In order to investigate the effects of E2 on retinal sensitivity, scotopic ERGs were conducted with female túngara frogs in three treatment groups: 1, saline-injected control (n = 7); 2, injected with hCG (n = 7); and 3, injected with a combination of hCG and fadrozole (hCG + fadrozole) (n = 7). The injections for the hCG + fadrozole group followed the protocol of Chakraborty and Burmeister (2009) (link), which used fadrozole to block estradiol production in hCG injected túngara frogs. This blocking effect likely results from inhibiting aromatase (Ankley et al. 2002 (link)), an enzyme that converts androgens to estrogens in vertebrate brains and gonads (Callard et al. 1978a , 1978b (link)). Injection protocols were as follows: Day 1: group 3 received a subcutaneous injection of a single dose of fadrozole (50 μg; Sigma–Aldrich, St. Louis, MO); groups 1 and 2 received saline. Day 2: group 3 received a second dose of fadrozole along with a dose of hCG (500 IU; Sigma) in two sequential subcutaneous injections. Group 2 received an injection of hCG (500 IU; Sigma). Group 1 received saline. All animals were then placed in dark adaptation containers (minimum 16 h). Day 3: ERGs were run 16–18 h after the last injection. Each injection was dissolved in (50 µL) saline solution (in mM): 126 NaCl, 0.5 KCl, 2.8 CaCl2, 2.2 MgCl2, and 10 NaHEPES, pH 7.4 (274 mOsm).
Fadrozole
Manufactured by Merck Group
Sourced in United States
Fadrozole is a laboratory equipment product manufactured by Merck Group. It is an aromatase inhibitor, a class of compounds that block the aromatase enzyme responsible for the conversion of androgens to estrogens. Fadrozole is used in research applications to study the role of estrogen in various biological processes.
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Lab products found in correlation
Ym298198, by Bio-Techne (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
17β estradiol, by Merck Group (1 mentions)
β oestradiol, by Merck Group (1 mentions)
Human chorionic gonadotropin (hcg), by Merck Group (1 mentions)
Osilodrostat, by Selleck Chemicals (1 mentions)
Progesterone, by Merck Group (1 mentions)
11 deoxycorticosterone, by Merck Group (1 mentions)
Corticosterone, by Merck Group (1 mentions)
Dihydrotestosterone, by Merck Group (1 mentions)
Flutamide, by Merck Group (1 mentions)
Estradiol, by Merck Group (1 mentions)
Corn oil, by Merck Group (1 mentions)
Testosterone, by Merck Group (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
10 protocols using fadrozole
1
Estradiol Effects on Retinal Sensitivity in Túngara Frogs
2
Modulating Gonadal Development in Fish
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XX females and XY males were treated with inhibitor of RA synthase DEAB and inhibitor of RA catabolic enzyme KET between 5 to 30 dah and 5 to 90 dah, respectively. Additionally, all-XX fish were treated with the aromatase inhibitor fadrozole and all-XY fish were treated with E2 between 5 to 30 dah. Drug treatments were administered by feeding. DEAB, KET, E2 and fadrozole were purchased from Sigma (Natick, USA). The food was sprayed with 100% ethanol containing 1 mM/kg DEAB; 200 μM/kg KET; 1 mM/kg E2; 750 μM/kg fadrozole. Control fish were fed a 100% ethanol sprayed diet. Experimental and control fish were reared in the same way apart from their food.
3
Intrathecal Administration of Selective Pharmacological Agents
4
Oral Fadrozole Administration in Fish
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Fadrozole was orally administered with an artificial diet (Love Larva; Maruha Co., Yamaguchi, Japan) mixed with a 100 μg/g diet of Fadrozole (Sigma-Aldrich, St. Louis, MO, USA) at 18 °C from 30 to 100 dah as described previously [7 (link)]. Phenotypic sex was determined at 200 dah by histological observation.
5
Intraspinal Drug Delivery and EM2 Quantification
6
Estrogen and Aromatase Inhibitor Treatments
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17β-estradiol (E2, CAS: 50-28-2, ≥ 99%) and fadrozole (CAS: 102676-31-3, ≥ 98%), a nonsteroidal aromatase inhibitor, were purchased from Sigma-Aldrich (St. Louis, Missouri). The bmp15-null female fish were treated with E2 for 20 days (from 80 to 100 dpf) continuously. We used two treatment methods: water-borne exposure and oral administration by feeding. For water-borne exposure, the bmp15 mutant fish were placed in a clean tank with 10 L water. E2 stock solution or the vehicle ethanol was added to the water to the final concentrations of 0 and 10 nM. The water was renewed daily during the exposure period to maintain relatively constant concentrations. For oral administration, the Otohime fish feed was mixed with E2 stock solution and dried overnight in an oven at 60°C. The fish were fed twice a day with the dried feed containing E2 at different concentrations (0, 2, 20 and 200 μg/g) and supplemented with artemia. The total amount of feed administered was 10% (W/W) of fish body weight per day (5% per meal). For the double mutant females (bmp15-/-;inha-/-), they were treated similarly from 80 to 100 dpf with dried powder feed containing fadrozole (0 and 200 μg/g).
7
Modulating Avian Gonadal Oestrogen
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For manipulation of oestrogen levels after sex determination (late treatment), eggs were incubated at 37.7°C pointed end down. At D7-7.5 (HH31) or D9 (HH35) a first injection in the air chamber was performed through a hole made at the rounded end. To downregulate oestrogen, the P450 aromatase inhibitor fadrozole (Sigma-Aldrich, F3806) was injected at 0.5 mg/egg in 50 μl of PBS and then re-injected at 0.3 mg/egg every other day. To increase oestrogen, β-oestradiol (Sigma-Aldrich, E2758) was injected at 120 µg/egg in 25 µl of 95% ethanol; embryos collected at D17 (HH43) were injected once more at D13 (HH39). For manipulation of oestrogen levels before sex determination (early treatment) eggs were injected once at D4 (HH23) with 0.5 mg or 1 mg/egg of fadrozole, or 120 µg/egg of β-oestradiol. Three or more gonadal pairs were analysed from each type of experimental treatment, per developmental stage. A minimum of two stages were assessed (D10 and D17).
8
Medaka Sex Steroid Manipulation
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For both male and female fish, the gonad was removed under tricaine methane sulfonate anesthesia (0.02%) through a small incision made in the ventrolateral abdominal wall. Immediately after removal of the gonad, the incision was sutured with nylon thread. Sham-operated fish received the same surgical treatment as gonadectomized fish, except for removal of the gonad. After a recovery period of 3 days, gonadectomized males and females were immersed in water containing 100 ng/ml of KT or E2, or vehicle (ethanol) alone for 6 days and then sampled. Sham-operated fish were treated with vehicle alone and used as controls.
In another experiment, females with intact ovaries were treated with 100 ng/ml of KT or AI (Fadrozole; Sigma-Aldrich, St. Louis, MO) by immersion in water for 9 days. Similarly, males with intact testes were treated with 100 ng/ml of E2 by immersion in water for 9 days. These fish were sampled on days 0 (untreated controls), 2, 5, and 9. The sex steroid concentration used was based on previously reported serum steroid levels in medaka (Foran et al., 2002 (link); Foran et al., 2004 (link); Tilton et al., 2003 (link)).
In another experiment, females with intact ovaries were treated with 100 ng/ml of KT or AI (Fadrozole; Sigma-Aldrich, St. Louis, MO) by immersion in water for 9 days. Similarly, males with intact testes were treated with 100 ng/ml of E2 by immersion in water for 9 days. These fish were sampled on days 0 (untreated controls), 2, 5, and 9. The sex steroid concentration used was based on previously reported serum steroid levels in medaka (Foran et al., 2002 (link); Foran et al., 2004 (link); Tilton et al., 2003 (link)).
9
Osilodrostat Synthesis and Characterization
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LCI699 (osilodrostat or Isturisa) (CAS 928134-65-0) was purchased from Selleckchem. Progesterone (CAS 57-83-0), corticosterone (CAS 50-22-6), 11-deoxycorticosterone (CAS 64-85-7), and fadrozole (CAS 102676-31-3) were purchased from Sigma-Aldrich.
10
Intranasal Steroid Administration Protocol
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Testosterone, dihydroTestosterone, estradiol, flutamide and fadrozole were provided by Sigma-Aldrich (France). Testosterone (0.20 mg/day under a volume of 2.5 µl in each nostril), dihydroTestosterone (0.04 mg/day under a volume of 2.5 µl in each nostril due to its much higher potency in transactivating AR target genes than Testosterone66 (link)) and estradiol (0.0375 mg/day under a volume of 2.5 µl in each nostril) were administered daily per the intranasal route via a proprietary oleogel (MetP Pharma AG, Emmetten, Switzerland)67 (link). flutamide (20 mg/kg) and fadrozole (250 μg/kg) were administered daily per gavage. Tamoxifen (Sigma-Alrich; 30 mg/ml) was dissolved in corn oil (Sigma-Aldrich) and administered by gavage (3 mg/day for 5 days) 2 weeks before inclusion of the animals in any experimental protocol.
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