Open Access

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

  • Benjamin J Reading1,
  • Robert W Chapman2,
  • Jennifer E Schaff3,
  • Elizabeth H Scholl4,
  • Charles H Opperman4 and
  • Craig V Sullivan1, 5Email author
Contributed equally
BMC Research Notes20125:111

DOI: 10.1186/1756-0500-5-111

Received: 25 October 2011

Accepted: 21 February 2012

Published: 21 February 2012

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, 912]. 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) [1316], 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 [2325]. 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 [2730]. 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.
https://static-content.springer.com/image/art%3A10.1186%2F1756-0500-5-111/MediaObjects/13104_2011_Article_1417_Fig1_HTML.jpg
Figure 1

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

    

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, 1320, 27, 28, 3638]]; 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) [4649], 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

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/.

Notes

Declarations

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.

Authors’ Affiliations

(1)
North Carolina State University, Department of Biology
(2)
South Carolina Department of Natural Resources
(3)
North Carolina State University, Genomic Sciences Laboratory
(4)
North Carolina State University, Department of Plant Pathology
(5)
Department of Biology, North Carolina State University

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