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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

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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.


Gonad development at 20 dpf in the control [Tg(vas:EGFP);cyp19a1a+/−; fish A–D] and mutant [Tg(vas:EGFP);cyp19a1a−/−; fish E–H]. Similar GFP signals (boxed in the photo) were observed in the two groups and histological examination showed no significant difference. Meiotic germ cells (arrowhead) with condensed chromatin were often seen in both the mutant and control gonads.
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f4: Gonad development at 20 dpf in the control [Tg(vas:EGFP);cyp19a1a+/−; fish A–D] and mutant [Tg(vas:EGFP);cyp19a1a−/−; fish E–H]. Similar GFP signals (boxed in the photo) were observed in the two groups and histological examination showed no significant difference. Meiotic germ cells (arrowhead) with condensed chromatin were often seen in both the mutant and control gonads.

Mentions: GFP signals were observed in both cyp19a1a mutant fish (−/−) and their sibling control (+/−) at 20 dpf, and individuals of the same genotype showed similar GFP intensity (Fig. 4). Interestingly, the GFP signal in the mutant fish (Fig. 4E–H) seemed to be slightly weaker than that of the control (Fig. 4A–D). Despite this, histological analysis showed little difference in gonadal development between the two genotypes. Although the meiotic cells were abundant in both genotypes, the gonocytes or gonial germ cells in both the control and mutant gonads were basically undifferentiated at the histological level with no obvious signs of sex differentiation (Fig. 4). Since we could not distinguish male and female germ cells based on morphological features, we would consider the gonads at this stage indifferent as suggested previously25 rather than ovary-like although the entry of the germ cells into meiosis suggests that the gonads might have committed femaleness at molecular level23. Although the term oocyte-like germ cell is frequently used in the literature, very few studies have clearly defined their characteristics24252658. In this study, we consider the germ cells to be oocyte-like only when they exhibited typical perinucleolar features with diameters being 20 μm or bigger59 and with basophilic cytoplasm and prominent nucleoli located at the periphery of the nuclei25.


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 20 dpf in the control [Tg(vas:EGFP);cyp19a1a+/−; fish A–D] and mutant [Tg(vas:EGFP);cyp19a1a−/−; fish E–H]. Similar GFP signals (boxed in the photo) were observed in the two groups and histological examination showed no significant difference. Meiotic germ cells (arrowhead) with condensed chromatin were often seen in both the mutant and control gonads.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Gonad development at 20 dpf in the control [Tg(vas:EGFP);cyp19a1a+/−; fish A–D] and mutant [Tg(vas:EGFP);cyp19a1a−/−; fish E–H]. Similar GFP signals (boxed in the photo) were observed in the two groups and histological examination showed no significant difference. Meiotic germ cells (arrowhead) with condensed chromatin were often seen in both the mutant and control gonads.
Mentions: GFP signals were observed in both cyp19a1a mutant fish (−/−) and their sibling control (+/−) at 20 dpf, and individuals of the same genotype showed similar GFP intensity (Fig. 4). Interestingly, the GFP signal in the mutant fish (Fig. 4E–H) seemed to be slightly weaker than that of the control (Fig. 4A–D). Despite this, histological analysis showed little difference in gonadal development between the two genotypes. Although the meiotic cells were abundant in both genotypes, the gonocytes or gonial germ cells in both the control and mutant gonads were basically undifferentiated at the histological level with no obvious signs of sex differentiation (Fig. 4). Since we could not distinguish male and female germ cells based on morphological features, we would consider the gonads at this stage indifferent as suggested previously25 rather than ovary-like although the entry of the germ cells into meiosis suggests that the gonads might have committed femaleness at molecular level23. Although the term oocyte-like germ cell is frequently used in the literature, very few studies have clearly defined their characteristics24252658. In this study, we consider the germ cells to be oocyte-like only when they exhibited typical perinucleolar features with diameters being 20 μm or bigger59 and with basophilic cytoplasm and prominent nucleoli located at the periphery of the nuclei25.

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.