Human chorionic gonadotropin (hcg)

For ZP isolation, wild-type female mice were super-ovulated by intraperitoneal injection of 10 I.U. hCG (human Chorionic Gonadotropin; ProSpec, Rehovot, Israel) 3 days before the experiment. 14 h before oocyte isolation, mice were injected with 10 I.U. PMSG (Pregnant Mare’s Serum Gonadotropin; ProSpec). Mice were killed by cervical dislocation and oviducts were dissected. Cumulus-enclosed oocytes were prepared from the oviducts in TYH buffer containing 300 μg/ml hyaluronidase (Sigma). After 15 min, cumulus-free oocytes were transferred into fresh buffer and washed twice. Zonae pellucidae and oocytes were separated by shear forces generated by expulsion from 50 nm pasteur pipettes. Zona pellucidae were counted, transferred into fresh buffer, diluted to a concentration of 1 ZP per ul, and solubilized by incubation at 75°C for 15 min (Thaler and Cardullo, 1996 (link)). Animal experiments were performed in accordance with the relevant guidelines and regulations and approved by the local authorities (LANUV) AZ84-02.05.40.13.127.

At least 2-month-old female CD1 or C57BL/6J mice were administrated with 2.5 IU of PMSG (Cat# HOR-272, Prospecbio) at 5:30 p.m. 3 d before artificial insemination, followed by 2.5 IU of hCG (Cat# HOR-250, Prospecbio) at 5:00 p.m. 1 d prior to AI. The next morning at 8:00 a.m., ~2-month-old mTmG and quinKO male mice were sacrificed, the cauda epididymis was dissected, and fat tissue and blood were removed before placing the cauda epididymis into 500 μl or 150 μl of EmbryoMax Human Tubal Fluid (HTF) (1×) (Cat# MR-070-D, MilliporeSigma) containing 4 mg/ml BSA (Cat# 12659-250GM, EMD Millipore Corp) (HTF-BSA) covered with 4 ml of mineral oil (Cat# M8410-500ML, Sigma). Three incisions were made on the cauda to allow sperm to swim out and to get capacitated for at least 30 min. 25 μl of mTmG and 25 μl quinKO sperm suspensions were mixed, and 40 μl (if using 500 μl HTF-BSA) or 25 μl (if using 150 μl HTF-BSA) of the mixed sperm were delivered to superovulated females using C&I Device for Mice (Cat# 60020, Paratech) at 9:00 a.m. Recipients were immediately paired with vasectomized males overnight. The next day, the plug was checked and the female mice with plugs were used for collecting embryonic day 10 (E10) embryos, and the ones without plugs were used for collecting zygotes, two-cell embryos, morulae, or blastocysts embryos.

Mature female golden hamsters 8–12 weeks old were injected with 30 IU of pregnant mare serum gonadotropin (PMSG, ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel) intraperitoneally, followed by an injection of 37 IU of human chorionic gonadotropin (hCG, ProSpec-Tany TechnoGene Ltd., Ness-Ziona, Israel) 56 h later. The hamsters were euthanized using a CO₂ chamber 17 h after administration of hCG injection. The oviducts were excised and placed in culture dishes containing saline. The ampule was tore and cumuli-oocyte complexes were collected and treated with hyaluronidase (SAGE, Trumbull, United States) for 1–2 min of incubation at 37°C. The remained cumulus cells were mechanically denuded with stripped pipette in 10% HEPES (SAGE, Trumbull, United States).

After the evaluation of oocyte quality, COCs were washed three times in Dulbecco´s modified Eagle´s medium (DMEM supplemented with 10 IU/mL PMSG (Prospec, Israel) and hCG (Prospec, Israel), 50 ng/ mL EGF, 100 ng/mL IGF1 and 5 ng/ mL FGF). The DMEM medium was pre-warmed for 1 hour in a Petri dish. COCs were cultured and placed in groups of 30 in 500 µL DMEM, for 44 hours at 38.5 °C under 5% CO₂ in air.

To obtain germinal vesicle stage (GV) oocytes, females were superovulated with 5 IU equine chorionic gonadotropin (eCG, Prospec, Sturgeon County, AB, Canada) administered by intraperitoneal (IP) injection. Ovaries were excised and minced in Hepes-KSOM at 44–46 h post-eCG. Cumulus-oocyte complexes (COCs) were collected, and cumulus cells were removed by repeated pipetting to obtain denuded oocytes.
One-cell stage embryos were obtained by inducing ovulation with an IP injection of 5 IU human chorionic gonadotropin (hCG, Prospec) at 47 h post-eCG. Females were then caged overnight with BDF1 males (Charles River Canada) following hCG injection. Embryos were obtained 21–24 h post-hCG by flushing oviducts with Hepes-KSOM medium using a blunt-end syringe.

IVF was performed by the method of Nakao et al. (61 (link)). Female mice were intraperitoneally injected 7.5 IU/100 μl pregnant mare serum gonadotropin (PMSG, Prospec-Tany Technogene). At 48 h after PMSG injection, mice were intraperitoneally injected 7.5 IU/100 μl human chorionic gonadotropin (hCG, Prospec-Tany Technogene). At 15 h after the hCG injection, their oviducts were collected and transferred to human tubal fluid medium on Ovoil in 60 mm³ dish. Cumulus–oocyte complexes were collected from the oviducts and transferred into a drop of sperm suspension (5 × 10⁵ sperm) and covered with Ovoil. After 6 h incubation at 37 °C in 5% CO₂, the oocytes were collected and washed three times in 100 μl drops of human tubal fluid covered with Ovoil. After 24 h co-incubation, the fertilization rate was calculated by the formula: fertilization rate (%) = the total number of two-cell embryos/the total number of oocytes. Groups of male mice were treated with 40 mg/l ramipril or 600 mg/l losartan in drinking water for a week before sperm isolation. For PPARγ blockade, isolated sperm were pretreated with GW9662 for 12 h before inoculation with oocytes.

C57BL/6 mice were obtained from Koatech (PyeongTaek, Korea) and maintained under specific pathogen-free conditions. Female mice were injected with 5 IU of serum gonadotropin (Prospec Bio, East Brunswick, NJ, USA) and 5 IU of hCG (Prospec) at 48 h intervals for estrus synchronization and superovulation. The female mice were then mated with the sperm donor mice at 2 p.m. Embryos were collected from the oviducts 20 h later at 10 a.m. the next day, and were cultured in KSOM medium (Merck Millipore, Billerica, MA, USA). This study was approved by the Institutional Animal Care and Use Committees of Seoul National University (SNU-180315-4, SNU-1708164, and SNU-180827-4) and was conducted in accordance with recommended guidelines.