Small-scale transcriptomics reveals differences among gonadal stages in Asian seabass (Lates calcarifer)

Abstract

Background: The Asian seabass (Lates calcarifer) is a protandrous hermaphrodite that typically matures as a male at approximately 2-4 years of age and then changes sex in subsequent years. Although several sexual maturation stages have been described histologically for both testis and ovary, the underlying gene expression profiles remain lacking. The development of a gene expression platform is therefore necessary to improve our understanding of the gonad development of this cultured teleost species.

Methods: Thirty Asian seabass gonads were collected from farms in Singapore, examined histologically and staged according to their sex and gonadal maturation status. Partial coding sequences of 24 sex-related genes were cloned using degenerate primers and were sequenced. Additional 13 cDNA sequences were obtained through next-generation sequencing. A real-time qPCR was then performed using the microfluidic-based Fluidigm 48.48 Dynamic arrays.

Results: We obtained 17 ovaries and 13 testes at various stages of sexual maturation. Of the 37 genes that were tested, 32 (86%) showed sexually dimorphic expression. These genes included sex-related genes, sox9, wt1, amh, nr5a2, dmrt1 and nr0b1, which showed testis-enhanced expression similar to other vertebrate species. Known male- and female-enhanced germ cells markers, which were established from studies in other species, similarly showed testis- and ovary-enhanced expression, respectively, in the Asian seabass. Three pro-Wnt signaling genes were also upregulated in the ovary, consistent with existing studies that suggested the role of Wnt signaling in ovarian differentiation in teleosts and mammals. The expression patterns of genes involved in steroidogenesis, retinoic acid metabolism, apoptosis and NF-κB signaling were also described. We were able to classify gonads according to sex and gonadal maturation stages, based on their small-scale transcriptomic profiles, and to uncover a wide variation in expression profiles among individuals of the same sex.

Conclusions: The analysis of a selected set of genes related to reproduction and in sufficient number of individuals using a qPCR array can elucidate new insights into the molecular mechanisms involved in Asian seabass gonad development. Given the conservation of gene expression patterns found in this study, these insights may also help us draw parallels with other teleosts.

Figure 1

Separation of the male and female Asian seabass gonads under the hierarchical clustering map. The expression profiles of 36 sex-related genes with p-value (gonad type) < 0.01 under a one-way ANOVA analysis were used to generate the hierarchical clustering map. Male and female gonads were clustered into two different clades. Within the female gonad clade (top section), F3 and F4 ovaries were further divided into two sub-clades, which indicated that their gene expression profiles were different. Red boxes indicate high expression, whereas blue boxes indicate low expression. White boxes within the clustering map indicate missing values for a particular sample and gene.

Figure 2

M1 testes of the Asian seabass showed female-like expression levels. amh(A), odf3 (B), sycp3l (C), sept6 (D) and tdrd7 (E) genes. Dot plots overlaid with box plots of the relative log₂ gene expression values are shown (reference genes: rpl8, ef1a and ubq). The M1 testes are labeled with cyan dots.

Figure 3

The expression of zp2, which is an oocyte marker, showed a wide variation and an increased level of expression in some M3-type testes in Asian seabass. The relative log₂ gene expression values of zp2 are shown. Female gonads (F1, F3 & F4; pink, red & orange) are on the left, whereas male gonads (M1-M4; light to dark blue) are on the right.

Figure 4

Pre-vitellogenic oocytes can be found lining the tubular walls in the M3 testis of an Asian seabass individual (SB335). Abbreviations: po – previtellogenic oocytes; sp – spermatozoa; st – spermatids; ssc – secondary spermatogonia; psc – primary spermatogonia. Pre-vitellogenic oocytes are indicated with asterisks.

Figure 5

The expression levels. cyp11c1 (A) and esr1 (B) are distinctly sexually dimorphic between male and female gonads in Asian seabass, regardless of their sexual maturation stage. Dot plots that are overlaid with box plots of the relative log₂ gene expression values are shown (reference genes: rpl8, ef1a and ubq).

Figure 6

Working hypothesis for the gonad transformation process. All zebrafish develop a juvenile ovary before the future males undergo gonadal transformation to form the testis. Several pathways and genes have been known to be involved in this process. Based on our results, we proposed that the same pathways and genes are also involved in the testis-to-ovary transformation process in the Asian seabass, despite the reversal of direction. The Asian seabass system is, hence, a mirror image of the zebrafish model. The arrows show the observed (zebrafish; top - blue) and/or expected (Asian seabass; bottom – pink) up- or downregulation of the genes or pathways during the gonadal transformation process.