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Inhibition, Aromatase leads to Reduction, E2 Synthesis by the undifferentiated gonad
Key Event Relationship Overview
AOPs Referencing Relationship
|AOP Name||Adjacency||Weight of Evidence||Quantitative Understanding||Point of Contact||Author Status||OECD Status|
|Aromatase inhibition leads to male-biased sex ratio via impacts on gonad differentiation||adjacent||High||Kelvin Santana Rodriguez (send email)||Under Development: Contributions and Comments Welcome|
Life Stage Applicability
|before or during gonadal sex differentiation||High|
Key Event Relationship Description
Aromatase (cyp191a) is a cytochrome P450-based enzyme that is rate limiting for the synthesis of 17ß-estradiol (E2) from testosterone in vertebrates (Simpson et al. 1994; Miller 1988; Payne and Hale 2004). The expression and activity of aromatase in the bipotential gonad of developing organisms, and subsequent autocrine and/or paracrine signaling mediated by E2 interactions with the estrogen receptor (or lack thereof), are thought to be key regulators of sex determination and gonadal differentation in vertebrates (Angelopoulou et al. 2012; Nakamura 2010).
Evidence Collection Strategy
Evidence Supporting this KER
There is little direct evidence of E2 production by the bipotential gonad, or that inhibition of aromatase decreases in E2 production in same. However, given the well-established role of aromatase in E2 production (Simpson et al. 1994; Payne and Hale, 2004) and the close association between aromatase expression and activity and gonadal sex determination/differentiation (Angelopoulou et al. 2012; Nakamura 2010), it is highly plausible that local estrogen production in the bipotential gonad plays a significant role in gonadal differentiation. However, particularly for species with genetic sex determination, it is just one of multiple determinants that ultimately influences differentiation of the gonad (Angelopoulou et al. 2012).
Multiple lines of empirical evidence support a link between aromatase inhibition and decreased E2 synthesis in bipotential gonads of developing fish.
- In Nile Tilapia (Oreochromis niloticus) reared at the 27°C, genetic males exhibited lower levels of aromatase gene expression and E2 levels during the critical period of sexual differentiation (18-26 days post fertilization) than genetic females. This correlation suggests that aromatase repression at the onset of sexual differentiation reduces the biosynthesis of E2 in the undifferentiated gonad. (D'Cotta et al. 2001)
- Generation of cyp19a1a and cyp19a1b (gonadal and brain forms of aromatase, respectively) gene mutant lines and a cyp19a1a;cyp19a1b double knockout line in zebrafish using transcription activator like effector nucleases (TALENs) showed that in both cyp19a1a-deficient and double knockout fish, E2 levels were significantly lower than in wild-type and cyp19a1b-deficient fish (Yin et al. 2017).
- Control XY and cyp19a1a -/- (deficient and double knockout) XX Nile tilapia had significantly lower levels of serum E2 when compared to the control XX and cyp19a1a+/- XX fish suggesting a decrease in E2 due to the cyp19a1a deficiency. (Zhang et al. 2017)
Uncertainties and Inconsistencies
As noted below it is difficult to predict the full suite of vertebrate species this KER might apply to. In addition, studies directly examining synthesis of E2 by bipotential gonads in organisms exposed to aromatase inhibitors are lacking.
Known modulating factors
Aromatase expression during gonadal differentiation is subject to both environmental and genetic controls to various degrees depending on species (Angelopoulou et al. 2012, Sarre et al. 2004). However, generalizable relationships that account for effects of specific parameters in the response-response relationships underlying this KER are currently unknown.
Quantitative Understanding of the Linkage
Quantitative understanding of this linkage is currently weak.
To date, none of the studies reviewed have offered insights into the quantitative relationship between the degree of aromatase inhibition and E2 synthesis by the undifferentiated, bipotential gonad.
- Based on studies in mature adult fish (fathead minnows, Pimephales promelas) effects of model aromatase inhibitors on E2 production (e.g., plasma concentrations) can be detected within a few hours of exposure in vivo (Schroeder et al. 2017; Skolness et al. 2011).
- Based on in vitro studies, significant reductions in aromatase activity and associated E2 synthesis can be detected in 90 min or less (Villeneuve et al. 2006).
Known Feedforward/Feedback loops influencing this KER
Aromatase expression and E2 synthesis in adult fish of several species are subject to feedback regulation via the brain-pituitary-gonadal axis (e.g., Villeneuve et al. 2009; 2013; Ankley et al. 2009; Yu et al. 2020; Norris 1997; Miller 1988; Callard et al. 2001).
However, it is unclear whether these feedback mechanisms are active during gonadal differentiation.
Domain of Applicability
The life stage applicable to this KER is developing embryos and juveniles during the gonadal differentiation. This KER is not applicable to sexually differentiated adults.
Because this KER occurs during differentiation, the relationship is relevant to animals with an undetermined (non-specific) sex.
Sequencing studies studies with mammalian, amphibian, reptile, bird, and fish species have shown that aromatase is well conserved among all vertebrates (Wilson et al. 2005; LaLone et al. 2018).
However, it is difficult to predict the biological domain of applicability of this KER based on phylogenetic characteristics. There is considerable within class variability, for example, among both fish and reptile species as to the role of aromatase expression and estrogen signaling in determining gonadal sex (Angelopoulou et al. 2012; Sarre et al. 2004). Thus susceptibility and relative sensitivities may vary considerably between species.
Angelopoulou, R., Lavranos, G., & Manolakou, P. (2012). Sex determination strategies in 2012: towards a common regulatory model?. Reproductive biology and endocrinology : RB&E, 10, 13. https://doi.org/10.1186/1477-7827-10-13
Ankley, G. T., Bencic, D. C., Cavallin, J. E., Jensen, K. M., Kahl, M. D., Makynen, E. A., Martinovic, D., Mueller, N. D., Wehmas, L. C., & Villeneuve, D. L. (2009). Dynamic nature of alterations in the endocrine system of fathead minnows exposed to the fungicide prochloraz. Toxicological sciences : an official journal of the Society of Toxicology, 112(2), 344–353. https://doi.org/10.1093/toxsci/kfp227
Callard, G. V., Tchoudakova, A. V., Kishida, M., & Wood, E. (2001). Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish. The Journal of steroid biochemistry and molecular biology, 79(1-5), 305–314. https://doi.org/10.1016/s0960-0760(01)00147-9
D'Cotta, H., Fostier, A., Guiguen, Y., Govoroun, M., & Baroiller, J. F. (2001). Aromatase plays a key role during normal and temperature-induced sex differentiation of tilapia Oreochromis niloticus. Molecular reproduction and development, 59(3), 265–276. https://doi.org/10.1002/mrd.1031
LaLone, C.A., D.L. Villeneuve, J.A. Doering, B.R. Blackwell, T.R. Transue, C.W. Simmons, J. Swintek, S.J. Degitz, A.J. Williams and G.T. Ankley. 2018. Evidence for cross-species extrapolation of mammalian-based high-throughput screening assay results. Environ. Sci. Technol. 52, 13960-13971.
Miller W. L. (1988). Molecular biology of steroid hormone synthesis. Endocrine reviews, 9(3), 295–318. https://doi.org/10.1210/edrv-9-3-295
Nakamura M. (2010). The mechanism of sex determination in vertebrates-are sex steroids the key-factor?. Journal of experimental zoology. Part A, Ecological genetics and physiology, 313(7), 381–398. https://doi.org/10.1002/jez.616
Norris, D. O. Vertebrate Endocrinology, 3rd ed.; Academic Press: San Diego, CA, 1997.
Payne, A. H., & Hales, D. B. (2004). Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine reviews, 25(6), 947–970. https://doi.org/10.1210/er.2003-0030
Sarre, S. D., Georges, A., & Quinn, A. (2004). The ends of a continuum: genetic and temperature-dependent sex determination in reptiles. BioEssays : news and reviews in molecular, cellular and developmental biology, 26(6), 639–645. https://doi.org/10.1002/bies.20050
Schroeder, A. L., Ankley, G. T., Habib, T., Garcia-Reyero, N., Escalon, B. L., Jensen, K. M., Kahl, M. D., Durhan, E. J., Makynen, E. A., Cavallin, J. E., Martinovic-Weigelt, D., Perkins, E. J., & Villeneuve, D. L. (2017). Rapid effects of the aromatase inhibitor fadrozole on steroid production and gene expression in the ovary of female fathead minnows (Pimephales promelas). General and comparative endocrinology, 252, 79–87. https://doi.org/10.1016/j.ygcen.2017.07.022
Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., Graham-Lorence, S., Amarneh, B., Ito, Y., Fisher, C. R., & Michael, M. D. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine reviews, 15(3), 342–355. https://doi.org/10.1210/edrv-15-3-342
Skolness, S. Y., Durhan, E. J., Garcia-Reyero, N., Jensen, K. M., Kahl, M. D., Makynen, E. A., Martinovic-Weigelt, D., Perkins, E., Villeneuve, D. L., & Ankley, G. T. (2011). Effects of a short-term exposure to the fungicide prochloraz on endocrine function and gene expression in female fathead minnows (Pimephales promelas). Aquatic toxicology (Amsterdam, Netherlands), 103(3-4), 170–178. https://doi.org/10.1016/j.aquatox.2011.02.016
Villeneuve, D. L., Breen, M., Bencic, D. C., Cavallin, J. E., Jensen, K. M., Makynen, E. A., Thomas, L. M., Wehmas, L. C., Conolly, R. B., & Ankley, G. T. (2013). Developing predictive approaches to characterize adaptive responses of the reproductive endocrine axis to aromatase inhibition: I. Data generation in a small fish model. Toxicological sciences : an official journal of the Society of Toxicology, 133(2), 225–233. https://doi.org/10.1093/toxsci/kft068
Villeneuve, D. L., Knoebl, I., Kahl, M. D., Jensen, K. M., Hammermeister, D. E., Greene, K. J., Blake, L. S., & Ankley, G. T. (2006). Relationship between brain and ovary aromatase activity and isoform-specific aromatase mRNA expression in the fathead minnow (Pimephales promelas). Aquatic toxicology (Amsterdam, Netherlands), 76(3-4), 353–368. https://doi.org/10.1016/j.aquatox.2005.10.016
Villeneuve, D. L., Mueller, N. D., Martinović, D., Makynen, E. A., Kahl, M. D., Jensen, K. M., Durhan, E. J., Cavallin, J. E., Bencic, D., & Ankley, G. T. (2009). Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor. Environmental health perspectives, 117(4), 624–631. https://doi.org/10.1289/ehp.11891
Wilson, J. Y., McArthur, A. G., & Stegeman, J. J. (2005). Characterization of a cetacean aromatase (CYP19) and the phylogeny and functional conservation of vertebrate aromatase. General and comparative endocrinology, 140(1), 74–83. https://doi.org/10.1016/j.ygcen.2004.10.004
Yin, Y., Tang, H., Liu, Y., Chen, Y., Li, G., Liu, X., & Lin, H. (2017). Targeted Disruption of Aromatase Reveals Dual Functions of cyp19a1a During Sex Differentiation in Zebrafish. Endocrinology, 158(9), 3030–3041. https://doi.org/10.1210/en.2016-1865
Yu, Q., Peng, C., Ye, Z., Tang, Z., Li, S., Xiao, L., Liu, S., Yang, Y., Zhao, M., Zhang, Y., & Lin, H. (2020). An estradiol-17β/miRNA-26a/cyp19a1a regulatory feedback loop in the protogynous hermaphroditic fish, Epinephelus coioides. Molecular and cellular endocrinology, 504, 110689. https://doi.org/10.1016/j.mce.2019.110689
Zhang, X., Li, M., Ma, H., Liu, X., Shi, H., Li, M., & Wang, D. (2017). Mutation of foxl2 or cyp19a1a Results in Female to Male Sex Reversal in XX Nile Tilapia. Endocrinology, 158(8), 2634–2647. https://doi.org/10.1210/en.2017-00127