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Event: 3

Key Event Title

The KE title should describe a discrete biological change that can be measured. It should generally define the biological object or process being measured and whether it is increased, decreased, or otherwise definably altered relative to a control state. For example “enzyme activity, decreased”, “hormone concentration, increased”, or “growth rate, decreased”, where the specific enzyme or hormone being measured is defined. More help

Reduction, 17beta-estradiol synthesis by ovarian granulosa cells

Short name
The KE short name should be a reasonable abbreviation of the KE title and is used in labelling this object throughout the AOP-Wiki. The short name should be less than 80 characters in length. More help
Reduction, 17beta-estradiol synthesis by ovarian granulosa cells

Biological Context

Structured terms, selected from a drop-down menu, are used to identify the level of biological organization for each KE. Note, KEs should be defined within a particular level of biological organization. Only KERs should be used to transition from one level of organization to another. Selection of the level of biological organization defines which structured terms will be available to select when defining the Event Components (below). More help
Level of Biological Organization
Cellular

Cell term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help
Cell term
granulosa cell

Organ term

Further information on Event Components and Biological Context may be viewed on the attached pdf.The biological context describes the location/biological environment in which the event takes place.  For molecular/cellular events this would include the cellular context (if known), organ context, and species/life stage/sex for which the event is relevant. For tissue/organ events cellular context is not applicable.  For individual/population events, the organ context is not applicable. More help

Key Event Components

Further information on Event Components and Biological Context may be viewed on the attached pdf.Because one of the aims of the AOP-KB is to facilitate de facto construction of AOP networks through the use of shared KE and KER elements, authors are also asked to define their KEs using a set of structured ontology terms (Event Components). In the absence of structured terms, the same KE can readily be defined using a number of synonymous titles (read by a computer as character strings). In order to make these synonymous KEs more machine-readable, KEs should also be defined by one or more “event components” consisting of a biological process, object, and action with each term originating from one of 22 biological ontologies (Ives, et al., 2017; See List). Biological process describes dynamics of the underlying biological system (e.g., receptor signalling). The biological object is the subject of the perturbation (e.g., a specific biological receptor that is activated or inhibited). Action represents the direction of perturbation of this system (generally increased or decreased; e.g., ‘decreased’ in the case of a receptor that is inhibited to indicate a decrease in the signalling by that receptor).Note that when editing Event Components, clicking an existing Event Component from the Suggestions menu will autopopulate these fields, along with their source ID and description. To clear any fields before submitting the event component, use the 'Clear process,' 'Clear object,' or 'Clear action' buttons. If a desired term does not exist, a new term request may be made via Term Requests. Event components may not be edited; to edit an event component, remove the existing event component and create a new one using the terms that you wish to add. More help
Process Object Action
estrogen biosynthetic process 17beta-estradiol decreased

Key Event Overview

AOPs Including This Key Event

All of the AOPs that are linked to this KE will automatically be listed in this subsection. This table can be particularly useful for derivation of AOP networks including the KE. Clicking on the name of the AOP will bring you to the individual page for that AOP. More help
AOP Name Role of event in AOP Point of Contact Author Status OECD Status
Aromatase (Cyp19a1) reduction leading to reproductive toxicity KeyEvent Elise Grignard (send email) Open for citation & comment EAGMST Under Review
Aromatase inhibition leading to reproductive dysfunction KeyEvent Dan Villeneuve (send email) Open for citation & comment TFHA/WNT Endorsed
Androgen receptor agonism leading to reproductive dysfunction KeyEvent Dan Villeneuve (send email) Open for citation & comment TFHA/WNT Endorsed
Prolyl hydroxylase inhibition KeyEvent Dalma Martinovic-Weigelt (send email) Under Development: Contributions and Comments Welcome
Unknown MIE leading to reprodl KeyEvent Dalma Martinovic-Weigelt (send email) Under Development: Contributions and Comments Welcome
AHR mediated epigenetic reproductive failure KeyEvent Jon Doering (send email) Under development: Not open for comment. Do not cite

Stressors

This is a structured field used to identify specific agents (generally chemicals) that can trigger the KE. Stressors identified in this field will be linked to the KE in a machine-readable manner, such that, for example, a stressor search would identify this as an event the stressor can trigger. NOTE: intermediate or downstream KEs in one AOP may function as MIEs in other AOPs, meaning that stressor information may be added to the KE description, even if it is a downstream KE in the pathway currently under development.Information concerning the stressors that may trigger an MIE can be defined using a combination of structured and unstructured (free-text) fields. For example, structured fields may be used to indicate specific chemicals for which there is evidence of an interaction relevant to this MIE. By linking the KE description to a structured chemical name, it will be increasingly possible to link the MIE to other sources of chemical data and information, enhancing searchability and inter-operability among different data-sources and knowledgebases. The free-text section “Evidence for perturbation of this MIE by stressor” can be used both to identify the supporting evidence for specific stressors triggering the MIE as well as to define broad chemical categories or other properties that classify the stressors able to trigger the MIE for which specific structured terms may not exist. More help

Taxonomic Applicability

Latin or common names of a species or broader taxonomic grouping (e.g., class, order, family) can be selected from an ontology. In many cases, individual species identified in these structured fields will be those for which the strongest evidence used in constructing the AOP was available in relation to this KE. More help
Term Scientific Term Evidence Link
fathead minnow Pimephales promelas High NCBI
Fundulus heteroclitus Fundulus heteroclitus High NCBI

Life Stages

The structured ontology terms for life-stage are more comprehensive than those for taxa, but may still require further description/development and explanation in the free text section. More help
Life stage Evidence
Adult, reproductively mature High

Sex Applicability

No help message More help
Term Evidence
Female High

Key Event Description

A description of the biological state being observed or measured, the biological compartment in which it is measured, and its general role in the biology should be provided. For example, the biological state being measured could be the activity of an enzyme, the expression of a gene or abundance of an mRNA transcript, the concentration of a hormone or protein, neuronal activity, heart rate, etc. The biological compartment may be a particular cell type, tissue, organ, fluid (e.g., plasma, cerebrospinal fluid), etc. The role in the biology could describe the reaction that an enzyme catalyses and the role of that reaction within a given metabolic pathway; the protein that a gene or mRNA transcript codes for and the function of that protein; the function of a hormone in a given target tissue, physiological function of an organ, etc. Careful attention should be taken to avoid reference to other KEs, KERs or AOPs. Only describe this KE as a single isolated measurable event/state. This will ensure that the KE is modular and can be used by other AOPs, thereby facilitating construction of AOP networks. More help

Like all steroids, estradiol is a cholesterol derivative. Estradiol synthesis in ovary is mediated by a number of enzyme catalyzed reactions involving cyp11 (cholesterol side chain cleavage enzyme), cyp 17 (17alpha-hydroxylase/17,20-lyase), 3beta hydroxysteroid dehyrogenase, 17beta hydroxysteroid dehydrogenase, and cyp19 (aromatase). Among those enzyme catalyzed reactions, conversion of testosterone to estradiol, catalyzed by aromatase, is considered to be rate limiting for estradiol synthesis. Within the ovary, aromatase expression and activity is primarily localized in the granulosa cells (reviewed in (Norris 2007; Yaron 1995; Havelock et al. 2004) and others). Reactions involved in synthesis of C-19 androgens are primarily localized in the theca cells and C-19 androgens diffuse from the theca into granulosa cells where aromatase can catalyze their conversion to C-18 estrogens.

How It Is Measured or Detected

One of the primary considerations in evaluating AOPs is the relevance and reliability of the methods with which the KEs can be measured. The aim of this section of the KE description is not to provide detailed protocols, but rather to capture, in a sentence or two, per method, the type(s) of measurements that can be employed to evaluate the KE and the relative level of scientific confidence in those measurements. Methods that can be used to detect or measure the biological state represented in the KE should be briefly described and/or cited. These can range from citation of specific validated test guidelines, citation of specific methods published in the peer reviewed literature, or outlines of a general protocol or approach (e.g., a protein may be measured by ELISA).Key considerations regarding scientific confidence in the measurement approach include whether the assay is fit for purpose, whether it provides a direct or indirect measure of the biological state in question, whether it is repeatable and reproducible, and the extent to which it is accepted in the scientific and/or regulatory community. Information can be obtained from the OECD Test Guidelines website and the EURL ECVAM Database Service on Alternative Methods to Animal Experimentation (DB-ALM). ?

Due to the importance of both theca and granulosa cells in ovarian steroidogenesis, it is generally impractical to measure E2 production by isolated granulosa cells (Havelock et al. 2004). However, this key event can be evaluated by examining E2 production by intact ovarian tissue explants either exposed to chemicals in vitro (e.g., (Villeneuve et al. 2007; McMaster ME 1995) or in vivo (i.e., via ex vivo steroidogenesis assay; e.g., (Ankley et al. 2007)). Estradiol released by ovarian tissue explants into media can be quantified by radioimmunoassay (e.g., Jensen et al. 2001), ELISA, or analytical methods such as LC-MS (e.g., Owen et al. 2014).

OECD TG 456 (OECD 2011) is the validated test guideline for an in vitro screen for chemical effects on steroidogenesis, specifically the production of 17ß-estradiol (E2) and testosterone (T).

The synthesis of E2 can be measured in vitro cultured ovarian cells. The methods for culturing mammalian ovarian cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM): Culture of Human Cumulus Granulosa Cells (EURL ECVAM Protocol No. 92), Granulosa and Theca Cell Culture Systems (EURL ECVAM Method Summary No. 92).

Domain of Applicability

This free text section should be used to elaborate on the scientific basis for the indicated domains of applicability and the WoE calls (if provided). While structured terms may be selected to define the taxonomic, life stage and sex applicability (see structured applicability terms, above) of the KE, the structured terms may not adequately reflect or capture the overall biological applicability domain (particularly with regard to taxa). Likewise, the structured terms do not provide an explanation or rationale for the selection. The free-text section on evidence for taxonomic, life stage, and sex applicability can be used to elaborate on why the specific structured terms were selected, and provide supporting references and background information.  More help

Key enzymes needed to synthesize 17β-estradiol first appear in the common ancestor of amphioxus and vertebrates (Markov et al. 2009; Baker 2011). Consequently, it is plausible that this key event is applicable to most vertebrates. This key event is not applicable to invertebrates, which lack the enzymes required to synthesize 17ß-estradiol.

References

List of the literature that was cited for this KE description. Ideally, the list of references, should conform, to the extent possible, with the OECD Style Guide (https://www.oecd.org/about/publishing/OECD-Style-Guide-Third-Edition.pdf) (OECD, 2015). More help
  • Ankley GT, Jensen KM, Kahl MD, Makynen EA, Blake LS, Greene KJ, et al. 2007. Ketoconazole in the fathead minnow (Pimephales promelas): reproductive toxicity and biological compensation. Environ Toxicol Chem 26(6): 1214-1223.
  • Baker ME. 2011. Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors. Molecular and cellular endocrinology 334(1-2): 14-20.
  • EURL ECVAM Method Summary no 92. Granulosa and Theca Cell Culture Systems - Summary
  • EURL ECVAM Protocol no 92 Culture of Human Cumulus Granulosa Cells. Primary cell culture method. Contact Person: Dr. Mahadevan Maha M.
  • Havelock JC, Rainey WE, Carr BR. 2004. Ovarian granulosa cell lines. Molecular and cellular endocrinology 228(1-2): 67-78.
  • Jensen K, Korte J, Kahl M, Pasha M, Ankley G. 2001. Aspects of basic reproductive biology and endocrinology in the fathead minnow (Pimephales promelas). Comparative Biochemistry and Physiology Part C 128: 127-141.
  • McMaster ME MK, Jardine JJ, Robinson RD, Van Der Kraak GJ. 1995. Protocol for measuring in vitro steroid production by fish gonadal tissue. Canadian Technical Report of Fisheries and Aquatic Sciences 1961 1961: 1-78.
  • Norris DO. 2007. Vertebrate Endocrinology. Fourth ed. New York: Academic Press.
  • OECD (2011), Test No. 456: H295R Steroidogenesis Assay, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris.DOI: http://dx.doi.org/10.1787/9789264122642-en
  • Owen LJ, Wu FC, Keevil BG. 2014. A rapid direct assay for the routine measurement of oestradiol and oestrone by liquid chromatography tandem mass spectrometry. Ann. Clin. Biochem. 51(pt 3):360-367.
  • Villeneuve DL, Ankley GT, Makynen EA, Blake LS, Greene KJ, Higley EB, et al. 2007. Comparison of fathead minnow ovary explant and H295R cell-based steroidogenesis assays for identifying endocrine-active chemicals. Ecotoxicol Environ Saf 68(1): 20-32.
  • Villeneuve DL, Mueller ND, Martinovic D, Makynen EA, Kahl MD, Jensen KM, et al. 2009. Direct effects, compensation, and recovery in female fathead minnows exposed to a model aromatase inhibitor. Environ Health Perspect 117(4): 624-631.
  • Yaron Z. 1995. Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture 129: 49-73.