EXPRESSION AND FUNCTIONAL ANALYSIS OF GENES AND PROTEINS INVOLVED IN OOCYTE MATURATION OF THE GIANT TIGER SHRIMP Penaeus monodon

Abstract

Identification and characterization of genes and proteins differentially expressed in ovaries is necessary for understanding mechanisms involving ovarian developmental processes of the giant tiger shrimp (Penaeus monodon). The full-length cDNA of PmRpd3, PmCdc16, PmCdk2 and PmCdk5 were characterized. They were 1949, 2068, 1763 and 1758 bp in length with an ORFs of 1452, 1332, 921 and 1524 bp corresponding to polypeptides of 483, 443, 306 and 507 amino acids, respectively. Quantitative real-time PCR indicated that the expression level of PmCdc2, PmCdk2, PmCdk7, PmChk1, PmBystin1, and PmRpd3 were more abundantly expressed in ovaries of broodstock than juveniles (P < 0.05) while the expression of PmCdc16 was in the opposite direction (P < 0.05). Eyestalk ablation promoted the expression level of PmCdc2, PmCdk7, PmChk1 and PmBystin1 (P < 0.05) but resulted in a decreased expression level of PmCdk2 and PmRpd3 (P < 0.05) relative to that in intact broodstock. However, it had no effect on the expressed profile of PmCdc16. Expression level of PmBystin1 in ovaries of 18-month-old P. monodon upon 5-HT injection (5-HT, 50 µg/g body weight) were significantly increased at 6 - 48 hours post injection (hpi, P < 0.05). PmCdc2 was immediately up-regulated at 1 hpi (P < 0.05) and its expression returned to the normal level at 3-72 hpi. The expression level of PmCdk7 was increased at 6 - 12 hpi (P < 0.05) and reduced to the previous level at 24 - 48 hpi. However, the expression level of PmCdc2 in cultured ovarian explants was not affected by different concentrations of 5-HT and 17α, 20β-DHP treatment (P > 0.05). In situ hybridization indicated that PmCdc2 and PmCdk7 were localized in ooplasm of previtellogenic oocytes in both intact and eyestalk-ablated broodstock while PmBystin1 was localized in the ooplasm of previtellogenic and vitellogenic oocytes but not in more mature stages of oocytes. Recombinant (r) PmApc11, rPmBystin1, rPmCdc2, rPmCdc20, rPmCdk7, rPmChk1 and rPmRpd3 were successfully expressed in vitro. Polyclonal antibodies against these proteins except PmApc11 and PmChk1 were successfully produced in rabbit. PmBystin1 (52 kDa) was expressed in stages II-IV and after spawning in both intact and eyestalk-ablated. PmCdk7 (67 kDa) was differential expressed in stages II-IV ovaries when compared with stage I ovaries in intact broodstock but it was comparably expressed among different ovarian developmental stages in eyestalk-ablated broodstock. This protein was not observed in premature ovaries of juveniles. In addition, western blot analysis revealed the expected 34 kDa band (PmCdc2) along with a smaller band of 23 kDa (ribosomal protein S3) in all stages of ovaries. Using phospho-Cdc2 (Thr161) polyclonal antibody, the positive signal of 34 kDa was observed in all ovarian stages but the most intense signal was found in stage IV ovaries. Immunofluorescence revealed the positive signals of PmCdk7 in ooplasm of previtellogenic oocytes and its subsequent nucleo-cytoplasmic translocation during oocyte development. Moreover, Anti-Phospho-Cdc2 (Thr161) PcAb gave the positive imunoreactive signals in ooplasm of follicular cells, previtellogenic and vitellogenic oocyes and around cortical rods of nearly mature and mature oocytes in both intact and eyestalk-ablated shrimp.