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Knockout of Zebrafish Ovarian Aromatase Gene ( cyp19a1a ) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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(A) Gonad development at 47 dpf in the control (cyp19a1a+/−) and mutant (cyp19a1a−/−). The gonadal differentiation had finished in the control fish with the ovary and testis well developed. The ovary contained both PG and PV follicles, indicating puberty onset in the female. All mutant fish were males with well-developed testes containing all stages of spermatogenic cells without any oocytes. (B) Rescue of mutant phenotype by E2 treatment. The juvenile mutant fish were treated with E2 (0.05 nM) from 15 to 30 dpf, and the fish were sampled for histological examination at 35 dpf. E2 treatment could induce normal ovarian formation in some mutant fish as shown by fish E. Other fish (e.g., fish F) still had undifferentiated gonads that were likely destined to males.
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f8: (A) Gonad development at 47 dpf in the control (cyp19a1a+/−) and mutant (cyp19a1a−/−). The gonadal differentiation had finished in the control fish with the ovary and testis well developed. The ovary contained both PG and PV follicles, indicating puberty onset in the female. All mutant fish were males with well-developed testes containing all stages of spermatogenic cells without any oocytes. (B) Rescue of mutant phenotype by E2 treatment. The juvenile mutant fish were treated with E2 (0.05 nM) from 15 to 30 dpf, and the fish were sampled for histological examination at 35 dpf. E2 treatment could induce normal ovarian formation in some mutant fish as shown by fish E. Other fish (e.g., fish F) still had undifferentiated gonads that were likely destined to males.

Mentions: At 47 dpf, the gonads had completed the process of differentiation. Both females and males were present in the control group. The males had well-developed testis showing the entire process of spermatogenesis, whereas the females started to enter puberty as marked by the appearance of the previtellogenic follicles (PV, or stage II with cortical alveoli). No females were found in the aromatase mutant fish, and all individuals had well-developed testis without EPOs (Fig. 8A).


Knockout of Zebrafish Ovarian Aromatase Gene ( cyp19a1a ) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation
(A) Gonad development at 47 dpf in the control (cyp19a1a+/−) and mutant (cyp19a1a−/−). The gonadal differentiation had finished in the control fish with the ovary and testis well developed. The ovary contained both PG and PV follicles, indicating puberty onset in the female. All mutant fish were males with well-developed testes containing all stages of spermatogenic cells without any oocytes. (B) Rescue of mutant phenotype by E2 treatment. The juvenile mutant fish were treated with E2 (0.05 nM) from 15 to 30 dpf, and the fish were sampled for histological examination at 35 dpf. E2 treatment could induce normal ovarian formation in some mutant fish as shown by fish E. Other fish (e.g., fish F) still had undifferentiated gonads that were likely destined to males.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120357&req=5

f8: (A) Gonad development at 47 dpf in the control (cyp19a1a+/−) and mutant (cyp19a1a−/−). The gonadal differentiation had finished in the control fish with the ovary and testis well developed. The ovary contained both PG and PV follicles, indicating puberty onset in the female. All mutant fish were males with well-developed testes containing all stages of spermatogenic cells without any oocytes. (B) Rescue of mutant phenotype by E2 treatment. The juvenile mutant fish were treated with E2 (0.05 nM) from 15 to 30 dpf, and the fish were sampled for histological examination at 35 dpf. E2 treatment could induce normal ovarian formation in some mutant fish as shown by fish E. Other fish (e.g., fish F) still had undifferentiated gonads that were likely destined to males.
Mentions: At 47 dpf, the gonads had completed the process of differentiation. Both females and males were present in the control group. The males had well-developed testis showing the entire process of spermatogenesis, whereas the females started to enter puberty as marked by the appearance of the previtellogenic follicles (PV, or stage II with cortical alveoli). No females were found in the aromatase mutant fish, and all individuals had well-developed testis without EPOs (Fig. 8A).

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus