and those containing cloning vector), RepeatMasker (masking of the sequences when matched with sequences in the largest repeat library, the RepBase, that covers a number of organisms including human, rodent, zebrafish, Drosophila, and Arabidopsis thaliana) (University of Washington Genome Center, Seattle; http://ftp.genome.washington.edu/cgi-bin/RepeatMasker), and CAP3 (assembly and clustering of EST sequences) . The post-processed sequences were further annotated for biological activities by comparison with the NCBI nr database using BlastN (a nucleotide-level annotation, E-value < 10-5), BlastX (a protein-level annotation, E-value < 10-5), BLAST2GO (Gene Ontology prediction of the annotated proteins from BlastX program), and InterProScan (protein signature identification).
To identify ESTs found only in the testis cDNA library of P. monodon, all newly sequenced contigs and singletons were compared against all sequences (38,429 ESTs) from the P. monodon database (PmDB: http://pmonodon.biotec.or.th). Moreover, to identify novel transcripts that have not been previously reported in shrimp, the sequenced data were also compared against all available Penaeidae ESTs (207,852 ESTs) from NCBI http://ncbi.nlm.nih.gov retrieved on 01/11/09.
Microarray analysis for identification of differential expression transcripts in testis of P. monodon
A cDNA microarray was constructed from various EST libraries of P. monodon, consisting of 5,568 features (1,076 unique cDNA features were amplified from testis EST libraries). The arrays were post-processed according to  immediately before hybridization.
Summary of microarray experiments in this study
I. Comparison of gene expression levels between ovary and testis in individual juveniles
Ovary from juvenile 1
Testis from juvenile 1
Ovary from juvenile 2
Testis from juvenile 2
Ovary from juvenile 3
Testis from juvenile 3
II. Comparison of gene expression levels between ovaries and testes pooled from juveniles
Pooled ovaries from juveniles (n = 98)
Pooled testes from juveniles (n = 114)
Pooled ovaries from juveniles (n = 98)
Pooled testes from juveniles (n = 114)
Pooled ovaries from juveniles (n = 98)
Pooled testes from juveniles (n = 114)
III. Comparison of gene expression levels between ovaries and testes pooled from broodstock
Ovary from broodstock 1 (GSI*: 12.4)
Testis from broodstock 1 (GSI: 1.1)
Ovary from broodstock 2 (GSI: 11.2)
Testis from broodstock 2 (GSI: 1.1)
Ovary from broodstock 3 (GSI: 12.6)
Testis from broodstock 3 (GSI: 1.1)
Microarray imaging and data analysis
The hybridized slides were scanned with a GenePix 4000B (Molecular Devices, Sunnyvale, CA) and processed using GenePix Pro version 6.1. Only spots with intensities greater than one standard deviation above the background intensity were further analyzed. The processed data were normalized within each array by the scaled print-tip (Lowess) method, and across arrays, using the Aroma package , (available from http://www.maths.lth.se/help/R/aroma/, run in R project environment http://cran.r-project.org. The microarray data have been deposited in NCBIs Gene Expression Omnibus http://www.ncbi.nlm.nih.gov/geo/ with GEO accession number GSE19037 at . The average logarithmic base 2 values of relative intensities between Cy3- and Cy5- samples (M values) were subjected to hierarchical clustering analysis and illustrated using the Treeview software . To identify differential expressed transcripts, only transcripts with relative expression level changes more than the median value ± 1SD in at least 1 array and present in at least 8 of the 9 arrays were further considered. A 3-fold change in at least 6 of the 9 arrays was considered as criteria for differential expression.
Examination of full-length cDNA sequences of Saposin and Dmc1by Rapid Amplification of cDNA Ends-Polymerase Chain Reaction (RACE-PCR)
Primer sequence, melting temperature and the expected amplification product of PmSap and PmDmc1
Gene expression analysis of PmSap and PmDmc1by quantitative real-time PCR
Gene-specific primers were designed for PmSap, PmDmc1 and Elongation factor 1 alpha (EF-1α, Table 3). For construction of the standard curve of each transcript, a plasmid containing the transcript was constructed by cloning PCR product into pGEM-Teasy vector and transforming the resulting vector into E. coli JM109. The plasmid was extracted and used as the template for construction of the standard curve by 10-fold serial dilutions (103 - 109 copy numbers). Real-time PCR reactions were carried out in a 96-well plate in triplicate.
The expression levels of PmSap and PmDmc1 in testis from juvenile (J-TT, N = 5), domesticated broodstock (DB-TT, N = 5), and wild broodstock (WB-TT, N = 5) and ovary from juvenile (J-OV, N = 4) and wild broodstock (WB-OV, N = 4) were further analyzed by quantitative real-time PCR analysis. EF-1α was used as an internal control. Each primer set was designed to generate 120 to 150 bp amplicons (Table 3). Each reaction was performed in a 15 μl reaction volume containing 2× LightCycler 480 SYBR Green I Master (Roche), 50 ng of first strand cDNA template, and 0.2 μM or 0.4 μM of each primer pair. Cycling parameters were 95°C for 10 min, 40 cycles of 95°C for 15 sec, 58°C for 30 sec, and 72°C for 30 sec. The specificity of PCR products was confirmed by melting curve analysis by heating at 95°C for 15 sec, 65°C for 1 min before heating to 97°C and gradually cooling down to 40°C within 10 sec. Real-time PCR of each shrimp was tested in duplicate. Relative expression levels (copy number of the target gene/that of EF-1α) of different sample groups were statistically tested by ANOVA followed by Duncan's new multiple range test or Tukey test (P < 0.05).
This research is supported by the National Center for Genetic Engineering and Biotechnology (BIOTEC). Part of this research (library construction, RACE-PCR and real-time-PCR) was carried out by RL during the PhD program and supported by the Royal Golden Jubilee PhD program, the Thailand Research Funds (TRF), Thailand. We would like to thank Dr. Amy Lum for reading and improving the manuscript. Also, we would like to thank Prof. Morakot Tanticharoen for her mentorship of this research program.
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