Identification of genes related to ovarian development of the giant tiger shrimp Penaeus monodon

Abstract

Reproduction-related genes in ovaries of the giant tiger shrimp (Penaeus monodon) were identified and characterized. A total of 4560 clones (2330 clones from typical, 1778 clones from normalized and 452 clones from subtraction ovarian cDNA libraries) were unidirectional sequenced and 2167 (47.5%) ESTs significant matched known genes previously deposited in the GenBank (E-value < 10-4) were obtained. TSP (452 clones, 9.9% of ESTs sequenced) and peritrophin (470 clones, 10.3%) predominated among known transcripts. Sequence assembly of all ESTs resulted in 441 contigs and 1231 (27.0%) singletons. RNA-arbitrary primed (RAP)-PCR was carried out for identification of differentially expressed transcripts during ovarian development of P. monodon. Fourteen candidate RAP-PCR markers were cloned and sequenced. Seven genes (B cell RAG associated protein, hypothetical protein W02B8.3, DNA replication licensing factor MCM5, APEX nuclease and U5 snRNP-specific protein, ABC transporter and moira CG18740-PA) showed a trend of differential expression between different ovarian developmental stages. Semiquantitative RT-PCR indicated that the expression level of B cell RAG associated protein and APEX nuclease at stage I ovaries was greater than that of other stages (P < 0.05). In contrast, U5 snRNP-specific protein was down-regulated at stage IV ovaries (P < 0.05). Accordingly, these transcripts should functionally contribute in ovarian development of P. monodon. The full length cDNA of SARIP, PGMRC1, p23 and Cdc42 transcripts and other functional important transcripts; phosphatidyl serine receptor (PSR), bystin, chk1, carbonyl reductase, protein disulfide-isomerase precursor (PDI) (prolyl 4-hydroxylase subunit beta) (cellular thyroid hormone-binding protein) (p55) (Erp59), gonadotropin-regulated long chain acyl-CoA synthetase, Cdc20, protein mago nashi, actin depolymerizing factor, f-box and wd-40 domain protein, cyclin-dependent kinase 7, selenoprotein M precursor and anaphase promoting complex subunit 11) were successfully identified by RACE-PCR or sequencing of the original cDNA clones. Quantitative real-time PCR indicated that Pm-p23 was up-regulated at the early stage (P < 0.05) whereas Cdc42 was down-regulated at stage III (P < 0.05) of ovarian development. Eyestalk ablation did not alter the expression pattern of Pm-p23 (P > 0.05) but caused increasing levels of Pm-Cdc42 at stage III and IV ovaries of P. monodon (P < 0.05). Pm-SARIP and Pm-PGMRC1 were differentially expressed in ovaries of normal broodstock (P < 0.05). Eyestalk ablation resulted in earlier and greater upregulation of these genes in ovaries of eyestalk ablated broodstock than normal broodstock (P < 0.05). This indicated that expression of Pm-SARIP and Pm-PGMRC1 was influenced by the downstream effects of GIH. Results suggested that Pm-SARIP and Pm-PGMRC1 may be used as bioindicators for monitoring progression of oocyte maturation as a consequent effects of maturation-inducing feed, hormone and/or neurotransmitter administration in P. monodon broodstock. In situ hybridization and immunohistochemistry were applied to examine localization of mRNA and proteins during ovarian development of P. monodon. All transcripts (Pm-Cdc42, Pm-PGMRC1, Pm-SARIP and Pm-p23) were localized in the ooplasm of previtellogenic oocytes. Immunohistochemistry revealed positive signals of the Pm-PGMRC1 protein only in the follicular layer and cell membrane of follicular cells. Nevertheless, the false positive signal was also detected when tissue sections were incubated with normal sera. Pm-Cdc42 was detected in oogonia, cytoplasm of previtellogenic oocytes and follicular layer. Recombinants Cdc42 and p23 proteins were successfully expressed in vitro. Polyclonal antibodies of these recombinant proteins were successfully produced. Western blot analysis indicated that the level of p23 was increased at the previtellogenic stage and decreased as oocyte development progressed. Results confirmed functional important of p23 gene products during oogenesis of P. monodon.