Isolation and characterization of genes functionally involved in ovarian development of the giant tiger shrimp Penaeus monodon by suppression subtractive hybridization (SSH).

Suppression subtractive hybridization (SSH) libraries between cDNA in stages I (previtellogenic) and III (cortical rod) ovaries of the giant tiger shrimp (Penaeus monodon) were established. In all, 452 ESTs were unidirectionally sequenced. Sequence assembly generated 28 contigs and 201 singletons, 109 of which (48.0%) corresponding to known sequences previously deposited in GenBank. Several reproduction-related transcripts were identified. The full-length cDNA of anaphase promoting complex subunit 11 (PmAPC11; 600 bp with an ORF of 255 bp corresponding to a polypeptide of 84 amino acids) and selenoprotein Mprecursor (PmSePM; 904 bp with an ORF of 396 bp corresponding to a polypeptide of 131 amino acids) were characterized and reported for the first time in penaeid shrimp. Semiquantitative RT-PCR revealed that the expression levels of PmSePM and keratinocyte-associated protein 2 significantly diminished throughout ovarian development, whereas Ser/Thrcheckpoint kinase 1 (Chk1), DNA replication licensing factor mcm2 and egalitarian were down-regulated in mature ovaries of wild P. monodon (p < 0.05). Accordingly, the expression profiles of PmSePM and keratinocyte-associated protein 2 could be used as biomarkers for evaluating the degree of reproductive maturation in domesticated P. monodon.

Introduction

The giant tiger shrimp (Penaeus monodon) is one of the most economically important cultured species (Bailey-Brock and Moss, 1992; Rosenberry, 2001). Breeding P. monodon in captivity, besides being difficult (Withyachumnarnkul ; Wongprasert ), is very much restricted by the current dependency on wild-caught broodstock, with the consequential overexploitation of high-quality sources in the wild. As a result, aquacultural production of P. monodon has undergone a significant decline over the last several years (Limsuwan, 2004).

The low degree of reproductive maturation of captive P. monodon has also limited the ability to genetically improve this important species by domestication and selective breeding programs (Withyachumnarnkul ; Kenway ; Preechaphol ). Eyestalk ablation is used commercially to induce ovarian maturation in penaeid shrimp but the technique leads to an eventual loss in egg quality and death of the spawners (Benzie, 1998). Therefore, predictable maturation and spawning of captive penaeid shrimp without the use of eyestalk ablation is a long-term goal for the industry (Quackenbush, 1992).

Basic information on ovarian development is somewhat limited in this shrimp. Initial steps towards an understanding of the molecular mechanisms involved in ovarian and oocyte development in this economically important species, are the identification and characterization of genes differentially expressed in the diverse stages of the process (Preechaphol ).

Recently, genes expressed in the shrimp's vitellogenic ovaries were identified and characterized. A total of 1051 clones from a conventional cDNA library were unidirectionally sequenced from the 5' terminus. The nucleotide sequences of 743 EST (70.7%) significantly matched known genes previously deposited in GenBank (E-value < 10-4), whereas 308 ESTs (29.3%) were regarded as newly unidentified transcripts (E-value > 10-4). A total of 559 transcripts (87 contigs and 472 singletons) were obtained after sequence assembly. Several reproduction-related genes, viz., chromobox protein, ovarian lipoprotein receptor, progestin membrane receptor component 1 and ubiquitin-specific proteinase 9, X chromosome, were isolated and characterized (Preechaphol ).

Suppression subtractive hybridization (SSH) is widely used for isolating differentially expressed genes in any two closely related samples, specimens or species (Diatchenko ). This technique should facilitate the identification of genes involved in ovarian (and oocyte) development. The genes identified could further assist in the domestication and selective breeding programs of P. monodon.

In order to provide a further insight into the molecular mechanisms involved in the reproductive maturation processes of P. monodon, we carried out SSH of genes expressed in stages I and III ovaries of wild P. monodon. The expression profiles of five reproduction-related genes during ovarian development in wild P. monodon broodstock were further examined using semiquantitative RT-PCR. Candidate biomarkers for evaluating the degrees of reproductive maturation in captive shrimp are reported herein.

Materials and Methods

Experimental animals

Four-month-old juveniles of P. monodon, with body weights of approximately 25-30 g, were purchased from a commercial farm in Chachoengsao (eastern Thailand). These were cultured in 15 ppt seawater at ambient temperature (28-32 °C) and a natural daylight cycle. Broodstock shrimp, with body weights of > 200 g, were wild-caught from Satun, located in the Andaman Sea, west of peninsular Thailand. Prior to SSH library construction, ovaries were dissected out from two broodstock and weighed. The gonadosomatic index (GSI), i.e., ovarian weight/body weight x 100, of each shrimp was calculated. In order to determine expression profiles of reproduction-related genes during P. monodon ovarian development, female juveniles and the broodstock were acclimated at normal farm conditions (28-30 °C, natural daylight and 35 ppt seawater) for 2-3 days. Ovarian developmental stages of broodstock were classified according to GSI: < 1.5, 2-4, > 4-6 and > 6% for previtellogenic (I), vitellogenic (II), early cortical rod (III) and mature (IV) ovaries (N = 4 for each stage), respectively. Ovaries were dissected from each shrimp immediately after collection and kept at -80 °C until use.

Isolation of total RNA and mRNA

Total RNA was extracted from various tissues of each individual with TRI-Reagent (Molecular Research Center) and mRNA was further purified using a QuickPrep Micro mRNA Purification Kit (GE Healthcare). Total RNA and mRNA were kept under absolute ethanol at -80 °C, prior to reverse transcription.

Construction of suppression subtractive hybridization (SSH) cDNA libraries and EST analysis

Initially, two micrograms of mRNA from the ovaries of the P. monodon broodstock were reverse-transcribed. Suppression subtractive hybridization (SSH) between cDNA from stages III (GSI = 5.69%) and I (1.43%) and vice versa (Diatchenko ) was carried out using a PCR-Select cDNA Subtraction Kit (BD Clontech). The subsequent products were ligated to pGEM-T Easy vector and transformed into E. coli JM109. Plasmid DNA was extracted from clones carrying > 300 bp inserts and unidirectionally sequenced using the M13 reverse primer. Sequencing data were pre-processed to remove low-quality sequences (sequence length < 100 bp, the percentage of undetermined bases > 3% and low complexity), by using SeqClean with option-A to disable the trimming of poly A tail. Repetitive sequences matching the RepBase dataset were masked by using RepeatMasker. Sequence clustering and assembly was done using TIGR Gene-Indices Clustering Tools (TGICL) (Pertea ) with CAP3 (Huang and Madan, 1999). Nucleotide sequences of assembled and non-assembled ESTs were compared with GenBank data using BlastN and BlastX (Altschul ). Significantly matches to nucleotides/proteins were considered when the E-value was < 1 x 10-4. Blast2GO was used for the additional annotation of biological activities in BlastX matched sequences, thereby enabling Gene Ontology (GO) prediction of sequence data for which no GO annotation is, as yet, available (Conesa ).

ESTs representing P. monodonselenoprotein M precursor (PmSePM) and anaphase promoting complex subunit 11 (PmAPC11) were further sequenced from the reverse direction of the original cDNA clones by using a M13 forward primer.

Semiquantitative RT-PCR

Expression profiles of keratinocyte-associated protein 2, Ser/Thrcheckpoint kinase 1, DNA replication licensing factor mcm2, PmSePM and egalitarian during ovarian development of P. monodon broodstock were analyzed by way of semiquantitative RT-PCR. EF-1α was included as the positive control. Initially, nonquantitative RT-PCR (Klinbunga ) was carried out using 100 ng of first-strand cDNA as the template, with varying concentrations of primers (0.05, 0.10, 0.15, 0.20, 0.25, 0.30 and 0.40 μM, respectively). Primer sequences are listed in Table 1. Optimal concentrations of MgCl2 (1.0, 1.5, 2.0, and 3.0 mM) were further selected using an optimized primer concentration. Finally, RT-PCR of these genes was undertaken with an optimized primer and MgCl2 concentrations for 20, 22, 24, 27, 30 and 35 cycles. The number of cycles before the product reached an amplification plateau was chosen.

Table 1

Nucleotide sequences of primers used for expression analysis of keratinocyte-associated protein 2, Ser/Thrcheckpoint kinase 1, DNA replication licensing factor mcm2, selenoprotein M precursor and egalitarian in ovaries of wild P. monodon broodstock.

Gene	Primer sequence
Keratinocyte-associated protein 2	F: 5'-CTGCTGTAAACAATCTGGAAAAC-3'
	R: 5'-GGGACACCTGAGCGGAAGT-3'
Ser/Thrcheckpoint kinase 1 (Chk1)	F: 5'-CTCCCCAGTGTCCTTATTGATTAG-3'
	R: 5'-TGGCTTTCATTCCCTCGCTG-3'
DNA replication licensing factor mcm2	F: 5'-TCAAGCGAGACAACAACGAACT-3'
	R: 5'-TTGGACCATCACTGGGCATC-3'
Selenoprotein M precursor (PmSePM)	F: 5'-GACATCCCACTCTTCCATAAT-3'
	R: 5'-TTTCATCTACAGTTCTTCCCTC-3'
Egalitarian	F: 5'-CACTTGTGCCCACTGTCTATG-3'
	R: 5'-CCTCCACTGCCAACACTACTC-3'
EF-1α	F: 5'-ATGGTTGTCAACTTTGCCCC-3'
	R: 5'-TTGACCTCCTTGATCACACC-3'

Semiquantitative RT-PCR was undertaken with 1.5 mM of MgCl2 and 0.2 μM of primers for the respective target genes, 0.15 μM of primers for egalitarian and 0.10 μM of those for EF 1-α, as follows: 94 °C for 3 min followed by appropriate cycles (22, 27, 24, 22 and 24 cycles for the target genes and 22 cycles for EF 1-α, respectively) of 94 °C for 30 s, 53 °C for 45 s and 72 °C for 45 s and a final extension at 72 °C for 7 min. The amplicon was electrophoretically analyzed through 1.5% agarose gels, and visualized with a UV transilluminator after ethidium bromide staining (Sambrook and Russell, 2001). The intensities of the targets and EF-1α were quantified from the gel photograph using the Quantity One software (BioRad), and relative expression levels of investigated transcripts (intensity of targets/intensity of EF-1α) in all experimental groups of P. monodon were statistically tested using analysis of variance (ANOVA), followed by the Duncan's new multiple range test. Results were considered significant when p < 0.05. The ovaries from five groups of shrimp (juveniles and stages I, II, III and IV broodstock, N = 4 for each group) were assayed for expression analysis.

Results and Discussion

An understanding of the roles of genes functionally involved in ovarian and oocyte development should ultimately lead to a plausible approach for inducing reproductive maturation in P. monodon. In this study, 220 and 232 clones, respectively, from the forward (cDNAs from stage III ovaries as the tester and those from stage I ovaries as the driver; GenBank accession no. GW775090-GW775309) and reverse (cDNAs from stage I ovaries as the tester and those from stage III ovaries as the driver; GenBank accession no. GW775310-GW775541) SSH ovarian libraries of P. monodon were unidirectionally sequenced and 136 (61.8%) and 133 (57.3%) ESTs, respectively, significantly matched known sequences in GenBank (E-value < 10-4, Tables 2 and 3). Homologues of thrombospondin (TSP; 39 ESTs accounting for 17.7% and 26 ESTs accounting for 11.2% of sequenced clones) and peritrophin (39 ESTs, 17.7% and 27 clones, 11.6%) were abundantly represented in both libraries similar to results from analyses of the conventional cDNA library of vitellogenic ovaries of P. monodon (79 and 87 clones accounting for 7.5 and 8.3% of clones sequenced, respectively; Preechaphol ).

Table 2

Examples of known transcripts excluding ribosomal proteins found in the forward ovarian SSH library (cDNAs from stage III ovaries as the tester and those from stage I ovaries as the driver) of P. monodon.

Transcript*	Species	Accession number	E-value	Size (bp)
Peritrophin 2	Penaeus monodon	AAM44050.1	5 x 10-86	454
Peritrophin 1	Penaeus monodon	AAM44049.1	4 x 10-41	381
Thrombospondin	Penaeus monodon	AAN17670	1 x 10-107	563
Thrombospondin	Marsupenaeus japonicus	BAC92764.1	3 x 10-44	502
Keratinocyte-associated protein 2	Rattus norvegicus	NP_001099914.1	8 x 10-25	470
Eukaryotic translation initiation factor 2, subunit 2 beta	Rattus norvegicus	AAH62402.1	7 x 10-11	605
Ser/Thr Checkpoint kinase 1 (Chk1), CG17161-PA	Drosophila melanogaster	AAF53552	2 x 10-22	417
Methionyl-tRNA formyltransferase, mitochondrial precursor (MtFMT)	Homo sapiens	NP_640335	4 x 10-7	483
Nucleolin	Xenopus laevis	NP_001081557.1	8 x 10-4	380
Eukaryotic initiation factor eIF-4A	Marsupenaeus japonicus	BAB78485	1 x 10-41	279
26S proteasome regulatory subunit rpn2	Culex quinquefasciatus	XP_001862500	3 x 10-52	468
Cytochrome c oxidase polypeptide IV	Bombyx mori	NP_001073120.1	3 x 10-38	405
Hypothetical protein DKFZp434J1672.1	Homo sapiens	CAB63724	6 x 10-24	525
Coatomer protein complex, subunit beta	Gallus gallus	NP_001006467.1	1 x 10-67	588
Chaperonin containing T-complex polypeptide 1	Carassius auratus	BAA89277	8 x 10-44	627
ATP synthase oligomycin sensitivity conferral protein	Toxoptera citricida	AAU84928	3 x 10-9	538
Cyclin A	Asterina pectinifera	BAA14010	4 x 10-42	368
Non-muscle myosin-II heavy chain	Apis mellifera	XP_393334	8 x 10-99	712
Procollagen-proline, 2-oxoglutarate 4-dioxygenase (protein disulfide isomerase-associated 1)	Xenopus tropicalis	CAJ83276	2 x 10-47	663
Chaperonin containing TCP1, subunit 7	Danio rerio	NP_775355.1	4 x 10-24	249
Isocitrate dehydrogenase 2	Tribolium castaneum	EFA04299	1 x 10-37	231
CD53 antigen	Homo sapiens	NP_001035122.1	4 x 10-04	394
Calreticulin	Galleria mellonella	BAB79277	5 x 10-103	714
DNA replication licensing factor mcm2	Xenopus tropicalis	AAH75567	2 x 10-47	490
RNA binding motif protein 4	Aedes aegypti	XP_001657237.1	6 x 10-38	563
Domino isoform D, CG9696-PD	Apis mellifera	XP_396786	9 x 10-61	350
Eukaryotic translation initiation factor 2B, subunit 5 epsilon, isoform 3	Macaca mulatta	XP_001103944	5 x 10-32	713
Translation initiation factor	Anopheles gambiae	CAD27760.1	2 x 10-66	708
Secreted nidogen domain protein	Strongylocentrotus purpuratus	XP_001196268.1	8 x 10-09	466
Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase	Danio rerio	NP_001009884.1	4 x 10-18	611
DEAD (Asp-Glu-Ala-Asp) box polypeptide 5	Tribolium castaneum	XP_972501.1	3 x 10-07	354
Deleted in malignant brain tumors 1	Strongylocentrotus purpuratus	XP_001180356.1	2 x 10-04	486
ATPase, H+ transporting, lysosomal accessory protein 2, CG8444-PA	Tribolium castaneum	XP_973593.1	1 x 10-07	562
Kinesin-like protein 2	Ciona intestinalis	NP_001011659	5 x 10-04	449
Elongation factor-1 alpha	Libinia emarginata	AAC03149	3 x 10-102	713
Chromosome-associated protein, CG9802-PA, isoform A	Apis mellifera	XP_393700	2 x 10-74	652
CWF19-like 2, cell cycle control	Xenopus tropicalis	NP_001039121.1	1 x 10-58	600
Myosin II essential light chain	Tribolium castaneum	XP_973734	6 x 10-15	516
Gastrula zinc finger protein XLCGF57.1	Danio rerio	XP_001344037.1	4 x 10-30	568
SJCHGC09076 protein	Schistosoma japonicum	AAW26562	6 x 10-06	559
Citrate synthase	Aedes aegypti	EAT45772.1	4 x 10-75	478
Zinc finger protein 146	Strongylocentrotus purpuratus	XP_788425.2	2 x 10-20	654
Sec23 protein	Drosophila melanogaster	NP_730978.1	6 x 10-63	465
Elongation factor-2	Libinia emarginata	AAR01298	8 x 10-82	538
Hypothetical protein TTHERM_00449680	Tetrahymena thermophila	XP_001013363.1	2 x 10-10	506
Calreticulin	Bombyx mori	AAP50845.1	1 x 10-128	695
RNA-binding protein 5	Apis mellifera	XP_394165.3	4 x 10-43	713
Mitochondrial ATP synthase e chain	Aedes albopictus	AAV90734	9 x 10-16	403
Zgc:113377	Danio rerio	NP_001025397	4 x 10-29	697
Inhibitor of Bruton agammaglobulinemai tyrosine kinase	Canis familiaris	XP_539018.2	2 x 10-12	634

*Accession no. GW775090-GW775309 for ESTs from the forward SSH library.

Table 3

Examples of known transcripts excluding ribosomal proteins found in the reverse ovarian SSH library (cDNAs from stage I ovaries as the tester and those from stage III ovaries as the driver) of P. monodon.

Transcript*	Species	Accession number	E-value	Size (bp)
Peritrophin 1	Penaeus monodon	AAM44049.1	2 x 10-53	412
Peritrophin 2	Penaeus monodon	AAM44050.1	1 x 10-72	406
Thrombospondin	Penaeus monodon	AAN17670	3 x 10-63	368
Thrombospondin	Marsupenaeus japonicus	BAC92764.1	9 x 10-61	405
Translation initiation factor eIF4A	Spisula solidissima	AAK85401	1 x 10-47	326
CG10527-like methyltransferase	Mesobuthus gibbosus	CAE53527.1	1 x 10-28	458
Selenoprotein M precursor	Homo sapiens	NP_536355.1	7 x 10-24	560
Stress-70 protein, mitochondrial precursor (75 kDa glucose-regulated protein)	Gallus gallus	NP_001006147.1	1 x 10-26	577
Neuralized protein	Drosophila virilis	AAB60619.1	4 x 10-27	575
Secreted nidogen domain protein	Strongylocentrotus purpuratus	XP_001196268.1	3 x 10-6	480
Thioesterase superfamily member 2	Gallus gallus	XP_419092.1	3 x 10-13	511
Hypothetical protein MGC75603	Xenopus tropicalis	NP_989388	2 x 10-6	642
Carbonyl reductase	Plecoglossus altivelis	BAB92960	2 x 10-20	589
Ovarian lipoprotein receptor	Penaeus semisulcatus	AAL79675	4 x 10-17	618
Allatotropin neuropeptide precursor	Spodoptera frugiperda	CAD98809.1	6 x 10-9	402
Chitin deacetylase-like 9, CG15918-PA	Drosophila melanogaster	NP_611192.1	1 x 10-17	353
Replication factor C/activator 1 subunit	Gallus gallus	AAA20552.1	5 x 10-58	583
Nuclease diphosphate kinase B	Danio rerio	AAF60971	9 x 10-34	430
Acyl-CoA synthase	Oceanicola batsensis	ZP_01000658.1	9 x 10-51	518
70 kD heat shock-like protein	Procambarus clarkia	ABC01063	1 x 10-103	692
Signal sequence receptor	Bombyx mori	NP_001091760.1	3 x 10-04	600
ATP synthase, CG11154-PA isoform A	Apis mellifera	XP_624156	6 x 10-115	690
Ubiquitin-like 1 activating enzyme E1B (SUMO-1 activating enzyme subunit 2)	Strongylocentrotus purpuratus	XP_001195210.1	4 x 10-24	473
Ribonuclease P 40kDa subunit isoform 3	Macaca mulatta	XP_001095772	6 x 10-19	688
Selenophosphate synthetase(selenium donor protein)	Drosophila melanogaster	NP_725374.1	5 x 10-103	710
Peptidylprolyl isomerase D	Danio rerio	NP_001002065.1	1 x 10-24	589
Egalitarian	Drosophila melanogaster	AAF47054.4	3 x 10-37	704
CCR4-NOT transcription complex, subunit 10	Tribolium castaneum	XP_974052	2 x 10-29	585
Protein phosphatase 2c gamma	Aedes aegypti	EAT47444.1	2 x 10-56	711
RNA polymerase I associated factor 53 isoform 1	Canis familiaris	XP_531998	5 x 10-16	710
Splicing factor, arginine/serine-rich 7	Apis mellifera	XP_001122800	2 x 10-41	633
Interleukin enhancer binding factor 2	Mus musculus	NP_080650.1	4 x 10-31	332
Nuclear autoantigenic sperm protein	Danio rerio	NP_956076.1		700
Cyteine-rich with EGF-like domain 2, CG11377-PA	Tribolium castaneum	XP_971778.1	6 x 10-25	510
Eukaryotic initiation factor 4A	Callinectes sapidus	ABG67961	1 x 10-64	569
ATP lipid-binding protein like protein	Marsupenaeus japonicus	BAB85212	9 x 10-30	588
TRI1, CG7338-PA	Apis mellifera	XP_624169	3 x 10-41	708
Ferritin	Litopenaeus vannamei	AAX55641.1	3 x 10-31	306
Deleted in malignant brain tumors 1	Strongylocentrotus purpuratus	XP_001180356.1	2 x 10-05	713
Transmembrane 4 superfamily member 8 isoform 1/ Tetraspanin 3	Homo sapiens	NP_005715	3 x 10-10	596
Neutral alpha-glucosidase AB precursor (Glucosidase II subunit alpha)	Sus scrofa	NP_999069.1	2 x 10-49	707
Calreticulin precursor (CRP55) (Calregulin)	Oryctolagus cuniculus	NP_001075704.1	4 x 10-19	300
Ataxin1 ubiquitin-like interacting protein	Gallus gallus	NP_001026544	5 x 10-41	612
Hypothetical protein	Mus musculus	XP_922736.3	2 x 10-15	403
HLA-B-associated transcript 3	Apis mellifera	XP_001121013.1	8 x 10-25	261
Cyclin B3, CG5814-PA	Apis mellifera	XP_397108	6 x 10-46	427
Hypothetical protein cgd5_1220	Cryptosporidium parvum	EAK88123.1	2 x 10-08	460
Ring finger protein 2, CG15814-PA, isoform A	Tribolium castaneum	XP_975438.1	9 x 10-40	431
2-Cys thioredoxin peroxidase	Aedes aegypti	AAL37254	1 x 10-56	564

*Accession no. GW775310-GW775541 for ESTs from the reverse SSH library.

Relatively high percentages of unknown transcripts were found in both the forward and reverse SSH ovarian libraries of P. monodon (84 and 99 ESTs accounting for 38.2% and 42.7%, respectively; Tables 2 and 3). The percentage of unknown transcripts in these SSH libraries was greater than that in the conventional ovarian (308/1051 clones, 29.3%; Preechaphol ) and testicular (290/889 clones, 32.6%; Leelatanawit ) cDNA libraries but lower than those found in the forward (112/178 ESTs, 62.9%) and reverse (87/187 ESTs, 46.5%) SSH testicular libraries of P. monodon, respectively (Leelatanawit ).

After sequence assembly, 16 contigs (from 97 ESTs) and 123 singletons were obtained for the forward and 14 contigs (from 142 ESTs) and 90 singletons for the reverse SSH libraries, respectively. In all, 229 transcripts (28 contigs from 251 transcripts and 201 singletons, i.e., 44.5%) were obtained when both libraries were analyzed simultaneously, of which 109 significantly matched known genes in GenBank (E-value < 10-4). Disregarding contigs representing thrombospondin/peritrophin (8 contigs) and unknown proteins (12 contigs), 8 contigs matched ribosomal protein S6, elongation factor 1-α, elongation factor 2, calreticulin, ficolin, selenophosphate synthetase, 70 kDa heat shock-like protein and a hypothetical protein, AGAP006171-PA.

The percent distribution of nucleotide sequences, according to GO categories of SSH ovarian cDNA libraries of P. monodon, was analyzed (Figure 1). In the category `biological process', ESTs involved in metabolic processes were predominant (e.g.anaphase promoting complex subunit 11, S-adenosylmethionine synthetase and T-complex protein 1 subunit epsilon, i.e., 35.0% of the examined ESTs), followed by those involved in cellular processes (e.g.acidic p0 ribosomal protein, DNA replication licensing factor mcm2 and coatomer protein complex subunit beta, i.e., 25.2% of the examined ESTs). Reproduction-related ESTs (e.g RNA binding motif protein 4, neuralized protein, dynein and egalitarian) were found in 2.4% of the examined sequences of combined SSH data. This discovery rate is higher than that of the conventional ovarian cDNA libraries of P. monodon (1.7%; Preechaphol ).

Figure 1

The percent distribution of nucleotide sequences in the SSH ovarian cDNA library of P. monodon according to three principal GO categories: A, biological process; B, cellular components and C, molecular functions, respectively.

As for the category `cellular component', ESTs functionally involved in the cell part (e.g.myosin II essential light chain, ATP synthase E chain and Ser/Thr checkpoint kinase 1, i.e., 35.5% of the examined ESTs) predominated, followed by those functionally displayed in organelles (e.g.selenoprotein M precursor, keratinocyte-associated protein 2 and interleukin enhancer binding factor 2; 25.5% of the examined ESTs).

In the category `molecular function', ESTs involved in binding (e.g.carbonyl reductase, translation initiation factor eif-2b, RNA binding motif protein 5 isoform 9 and selenophosphate synthetase, i.e., 50.5% of the examined ESTs) predominated followed by those displaying catalytic activity (e.g.MGC80929 protein isoform 1, oncoprotein nm23 and eukaryotic initiation factor 4A, i.e., 30.5% of the examined ESTs).

The highly organized eukaryotic cilia and flagella contain approximately 250 proteins (Inaba, 2003). They are constructed around evolutionarily conserved microtubule-based structures called axonemes (nine outer doublet microtubules, dynein arms, a central pair of microtubules and radial spokes) (Luck, 1984; Dutcher, 1995; King, 2000). Dynein is functionally related to the transport of various cytoplasmic organelles (Aniento ). In Drosophila, egalitarian binds to the dynein light chain. Point mutations that specifically inhibit Egl-Dlc association disrupt microtubule-dependant trafficking both to and within the oocyte, thereby resulting in a loss of oocyte fate maintenance and polarity (Carpenter, 1994).

The physiological role of carbonyl reductase was thought to be an NADPH-dependent reduction in a variety of endogenous and foreign carbonyl compounds. However, increasing evidence indicates its involvement in steroid metabolism. In ayu, its localization in ovaries, enzymatic characteristics and transcriptional increase with oocyte maturation, infer its additional function as 20β-HSD in the production of maturation inducing hormones (MIH) (Tanaka ).

The DNA replication (or origin) licensing system is prominant in ensuring precise duplication of the genome in each cell cycle, besides being a powerful regulator of metazoan cell proliferation (Eward ). The protein kinase Chk1 plays a role in checkpoint control. Recombinant Xenopus Chk1 phosphorylates the mitotic inducer Cdc25 in vitro at multiple sites. Nevertheless, only XChk1-catalyzed phosphorylation of Cdc25 at Ser-287 is sufficient to confer the binding of 14-3-3 proteins (Kumagai ). Moreover, the meiotic maturation of oocytes is regulated by the maturation promoting factor (MPF), a complex of cdc2 (Cdk1), cyclin B and other Cdk/cyclin complexes (Kobayashi ; Kishimoto, 1999, 2003). Chk1-dephophorylated Cdc25 activates MPF, thereby causing meiotic resumption in oocytes (Kishimoto, 2003).

Recently, the full length cDNA of keratinocyte-associated protein 2 was isolated in the Pacific white shrimp (Litopenaeus vannamei), although the function of this protein is still unknown. Moreover, its expression was altered following infection by the White Spot Syndrome Virus, WSSV (Clavero-Salas ).

The full length cDNAs of anaphase promoting complex subunit 11 (biological process GO:0008152; GenBank accession no. GW775392) and selenoprotein M precursor (cellular component GO:0005783; GenBank accession no. GW775333) were hereby reported and identified for the first time in penaeid shrimp.

The anaphase promoting complex subunit 11 of P. monodon (PmAPC11) was 600 bp in length, and consisted of an ORF of 255 bp corresponding to a polypeptide of 84 amino acids, with 5' and 3' UTRs of 1 and 387 bp, respectively (Figure 2A). The closest similar sequence to PmAPC11 was the anaphase promoting complex subunit 11 homolog of Tribolium castaneum (E-value = 1 x 10-41). The predicted molecular mass and pI of the deduced PmAPC11 was 9.84 kDa and 7.99, respectively. Activation of the anaphase-promoting complex (APC) by Cdc20 enables anaphase initiation and exit from mitosis (Kramer ; Lorca ).

Figure 2

The full length cDNA and deduced protein sequences of PmAPC11 (600 bp, ORF of 255 bp corresponding to a deduced polypeptide of 84 amino acids; GenBank accession no. GW775392) and PmSePM (904 bp, ORF of 396 bp corresponding to a deduced polypeptide of 131 amino acids; GenBank accession no. GW775333). The putative start and stop codons are illustrated in boldface and underlined. The predicted signal peptide and poly A additional signals of PmSePM are underlined and italicized and underlined, respectively. The predicted Sep15_SelM domain (positions 31-107) found in the deduced PmSePM protein is highlighted.

The selenoprotein M precursor of P. monodon (PmSePM) was 904 bp in length, and consisted of an ORF of 396 bp, corresponding to a polypeptide of 131 amino acids, and 5' and 3' UTRs of 6 and 502 bp, respectively (Figure 2B). It significantly matched the selenoprotein M precursor of L. vannamei (E-value = 2 x 10-58). The predicted molecular mass and pI of the deduced PmSePM protein was 15.10 kDa and 7.75, respectively. PmSePM contained a signal peptide located between A21 and E22, as well as a Sep15_SelM domain (positions 31-107, E-value = 1.9 x 10-34) that exerts the thiol-disulphide isomerase activity functionally involved in disulphide bond formation of proteins in the endoplasmic reticulum (ER) (Ferguson ).

In addition, the EST representing selenophosphate synthetase, an enzyme involved in selenocysteine biosynthesis, was also identified. In humans, selenium deficiency leads to male infertility and susceptibility to viral infections. More than 20 selenoproteins have been identified in higher eukaryotes (Guimaraes ; Rayman, 2000; Korotkov ) but their functions in ovarian/oocyte development of P. monodon remain unknown. The analysis of baseline information, acquired as part of this study addresses the paucity of data and should provide a better understanding of reproductive maturation in cultured female P. monodon.

To address the functional involvement of various genes during ovarian development of P. monodon, the expression profiles of keratinocyte-associated protein 2, Ser/ThrChk1, DNA replication licensing factor mcm2, PmSePM and egalitarian were examined by semiquantitative RT-PCR analysis. The control gene (EF-1α) seemed to be comparably expressed in all the groups of samples examined, thereby inferring its acceptability for use in normalizing target gene expression. All transcripts were more abundantly expressed in the ovaries of broodstock than juveniles (p < 0.05, Figure 3). The expression level of PmSePM peaked in stage I (previtellogenic) of development (GSI < 1.5), to progressively and significantly decrease in stages II (vitellogenic), III (cortical rod) and IV (mature) (p < 0.05). Likewise, keratinocyte-associated protein 2 was initially down-regulated in stage III, and subsequently, stage IV (p < 0.05). The expression of Ser/ThrChk1, DNA replication licensing factor mcm2 and egalitarian during stages I, II and III, was comparable (p < 0.05), although down-regulated in the final stage of ovarian development in wild P. monodon broodstock (p < 0.05, Figure 3).

Figure 3

Histograms showing relative expression levels of keratinocyte-associated protein 2 (A), Ser/ThrChk1 (B), DNA replication licensing factor mcm2 (C), selenoprotein M precursor (PmSePM; D) and egalitarian (E) in different ovarian developmental stages of P. monodon. For expression analysis, ovaries from 5 groups of shrimp (juveniles and stages I, II, III and IV broodstock, N = 4 for each group) were assays. The same letters indicate that the relative expression levels were not significantly different (p > 0.05).

In various animals, a wide variety of maternal mRNA is generally transcribed at the early oogenesis stage, to then be stored in oocytes and carried into fertilized eggs (Qiu ; Nishimura ). Several reproduction-related genes that are up-regulated during the ovarian development of P. monodon, for example, Ovarian-Specific Transcript 1 (Pm-OST1) and cyclin B (PmCyB), have been previously reported (Klinbunga ; Visudtiphole ). The down-regulation of keratinocyte-associated protein 2, Ser/ThrChk1, DNA replication licensing factor mcm2, PmSePM and egalitarian implied that lower levels of these gene products may be necessary for the development and final maturation of P. monodon oocytes. The findings facilitate the possible use of RNA interference (RNAi) for studying their functional involvement in P. monodon ovarian development. Moreover, the expression profiles of keratinocyte-associated protein 2 and selenoprotein M precursor are potentially applicable as biomarkers to indicate degrees of reproductive maturation in the domesticated shrimp.

In this study, genes expressed in ovaries of P. monodon were identified by SSH analysis. The expression profiles of reproduction-related transcripts were examined. Further studies of the molecular mechanisms of those genes and proteins involved in controlling each stage of oocyte maturation should be carried out, to reach a better understanding of the reproductive maturation of P. monodon in captivity.