Transcriptome display during tilapia sex determination and differentiation as revealed by RNA-Seq analysis

Abstract

Background: The factors determining sex in teleosts are diverse. Great efforts have been made to characterize the underlying genetic network in various species. However, only seven master sex-determining genes have been identified in teleosts. While the function of a few genes involved in sex determination and differentiation has been studied, we are far from fully understanding how genes interact to coordinate in this process.

Results: To enable systematic insights into fish sexual differentiation, we generated a dynamic co-expression network from tilapia gonadal transcriptomes at 5, 20, 30, 40, 90, and 180 dah (days after hatching), plus 45 and 90 dat (days after treatment) and linked gene expression profiles to both development and sexual differentiation. Transcriptomic profiles of female and male gonads at 5 and 20 dah exhibited high similarities except for a small number of genes that were involved in sex determination, while drastic changes were observed from 90 to 180 dah, with a group of differently expressed genes which were involved in gonadal differentiation and gametogenesis. Weighted gene correlation network analysis identified changes in the expression of Borealin, Gtsf1, tesk1, Zar1, Cdn15, and Rpl that were correlated with the expression of genes previously known to be involved in sex differentiation, such as Foxl2, Cyp19a1a, Gsdf, Dmrt1, and Amh.

Conclusions: Global gonadal gene expression kinetics during sex determination and differentiation have been extensively profiled in tilapia. These findings provide insights into the genetic framework underlying sex determination and sexual differentiation, and expand our current understanding of developmental pathways during teleost sex determination.

Keywords: RNA-seq analysis; Sex determination and differentiation; Tilapia.

Fig. 1

Histological analysis of characteristic gonadal stages in tilapia ovary (XX) and testsis (XY). Panels indicate gonads sampled from fish at different days after hatch (dah) as follows: a and b at 5 dah, c and d at 20 dah, e and f at 30 dah, g and h at 40 dah, i and j at 90 dah, and k and l at 180 dah. OG, oogonia; SG, spermatogonia; OC, oocytes; CV, ovarian cavity; ST, spermatids; SC, spermatocytes

Fig. 2

Global gene expression profiles in XX and XY gonads from 5 to 180 dah. Two different groups were clearly identified. One consisted of samples at earlier stages (Branch I: 5, 20, 30, and 40 dah), and the other consisted samples at later developmental stages (Branch II: 90, 180 dah). Intersex_45 dat XX and Intersex_90 dat were gonadal samples of 90dah XX fish treated with Fadrozole for 45 and 90 days, respectively [29]. F, female; M, male

Fig. 3

Gene expression modules constructed by the WGCNA. a the clustering dendrogram of genes expressed in the XX and XY gonads is shown. WGCNA identifies gene modules using the cutreeDynamic function, which detects clusters in a dendrogram depending on their shape [32]. Original modules of very similar coexpression are merged. Color name-based module labels are generated automatically by WGCNA and are shown along with the number of genes belonging to each module. b Association between modules and traits (such as Genotype, Phenotype, Developmental stages, and Key genes) is depicted. Rows correspond to module eigengenes (shown on the left with the colored boxes), columns to a trait (indicated below each column). Each cell contains the P value of the trait-module association. Large positive values indicate a strong correlation, while large negative values indicate a strong negative association

Fig. 4

Heatmap analysis of key genes involved in tilapia sex determination and differentiation. Each row represents a gene listed on the right. Each column stands for a gonadal sample specified in Fig. 1. The expression of genes is color coded from low (green) to high (red). Full gene names are shown in Additional file 1

Fig. 5

Cellular localization of Borealin, Gtsf1 and ZarI in tilapia testis and ovary by ISH. Borealin was weakly expressed in the oogonia, highly expressed in the phase I and II oocytes of the ovary, but was not expressed in the phase III and IV oocytes (a), while no Borealin expression was found in the testis (d). Gtsf1 was weakly expressed in the oogonia and phase I oocytes, highly expressed in the phase II oocytes, and moderately expressed in the phase II oocytes of the ovary (b), while no Gtsf1 expression were detected in the testis (e). ZarI was highly expressed in the phase I and II oocytes, and weakly expressed in the phase III oocytes in the ovary (c), and moderately expressed in the spermatocytes in the testis (f). OG, oogonia; OC, oocytes; I-IV, phase I to phase IV oocytes; SG, spermatogonia; SC, spermatocytes; Arrowheads indicate the positive signal