Limits...
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.


Related in: MedlinePlus

Gonad development at 25 dpf in the control (cyp19a1a+/−; fish A–F) and mutant (cyp19a1a−/−; fish G–L). The GFP intensity in the gonads (boxed in the photo) started to diverge in the control (A–C vs. D–F) but not the mutant fish (G–L), suggesting the start of gonadal differentiation. Two groups of fish could be distinguished by histological analysis. One group had well-developed EPOs (asterisks) in the gonads (fish A–C) in the control and (G–I) in the mutant) and the other contained mostly undifferentiated germ cells (fish D–F) in the control and (J–L) in the mutant. The EPOs in the control fish (A–C) were generally larger with stronger GFP signal than those in the mutant fish (G–I). Numerous meiotic germ cells (arrowheads) were present in the gonads of both control and mutant fish. The dark-stained condensed apoptotic germ cells (arrows) were often observed at this stage, indicating the process of juvenile ovary-testis transformation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120357&req=5

f5: Gonad development at 25 dpf in the control (cyp19a1a+/−; fish A–F) and mutant (cyp19a1a−/−; fish G–L). The GFP intensity in the gonads (boxed in the photo) started to diverge in the control (A–C vs. D–F) but not the mutant fish (G–L), suggesting the start of gonadal differentiation. Two groups of fish could be distinguished by histological analysis. One group had well-developed EPOs (asterisks) in the gonads (fish A–C) in the control and (G–I) in the mutant) and the other contained mostly undifferentiated germ cells (fish D–F) in the control and (J–L) in the mutant. The EPOs in the control fish (A–C) were generally larger with stronger GFP signal than those in the mutant fish (G–I). Numerous meiotic germ cells (arrowheads) were present in the gonads of both control and mutant fish. The dark-stained condensed apoptotic germ cells (arrows) were often observed at this stage, indicating the process of juvenile ovary-testis transformation.

Mentions: At 25 dpf, the gonadal GFP signals started to diverge among individuals in the control fish, which could be divided into two groups with one having significantly increased GFP signal. Histological analysis showed that the fish with enhanced GFP signal all had typical perinucleolar oocytes (PO; primary growth stage, PG) in the gonads (Fig. 5A–C), whereas those with similar or reduced GFP signal had small oocyte-like early POs (EPOs) or undifferentiated germ cells (Fig. 5D–F). In the mutant fish without aromatase, the GFP signal in all individuals remained more or less the same. In some mutant fish, the gonads contained EPOs, which were obviously smaller in size than those in the control fish (Fig. 5G–I). Both meiotic and apoptotic germ cells were commonly seen in the mutant gonads at this stage of development. In some individuals of both control and mutant groups, the gonads had increased amount of stromal cells among germ cells or germ cell cysts, a sign of testis development in genetically male fish25.


Knockout of Zebrafish Ovarian Aromatase Gene ( cyp19a1a ) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation
Gonad development at 25 dpf in the control (cyp19a1a+/−; fish A–F) and mutant (cyp19a1a−/−; fish G–L). The GFP intensity in the gonads (boxed in the photo) started to diverge in the control (A–C vs. D–F) but not the mutant fish (G–L), suggesting the start of gonadal differentiation. Two groups of fish could be distinguished by histological analysis. One group had well-developed EPOs (asterisks) in the gonads (fish A–C) in the control and (G–I) in the mutant) and the other contained mostly undifferentiated germ cells (fish D–F) in the control and (J–L) in the mutant. The EPOs in the control fish (A–C) were generally larger with stronger GFP signal than those in the mutant fish (G–I). Numerous meiotic germ cells (arrowheads) were present in the gonads of both control and mutant fish. The dark-stained condensed apoptotic germ cells (arrows) were often observed at this stage, indicating the process of juvenile ovary-testis transformation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Gonad development at 25 dpf in the control (cyp19a1a+/−; fish A–F) and mutant (cyp19a1a−/−; fish G–L). The GFP intensity in the gonads (boxed in the photo) started to diverge in the control (A–C vs. D–F) but not the mutant fish (G–L), suggesting the start of gonadal differentiation. Two groups of fish could be distinguished by histological analysis. One group had well-developed EPOs (asterisks) in the gonads (fish A–C) in the control and (G–I) in the mutant) and the other contained mostly undifferentiated germ cells (fish D–F) in the control and (J–L) in the mutant. The EPOs in the control fish (A–C) were generally larger with stronger GFP signal than those in the mutant fish (G–I). Numerous meiotic germ cells (arrowheads) were present in the gonads of both control and mutant fish. The dark-stained condensed apoptotic germ cells (arrows) were often observed at this stage, indicating the process of juvenile ovary-testis transformation.
Mentions: At 25 dpf, the gonadal GFP signals started to diverge among individuals in the control fish, which could be divided into two groups with one having significantly increased GFP signal. Histological analysis showed that the fish with enhanced GFP signal all had typical perinucleolar oocytes (PO; primary growth stage, PG) in the gonads (Fig. 5A–C), whereas those with similar or reduced GFP signal had small oocyte-like early POs (EPOs) or undifferentiated germ cells (Fig. 5D–F). In the mutant fish without aromatase, the GFP signal in all individuals remained more or less the same. In some mutant fish, the gonads contained EPOs, which were obviously smaller in size than those in the control fish (Fig. 5G–I). Both meiotic and apoptotic germ cells were commonly seen in the mutant gonads at this stage of development. In some individuals of both control and mutant groups, the gonads had increased amount of stromal cells among germ cells or germ cell cysts, a sign of testis development in genetically male fish25.

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