Mr 070 d

Spontaneously cycling adult females were housed singly with a B6D2F1/J male overnight. Zygotes were collected from the oviduct at 11:00 am on 0.5 dpc. Two-cell embryos were collected from the oviduct at 9:00 am on 1.5 dpc. Unless otherwise specified, embryos from each mouse were cultured separately in microdrops of potassium simplex optimized medium with amino acids (KSOM/AA; Millipore, Billerica, MA) covered with mineral oil and embryo morphological appearance was documented daily. To test the effects of PG E₂ (PGE₂) on fertilization and embryo development, oocytes were fertilized in Nunc four-well culture plates (Thermo Scientific, Grand Island, NY) in 400 µL human tubal fluid medium (MR-070-D, Millipore) containing 0.1% ethanol (vehicle control) or 1 μM PGE₂ (Cayman Chemical, Ann Arbor, MI). Alternatively, pronuclear stage zygotes from superovulated and mated CF-1 females were cultured in 400 µL KSOM/AA containing 0.1% ethanol or 1 μM PGE₂. For these experiments, the medium was not covered with mineral oil to avoid loss of the PGE₂ from the aqueous phase.

Cumulus-oocyte complexes from superovulated C57Bl6 mice were collected by piercing oviducts placed in human tubal fluid (HTF; Millipore catalogue no. MR-070-D) 20 h after hCG injection. Oocytes were immediately inseminated after collection, as described by Ferrer et al. [84 (link)]. Spermatozoa from an adult male (3 Hgsnat+/+ and 3 Hgsnat−/−) mice 7 month-old were squeezed out of freshly isolated mouse cauda epididymides in HTF media using sterile forceps and allowed to ‘swim out’ for 10 min at 37°C. Sperm were counted using a hemocytometer and inseminated with an equal number of oocytes. Insemination and subsequent wash and rest steps occurred in 50 μl HTF drops overlaid with sterile mineral oil in a culture dish. Cumulus-oocyte complexes were inseminated with 10⁶ sperm/ml in HTF. After 20 h incubation (37°C, 5% CO2), oocytes successfully fertilized were assessed by the presence of a second polar body or cleavage. Trials for each group were repeated at least three times. A chi-square homogeneity test with the Bonferroni correction was performed to assess the significant difference between the groups.

ICSI for human subjects was performed at the Reproductive and Genetic Hospital of CITIC-Xiangya, as described in our earlier study (Tan et al., 2022 (link)). ICSI with mouse sperm was performed as previously described (Liu et al., 2021 (link); Cong et al., 2022 (link)). Briefly, cauda epididymal sperm were obtained and maintained in HTF medium (Millipore, MR-070-D, Billerica, MA, USA) (Wang et al., 2023b ). Two-month-old B6D2F1 WT female mice were superovulated using 7.5 IU of pregnant mare serum gonadotropin, followed by 7.5 IU of hCG 48 h later, and then oocytes were collected. Sperm were injected into oocytes, and the ICSI embryos were then cultured in KSOM+AA WITH D-GLUCOSE medium (Millipore, MR-107-D) at 37°C under 5% CO₂.