Morphological changes and regulation of the genes dmrt1 and cyp11b during the sex differentiation of barramundi (Lates calcarifer Bloch)

Elucidating teleost sex differentiation is not only important for a broad understanding of vertebrate sexual development, but also to aid sex control in aquaculture. Barramundi, Lates calcarifer, is an important aquaculture species which exhibits protandry, whereby fish mature initially as male and subsequently sex change to the female gonadal state several years later. This late female sex change impedes efficient breeding programs, as males and females of the same generation cannot be mated. In order to further understand how sex development is controlled and possibly manipulated in barramundi, the timing and process of testicular differentiation from hatching through to spermatogenesis was morphologically described by histological analyses. Furthermore, expression of two key male genes, dmrt1 and cyp11b were also examined using RT-qPCR. The barramundi proto-testes originates as a strip of cells attached to the dorsal coelemic cavity at 4 days post hatch (dph), with the formation of a slit-like lumen by 44 dph (Fish Total length (TL) 25.8 ± 3.3 mm) which is the first morphological indication of testicular differentiation in the species. This slit-like lumen later develops into the testes efferent duct. Other major indicators of testicular development were invagination among epithelial cells and the formation of capillary vessels at 60 dph, the organization of primary germ cells into lobules at 90 dph and differentiation of the somatic cells into Sertoli cells at 120 dph. Final testicular differentiation was histologically discernible at 140 dph when the somatic cells had differentiated into Sertoli cells and germ cells into spermatocytes. The gene dmrt1 was detected exclusively to be expressed in Sertoli cells by in situ hybridization using an mRNA probe. Cyp11b and dmrt1 were expressed highest in the gonad at 90 and 120 dph, respectively, with dmrt1 expression observed to rapidly increase from 70 to 120 dph. Beginning of sex differentiation to the finalisation of testicular development in barramundi was determined in this study to occur from 44 to 140 dph (TL 169.4 ± 40.3 mm). As it has been possible for other teleosts, determination of the timing and duration of testes development provides an opportunity to manipulate sex through intervening with early developmental processes in this important aquaculture species