ReproArray(GTS): a cDNA microarray for identification of reproduction-related genes in the giant tiger shrimp Penaeus monodon and characterization of a novel nuclear autoantigenic sperm protein (NASP) gene.

Expressed sequence tags (ESTs) were established from various tissues of the giant tiger shrimp (Penaeus monodon). To simultaneously examine expression patterns of a large number of transcripts in ovaries and testes of P. monodon, a cDNA microarray (ReproArray(GTS)) containing 4992 features amplified from cDNAs of ovary (1920) and testis (3072) EST libraries was constructed and subjected to high-throughput gene expression analysis in four different stages of ovarian development (previtellogenesis, vitellogenesis, early cortical rod and late cortical rod stages). Several transcripts were found to be differentially expressed during P. monodon ovarian development. Among many important reproduction-related genes with differential expression from microarray data, nuclear autoantigenic sperm protein (NASP) was further characterized by RACE-PCR. The full-length cDNA of P. monodon NASP (PmNASP) was 2126 bp in length containing an open reading frame (ORF) of 1812 bp corresponding to a deduced protein of 603 amino acids with 5? and 3?UTRs of 93 and 202 bp (excluding the poly A tail), respectively. Higher PmNASP transcript levels at later stages of ovarian development was consistently confirmed by quantitative real-time PCR. This study indicated that ReproArray(GTS) is effective for high-throughput screening of genes that play important roles in ovarian development of P. monodon.

Identification, characterization and expression of sex-related genes in testes of the giant tiger shrimp Penaeus monodon.

Isolation and characterization of genes involving gonadal development are an initial step towards understanding reproductive maturation and sex determination of the giant tiger shrimp (Penaeus monodon). In the present study, 896 clones from the testis cDNA library were sequenced. A total of 606 ESTs (67.6%) significantly matched sequences in the GenBank (E-value <1e-04) whereas 290 ESTs (32.4%) were newly unidentified transcripts. The full length cDNA of genes functionally involved in testicular development including cyclophilin A (PMCYA), small ubiquitin-like modifier 1 (PMSUMO-1), ubiquitin conjugating enzyme E2, dynactin subunit 5, cell division cycle 2 (cdc2) and mitotic checkpoint BUB3 were discovered. In addition, Tra-2, a gene involving sex determination cascades, was successfully characterized by RACE-PCR and first reported in crustaceans. Expression analysis indicated that a homologue of low molecular weight neurofilament protein XNF-L (termed P. monodon testis-specific transcript 1, PMTST1; N=8 for each sex) was only expressed in testes but not ovaries. PMCYA, thyroid hormone receptor-associated protein complex 240 kDa component (Trap240), multiple inositol polyphosphate phosphatase 2 (MIPP2) and heat shock-related 70 kDa protein 2 (HSP70-2), but not PMSUMO-1, PMTra-2 and prohibitin2 were differentially expressed between ovaries and testes of P. monodon. Expression of PMTST1 was up-regulated but that of the remaining genes in testes of P. monodon broodstock was down-regulated after shrimp were molted (P<0.05). Significant reduction of PMSUMO-1 and increment of prohibitin2 transcripts in domesticated broodstock (P<0.05) suggested that these reproductively related genes may be used as biomarkers to evaluate reduced degrees of the reproductive maturation in domesticated P. monodon.

Expressed sequence tag analysis for identification and characterization of sex-related genes in the giant tiger shrimp Penaeus monodon.

Sex-related genes expressed in vitellogenic ovaries of the giant tiger shrimp, Penaeus monodon, were identified by an EST approach. A total of 1051 clones were unidirectionally sequenced from the 5 terminus. Nucleotide sequences of 743 EST (70.7%) significantly matched known genes previously deposited in the 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. Thrombospondin (TSP) and peritrophin (79 and 87 clones accounting for 7.5 and 8.3% of clones sequenced, respectively) predominated among characterized transcripts. Several full length transcripts (e.g. cyclophilin, profillin and thioredoxin peroxidase) were also isolated. A gene homologue encoding chromobox protein (PMCBX, ORF of 567 nucleotides encoding a protein of 188 amino acids) which is recognized as a new member of the HP1 family was identified. Expression patterns of 14 of 25 sex-related gene homologues in ovaries and testes of P. monodon broodstock were examined by RT-PCR. Female sterile and ovarian lipoprotein receptor homologues were only expressed in ovaries whereas the remaining transcripts except disulfide isomerase related P5 precursor and adenine nucleotide translocator 2 were higher expressed in ovaries than testes of P. monodon broodstock. A homologue of ubiquitin specific proteinase 9, X chromosome (Usp9X) revealed a preferential expression level in ovaries than testes of broodstock-sized P. monodon (N = 13 and 11, P <0.05) but was only expressed in ovaries of 4-month-old shrimp (N = 5 for each sex).