This AOP is licensed under a Creative Commons Attribution 4.0 International License.
Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation
Point of Contact
- Kelvin Santana Rodriguez
- Brendan Ferreri-Hanberry
|Author status||OECD status||OECD project||SAAOP status|
|Under Development: Contributions and Comments Welcome|
This AOP was last modified on April 05, 2021 18:16
|Inhibition, Aromatase||September 16, 2017 10:14|
|Reduction, 17beta-estradiol synthesis by the undifferentiated gonad||September 15, 2020 13:49|
|Increased, Differentiation to Testis||September 15, 2020 14:01|
|Increased, Male Biased Sex Ratio||August 07, 2020 18:17|
|Decrease, Population trajectory||September 26, 2017 11:33|
|Inhibition, Aromatase leads to Reduction, E2 Synthesis by the undifferentiated gonad||September 30, 2020 09:42|
|Reduction, E2 Synthesis by the undifferentiated gonad leads to Increased, Differentiation to Testis||September 30, 2020 10:15|
|Inhibition, Aromatase leads to Increased, Differentiation to Testis||September 30, 2020 09:34|
|Increased, Differentiation to Testis leads to Increased, Male Biased Sex Ratio||September 17, 2020 09:03|
|Increased, Male Biased Sex Ratio leads to Decrease, Population trajectory||September 17, 2020 06:34|
|Fadrozole||November 29, 2016 18:42|
|Letrozole||November 29, 2016 18:42|
|Exemestane||November 12, 2020 01:53|
|Stressor:292 Clotrimazole||November 12, 2020 01:55|
|Prochloraz||November 29, 2016 18:42|
This adverse outcome pathway links inhibition of aromatase activity in teleost fish during gonadogenesis leading to a male-biased sex determination and successively, reduced population sustainability. Most gonochoristic fish species, develop either as males or females, and do not change sex throughout their entire life spans. However, there’s a developmental window in which their sex determination can be sensitive to environmental conditions or chemical pollutants. Treatment with steroid hormones prior to sexual differentiation has shown to induce ovary or testis development according to the type of steroid that is administered. For most vertebrate taxa, aromatase (Cyp19a1) is the rate-limiting enzyme for the biosynthesis of 17β - estradiol from testosterone. Many endocrine disrupting chemicals such as fadrozole, letrozole and exemestane are well known chemicals that inhibit the activity aromatase. Exposure during the critical period of sex differentiation in gonochoristic teleost fish with an aromatase inhibitor that blocks estrogen biosynthesis can induce phenotypic males. Given that females carry the major reproductive production of the population, a male-biased sex ratio can result in a reduced population fitness, particularly for those species present in ecosystems that are heavily impacted by human activities.
Summary of the AOP
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
|Sequence||Type||Event ID||Title||Short name|
|MIE||36||Inhibition, Aromatase||Inhibition, Aromatase|
|KE||1789||Reduction, 17beta-estradiol synthesis by the undifferentiated gonad||Reduction, E2 Synthesis by the undifferentiated gonad|
|KE||1790||Increased, Differentiation to Testis||Increased, Differentiation to Testis|
|KE||1791||Increased, Male Biased Sex Ratio||Increased, Male Biased Sex Ratio|
|AO||360||Decrease, Population trajectory||Decrease, Population trajectory|
Relationships Between Two Key Events (Including MIEs and AOs)
Life Stage Applicability
Overall Assessment of the AOP
Domain of Applicability
The life stage applicable to this AOP is developing embryos and juveniles prior to- or during the gonadal developmental stage. Since the sexually dimorphic expression of aromatase plays a crucial role in the differentiation to either testis or ovaries in the undifferentiated bipotential gonad, this key event relationship can be applicable to the exact stage of development at which the aromatase enzyme works to influence gonadal differentiation. This AOP is not applicable to sexually differentiated adults.
Studies with zebrafish have shown that both brain and gonadal aromatase expression can be observed at 20 days post-fertilization with and increase in expression at 25 days post-fertilization in zebrafish destined to become females which also coincided with onset of gonadal differentiation period (Lau et al. 2016). In tilapia, aromatase expression can be observed as early as 3-4 days post fertilization with and increase in expression starting at 11 days post-fertilization in genetic females (Kwon, J. et al. 2001). Additionally, it has been shown that the period of 7-14 days post-fertilization is the most sensitive towards an aromatase inhibitor and that a consecutive exposure of 2-3 weeks is sufficient for the masculinization of the majority of genetic female tilapia fish (Kwon, J. et al. 2000). This suggest that to redirect the sexual differentiation pathway from ovary to testis, an alteration of aromatase expression will be most effective during the early developmental stage prior and during the critical sex differentiation period.
The molecular initiation event for this AOP occurs prior to gonad differentiation. Therefore, this AOP is only applicable to sexually undifferentiated individuals.
The taxonomic applicability of this AOP is the class Osteichthyes. However, phylogenetic analysis among mammalian, amphibian, reptile, bird, and fish has shown that aromatase is well conserved among all vertebrates (Wilson JY et al., 2005). Additionally, CYP19 was detected in the amphioxus suggesting that it has possible origin in primitive chordates. Therefore, because all key events in the present AOP can be applicable to most non-mammalian vertebrates, it is probable that this AOP could be relevant to amphibians, reptiles and birds as well. Though, the outcomes mind differ due to species-specific differences.
Essentiality of the Key Events
Support for the essentiality of several of the Key Events in the AOP was provided mainly by gene knockout of the cyp1a1 gene in zebrafish and tilapia. Teleost fish have two genes encoding for aromatase; cyp1a1a that is mainly expressed in the gonads and cyp1a1b expressed in the brain. Studies have demonstrated that mutant lines of cyp1a1b develop as females while cypa1a mutants develop as males suggesting that gonadal aromatase inhibition is crucial step for the subsequent key events to occur.
- Lau et al. 201613 generated indel mutations in zebrafish cyp19a1a gene using TALEN and CRISPR/Cas9 approaches. All mutant cyp19a1a-/- developed as males. Histological examination (at 120 days post-fertilization) of the cyp1a1a-/- mutant showed that all exhibited normal spermatogenesis in the testis with no observable difference to the wild type (+/+) and heterozygous (+/-) males. However, to prove the role of E2 synthesis for ovarian differentiation, they performed an experiment to rescue the phenotype of cyp19a1a mutant by E2 treatment (0.05, 0.50 and 5.00 nM) over the time of gonadal differentiation (15–30 days port-fertilization). The result showed that exposure to E2 caused normal ovarian formation with fully developed perinucleolar oocytes and little amount of stromal tissues, and the effect could be observed in some individuals even at the lowest concentration (0.05 nM). This supports the essentiality of aromatase inhibition relative to E2 synthesis reduction as a critical step for testis differentiation.
- On a similar study with zebrafish, Muth-Köhne et al. 20168 generated cyp19a1a and cyp19a1b gene mutant lines and a cyp19a1a;cyp19a1b double-knockout line in zebrafish using transcription activator-like effector nucleases (TALENs). All cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants developed as males whereas cyp1a1b double mutant (-/-) had a 1:1 sex ratio similar to the wild type controls. This supports the essentiality of gonadal aromatase inhibition for testis differentiation leading to a male biased sex ratio population. Additionally, a rescue experiment was performed using 17 β-estradiol on all male mutant cyp1a1a-/- and the results suggested that treatment could rescue the sex ratio defect (9 females among 14 fish).
- Similar support using Nile tilapia (Oreochromis niloticus) was provided in a study by Zhang et al. 201712. Using genetic female mutant for cypa1a and cyp1a1b. Results showed that all cyp19a1a+/- XX and cyp19a1a+/+ XX fish developed as females, whereas all cyp19a1a-/- XX and cyp19a1a-/- XY fish developed as males. The cyp19a1a-/- XX tilapia shifted to the male pathway at as early as 5 days after hatch (dah), as reflected by the gonadal expression and were fertile. This supports the essentiality of gonadal aromatase inhibition during early development for a testis differentiation pathway to be induced.
There is good evidence from gene knockout experiments of the two different isoforms of aromatase that support the specificity of gonadal aromatase inhibition for the subsequent key events to occur.
E2 Synthesis by the undifferentiated gonad
There is evidence from a stop (by cyp19a1knockout) and recovery (through compensation) experiment where E2 can rescue the sex ratio altered due to the gonadal aromatase gene knockout suggesting that E2 depletion is necessary for the subsequent key events to occur.
Differentiation to Testis
Biological plausibility provides strong support for the essentiality of this event for the subsequent key events to occur.
Male Biased Sex Ratio
Breeding females (and both sexes) are necessary for population sustainability. A male biased sex population suggests a reduced offspring production and consequentially reduced population sustainability.
Aromatase is the key enzyme in the conversion of C19 androgens to C18 estrogens and the biological plausibility linking aromatase inhibition to E2 reduction is very solid. Additionally, the role of E2 as a major regulator for downstream estrogen-responsive genes necessary for proper female gonad development is well documented in literature (Gorelick et al. 2011; Guiguen et al. 2010). The link between E2 reduction for the undifferentiated gonad leading to an increased differentiation to testis is highly plausible. As the levels of estradiol are reduced, ER responsive genes required for proper ovarian differentiation will be downregulated in the bipotential gonad and instead allowing gene expression that leads to the morphological development of the testes due to an imbalance in the androgen to estrogen ratio (Shi et al., 2018; Yin et al. 2017; Zhang et al. 2017). Therefore, it is plausible that estradiol reduction in the undifferentiated gonad at the onset of sexual differentiation promotes testis differentiating in a concentration dependent manner (Baumann et al., 2015; Morthorst et al., 2010). The direct link between increased differentiation to testis leading to a male biased sex ratio is also well supported by biological plausibility. If the conditions that favored a male producing phenotype (in this case, the aromatase inhibitor) overlap with the critical period of sex differentiation in a given population, it is reasonable that more male offspring will be produced (D'Cotta et al., 2001, Kwon et al., 2000; Luzio et al. 2016). Therefore, persistence of such conditions for repeated or prolong periods of times within the habitat of given species, can result in a male-biased population. Empirical evidence supporting the direct link between male biased population and a reduced population sustainability in fish species is lacking. However, increasing or permanent biased sex ratios can definitely have significant effects in sustainable fish populations (Marty et al. 2017). A male-biased sex ratio already suggests that the number of breeding females is reduced. If the male-biased sex ratio persists and/or increases over time, the offspring production for such population could eventually decrease and consequently, population productivity would be reduced (Brown et al. 2015; Grayson et al. 2014).
Temporal concordance of the AOP from aromatase inhibition to decreased E2 production, increased differentiation to testes and increased male-biased sex ratio (e.g., (Ruksana et al., 2010; Yin et al. 2017; Zhang et al. 2017) has been established. However, beyond that key event, temporal concordance has not yet been established possibly due limiting capability to test and/or document particular population viability in situ. From the evidence gathered for this specific AOP, the best wat to determine population viability is via multifactorial population viability analyses that generate the distribution of likely fates for a population exposed to endocrine disrupting chemicals that affect aromatase activity at the developmental stage.