Experimental mice were used in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals using institutional care and committee-approved protocols. To disrupt KDM1A in oocytes, mice bearing the Kdm1a conditional allele Lsd1fl (Kerenyi et al. 2013 (link)) were crossed with ZP3-Cre transgenic mice. Lsd1fl/fl/ZP3-Cre+ female mice were referred to as KDM1A knockout; Lsd1fl/+/ZP3-Cre− female littermates were used as controls. Generation of KDM1B knockout (Aof11lox/1lox) mice was described previously (Ciccone et al. 2010 (link)), and wild-type littermates were used as controls. To obtain MII oocytes, 6- to 8-wk-old mice were injected intraperitoneally with 5 IU of pregnant mare's serum gonadotrophin (PMSG) (Sigma) and, 48 h later, 5 IU of human chorionic gonadotrophin (hCG) (Sigma). Superovulated mice were euthanized 16 h after hCG injection, and oocytes were collected from the oviducts and released into a hyaluronidase/M2 solution for removal of cumulus cells.
Human chorionic gonadotrophin hcg
Manufactured by Merck Group
Sourced in United States
Human chorionic gonadotrophin (hCG) is a hormone produced during pregnancy. It is a glycoprotein that consists of an alpha and a beta subunit. hCG is primarily used in laboratory settings for pregnancy testing and monitoring.
Automatically generated - may contain errors
Lab products found in correlation
Milrinone, by Merck Group (2 mentions)
Collagenase type 5, by Merck Group (2 mentions)
M2 medium, by Merck Group (2 mentions)
Human chorionic gonadotropin (hcg), by Merck Group (2 mentions)
Forskolin, by Merck Group (1 mentions)
S adenosylmethionine sam, by New England Biolabs (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Ksom medium, by Merck Group (1 mentions)
Accutase solution, by Merck Group (1 mentions)
Pregnant mare serum gonadotropin, by Merck Group (1 mentions)
L cysteine hydrochloride, by Merck Group (1 mentions)
5 protocols using human chorionic gonadotrophin hcg
1
Generating Oocytes from Transgenic Mice
2
Evaluating Cell Signaling Modulators
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DBP and MBP were purchased from Tokyo Kasei Kogyo Co Ltd. (Tokyo, Japan). Human chorionic gonadotrophin (hCG) and forskolin were obtained from Sigma (St. Louis, MO, USA). RPMI 1640 medium, fetal bovine serum (FBS), streptomycin sulfate, antibiotic penicillin G sodium (10,000 U/ml), and phosphate-buffered saline with Ca2+ and Mg2+ were obtained from Gibco (Grand Island, NY, USA). S-adenosylmethionine (SAM) was purchased from New England BioLabs (Ipswich, MA, USA). All other chemicals used were of analytical grade.
3
Collecting Mouse Oocytes, Embryos, and Follicles
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Eight-week-old female Mfn2-/- and WT mice were used to collect oocytes, embryos, and follicles. To collect germinal vesicle (GV) stage oocytes, 5 IU PMSG (Sigma, St. Louis, MO) was injected intraperitoneally and ovaries were extracted 44-48 h after the injection. To retrieve the oocytes, ovaries were punctured with a 26-gauge needle. Collected oocytes were placed in M2 medium (Sigma, St. Louis, MO) and 10 μM milrinone (Sigma, St. Louis, MO) to prevent meiotic resumption. To obtain mature oocytes, an additional injection of 5 IU of human chorionic gonadotrophin (hCG; Sigma, St. Louis, MO) to induce oocyte maturation and ovulation was given 48 h after the PMSG injection. Unfertilized oocytes at metaphase of the second meiotic division (MII) were collected from oviducts 14 h after the hCG injection. To collect fertilized embryos, females were mated with WT males immediately after the hCG injection. The following morning, mating was confirmed by the presence of a vaginal plug. Two-cell embryos were collected 44 h after hCG injection from the oviducts in KSOM medium (Millipore, Darmstadt, Germany). Blastocysts were collected 92 h after hCG injection from uterus into M2 medium (Sigma, St. Louis, MO). Secondary follicles were collected by digesting the ovaries with 1.5 mg/mL collagenase type V (Sigma, St. Louis, MO) for 1 h at 37°C in M2 medium (Sigma, St. Louis, MO) [16 (link)].
4
Isolation of Mouse Oocytes and Granulosa Cells
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Secondary follicles were collected from 8-week-old Mfn1−/− and WT mice by digesting the ovaries with 1.5 mg/mL collagenase type V (Sigma, St. Louis, MO) for 1 h at 37 °C in M2 medium (Sigma, St. Louis, MO)5 (link). Secondary follicle-enclosed oocytes (referred as the oocytes) and granulosa cells were collected by further digesting the harvested secondary follicle with Accutase solution (Sigma, St. Louis, MO) for 15 min. To collect germinal vesicle (GV) stage oocytes, ovaries were obtained from 8-week-old Mfn1−/− and WT mice51 (link) 44–48 h after intraperitoneal injection of 5 IU PMSG (Sigma, St. Louis, MO). Ovaries were then punctured with a 26-gauge needle, and GV stage oocytes were collected in M2 medium (Sigma, St. Louis, MO) and 10 μM milrinone (Sigma, St. Louis, MO) to prevent meiotic resumption. To obtain mature oocytes, an additional injection of 5 IU of human chorionic gonadotrophin (hCG; Sigma, St. Louis, MO) to induce oocyte maturation and ovulation was given 48 h after the PMSG injection. Unfertilized oocytes at metaphase of the second meiotic division (MII) were collected from oviducts 14 h after the hCG injection.
5
Xenopus Embryo Harvesting and Fertilization
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The procedure used to harvest the embryos was as described previously (Kay and Peng, 1991) . Eggs were obtained from female X. laevis that had been primed between ~3 to 5 days prior to ovulation with 75 IU pregnant mare serum gonadotropin (Sigma-Aldrich Corp., St. Louis, MO, USA) to reinitiate oocyte meiosis. The females were then given a second injection of 500 IU human chorionic gonadotrophin (hCG; Sigma-Aldrich Corp.) at ~18-22 h prior to ovulation. The eggs were fertilized in vitro with macerated testis, and then dejellied with 0.1× Marc's Modified Ringer's solution (MMR: 100 mM NaCl, 2 mM KCl, 1 mM MgSO 4 , 2 mM CaCl 2 , 5 mM HEPES, pH 7.4) containing 2-3% L-cysteine hydrochloride (Sigma-Aldrich Corp.; pH 8.0) for ~3-5 min with gentle shaking. After dejellying, the embryos were rinsed five times with 0.1× MMR to remove the L-cysteine hydrochloride and then incubated in 0.1× MMR at 23°C until required. Staging was according to Nieuwkoop and Faber (1967) .
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