EFFECTS OF HORMONES ON EXPRESSION OF REPRODUCTION-RELATED GENES OF THE GIANT TIGER SHRIMP Penaeus monodon

Abstract

Knowledge on molecular mechanisms and functional involvement of reproduction-related genes in ovarian development are necessary for better understanding the reproductive maturation of P. monodon in captivity. Here is the full-length cDNA of P. monodon ring finger protein 121 (PmRnf121). Effects of hormone and neurotransmitter administration on expression levels of PmRnf121, non-receptor tyrosine kinase (PmnRTK) and receptor tyrosine kinase (PmRTK) were also examined. The full-length cDNA of PmRnf121 was characterized by RACE-PCR and it contained a complete ORF of 1023 bp in length deducing to a polypeptide of 341 amino acids. The partial cDNA sequence of PmRTK containing the ORF of 587 bp in length deducing to 195 amino acids was also isolated.

The expression levels of PmRnf121, PmnRTK and PmRTK during ovarian development were examined by quantitative real-time PCR. In intact broodstock, PmRnf121 was not differentially expressed during ovarian development of wild P. monodon (P > 0.05) but PmnRTK and PmRTK were differentially expressed during ovarian development of wild P. monodon (P < 0.05). Eyestalk ablation did not affect the expression level of PmRnf121 (P > 0.05) but promoted the expression level of PmnRTK in stages IV and post-spawning ovaries and that of PmRTK in the post-spawning ovaries (P < 0.05).

Effects of exogenous injection of hormones and neurotransmitters were determined. Serotonin injection (50 mg/g BW) did not affect the expression level of PmRnf121 (P > 0.05) but resulted in up-regulation of PmnRTK (at 6 hpi) and PmRTK (at 6 and 48 hpi) (P < 0.05). Progesterone (0.1 µg/g BW) did not affect to expression levels of these genes (P > 0.05). Exogenous injection of 17ß-estradiol (0.01 µg/g BW) increased the expression levels of PmRnf121 at 28 dpi compare to the negative control (P < 0.05) but it did not affect PmnRTK and PmRTK expression (P > 0.05). Interestingly, the expression levels of PmnRTK and PmRTK in eyestalk-ablated group were significantly induced at 28 dpi (P < 0.05).

Moreover, shrimp were fed with diets supplemented with 17ß-estradiol (1 and 10 mg/kg) and the diets affected the expression levels of PmRnf121 and PmRTK at 28 and 35 days of treatment compared to the negative control (P < 0.05). In contrast, the expression level of PmnRTK was lower than that of the negative control at 7 and 35 days of treatment (P > 0.05).

Recombinant clones for expression of PmRnf121 and PmRTK were constructed. The former was not expressed but the latter was expressed in the insoluble form in E. coli (23.83 kDa). Western blot analysis of total ovarian proteins was carried out against commercially available anti-Rnf121 MAb but results need to be confirmed by mass spectrometry of the positive immunoreactive band (58 kDa).