This Key Event Relationship is licensed under the Creative Commons BY-SA license. This license allows reusers to distribute, remix, adapt, and build upon the material in any medium or format, so long as attribution is given to the creator. The license allows for commercial use. If you remix, adapt, or build upon the material, you must license the modified material under identical terms.

Relationship: 2146


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

Increased, Differentiation to Testis leads to Increased, Male Biased Sex Ratio

Upstream event
The causing Key Event (KE) in a Key Event Relationship (KER). More help
Downstream event
The responding Key Event (KE) in a Key Event Relationship (KER). More help

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 adjacent High Kelvin Santana Rodriguez (send email) Under Development: Contributions and Comments Welcome WPHA/WNT Endorsed
Androgen receptor agonism leading to male-biased sex ratio adjacent Dan Villeneuve (send email) Open for citation & comment WPHA/WNT Endorsed

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
Odontesthes bonariensis Odontesthes bonariensis Low NCBI
Oreochromis niloticus Oreochromis niloticus NCBI
zebrafish Danio rerio High NCBI
fathead minnow Pimephales promelas Low NCBI

Sex Applicability

An indication of the the relevant sex for this KER. More help
Sex Evidence
Male Moderate

Life Stage Applicability

An indication of the the relevant life stage(s) for this KER.  More help
Term Evidence
Juvenile Moderate
Development Moderate

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 gonadal sex determination in vertebrates, the developing organism has a primordial bipotential gonad that can be fated to either sex depending on the genetic makeup of the embryo (genetic sex determination; GSD) or environmental conditions (environmental sex determination; ESD) or a combination of both factors.

Regardless of whether gonadal development is controlled via GSD or ESD (or both), the operational definition of male versus female in terms of function usually is defined by the presence, respectively, of testes versus ovaries. For species exhibiting sex-specific secondary sexual characteristics preferential differentiation to testis can be accompanied by easily discerned external phenotypic changes as well. If there is increased differentiation to testis in individuals of a population of organisms this will by default produce a male biased sex ratio as defined by what would be considered normal for that species. 

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

It is highly plausible that as a gonadal phenotype increases toward testis formation, male-biased sex ratios in a defined cohort of organisms will occur. If this condition persists for repeated or prolonged periods of times within the habitat of given species, this will result in a male-biased sex ratio.

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

A major uncertainty for this KER involves what would be defined as "normal" for degree of testis differentiation and by extension sex ratio. There needs to be knowledge as to baseline expectations for testis differentiation for a given species in a given habitiat (or lab setting) to ascertain whether increases are occurring. Baseline information of this type is available or can be inferred for some species but certainly not for all that might be considered.

A second significant uncertainty involves situations where the gonad cannot be clearly defined as either testis or ovary. This can occur in some fish and amphibian species, where the gonad has cell types indicative of both testes and ovaries (Abdul-moneim et al. 2015). In these instances classification of individuals as male versus female may not be possible, requiring a third category related to an intersex condition. There are seemingly multiple underlying causes of intersex, one of which appears to be exposure to estrogenic chemicals during gonad differentiation (Jobling et al. 1998; Norris et al. 2018; Grim et al. 2020).

A third uncertainty involves whether all individuals defined as males based on gonad phenotype will have the same degree of function in terms of producing viable gametes. It is possible, for example, that genotypic females which develop a male phenotype due to an environmental factor such as exposure to an endocrine-active chemical may not be functionally equivalent to a genetic male relative to sperm production/viability. This could be an important consideration relative to the types of predictions attempted based on a male-biased sex ratio in a population.

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

Not applicable.

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

Not applicable.

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

Timescales will vary based on species-specific developmental rates, but since one KE often will define the second (i.e., an animal is defined as a male based on the presence of testis) timescale may not be a relevant consideration.  

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

Not applicable. 

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

This KER is applicable to any species in which males are defined by the occurrence of testis and/or associated male secondary sexual characteristics. 


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

Abul-moneim, A, DP Coulter, CT Mahapatra and MS Sepulveda. 2015. Intersex in fishes and amphibians: Population implications, prevalance, mechanisms and molecular biomarkers. J Appl Toxicol 35:1228-1240.

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. 

Grim, KC, EE Henderson, MJ Wolfe and JC Wolfe. 2020. Histopathological prevalance and severity of testicualr oocytes in smallmouth bass from two archival collections. J Aquat Anim Health 32:32-43.

Jobling, M Nolan, CR Tyler, G. Brighty and JP Sumpter. 1998. Widespread sexual disruption in wild fish. Environ Sci Technol 32:2498-2506.

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. (2016a). Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures. Aquatic toxicology (Amsterdam, Netherlands), 177, 269–284.

Luzio, A., Monteiro, S. M., Rocha, E., Fontaínhas-Fernandes, A. A., & Coimbra, A. M. (2016b). 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.

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.

Norris, DO, AL Bolden and AM Vajda. 2018. The occurrence of intersex fishes in Boulder Creek, Colorado is a recent phenomenon. Gen. Comp. Endocrinol. 265:56-60.

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.

Shen ZG, Fan QX, Yang W, Zhang YL, Hu PP, Xie CX. Effects of non-steroidal aromatase inhibitor letrozole on sex inversion and spermatogenesis in yellow catfish Pelteobagrus fulvidraco. Biol Bull. 2013 Sep;225(1):18-23. doi: 10.1086/BBLv225n1p18. PMID: 24088793.

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., and Michael, M. D. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine reviews15(3), 342–355.

Uchida, D., Yamashita, M., Kitano, T., & Iguchi, T. (2004). An aromatase inhibitor or high water temperature induce oocyte apoptosis and depletion of P450 aromatase activity in the gonads of genetic female zebrafish during sex-reversal. Comparative biochemistry and physiology. Part A, Molecular & integrative physiology, 137(1), 11–20.