MOLECULAR CLONING, EXPRESSION ANALYSIS AND POLYCLONAL ANTIBODY PREPARATION OF ANTI-MÜLLERIAN HORMONE (amh) GENE IN LARGEMOUTH BASS MICROPTERUS SALMOIDES

In order to study the characteristics of amh gene in largemouth bass Micropterus salmoides and its potential role during gonadal development, the cDNA full-length sequence of the amh gene was cloned by RACE technology, and the Amh polyclonal antibody was prepared. Quantitative real-time PCR (qRT-PCR) and Western Blot technology was used to analyze the expression pattern of amh in different tissues and different developmental stages. Finally, HE staining and immunohistochemistry were used to observe the morphological and histological changes of gonads at different developmental stages and their potential relationship with Amh expression. The results showed that the cDNA full-length sequence of the amh gene in largemouth bass was 2050 bp in length, including 24 bp at the 5′-UTR, 394 bp at the 3′-UTR, and a 1632 bp open reading frame (ORF) encoding 543 amino acids. The amh mRNA was found expressed in eleven tissues. The expression level in testis was the highest for male fish, then followed by muscle, and the expression level in ovary was the highest for female fish, followed by muscle. There were significant differences in the expression of amh gene in gonads of male and female fish at different developmental stages, and the expression in testis was significantly higher than that in ovary (P<0.05). Western Blot showed that Amh protein was highly expressed in testis. The expression in testis increased first and then decreased, and reached the highest after hatching 65d (P<0.05). Immunohistochemical results showed that Amh was expressed in Sertoli cells of early testis; the expression in male testis decreased significantly at 135d and 215d after hatching. HE staining showed that the testis of male fish was in the stage of spermatogenesis and sperm deformation, Amh was mainly expressed in Sertoli cells. Sperm occupied most of the space of testis, and the proportion of Sertoli cells decreased. The expression was low in female ovaries at 45d, 65d and 135d after hatching, and increased at 215d after hatching, and Amh positive signals were detected in the edge of oocyte membrane and peripheral follicular cells and granulosa cells. The results showed that Amh gene may be involved in the early testicular development in testis; in the ovary, it may participate in the development of granulosa cells and follicular cells. In conclusion, Amh played a key regulatory role in gonadal development of Micropterus salmoides.