Karyotypic analysis was performed using standard protocols for mouse chromosome analysis. After culture for 3 days, SSCs were treated with culture medium containing colchicine (100 ng/ml; Sigma) for 3 h, hypotonically treated with 75 mM KCl for 15 min at 37 °C, immersed twice in methanol: acetic acid (3: 1) for 30 min at −30 °C, dried in air for 3–4 days, digested with 0.025% trypsin, and then stained with Giemsa. To verify the chromosomal type of recipient mouse oocytes, karyotypic analysis of mature oocytes from recipients was performed. To collect mature oocytes, recipient mice were superovulated with 10 IU pregnant mare serum gonadotropin (PMSG; ProSpec-Tany) for 48 h, followed by 10 IU human chorionic gonadotropin (hCG; ProSpec-Tany). These oocytes were hypotonically treated with 75 mM KCl at 37 °C for 15 min and then fixed with two solutions consisting of methanol/acetic acid/water (5:1:2) for 5–10 min and methanol/acetic acid (3:1) for 15 min at room temperature. Fixed cells were mounted on slides and immediately exposed to steam from boiling water (90–100 °C) for 30 secto cause expansion of the cells, followed by drying at 37 °C and Giemsa staining (Amresco) [25] (link).
Pregnant mare serum gonadotropin
Manufactured by ProSpec
Sourced in Israel
Pregnant mare serum gonadotropin is a hormone extracted from the serum of pregnant mares. It contains a mixture of follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which play a crucial role in the regulation of reproductive processes.
Automatically generated - may contain errors
Lab products found in correlation
Human chorionic gonadotropin hcg, by ProSpec (3 mentions)
Human chorionic gonadotropin (hcg), by ProSpec (3 mentions)
Human chorionic gonadotropin (hcg), by Merck Group (3 mentions)
Female balb c mice, by Central Lab Animal (2 mentions)
Colchicine, by Merck Group (1 mentions)
G mopsplus medium, by Vitrolife (1 mentions)
G ivf plus medium, by Vitrolife (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Anti α tubulin, by Cell Signaling Technology (1 mentions)
Alexa 488, by Thermo Fisher Scientific (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Benzylpenicillin potassium, by Fujifilm (1 mentions)
B6c3f1 females, by Charles River Laboratories (1 mentions)
Tamoxifen, by Merck Group (1 mentions)
Phosphor akt ser473, by Cell Signaling Technology (1 mentions)
C fos, by Abcam (1 mentions)
Human tubal fluid medium, by Irvine Scientific (1 mentions)
Vectashield antifade mounting medium with 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
Rabbit anti α tubulin antibody, by Cell Signaling Technology (1 mentions)
11 protocols using pregnant mare serum gonadotropin
1
Karyotypic Analysis of Mouse Chromosomes
2
Ovarian mRNA and miRNA Profiling
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All experiments and analyses were conducted in accordance with the ARRIVE guidelines and regulations. The animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Oriental Medicine, Daejeon, Korea (approval number 20‐090). Female BALB/c mice aged 12 and 44 weeks (Central Lab Animal Inc., Seoul, Korea) were housed under specific pathogen‐free conditions.
The mice were treated with 5 IU of pregnant mare serum gonadotropin (Prospec, Rehovot, Israel) and 5 IU of human chorionic gonadotropin (hCG; Prospec) to induce superovulation of oocytes for assessment. Hormonally stimulated ovaries were removed post‐ovulation and immediately placed in liquid nitrogen, prior to processing for mRNA and small RNA sequencing. RNA profiling was performed using gonadotropin‐stimulated ovaries to analyze mRNAs and miRNAs contributing to the clinical phenotypes of poor oocyte quantity and quality.
The mice were treated with 5 IU of pregnant mare serum gonadotropin (Prospec, Rehovot, Israel) and 5 IU of human chorionic gonadotropin (hCG; Prospec) to induce superovulation of oocytes for assessment. Hormonally stimulated ovaries were removed post‐ovulation and immediately placed in liquid nitrogen, prior to processing for mRNA and small RNA sequencing. RNA profiling was performed using gonadotropin‐stimulated ovaries to analyze mRNAs and miRNAs contributing to the clinical phenotypes of poor oocyte quantity and quality.
3
Oocyte Maturation and Collection Protocol
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Ovulation was induced as previously described, with modifications (Cornejo-Cortes et al., 2006 (link)). Briefly, after four-week GH treatment, rats were super-ovulated with 50 IU pregnant mare serum gonadotropin (ProSpec, Israel). After 48 h, 50 IU human chorionic gonadotropin (ProSpec, Israel) was administered intraperitoneally to trigger oocytes maturation. Oviducts were harvested 24 h after hCG administration. Oocytes were then released into pre-heated G-MOPSplus medium (Vitrolife, Sweden) by tearing oviducts with a needle. Cumulus-free oocytes were harvested from the oocyte-corona-cumulus complex after removing granulosa cells with the addition of 0.3 mg/ml hyaluronidase (Sigma-Aldrich). They were washed thrice with G-MOPSplus and finally incubated at 37°C in G-IVFplus medium (Vitrolife, Sweden).
4
Xenopus Laevis Frog Breeding and Embryo Collection
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Adult albino Xenopus laevis frogs (RRID:XEP_Xla200 ) were maintained and bred at 18°C. Female frogs were primed by injection of 50 IU pregnant mare serum gonadotropin (ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Isreal). After 3 days, male and primed female frogs were injected with 150 IU and 400 IU of human chorionic gonadotropin (Sigma-Aldrich, Oakville, CA) into the dorsal lymph sac, respectively. The injected male and female frogs were placed in isolated tanks for mating. Embryos were collected the following day and maintained in Modified Barth’s Saline with HEPES (MBSH) in an incubator at 20°C with LED illumination set to a 12 hr/12 hr day-night cycle and staged according to Nieuwkoop and Faber (NF) developmental stages (Nieuwkoop and Faber, 1994 ). All experiments were conducted according to protocol application number 2015–7728 approved by The Animal Care Committee of the Montreal Neurological Institute and in accordance with Canadian Council on Animal Care guidelines. Xenopus laevis sex cannot be determined visually pre-metamorphosis, and thus the sex of experimental animals was unknown.
5
Superovulation of ICR Mice
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Our study was approved by the ethics committee at the Eulji University Institutional Animal Care and Use Committee (EUIACUC 19-19, EUIACUC 20-12). The protocol for superovulation was applied according to our previous report [14 (link)]. Briefly, female ICR mice (aged 5-10 weeks) and male ICR mice (aged 10 weeks-3 months) were mated under controlled light-dark cycle (lights on at 8:00 AM; lights off at 8:00 PM). Before mating, female mice were superovulated with an intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (Prospec, Israel) and 5 IU human chorionic gonadotropin (hCG; Prospec, Israel) 47-48 h later.
6
Analyzing Meiotic Spindle in Ovulated Oocytes
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The chromosome and meiotic spindle in vivo or in vitro ovulated oocytes were localized to evaluate meiotic maturation [48 (link)]. For the in vivo control, six-week-old female ICR mice were superovulated with 7.5 IU pregnant mare serum gonadotropin (Prospec, Israel) and 7.5 IU human chorionic gonadotropin (hCG; Sigma-Aldrich, USA) with a 48-h interval. Then, oocytes within the polar body thought to be in MII were retrieved for further staining. Oocytes were frozen in 4% PFA for 30 min before being permeabilized for 15 min in DPBS with 0.1% Triton X-100 and 0.3% bovine serum albumin (BSA). The oocytes were then blocked for 1 h in a 3% BSA solution at room temperature. The oocytes were incubated with primary antibodies as 1:200 anti-alpha tubulin (Cell Signaling Technologies, USA) solution for 2 h at room temperature and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies as 1:1000 Alexa 488 (Life Technologies, USA). Finally, oocytes were mounted with Vectorshield mounting solution (Vector Laboratories, Burlingame, USA) containing 4′6-diamidino-2-phenylindole (DAPI, Sigma, MO, USA). Images were taken using an upright fluorescence microscope (Axio Imager M2, Carl Zeiss, Germany) at 400 × magnification [49 (link),50 (link)].
7
Porcine Oocyte Maturation In Vitro
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Porcine ovaries were obtained from a local slaughterhouse and transported to the laboratory in 0.9% saline containing 0.75 µg/mL benzyl-penicillin potassium (Wako, Osaka, Japan) and 0.5 µg/mL streptomycin sulfate salt at 37–38 °C. After the ovaries had been washed, cumulus oocyte complexes (COCs) were aspirated from follicles (diameter: 3–8 mm) using a 10 mL syringe with an 18-gauge needle. COCs with three or more layers of cumulus cells and homogeneous cytoplasm were selected and washed three times in 0.9% saline with 1 mg/mL bovine serum albumin (BSA). Washed COCs were incubated in IVM I medium for 22 h at 38.5 °C and 5% CO2 in air. During the first period of maturation (0–22 h), the IVM I medium consisted of 10% porcine follicular fluid, 0.57 mM cysteine, 25 μM β-mercaptoethanol, 10 ng/mL epidermal growth factor, 10 IU/mL pregnant mare serum gonadotropin (Prospec, East Brunswick, NJ, USA), and 10 IU/mL human chorionic gonadotropin (Prospec). After the first maturation period, a second period (from 22 to 44 h) was initiated. The same media was used (without hormone).
8
Conditional RUNX1 Expression in Endothelial Cells
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This study was performed in accordance with the approved institutional animal care and use committee (IACUC) protocol 803789 of the University of Pennsylvania. We obtained Tg(Cdh5-CreERT) [Tg(Cdh5-cre/ERT2)1Rha] mice from Ralf Adams (Sörensen et al. 2009 (link)), and RUNX1 conditional knock-in mice (Rosa26Runx1/Runx1) from Qiufu Ma (Qi et al. 2017 (link)). Timed matings were performed between Rosa26Runx1/Runx1, Rosa26Runx1/+, or +/+ females and Tg(Cdh5-CreERT); Rosa26Runx1/Runx1 males to obtain E13.5 fetuses. B6C3F1 females were purchased from Charles River Laboratories, superovulated, and mated to Tg(Cdh5-CreERT); Rosa26Runx1/Runx1 males to obtain E9.5 embryos. For superovulation, 3-wk-old B6C3F1 females were injected intraperitoneally with 5 IU of pregnant mare serum gonadotropin (Prospec Protein Specialists) 2 d prior to mating. Forty-eight hours later, the B6C3F1 females were injected intraperitoneally with 5 IU of human chorionic gonadotropin (Sigma-Aldrich) and placed in cages with males overnight for mating. For embryonic and fetal experiments, pregnant dams were injected intraperitoneally with 2 mg of tamoxifen (Sigma-Aldrich). One-month-old mice received 2 mg of tamoxifen via oral gavage.
9
Traditional Chinese Medicine Granules
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XYS granules was provided by Guangdong Yi Fang Pharmaceutical Co Ltd. (Guangzhou, China), which consists of radix bupleuri (chaihu) (16.2%), angelica (danggui) (16.2%), radix paeoniae alba (baishao) (16.2%), rhizoma atractylodis macrocephalae (baizhu) (16.2%), poria (fuling) (16.2%), ginger (shengjiang) (5.4%), mint (bohe) (5.4%), and glycyrrhizae (gancao) (8.1%). Antibodies against beta 2 adrenergic receptor (β2R), S6K I, phosphor-p70 S6K I (Thr229) and c-fos were purchased from Abcam (Cambridge, UK). Antibodies against Bcl-2 (B-cell lymphoma-2), Bax (B-cell lymphoma-2 associated X protein), cleaved caspase-3 (cleaved cysteinly aspartate specific proteinase-3), microtubule-associated protein light chain 3A (LC3A), LC3B, Akt and phosphor-Akt (Ser473) were purchased from Cell Signaling (Danvers, MA, USA). Antibody against dopamine beta hydroxylase (DβH) was purchased from Thermo Scientific (Rockford, IL, USA). (-)-Noradrenaline (NE) was from Sigma Chemical Co (St Louis, MO, USA). Pregnant mare serum gonadotropin was from ProSpec (Ness Ziona, Israel).
10
Aged Mice Endometrial Responses
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All experiments and analyses were conducted in accordance with the relevant guidelines and regulations. The animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the Korea Institute of Oriental Medicine, Daejeon, Korea (approval number 20–090). Female BALB/c mice aged 8 weeks (young, n = 6) and 40 weeks (old, n = 6) (Central Lab Animal Inc., Seoul, Korea) were housed under specific pathogen-free conditions and orally administered either distilled water or 2.5 g/kg SM (Hanpoong, Iksan, Korea) five times per week for 4 weeks. The mice were administered 5 IU of pregnant mare serum gonadotropin (Prospec, Rehovot, Israel) and 5 IU of human chorionic gonadotropin (Prospec) to mimic the gonadotropin releasing hormone-stimulated endometrial cycle before embryo transfer in in-vitro fertilization patients. The hormonally stimulated uteri from the mice were removed and immediately placed in liquid nitrogen until mRNA sequencing.
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