Analysis of differential expression and characterization of PIN in the gonads during sex reversal in the red-spotted grouper

The red-spotted grouper, Epinephelus akaara, is a protogynous hermaphroditic fish that shows the characteristic of natural sex change. In this study, 2-year-old female groupers were successfully reversed to functional males by oral administration of 17α-methyltestosterone (MT) for 42 days. The protein inhibitor of the neuronal nitric oxide synthase (PIN) gene was cloned from sex-reversed male gonads using modern suppression subtractive hybridization (SSH), cDNA synthesis and rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR). The full-length cDNA of PIN is 499 bp containing a 270 bp open reading frame (ORF) that encodes 89 amino acids. Virtual Northern blotting and reverse transcription-PCR (RT-PCR) analysis revealed that PIN was specifically transcribed in sex-reversed male gonads. Tissue-specific expression analysis showed that PIN gene was expressed in the brain, heart, liver, spleen, and kidney but not in the muscle tissue. Analyses of the expression pattern by RT-PCR and Western blotting indicated that transcription and the level of expression of PIN in the gonads increased gradually during the transformation from female to male. The results showed that PIN is strongly expressed in the sex-reversed male gonad but scarcely in the female gonad, and that its expression is upregulated as the change of sex proceeds. Taken together, these findings demonstrate that PIN is associated with the MT-induced sex transition of the red-spotted grouper, but the precise role of the gene in this process remains to be further investigated.