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Relationship: 2167

Title

A descriptive phrase which clearly defines the two KEs being considered and the sequential relationship between them (i.e., which is upstream, and which is downstream). More help

Inhibition, Aromatase leads to Increased, Differentiation to Testis

Upstream event

The causing Key Event (KE) in a Key Event Relationship (KER). More help

Inhibition, Aromatase

Downstream event

The responding Key Event (KE) in a Key Event Relationship (KER). More help

Increased, Differentiation to Testis

Key Event Relationship Overview

The utility of AOPs for regulatory application is defined, to a large extent, by the confidence and precision with which they facilitate extrapolation of data measured at low levels of biological organisation to predicted outcomes at higher levels of organisation and the extent to which they can link biological effect measurements to their specific causes. Within the AOP framework, the predictive relationships that facilitate extrapolation are represented by the KERs. Consequently, the overall WoE for an AOP is a reflection in part, of the level of confidence in the underlying series of KERs it encompasses. Therefore, describing the KERs in an AOP involves assembling and organising the types of information and evidence that defines the scientific basis for inferring the probable change in, or state of, a downstream KE from the known or measured state of an upstream KE. More help

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	non-adjacent	High		Kelvin Santana Rodriguez (send email)	Under Development: Contributions and Comments Welcome

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) that help to define the biological applicability domain of the KER.In general, this will be dictated by the more restrictive of the two KEs being linked together by the KER. More help

Term	Scientific Term	Evidence	Link
zebrafish	Danio rerio	High	NCBI
Oreochromis niloticus	Oreochromis niloticus	High	NCBI
red-eared slider	Trachemys scripta	Low	NCBI
African clawed frog	Xenopus laevis	Low	NCBI
Gallus gallus	Gallus gallus	Low	NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help

Sex	Evidence
Unspecific	High

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER. More help

Term	Evidence
Development	High

Key Event Relationship Description

Provides a concise overview of the information given below as well as addressing details that aren’t inherent in the description of the KEs themselves. More help

Prior to sex determination, many vertebrates have a bipotential gonad that can develop into testis or ovary depending on genetic makeup (genetic sex determination), environmental conditions during development (environmental sex determination) or a combination of both (Trukhina et al. 2013).

A key variable influencing gonad differentiation is the production of sex steroids such as 17ß-estradiol (E2) and testosterone (T). In many vertebrates, including a variety of fish species, the "default" gonadal sex is male, with the presence of E2 (or perhaps the relative relationship between E2 and T production/levels) controlling the alternative path to development of ovaries (Angelopoulou et al. 2012).

Cytochrome P450 aromatase (CYP191a) is the enzyme responsible for the conversion of T to E2 in gonadal tissues of vertebrates (Miller 1988; Simpson et al. 1994). Consequently, inhibition of CYP191a expression/activity during gonadal differentiation can lead to an increased occurrence of testis.

Evidence Collection Strategy

Include a description of the approach for identification and assembly of the evidence base for the KER. For evidence identification, include, for example, a description of the sources and dates of information consulted including expert knowledge, databases searched and associated search terms/strings. Include also a description of study screening criteria and methodology, study quality assessment considerations, the data extraction strategy and links to any repositories/databases of relevant references.Tabular summaries and links to relevant supporting documentation are encouraged, wherever possible. More help

Evidence Supporting this KER

Addresses the scientific evidence supporting KERs in an AOP setting the stage for overall assessment of the AOP. More help

See below.

Biological Plausibility

Addresses the biological rationale for a connection between KEupstream and KEdownstream. This field can also incorporate additional mechanistic details that help inform the relationship between KEs, this is useful when it is not practical/pragmatic to represent these details as separate KEs due to the difficulty or relative infrequency with which it is likely to be measured. More help

Plausibility is high. CYP19a1 aromatase is rate-limiting for the synthesis of E2 in vertebrates (Simpson et al. 1994; Payne et al. 2004), so inhibition of the enzyme reduces E2 levels. Gonadal differentiation of many non-mammalian vertebrates, including a number of fish species, is dependent upon signaling associated with the sex steroids T and E2 (Guiguen et al. 2010; Nakamura 2010). In many of these species there exists a bipotential gonad during early development that, based on steroidal signaling, can differentiate into either testis of ovary. When the "default" differentiation pathway is to testis, as is often the case (Angelopoulou et al. 2012), decreases in E2 plausibly favor the development of testis.

Empirical Evidence

Provides specific (citable) evidence that supports the idea of a change in the upstream KE (KEupstream) leading to, or being associated with, a subsequent change in the downstream KE (KEdownstream), assuming the perturbation of KEupstream is sufficient. More help

There is empirical evidence in several species representing different vertebrate classes that aromatase inhibition leads to increased differentiation to testis.

Fish

An established chemical inhibitor of CYP19a1, fadrozole, has been shown to cause a concentration-dependent inhibition of aromatase activity in zebrafish (Danio rerio) during gonadal differentiation resulting in a shift towards male development (Frenske et al. 2004; Luzio et al. 2015; Luzio et al. 2016; Muth-Köhne et al. 2016; Luzio et al. 2016)
Generation of cyp19a1a (brain form of aromatase) and cyp19a1b (gondadal form of aromatase) gene mutant lines and a cyp19a1a;cyp19a1b double knockout line in zebrafish using transcription activator like effector nucleases (TALENs) has shown that cyp19a1a mutants and cyp19a1a;cyp19a1b double mutants result in all male gonadal phenotypes (Lau et al. 2016; Yin et al. 2017). This was characterized by a high number of apoptotic cells and stromal cells by 29 days post fertilization (dpf) and by 40 dpf a typical testicular structure had appeared showing cystic spermatogeic cells.
Nile tilapia (Oreochromis niloticus) treated with the aromatase inhibitor exemestane during sexual differentiation (from 9 through 35 dph) all had well developed testes by 120 dph (Ruksana et al., 2010)
Additional studies with the Nile tilapia have shown that aromatase repression in the gonad is required to favor sexual differentiation to testis (Kwon et al., 2000; D’Cotta et al., 2001; Kwon et al. 2001)

Birds

Studies with chicken (Gallus g. domesticus) embryos with the aromatase inhibitor letrozole on the first day of embryonic development has shown that the gonad of genetic females had poorly developed seminiferous tubules suggesting that they had undergone testicular sexual differentiation (Trukhina et al. 2016).
Gonads of genetic female chickens treated at embryonic day 3.5 with the aromatase inhibitor Fadrozole were masculinized by the embryonic day 9.5 (Bannister et al., 2011).

Reptiles

Administration of aromatase inhibitors CGS16949A and CGS20267 to red-eared slider turtle (Trachemys scripta) eggs incubated at female producing temperatures resulted in all male offspring (Crews and Bergeron 1994).

Amphibians

In vitro exposure of Xenopus laevis (African clawed frog) gonads treated with the aromatase inhibitor CGS 16949A resulted in histological characteristics indicative of a male phenotype (Miyata and Kubo 2000).

Uncertainties and Inconsistencies

Addresses inconsistencies or uncertainties in the relationship including the identification of experimental details that may explain apparent deviations from the expected patterns of concordance. More help

As noted below, it is uncertain as to the full range of species this key event relationship might be applicable due to susbtantial taxonomic variation in the role that steroid signaling plays in gonadal differentiation.

Known modulating factors

This table captures specific information on the MF, its properties, how it affects the KER and respective references.1.) What is the modulating factor? Name the factor for which solid evidence exists that it influences this KER. Examples: age, sex, genotype, diet 2.) Details of this modulating factor. Specify which features of this MF are relevant for this KER. Examples: a specific age range or a specific biological age (defined by...); a specific gene mutation or variant, a specific nutrient (deficit or surplus); a sex-specific homone; a certain threshold value (e.g. serum levels of a chemical above...) 3.) Description of how this modulating factor affects this KER. Describe the provable modification of the KER (also quantitatively, if known). Examples: increase or decrease of the magnitude of effect (by a factor of...); change of the time-course of the effect (onset delay by...); alteration of the probability of the effect; increase or decrease of the sensitivity of the downstream effect (by a factor of...) 4.) Provision of supporting scientific evidence for an effect of this MF on this KER. Give a list of references. More help

There are almost certainly many factors that could modulate this KER, but a systematic description of these is not currently possible.

Quantitative Understanding of the Linkage

Captures information that helps to define how much change in the upstream KE, and/or for how long, is needed to elicit a detectable and defined change in the downstream KE. More help

There are too few data to develop a quatitative understanding of the linkage between aromatase inhibition and increased differentiation to testis.

Response-response Relationship

Provides sources of data that define the response-response relationships between the KEs. More help

Not applicable.

Time-scale

Information regarding the approximate time-scale of the changes in KEdownstream relative to changes in KEupstream (i.e., do effects on KEdownstream lag those on KEupstream by seconds, minutes, hours, or days?). More help

The timeframe for differentiation of the bipotential gonad is species-dependent occurring, for example, over the course of days to weeks in most fishes. However, this period of time could be substantially longer in long-lived species.

Known Feedforward/Feedback loops influencing this KER

Define whether there are known positive or negative feedback mechanisms involved and what is understood about their time-course and homeostatic limits. More help

None known.

Domain of Applicability

A free-text section of the KER description that the developers can use to explain their rationale for the taxonomic, life stage, or sex applicability structured terms. More help

Life Stage

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.

Sex

Because this KER occurs during differentiation, the relationship is relevant to animals with an undetermined (non-specific) sex.

Taxonomic Applicability

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 among species.

References

List of the literature that was cited for this KER description. More help

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

Bannister, S. C., Smith, C. A., Roeszler, K. N., Doran, T. J., Sinclair, A. H., & Tizard, M. L. (2011). Manipulation of estrogen synthesis alters MIR202* expression in embryonic chicken gonads. Biology of reproduction, 85(1), 22–30. https://doi.org/10.1095/biolreprod.110.088476

Crews, D., & Bergeron, J. M. (1994). Role of reductase and aromatase in sex determination in the red-eared slider (Trachemys scripta), a turtle with temperature-dependent sex determination. The Journal of endocrinology, 143(2), 279–289. https://doi.org/10.1677/joe.0.1430279

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

Fenske, M. & Segner, H. (2004). Aromatase modulation alters gonadal differentiation in developing zebrafish (Danio rerio). Aquatic toxicology (Amsterdam, Netherlands). 67. 105-26. DOI 10.1016/j.aquatox.2003.10.008.

Guiguen, Y., Fostier, A., Piferrer, F., & Chang, C. F. (2010). Ovarian aromatase and estrogens: a pivotal role for gonadal sex differentiation and sex change in fish. General and comparative endocrinology, 165(3), 352–366. https://doi.org/10.1016/j.ygcen.2009.03.002

Kwon, J. Y., Haghpanah, V., Kogson-Hurtado, L. M., McAndrew, B. J., & Penman, D. J. (2000). Masculinization of genetic female nile tilapia (Oreochromis niloticus) by dietary administration of an aromatase inhibitor during sexual differentiation. The Journal of experimental zoology, 287(1), 46–53.

Kwon, J. Y., McAndrew, B. J., & Penman, D. J. (2001). Cloning of brain aromatase gene and expression of brain and ovarian aromatase genes during sexual differentiation in genetic male and female Nile tilapia Oreochromis niloticus. Molecular reproduction and development, 59(4), 359–370. https://doi.org/10.1002/mrd.1042

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.

Lau, E. S., Zhang, Z., Qin, M., & Ge, W. (2016). Knockout of Zebrafish Ovarian Aromatase Gene (cyp19a1a) by TALEN and CRISPR/Cas9 Leads to All-male Offspring Due to Failed Ovarian Differentiation. Scientific reports, 6, 37357. https://doi.org/10.1038/srep37357

Luzio, A.,Monteiro, S., Garcia Santos, S., Rocha, E., Fontainhas-Fernandes, A.,& Coimbra, A. (2015). Zebrafish sex differentiation and gonad development after exposure to 17α-ethinylestradiol, fadrozole and their binary mixture: A stereological study. Aquatic Toxicology. 166. 83-95. DOI 10.1016/j.aquatox.2015.07.015.

Luzio, A., Matos, M., Santos, D., Fontaínhas-Fernandes, A. A., Monteiro, S. M., & Coimbra, A. M. (2016). Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures. Aquatic toxicology (Amsterdam, Netherlands), 177, 269–284. https://doi.org/10.1016/j.aquatox.2016.05.029

Luzio, A., Monteiro, S. M., Rocha, E., Fontaínhas-Fernandes, A. A., & Coimbra, A. M. (2016). Development and recovery of histopathological alterations in the gonads of zebrafish (Danio rerio) after single and combined exposure to endocrine disruptors (17α-ethinylestradiol and fadrozole). Aquatic toxicology (Amsterdam, Netherlands), 175, 90–105. https://doi.org/10.1016/j.aquatox.2016.03.014

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

Miyata, S., & Kubo, T. (2000). In vitro effects of estradiol and aromatase inhibitor treatment on sex differentiation in Xenopus laevis gonads. General and comparative endocrinology, 119(1), 105–110. https://doi.org/10.1006/gcen.2000.7497

Muth-Köhne, E., Westphal-Settele, K., Brückner, J., Konradi, S., Schiller, V., Schäfers, C., Teigeler, M., & Fenske, M. (2016). Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test. Aquatic toxicology (Amsterdam, Netherlands), 176, 116–127. https://doi.org/10.1016/j.aquatox.2016.04.018

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

Ruksana, S., Pandit, N. P., & Nakamura, M. (2010). Efficacy of exemestane, a new generation of aromatase inhibitor, on sex differentiation in a gonochoristic fish. Comparative biochemistry and physiology. Toxicology & pharmacology : CBP, 152(1), 69–74. https://doi.org/10.1016/j.cbpc.2010.02.014

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

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

Trukhina, A. V., Lukina, N. A., Wackerow-Kouzova, N. D., & Smirnov, A. F. (2013). The variety of vertebrate mechanisms of sex determination. BioMed research international, 2013, 587460. https://doi.org/10.1155/2013/587460

Trukhina, Antonina & Lukina, Natalia & Smirnov, Aleksandr. (2016). Experimental Sex Inversion of Chicken Embryos at Aromatase Inhibition, Estrogen Receptor Modulation, DNA Demethylation and Progesterone Treatment. Natural Science. 08. 451-459. 10.4236/ns.2016.811047.

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