Key Event Overview
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AOPs Including This Key Event
|AOP Name||Event Type||Essentiality|
|PPARα activation leading to impaired fertility in adult male rodents||KE||Weak|
|PPARα activation in utero leading to impaired fertility in males||KE||Moderate|
Level of Biological Organization
How this Key Event works
Steroidogenesis begins with the transport of cholesterol from intracellular stores into mitochondria. This process involves a series of protein-protein interactions involving cytosolic and mitochondrial proteins located at both the outer and inner mitochondrial membranes. In steroidogenic cells the cholesterol import to the mitochondrial inner membrane is crucial for steroid synthesis (Rone, Fan, and Papadopoulos 2009). This process is facilitated by the Scavenger Receptor Class B, type 1 (SR-B1) [more relevant for rodents, than for humans] that mediates the selective uptake of cholesterol esters from high-density lipoproteins. Steroidogenic acute regulatory protein (STAR) and the translator protein (TSPO) [former peripheral benzodiazepine receptor (PBR)] mediate cholesterol transport from the outer to the inner mitochondrial membrane. The conversion of cholesterol to pregnenolone is done by Cholesterol side-chain cleavage enzyme (P450scc), the start of steroidogenesis [reviewed in (Miller and Auchus 2011)].
In mitochondria of steroidogenic tissues there are two specialized mechanisms related to hormone synthesis: one by which cholesterol is delivered to the mitochondria and the other by which specialized intra-mitochondrial enzymes participate in the synthesis of hormonal steroids.
General role in biology
Systemic steroid hormones are primarily formed by the gonads, adrenal glands, and during in utero development by the placenta. Some other organs like brain (Baulieu 1998), and heart (Kayes-Wandover and White 2000) have also been identified as steroid-producing tissues, mainly for local needs. The steroid hormones are indispensable for mammalian life. They are made from cholesterol via complex biosynthetic pathways that are initiated by specialized, tissue-specific enzymes in mitochondria. These hormones include glucocorticoids (cortisol, corticosterone) and mineralocorticoids (aldosterone) produced in the adrenal cortex, estrogens (estradiol), progestins (progesterone) and androgens (testosterone, dihydrotestosterone) produced in the gonads, and calciferols (1,25-dihydroxy vitamin D [1,25OH2D]) produced in the kidneys (Miller and Auchus 2011). Cholesterol is the precursor for the synthesis of steroid hormones in mitochondria. Steroidogenesis begins with the metabolism of cholesterol to pregnenolone facilitated by P450scc. The rate of steroid formation depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and the loading of P450scc with cholesterol (Miller and Auchus 2011). Interference with one or more of these reactions leads to reduced steroid production.
How it is Measured or Detected
This KE can be indirectly measured by:
1. Expression of the proteins involved in cholesterol transport by qPCR or Western blot.
3. Cholesterol transport to the mitochondrial inner membrane in intact cells:
- Indirectly as pregnenolone formation by cells. The pregnenolone concentration is assayed by commercially available radioimmunoassays and reflects the amount of cholesterol transported to the mitochondrial inner membrane (Charman et al. 2010).
- Filipin staining is one of the most widely used tools for studying intracellular cholesterol distribution. The fluorescent detergent filipin binds selectively to cholesterol (and not to cholesterol esters) (Schroeder, Holland, and Bieber 1971). Filipin can be only used for the qualitative analysis of cholesterol distribution, since its fluorescence intensity is not necessarily linearly related to cholesterol content.
The cholesterol transport can be measured in vitro cultured Leydig cells. The methods for culturing Leydig cells can be found in the Database Service on Alternative Methods to animal experimentation (DB-ALM): Leydig Cell-enriched Cultures  Testicular Organ and Tissue Culture Systems 
Evidence Supporting Taxonomic Applicability
The enzymes needed for cholesterol transport were found in amphioxus and are present in vertebrates (Albalat et al. 2011).
Albalat, Ricard, Frédéric Brunet, Vincent Laudet, and Michael Schubert. 2011. “Evolution of Retinoid and Steroid Signaling: Vertebrate Diversification from an Amphioxus Perspective.” Genome Biology and Evolution 3: 985–1005. doi:10.1093/gbe/evr084.
Baulieu, E E. 1998. “Neurosteroids: A Novel Function of the Brain.” Psychoneuroendocrinology 23 (8) (November): 963–87.
Charman, Mark, Barry E Kennedy, Nolan Osborne, and Barbara Karten. 2010. “MLN64 Mediates Egress of Cholesterol from Endosomes to Mitochondria in the Absence of Functional Niemann-Pick Type C1 Protein.” Journal of Lipid Research 51 (5) (May): 1023–34. doi:10.1194/jlr.M002345.
Kayes-Wandover, K M, and P C White. 2000. “Steroidogenic Enzyme Gene Expression in the Human Heart.” The Journal of Clinical Endocrinology and Metabolism 85 (7) (July): 2519–25. doi:10.1210/jcem.85.7.6663.
Miller, Walter L, and Richard J Auchus. 2011. “The Molecular Biology, Biochemistry, and Physiology of Human Steroidogenesis and Its Disorders.” Endocrine Reviews 32 (1) (February): 81–151. doi:10.1210/er.2010-0013.
Rone, Malena B, Jinjiang Fan, and Vassilios Papadopoulos. 2009. “Cholesterol Transport in Steroid Biosynthesis: Role of Protein-Protein Interactions and Implications in Disease States.” Biochimica et Biophysica Acta 1791 (7) (July): 646–58. doi:10.1016/j.bbalip.2009.03.001.
Schroeder, F, J F Holland, and L L Bieber. 1971. “Fluorometric Evidence for the Binding of Cholesterol to the Filipin Complex.” The Journal of Antibiotics 24 (12) (December): 846–9.
Steer, C. 1984. “Detection of Membrane Cholesterol by Filipin in Isolated Rat Liver Coated Vesicles Is Dependent upon Removal of the Clathrin Coat.” The Journal of Cell Biology 99 (1) (July 1): 315–319. doi:10.1083/jcb.99.1.315.