Abstract
Sexual or gonadal differentiation is a complex event and its mechanism remains elusive in teleosts. Despite its complexity and plasticity, the process of ovarian differentiation is believed to involve gonadal aromatase (cyp19a1a) in nearly all species studied. However, most data concerning the role of aromatase have come from gene expression analysis or studies involving pharmacological approaches. There has been a lack of genetic evidence for the importance of aromatase in gonadal differentiation, especially the timing when the enzyme starts to exert its effect. This is due to the lack of appropriate loss-of-function approaches in fish models for studying gene functions. This situation has changed recently with the development of genome editing technologies, namely TALEN and CRISPR/Cas9. Using both TALEN and CRISPR/Cas9, we successfully established three mutant zebrafish lines lacking the ovarian aromatase. As expected, all mutant fish were males, supporting the view that aromatase plays a critical role in directing ovarian differentiation and development. Further analysis showed that the ovarian aromatase did not seem to affect the formation of so-called juvenile ovary and oocyte-like germ cells; however, it was essential for further differentiation of the juvenile ovary into the true ovary.
Highlights
Similar change has been induced by blocking estrogen signaling with estrogen receptor antagonists[20]
Zebrafish is a gonochoristic species at adult stage; it is considered a juvenile hermaphrodite because the fish first develops ovary-like gonads or juvenile ovaries, which are characterized by initiation of meiosis around 10 dpf followed by development of perinucleolar oocytes (POs) and ovarian cavity[23]
All the information accumulated points to the importance of aromatase in ovarian differentiation and development in fish models[8], there is a lack of genetic data on its importance in the event, especially the timing of its action during gonadal differentiation
Summary
Similar change has been induced by blocking estrogen signaling with estrogen receptor antagonists[20]. High temperature can significantly influence sex ratio in the zebrafish This effect seemed to be mediated by inhibiting cyp19a1a gene expression, leading to oocyte apoptosis and development of testicular tissue during sex differentiation[39]. Environmental hypoxia has been reported to cause male-dominant population in the zebrafish, and again it is associated with a decreased expression of cyp19a1a40 All these lines of evidence from physiological, pharmacological and environmental studies indicate that aromatase is a key factor in driving ovarian differentiation in fish including the zebrafish. CRISPR/Cas[9] is simpler and easier to construct than TALEN; due to the short recognition site of only 18 nucleotides, off-targeting effect may occur[51] With these convenient and powerful genetic tools, zebrafish has become one of the top models for investigating functions of interested genes in vivo using gene knockout approach, which has been restricted mostly to the mouse model. Three null mutant zebrafish lines were established using TALEN and CRISPR/Cas[9] respectively for phenotype analysis on sex ratio and gonadal development
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