Key Event Title
|Level of Biological Organization|
Key Event Components
|estrogen biosynthetic process||17beta-estradiol||decreased|
Key Event Overview
AOPs Including This Key Event
|AOP Name||Role of event in AOP|
|Aromatase (Cyp19a1) reduction leading to reproductive toxicity||KeyEvent|
|Aromatase inhibition leading to reproductive dysfunction||KeyEvent|
|Androgen receptor agonism leading to reproductive dysfunction||KeyEvent|
|Prolyl hydroxylase inhibition||KeyEvent|
|Unknown MIE leading to reprodl||KeyEvent|
|AHR mediated epigenetic reproductive failure||KeyEvent|
|fathead minnow||Pimephales promelas||High||NCBI|
|Fundulus heteroclitus||Fundulus heteroclitus||High||NCBI|
|Adult, reproductively mature||High|
Key Event Description
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
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
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.
- 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.