Abstract
Zebrafish sex determination and sexual differentiation is complex. Neither sex chromosomes, major sex-determining genes nor universal sex-linked markers have been identified, and a variety of environmental factors can influence sex ratios. It is thus unsurprising that the molecular pathways underpinning zebrafish sexual development remain poorly understood. A few key genes such as, SRY-box containing gene 9a (sox9a), anti-Müllerian hormone (amh), cytochrome P450, family 19, subfamily A, polypeptide 1a (cyp19a1a) are known to play roles in zebrafish gonadal differentiation. However, at this time, there are many more questions than answers about sex determination and differentiation in this ubiquitious, and immensely well-studied, species.
As with many other vertebrates, sex hormone treatment can induce sex reversal of gonadal phenotype and sexual behaviour in zebrafish. In this study, we take advantage of hormonal manipulation of sex in developing zebrafish and RNA-Seq to unravel the unknown genetic pathways that underlie sex determination and sexual differentiation. We conducted global transcriptomic profiling of juvenile zebrafish brains and gonads at two important developmental stages in gonadal differentiation: (1) juvenile ovary-to-testis transformation (40 days post fertilization) and (2) the completion of testicular and ovarian differentiation (60 days post fertilization). Gene expression profiles from 17α-methyltestosterone masculinised juvenile zebrafish were also compared with untreated zebrafish to improve understanding of androgenic effects on zebrafish sexual differentiation pathways.
To our knowledge, this is the first study profiling transcriptome-wide sex dimorphic gene expression in teleost brains during gonadal differentiation. We identified modest sex differences in developing zebrafish brains; 33 and 185 sex dimorphic genes were identified in the brains of 40 dpf and 60 dpf zebrafish respectively. Methyltestosterone treatment significantly altered expression patterns at both timepoints. We observed a total of 1,554 genes differentially expressed between control female brains and MT-treated brains at 40 dpf. 1,379 genes were differentially expressed between 40 dpf male brains and 40 dpf MT-treated brains. At 60 dpf, 728 genes exhibited differential expression between control female brains and MT-treated brains. Interestingly, significantly fewer genes (269) were differentially expressed between control male brains and MT-treated brains at 60 dpf.
In contrast, we observed extensive sex differences in zebrafish gonadal transcriptomes. A total of 5,039 genes were sexually dimorphic at 40 dpf. 2,502 and 2,537 genes were up-regulated in transforming testes and juvenile ovaries respectively. At 60 dpf, we identified 4,190 testis-biased genes and 4,267 ovary-biased genes. Many of the sex dimorphic genes identified have no previous links with gonadal differentiation.
Despite accelerated testicular development in MT-treated zebrafish, the numbers of differentially expressed genes identified when control ovaries were compared to control testes or MT-treated testes were similar. We identified 5,237 differentially expressed genes between 40 dpf juvenile ovaries and 40 dpf MT-treated testes. 7,513 genes were differentially expressed between ovaries and MT-treated testes at 60 dpf. There were considerably fewer differences between control testes and MT-treated testes. While 1,222 genes were differentially expressed between control testes and MT-treated testes at 40 dpf, only 20 genes separated the transcriptomes of 60 dpf control testes and MT-treated testes. It appears that androgen-induced masculinization shares key molecular regulators with normal testicular differentiation.
A pair of rec8 genes were up-regulated in control testes and MT-treated testes. Testis-biased Rec8 expression was previously reported in mammals. Rec8 is required for sister chromatid cohesion, formation of synaptonemal complexes and homologous recombination during meiosis. To elucidate the functional significance of rec8 genes in testicular development, we characterized rec8 genes in adult zebrafish. Sequence homology, synteny and phylogenetic relationships between the zebrafish rec8 genes were explored in silico. Analysis of rec8a and rec8b gene expression in adult zebrafish confirmed testis-biased expression. Our work sets up a framework for which we can explore further functional testing of zebrafish rec8 genes using morpholino knockdown and CRISPR/Cas knockout technologies.