Pregnenolone

Manufactured by Merck Group

Sourced in United States, Germany, China, Italy

Pregnenolone is a steroid compound that serves as a precursor in the biosynthesis of various hormones, including progesterone, cortisol, and testosterone. It is an important intermediate in the production of these vital biological molecules.

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Lab products found in correlation

Close spelling variants of this product

Pregnenolone sulfate (14 citations) Pregnenolone 16α-carbonitrile (5 citations) Pregnenolone 16α-carbonitrile (PCN) (4 citations) Pregnenolone sulfate sodium salt (3 citations) Pregnenolone sulphate (3 citations) 17α-OH pregnenolone (2 citations) Pregnenolone (PREG), (2 citations)

30 protocols using pregnenolone

Nocodazole and Pregnenolone Treatments

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For nocodazole treatment, the dechorionated embryos were incubated in 1 μg/mL nocodazole (Sigma, USA), which was diluted in 1 x Danieau buffer as previously described 6 (link). For pregnenolone treatment, the dechorionated embryos were incubated with 0.1, 1, 10 or 20 μM pregnenolone (Sigma, USA) from 1k-cell stage until the late epiboly stage.

Xiao Q., Xia J.H., Zhang X.J., Li Z., Wang Y., Zhou L, & Gui J.F. (2014). Type-IV Antifreeze Proteins are Essential for Epiboly and Convergence in Gastrulation of Zebrafish Embryos. International Journal of Biological Sciences, 10(7), 715-732.

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Steroid Extraction and Quantification

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Pregnenolone (PREG), Pregnenolone-20,21-¹³C₂-16,16 D₂ (¹³C₂ D₂–PREG), progesterone (PROG), progesterone-2,3,4,20,25-¹³C₅ (¹³C₅–PROG), 17β-Estradiol (17β-E), 17β-Estradiol-2,3,4-¹³C₃ (¹³C₃-17β-E) dihydroprogesterone (DHP), allopregnanolone (ALLO), isoallopregnanolone (ISOALLO), testosterone (T), dihydrotestosterone (DHT), 5α-androstane-3α,17β-diol (3α-diol) and dehydroepiandrosterone (DHEA) were purchased from Merck Life Science, Italy. Acetonitrile, acetic acid, formic acid, methanol, 2-propanol and water were HPLC grade (Merck Life Science, Milano, Italy).

Diviccaro S., Caputi V., Cioffi L., Giatti S., Lyte J.M., Caruso D., O’Mahony S.M, & Melcangi R.C. (2021). Exploring the Impact of the Microbiome on Neuroactive Steroid Levels in Germ-Free Animals. International Journal of Molecular Sciences, 22(22), 12551.

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Purification of Steroid Metabolizing Enzymes

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Progesterone, pregnenolone, 17α-OH Progesterone, and 4-androstene-3,17-dione were purchased from Sigma-Aldrich. 17α-OH pregnenolone was purchased from Cayman Chemical (Ann Arbor, MI, USA). Dehydroepiandrosterone (DHEA) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). 3-[(3-Cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) was purchased from GoldBio (St. Louis, MO, USA). HisPurTM Ni-NTA resin was purchased from Thermo Fisher (Waltham, MA, USA). A HiTrap^® DEAE Fast Flow column was purchased from Cytiva (Marlborough, MA). E. coli JM109 cells were purchased from Enzynomics (Daejeon, Korea). Rat NADPH-cytochrome P450 reductase (POR) was heterologously expressed in E. coli HMS174 (DE3) and purified as described elsewhere [27 (link)]. Recombinant human b₅ was expressed in E. coli JM109 cells from a plasmid [pSE420 (Amp)] and the protein was solubilized and purified by DEAE-Sepharose ion exchange chromatography [28 (link)].

Lee S.G., Kim V., Lee G.H., Kim C., Jeong E., Guengerich F.P, & Kim D. (2023). Hydroxylation and Lyase Reactions of Steroids Catalyzed by Mouse Cytochrome P450 17A1 (Cyp17a1). Journal of inorganic biochemistry, 240, 112085.

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Quantification of Sterol Sulfates

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All consumables were from VWR (Ismaning, Germany). Derivatization reagents trifluoroacetic anhydride (TFAA), 1-(trimethylsilyl)imidazole (TSIM), and N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) were from Macherey-Nagel (Düren, Germany). Deionized water was prepared with an in-house ion-exchanger. 1,4-Dioxane and methyl tert-butyl ether (MtBE) were distilled before use. β-Glucuronidase/sulfatase from Helix pomatia type HP-2, 5α-cholestane (≥97%), pregnenolone (>98%), pregnenolone sulfate sodium salt (>98%), and cholesteryl sulfate sodium salt (>99%) were purchased from Sigma-Aldrich (Schnelldorf, Germany). Dehydroepiandrosterone sulfate sodium salt (>99%) and 25-hydroxycholesteryl sulfate sodium salt (>99%) were from Avanti Polar Lipids (Alabaster, AL, USA). All other sterol sulfate sodium salts were from Steraloids (Newport, RI, USA). All other reagents and solvents were purchased in HPLC grade or in pro analysis quality from Sigma-Aldrich (Schnelldorf, Germany).

Junker J., Chong I., Kamp F., Steiner H., Giera M., Müller C, & Bracher F. (2019). Comparison of Strategies for the Determination of Sterol Sulfates via GC-MS Leading to a Novel Deconjugation-Derivatization Protocol. Molecules, 24(13), 2353.

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Steroid Sulfatase Enzyme Assays

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All chemicals used for the assays were obtained from commercial suppliers and used without further purification. PAPS, PAP, MUS, MU, steroid substrates (i.e. cholesterol, pregnenolone, DHEA), and two steroid sulfates (cholesterol and pregnenolone sulfate) were purchased from Sigma Aldrich (St. Louis, MO, USA). DHEA sulfate was obtained from Cayman Chemical (Ann Arbor, MI, USA). Galeterone and amoxicillin were purchased from Selleck Chemicals (Houston, TX, USA). Other chemicals used for enzyme purifications were of the highest purity commercially available. Methanol Chromasolv LC-MS grade (Riedel-de Haën - Honeywell, Muskegon, MI, USA) was used for DESI-MS analysis.

Kulathunga S.C., Morato N.M., Zhou Q., Cooks R.G, & Mesecar A.D. (2022). Novel Desorption Electrospray Ionization Mass Spectrometry Assay for Label-Free Characterization of SULT2B1b Enzyme Kinetics. ChemMedChem, 17(9), e202200043.

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Steroid Hormone Analytical Protocols

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The following were purchased from Sigma-Aldrich (St. Louis, MO, USA): the steroid hormone corticosterone and its precursors including pregnenolone, progesterone, 11-deoxycorticosterone, dehydrocorticosterone, testosterone and its precursor, 17-hydroxyprogesterone, androstenedione and dihydrotestosterone; and stable isotopes, including testosterone-d3. The stable isotope corticosterone-d8 was purchased from Otsuka Pharmaceutical (Tokyo, Japan). LC-MS-grade acetonitrile and formic acid were purchased from Supelco (Bellefonte, PA, USA).

Maeda N., Tahata S., Yagi T., Tanaka E., Masu K., Sato M., Haeno S., Onaga T, & Yokota H. (2015). Assessment of Testicular Corticosterone Biosynthesis in Adult Male Rats. PLoS ONE, 10(2), e0117795.

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Comprehensive Steroid Analysis Workflow

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The internal standards (d3-testosterone, d4-cortisol, d9-progesterone, d7-cholesterol, 13c3-androstene-3,17-dione, and 13c3-estrone) were purchased from Cerilliant (Round Rock, TX, USA). L-ascorbic acid, sodium acetate, acetic acid, β-glucosaldosidase/arylsulfatase, ammonium iodide (NH4I), dithioerythritol (DTE), and N-methyl-n-(trimethylsilyl)trifluoroacetamide (MSTFA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Commercial standards of steroids (dehydroepi androsterone, testosterone, 11-deoxy corticosterone, cortisone, cortisol, 16α-hydroxy estrone, pregnenolone, 17α-hydroxypregnenolone, epipregnanolone, dihydrotestosterone, 21-hydroxyprogesterone, androsterone, epiandrosterone, corticosterone, estrone, 17β-estradiol, estriol, 16-epiestriol, progesterone, 17α-hydroxyprogesterone, desmosterol, cholesterol, etc.) were purchased from Sigma-Aldrich, J&K Chemical Ltd. (Beijing, China), or Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Chromatographic pure hexane, methanol, and ethyl acetate were purchased from Merck (Fairfield, OH, USA). The Oasis HLB SPE cartridge was obtained from Waters (1.5 ml, 60 mg; Waters, Milford, MA, USA).

Wu D., Ye L., Zhang X., Yin M., Guo Y, & Zhou J. (2023). Characteristics of steroid hormones in systemic lupus erythematosus revealed by GC/MS-based metabolic profiling. Frontiers in Endocrinology, 14, 1164679.

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Analytical Characterization of Compounds

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Optical rotations were obtained on a JASCO P200 polarimeter. Melting points were determined using a Büchi^® B-540 melting point apparatus. IR spectra were measured on a Perkin-Elmer FTIR Spectrum 2. NMR spectra were recorded on a Bruker Avance III - 500 MHz NMR spectrometer equipped with a multi-nucleus probe BBFO (5 mm). UV spectra were recorded on the PDA detector of the Varian LC-920 system. LC-HRESIMS data were recorded on an Agilent 6520 Accurate-Mass Q-TOF hyphenated to an Agilent 1200 system equipped with a Zorbax Agilent C18 column (50 mm × 2.1 mm, 1.8 μm). Compounds were purified by semi-preparative HPLC with a Gilson 322 system equipped with an Axia C18 column (21.2 mm × 100 mm, 5 μm). HPLC analyses were performed on a Varian LC-920 system with a Kinetex column C18 100 Å (100 × 3.0 mm, 2.6 μm). Solid-phase extractions were performed on Chromabond^® SPE cartridges (Macherey-Nagel). The reading of the microplates was performed on an ELISA Versamax plus^® (Molecular Devices) plate reader. Solvents for extraction, fractionation and HPLC were supplied by Sigma-Aldrich. The bioassay material was purchased as following: GLP-1 ELISA kit (Active GLP-1, EGLP-35K Millipore), DMEM and PBS (Gibco), BSA (Sigma-Aldrich) and MTS mother solution CellTiter 96 (Promega), compounds standards pregnenolone and pregnenolone sulphate (Sigma-Aldrich).

Tsoukalas M., Psichas A., Reimann F., Gribble F.M., Lobstein A, & Urbain A. (2017). Pregnane glycosides from Cynanchum menarandrense. Steroids, 125, 27-32.

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Modulating Leydig Cell Steroidogenesis

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The MA-10 mouse Leydig cells were purchased from American Type Culture Collection (ATCC) and cultured in DMEM/F12 medium (Sigma-Aldrich; D8900) supplemented with 15% horse serum (Gibco), 1% penicillin–streptomycin (Gibco), 20 mM HEPES and 1.2 g/L of sodium bicarbonate. After pre-coating 0.1% gelatin on the dishes and plates for 4 h, the cells were maintained at 37℃ with 5% CO₂ in a humidified incubator. Palmitic acid (PA) and oleic acid (OA) were separately dissolved in pure ethanol (100 mM) and diluted to a working concentration with the culture medium containing 1% BSA to conjugate fatty acids. To stimulate steroidogenesis, the cells were treated with 50 μM 8-Br-cAMP (Tocris) for 4 h, and then the conditioned medium was collected for further measurements. We treated the cells with 10 µg/mL of 22(R)-hydroxycholesterol (22R-OHC; Sigma) to assay CYP11A1 activity and 1 µg/mL of pregnenolone (Sigma) to assay 3β-HSD activity for 4 h and measured the progesterone production. To modulate autophagic activities, the cells were treated with 50 nM bafilomycin (Tocris), 20 μM chloroquine (Sigma-Aldrich), and different concentrations of rapamycin (Tocris) for 24 h before analysis. For the knockdown experiments, SMARTpool specific siRNAs (Dharmacon) were reversely transfected using Lipofectamine RNAiMAX Transfection Reagent (Thermo).

Huang C., Hsu H.J., Wang M.E., Hsu M.C., Wu L.S., Jong D.S., Jiang Y.F, & Chiu C.H. (2021). Fatty acids suppress the steroidogenesis of the MA-10 mouse Leydig cell line by downregulating CYP11A1 and inhibiting late-stage autophagy. Scientific Reports, 11, 12561.

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Sterol Biosynthesis Pathway Optimization

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Cholesterol (cholest-5-en-3β-ol) was obtained from AppliChem (Germany); phytosterol (total sterols content—95.47%: β-sitosterol—42.39%, stigmasterol—26.08%, campesterol—23.48%, brassicasterol—3.52%)—from Jiangsu Spring Fruit Biological Products Co., Ltd. (China); pregn-4-ene-3,20-dione (progesterone); 3β-hydroxypregn-5-en-20-one (pregnenolone); 3β-hydroxyandrost-5-en-17-one (dehydroepiandrosterone, DHEA); acetamide—from Sigma-Aldrich (USA); yeast extract, bacto-peptone—from Difco (USA); randomly methylated β-cyclodextrin (MCD)—from Wacker-Chemie GmbH (Germany). Hygromycin B and DNA modifying enzymes were obtained from Thermo Fisher Scientific (USA); Tween 80—from Serva (Germany); DNA purification Kits—from Qiagen (Germany). The DNA manipulations were carried out according to the manufacturer’s instructions. All other reagents were of the highest purity grade and were purchased from domestic commercial suppliers (Russia).

Karpov M., Strizhov N., Novikova L., Lobastova T., Khomutov S., Shutov A., Kazantsev A, & Donova M. (2024). Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system. Microbial Cell Factories, 23, 105.

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