Human chorionic gonadotropin (hcg)

In order to keep the quality of the sperm, male mice were housed with female mice (do not belong to NC, POI, or EVs groups) for 1 wk, then housed alone for 1 wk before sperm collection. Six female mice in each group (NC, POI, and EVs groups) were superovulated via a single intraperitoneal injection of 10 IU pregnant mare serum gonadotropin (PMSG, Zhejiang, China) 4 wk after treatment, followed by injection of 10 IU human chorionic gonadotropin (hCG, Sansheng, Ningbo, Zhejiang, China) 48 h later. The cumulus oocyte complexes were collected from the ampulla portion of the oviduct under stereomicroscope 12–14 h after hCG administration and placed in IVF culture media (Vitrolife, Stockholm, Sweden) at 37°C in 5% CO₂ air. Meanwhile, the 12–13-wk-old male mice were sacrificed by cervical dislocation; the sperm in epididymides were collected through 5–7 longitudinal cuts on epididymis with a syringe needle, and then incubated for at least 1 h at 37°C in 5% CO₂ air for capacitation in IVF culture media. The metaphase II oocytes were incubated with sperm to facilitate fertilization in IVF culture media for 5–6 h, and fertilized zygotes were subsequently cultured in G1 and G2 culture media (Vitrolife) at 37°C in 5% CO₂ air²⁰ . The development of the fertilized zygote was observed and recorded.