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Event Title

17beta-estradiol synthesis by ovarian granulosa cells, Reduction
Short name: 17beta-estradiol synthesis by ovarian granulosa cells, Reduction

Key Event Overview

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AOPs Including This Key Event

AOP Name Event Type Essentiality
Aromatase (Cyp19a1) reduction leading to impaired fertility in adult female KE Strong
Aromatase inhibition leading to reproductive dysfunction KE Strong
Androgen receptor agonism leading to reproductive dysfunction KE Moderate
Prolyl hydroxylase inhibition leading to reproductive dysfunction via increased HIF1 heterodimer formation KE Moderate
Unknown MIE leading to reproductive dysfunction via increased HIF-1alpha transcription KE

Taxonomic Applicability

Name Scientific Name Evidence Links

Level of Biological Organization

Biological Organization

How this Key Event works

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 RIA (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).

Evidence Supporting Taxonomic Applicability

Key enzymes needed to synthesize 17β-estradiol first appear in the common ancestor of amphioxus and vertebrates (Baker 2011). Consequently, it is plausible that this key event is applicable to most vertebrates.


  • Norris DO. 2007. Vertebrate Endocrinology. Fourth ed. New York: Academic Press.
  • Havelock JC, Rainey WE, Carr BR. 2004. Ovarian granulosa cell lines. Molecular and cellular endocrinology 228(1-2): 67-78.
  • Yaron Z. 1995. Endocrine control of gametogenesis and spawning induction in the carp. Aquaculture 129: 49-73.
  • 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.
  • 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.
  • 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.
  • 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.
  • Baker ME. 2011. Origin and diversification of steroids: co-evolution of enzymes and nuclear receptors. Molecular and cellular endocrinology 334(1-2): 14-20.
  • 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.
  • 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.
  • 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

  • EURL ECVAM Protocol no 92 Culture of Human Cumulus Granulosa Cells. Primary cell culture method. Contact Person: Dr. Mahadevan Maha M.
  • EURL ECVAM Method Summary no 92. Granulosa and Theca Cell Culture Systems - Summary