G ivf media

Ovarian stimulation was carried out using a long luteal gonadotropin-releasing hormone (GnRH) agonist protocol or an antagonist protocol, as described previously[18 (link)], Briefly, once two-thirds of the follicles reached 18 mm, 5,000–10,000 IU of human chorionic gonadotropin (hCG; Pregnyl, Merck) was injected. At 35–36 h after hCG administration, oocytes were collected in G-IVF media (Vitrolife) under transvaginal ultrasound guidance.

Donor sperm were first collected from male mice into G-IVF media (Vitrolife, Goteborg, Sweden) and incubated under oil for 1 h at 37 ℃ in 5% CO
₂ for capacitation. Metaphase Ⅱ oocytes were then placed into 250 μL of media with sperm (2 × 10
⁵/mL to 3 × 10
⁵/mL) for fertilization. Six hours later, zygotes with clear pronuclei were transferred into fresh G-1 media (Vitrolife) overnight until the two-cell embryonic. The fertilized embryos were cultured in G-1 media for five days to the blastocyst stage.

PMSG‐primed mice were received one injection of mTOR inhibitor, rapamycin (2 mg/kg, Sigma) 1 h before HCG treatment and ovaries and oocytes were collected, respectively, 14–16 h later. Collected ovaries were used to evaluate the effect of rapamycin on primordial follicle activation and oocytes were used to check its effect on oocyte maturation, fertilisation and early embryonic development. For IVF studies, donor sperm were first collected from male mice into G‐IVF media (Vitrolife) and incubated under oil for 1 h at 37°C in 5% CO₂ for capacitation. MII oocytes were then placed into 250 μl of media with sperm (2–3 × 10⁵/ml) for fertilisation. Six hours later, zygotes with clear pronuclei were transferred into fresh G‐1 media (Vitrolife, Sweden) overnight until the two‐cell embryonic stage.