Pregnant mare serum gonadotropin

To induce a preovulatory surge of plasma estradiol levels that sets in motion the LP phase of puberty, we injected 26-day-old female rats intraperitoneally (i.p.) with 5 IU of pregnant mare serum gonadotropin (Sigma-Aldrich, St. Louis, MO, USA), as previously described^{48 (link)}. The MBH from each animal was collected 48 h later for molecular analysis.

To generate immunodeficient mice using CRISPR/Cas9, microinjection of fertilized embryos was performed: initially, 6–8-week-old BALB/c, B6, and FVB mice were super-ovulated by intra-peritoneal injections of 5 IU pregnant mare serum gonadotropin (Sigma) and 5 IU human chorionic gonadotropin (Sigma) at 48-h intervals. The fertilized embryos were then collected from the super-ovulated mice crossed with stud males. A mixture of 50 ng/μL of Cas9 mRNA and 250 ng/μL of sgRNA was microinjected into the cytoplasm of zygotes, using a piezo-driven manipulator (Prime Tech) to induce mutations, and the resulting embryos were transferred into the oviducts of ICR pseudo-pregnant foster mothers to produce live mice.

Both oocytes and cumulus cells were prepared by the superovulation of 8‐ to 10‐week‐old B6D2F1 female mice. Superovulation was induced by sequential injection 5 IU of pregnant mare serum gonadotropin (Sigma‐Aldrich), followed by 5 IU of human chorionic gonadotropin (hCG, Sigma‐Aldrich) with an interval of 48 hours. Cumulus‐oocyte complexes (COCs) were collected from oviducts in M2 medium (Sigma‐Aldrich) at 14 hours after hCG injection and were treated with M2 containing 0.1% bovine testicular hyaluronidase (Sigma‐Aldrich) to obtain dissociated cumulus cells and oocytes. The cumulus‐free oocytes were then cultured in potassium simplex optimized medium (KSOM; Millipore) at 37°C under 5% CO₂ until further use. Dispersed cumulus cells were dissociated by hyaluronidase treatment, diluted in M2 medium and collected. The pellet was then resuspended in a small volume of polyvinylpyrrolidone (PVP) in M2 medium in a manipulator chamber.

Pregnant mare serum gonadotropin (PMSG), bovine serum albumin (BSA), sodium pyruvate, citrinin and liquiritigenin were purchased from Sigma (St. Louis, MO, USA). Human chorionic gonadotropin (hCG) was obtained from Serono (NV Organon, Oss, The Netherlands). The TUNEL in situ cell death detection kit was purchased from Roche (Mannheim, Germany) and CMRL-1066 medium from Gibco Life Technologies (Grand Island, NY, USA).

NOD.CB17/Prkdcscid/JKrb (NOD/SCID) mice have been bred in KRIBB for more than 20 years after receiving them from Jackson Laboratory in 1999. Female NOD/SCID mice (4–5 weeks of age) were superovulated by intraperitoneal injection with 5 IU pregnant mare serum gonadotropin (sigma, St. Louis, USA), followed 46 h later by injection of 5 IU human chorionic gonadotropin (hCG, sigma, USA). The animals were sacrificed 14 h following hCG administration and oviducts were collected. Cumulus oocyte complexes were released from the oviducts and placed in pre-equilibrated fertilization drops consisting of HTF Media. Fresh sperm was isolated from the epididymis and vas deferens of male NOD/SCID mice (5 months of age) into HTF medium. Fertilization was carried out in HTF medium by transferring cumulus oocyte complexes to fertilization drops which contained the activated sperm and was incubated for 8 h at 37 °C (5% CO₂). After incubation, the oocytes were washed in fresh HTF medium to remove excess sperm, incubation for 7 h at 37 °C (5% CO₂) in M16 medium and microinjection performed in M2 medium.