Oocytes at GV stage were divided into three groups: (1) Antioxidants group: oocytes were exposed to pre-maturation aging in M16/M2 medium (Sigma) containing 0.5 mM IBMX (Sigma, St. Louis, MO, USA) and 10 μmol sodium citrate (Sigma), 25 μmol ALA (Sigma) and 20 μmol ALCAR (Sigma); (2) Control group: oocytes were exposed to pre-maturation aging in M16/M2 medium with 0.5 mM IBMX but without any antioxidant; (3) Fresh group: oocytes were cultured in M16/M2 media for IVM without aging. For pre-maturation aging, oocytes in antioxidant group and control group were cultured in 20 μl of droplets at 37 °C in a humidified atmosphere of 5.0% CO2 for 12, 24, and 36 h, respectively. After pre-maturation aging, oocytes were cultured without IBMX for 14 h in M2 medium supplemented with 10% FBS, recombinant 75 mIU/mL follicle-stimulating hormone (Sigma), 0.5 IU/mL human chorionic gonadotropin (Sigma), and 5 ng/mL recombinant epidermal growth factor (Sigma). Oocytes in fresh group were directly cultured for 14 h in the IVM medium without pre-maturation aging. After IVM, oocytes with extrusion of the first polar body were considered as MII stage. Oocytes at MII stage were counted and then used for assessment of mitochondrial distribution and spindle morphology. The oocytes arrested at GV stage after IVM were used for examination of DNA DSBs.
Follicle stimulating hormone
Manufactured by Merck Group
Sourced in United States, Switzerland
Follicle-stimulating hormone is a laboratory product that measures the levels of the follicle-stimulating hormone in the body. Follicle-stimulating hormone is a glycoprotein hormone produced by the anterior pituitary gland that regulates the development, growth, pubertal maturation, and reproductive processes of the body.
Automatically generated - may contain errors
Lab products found in correlation
Luteinizing hormone, by Merck Group (5 mentions)
Sodium pyruvate, by Merck Group (2 mentions)
Human chorionic gonadotropin (hcg), by Merck Group (2 mentions)
M16 m2 medium, by Merck Group (1 mentions)
Alcar, by Merck Group (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Sodium citrate, by Merck Group (1 mentions)
Recombinant epidermal growth factor, by Merck Group (1 mentions)
Rhodamine 123, by Merck Group (1 mentions)
Anti actin antibody, by Santa Cruz Biotechnology (1 mentions)
Western enhanced chemiluminescence ecl detection reagent, by Bio-Rad (1 mentions)
Anti rabbit and mouse igg conjugated with horseradish peroxidase, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Tissue culture medium 199, by Thermo Fisher Scientific (1 mentions)
Hepatocyte growth factor (hgf), by R&D Systems (1 mentions)
Recombinant human bmp4, by R&D Systems (1 mentions)
Testosterone, by Steraloids (1 mentions)
Activin a, by Merck Group (1 mentions)
Recombinant human bmp 7, by R&D Systems (1 mentions)
Knockout serum replacement (ksr), by Thermo Fisher Scientific (1 mentions)
14 protocols using follicle stimulating hormone
1
Antioxidant Effects on Oocyte Maturation
2
Molecular Profiling of Cellular Responses
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3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium
iodide (PI), and Rhodamine 123, and follicle stimulating hormone (FSH) were
obtained from Sigma-Aldrich (St. Louis, MO, USA). Complete Protease Inhibitor
Cocktail Tablet was from Roche Applied Science (Mannheim, Germany).
Dichlorofluorescein diacetate (DCF-DA) fluorescent probe was purchased from
Invitrogen (Carlsbad, CA, USA). The western enhanced chemiluminescence (ECL)
detection reagent was purchased from Bio-Rad (Hercules, CA, USA). Anti-actin
antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-aromatase antibody was obtained from Acris Antibodies (SM2222P; San Diego,
CA, USA). Anti-StAR antibody was purchased from Abcam (ab96637; Cambridge, UK).
Anti-rabbit and mouse IgG-conjugated with horseradish peroxidase were purchased
from Cell Signaling Technology (Beverly, MA, USA).
iodide (PI), and Rhodamine 123, and follicle stimulating hormone (FSH) were
obtained from Sigma-Aldrich (St. Louis, MO, USA). Complete Protease Inhibitor
Cocktail Tablet was from Roche Applied Science (Mannheim, Germany).
Dichlorofluorescein diacetate (DCF-DA) fluorescent probe was purchased from
Invitrogen (Carlsbad, CA, USA). The western enhanced chemiluminescence (ECL)
detection reagent was purchased from Bio-Rad (Hercules, CA, USA). Anti-actin
antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Anti-aromatase antibody was obtained from Acris Antibodies (SM2222P; San Diego,
CA, USA). Anti-StAR antibody was purchased from Abcam (ab96637; Cambridge, UK).
Anti-rabbit and mouse IgG-conjugated with horseradish peroxidase were purchased
from Cell Signaling Technology (Beverly, MA, USA).
3
Porcine Oocyte Maturation Protocol
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All animal handling and experiments were performed according to a protocol approved by the Animal Research Committee of Chungbuk National University, Korea. All chemicals used in this study were purchased from Sigma-Aldrich, unless otherwise indicated. Porcine ovaries were provided by a local slaughterhouse (Umsung, Cheongju, Korea). Cumulus-oocyte complexes (COCs) were aspirated from the follicles (3–8 mm in diameter) of porcine ovaries and washed three times with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered Tyrode’s medium containing 0.1% (w/v) poly(vinyl alcohol) (HEPES-TL-PVA). The collected COCs were incubated with in vitro maturation (IVM) medium for 44 h at 38.5°C, 5% CO2. The IVM medium comprised tissue culture medium 199 (Gibco) supplemented with 0.1 g/l sodium pyruvate, 0.6 mM L-cysteine, 10 ng/ml epidermal growth factor, 10% porcine follicular fluid (v/v), 10 IU/ml luteinizing hormone (Sigma-Aldrich), and 10 IU/ml follicle-stimulating hormone (Sigma-Aldrich). The COCs were pipetted in TL-HEPES supplemented with 1 mg/ml hyaluronidase (w/v) for 3 min.
4
Superovulation in C57BL/6 Mice
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Pubertal female C57BL/6 mice were injected intraperitoneally with 30 IU gonadotropins, 15 IU Follicle-stimulating hormone (FSH) and 15 IU luteinizing hormone (LH) followed by 5 IU menotropin (hCG) after 48 h (Sigma-Aldrich, Gillingham, UK). After the imaging studies, the ovaries were removed and snap-frozen in liquid nitrogen for later analysis of protein expression by western blot.
5
Directed Differentiation of Testicular Organoids
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After culturing the organoids for 3 days, which were designated as day 0 undifferentiated organoids, the OFM media was completely removed, and the culture was continued in organoid differentiation medium (ODM). ODM was composed of Minimum Essential Medium α (ThermoFisher Scientific, cat# 12571063) supplemented with 10% KnockOut Serum Replacement (ThermoFisher Scientific, cat# 10828028), hepatocyte growth factor (5 ng/mL) (R&D Systems, cat# 294-HG), activin A (100 ng/mL) (Sigma-Aldrich, cat# a4941), follicle stimulating hormone (1 ng/mL) (Sigma-Aldrich, cat# F4021), luteinizing hormone (1 ng/mL) (Sigma-Aldrich, cat# L5259), testosterone (1 μM) (Steraloids, cat# A6950-000), recombinant human BMP-4 (20 ng/mL) (R&D Systems, cat# 314-BP), recombinant human BMP-7 (20 ng/mL) (R&D Systems, cat# 354-BP), 3,3’,5-triodo-L-thyronine sodium (2 ng/mL) (Sigma-Aldrich, cat# T6397), l-ascorbic acid-2-glucoside (1 mM) (Matrix Scientific, cat# 092375) and 1% Penicillin-Streptomycin (22 (link), 23 (link)). Culture was carried out for an additional 28 days, with full media changes every second day. The organoids were sampled every 7 days, including day 0, for analysis.
6
Porcine Oocyte In Vitro Maturation
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Porcine ovaries were obtained from a local slaughterhouse and placed in 38 °C saline supplemented with 50 mg/mL streptomycin sulfate and 75 mg/mL penicillin G. Follicular fluid was collected using a syringe with a 6-gauge needle, and cumulus–oocyte complexes with at least three layers of dense cumulus cells and a uniformly granular ooplasm were collected under a microscope. A total of 500 μL of in vitro maturation medium [TCM-199 (Invitrogen, Carlsbad, CA, USA) supplemented with 0.57 mM L-cysteine (Sigma), 10 IU/mL follicle-stimulating hormone (Sigma), 10 ng/mL epidermal growth factor (Sigma), 10 IU/mL luteinizing hormone (Sigma), 0.1 mg/mL sodium pyruvate (Sigma), and 10% (v/v) porcine follicular fluid (Sigma)] per well was added to a 4-well plate. After rinsing in TL-HEPES, approximately 80 COCs were transferred to each well. The 4-well plate (30004, SPL Life Sciences, Seoul, Republic of Korea) was placed in an incubator maintained at 38.5 °C in an atmosphere of 5% CO2 and 100% humidity after covering it with mineral oil (370 μL/well).
7
Quantitative Proteomic Analysis of Cells
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Tissue culture medium 199 (TCM 199) and fetal calf serum were purchased from Gibco BRL (Paisley, Scotland, UK). Follicle-stimulating hormone, formic acid (FA), bis-acrylamide and acetonitrile (ACN) were supplied by Sigma (St, Louis, MO, USA). Urea, iodoacet-amide (IAA), ammonium bicarbonate (NH4HCO3) were obtained from Amresco (Solon, OH, USA). Protease inhibitor complete tablets was purchased from Roche. iTRAQ-4plex Regent kit was obtained from Applied Biosystems (Forster City, CA, USA). Trypsin was from Promega (Madison, WI, USA). All the water was prepared using a MilliQ system (Bedford, MA, USA).
8
Ovarian Follicle 3D Culture and IVM
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Under a stereomicroscope, the individual follicles were isolated from ovarian organoids using 27-gauge needles and cultured with follicle 3D culture medium (α-MEM supplemented with 5% FBS, 2% polyvinylpyrrolidone (Cat. No. 9003-39-8, Sigma-Aldrich),2 mM Glutamax, 200 μM ascorbic acid, 50 mM β-mercaptoethanol, 100 IU/mL Penicillin-streptomycin, 20 ng/mL EGF, 1 mM Sodium Pyruvate, 0.2 IU/mL follicle-stimulating hormone (Cat. No. 2413405A1023, MSD, New Jersey, NJ, USA), 20 ng/mL BMP15 (Cat. No. 5096-BM-005, R&D, Santa Clara, CA, USA), and 20 ng/mL GDF9 (Cat. No. 739-G9-010, R&D) at 37 °C with 5% CO2. After 2 days of follicle 3D culture, follicles were treated with 0.1% type IV collagenase for 5 min, and further cultured in follicle 3D culture medium continued for 9 d, while changing the medium every other day. Following this, in vitro-grown follicles were subjected to IVM.
9
Follicle Stimulation and LPA Treatment
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The control group culture medium comprised of 75 mIU/mL follicle-stimulating hormone (Livzon Co., Zhuhai, China), 75 mIU/mL luteinizing hormone (Livzon Co., Zhuhai, China), and 10 ng/mL human epidermal growth factor (Sigma, St. Louis, MO, USA). The LPA treatment group culture medium contained of 75 mIU/mL follicle-stimulating hormone, 75 mIU/mL luteinizing hormone, 10 ng/mL human epidermal growth factor, and 10 µM LPA (Sigma, St. Louis, MO, USA).
10
Mammalian Cell Culture Reagents
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Follicle-stimulating hormone, verapamil, Ham's F12/DMEM medium, penicillin, streptomycin, kanamycin and amphotericin B, protease inhibitors, Serum Replacement 3, sodium pyruvate, D-glucose, HEPES and sodium bicarbonate were obtained from Sigma Aldrich Chemical Company (St. Louis, MO, USA). All salts used for buffer solutions were of analytical grade.
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