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. 2012 Feb 21;5:111. doi: 10.1186/1756-0500-5-111

An ovary transcriptome for all maturational stages of the striped bass (Morone saxatilis), a highly advanced perciform fish

Benjamin J Reading 1,#, Robert W Chapman 2,#, Jennifer E Schaff 3, Elizabeth H Scholl 4, Charles H Opperman 4, Craig V Sullivan 1,5,✉
PMCID: PMC3305648  PMID: 22353237

Abstract

Background

The striped bass and its relatives (genus Morone) are important fisheries and aquaculture species native to estuaries and rivers of the Atlantic coast and Gulf of Mexico in North America. To open avenues of gene expression research on reproduction and breeding of striped bass, we generated a collection of expressed sequence tags (ESTs) from a complementary DNA (cDNA) library representative of their ovarian transcriptome.

Results

Sequences of a total of 230,151 ESTs (51,259,448 bp) were acquired by Roche 454 pyrosequencing of cDNA pooled from ovarian tissues obtained at all stages of oocyte growth, at ovulation (eggs), and during preovulatory atresia. Quality filtering of ESTs allowed assembly of 11,208 high-quality contigs ≥ 100 bp, including 2,984 contigs 500 bp or longer (average length 895 bp). Blastx comparisons revealed 5,482 gene orthologues (E-value < 10-3), of which 4,120 (36.7% of total contigs) were annotated with Gene Ontology terms (E-value < 10-6). There were 5,726 remaining unknown unique sequences (51.1% of total contigs). All of the high-quality EST sequences are available in the National Center for Biotechnology Information (NCBI) Short Read Archive (GenBank: SRX007394). Informative contigs were considered to be abundant if they were assembled from groups of ESTs comprising ≥ 0.15% of the total short read sequences (≥ 345 reads/contig). Approximately 52.5% of these abundant contigs were predicted to have predominant ovary expression through digital differential display in silico comparisons to zebrafish (Danio rerio) UniGene orthologues. Over 1,300 Gene Ontology terms from Biological Process classes of Reproduction, Reproductive process, and Developmental process were assigned to this collection of annotated contigs.

Conclusions

This first large reference sequence database available for the ecologically and economically important temperate basses (genus Morone) provides a foundation for gene expression studies in these species. The predicted predominance of ovary gene expression and assignment of directly relevant Gene Ontology classes suggests a powerful utility of this dataset for analysis of ovarian gene expression related to fundamental questions of oogenesis. Additionally, a high definition Agilent 60-mer oligo ovary 'UniClone' microarray with 8 × 15,000 probe format has been designed based on this striped bass transcriptome (eArray Group: Striper Group, Design ID: 029004).

Background

The striped bass and its relatives in the genus Morone (the temperate basses) are ecologically and economically important aquaculture and fisheries species native to estuaries and rivers of the Atlantic coast and Gulf of Mexico in North America [1,2]. Although the striped bass and its hybrids have been reared as commercial aquaculture products in the United States since the late 1980s, little genetic information is available for these species in public databases at the National Center for Biotechnology Information (NCBI) or elsewhere, consisting only of microsatellite DNA markers [3,4], the mitochondrial genome (GenBank: HM447585), and a medium density genetic linkage map [5]. A major factor contributing to restricted growth of hybrid striped bass farming nationwide is reproductive dysfunction of female striped bass, resulting in non-viable eggs, embryos, and larvae [6]. These reproductive failures hamper selective breeding efforts required for species domestication and improvement. The exact cause(s) of poor egg quality and embryonic mortality in farmed fishes, however, still remain to be discovered, making appropriate and timely corrective measures difficult to achieve [review: [7,8]].

Functional genomics has emerged as a major research field and gene expression (transcriptomics) and proteomics studies are promising approaches to gain new insights into reproductive molecular biology [7,9-12]. Marked advancement in striped bass reproductive technology based on such "Omic" analyses is, however, currently restricted due to the lack of an available, comprehensive sequence database for this species or for other members of the genus Morone that are important in aquaculture (e.g. hybrid striped bass) or as research models (e.g. white perch, M. americana). Transcriptome resources are currently available for other commercially important fishes, including rainbow trout (Oncorhynchus mykiss) [13-16], coho salmon (Oncorhynchus kisutch) [17], tilapia (Oreochromis mossambicus) [18], Atlantic halibut (Hippoglossus hippoglossus) [19], Senegalese sole (Solea senegalensis) [20], Atlantic salmon (Salmo salar) [21], and cod (Gadus morhua) [22].

The emergence of pyrosequencing and later generation DNA sequencing technologies has made acquisition of significant genomic resources accessible and affordable for non-model organisms [23-25]. Vast numbers of expressed sequence tags (ESTs) can readily be generated using these methods, providing direct evidence of gene transcription, and collections of such EST sequences are presently the most important resources used for transcriptome exploration [26]. Depending on the number of ESTs sequenced, resulting databases can represent a high proportion of the total number of gene transcripts expressed by a given tissue (i.e. transcriptome), making downstream procedures for transcriptome profiling, such as oligo microarray or real-time quantitative reverse transcription PCR, tractable without the need for an entire genome sequence.

When sequencing depth is limited, organ specific EST collections permit more efficient gene expression analyses using 'UniClone' microarrays, which are comprised of probe sequences isolated from a single organ type [27-30]. UniClone arrays represent a larger proportion of a target organ transcriptome and have reduced redundancy when compared to arrays comprised of ESTs derived from several different tissue types. Additionally, to realize the full benefits of proteomic analyses based on mass spectrometry, species-specific ESTs are required, since algorithms used for spectral analyses (e.g. SEQUEST, Proteome Discoverer Software, Thermo Scientific, West Palm Beach, FL) require a homologous reference sequence database. For non-model organisms, sequence information from even closely related species can be insufficient for the accurate identification of peptides, since these algorithms tend to be conservative and heterospecific amino acid substitutions can result in peptide misidentification or an inability to detect orthologues [31].

Therefore, the goal of the present study was to provide an ovary transcriptome database representative of all stages of oogenesis and atresia in striped bass, one that could provide the requisite foundation for functional genomics and proteomics investigations of reproduction and egg quality in this species and that would support similar studies in the other temperate basses.

Results

A total of 230,151 EST short read sequences with a combined length of 51,259,448 bp (average length 224 bp) were generated from cDNA pooled from ovarian tissues and eggs encompassing the various stages of ovary growth, maturation and atresia. A total of 11,208 high-quality contigs with a length of at least 100 bp were assembled and these included 2,984 contigs that were 500 bp or longer (average length 895 bp; total length 5,068,343 bp) (Additional File 1). Blastx comparisons revealed 5,482 orthologues, of which 4,120 (36.7%) were annotated with Gene Ontology (GO) terms. The number of unknown, unique sequences was 5,726 (51.1%). The breakdown of GO annotation classes within the three categories of GO terms for all annotated sequences is shown in Figure 1: Biological Process (2nd level) and Molecular Function and Cellular Component (3rd level). A complete list, in FASTA format, of the contig assemblies identified by their annotations are included as Additional File 2 and a list of the assemblies and their GO terms are included as Additional File 3.

Figure 1.

Figure 1

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Gene ontology graph of A. Cellular Component (3rd level GO terms), B. Molecular Function (3rd level GO terms), and C. Biological Process (2nd level GO terms) of annotated genes in the striped bass ovary transcriptome. The number of GOs in each class is shown and sections that contained 50-150 entities are represented in black, 151-500 by dark gray, 500 and up by light gray, and the predominant class is indicated in white.

There were 66 contigs that were each assembled from groups of ESTs that comprised ≥ 0.15% of the total 230,151 reads (i.e. ≥ 345 reads per contig) and these contigs were considered to have abundant ovary expression. These contigs were identified by NCBI UniGene cluster and compared to zebrafish, Danio rerio, orthologues evaluated by Digital Differential Display (DDD) (Table 1). Twenty-two striped bass genes from this list (33.3% of the total listed) either had no blastx returns (i.e. were novel), or were identified as being unnamed gene products, or had gene names but no zebrafish UniGene orthologues. These were excluded from further evaluation. Of the remaining informative 44 genes, 23 (52.5%) are predicted to have predominant ovary expression based on DDD of zebrafish orthologues, 11 (25.0%) would be expected to have no difference in expression between ovary and other tissues of the body based on the DDD results, and 10 (22.7%) would likely have predominant expression in other tissues of the body based on the DDD comparison. Overall, the estimated 66 most abundantly expressed striped bass ovary genes were assembled from ~1/6 of the total number of short read sequences (Table 1).

Table 1.

Transcripts abundantly expressed in the striped bass ovary.

Contig Number BLAST 2GO Annotation Gene GeneID zebrafish taxid: 7955 orthologue Assembled contig length (bp) Number of observe sequence reads % Total sequence reads (230,151) Fraction of ESTs that mapped to the zebrafish UniGene by DDD Zebrafish UniGene
Ovary Body
1 10186 cyclin b2 ccnb2 368316 1284 1146 0.4979340 0.0025 > 0.0001 Dr.80580
2 10415 zona pellucida glycoprotein zp2.3 114439 1329 1076 0.4675192 0.0429 > 0.0012 Dr.143785
3 10181 novel protein with zona pellucida-like domain si: ch211-14a17.7 368669 646 1001 0.4349318 0.0015 > 0.0001 Dr.75717
4 9349 zona pellucida c zpcx 334011 2036 923 0.4010411 0.0013 > 0.0001 Dr.80433
5 146 nad h quinone 1 nqo1 322506 916 908 0.3945236 n.d. = n.d. Dr.4189
6 8878 tubulin beta 2c zgc: 123194 641421 1510 869 0.3775782 n.d. = n.d. Dr.52550
7 9768 egg envelope component zpax si: dkeyp-50f7.2 334036 2890 864 0.3754057 0.0017 > 0.0003 Dr.105787
8 10472 fatty acid binding protein liver fabp1b.1 554095 419 848 0.3684538 n.d. = n.d. Dr.24261
9 9294 --NA-- -- -- 812 839 0.3645433 -- -- --
10 10137 choriogenin 1 zp3b 64692 1389 817 0.3549843 0.0029 > 0.0003 Dr.75734
11 11102 hypothetical protein LOC100049339 polr2a 553347 774 767 0.3332595 * * Dr.79109
12 11074 --NA-- -- -- 181 762 0.3310870 -- -- --
13 10663 zgc: 175135 protein zgc: 165551 100003969 636 706 0.3067551 0.0039 > 0.0003 Dr.106137
14 9917 heat shock protein 8 hspa8 573376 2266 699 0.3037136 0.0011 < 0.0029 Dr.75087
15 11091 novel protein with zona pellucida-like domain LOC100331707 100331707 1219 675 0.2932857 -- -- --
16 3 --NA-- -- -- 1585 654 0.2841613 -- -- --
17 11147 fatty acid-binding heart fabp11a 447944 581 638 0.2772093 n.d. = n.d. Dr.78045
18 10883 mgc86501 protein wu: ft38e01 798996 568 623 0.27069919 0.0024 > 0.0002 Dr.106837
19 9329 histone h3f3c 336231 945 619 0.2689539 0.0001 < 0.0003 Dr.75577
20 10302 voltage gated chloride channel domain-containing protein -- -- 996 616 0.2676504 -- -- --
21 11112 egg envelope component zpc zp3c 563179 1527 610 0.2650434 0.0002 > 0 Dr.113688
22 30 histone h2a LOC573838 (h2af1o) 100332229 447 607 0.2637399 0.0024 > 0.0002 Dr.75698
23 10079 --NA-- -- -- 811 585 0.2541810 -- -- --
24 10058 beta-actin bactin2 57935 1874 578 0.2511395 0.0026 < 0.0077 Dr.75125
25 10823 apolipoprotein d zgc: 123339 567972 816 560 0.2433185 * * Dr.15815
26 10825 --NA-- -- -- 154 555 0.2411460 -- -- --
27 10773 hypothetical protein LOC100049339 -- 30705 756 555 0.2411460 -- -- --
28 6635 h1 histone member oocyte-specific h1m 327403 823 523 0.2272421 n.d. = n.d. Dr.75735
29 11098 adp atp translocase slc25a5 192321 1243 515 0.2237661 0.0015 < 0.0078 Dr.30295
30 127 nucleoside diphosphate kinase b nme2b.1 30083 834 511 0.2220281 n.d. = n.d. Dr.11052
Contig Number BLAST 2GO Annotation Gene GeneID zebrafish taxid: 7955 Orthologue Assembled contig length (bp) Number of observe sequence reads % Total sequence reads (230,151) Fraction of ESTs that mapped to the zebrafish UniGene by DDD Zebrafish UniGene
Ovary Body
31 10309 60 s acidic ribosomal protein p0 rplp0 58101 932 497 0.2159452 0.0008 < 0.0033 Dr.55617
32 11081 loc494706 protein (oogenesis-related gene) org 100001110 601 495 0.2150762 0.0016 > 0.0001 Dr.80745
33 10120 elongation factor 1 alpha efla 30516 1744 492 0.2137727 0.0032 < 0.0108 Dr.31797
34 10015 heat shock protein 90 hsp90ab1 30573 1900 485 0.2107312 0.0006 < 0.0020 Dr.35688
35 11073 unnamed protein product -- -- 414 481 0.2089932 -- -- --
36 10797 complement component (3b 4b) receptor 1 LOC565541 565541 1696 470 0.2042138 * * Dr.91858
37 92 cyclin b1 ccnb1 58025 738 470 0.2042138 0.0035 > 0.0002 Dr.121261
38 10403 --NA-- -- -- 327 469 0.2037793 -- -- --
39 126 karyopherin alpha 2 (rag cohort importin alpha 1) zgc: 55877 406343 1085 469 0.2037793 0.0010 > 0.0002 Dr.20877
40 10948 --NA-- -- -- 248 465 0.2020413 -- -- --
41 10900 zpb protein LOC100334275 100334275 1561 461 0.2003033 * * Dr.141250
42 36 claudin 4 cldnd 81583 731 456 0.1981308 0.0004 > 0.0001 Dr.75663
43 216 stathmin 1 oncoprotein 18 variant 8 stmn1b 550548 964 450 0.1955238 0 < 0.0004 Dr.105609
44 10949 --NA-- -- 550134 151 420 0.1824889 -- -- --
45 9337 Securin [Anoplopoma fimbria] LOC566690 566690 435 414 0.1798819 0.0002 > 0 Dr.118007
46 9321 dna replication inhibitor gmnn 368320 1121 412 0.1790129 n.d. = n.d. Dr.119358
47 10986 cell division cycle 20 homolog (cerevisiae) cdc20 406353 1597 410 0.1781439 0.0005 > 0.0001 Dr.105018
48 11071 --NA-- -- -- 215 402 0.1746679 -- -- --
49 10743 --NA-- -- -- 273 398 0.1729299 -- -- --
50 1174 cyclin k LOC100331304 100331304 3331 397 0.1724954 0.0009 > 0 Dr.148591
51 10438 ribonucleotide reductase m2 polypeptide rrm2 30733 1621 396 0.1720610 0.0018 > 0.0003 Dr.75098
52 11198 ribosomal protein s20 rps20 406485 477 393 0.1707575 0.0014 > 0.0008 Dr.18943
53 11014 karyopherin alpha 2 (rag cohort importin alpha 1) kpna2 436607 534 380 0.1651090 0.0009 > 0.0002 Dr.75709
54 10351 --NA-- -- -- 299 375 0.1629365 -- -- --
55 10265 unnamed protein product -- -- 1075 375 0.1629365 -- -- --
56 771 cytochrome c oxidase copper chaperone cox17 447914 410 375 0.1629365 0.0007 > 0.0001 Dr.82168
57 10107 tubulin, alpha 1c MGC171407 573122 697 374 0.1625020 n.d. = n.d. Dr.120425
58 161 --NA-- -- -- 2532 371 0.1611985 -- -- --
59 231 epididymal secretory protein e1 precursor npc2 282673 728 360 0.1564190 -- -- --
60 11090 --NA-- -- -- 308 356 0.1546811 -- -- --
Contig Number BLAST 2GO Annotation Gene GeneID zebrafish taxid: 7955 orthologue Assembled contig length (bp) Number of observed sequence reads % Total sequence reads (230,151) Fraction of ESTs that mapped to the zebrafish UniGene by DDD Zebrafish UniGene
Ovary Body
61 10741 ppia protein (pepitidylprolyl isomerase A) ppia 336612 825 356 0.1546811 0.0005 < 0.0011 Dr.104642
62 9354 superoxide dismutase sod1 30553 795 356 0.1546811 n.d. = n.d. Dr.75822
63 10048 ubiquitin b ubb 550134 169 355 0.1542466 n.d. = n.d. Dr. 104259
64 10083 cyclin a2 ccna2 192295 2108 351 0.1525086 n.d. = n.d. Dr.121874
65 10746 eukaryotic translation elongation factor 1 gamma eef1g 195822 1533 350 0.1520741 0.0006 < 0.0011 Dr.75657
66 10761 egg envelope component zpax si: dkeyp-50f7.2 334036 2731 347 0.1507706 0.0017 > 0.0003 Dr.105787
TOTALS 69173 36532 15.8730570
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Genes are ranked (1-66) by number of observed 454 short read sequences used in each contig assembly. Digital Differential Display (DDD) results of orthologous sequences in zebrafish are also shown

Annotation "--NA--"indicates no blastx return; Dashes (--) indicate unknown or data not available; asterisks (*) indicate the UniGene was not present in the EST libraries used for DDD. Sequences with expression differences evaluated by DDD (FET, P ≤ 0.05) are indicated by ">" (enhanced ovary expression) or "<" (enhanced body expression); "n.d." indicates no significant difference in expression between ovary and body (=)

All of the high-quality ESTs have been deposited in the NCBI Short Read Archive (GenBank: SRX007394) and annotated contigs are posted under "Resources" on the National Animal Genome Research Program Aquaculture Genome Projects website (http://www.animalgenome.org/aquaculture/database/) [32]. These contigs also have been submitted to Agilent Technologies eArray (Santa Clara, CA) for ovary UniClone microarray design (http://www.chem.agilent.com/). We designed a high definition 60-mer SurePrint oligo array with 8 × 15,000 probe format comprised of 11,145 UniGene probes from the transcriptome, plus an additional 3,854 probes printed in duplicate or selected from Morone cDNAs available from NCBI or from our own unpublished results (B.J. Reading and C.V. Sullivan, unpublished data) and datasets (eArray Group: Striper Group, Design ID: 029004).

Discussion

This collection of ESTs represents the first contribution of a large reference sequence database for species of the genus Morone and provides a basis for future gene expression studies in these temperate basses. Availability of characterized ovarian transcriptomes from fishes other than zebrafish is limited. Partial transcriptomes have been reported for tilapia (474 EST assemblies) [18] and for cod (1,361 EST assemblies) [22]. Several thousand ovarian ESTs have been reported for salmonid fishes [[13,15,33] and references therein], but to our knowledge these have not been assembled into a comprehensive ovarian transcriptome. Numbers of total ESTs currently available in the NCBI EST database for some other commercially important finfishes are as follows: rainbow trout (287,967), coho salmon (4,942), tilapia (Genus Oreochromis, 121,346), Atlantic halibut (20,836), Senegalese sole (10,631), Atlantic salmon (498,212), and cod (229,094). Therefore, the 230,151 ESTs reported herein represent a comparatively valuable transcriptome resource for striped bass.

If the 11,208 contigs are considered to be UniGenes, this represents a substantial proportion of the estimated total protein-coding gene transcripts expressed by the striped bass ovary (i.e. transcriptome) as the average number of mRNA transcripts expressed by a single tissue type is estimated to be between 10,000-15,000 [34], but can be as low as 8,200 [35]. Since over 1,300 GOs from Biological Process classes of Reproduction (121), Reproductive process (55), and Developmental process (1,188) were assigned to the annotated contigs (Figure 1), this sequence collection should prove to be a powerful tool for analysis of ovarian gene expression related to fundamental questions of oogenesis.

Approximately 52.5% of the informative contigs considered to have abundant ovary expression (i.e. those with ≥ 345 reads per contig) were also predicted to have predominant expression in striped bass ovary through DDD comparisons to zebrafish orthologues (Table 1). These include cyclin B2 (ccnb2, contig10186), several egg envelope and zona pellucida proteins, histone H2A (h2af1o, contig00030), oogenesis-related gene (org, contig11081), cyclin B1 (ccnb1 contig00092), karyopherin alpha 2 (kpna2, contigs 00126 and 11014), claudin 4 (cldnd, contig00036), securin (LOC566690, contig 09337), cell division cycle 20 homolog (cdc20, contig10986), cyclin K (LOC100331304, contig11174), ribonucleotide reductase M2 polypeptide (rrm2, contig10438), ribosomal protein S20 (rps20, contig11198), cytochrome C oxidase copper chaperone (cox17, contig00771), and epididymal secretory protein E1 (npc2, contig00231). Many of these are well-characterized ovary transcripts and several recent and informative papers have been published detailing the functions of these genes and their protein products in fish oocytes and embryos [see: [7,8,13-20,27,28,36-38]]; others are briefly detailed below.

The remaining 47.5% of abundant striped bass ovary genes that were compared to zebrafish orthologues in the DDD were predicted to have indifferent or predominant expression levels in other tissues of the body relative to the ovary. These may represent constitutively expressed genes or those expressed at high levels in the ovary albeit comparatively lower than in other tissues of the body, respectively. Examples of potential genes with constitutive expression include NADH quinone 1 (nqo1, contig00146), tubulin (zgc:123194, contig08878 and MGC171407, contig10107), fatty acid binding proteins (fabp1b, contig10472 and fabp11a, contig11147), H1 histone member oocyte-specific (h1m, contig06635), nucleoside diphosphate kinase B (nme2b, contig00127), geminin DNA replication inhibitor (gmnn, contig09321), superoxide dismutase (sod1, contig09354), ubiquitin B (ubb, contig10048), and cyclin A2 (ccna2, contig10083). Of these, fatty acid-binding protein heart (fabp11a) has been shown to be up-regulated in ovary of rainbow trout females that mature precociously [13] and an orthologue of h1m (H1foo) is generally considered to be an oocyte specific histone in mouse (Mus musculus) [39,40], contrary to the DDD prediction. The UniGene EST Profile of zebrafish h1m (Dr. 75735) indicates that it is predominantly expressed in skin, however the second most abundant site of expression is the reproductive system.

The following genes expressed in striped bass ovary are also expressed in zebrafish ovary, however the DDD indicates that they are predominantly expressed in other tissues of the body (Table 1): histone (h3f3c, contig09329), beta-actin (bactin2, contig10058), ADP/ATP translocase (slc25a5, contig11098), 60S acidic ribosomal protein P0 (rplp0, contig10309), elongation factor 1 alpha (ef1a, contig10120), peptidylprolyl isomerase A (ppia, contig10741), eukaryotic translation elongation factor 1 gamma (eef1g, contig10746), stathmin 1 oncoprotein 18 variant 8 (stmn1b, contig00216), and heat-shock proteins 8 (hspa8, contig09917) and 90 (hsp90ab1, contig10015). Ovarian representation of gene transcripts that show predominant expression in other tissues of the body is not surprising given the heterogeneous complexity of the ovary, which is comprised of vasculature, blood and other connective tissues, the somatic follicle, and germ cells. Furthermore, most of these genes, for example ef1a and bactin2, are considered to have constitutively high expression in most tissues, and this is supported by the corresponding zebrafish UniGene EST Profiles (Dr. 31797 and Dr.75125, respectively). There were, however, three exceptional genes whose expression, although considered to be lower in comparison to other tissues of the body by DDD, have been shown to be highly expressed in ovary. Stathmin (stmn) is expressed in oocytes and pre-implantation embryos of mice [41] and in cod ovary [22], and Stmn proteins have been detected in zebrafish ovary [36]. Contig00216 encodes a full-length, 147 amino acid Stmn and has been putatively identified as stmn1b, however it is highly similar to two zebrafish stmn isoforms (95% and 94% amino acid identity with stmn1b and stmn1a, respectively). Although stmn1b has body predominant expression in zebrafish by DDD (Table 1), zebrafish stmn1a (UniGene Dr.52664) shows ovary predominant expression and, therefore, contig00216 may actually be orthologous to stmn1a. Given the high similarity of this sequence to both zebrafish stmn1 isoforms, it is not possible to definitively assign identity without comparison to the other striped bass stmn isoform, which is unavailable. Recently, hsp8 and hsp90 (corresponding to striped bass hspa8 and hsp90ab1, respectively) have been characterized as some of the most abundant genes expressed in mouse and fish eggs at both the transcript and protein levels [36,37,42].

This inconsistent result may relate to the inherent weaknesses of DDD, since only highly expressed genes are adequately represented in the EST libraries used to conduct the in silico comparisons and the Fisher's exact test (FET) is conservative [43]. Although this method does not offer quantitation, ranking of the striped bass contigs by number of short reads used in assembly paired with comparisons to zebrafish orthologues evaluated by DDD proved to be a useful tool for estimating relative ovarian abundance of the striped bass gene transcripts. Reservation must be taken when considering such interspecific DDD comparisons for the purpose of excluding genes that are predicted to have less predominant expression in one tissue compared to another, since they may be highly expressed in both. This is a promising approach for characterization of novel gene transcripts from EST libraries and has recently been used to identify ovary specific genes in zebrafish [44] and rainbow trout [15], however such results should be further validated using an experimental evaluation of gene expression.

The growing oocyte is considered to be largely transcriptionally inactive, acting as a storehouse of specific maternal RNAs, proteins, and other molecules required for competency for fertilization, initiation of zygotic development, and transition to embryonic gene expression [review: [37,38]]. These maternal factors may be stored in oocytes for extended periods of time until use (e.g. months to years). Therefore, a system of regulatory proteins and RNAs must mediate the oocyte cell cycle during growth, ovarian maturation (OM), and zygotic development from fertilization until activation of the embryonic genome at the mid-blastula transition [45]. A number of known cell-cycle regulators and proteins critical for these processes have been identified as predominantly expressed in striped bass ovary (Table 1). Examples include cyclins B1 and B2 (ccnb1, ccnb2) [46-49], cyclin K (ccnk) [50], securin [51], cdc20 [27], kpna2 [22,52], gmnn [53], h2af1o [54] and org [44]. Transcripts encoding several different cell division and cell cycle regulatory proteins were similarly reported in the ovaries of cod [22] and rainbow trout [13].

Solute carrier protein (SLC) family members are selected to illustrate representation of sequences in the striped bass ovary transcriptome encoding proteins from a large gene series. The SLCs are a diverse group of eukaryotic membrane proteins that control cellular influx and efflux of solutes, including ions, fatty acids, amino acids, sugars, drugs, and vitamins [55,56]. The Human Genome Gene Nomenclature Committee [57] classifies approximately 400 different human SLCs into 47 families. At least one representative protein from 19 (~40.4%) of these families was identified in the striped bass ovary transcriptome (Table 2). Characterization of SLC gene expression in growing oocytes and during OM would be of direct importance to understanding mechanisms of oogenesis and egg quality in light of what is known of oocyte and egg physiology. Due to osmoregulatory requirements imposed by both fresh and marine waters, embryos of egg-laying fishes develop within the confines of an established chorion that becomes osmotically closed after fertilization. Therefore, ovulated eggs must contain all of the water required during embryogenesis as a medium and substrate for biochemical reactions and as a diluent for waste products (e.g. ammonia). Furthermore, water contributes to appropriate egg buoyancy, especially in marine fishes that spawn pelagic eggs. Prior to ovulation, a hyperosmotic solute concentration develops within the oocytes of these species, followed by passive influx of water through aquaporin membrane channels [review: [58,59]]. Inorganic ions have primarily been implicated in this phenomenon, however the exact mechanisms of their entry have not been verified. Bobe et al. [14] demonstrated up regulation of slc26 (Pendrin) and aqp4 (aquaporin 4) expression in ovary of rainbow trout during OM. Gene transcripts encoding a slc26a6-like protein, along with several other ion transporters (Table 2) and aquaporin 1 (contig08717) were identified in striped bass ovary. This indicates the potential for discovery of previously unknown mechanisms of teleost oocyte hydration by gene expression analyses of these particular SLCs and water transport genes in the striped bass and related species (genus Morone), which can tolerate a wide range of environmental salinities.

Table 2.

Solute carrier family members identified in the striped bass ovary transcriptome

Contig Gene Gene ID Danio orthologue Contig Length (bp) Solute carrier family function
04292a slc3a2 796322 629 Heavy subunit of the heteromeric amino acid transporters (Na+-independent, transport of large neutral amino acids: phenylalanine, tyrosine, leucine, arginine and tryptophan)
10145 slc3a2-like 100003805 1740 Heavy subunit of the heteromeric amino acid transporters (Na+-independent, transport of large neutral amino acids: phenylalanine, tyrosine, leucine, arginine and tryptophan)
09132b slc4a7 568872 563 Electroneutral Na+ and HCO3--dependent cotransporter
11036c slc7a2 100007793 815 Cationic amino acid transporter/glycoprotein-associated amino-acid transporter (transport of the cationic amino acids including arginine, lysine and ornithine)
00672d slc7a8 100007704 987 Na+-independent, transporter of small and large neutral amino acids such as alanine, serine, threonine, cysteine, phenylalanine, tyrosine, leucine, arginine and tryptophan; when associated with Slc3a2, acts as an amino acid exchanger
05979 slc7a10 567420 240 Na+-independent, high affinity transport of small neutral D- and L-amino acids
04450 slc9a3r1 327272 385 Na+/H+ exchanger
02807 slc10a3 406519 692 Na+/bile acid cotransporter
06556 slc10a4 556491 249 Na+/bile acid cotransporter
03289 slc12a5-like 572215 251 Electroneutral cation/Cl- cotransporter (K+/Cl- transporter)
04100 slc19a2-like 100329244 778 Thiamine transporter
00585 slc20a1a 406458 2129 Na+-dependent PO43- transporter
05003 slc20a1b 321541 246 Na+-dependent PO43- transporter
00176 slc25a3 322362 1448 Mitochondrial carrier (PO43- transporter)
01147 slc25a5 192321 1302 Mitochondrial carrier (ADT/ATP translocator)
01400e slc25a12 337675 693 Mitochondrial carrier (aspartate/glutamate transporter)
01037 slc25a26 560478 349 Mitochondrial carrier (S-adenosylmethionine transporter)
09234 slc25a29 569608 579 Mitochondrial carrier (carnitine/acylcarnitine transporter)
06849 slc25a43 796731 254 Mitochondrial carrier
07197f slc25a46 436831 251 Mitochondrial carrier
08784 slc26a6-like 557779 215 Multifunctional anion exchanger (Pendrin-like; Cl-, oxalate, SO42-, and HCO3- transporter)
04105 slc27a1 541410 265 Fatty acid transporter (FATP-1; long-chain fatty acid translocator)
01322g slc29a1 563580 260 Facilitative nucleoside transporter (cellular uptake of nucleosides)
05237 slc30a2 563540 293 Zinc transporter
06016 slc30a2-like 560642 608 Zinc transporter
05293h slc30a5 436594 506 Zinc transporter
03716 slc30a7 327439 392 Zinc transporter (zinc efflux transporter)
09883 slc31a2 -- 2142 Copper transporter (low affinity copper uptake)
02632 slc35a2 368487 186 Nucleoside-sugar transporter (UDP-galactose transporter)
07709 slc35e1-like 100332364 249 Nucleoside-sugar transporter
04693 slc38a8-like 795255 414 Na+-coupled neutral amino acid transporter
05870 slc38a9 562137 243 Na+-coupled neutral amino acid transporter
02706 slc38a11 550337 347 Na+-coupled neutral amino acid transporter
02072 slc39a3 321324 414 Metal ion transporter (zinc influx transporter)
08253i slc39a13 368686 239 Metal ion transporter (zinc influx transporter)
05275j slc44a1 100333377 256 Choline transporter
02670 slc44a2-like 321056 269 Choline transporter
07718 slc44a4-like 393385 255 Choline transporter
05152 slc48a1a 436697 853 Heme transporter hrg1-B
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For contigs with superscripted letters, see also the following corresponding contigs: a02586 (slc3a2); b07936, b03956 (slc4a7); c00241, c09351, c04452 (slc7a2); d04260 (slc7a8); e07750 (slc25a12); f06248 (slc25a46); g04062 (slc29a1); h09154 (slc30a5); i06486 (slc39a13); j02053 (slc44a1)

Conclusions

In summary, as we continue to advance our understanding of reproduction in temperate basses of the genus Morone, this reference sequence database of ovarian transcripts will provide the requisite foundation for gene expression studies and will open avenues of research related to reproduction and egg quality. Several important candidate genes have already been identified for future study. Furthermore, these sequences have been used to design an ovary UniClone oligo microarray for assessing changes in gene expression during oogenesis and in female striped bass spawning good and poor quality eggs. Our recent deployment of this microarray in a study of striped bass egg quality has allowed us to detect differences in ovarian gene expression explaining and predicting most of the eventual variance in early embryo mortality among good and poor quality spawners.

Methods

Sample collection and preparation

Striped bass were reared in outdoor tanks at the N.C. State University Pamlico Aquaculture Field Laboratory [60]. As the striped bass is a group synchronous, single clutch, iteroparous spawner, ovarian tissues were collected by dissection or through ovarian biopsy [61] from females whose most advanced clutch of oocytes/eggs represented one of several stages (≥ 3 females/stage) of oocyte growth (early primary growth oocytes, diameter 49-81 μm; late primary growth oocytes showing evidence of lipid droplet accumulation, diameter 162-184 μm; vitellogenic growth oocytes, diameter 558-764 μm [see:[62][63]]), oocyte maturation (post-vitellogenic and maturing oocytes, diameter > 900 μm), and atresia [64], and ovulated eggs. All samples were preserved in RNAlater® (Applied Biosystems/Ambion; Austin, TX). Tissues were pooled in equal weight by oocyte/egg stage and total RNA was extracted in TRIzol® Reagent (Invitrogen; Carlsbad, CA). RNA quality was assessed by agarose gel electrophoresis and NanoDrop™ spectrophotometry (Fisher Scientific; Pittsburgh, PA). Dynabeads® (Invitrogen) were used to purify mRNA as described by the manufacturer.

cDNA library construction and sequencing

Ovary mRNA was submitted for cDNA synthesis at the N.C. State University Genomic Sciences Laboratory (Raleigh, NC). First and second strand cDNA was synthesized from 2.5 μg of Dnase treated mRNA using the SuperScript™ Double-Stranded cDNA Synthesis Kit (Invitrogen) and oligo (dT)17 according to the manufacturer. Approximately 2 μg of cDNA was prepared for FLX sequencing using standard Roche protocols [65]. Briefly, cDNA was nebulized to generate fragments averaging ~500 bp in length, fragment ends were repaired, and adapters containing PCR and sequencing primer annealing sites were ligated. Fragments were immobilized on beads, clonally amplified and then sequenced on a 1/2 plate using standard FLX platform (Roche; Indianapolis, IN).

Sequence assembly and annotation

Short reads were assembled into contigs using Roche's Newbler software (gsAssembler) with default settings except that the minimum overlap was set to 30 bp. Parameters were set to generate files for large contigs (> 500 bp) and for all contigs > 100 bp. High quality contig assemblies were subjected to BLAST (blastx) [66] of the NCBI database and annotated according to the Gene Ontology Consortium [67] using Blast2GO 2048 M version 12.2.0 [10,68,69]. Parameters for blastx were: Expect value 1.0E-3 and HSP Length Cutoff 33. Parameters for the GO annotations were: E-value-hit-filter 1.0E-6, Annotation Cutoff 55, GO Weight 5, and HSP-Hit Coverage Cutoff 0. Combined GO graphs for the annotated sequences (4,120 total) were created using percentages of 2nd level GO terms for Biological Process and 3rd level GO terms for Molecular Function and Cellular Component. Represented GO classes were restricted to those with 50 or more entities (sequence cutoff = 50.0); Sequence Filter = 50, Score alpha = 0.6, Node Score Filter = 10. Parameters for the Combined Graphs, Level Pie Configuration were: Ontology Level = Level 2 or 3 as described above.

Estimation of abundant gene transcripts

Contigs that were assembled from a number of ESTs comprising ≥ 0.15% of the total 230,151 short reads (i.e. those having ≥ 345 reads per contig) were considered to be abundant [see: [38]]. These contigs were ranked by relative abundance and compared to zebrafish orthologues shown to be ovary predominant by NCBI UniGene DDD [70], see: [15,44]. Zebrafish EST libraries were used to determine relative representation by DDD of orthologous UniGene clusters in ovary (104, 986 ESTs; Lib.IDs 20503, 15519, 20772, 20502, 19214, 15930, 9874, 9767) and body tissues excluding gonads (714, 604 ESTs; Lib.IDs 1520, 1521, 15438, 1028, 17704, 17768, 19753, 1522, 19745, 19746, 20694, 20725, 15518, 21372, 19747, 19748, 4913, 9766, 21371, 19741, 19749, 20771, 19739, 19740, 10504, 19737, 13027, 1029, 17276, 15077, 19752, 15517, 2387, 17282, 17284, 19738, 9968, 9993, 14182, 14249, 19217, 24670, 20072, 20071, 19253, 19219, 19218, 19215, 17283, 17275, 14410, 14409, 13866, 12106, 9706, 4264, 1727). Libraries with sequences derived from embryos, larvae, or whole bodies including gonads were excluded. The Fisher's exact test (FET) was used to determine difference between the number of times sequences from the ovary or body libraries were assigned to a specific UniGene cluster (P ≤ 0.05). Numerical DDD scores of genes with significantly different expression profiles were reported as the fraction of sequences within the EST libraries that mapped to the UniGene cluster.

Availability of supporting data

The data sets supporting the results of this article are available in the National Center for Biotechnology Information repository, Short Read Archive: SRX007394 and the National Animal Genome Research Program Aquaculture Genome Projects repository, http://www.animalgenome.org/aquaculture/database/.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

BJR conducted the sample preparation, DDD statistical analyses, and drafted the manuscript. JES performed the FLX pyrosequencing and contig sequence assemblies. RWC performed the GO annotations. RWC, EHS, and CHO participated in design of the study and critical review of the manuscript. CVS conceived the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.

Supplementary Material

Additional file 1

Striped bass ovary contig assemblies in FASTA format.

Click here for file (1.8MB, ZIP)
Additional file 2

Striped bass ovary contig assemblies identified by their annotations in FASTA format.

Click here for file (1.6MB, ZIP)
Additional file 3

List of striped bass ovary contig assemblies and their GO terms.

Click here for file (964.1KB, TXT)

Contributor Information

Benjamin J Reading, Email: bjreadin@ncsu.edu.

Robert W Chapman, Email: ChapmanR@dnr.sc.gov.

Jennifer E Schaff, Email: jeschaff@ncsu.edu.

Elizabeth H Scholl, Email: scholl@pngg.org.

Charles H Opperman, Email: warthog@ncsu.edu.

Craig V Sullivan, Email: craig_sullivan@ncsu.edu.

Acknowledgements

The authors are indebted to Zhanjiang (John) Liu (Auburn University) and Zhiliang Hu (Iowa State University) for organizing the online transcriptome posted to the National Aquaculture Genome Project (NAGP) webpage. We thank Andy S. McGinty and Michael S. Hopper (N.C. State University Pamlico Aquaculture Field Laboratory) for care and maintenance of the striped bass and Marion Beal (The Hollings Marine Laboratory) for posting the short read archive to NCBI. This is an NAGRP Aquaculture Genome (NRSP-8) Project and one of the authors (CVS) is the striped bass NRSP-8 species coordinator. This work was supported by research grants R/AF-46 and MG-XX from the North Carolina Sea Grant Program to C.V.S. and by a special grant NC09211 from the U.S. Department of Agriculture (USDA) National Institute of Food and Agriculture (NIFA) to C.V.S. and three other Co-principal investigators.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional file 1

Striped bass ovary contig assemblies in FASTA format.

Click here for file (1.8MB, ZIP)
Additional file 2

Striped bass ovary contig assemblies identified by their annotations in FASTA format.

Click here for file (1.6MB, ZIP)
Additional file 3

List of striped bass ovary contig assemblies and their GO terms.

Click here for file (964.1KB, TXT)

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