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 40 dpf in the control (cyp19a1a+/−; fish A–D) and mutant (cyp19a1a−/−; fish E–L). The control fish had well-differentiated ovary (A and B) and testis (C and D), whereas all the mutant individuals were undergoing or had completed ovary-testis transformation with typical testicular tissues containing different stages of spermatogenic cells. Some individuals (E–H) still contained a few typical EPOs (asterisks) scattered among the testicular tissues. Arrows indicate the apoptotic germ cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Gonad development at 40 dpf in the control (cyp19a1a+/−; fish A–D) and mutant (cyp19a1a−/−; fish E–L). The control fish had well-differentiated ovary (A and B) and testis (C and D), whereas all the mutant individuals were undergoing or had completed ovary-testis transformation with typical testicular tissues containing different stages of spermatogenic cells. Some individuals (E–H) still contained a few typical EPOs (asterisks) scattered among the testicular tissues. Arrows indicate the apoptotic germ cells.

Mentions: The typical testis structure (cystic spermatogenic cells) started to appear in the gonads at 40 dpf (could be earlier). In the control fish, the GFP signal continued to polarize with stronger signal in females (Fig. 7A and B) and weak signal in males (Fig. 7C and D). Most mutant fish had spermatogenic cells present in the gonads (Fig. 7E–L) with some still in the process of juvenile ovary-to-testis transformation as indicated by the presence of EPOs among the spermatogenic cysts (Fig. 7E–H).


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 40 dpf in the control (cyp19a1a+/−; fish A–D) and mutant (cyp19a1a−/−; fish E–L). The control fish had well-differentiated ovary (A and B) and testis (C and D), whereas all the mutant individuals were undergoing or had completed ovary-testis transformation with typical testicular tissues containing different stages of spermatogenic cells. Some individuals (E–H) still contained a few typical EPOs (asterisks) scattered among the testicular tissues. Arrows indicate the apoptotic germ cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Gonad development at 40 dpf in the control (cyp19a1a+/−; fish A–D) and mutant (cyp19a1a−/−; fish E–L). The control fish had well-differentiated ovary (A and B) and testis (C and D), whereas all the mutant individuals were undergoing or had completed ovary-testis transformation with typical testicular tissues containing different stages of spermatogenic cells. Some individuals (E–H) still contained a few typical EPOs (asterisks) scattered among the testicular tissues. Arrows indicate the apoptotic germ cells.
Mentions: The typical testis structure (cystic spermatogenic cells) started to appear in the gonads at 40 dpf (could be earlier). In the control fish, the GFP signal continued to polarize with stronger signal in females (Fig. 7A and B) and weak signal in males (Fig. 7C and D). Most mutant fish had spermatogenic cells present in the gonads (Fig. 7E–L) with some still in the process of juvenile ovary-to-testis transformation as indicated by the presence of EPOs among the spermatogenic cysts (Fig. 7E–H).

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