Molecular cloning and mRNA expression pattern of Sox9 during sex reversal in orange-spotted grouper ( Epinephelus coioides)

Abstract

Sox9 is a key gene in male sex determination and gonad development. To study its potential function in the female-to-male sex reversal in orange-spotted grouper, we conducted the following studies. The Sox9 gene was cloned and full-length sequence of Sox9 mRNA was determined using the rapid amplification of cDNA ends method. The Sox9 mRNA consists of 3277 bp in size with a 328 bp 5′ untranslated region and a 1511 bp 3′ untranslated region. The 1437 bp opening reading frame encodes a 479 amino acid protein. The Sox9 gene contains 3 exons and 2 introns; the beginning and end of both introns conform to the “GT-AG” rule. RT-PCR analysis indicated that Sox9 mRNA was expressed in brain, kidney, heart, liver, muscle, stomach, intestine, spleen, testis and ovary in adult orange-spotted grouper. Artificial sex reversal was successfully performed by implanting a medicinal strip containing 17α-methyltestosterone into the groupers. By real-time PCR, we found that Sox9 was weakly expressed in the ovary-stage gonads (before treatment). Once the sex reversal began (1 week after treatment), Sox9 mRNA expression level significantly increased. However, at 2 weeks after treatment, Sox9 mRNA expression level significantly decreased to a level that was only slightly higher than that before treatment. Sox9 mRNA expression increased again at 4 weeks after treatment, and at 6 and 8 weeks, it was maintained at a high level close to that at 1 week after treatment. The results suggest that Sox9 may be one of the important factors initiating and maintaining the masculinization of orange-spotted grouper during sex reversal.